Plant- and Marine-derived n-3 Polyunsaturated Fatty Acids ...
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Plant- and Marine-derived n-3 Polyunsaturated Fatty Acids Prevent Mammary Tumor Development by Jiajie Liu A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Human Health and Nutritional Sciences Guelph, Ontario, Canada © Jiajie Liu, August, 2015
Transcript of Plant- and Marine-derived n-3 Polyunsaturated Fatty Acids ...
Plant- and Marine-derived n-3 Polyunsaturated Fatty Acids Prevent
Mammary Tumor Development
by
Jiajie Liu
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Master of Science
in
Human Health and Nutritional Sciences
Guelph, Ontario, Canada
© Jiajie Liu, August, 2015
ABSTRACT
PLANT- AND MARINE-DERIVED N-3 POLYUNSATURATED FATTY ACIDS
PREVENT MAMMARY TUMOR DEVELOPMENT
Jiajie Liu Advisor:
University of Guelph, 2015 Dr. David WL Ma
Marine-derived n-3 polyunsaturated fatty acids (PUFA) are shown to inhibit
mammary carcinogenesis. However, evidence regarding α-linolenic acid (ALA), a plant-
based and major n-3 PUFA in Western diet, remains equivocal. This study examined the
inhibitory potency of plant- versus marine-derived n-3 on mammary tumorigenesis. Female
MMTV-neu(ndl)YD5 mice were lifelong exposed to one of four oil diets: 1) 10% safflower
(n-6, control), 2) 10% flaxseed, or 7% safflower plus either 3) 3% flaxseed, or 4) 3%
menhaden. Compare to control, 10% flaxseed and 3% menhaden oil had similar inhibitory
effects, which significantly reduced terminal end buds, and decreased overall tumor
outcomes by regulating tumor protein expression (p<0.05). A significant dose-dependent
reduction on tumor outcomes were observed in mice fed 3% and 10% flaxseed oil (p<0.05).
However, 3% flaxseed had weaker inhibitory potency compared to 3% menhaden oil
(p<0.05). Overall, marine-based n-3 PUFA are more potent than plant-derived ALA in
mitigating mammary tumorigenesis.
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my parents for supporting my all the way
through my undergraduate and master studies. Without your support, I cannot study
abroad and have different life experiences on the other side of the world. Thank you so
much for the encouragement and accompanying me through the good and bad days. None
of this would have been possible without your love and patience. I love you.
Second, I would like to express my deep and sincere gratitude to my supervisor,
Dr. David Ma for his advice, support and guidance throughout the entirety of this project.
I would like to thank him for giving me the incredible opportunity to work in his lab and
conduct this research project. It has been an unforgettable experience. To my committee
members, Dr. Lindsay Robinson and Dr. Krista Power, thank you for your counsel
throughout this process.
A big thank you to my wonderful lab mates, past and present, with whom I spent
many of my working hours over the past two years:
• Lyn Hillyer---Thank you so much for taking the Ma Lab under your motherly
wing, for managing to take care of all of us, from protocols to statistics to treats in
the lab. I am truly thankful for your extensive guidance and patience.
• Mike Leslie, Daniel Cohen, Elie Chamoun, Salma Abdelmagid, Chris Pinelli---
Thank you so much for the lab training, editing my report and making me laugh
all the time. You gave me lots of happy moments during these two years although
I still don't understand your jokes sometime. Love you all.
Last but not least, thank you to Dr. Chaowu, Dr. Tyler Avis and Dr. Veronic
Bezaire, who always made time to chat, advice, write reference letters and put me in
contact whenever research opportunities arise. It was a great privilege to get to know you
as mentors and friends.
My thesis work was funded by Canadian Institutes of Health Research and
Ontario Graduate Scholarship.
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TABLE OF CONTENTS
Title Page
Abstract
Acknowledgements ........................................................................................................................ iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................. ix
List of Figures ................................................................................................................................. x
List of Abbreviations ..................................................................................................................... xi
Chapter 1. Literature Review: Individual protective effect of n-3 Polyunsaturated
Fatty acids on Breast Cancer Development ............................................................ 1
1.1 Abstract .............................................................................................................................. 2
1.2 Introduction ........................................................................................................................ 2
1.3 The effects of n-3 PUFA in Human Breast Cancer studies ................................................ 4
1.4 PUFA – Potential Mechanisms of Action ....................................................................... 11
1.4.1 Influence on Cell Plasma Membrane Composition ............................................... 11
1.4.2 Inhibition of Arachidomic Acid Derived Eicosanoid Biosynthesis ....................... 12
1.4.3 Influence on Gene Expression and Signaling Transduction ................................... 13
1.5 The Effect of n-3 PUFA Mixtures on Breast Cancer Development ................................ 19
1.5.1 Animal Models ........................................................................................................ 19
1.5.1.1 Breast Cancer Studies in Xenograft Rodent Models ................................. 19
1.5.1.2 Breast Cancer Studies in Transgenic Rodent Models ................................ 20
1.5.1.3 Breast Cancer Studies in Chemically-Induced Rodent Models ................. 24
1.5.2 Cell Culture Studies ................................................................................................ 25
1.6 The effect of Individual n-3 PUFA on Breast Cancer Development ............................... 31
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1.6.1 ALA and Breast Cancer .......................................................................................... 31
1.6.1.1 Inefficient Conversion from ALA to EPA and DHA ................................. 31
1.6.1.2 Individual Effect of ALA on Breast Cancer............................................... 32
1.6.2 Individual Effect of EPA on Breast Cancer ............................................................. 34
1.6.3 Individual Effect of DHA on Breast Cancer ........................................................... 35
1.7 Plant-Derived n-3 (ALA) vs. Marine-Based n-3 (EPA, DHA) ........................................ 37
1.8 Conclusion ........................................................................................................................ 38
Chapter 2. Updated Mini Literature Review - Role of Flaxseed Oil in the Prevention
and Treatment of Breast Cancer ............................................................................ 43
2.1 Introduction ...................................................................................................................... 44
2.2 Role of Flaxseed in Mammary Gland Development and Breast Cancer Risk ................. 45
2.2.1 Animal Study............................................................................................................ 45
2.2.1.1 Early Life Exposure ..................................................................................... 45
2.2.1.2 Adulthood Exposure Once Breast Cancer is Established ............................ 47
2.2.2 Cell Culture Study .................................................................................................... 49
2.2.3 Human Observational Study .................................................................................... 50
2.2.4 Human Intervention Study ....................................................................................... 50
2.3 Role of Flaxseed Oil in Mammary Gland Development and Breast Cancer Risk ........... 51
2.3.1 Animal Study............................................................................................................ 51
2.3.1.1 Early Life Exposure ..................................................................................... 51
2.3.1.2 Adulthood Exposure Once Breast Cancer is Established ............................ 52
2.3.2 Cell Culture Study .................................................................................................... 55
2.3.3 Human Observational Study .................................................................................... 56
2.4 Conclusion and Future Directions .................................................................................... 56
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Chapter 3: Rationale, Hypothesis, Objective and Experimental Design ............................... 63
3.1 Rationale .......................................................................................................................... 64
3.2 Mouse Model: MMTV-neu (ndl) YD5 ............................................................................ 64
3.3 Hypothesis ........................................................................................................................ 66
3.4 Objective .......................................................................................................................... 66
3.5 Experimental Design ........................................................................................................ 66
Chapter 4. Plant- and Marine-derived n-3 Polyunsaturated Fatty Acids Prevent
Mammary Tumor Development ............................................................................. 68
4.1 Abstract ............................................................................................................................ 69
4.2 Introduction ...................................................................................................................... 70
4.3 Material and Methods....................................................................................................... 72
4.3.1 Animals and Diets ................................................................................................... 72
4.3.2 Food Intake, Body Weights, Puberty Onset ........................................................... 73
4.3.3 Early Mammary Gland Developmental Period (6-week-timepoint) ...................... 76
4.3.3.1 Euthanization and Tissue Collection .......................................................... 76
4.3.3.2 Mammary Gland Whole Mounts................................................................ 76
4.3.3.3 Counting of TEB structures ....................................................................... 77
4.3.4 Mammary Tumor Developmental Period (20-week-timepoint) ........................... 77
4.3.4.1 Tumor Palpation ........................................................................................ 77
4.3.4.2 Euthanization and Tissue Collection ......................................................... 78
4.3.4.3 Fatty Acid Anlysis ..................................................................................... 78
4.3.4.4 Western Blot Analysis ............................................................................... 80
4.3.4.5 ELISA........................................................................................................ 81
4.3.4.6 Immunohistochemistry .............................................................................. 81
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4.3.5 Statistical Analysis ............................................................................................... 82
4.4 Results .............................................................................................................................. 83
4.4.1 Food Intake, Body Weight Gain and Puberty Onset ............................................... 83
4.4.2 TEB Structure in 6-week-old Mice ......................................................................... 85
4.4.3 Tumor Latency and Tumor Free Status................................................................... 88
4.4.4 Tumor Volume ........................................................................................................ 92
4.4.5 Tumor Multipicity ................................................................................................... 92
4.4.6 Final Total Tumor Weight....................................................................................... 93
4.4.7 Fatty Acid Composition .......................................................................................... 94
4.4.7.1 Fatty Acid Composition in Serum ................................................................ 94
4.4.7.2 Fatty Acid Composition in Mammary Gland ............................................... 94
4.4.7.2 Fatty Acid Composition in Mammary Tumors ............................................ 97
4.4.8 Tumor Protein Analysis ......................................................................................... 102
4.4.8.1 Her2 and pHer2 ........................................................................................... 102
4.4.8.2 Akt and pAkt ............................................................................................... 103
4.4.8.3 Ki67 and Cleaved-caspase-3 ....................................................................... 104
4.5 Discussion ....................................................................................................................... 107
4.5.1 Role of Plant- and Marine-derived n-3 PUFA in Mammary Gland
Development and Breast Cancer Risk .................................................................... 107
4.5.2 Role of Plant- and Marine-derived N-3 PUFA in Mammary Tumor
Development ........................................................................................................... 109
4.5.3 Potential Mechanisms of Anti-tumorigenic Actions .............................................. 111
4.5.3.1 Change Membrane Fatty Acid Composition ............................................. 111
4.5.3.2 Modulate Cellular Signaling Molecules .................................................... 113
4.5.4 Physiological Relevance to Human Intake............................................................. 116
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4.6 Conclusion ...................................................................................................................... 117
Chpater 5. General Discussion and Future Directions .......................................................... 118
5.1 General Doscission ......................................................................................................... 119
5.2 Limitations ...................................................................................................................... 120
5.3 Future Directions ............................................................................................................ 121
5.4 Concluding Remarks ...................................................................................................... 122
Chapter 6. References and Appendix...................................................................................... 123
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LIST OF TABLES
Table 1.1 N-3 PUFA and breast cancer risk: prospective cohort studies ....................................... 6
Table 1.2 N-3 PUFA and breast cancer risk: case-control studies ............................................. 7-8
Table 1.3 N-3 PUFA and breast cancer risk: xenograft rodent models ....................................... 22
Table 1.4 N-3 PUFA and breast cancer risk: transgenic rodent models ...................................... 23
Table 1.5 N-3 PUFA and breast cancer risk: chemically-induced rodent models .................. 27-28
Table 1.6 N-3 PUFA and breast cancer risk: cell culture studies ........................................... 29-30
Table 1.7 Individual role of ALA, EPA and DHA on BC ...................................................... 39-41
Table 1.8 Individual effect of ALA, EPA and DHA on different types of BC ........................... 42
Table 2.1 Early exposure to flaxseed and breast cancer risk ....................................................... 57
Table 2.2 Late life exposure to flaxseed and breast cancer risk ............................................. 58-59
Table 2.3 Late life exposure to flaxseed oil and breast cancer risk ........................................ 60-62
Table 4.1 Composition of purified diets ...................................................................................... 74
Table 4.2 Fatty acid composition of diets .................................................................................... 75
Table 4.3 Serum fatty acid composition of mice at 6 or 20 weeks of age ................................... 95
Table 4.4 Fatty acid composition of MG phospholipids ............................................................... 98
Table 4.5 Fatty acid composition of mammary tumor phospholipids ........................................ 101
x
LIST OF FIGURES
Figure 1.1 Synthetic pathways of long-chain PUFA and eicosanoids .......................................... 14
Figure 1.2 Hypothetical scheme showing how n-3 PUFA modulates cell fuctions ..................... 15
Figure 4.1 Mice body weight changes throughout 3 to 20 weeks of age...................................... 84
Figure 4.2 Average pubertal onset ................................................................................................ 85
Figure 4.3 Representative stereoscopic wholemount images ....................................................... 86
Figure 4.4 Average total number and density of terminal end buds at 6 weeks of age ................ 87
Figure 4.5 Average tumor latency ................................................................................................ 88
Figure 4.6 The proportion of mice tumor free throughout 20-week study ................................... 89
Figure 4.7 Tumor volume. ............................................................................................................ 90
Figure 4.8 Tumor multiplicity....................................................................................................... 91
Figure 4.9 Final total tumor weight .............................................................................................. 93
Figure 4.10 Mammary gland fatty acid composition .................................................................... 96
Figure 4.11 Mammary Tumor fatty acid composition. ............................................................... 100
Figure 4.12 Effect of plant- versus marine-derived n-3 PUFA on tumor protein
expression of HER-2 and pHER-2 .......................................................................... 102
Figure 4.13 Effect of plant- versus marine-derived n-3 PUFA on tumor protein
expression of total Akt and pAkt
Ser 473 ..................................................................... 103
Figure 4.14 Effect of plant- versus marine-derived n-3 PUFA on tumor protein
expression of Ki-67 and cleaved-caspase-3 ............................................................. 104
Figure 4.15 The photomicrographs of representative immunohistochemical stained
mammary tumor section with Ki67 ......................................................................... 105
Figure 4.16 The photomicrographs of representative immunohistochemical stained
mammary tumor section with cleaved-caspase-3 .................................................... 106
xi
LIST OF ABBREVIATIONS
AA: arachidonic acid
AB: alveolar buds
ALA: alpha-linolenic acid
Akt: protein kinase B
BC: breast cancer
BMI: body mass index
COX: cyclooxygenase
DHA: docosahexaenoic acid
DMBA: 7, 12-dimethylbenz(a)anthracene
EPA: eicosapentaenoic acid
FASN: fatty acid synthase
GC: gas chromatography
HER-2: human epidermal growth factor-2
LA: linoleic acid
5-LOX: 5-lipoxygenase
LTB4: leukotriene B4
MG: mammary gland
MMTV: mouse mammary tumor virus
MUFA: monounsaturated fatty acids
MNU: N-methyl-N-nitrosourea
NFĸB: nuclear factor kappa B
N-3 PUFA: omega-3 polyunsaturated fatty acids
N-6 PUFA: omega-6 polyunsaturated fatty acids
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pAkt: phosphorylated- protein kinase B
PCR: polymerase chain reaction
PC: phosphatidylcholine
PE: phosphatidylethanolamine
PGE2: prostaglandin E2
PPARγ: peroxisome proliferator-activated receptor gamma
PUFA: polyunsaturated fatty acids
TEB: terminal end bud
TG: transgenic
WT: wild type
1
Chapter One
Literature Review
Individual Protective Effect of n-3 Polyunsaturated Fatty Acids on
Breast Cancer Development
Published as an open access article in Nutrients 2014, 6:5184-5223- “The Role of n-3
Polyunsaturated Fatty Acids in the Prevention and Treatment of Breast Cancer” by Jiajie
Liu and David W.L. Ma
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1.1 Abstract
Breast cancer (BC) is the most common cancer among women worldwide.
Dietary fatty acids, especially n-3 polyunsaturated fatty acids (PUFA), are believed to
play a role in reducing BC risk. Evidence has shown that fish consumption or intake of
long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA), are beneficial for inhibiting mammary carcinogenesis. The evidence regarding α-
linolenic acid (ALA), however, remains equivocal. It is essential to clarify the relation
between ALA and cancer since ALA is the principal source of n-3 PUFA in the Western
diet and the conversion of ALA to EPA and DHA is not efficient in humans. In addition,
the specific anticancer roles of individual n-3 PUFA, alone, have not yet been identified.
Therefore, the present review evaluates ALA, EPA and DHA consumed individually as
well as in n-3 PUFA mixtures. Also, their role in the prevention of BC and potential
anticancer mechanisms of action are examined. Overall, this review suggests that each n-
3 PUFA has promising anticancer effects and warrants further research.
1.2 Introduction
Breast cancer (BC) is a major health problem among women worldwide, and is
the second leading cause of death for women in Canada and the United States (1, 2). On
average, 65 Canadian women will be diagnosed with BC per day, with 1 in 9 females
expected to develop BC in their lifetime (1, 2). Both genetic and environmental factors
are believed to play a role in a woman’s risk of developing BC (3, 4). Most anticancer
drugs, developed to date, aim to kill cancer cells and decrease tumor burden but are
relatively ineffective against some phases of tumorigenesis (5, 6). Thus, alternate
strategies to prevent tumorigenesis are urgently required. In the past few decades,
epidemiological studies have suggested that a healthy diet and lifestyle are critical for the
prevention of BC. Dietary fatty acids are one of the most intensively studied dietary
factors (7-9).
Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and trans fatty
acids (TFA) have been found to increase cancer risk; while specific polyunsaturated fatty
acids (PUFA) are indicated to have anticancer effects (9, 10). There are two major classes
3
of PUFA: n-6 PUFA and n-3 PUFA. In mammals, n-6 and n-3 PUFA are both essential
fatty acids for health and must be consumed as part of the diet because they cannot be
endogenously synthesized (11). Linoleic acid (LA, 18:2n-6) and arachidonic acid (AA,
20:4n-6) are the two most common n-6 PUFA in typical Western diets; LA can be found
in some plant oils such as corn and safflower oils, and AA usually comes from dietary
animal sources or can be synthesized from LA (12, 13). α-linolenic acid (ALA, 18:3n-3)
is the precursor of then-3 PUFA family which can be further elongated and desaturated to
two important long chain n-3 PUFA, eicosapentaenoic acid (EPA, 20:5n-3) and
docosahexaenoic acid (DHA, 22:6n-3) (11). ALA is a plant-derived n-3 PUFA, which is
present in flaxseed, canola and soybean oils (14). The longer chain n-3 PUFA, EPA and
DHA can be obtained directly from marine sources such as seafood and fish oils, and are
widely known for their cardioprotective benefit (13). ALA is the major n-3 PUFA
consumed in the Western diet, whereas intakes of EPA and DHA are typically low. It is
estimated that the typical North American diet provides approximately 1.4 g of ALA and
0.1–0.2 g of EPA plus DHA per day (15). With regard to dietary reference intake of n-3
PUFA, the Institute of Medicine (IOM) recommends since 2005 a daily intake 1.1 g ALA
for women and 1.6 g ALA for men to prevent some chronic diseases, and up to ten
percent of this can be consumed as EPA and/or DHA (16). While current intakes meet
IOM recommendations, in a 2014 report by the Academy of Nutrition and Dietetics, 500
mg EPA plus DHA per day is required for the general healthy adult population (17).
Results from both in vivo and in vitro studies suggest that n-6 PUFA accelerate
tumorigenesis, in contrast, n-3 PUFA may have anticancer effects (18-22). Western diets
are typically deficient in n-3 PUFA and high in n-6 PUFA compared with traditional
Asian diets (8, 23, 24). Migration studies have shown that Asian women, who typically
have a lower rate of BC and higher fish consumption exhibited an increased incidence of
BC within one generation after migration to the Western countries (24, 25). Historically,
the intake of n-6 and n-3 PUFA has been estimated to be approximately equal. However,
in recent years, the content of Western diets has significantly increased in n-6 PUFA
resulting in an increase in the n-6/n-3 PUFA ratio (8). Excessive amounts of n-6 PUFA
and a very high n-6/n-3 ratio (16:1 or higher), as is currently found in Western diets, have
been suggested to promote the pathogenesis of many diseases such as cardiovascular
4
disease, autoimmune diseases and some types of cancer; whereas increased levels of n-3
PUFA (a low n-6/n-3 ratio) have been shown to exert suppressive effects (8). However, it
remains to be resolved whether it is simply the reduced n-3 PUFA or the changing n-6/n-
3 ratio that is relevant to these outcomes.
Substantial evidence from cell culture and rodent studies indicate that increased
fish consumption or intake of n-3 PUFA inhibits BC cell proliferation and reduces BC
risk relative to n-6 PUFA (26-30). Nevertheless, there has been longstanding controversy
in epidemiological and observational studies regarding the potential anticancer effects
of n-3 PUFA due to the inability to show causality. Previous published reviews have
focused on the role of fish and marine n-3 fatty acids in BC prevention, whereas the
evidence for ALA and individual effects of long chain n-3 PUFA is lacking (3, 8, 11, 23,
31, 31, 31-34). Since a typical North American diet is mainly comprised of ALA as the
source of n-3 PUFA, it is necessary to elucidate the specific effects of ALA and cancer
risk. Therefore, the purpose of the present review is to evaluate the preventative role of
ALA, EPA and DHA in BC development when consumed individually, as well as in n-3
PUFA mixtures through dietary and supplemental forms. In addition, the potential
mechanisms by which they exert anticancer effects will also be discussed.
1.3 The Effects of n-3 PUFA in Human BC Studies
Dietary n-3 PUFA may influence breast cancer (BC) progression and prognosis.
In the past ten years, six prospective cohort studies (Table 1.1) and nine case-control
studies (Table 1.2) have examined the association between the consumption of either fish
or fish oil supplements and BC risk, showing a protective effect of n-3 PUFA. These
studies were conducted in many different geographic areas with mixed findings. In
general, Asian populations with a low total fat intake and high fish consumption,
associated n-3 PUFA intake with a reduced risk of BC (35-39). There was also a weak
association with reduced BC risk in US studies involving women whose diets had
higher n-6 PUFA content combined with fish oil supplements (18, 40, 41). European
studies were less consistent and somewhat contradictory (42-45).
5
In the Japan Collaborative Cohort (JACC) study, a significant decrease in the risk
of BC was detected in women with the highest dietary intake of fish fat and the long-
chain n-3 PUFA (35). Similar results were observed in a large prospective study of
35,298 Singapore women, indicating an inverse association between dietary n-3 PUFA
from marine sources and BC risk (36). Relative to the lowest quartile of n-3 PUFA intake,
individuals in the top three quartiles exhibited a 26% reduction in BC risk (relative risk =
0.74; 95% confidence interval = 0.58–0.94). Additionally, a recent analysis from the
VITamins And Lifestyle (VITAL) cohort carried out in US indicated that the current use
of fish oil supplements was associated with a decreased risk of localized invasive ductal
carcinomas in postmenopausal women (40).
The association between dietary intakes of n-3 PUFA or fish oil supplements with
overall survival was also examined. Patterson et al. demonstrated that women with higher
intakes of EPA and DHA from food, but not from fish oil supplements, had a dose-
dependent reduction in all-cause mortality (46). They also showed a reduced risk of
additional BC events of approximately 25% when compared with the lowest tertile of
intake (tertile 3:hazard ratio = 0.72; 95% confidence interval = 0.57–0.90) (46). In
support of these self-reported intake studies, Zheng et al. performed a comprehensive
analysis of 21 independent prospective cohort studies and found that marine n-3 PUFA
were associated with a 14% risk reduction of BC, and the relative risk remained similar
whether marine n-3 PUFA was measured as dietary intake or as tissue biomarkers (7).
Further, a dose-response analysis indicated a 5% lower risk of BC per 0.1 g/day (0.95,
0.90 to 1.00, I2 = 52%) increment of dietary marine n-3 PUFA (7). Conversely, a French
study comprising over 56,000 women found no association between total n-3 or n-6
PUFA intake and BC risk (42). Also, a large study of postmenopausal women in
Denmark concluded that increased fish consumption was associated with elevated
incidence rates of BC, but this association was present only for development of estrogen
positive BC (43). These null studies were mostly conducted in European populations with
relatively low per capita intake of n-3 PUFA (32, 44).
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Table 1.1 n-3 PUFA and breast cancer risk: Prospective cohort studies.
Year Country Subjects Method of
Assessment n-3/n-6 PUFA Source BC Risk Ref
2005 Japan
26,291 women
40–79 years
129 BC cases FFQ
1
Animal and fish fat, vegetable
oil, SFA, MUFA and PUFA ↑ fish fat, EPA + DHA ↓ BC risk (35)
2003 Singapore
35,298 women
45–74 years
342 BC cases FFQ
Fish/shellfish, saturated,
monounsaturated and
polyunsaturated fat
↑ n-3 PUFA from fish/shellfish ↓ BC risk
↑ n-6 PUFA (low marine n-3) ↑ BC risk (36)
2010 US
35,016
postmenopausal
50–76 years
880 BC cases
FFQ Dietary fish oil supplement ↑ fish oil ↓ risk of invasive ductal
carcinomas (40)
2009 France
56,007 women
40–65 years
1650 BC case
FFQ
ALA and n-6 PUFA from fruit,
nuts and vegetable oils; Long
chain n-3 PUFA from meals
No association between total n-3 and BC risk
↑ ALA ↓BC risk
↑ long chain n-3 PUFA ↓ BC risk (at highest
quintile of n-6 PUFA)
(42)
2003 Denmark
23,693
postmenopausal
50–64 years
424 BC cases
FFQ Fish ↑ intake of fish ↑ ER+ BC incidence (43)
2011 China
72,571 women
40–70 years
712 BC cases
FFQ Fish, marine-derived
n-3 PUFA red meat ↑ n-6/n-3 PUFA ratio ↑ BC risk (37)
1 FFQ: food frequency questionnaire; ↑: increase; ↓: decrease
7
Table 1.2 n-3 PUFA and breast cancer risk: Case-control studies.
Year Country Subjects
Characteristics
Method of
Assessment n-3/n-6 PUFA Source BC Risk Ref
2007 Japan 103 incident BC cases
309 controls
erythrocyte
membrane
FFQ
dietary food intake
including soy and meat
products, fish and other
seafood, vegetables
↑ dietary intake of n-3 fatty acids ↓ BC risk
↑ long chain n-3 PUFA in erythrocyte ↓ BC risk
↑ saturated fatty ↑ BC risk
(46)
2007 China 322 incident BC cases
1030 controls
erythrocyte
membrane ↑ total n-3 fatty acids and EPA ↓ BC risk (47)
2009 China
155 NPFC 1
185 PFC 2
241 BC, 1030 controls
erythrocyte
membrane
FFQ
dietary food intake
↑ EPA ↓ risk of NPFC
↓ progression of PFC to BC
↑ γ-linolenic acid ↑ risk of NPFC, PFC and BC
(38)
2002 US 73 BC patients
74 controls
breast
adipose tissue
↑ EPA and DHA ↓ n-6/n-3 PUFA ratio ↓ BC
risk
↑ n-6 PUFA ↑ BC risk
(18)
1 Benign proliferative fibrocystic conditions (PFC);
2 non-proliferative fibrocystic conditions (NPFC);
↑: increase; ↓: decrease.
8
Table 1.2 n-3 PUFA and breast cancer risk: Case-control studies (continue)
Year Country Subjects
Characteristics
Method of
Assessment
n-3/n-6 PUFA
Source BC Risk Ref
2003 US 565 incident BC
554 controls FFQ
Daily fat
intake
↓ n-6/n-3 PUFA ratio ↓ BC risk (premenopausal)
↑EPA, DHA ↓ BC risk (21% and 18%,
respectively)
(41)
2009 Denmark 463 BC cases
1098 controls
Gluteal
adipose tissue
biopsy
Dietary food
intake
No association between total or individual marine
n-3 PUFA in adipose tissue and risk of BC (44)
2002 France
241 invasive BC cases
88 controls-benign
breast disease
Breast
adipose tissue ↑ ALA ↑ DHA ↓ n-6/n-3 PUFA ratio ↓ BC risk (45)
2012 Mexican 1000 incident BC cases
1074 controls
Interview
and FFQ
Dietary food
intake
↑ n-3 PUFA ↓ BC risk (obese women)
↑ n-6 PUFA ↑ BC risk (premenopausal) (19)
9
In order to examine the relationship between n-3 PUFA exposure and BC risk,
several case-control studies have been conducted using different biomarkers (Table 1.2).
Kuriki et al. investigated the fatty acid compositions of erythrocyte membranes as a
biomarker and demonstrated that BC risk exhibited a significant inverse association with
dietary intake of n-3 PUFA derived from fish and high levels of long-chain n-3 PUFA in
erythrocyte membranes (47). Another assessment of erythrocyte fatty acid composition
found the inverse association significant only for EPA and total n-3 PUFA content (48).
Furthermore, Shannon et al. evaluated the role of n-3 and n-6 PUFA in the development
of benign proliferative fibrocystic conditions (PFC) and non-proliferative fibrocystic
conditions (NPFC) in the breast (38). They showed that women in the highest quartile of
erythrocyte EPA concentrations were 67% less likely to have NPFC alone or with BC,
and EPA significantly lowered the risk of progressing from PFC to BC by 43% (38).
However, γ-linolenic acid (n-6 PUFA) was found to be positively associated with nearly
all conditions (38). These results were consistent with an earlier meta-analysis showing
that total and individual n-3 PUFA, especially EPA and DHA, play a protective effect
against BC, while total SFA, MUFA, palmitic and oleic acids were associated with
increased BC risk (9).
In a Korean case control study, 358 patients with BC and 360 healthy controls
underwent dietary assessment by questionnaire and interview to determine their dietary
consumption of fish and n-3 PUFA derived from fish. Both pre- and postmenopausal
women in the highest quartile of fatty-fish intake had a lower incidence of BC (odds ratio
OR = 0.23, 95% CI = 0.13–0.42; p < 0.001), but the protective effect of EPA and/or DHA
intake was only observed for postmenopausal women (39). These findings were similarly
observed in a study of Mexican women where BC risk was lower in obese women (BMI
≥ 30) with high n-3 PUFA intake, not in women of normal weight (19). In contrast, a
case-cohort study of Danish women did not find any association between either total or
individual marine n-3 PUFA intake or BC risk (44). As the total levels of marine n-3
PUFA intake were low in Europe, this may account for observed discrepancies relative to
populations consuming marine-rich diets (32, 44, 45).
10
Other population studies have investigated interactions between n-3 and n-6
PUFA. Quantifying consumption of both types of fatty acids is an essential step in
isolating n-3 PUFA specific effects. The study of Bagga et al. showed that excessive
intake of n-6 PUFA contributed to the high risk of BC in US, while a decreased risk of
BC development was accompanied with higher EPA and DHA consumption (18). A
similar inverse relationship was also observed in regard to the n-6/n-3 PUFA ratio. In
another US study, when the analysis was restricted to pre-menopausal women, the
consumption of the lowest ratio of n-6 to n-3 was associated with a 41% reduction of BC
risk, although it was not significant (41). This observation was also observed in studies
conducted in China and France, although there was no association between n-3 PUFA
intake and BC risk, low n-3 PUFA intake by women who had the highest n-6 PUFA was
correlated with elevated BC risk (37, 45). However, based on the ratio, it is not possible
to determine whether it is increased n-6 or decreased n-3 PUFA that is the causal driver
of BC risk. Nevertheless, these studies indicate the necessity of higher n-3 PUFA intakes
given that n-6 intakes are adequate in all populations, and thus heightening the potential
value of n-3 PUFA as effective agents against BC.
Diet intervention by n-3 PUFA supplements as a mean of decreasing BC risk in
women still needs to be tested clinically. To date, few human intervention studies have
assessed the effectiveness of n-3 PUFA in BC prevention and treatment. One randomized
clinical trial tested the combined effects of n-3 PUFA (EPA + DHA = 3.36 g/day, 2 year)
and Raloxifene (anti-estrogen) in reducing risk of BC in postmenopausal women (49).
Although the plasma n-6/n-3 ratio significantly decreased among subjects after n-3 PUFA
intervention compared with the subjects without intervention, n-3 PUFA administration
did not affect any selected biomarkers that associated with BC risk (49). While in another
human intervention study, Thompson et al. demonstrated that daily intake of 25 g
flaxseed (ALA = 57% of total fatty acids) can significantly reduce cell proliferation and
increase cell apoptosis in tumors of postmenopausal BC patients. These limited results
provide encouragement for future study of n-3 PUFA as an adjuvant therapy in BC (50).
11
1.4 PUFA—Potential Mechanisms of Action
For more than 30 years, numerous studies have attempted to establish whether
there is a causal relationship between n-3 PUFA ingestion and a reduction in mammary
carcinogenesis. Mounting evidence shows that dietary n-3 PUFA may exert an anti-
carcinogenic action by altering the composition of cell membrane phospholipids,
inhibiting AA metabolism and decreasing AA derived eicosanoids, as well as modulating
the expression and function of numerous receptors, transcription factors and lipid derived
signaling molecules. However, studies of the effects of dietary n-3 PUFA on BC
progression and prognosis are limited in humans. Epidemiological studies do not allow
for the analysis of important cellular interactions and the specific molecular pathways
which are activated during the course of tumor initiation and cancer progression in the
mammary gland. Thus, the use of animal models (in vivo) and BC cell lines (in vitro)
provide crucial avenues for improving our understanding of the underlying biological
pathways involved in trigger BC development and potential therapeutic approaches for
BC treatment and prevention. Here we first introduce some potential mechanisms and
important downstream mediators that involved in the anticancer action of n-3 PUFA.
1.4.1 Influence on Cell Plasma Membrane Composition
Fatty acids play an important role in membrane biogenesis in the form of
glycerophospholipids, a major class of lipids found all cell membranes (51). Dietary
PUFA integrate into plasma membrane glycerophosholipids and influence the fatty acid
composition (52). The sn-1 position on the glycerol backbone of glycerophospholipids is
usually linked to saturated fatty acids, and the sn-2 position is linked to an n-6 PUFA
such as AA. Increased intake of dietary n-3 PUFA may replace n-6 with n-3 fatty acids at
the sn-2 position of glycerophospholipids (52). Since n-3 PUFA has a greater density
compared to n-6 PUFA, the aggregation of n-3 fatty acids tend to be closer to the lipid-
water interface of the membrane. This characteristic can significantly affect plasma
membrane fluidity and permeability (53). In addition, due to the high level unsaturation
of long chain n-3 PUFA, they have very poor affinity for cholesterol (52, 54). Membrane
12
cholesterol serves as a spacer for the hydrocarbon chains of sphingolipids and maintains
the assembled microdomains of lipid rafts (55). Thus, cholesterol depletion leads to the
disorganization of lipid raft structure (54). Lipid rafts are important membrane domains
for cell signaling since it is enriched with many regulatory proteins and some growth
factor receptors(54, 55). As a result, the incorporation of n-3 PUFA, especially EPA and
DHA, can disturb formation of lipid rafts and suppress raft-associated cell signal
transduction (22, 56, 57).
1.4.2 Inhibition of Arachidonic Acid (AA) Derived Eicosanoid Biosynthesis
One of the key cellular functions of PUFA is related to their enzymatic
conversion into eicosanoids. Eicosanoids are short-lived, hormone-like lipids, typically
comprised of 20 carbon atoms, which play a critical role in platelet aggregation, cellular
growth and cell differentiation (31). The most salient mechanism by which n-3 PUFA
reduce tumor development is through inhibiting the synthesis of inflammatory
eicosanoids derived from AA (31). As indicated in Figure 1.1, firstly, both ALA and LA
are initially converted to their long-chain metabolites (EPA and AA, respectively)
through the same desaturation/elongation pathway; therefore, there exists potential
competition between these two families of fatty acids for desaturases and elongases. The
initial conversion of ALA to stearidonic acid (18:4n-3) is the rate limiting reaction of the
pathway. The affinity of delta 6-desaturase for ALA is greater than for LA (58). As a
result, a higher intake of ALA reduces the synthesis of AA from LA and thus, less AA is
available for synthesis of inflammatory eicosanoids (59). Secondly, increase consumption
of n-3 PUFA results in their incorporation into membrane phospholipids, where they
partially replace AA, therefore reducing the substrate for AA derived eicosanoids. Healy
et al. showed that dietary supplements with four different concentrations of fish oil
resulted in the incorporation of EPA and DHA into human inflammatory cells occurs in a
dose-response fashion at the expense of AA (60). Thirdly, both AA and EPA are
substrate for eicosanoid synthesis such as prostaglandins (PG) and leukotrienes (LT) (31).
AA can be metabolized by two major pathways including the cyclooxyenase (COX) and
13
lipoxygenase (LOX) pathways. COX-2 catalyzes the rate limiting step in the formation of
2-series PGs. 5-LOX catalyzes the first step in oxygenation of AA to produce hydroxyl
derivatives and 4-series LTs. The overexpression of COX-2 has been detected in many
types of cancers and PGE2 has been shown to promote cell proliferation in mammary
tumor tissues (61). PGE2 stimulates the expression and activation of aromatase, the
enzyme that converts androgens to estrogens (62). Therefore, it is hypothesized that
estrogen levels can be lowered by decreasing n-6 PUFA intake. In contrast, EPA
metabolism results in 3-series PGs and 5-series LTs, with a slightly different structure
and anti-tumorigenic properties (58). N-3 PUFA supplementation has been shown to
lower production of PGE2 (by 60%) and LTB4 (by 75%) in human peripheral blood
mononuclear cells (63). Furthermore, n-3 PUFA has been suggested to suppress the
expression of COX-2 and 5-LOX. Feeding mice with high n-3 PUFA diets influences
mammary tumor development by down-regulating COX-2 and 5-LOX expression (20,
64). In addition to the inhibitory effects on the generation of inflammatory eicosanoids,
recent studies have identified a novel group of lipid mediators, termed resolvins, which
are formed from EPA and DHA (65, 66). These mediators appear to exert potent anti-
inflammatory actions and are considered as potential therapeutic interventions for some
chronic inflammatory diseases and cancer (67, 68).
In general, n-3 PUFA can not only block AA metabolism, but can also compete
with AA for eicosanoid synthesis. This anti-inflammatory effect of n-3 PUFA is of
interest since chronic inflammation has been linked to cancer initiation and progression
(69).
1.4.3 Influence on Gene Expression and Signaling Transduction
BC development is a multi-step process that requires the accumulation of several
genetic alterations in a single cell. Many studies have now demonstrated that the
regulation of intracellular signaling is a critical aspect of controlling cell functions in
various types of cancers. Neoplastic growth and progression are generally regarded as
dependent on a high rate of cell proliferation and a low rate of apoptosis. Dietary PUFA
14
and their metabolites may exert some of their anti-cancer or tumor-promoting effects by
affecting gene expression or activating signal transduction molecules involved in the
control of cell proliferation, differentiation apoptosis and metastasis (Figure 1.2).
Figure 1.1 Synthetic pathways of long-chain PUFA and eicosanoids. α-linolenic acid
(ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6) are essential PUFA obtained from the
diet, and involve in similar sequential desaturation and elongation steps, give rise to long
chain, more unsaturated PUFA eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic
acid (DHA; 22:6n-3), and arachidonic acid (AA; 20:4n-6). Relevant intermediates in
these pathways include SDA (stearidonic acid), ETA (eicosatetraenoic acid), DPA
(docosapentaenoic acid), GLA (γ-linolenic acid), DGLA (dihomo-γ-linolenic acid) and
AdA (adrenic acid). Both AA and EPA are substrates for the synthesis of eicosanoid
products such as prostaglandins (PG) and leukotrienes (LT). The products of n-6 PUFA
tend to promote cell proliferation while the products of n-3 PUFA have anti-tumorigenic
properties. N-3 PUFA may lower the risk of BC by disrupting the biosynthesis of AA-
derived inflammatory eicosanoids.
Dietary n-3 PUFA
ALA (18:3 n-3)
SDA (18:4 n-3)
ETA (20:4 n-3)
EPA (20:5 n-3)
DPA (22:5 n-3)
DHA (22:6 n-3)
LA (18:2 n-6)
GLA (18:3 n-6)
DGLA (20:3 n-6)
AA (20:4 n-6)
AdA (22:4 n-6)
DPA (22:5 n-6)
Dietary n-6 PUFA
COX-2 ↑
5-LOX↑
∆6-desaturase
elongase
∆5-desaturase
elongase
∆6-desaturase
Prostaglandin-3-series
Leukotrienes-5-series Prostaglandin-2-series
Leukotrienes-4-series Resolvins (E-series)
Resolvins (D-series)
15
Figure 1.2 Hypothetical scheme showing how n-3 PUFA modulates cell functions via
intracellular signaling molecules. Cell proliferation and cell apoptosis are the two
important fundamental processes integral to carcinogenesis. n-3 PUFA exerts anti-cancer
effects by reducing the expression of some growth factors including human epidermal
growth factor receptor-2 (HER-2), epidermal growth factor receptor (EGFR) and insulin-
like growth factor 1(IGF-1R); inhibiting cell proliferation by either activating PPARγ or
decreasing levels of fatty acid synthase (FAS) protein; and promoting cell apoptosis via
blocking PI3K/Akt pathways, downregulating phosphorylated Akt, inhibiting NF-κB
activity and lowering Bcl-2/Bax ratio.
1.4.3.1 EGFR and HER-2
Among numerous factors, carcinogenesis involves the activation of oncogenes
such as the epidermal growth factor receptor (EGFR) and human epidermal growth factor
receptor-2 (HER-2). EGFR is a receptor tyrosine kinase, which plays essential roles in
regulating a number of cellular processes including cell proliferation, survival and
migration (70). EGFR is usually activated in response to extracellular ligands (epidermal
growth factor [EGF)) by its phosphorylation [54]. Dysregulated EGFR activation is often
associated with overexpression of EGFR, which has been observed in several cancer
High n-3
Low n-6
HER-2↓
IGF-1R↓
EFGR↓
FASN↓
PPARγ↑
Bcl-2↓
Bax↑
pAkt↓
NF-kB↓
PI3K↓
16
types including breast carcinomas (71). Marine n-3 PUFA were able to inhibit EGFR
activity, in particular, DHA was found to induce apoptosis in BC cells by down-
regulating EGFR expression (72). Similar to EGFR, HER-2 (HER-2/neu or erbB-2) is a
185-kD transmembrane receptor tyrosine kinase that is involved in human mammary
oncogenesis (73). The overexpression of HER-2 occurs in 25%–30% of human invasive
BCs, and is associated with a more aggressive phenotype and poor patient prognosis (12,
74). HER-2 functions as a co-receptor and forms homodimers or heterodimers with
EGFR or insulin-like growth factor 1(IGF-1R) to activate downstream target signaling
cascades that involve cell survival and proliferation, such as phosphatidylinositol 3-
kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and inhibition of apoptotic
pathways such as Bcl-2-associated death promoter protein (75, 76). It should be noted
that the HER-2 receptors also activate lipogenic pathways mediated by the fatty acid
synthase (FAS) protein (6), a key lipogenic enzyme catalyzing the terminal steps in the
de novo biogenesis of fatty acids in cancer pathogenesis (51). Dietary n-3 PUFA were
demonstrated to inhibit the early stages of HER-2/neu-mediated mammary
carcinogenesis in rats (77). Notably, ALA alone was able to reduce HER-2 protein
expression by 79% in MCF-7 cell lines (78). Both EGFR and HER-2 are regarded as
important therapeutic targets against BC, and n-3 PUFA may be a dietary treatment for
controlling the growth factor-mediated oncogenesis.
1.4.3.2 Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear
receptor superfamily and function as ligand-activated transcription factors (79). PPARγ is
a subset of the PPAR family, it is mainly expressed in adipose tissue, mammary gland,
colon and the immune system (80, 81). PPARγ regulates the expression of target genes
by binding to DNA sequence elements, termed PPAR response elements (PPREs).
PPREs have been identified in the regulatory regions of a variety of genes that are
involved in lipid metabolism and homeostasis, but recently have appeared to be involved
in cell proliferation, cell differentiation, and inflammatory responses (79, 82). PPARγ
ligands include naturally occurring compounds such as PUFA and eicosanoids, as well as
synthetic activators, such as the hypolipidemic drugs (83). Clay et al. indicated that
17
induction of apoptosis is a biological response resulting from PPARγ activation in some
BC cells (84). n-3 PUFA are direct agonists for PPARγ, which have been shown to exert
anti-tumorigenic effects via the activation of PPARγ (82, 85). For instance, DHA was
found to attenuate MCF-7 cell proliferation by activation of PPARγ (86). In addition,
dietary supplementation with a low ratio of n-6/n-3 PUFA (1:14.6) was shown to increase
PPARγ protein content, which was paralleled with a reduction of tumor burden in rats
with induced mammary carcinogenesis (87). As a result, PPARγ activation is beneficial
for controlling BC, which suggests a potential role for PPARγ ligands in the treatment of
BC.
1.4.3.3 Bax/Bcl-2
Apoptosis is a form of cell death triggered during a variety of physiological
conditions and is tightly regulated by a number of gene products that promote or block
cell death at different stages (88). Bcl-2 is well-known as an important apoptosis-
regulator protein (89), normally blocking apoptosis and its overexpression contributes to
BC by prolonging cell survival (90). Bax is a pro-apoptotic member of the Bcl-2 family
of proteins. It is likely to have pore-forming activity to increase mitochondrial membrane
permeability, and can also form a homodimer with Bcl-2 to enhance the effects of
apoptotic stimuli (90, 91). Raisova et al. showed that the Bax/Bcl-2 ratio determines the
susceptibility of cells to apoptosis (92). Thus, a low Bax/Bcl-2 ratio is associated with
enhanced survival of BC cells and resistance to apoptosis, and vice versa. It has been
proposed that diets rich in n-3 PUFA, such as fish and canola oil, reduces the abundance
of Bcl-2 and up-regulates Bax expression to induce apoptosis, thereby reducing BC risk
(27, 93).
1.4.3.4 PI3K/Akt, NF-κB
Besides Bcl-2, the PI3K/Akt pathway also plays an important role in cell
apoptosis. Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric lipid kinase that is
composed of a regulatory and catalytic subunit that is encoded by different genes (93).
The primary consequence of PI3K activation is the generation of the second messenger
PtdIns (3,4,5) P3 (PIP3) in the membrane, which in turn recruits and activates Akt, a
18
downstream serine/threonine kinase (94). Akt activation is a dual regulatory mechanism
that requires translocation to the plasma membrane and phosphorylation at Thr308 and
Ser473 (52, 94, 95). Due to this mechanism, Akt functions as an anti-apoptotic signaling
molecule. Thus, upregulation of phosphorylated Akt is relevant to tumor cell growth and
resistance to cell apoptosis (6, 87, 96). HER-2 overexpression constitutively activates
survival and proliferation pathways by increasing activation of Akt, however, n-3 PUFA
was found to either modulate total Akt expression or interact with Akt to down-regulate its
phosphorylation (6, 97). Since Akt requires translocation to the plasma membrane for
activation, it is possible that tumor cell membrane enrichment of n-3 PUFA might affect the
phosphorylation of Akt that are recruited to the membrane for activation (96). In addition,
recent studies have demonstrated that PI3K/Akt promoted cell survival is mediated, in
part, through the activation of the nuclear factor kappa-B (NF-κB) transcription factor
(95, 98, 99). NF-κB is a key regulator of genes involved in cell proliferation, migration,
and angiogenesis (100, 101). In tumor cells, impaired regulation of NF-κB activation will
lead to deregulated expression of the anti-apoptotic genes under the control of NF-κB
(100). For instance, NF-κB has been shown to inhibit the activity of p53, a tumor
suppressor known to trigger apoptosis in cells with damaged DNA (102). As a result,
constitutive NF-κB expression may contribute to the development and progression of BC.
1.4.3.5 Cell Proliferation Marker: Ki-67 and PCNA
Cell proliferation is another fundamental process integral to carcinogenesis. Ki-67
is a nuclear protein, and being widely used as a prognostic or predictive marker in BC
and other malignant disease (103). The human Ki-67 protein is present during all active
phases of the cell cycle G(1), S, G(2), and mitosis, but is absent from resting cells G(0),
which makes Ki-67 an excellent marker for determining the growth fraction of a given
cell population (104). Ki-67 immunohistochemical staining has been used as an index of
tumor growth in numerous of cancer studies, especially prostate and breast carcinomas
(26, 87). Treatment with ALA-rich flaxseed oil markedly lowered tumor burden in rats
accompanied by reduced Ki-67 level(78, 105).
Similarly to Ki-67, Proliferating Cell Nuclear Antigen (PCNA) is also considered
a potential prognostic marker in BC (106). PCNA is a ring-like nuclear protein which
19
functions as the sliding clamp of DNA polymerases (107, 108). Thus, it is involved in
DNA replication and repair machinery of the cell (109). Expression of PCNA is a valid
cell proliferation marker since the distribution of PCNA was found to occur during G1, S
and G2 phase, but reaches low immunohistochemically detectable levels in M-phase of
the cell cycle (106, 108, 109). It has been shown that supplementation with n-3 PUFA
reduces the percentage of proliferating tumor cells by decreasing the expression of PCNA
(110).
The overall effect of high n-3 PUFA intake on cellular signaling process either
inhibits cell proliferation or promotes cell apoptosis (Figure 1.2). The changes in gene
expression, transcription factor activity and signaling transduction will be highlighted in
the following sections.
1.5 The Effect of n-3 PUFA Mixtures on BC Development
1.5.1 Animal Models
The inhibitory effects of n-3 PUFA on tumor growth have been well documented
in rodent models of BC. Xenograft, transgenic and chemically induced methods are the
three main approaches employed in rodent models.
1.5.1.1 Breast Cancer Studies in Xenograft Rodent Models
Xenograft rodent models involve the transplantation of human BC cells into
immunocompromised mice (78, 111). The type and concentration of dietary PUFA have
profound influences on the growth rate of transplantable human BC in rodents (Table
1.3). In one study, MDA-MB-435 BC cells xenografted into athymic nude mice in order
to compare intake of LA alone with diets containing LA and various proportions of
EPA/DHA (20). The diet rich in n-6 PUFA stimulated the growth and migration of
human BC cells in mice, whereas diets supplemented with EPA or DHA exerted
suppressive effects (20). Similarly, Karmail et al. also found an inhibition of R3230AC
20
mammary tumor growth in rat fed with Maxepa, a menhaden oil supplement that contains
approximately 18% EPA and 12% DHA (112). These observations were largely
attributed to the high incorporation of n-3 PUFA into the tumor phospholipids, which
further reduced pro-inflammatory eicosanoid synthesis from AA (20). More recently, a
diet supplemented with 3% w/w fish oil concentrate was shown to stimulate lipid
peroxidation in the tumor cells, and thereby slowed down the tumor growth rate in
athymic nude mice implanted with MDA-MB-231 (30). Similar effects were observed in
MCF-7 human breast cancer xenografts, although a higher percentage of fish oil (19%
w/w) was required to decrease tumor volume in the nude mice (113). These findings
supported an earlier study showed that n-6/n-3 PUFA consumed in a low ratio resulted in
prolonged tumor latency and reduced tumor growth rate in BALB/cAnN mice (114).
Therefore, diet enriched with n-3 PUFA can alter murine mammary tumorigenesis.
1.5.1.2 Breast Cancer Studies in Transgenic Rodent Models
In transgenic mouse models, tumor growth can be initiated in two different ways:
gain of function involving oncogenes responsible for cell proliferation or loss of function
involving cell apoptotic pathways (115, 116). For example, the mouse mammary tumor
virus (MMTV) promoter allows the cancer-causing virus to be activated and expressed in
mammary tissue, leading to the development of mammary tumors (117). Several studies
have employed MMTV-neu related models and provided strong evidence for protective
effects of n-3 PUFA towards HER-2 positive BC (Table 1.4).
One study examined mammary tumor development in MMTV-Her-2/neu mice
fed menhaden fish oil from 7 weeks of age onwards. The tumor incidence was
dramatically reduced as well as prolonged tumor latency (115). Further, Yee et al.
demonstrated that dietary n-3 PUFA downregulated COX-2 and Ki-67 to inhibit cell
proliferation, further reducing atypical hyperplasia to prevent HER-2/neu mammary
carcinogenesis at early stages (77). Moreover, we showed that lifelong n-3 PUFA
exposure can mitigate tumor development in mice expressing MMTV-neu(ndl)-YD5, a
more aggressive HER-2- positive BC model (116). In this study, MMTV-neu(ndl)-YD5
21
mice were crossed with fat-1 mice, yielding mice that were capable of endogenous
synthesis of n-3 from n-6 PUFA and had the susceptibility to mammary tumor growth
(116). Thus, this study provided a direct evidence for a protective effect of n-3 PUFA via
both complementary genetic and conventional dietary approaches (116). In order to
understand the dose-dependent effect of n-3 PUFA on mammary gland tumor
development, Leslie et al. further demonstrated a dose-response relationship of 0%, 3%
and 9% (w/w) menhaden oil on tumor volume reduction in MMTV-neu-YD5 mice (21).
In line with the previous study, the observed dose-dependent effects of n-3 PUFA were
associated with a dose-dependent change in the fatty acid profile, reflecting a decreased
n-6/n-3 ratio in the mammary glands and an increase of EPA and DHA in tumor
phospholipid classes (21). This is consistent with an earlier human clinical trial which
showed dietary DHA and EPA supplementation caused a dose-dependent increase of n-3
PUFA in serum and breast adipose tissues of BC patients (118).
Supplementing mice with 24% (w/w) menhaden oil delayed mammary tumor
development by 15 weeks relative to mice fed the same amount of corn oil (119). This
result heightened the potential value of n-3 as an effective agent against BC, while the
timing of exposure to n-3 PUFA will also influence future cancer risk (34). Mammary
gland research has identified critical periods of development, including early windows
such as in utero, lactation and pubescence. The mammary gland undergoes rapid growth
during these periods, and exposure to environmental agents may influence the long-term
health of mammary tissue. Thus, supplement with n-3 PUFA in early life stages may
protect against later BC development. In support of this, Su et al. conducted a study
exposing rats to maternal high n-6 PUFA diet with or without fish oil supplementation
during the perinatal period via maternal intake or during puberty or adulthood (29). They
found that fish oil intake during the perinatal period had a greater effect in preventing
mammary tumors than fish oil supplementation in later life (29). The decreased maternal
serum estradiol levels in pregnant rats with fish oil supplementation was thought to play a
role in reducing susceptibility to later BC development in the female offspring (29).
22
Table 1.3 n-3 PUFA and breast cancer risk: xenograft rodent models.
Animal Model n-3 PUFA Source Feeding
Period Main Findings Mechanism Ref
Athymic nu/nu mice
MDA-MB 231
3% w/w fish oil concentrate
(10.2 g/kg EPA, 7.2 g/kg
DHA, 3.0 g/kg ALA)
7-week
(fed after tumor
established)
↓ tumor growth rate
↑ effectiveness of
doxorubucin
↑ EPA incorporation into tumor
↑ lipid peroxidation in tumor (30)
Athymic nu/nu mice
(NCr-nu/nu)
MDA-MB 435
40 or 80 g/kg EPA, DHA 13-week
(fed before
transplantation)
↓ tumor growth, size
↓ tumor weight
↑ EPA, DHA in tumor
phospholipids
↓ LA, AA in tumor
phospholipids
↓ AA-derived eicosanoids
(20)
Inbred F44 rats
R3230AC
5% marine oil
supplementation
(18% EPA, 12% DHA)
4-week
(fed before
transplantation)
↓ tumor weight, volume ↑ EPA, DHA, AA incorporation
into tumor
↓ Prostaglandins 2 series
(112)
BALB/cAnN mice
Mouse BC cell
10% or 20% w/w menhaden
fish oil
7-week
(fed before
transplantation)
↑ tumor latency
↓ tumor growth rate NA (114)
Athymic nude mice
MCF-7
19% w/w menhaden oil
(1.9 g/kg ALA, 19.4 g/kg
EPA, 24.3 g/kg DHA)
6 or 8-week
(fed after tumor
established)
↓ tumor volume ↑ lipid peroxidation in tumor (113)
↑: increase; ↓: decrease; NA: not available
23
Table 1.4 n-3 PUFA and breast cancer risk: transgenic rodent models.
Animal Model n-3 PUFA Source Feeding Period Main Findings Mechanism Ref
MMTV-HER-
2/neu
22.50 kcal% menhaden oil
(15 g/kg EPA, 10.8 g/kg
DHA)
28-week
(fed before
tumor
development)
↓ atypical ductal
hyperplasia
↓ cell proliferation
prevented HER-2/neu at
early stages
↓ Ki-67 expression
↓ COX-2 expression (77)
MMTV-HER-
2/neu
22.50 kcal% menhaden oil
(15 g/kg EPA, 10.8 g/kg
DHA)
52-week
(fed before
tumor
development)
↓ tumor incidence and
multiplicity
↑ tumor latency
↓ mammary gland dysplasia
NA (115)
MMTV-neu
(ndl)-YD5 × fat1
3% w/w menhaden oil (0.5
g/kg ALA, 4.1 g/kg EPA,
3 g/kg DHA)
20-week
(lifelong
treatment, fed
before tumor
development)
↓ tumor volume and
multiplicity
↑ EPA, DHA and overall n-3 in
mammary tissues
↓ n-6/n-3 ratio in tumor
phospholipids
(116)
MMTV-neu
(ndl)-YD5
3% w/w menhaden oil (0.5
g/kg ALA, 4.1 g/kg EPA,
3 g/kg DHA)
9% w/w menhaden oil (1.3
g/kg ALA, 12.4 g/kg EPA,
9 g/kg DHA)
20-week
(lifelong
treatment, fed
before tumor
development)
↓ tumor volume and
multiplicity
↑ tumor latency
(all in a dose-dependent
manner)
↑ EPA, DPA in mammary tissues
↑ EPA, DHA in tumor
phospholipids
↓ LA, AA, n-6/n-3 PUFA ratio in
both mammary and tumor tissues
in a dose-dependent manner
(21)
↑: increase; ↓: decrease; NA: not available.
24
1.5.1.3 Breast Cancer Studies in Chemically-Induced Rodent Models
Evidence from chemical-induced BC studies consistently supports the anti-cancer
effect of n-3 PUFA. Induced mammary carcinoma in rats by the injection of carcinogenic
chemicals such as 7, 12-dimethylbenz (α) anthracene (DMBA) and N-methyl-N-
nitrosourea (MNU) have been widely used in various BC chemopreventive studies. When
provided in the diet through menhaden or fish oil concentrates, the incorporated n-3
PUFA, EPA and DHA in particular, lower tumor incidence as well as retard tumor
growth and metastasis of chemically-induced and transplantable mammary tumors
(Table 1.5). Olivo et al. compared the effects of low- or high-, n-3 or n-6 PUFA exposure
on mammary tumorigenesis in rats (110). Feeding rats a low-fat n-3 diet significantly
lowered the incidence of DMBA-induced mammary tumors compared to n-6 fed rats.
This was accompanied by reduced cell proliferation and elevated lipid peroxidation.
However, the high-fat n-3 diet resulted in a significant increase risk of mammary
tumorigenesis relative to rats fed with low fat n-6 diet (110). Further, an in vivo study
demonstrated that low-fat n-3 PUFA diets inhibited cell proliferation via activation of
PPARγ and/or down-regulation of COX-2 and PCNA expression; whereas high fat n-3
PUFA diets stimulated cell proliferation and were positively associated with levels of
phosphorylated Akt (110). Similar suppressive effects of n-3 PUFA were observed in
another DMBA-induced rat model (27). Supplementation of EPA and DHA (from
Maxepa) effectively suppressed cell proliferation, which was accompanied by down-
regulation of Ki-67 and HER-2/neu expressions. Meanwhile, EPA and DHA induced cell
apoptosis through modulating the expression of Bcl-2 and Bax in mammary tissue of
Sprague-Sawley rats (26-28). These findings suggest that gene-nutrient interactions are of
a critical importance in the development of BC.
It has been shown that the different dietary fatty acid composition and n-6/n-3
PUFA ratios can diversely influence the occurrence and progression of BC (8, 32, 64).
Wei et al. fed MNU-induced rats with various n-6/n-3 PUFA ratios and demonstrated the
1:1 ratio of n-6/n-3 PUFA in diet was more effective in the prevention of mammary
tumor development when compared with diets higher in n-6 PUFA (64). Replacement of
n-6 by n-3 PUFA in diets, reflected an increase in EPA and DHA in mammary tumor
25
tissue, which subsequently downregulated the expression of lipid metabolic-related genes
and inhibited cell proliferation (64). In agreement with this study, Jiang et al. reported
that low n-6/n-3 PUFA ratio (1:14.6) caused 80% reduction in tumor burden and 30%
decrease in tumor multiplicity in the same rodent model compared to a high n-6/n-3
PUFA ratio (1:0.7). These observations were mainly mediated by downregulating NF-κB,
pAkt and IGF-IR, and increased activity of PPARγ (87).
1.5.2. Cell Culture Studies
It has been well established that n-3 PUFA can suppress the development of
cancers by inhibiting cellular proliferation and inducing apoptosis. Cell culture studies
investigating the effects of n-3 PUFA on murine and human BC cells provide important
insights into the mechanisms underlying this inhibitory effect. Although there are few
studies describing the individual effects of ALA, EPA and DHA in vitro, the available
data consistently show that n-3 PUFA have direct growth inhibitory effects on several BC
cells lines (Table 1.6).
MDA-MB-231 human BC cell line was used to investigate the effect of various
classes of fatty acids (n-3, n-6 and n-9 PUFA)(120). EPA and DHA exhibited a dose-
dependent inhibition of cell growth, whereas LA and oleic acid (OA) stimulated cell
growth at very low concentration (121). Some in vitro studies have not included the
essential n-6 PUFA (i.e., LA) in the growth medium, which would be present in vivo and is
required for mammary tumorgenesis in animals. These studies are not able to explain the
tumor growth inhibition by n-3 PUFA in the presence of abundant LA. Schley et al.
examined the inhibitory effects of n-3 PUFA on BC in vitro in the presence or absence of
LA (22, 96). It was demonstrated that EPA and DHA induced apoptosis and increased
DNA fragmentation in MDA-MB-231 cells when provided in combination with LA.
Similar effects were observed in the MCF-7 BC cell line (72, 86, 105, 122). Barascu et
al. revealed that EPA and DHA decreased MCF-7 cell growth and increased the fraction
of apoptotic cells in a concentration-dependent manner, and particularly, a higher
efficiency noted for DHA (86). Specifically, they demonstrated that n-3 PUFA inhibited
cell proliferation by lengthening the cell cycle between the G2/M transition (86). In
26
accordance with this finding, a previous study demonstrated that DHA induced marked
G2-M and G1-S arrest of the MCF-7 cells (123). Furthermore, treatment with EPA and
DHA was also shown to induce cell differentiation and increase lipid peroxidation
product levels in MCF-7 cells (105, 123, 124).
Chajes et al. examined the effect of ALA, EPA, and DHA on the proliferation of
human estrogen-positive (ER+) and estrogen-negative (ER−) BC cell lines (124). EPA
and DHA displayed a significant inhibitory effect on the proliferation of all types of
tumor cells, whereas ALA significantly inhibited cell growth in (ER−) MDA-MB-231
and HBL-100 human breast tumor cells but not in (ER+) MCF-7 cells (124). The
efficiency of this inhibitory effect was found to be correlated with the generation of lipid
peroxidation products (124). Consistently, another in vitro study identified a dose-
dependent inhibitory effect of ALA, EPA, and DHA on MCF-7 cells; however, the cells
were dramatically inhibited by EPA and DHA, and moderately inhibited by ALA (122).
Rapid tumor cell proliferation is a critical feature for tumor aggressiveness.
Treatment with LA and OA increased MDA-MB-231 cell proliferation through
production of pro-inflammatory prostaglandins and leukotrienes (121). EPA and DHA
could mimic the effect of indomethacin, an inhibitor of both COX and LOX, which in
turn attenuated BC cell proliferation by inhibiting n-6 PUFA related eicosanoid synthesis
(125). In addition, treatment with EPA and DHA inhibited EGFR phosphorylation and
diminished EFGR levels in lipid rafts (22, 72). These observations were due to the
incorporation of n-3 PUFA into BC cell membrane, which subsequently altered
membrane structure, signal transduction and function of BC cells (72). Moreover, n-3
PUFA can exert anti-proliferative effects by limiting cell cycle progression, attributed to
an inhibition of CDK1-cyclin B1 complex, a master regulator required for the initiation
of mitosis (86). Furthermore, increasing evidence demonstrated that n-3 PUFA can
inhibit cell proliferation and increase cell differentiation by activating PPARγ, although
this effect was only observed with DHA treatment (105).
27
Table 1.5 n-3 PUFA and breast cancer risk: chemically-induced rodent models
↑: increase; ↓: decrease; NA: not available.
Carcinogen n-3 PUFA Source Feeding Period Main Findings Mechanism Ref
MNU Fish oil 2%–10% w/w
n-3 PUFA in diet
18-week (at the same
time as MNU
administration)
10% w/w n-3 PUFA (1:1n-6/n-3 ):
↓ body weight, no tumor occurrence
5% w/w n-3 PUFA:
↓ tumor incidence and multiplicity
↑ EPA, DHA in
mammary
↓ FAS, COX-2, 5-LOX
(64)
MNU
Fish oil concentrate
Low n-6/n-3 = 1:14.6
High n-6/n-3 = 1:0.7
2-week (at the same
time as MNU
administration)
Low vs. high ratio n-6/n-3 PUFA diet:
↓ tumor incidence (21%),
↓ tumor multiplicity (30%), tumor burden
(80%)
↑ apoptotic index (129%)
↓ Ki-67
↑ Bax, Bax/Bcl2, PPARγ
↓ NF-κB p65, pAkt, IGF-
IR
(87)
MNU
EPA/DHA alone: 95 g/kg
EPA/DHA EPA + DHA:
47.5 g/kg EPA + 47.5 g/kg
DHA
20-week (at the same
time as MNU
administration)
DHA alone vs EPA + DHA vs EPA alone:
↓ tumor incidence: 23%, 73%, 65%
↓ tumor multiplicity: 0.23, 1.67, 1.59
DHA is more effectively than EPA
NA (120)
28
Table 1.5 n-3 PUFA and breast cancer risk: chemically-induced rodent models (continue)
↑: increase; ↓: decrease; NA: not available.
Carcinogen n-3 PUFA Source Feeding Period Main Findings Mechanism Ref
DMBA
Maxepa (fish oil
concentrate):
90 mg EPA + 60 mg
DHA per day
24-week or 35-week
study (before
DMBA injection)
↓ DNA single-strand breaks
↓ cell proliferation ↓ Ki-67, Her-2/neu (26)
DMBA Maxepa: 90 mg EPA +
60 mg DHA per day
24-week study
35-week study
(before DMBA
injection)
↓ tumor incidence (23%), tumor
multiplicity (42%) ↑ cell apoptosis ↓cell
proliferation ↓ Bcl-2 ↑Bax ↑ p53 (27, 28)
DMBA
Fish oil (0.5%ALA,
16% EPA, 1.2% DPA,
8% DHA in fish oil) NA
↓ tumor incidence with fish oil
consumption: adulthood < puberty <
perinatal
↓ tumor multiplicity with fish oil
consumption: adulthood > puberty >
perinatal
↓ maternal serum
estradiol (29)
DMBA
Menhaden oil Low-fat
n-3 PUFA diet: 4.6 g/kg
EPA + 3.2 g/kg DHA
High fat n-3 PUFA diet:
9.1 g/kg EPA + 6.3 g/kg
DHA
20-day (before
DMBA injection)
Low n-3 diet: ↓ tumor incidence ↓ TEBs ↓
cell proliferation ↑ cell apoptosis;
High n-3 diets exert opposite effects
Low n-3 diet: ↓ COX-
2, PCNA ↑ PPARγ
↑ lipid peroxidation
High n-3 diet: ↑ pAkt
↑ lipid peroxidation
(110)
29
Table 1.6 n-3 PUFA and breast cancer risk: cell culture studies.
Cell Type n-3 PUFA Source Main Finding Mechanism Ref
MDA-MB-
231
EPA/DHA alone: 75 μM
100 μM EPA+DHA
combination: 45 μM EPA+30
μM DHA or 60 μM EPA+40 μM
DHA
(in presence/absence of LA)
↓ cell viability, cell proliferation
↑ DNA fragmentation, cell apoptosis
DHA was more potent than EPA
↓ pAkt
↓ NF-κB and DNA binding
activity
(96)
MDA-MB-
231
0.5–2.5 μg/mL of EPA, DHA
(1.7–8.2 μM EPA, 1.5–7.6
μMDHA)
↓ tumor cells growth
(DHA > EPA, dose-dependent)
↓ LA composition in cell lipids
↓ AA-derived eicosanoid
synthesis (121)
MDA-MB-
231
EPA/DHA alone: 75 μM
100 μM EPA+DHA
combination: 45 μM EPA+30
μM DHA or 60 μM EPA+40 μM
DHA
(in presence/absence of LA)
↓cell growth (48%–62%)
↑ EPA, DHA, DPA and total n-3 in
lipid rafts
↓ EGFR levels ↑ pEGFR
(22)
MDA-MB-
231 MCF-7 EPA (230 μM), DHA (200 μM) ↓ cell viability ↑ cell apoptosis
↓ Bcl-2 ↑pro-caspase-8
↓ pEGFR
↓ EGFR (only DHA)
↓ AA ↑ EPA, DPA, DHA in total
cell lipids
(72)
↑: increase; ↓: decrease; NA: not available
30
Table 1.6 n-3 PUFA and breast cancer risk: cell culture studies (continue)
Cell Type n-3 PUFA Source Main Finding Mechanism Ref
MDA-MB-231
MCF-7 3–100 μM of EPA, DHA
At 50 μM EPA, 30 μM DHA
↑ cell apoptosis ↓ cell growth
At 50 μM EPA, DHA
↑ G2/M duration (DHA was more potent than
EPA)
↓ phosphorylation of cyclin B1
↓ activity of CDK1-cyclin B1 (86)
MCF-7 100 μM of EPA, DHA
↓ cell growth (30% by EPA, 54% by DHA)
↑ cell differentiation (30% by EPA, 65% by
DHA)
No significant effects on cell apoptosis and cell
cycle DHA was more potent than EPA
↑ PPARγ (DHA only) (126)
MCF-7
MCF-10A 6–30 μM of ALA, EPA, DHA
All n-3 PUFA ↓ MCF-7 cell growth (EPA,
DHA > ALA, dose-dependent)
AA ↓ MCF-7 cell growth (similar as ALA)
NA (122)
ER+ and
ER− cells
20 μg/mL of ALA, EPA, DHA
(72 μM ALA,
66 μM EPA, 61 μM DHA)
EPA, DHA
↓ cell proliferation (all cell lines) ALA
↓ estrogen independent BC cell proliferation
↑ lipid peroxidation (124)
↑: increase; ↓: decrease; NA: not available
31
Dysregulation of apoptosis is also a hallmark of cancer cells, and thus agents that
activate apoptosis are highly desired. Treatment with EPA and DHA inhibited
phosphorylation of Akt as well as the NF-κB DNA binding activity (96). This represents
a novel mechanism by which n-3 PUFA induce apoptosis in MDA-MB-231 cells (96).
Additionally, it has been shown that n-3 PUFA decreased expression of Bcl-2 and
increased activity of pro-caspase-8, an apoptosis effector enzyme (72). As a result, n-3
PUFA can inhibit BC development in vitro by both suppressing tumor cell proliferation
and inducing tumor cell death.
1.6. The Effect of Individual n-3 PUFA on BC Development
1.6.1 ALA and BC
1.6.1.1 Inefficient Conversion from ALA to EPA and DHA
α-Linolenic acid (18:3n-3; ALA) is the major n-3 PUFA in the Western diet.
Typical consumption of ALA in Europe, Australia and North America ranges between
0.6 and 1.7 g per day in men and 0.5–1.4 g per day in women (58). This is about 10-fold
lower than the consumption of n-6 PUFA. ALA, regarded as the precursor for long-chain
PUFA, can be converted to EPA (20:5n-3), DPA (22:5n-3) and DHA (22:6n-3) by the
pathway shown in Figure 1.1. Whether the essentiality of ALA in the diet primarily
reflects the activity of ALA itself or of long-chain PUFA synthesized from ALA is a
matter of debate. The concentration of ALA in plasma phospholipids, cells and tissues is
found to less than 0.5% of total fatty acids (127). Although the dietary intake of EPA and
DHA are approximately 10-fold lower than those of ALA in North America, the
concentrations of these long-chain PUFA in plasma, cell and tissue phospholipids are
greater than those of ALA (127). This apparent mismatch between dietary intakes and
levels of incorporation further suggests that the primary biological role of ALA is for
EPA and DHA synthesis. However, it is also possible that the low concentration of ALA
may be due to negative selection in the incorporation of ALA into blood and cell
membrane lipid pools (58). Since consumption of EPA and DHA show a strong inverse
32
association with the risk of BC, this raises the question of whether conversion of ALA to
EPA and DHA in humans is a viable alternative to dietary sources of these long-chain
PUFA. The majorities of human studies estimate that ALA supplementation in human
adults generally lead to an increase in EPA and DPA, but have little or no effect on DHA
content (128-132). Conservatively, Pawlosky et al. estimated the overall efficiency of
conversion from ALA was 0.2% to EPA, 0.13% to DPA and 0.05% to DHA (129). This
inefficient conversion is due to the first rate limiting reaction catalyzed by Δ6-desaturase
(58). As a result, the extent of ALA conversion to EPA and DHA is inefficient and
limited. Thus, ALA may not be considered as an effective alternative source to fish for
providing EPA and DHA.
1.6.1.2 Individual Effect of ALA on Breast Cancer
Data derived from epidemiological and observational studies suggest that ALA
present in the Western diet has protective effects in BC (Tables 1.7 and 1.8). Two case
control studies compared the fatty acid composition in the adipose breast tissue from
women with invasive non-metastatic breast carcinoma and women with benign breast
disease (44, 133). Low ALA content in adipose tissue was found to be associated with an
increased risk of BC. This observation was consistent with a previous cohort study on
121 BC patients, which demonstrated a link between a low level of ALA in adipose
breast tissue and increased risk of metastatic development (134). In particular, the ratio of
n-6/n-3 PUFA was also shown to be positively correlated with BC in these patients,
highlighting the role of n-3 and n-6 PUFA balance in BC.
Dietary supplementation of ALA reduced the growth of established mammary
tumors in chemically-induced rats and xenograft rodent models. In OVX athymic mice
with high circulating estrogen level, flaxseed oil and its high ALA content, attenuated
(ER+) MCF-7 breast tumor growth by reducing cell proliferation and increasing
apoptotic index (78). This effect was probably due to the downregulation of tyrosine
kinase receptors such as EGFR and HER2, with a subsequent reduction in pAkt.
Compared to corn oil, consumption of ALA-rich flaxseed oil tends to modify the n-6/n-3
33
PUFA ratio by increasing serum ALA, EPA and DHA concentrations. This provides
evidence that ALA absorption and conversion may contribute to the observed tumor
reducing effect in the flaxseed oil diet (78). Further, an in vitro study showed that pure
ALA inhibited MCF-7 cell proliferation by 33%, which was in accordance with the in
vivo reduction in palpable tumor growth (33%) (78). This suggests that ALA itself, rather
than the generated EPA and DHA, exerts this anti-tumorigenic effect (78). Another in
vivo study was designed to elucidate which component(s) of flaxseed (lignan or ALA)
was responsible for enhancing tamoxifens effect on reducing the growth of established
MCF-7 breast tumors at low circulating estrogen levels (105). ALA-rich flaxseed oil had
a stronger effect in reducing the palpable tumor size of tamoxifen-treated tumors
compared with lignan-treated mice (105). More importantly, ALA was found to
downregulate HER2 expression, and subsequently modulates growth factor-mediated
signaling pathways by repressing IGF-1R and Bcl-2 (105).
Remarkably, long-term changes in dietary ALA and LA ratio significantly affect
mammary tumor outcomes. Feeding BALB/c mice with ALA-rich linseed oil inhibited
the development of mammary tumors compared to mice on corn oil diets (135). In a
recent study, use of canola oil (10% ALA) instead of corn oil (1% ALA) in the diet of
MDA-MB-231 implanted mice reduced tumor growth rate (136). Progression to
apoptosis is associated with a balance between pro-apoptotic Bax and anti-apoptotic Bcl-
2. Mice exposed to canola oil had an increased ratio of Bax/Bcl-2, which was responsible
for the apoptosis of defective epithelial cells (93). Furthermore, the maternal diets have a
life-long influence on development of BC in the daughter. Substitution of corn oil with
canola oil in the maternal diet increased n-3 PUFA incorporation into mammary glands,
which in turn delayed occurrence of mammary tumors and increased tumor cell apoptosis
in offspring (93). Elsewhere, maternal replacement of dietary soybean oil with canola oil
significantly lowered the burden of MNU-induced mammary tumors, along with increased
survival rate in the offspring (137). As a result, maternal ALA supplementation brings
about a stable epigenetic imprint of genes that are involved in the development and
differentiation of the mammary gland, which are passed on to female offspring where they
exert a protective effect against BC.
34
Notably, the protective role of ALA in BC was inconclusive in in vitro studies. In
BT-474 and SkBr-3 cancer cells that naturally amplify the HER-2 oncogene, exogenous
supplementation with ALA significantly suppressed HER-2 mRNA expression, thereby
reducing the probability of activation that leads to tumor growth (138). Moreover, ALA
co-exposure was reported to synergistically enhance trastuzumab (an anti-cancer therapy)
efficacy in HER2-overexpression BC cells (139). However, in a separate study, ALA
exerted minimal tumor-reducing effects in the presence of trastuzumab (140). In addition,
some studies had difficulties in characterizing the role of ALA in estrogen dependent and
independent BC cell lines (122, 124). This suggests a variable effect of ALA on cell
proliferation depending on the cell line assessed.
Overall, these findings suggest that diets rich in ALA can inhibit mammary tumor
development in animals and in vitro, however, it cannot be ruled out that some of the
effects are due in part to conversion of ALA to EPA and DHA, albeit limited.
1.6.2 Individual Effect of EPA on Breast Cancer
Fish oil is a mixture of EPA and DHA, which have been previously shown to
have protective effects against BC. However, whether EPA and DHA differentially or
similarly affect BC has not yet been determined. In support of the independent effect of
EPA on BC, several in vitro studies consistently demonstrated the ability of EPA to
induce apoptosis in human BC cells (Tables 1.7 and 1.8). Chiu et al. demonstrated that 40
μM EPA induced cell apoptosis through inhibition of anti-apoptotic regulator proteins,
such as Bcl-2 (141). Moreover, Akt was also shown to be susceptible to EPA (97).
Treatment with EPA lowered both total and phosphorylated Akt content in transfected
MCF-7 cells that overexpressing constitutively active Akt (97). Additionally, co-
treatment with EPA enhanced the growth inhibitory response to tamoxifen in MCF-7
cells, thus suggesting that EPA may be useful as a nutritional adjuvant in the treatment of
BC (97).
Diets rich in EPA have also been shown to inhibit the growth of spontaneous or
transplanted mammary carcinomas in animal models and human clinical trials. EPA was
35
observed to slow tumor growth and reduced metastasis in mice implanted with KPL-1
human BC cells (142). In a separate study, when compared with LA diet, intake of EPA
significantly inhibited tumor cell proliferation and development of lung metastasis in
mice with induced mammary tumorigenesis (20, 121, 125). These inhibitory effects were
attributed to high incorporation of EPA into tumor phospholipids, and subsequently,
disrupting inflammatory eicosanoid biosynthesis from AA (20). More recently, EPA was
found to regulate cell proliferation in MCF-7 xenografts via an inhibitory G protein-
coupled receptor-mediated signal transduction pathway (143). Furthermore, a human
clinical study demonstrated a strong positive correlation between plasma EPA
concentrations and PPARγ mRNA levels in adipose tissue of obese subjects (144). Since
activators of PPARγ are known to inhibit cell proliferation and tumorigensis, up-
regulation of PPARγ gene expression by EPA might be a potential mechanism of action.
These findings suggest that EPA can independently act to inhibit the development and
progression of human BC.
1.6.3 Individual Effect of DHA on Breast Cancer
Several independent reports have shown that DHA can inhibit mammary
carcinoma development and progression (Tables 1.7 and 1.8). However, the specific
mechanisms underlying these protective effects of DHA remain to be determined.
HER-2 signaling is central to many processes involved in cellular proliferation
and survival (6). Treatment with DHA alone in vitro was shown to be effective in
disrupting lipid rafts in HER-2 overexpressing cells, inhibiting HER-2 activity and its
downstream signaling molecules (Akt and FAS, etc.), and consequently led to cell death
(6). Menendez et al. demonstrated that exogenous supplementation with DHA was able
to downregulate HER-2/neu oncogene expression in SK-Br3 and BT-474 human BC cells
(5). This supports the therapeutic potential of DHA supplementation in the treatment of
HER-2 positive BC. On the other hand, recent evidence suggests that DHA itself is
capable of decreasing EGFR localization in the lipid rafts of the MDA-MB-231 BC cell
line (56). Moreover, DHA supplementation was reported to significantly enhance the
36
efficacy of EGFR inhibitors, which provides strong evidence for the potential
development of combination therapies targeting EGFR (56). The pre-exposure with DHA
has been shown to synergistically enhance the cytotoxicity of antimitotic drugs including
Taxane and Taxol against highly metastatic BC cells (5). This was attributed to the
incorporation of DHA into cellular lipids, and subsequently altered membrane fluidity
and function, thereby increasing drug intake (5). In agreement with this, a recent human
clinical trial demonstrated that addition of DHA into chemotherapy increased survival in
metastatic BC patients (145).
A number of earlier studies have suggested that the anti-cancer property of DHA
is attributable to its ability to inhibit cell growth and induce apoptosis. Kang et al.
demonstrated that DHA induced apoptosis in MCF-7 cells through a combination of
pathways (146). Mechanistically, it was largely due to increased lipid peroxidation,
followed by accumulation of reactive oxygen species in cancer cells and higher oxidative
stress, ultimately resulting in cell apoptosis (146). It was also possible that the elevated
intracellular levels of DHA stimulated activation of apoptosis effector enzyme, such as
caspase-8 and caspase-3, and thus inducing apoptotic cell death (146, 147). Furthermore,
Chiu et al. found that pure DHA inhibited growth of MCF-7 cells (148). Although DHA
did not affect pro-apoptotic Bax protein, it induced the downregulation of anti-apoptotic
Bcl-2 gene expression time-dependently, and thus increasing the Bax/Bcl-2 ratio (148).
DHA has also shown to suppress KPL-1 cell growth in vitro, accompanied by
downregulation of Bcl-2 (149). Since the ratio of Bax/Bcl-2 is positively associated with
apoptotic activity, the regulation of Bax and Bcl-2 can be considered an important step in
the apoptotic actions of DHA.
Animal studies also support the anti-cancer role of DHA in BC. Dietary
supplementation of pure DHA was showed to suppress tumor development in both
chemically-induced carcinoma and xenograft rodent models (150, 151). Low level DHA
administration markedly reduced tumor growth rates and tumor weight compared with
rats fed LA (150). These observed suppressive effects of DHA resulted from diminished
tumor eicosanoid concentrations and decreased cell proliferation (150). DHA has also
been shown to decrease mammary tumor incidence coinciding with a 60% increase in
37
BRCA1 protein, a major tumor suppressor (151). In complement, DHA supplementation
was shown to increase BRCA1 at the transcriptional level (152). The correlation between
the in vitro and in vivo observations adds weight to DHA’s potential mechanism and
supports a beneficial role for DHA against BC.
Altogether, ALA, EPA and DHA have various effects against mammary tumor
development in vivo. In terms of in vitro studies, EPA and DHA individually exert an
inhibitory effect on the proliferation of almost all types of tumor cells; whereas ALA only
has effects on ER− and HER-2 positive BC cells in vitro (Table 1.8). As little as 50 μM
of EPA or 30 μM of DHA can dramatically reduce tumor cell viability, while 72 μM of
ALA only cause moderate inhibition on ER− cell proliferation. However, it is not
appropriate to compare the effective dosage of individual n-3 PUFA from different type
of studies, since the amount of tumor cells and the duration of treatment are different.
Additional experimental studies are needed to compare the efficacy of each individual n-
3 PUFA under the same conditions.
1.7 Plant-Derived n-3 (ALA) vs. Marine-Based n-3 (EPA, DHA)
The potential health benefits of n-3 PUFA have been examined in various types of
studies. However, the relative potency of plant-based n-3 versus marine n-3 PUFA, as
well as EPA versus DHA in inhibiting tumor growth remains unclear. There are only two
studies that have compared the efficacy between EPA and DHA (120). The first study
compared the ability of dietary EPA or DHA to suppress MNU-induced mammary
carcinogenesis in a rat model (120). Although treatment with DHA or EPA alone was not
as effective in combination, DHA was more potent in delaying tumor onset and reducing
tumor multiplicity relative to EPA (120). In accordance, several BC cell line studies
indicated that DHA was more effective in inhibiting MDA-MB-231 and MCF-7 cell
proliferation and invasion than EPA at the same concentration (86, 96, 121, 125, 153)
In terms of the efficacy of plant-based n-3 PUFA, there has not yet been a human
clinical study directly comparing ALA versus EPA or DHA in BC. To the best of our
38
knowledge, only two cell culture studies have examined the effect of different types of n-
3 PUFA. ALA was less effective compared with EPA or DHA, but ALA had moderate
inhibitory effects in some BC cell lines (122). This may be due to the conversion of ALA
to EPA and DHA that would lower the amount of ALA incubated with cancer cells. In
addition, the lower incorporation of ALA into the cellular lipid pool may also be a
confounding factor.
1.8 Conclusions
In summary, the present review has assessed the anticancer effects of n-3 PUFA
when consumed or treated individually, as well as in n-3 PUFA mixtures. ALA, EPA and
DHA can differentially inhibit mammary tumor development by changing the cell
membrane fatty acid composition, suppressing AA-derived eicosanoid biosynthesis and
influencing signaling transcriptional pathways to inhibit cell proliferation and induce
apoptosis. This review also provided evidence for using n-3 PUFA as a nutritional
intervention in the treatment of BC to enhance conventional therapeutics, or potentially
lowering effective doses.
Overall, in order to provide definitive recommendations, additional human studies
are required. Long-term studies tracking fish or n-3 PUFA intake are needed to
demonstrate a role for n-3 PUFA in prevention. Also, additional clinical trials are needed
to evaluate the effect n-3 PUFA on BC outcomes. Nevertheless, evidence does not
indicate harm and all forms of n-3 PUFA may be included in a
healthy diet.
39
Table 1.7 Individual role of ALA, EPA and DHA on BC.
n-3 Amount of Fatty Acid Effect Mechanism Ref
ALA
NA Moderate decrease BC risk NA (45)
~22.8 g of ALA per kg diet Reduced tumor cell proliferation Inhibited HER2, EGFR expression (78)
~22.8 g of ALA per kg diet Inhibited MCF-7 cell proliferation (78)
~11 g ALA per kg diet Reduced tumor incidence and burden Increased BAX/Bcl-2 ratio (93)
10.6 g ALA per kg diet Decreased tumor growth rate Inhibited HER2 expression (105)
72 μM ALA Moderate inhibited ER-negative cell
proliferation, not affect MCF-7 NA (124)
30 μM of ALA Slightly inhibited MCF-7 NA (122)
NA Inversely associated with BC risk NA (133)
NA Inversely correlated with metastasis
development NA (134)
55.9 g ALA per kg diet Reduced tumor growth and metastasis NA (135)
8 g ALA per kg diet Decreased tumor growth rate NA (136)
10 g ALA per kg diet Reduced tumor burden, increased survival rate NA (137)
2.5-40 μM of ALA Enhanced cytotoxic effects of Trastuzumab
(at 10 μM of ALA) Down-regulated HER2 (at 20 μM of ALA) (138)
10 μM of ALA Diminished proteolytic cleavage of the
extracellular domain of HER2 Inhibited HER-2 activity (139)
~21.2 g of ALA per kg diet Minimal inhibited tumor growth w/wo
Trastuzumab NA (140)
52.8 g of ALA per kg diet Inhibited mammary tumor development NA (141)
NA: not available
40
Table 1.7 Individual role of ALA, EPA and DHA on BC (continue).
n-3 Amount of Fatty Acid Effect Mechanism Ref
EPA
40–80 g of EPA per kg diet Slowed down tumor growth, reduced tumor burden Decreased AA derived-eicosanoid (20)
3–100 μM of EPA Induced BC cell apoptosis (at 50 μM of EPA) NA (86)
40–200 μM of EPA Restored the growth inhibitory effect of Tamoxifen
(at 40 μM of EPA) Decreased pAkt (at 20 μM of EPA) (97)
20–80 g of EPA per kg diet Inhibited the development of lung metastasis NA (125)
100 μM of EPA Inhibited MCF-7 cell growth NA (126)
40 μM of EPA Induced apoptosis, inhibited cell proliferation, arrested
cell cycle at G0/G1 Down-regulated Bcl-2 expression (142)
95 g of EPA per kg diet Reduced KPL-1 cell proliferation rate and metastasis NA (143)
42 g of EPA per kg diet Suppressed cell proliferation in MCF-7 xenografts in rats NA (144)
50 μM of EPA Increased PPARγ at mRNA level NA (145)
0–200 μM of EPA Inhibited MCF-7 cell growth (at 60 μM of EPA) NA (146)
NA: not available
41
Table 1.7 Individual role of ALA, EPA and DHA on BC (continue).
n-3 Amount of Fatty
Acid Effect Mechanism Ref
DHA
120 μM of DHA Decreased cancer cell viability, enhanced the cytotoxic
activity of taxanes Decreased the expression of Her-2/neu (5)
100 μM of DHA Disrupted lipid rafts, induced apoptosis in HER-2
overexpressing cells Decreased Akt activity and FANS (6)
100 μM of DHA Decreased MDA-MB-231 cell proliferation, enhanced
EGFR inhibitors
Altered EGFR phosphorylation and
localization (56)
0–200 μM of DHA Reduced MCF-7 cell viability and DNA synthesis
(at 25 μM of DHA)
Increased lipid peroxidation, caspase 8
activation (146)
20 or 100 μM of
DHA
Inhibited MDA-MB-231 cell proliferation, promoted
nuclear condensation
Increased caspase-3 activity
(at 100 μM of DHA) (147)
10–160 μM of DHA Inhibited MCF-7 cell growth and induced apoptosis
(at 40 μM of DHA)
Downregulated Bcl-2, increased Bax/Bcl-2
ratio (148)
270 μM of DHA 50% inhibitory KPL-1 cell growth after 72 h treatment Downregulated Bcl-2, increased Bax/Bcl-2
ratio (149)
40 g of DHA per kg
diet
Decreased tumor growth rate and final tumor weight,
increased apoptosis Reduced tumor PGE2, decreased Ki-67 (150)
32 g of DHA per kg
diet Reduced tumor incidence Increased BRCA1 at protein level (151)
30 μM of DHA 50% inhibitory MCF7 cell growth after 96 h treatment Increased BRCA1/2 at transcriptional level (152)
NA Increased response of the tumor to chemotherapies,
increased survival rate (154)
NA: not available
42
Table 1.8 Individual effect of ALA, EPA and DHA on different types of BC.
BC Cell Type ALA EPA DHA
MDA-MB-231 (ER−)
MDA-MB 435 (ER−) NA
MCF-10A (ER−) —
HBL-100 (ER−)
MCF-7 (ER+) —
ZR-75 (ER+) —
T-47-D (ER+) —
SK-Br3 and BT-474
(HER-2/neu positive) NA
Have significant inhibitory effect on cell proliferation; — slightly
inhibit the cell growth; NA: not available.
43
Chapter Two
Mini review update
Role of Flaxseed Oil in the Prevention and Treatment of Breast
Cancer
44
2.1 Introduction
Flaxseed (Linum usitatissimum), also known as linseed, is widely cultivated
throughout the northern hemisphere for both human and animal consumption(155).
The composition of flaxseed (FS) varies by growth location, year, environmental
conditions and cultivar. Typically, FS contains approximately 30% fiber, 20% protein,
40% fat, 4% lignan and 6% moisture (33, 156). In the past several decades, FS has
been the focus of interest in the field of food supplements due to its potential health
benefits in reducing the risk of cardiovascular disease, type II diabetes and some types
of cancer, including BC (157-159).
Two components of FS that may play a role in breast cancer (BC) are the
lignan and oil. The oil is rich in α-linolenic acid (ALA, 18:3n-3)(14, 159). Although
many foods contain lignans, FS is unique in that it is the densest source of the
mammalian lignan precursors, namely secoisolariciresinol diglycoside (SDG), with
between 60 and 300mg lignans per 100g servings(160). SDG is known as a type of
phytoestrogen, which is suggested to have estrogenic or anti-estrogenic properties,
and thus has been studied in hormone-related diseases including BC (156, 161-163).
Flaxseed oil (FSO) is low in saturated (9%), moderate in monounsaturated
(18%), and rich in polyunsaturated fatty acid (73%) (156). Notably, the predominant
polyunsaturated fatty acid (PUFA) in FSO is ALA (approximately 57%), followed by
linoleic acid (16%) (155, 156). According to our previous review paper, both ALA
and its long-chain n-3 PUFA metabolites, eicosapentaenoic acid (EPA, 20:5n-3) and
docosahexaenoic acid (DHA, 22:6n-3), can differentially inhibit mammary
carcinogenesis (164). However, the conversion of ALA to EPA and DHA is highly
inefficient in humans. Several human clinical trials demonstrated that dietary
supplement with FSO significantly increased the levels of ALA and EPA in the breast
milk, plasma, and erythrocyte, but failed to increase DHA content (128, 165). Thus,
the health benefits and the anticancer effects of FSO may be related to ALA
specifically, the metabolites EPA, and DHA, or the overall fatty acid profile of the oil.
Substantial evidence from rodent studies suggests that dietary FS and its oil
components can prevent BC development and inhibit mammary tumorigenesis (78,
105, 159). The anti-tumorigenic properties of FS have received considerable attention
45
in Western countries. Many women and newly diagnosed BC patients change their
diet by increasing consumption of FSO and FS associated food products (166, 167).
Nevertheless, the epidemiologic studies examining the associations of FS and ALA-
rich FSO with the risk of development of cancer, however, have been inconclusive.
Previous published reviews mainly focused on the anticancer effects of fish oil,
whereas the evidence regarding a protective role for FSO in BC development remains
equivocal. Since FS is widely consumed in Western countries and ALA is the
principle source of n-3 PUFA in the Western diets, there is growing interest in
determining the role of FSO in the prevention and treatment of BC. Therefore, the
purpose of this mini review is to give an update on the protective role of ALA-rich
FSO as well as whole ground FS on BC development. Additionally, the potential
mechanisms of these anti-tumorigenic properties will be highlighted.
2.2 Role of flaxseed in mammary gland development and breast cancer risk
2.2.1 Animal Study
Rodent models have allowed for the study of the role of FS exposure at
various stages of the life cycle and carcinogenesis pathway. Time of exposure to FS is
an important factor in modulating cancer risk at multiple stages, including 1) early life
exposure, such as exposure in utero, during gestation and lactation; 2) adulthood
exposure; 3) exposure once mammary tumor is established.
2.2.1.1 Early life exposure
Development of the mammary gland (MG) is unique and continues throughout
life. Findings from experimental rodent models reveal that exposure to dietary factors
during the early life periods when the MG is undergoing extensive modeling and re-
modeling, alter susceptibility to develop mammary tumors during adulthood (168,
169). Two important structures that relate to MG development and risk of BC are
terminal end buds (TEB) and alveolar buds (AB). TEBs are the large bulbous
structures in a rodent MG that give rise to malignant mammary tumors upon exposure
46
to a chemical carcinogen (169). This was attributed to the presence of a cap of highly
proliferative stem cells on each bud, and thus TEB is related with high cell
proliferation (170, 171). Similar structures in a human breast called terminal ductal
lobular unit 1 (TDLU1), which appear to be the sites of BC initiation in most women
(169). The incidence of carcinomas in rodents is directly associated with the density
of TEBs in the MGs at the time of carcinogen administration (170-172). In response
to stimulation by endogenous hormone or some dietary components, such as n-3
PUFA and soy protein, the highly proliferative TEB structures can be differentiated to
AB structures, which are less susceptible to carcinogens (110, 169). Thus, the dietary
factors that promote the early differentiation of TEB to AB may be a potential
preventive agent for BC during adulthood, and research has been conducted to
determine whether FS is such an agent.
Findings from rodent studies that relate early life exposure to FS with BC risk
are variable (Table 2.1). Exposure of female rats to 10% (w/w) flaxseed in utero,
during suckling, from suckling to postnatal (PND) day 50, and throughout lifetime
significantly delayed puberty onset, reduced number of estrous cycles and enhanced
the differentiation of highly proliferative TEB structures to less proliferative ABs
(173-175). This enhanced differentiation was partially mediated via the modulation of
the epidermal growth factor receptor (EGFR) signaling pathway, which potentially
protects against mammary carcinogenesis at early adulthood (175). However, no
beneficial effects were observed when exposure to FS occurred post-weaning, which
suggested that gestation and lactation are the critical periods for inducing structural
changes in the MG (173). Furthermore, exposure to 10% (w/w) flaxseed during
suckling was shown to significantly inhibit dimethylbenz(α)anthracene (DMBA)
induced rat mammary tumorigenesis (176). This observation emphasized that
exposure to FS during early stages of MG development can reduce the risk of
mammary tumorigenesis in rats later in life. Moreover, SDG at a level present in a 10%
(w/w) FS diet, has a similar effect as FS, indicating the anti-tumorigenic effect of FS
may be partly dependent on its lignans (176). However, other studies concluded that
exposure to 10% (w/w) FS or 15% (w/w) defatted FS in utero and during suckling
failed to enhance MG morphogenesis, and subsequently increased DMBA-induced
mammary tumorigenesis among female offspring (177, 178). This increase was seen
both in mammary tumor incidence and multiplicity (177, 178). The reason for this
47
discrepancy may be due to the source of FS (159, 177). Studies showing cancer-
promoting effect of FS used FS from the US and Finland, whilst those showing a
protective effect used the FS from Canada. The different growth locations may have
led to differences in the level of cadmium in the seed; cadmium is a heavy metal that
activates the estrogen receptor (ER), and thus has estrogenic effects (159, 179).
2.2.1.2 Adulthood exposure once breast cancer is established
Many studies have investigated the late exposure effect of whole ground FS in
different rodent models of breast carcinogenesis (Table 2.2). Carcinogen-induced,
xenograft and transgenic mouse models are the three main approaches employed in
these animal studies.
A high fat diet has been shown to increase the proliferation of mammary
epithelial cells and mammary tumors in rodent models. Supplementation of a high-fat
diet with 5 or 10% (w/w) FS before DMBA administration reduced the risk for BC,
which was mediated by lowered the epithelial cell proliferation and nuclear aberration
in the rat MGs, particularly in the TEB structure (180). Nuclear aberrations are lethal
events that have been correlated with carcinogen exposure, and thus reductions in the
level of nuclear aberration indicate less carcinogen damage in MGs (180).
Interestingly, this inhibitory effect was more pronounced with FS flour (38% oil) than
with the defatted FS meal (2.8% oil), suggesting the oil components in FS may play a
role (180). Next, the same model was used to determine the effect of exposure of a 5%
(w/w) FS diet during initiation (before DMBA administration), promotion (after
DMBA administration), and throughout both of the two stages on mammary tumor
development after 25 weeks (181). Although there was no significant changes in
tumor incidence, the number of tumors was decreased in the groups fed 5% (w/w) FS
at the initiation stage and throughout the study (181). Moreover, the size of tumors
was significantly reduced in the group fed 5% (w/w) FS during the promotion stage.
These tumor-suppressive effects may partially be due to the high level of ALA in FS
oil, which altered the fatty acid composition of the rat MGs and tumor tissues (181).
Later on, Thompson et al. demonstrated that FS can reduce mammary tumor growth
at late stages of carcinogenesis (182). Feeding the mice with 2.5 and 5% (w/w) FS
48
diet 13 weeks after DMBA administration significantly inhibited the growth of tumors
that established at the start of the dietary treatment, and also reduced the numbers of
newly developed tumors (182). In contrast to the DMBA-induced mammary
carcinogenesis, feeding FS at the 2.5 or 5% levels had no significant effect on MNU-
induced mammary tumor incidence, size or multiplicity when compared with the rat
fed a 20% soybean oil control diet (183). The lack of a significant effect of FS in
comparison to previous studies may be due to differences in the experimental design,
type and dose of carcinogen, and the protective effects of ALA present in the control
diet (1.6% ALA) (183).
The next series of experiments evaluated the effect of FS on established BC
using the xenograft rodent model. Dietary intake of 10% (w/w) FS inhibited the
growth of human ER-negative breast tumors (MDA-MB-435) and reduced metastasis
to distant organs, including the lymph node and lungs (184). This inhibitory effect
was modulated through downregulation protein expression of IGF-1 and EGFR.
Similar effects were also observed in the ER-positive MCF-7 human breast tumors (3).
In ovarietomized anthymic mice, 5% and 10% (w/w) FS exposure significantly
regressed established MCF-7 tumors in the presence of low or high estrogen level,
which mimics the hormone status of post- and premenopausal women, respectively
(3). This tumor suppression was concurrent with a reduction in cell proliferation (Ki-
67) and an induction of apoptosis, which was mediated by decreasing the expression
of estrogen sensitive gene products such as ERα and cyclin D1, and receptors of
tyrosine kinase growth factors such as human epidermal growth factor receptor-2
(HER2), EFGR and IGF-IR (185, 186). ER-α and ER-β are the two ER subtypes
identified in the mammary gland, and have distinct roles in mammary development.
ER-α is associated with increased cell proliferation and BC risk, while activation of
ER-β may prevent proliferative effects (186). Cyclin D1 is known as a major player in
cell cycle regulation, and has been shown to be regulated by estrogen acting through
the ER via the AP-1 DNA response element (187). As a result, the potential
mechanisms of the anti-tumorgenic effect of FS may be through inhibition of ER- and
growth factor-mediated signaling pathways.
Tamoxifen (TAM) and trastuzumab (TRAS, Herceptin™) are the most
commonly prescribed drugs for BC. It has been shown that dietary 10% (w/w) FS did
49
not interfere with the effectiveness of TAM and TRAS, but strengthened the tumor
inhibitory effect of TAM under high or low estrogen concentration in both short- and
long-term treatment (105, 140, 188, 189). The 10% (w/w) FS used in these animal
studies is equivalent to human intake of about 25-50 g of FS per day, depending on
the person’s food consumption.
Furthermore, the Tg.NK transgenic mouse model that carries an activated
analogue, the c-neu oncogene, driven by the mouse mammary tumor virus (MMTV)
promoter was chosen as a relevant model for studying in vivo effects of FS on the
development of HER2-positive BC (190). Increasing levels of FS (60mg to 540mg/kg)
were incorporated into the diet and fed to female transgenic mice over a range of time
(190). Although post-weaning exposure to the FS diets had no effect on MG
differentiation, tumor incidence and number of large tumors (>6mm diameter) were
significantly lower in mice fed the highest FS diet (0.54% FS by weight) compared
with control after 23 weeks (190). However, none of the FS diets affected tumor
volume or multiplicity compared with control (190). This may be due to the dosage of
FS used in this study that was too low to have a significant inhibitory effect. Overall,
these findings support the role for dietary FS in the inhibition of mammary tumor
development in animal models.
2.2.2 Cell culture study
The protective role of FS has also been demonstrated in vitro. The cell growth
inhibitory effects of FS sprouts were examined over a wide range of doses and times
(191). FS sprout at 100 ug/ml resulted in a significant growth reduction of ER-
positive (MCF-7) and ER-negative (MDA-MB-231) cells in a time-dependent manner
(191). After 72 hours treatment, 100 ug/ml of FS sprouts significantly induced
apoptotic cell death, which was accompanied by an increase of p53 gene transcription
in both cell lines (191). p53 is known as a tumor suppressor gene involved in cell
apoptosis (192). Therefore, FS sprouts reduce BC cell growth by increasing p53-
mediated apoptosis.
50
2.2.3 Human observational study
The initial case control study by the Ontario Women’s Diet and Health Study
was conducted to evaluate phytoestrogen intake and BC risk. Based on food
frequency questionnaire responses from 3370 women aged between 25-74 years, FS
associated products were accounted for 91% of the total lignan intake (193). Among
all the participants in this study, a daily intake of ≥ 5.35 mg lignans was associated
with a 19% reduction in BC incidence (odds ratio =0.81; 95% confidence interval
=0.65-0.99) (193). However, following stratification by BMI, this significant
association was observed only in overweight (BMI > 25) women. When stratified by
pre- or post-menopausal status, no statistically significant associations were observed
between lignan intake and BC risk (193). In 2013, Lowcock et al. reported a second
study, looking specifically at FS intake. Consumption of FS, and of flax bread was
associated with statistically significant 20-30% reductions in BC risk (194). This
relationship remained significant for postmenopausal women, but not among
premenopausal women (194).
The association between intake of sesame/flaxseed on mortality was
investigated in a large German cohort study of 2653 postmenopausal BC
patients(195). There was no significant association with increasing intake of
sesame/flaxseeds and BC-specific survival or overall survival (all PTrend>0.1).
Although the women in the study consuming lower level of sesame/flaxseeds
(0.3g/day) had a 25% reduction in BC-specific mortality, those with higher daily
intake (3.6g FS /day) had no survival advantage (195).
2.2.4 Human intervention study
To date, only one randomized double-blind placebo-controlled trial has
investigated the effect of FS on the biological markers of BC. In this study, muffins
containing 25g of FS were consumed by postmenopausal BC patients for 32 days
prior to surgery (50). Daily intake of FS significantly reduced tumor cell proliferation
and increased tumor cell apoptosis in postmenopausal patients, indicating by lowering
Ki-67 index and HER2 protein expression by 34% and 71%, respectively (50). In
addition to this intervention study, several other clinical trials have indirectly assessed
51
the role of FS supplementation on BC risk by measuring changes in estrogen
metabolism (196-198). Since estrogen is involved in the development and progression
of BC, the ability to modulate estrogen metabolism and thereby affect tissue exposure
to biologically active estrogen may influence BC (196). 2-hydroxyestrone (2OHE1)
and 16α-hydroxyestrone (16αOHE1) are the two major metabolites of estrogen;
however, 2OHE1 has shown little biological activity with some anti-estrogenic
actions in vitro (199). Brooks et al. demonstrated that dietary supplementation with 25
g ground FS significantly alters the metabolism of estrogen in favor of the less
biologically active estrogen metabolite 2OHE1 in postmenopausal women (198).
Similar results were shown in a study with daily supplementation of 10g FS for 7
weeks (197). Furthermore, Hutchins et al. reported that daily consumption of 5 or 10
g ground FS for 7 weeks significantly reduced serum estrogen concentrations in
postmenopausal women (196). These findings consistently demonstrated the ability of
FS to modulate estrogen metabolism, which may be a potential mechanism for FS in
reducing the risk of BC.
Altogether, these data are supportive of findings from animal models and
confirm the protective role of FS in BC development.
2.3. Role of flaxseed oil in mammary gland development and breast cancer risk
2.3.1 Animal Study
2.3.1.1 Early life exposure
Few studies have specifically investigated the role of flaxseed oil (FSO) in the
development of MG and subsequent risk of BC. Gestation and lactation exposure to
1.82% (w/w) FSO (at levels present in 5% FS by weight) does not affect MG
differentiation as indicated by TEB and AB density (173). In a recent study, wild type
FVB/NHanHsd mice were exposed to a high fat diet containing 24% (w/w) corn oil or
FSO from preconception to 6 weeks of age (200). Exposure to FSO diet, but not corn
oil, resulted in differential expression of genes involved in energy metabolism,
immune response and inflammation (200). Since there were no detailed measurements
based on the histopathology of the MG or the fatty acid composition of the tissues, it
52
is difficult to associate these gene changes in the MGs with BC risk. However, these
findings may at least suggest that intake of FSO during early life has a lasting effect
on the MG at the level of the gene expression (200).
2.3.1.2 Adulthood exposures once breast cancer is established
A considerable number of animal studies have shown that diet rich in ALA
can inhibit BC development (Table 2.3, some studies reviewed in Liu & Ma, 2014)
(164). Since FSO is known as the most abundant plant source of ALA, clarification of
FSO's involvement in BC development is essential. Cameron compared the effects of
diets rich in various oils (all at 6% by weight) on mammary tumor growth in DMBA-
treated C3H/Heston mice (201). ALA-rich FSO reduced tumor incidence compared
with n-6 PUFA rich corn and safflower oils. While in a separate study, feeding
Sprague-Dawley rats with 1.82% FSO, the level present in 5% FS, had no effect on
newly developed tumors, but significantly inhibited the growth of established tumors
(182). This inhibitory effect was correlated with an increased incorporation of total n-
3 PUFA, particularly ALA, and its elongation product EPA and DHA, resulting in a
significant rise in the ratio of n-3 to n-6 in mammary tumor tissue (182). Thus, FSO
may be more effective when tumors have already been established.
Studies using ovariectomized athymic mice with implanted tumors have been
valuable in determine the effect of FSO in reducing established tumors. One study
compared the effects of diets rich in various oils (from corn, fish and linseed) at 10%
level on the growth of implanted tumors derived from mouse mammary tumors (410
and 410.4) (135). Feeding BALB/c mice with ALA-rich linseed oil significantly
reduced the growth of 410.4 mammary tumors when compared to mice on corn oil
(135). Metastasis data paralleled the tumor growth rate (135). These observations
were negatively associated with the enhanced incorporation of n-3 PUFA into tumors;
where n-3 PUFA exert their physiological effects primary through changes in AA
metabolism, specifically decreasing pro-inflammatory eicosanoid synthesis, which
was indicated as reduced tumor prostaglandins E production (135).
Xenograft studies have also determined the effect of ALA-rich FSO diets on
the growth of human breast tumors. Dietary supplementation with 4% (w/w) FSO
53
(levels equivalent in 10% FS diet) was shown to attenuate tumor growth and
metastasis in anthymic nude mice with established ER-negative human breast tumor
(MDA-MB-435) (202). Interestingly, when compared with mice fed with the same
amount of SDG present in 10% (w/w) FS diet, FSO was more potent in inhibiting
MDA-MB-435 tumor development. This in turn was related to a decrease in cell
proliferation and enhanced apoptosis, as indicated by lowered Ki67 and increased
apoptosis labeling index (202). Later on, Chen et al. used the same model to
determine the therapeutic effect of adjuvant FSO on the locoregional recurrence and
metastasis (203). 4% (w/w) FSO significantly reduced metastasis after the primary
tumor was surgically excised, and this was mediated by decreasing extracellular
vascular endothelial growth factor (VEGF) in breast tumor cells (204). However, FSO
had little effect on the tumor recurrence unless excision was made when the tumors
were small (< 0.9g) (203). Similar effects were also observed in ER-positive MCF-7
human breast tumors (refer to the review by Liu & Ma) (164). In support of this, a
recent study demonstrated dietary exposure to flaxseed cotyledon (FC), the primary
location of FSO in the seed (33.37g/kg diet), reduce the growth of MCF-7 breast
tumors at low circulating levels of estrogen (205). Notably, the regression of the
established tumor caused by the FC diet (56 %) was close to that caused by the 10%
(w/w) FS diet (62%) which was tested for 8 weeks under the same conditions,
suggesting that FC may potentially substitute for FS (206). These anti-tumorigenic
effects of FC was mainly mediated via downregulation of growth factor receptors,
such as EGFR, HER2 and IGF-1R, which further suppress the activation of protein
kinases MAPK and Akt downstream to the cascade of cyclin-dependent kinase (205).
BT-474, an ER-positive human BC cell line that exhibits amplified HER-2
was injected into athymic mice to investigate the interaction between FSO and TRAS,
since TRAS is a first line therapy for HER2-positive BC (159, 207). Although
feeding mice with 4 or 8% (w/w) of FSO alone did not cause significant effect on the
growth of HER2-overexpressing BT-474 tumors in athymic mice under high
concentrations of estrogen, it enhances the tumor-reducing effect of TRAS (159, 207).
Combining TRAS2.5 treatment (2.5 mg TRAS/kg body weight) with an FSO-rich diet
(4% by weight) for 4 weeks led to a greater reduction in tumor area compared to
treatment with this dose of TRAS alone (159). Tumor analysis showed that
FSO+TRAS2.5 treatment caused a greater reduction in cell proliferation and a greater
54
increase in apoptosis compared to TRAS2.5 treatment alone further confirming the
reduction in tumor growth (159, 207). Importantly, combining low dose TRAS with
dietary FSO was just as effective as high dose TRAS, indicating that with FSO
consumption, only lower doses of the drug may be required to exert the same effect
(207). This may help delay the onset of TRAS resistance and relieve some of the side
effects of TRAS treatment, although more research is needed in this area. These
interactive effects are suggested to through the alteration in the membrane fatty acid
profile, which consequently led to a greater reduction of HER2 signaling as indicated
by lower levels of pHER2, pMAPK and pAkt expression (159). Additionally, FSO
was also shown to improve the responsiveness of TAM in reducing MCF-7 tumor
growth (206). Since TAM functions as a selective ER modulator, and is the most
commonly treatment for ER-positive BC, the interaction effect between FSO and
TAM was believe to mediate through ER signaling pathway, which may also block
the crosstalk between ER and growth factor receptors to inhibit the activation of
transcription (205).
The MMTV-c-neu transgenic mouse model has also been used to investigate
the role of FSO in the prevention of HER2-positive BC (208). The Tg.NK transgenic
mice were fed with 0.2 ml oil diets that contained increased proportion of FSO (0.05,
0.1, and 0.2 ml) mixed into corn oil for 30 weeks (208). Although the low dose FSO
wasn’t able to affect mammary tumor growth, there was a trend towards reduced
tumor incident and overall tumor weight with higher dose of FSO. This result
suggests that the ratio of n-6:n-3 plays an important role in mediating the effect of
FSO on HER2-overexpressing mammary tumourigenesis (208). The high level of
ALA in FSO may be beneficial in delaying the growth of mammary tumors if the n-6:
n-3 PUFA ratio is close to 1.
Taken together, these findings suggest that FSO is capable of reducing
tumorigenesis in animal models, modulating HER2 expression and growth factor
receptor signaling pathways. They also provide support for clinical trials to explore
the use of FSO as a dietary complementary aid for BC treatment with TRAS or TAM.
55
2.3.2 Cell culture study
It has been shown that ALA-rich FSO can inhibit BC development in vitro by
both suppressing tumor cell proliferation and inducing apoptosis; however, this
protective effect may depend on the BC cell line being assessed (reviewed in Liu &
Ma, 2014) (164). BC can be divided into several molecular subtypes based on the
expression of cell ER, progesterone receptor (PR) and HER2 (209). Recently,
Wiggins et al. examine the effect of ALA on the growth of four BC cell lines with
varying ER, PR and HER2 status at both high and low circulation of estrogen (209).
As a result, ALA significantly reduced viable cell number (>50%) in all four cell lines
regardless of cancer subtype and estrogen status, at a dose as low as 50 µM treated for
96 h (209). Later on, those four cell lines were incubated with 75 µM ALA in
presence of estrogen to determine the potential mechanism of action of ALA (209).
Among the four cell lines, MCF-7 (ER+PR+low HER2) had the most changes in gene
expression after 24 h treatment, with the reduction in MAPK1 gene expression being
most notable. MAPK1codes a protein that is integral in the MAPK signaling cascade
leading to increased cell growth and crosstalk with other pathways such as PI3K/Akt
and estrogen signaling (98). This suggests the proposed mechanism of ALA is
through reducing the activation of MAPK cascade to decrease cell growth. Although
MDA-MB-468 (ER-PR-HER2-) has very little HER2 to begin with, there was a
significant reduction in HER2 expression after 24h incubation with ALA (209).
Interestingly, in BT474 (ER+PR+HER2+) cell line that overexpress HER2, no
significant changes in the expression of HER2 was observed; instead of it, there was
an unexpected decrease in BRCA1 (breast cancer 1, early onset), a tumor suppressor
gene (209). In the aggressive triple negative cell line MDA-MB-231 (ER-PR-low
HER2), there was a time-dependent reduction in the number of viable cell, and some
genes expressions that altered by ALA treatment were also dependent on time (209).
Overall, these results suggest the intake of ALA-rich FSO can reduce growth
of different BC cells and change the expression of a variety of genes; however, this
anticancer effect may depend on the molecular subtype of the BC cell lines.
56
2.3.3 Human observational study
A large body of literature composed of human observational studies
examining the protective role of ALA in BC was inconclusive (has been reviewed in
Liu & Ma, 2014) (164). Some limitations are identified in these observational studied
which may impact the findings; they include 1) different measurements were used to
determine ALA exposure; 2) differences in sources of ALA; 3) the baseline
characteristics of the participants varied in different studies, such as age and
menopausal status. More importantly, no clinical studies have been conducted to
specifically examine the association between FSO consumption and BC risk.
Therefore, the protective role of FSO in decreasing BC risk still needs to be tested
clinically. To our knowledge, no human intervention trials have been completed to
investigate the effect of ALA-rich FSO on BC prevention or treatment.
2.4 Conclusion and future directions
In summary, the present review has assessed the anticancer effects of both FS
and its oil components. The current evidence from human observational and dietary
intervention studies suggests that FS and FSO are safe for consumption by healthy
individuals to potentially prevent BC and by BC patients to potentially reduce tumor
growth. ALA-rich FSO may be more effective in established mammary tumors. The
potential mechanisms by which FSO exert anti-tumorigenic actions are mainly
through inhibition of ER- and growth factor receptor-mediated signaling pathways.
This review also provided evidence for the potential use of dietary FSO as a
complementary agent in the treatment of BC to enhance conventional therapeutics, or
potentially lowering effective doses.
Further research is still needed in a clinical setting before definitive
recommendations can be made regarding the use of FS or FSO as an alternative,
adjuvant therapy for BC. For instance, long term studies tracking intake of FS and its
oil components intake are needed to demonstrate the relationship between FS
exposure and BC outcome. Also, additional large human intervention trails are
required to investigate the beneficial interactions of ALA-rich FSO with current BC
therapies.
57
Table 2.1 Early exposure to flaxseed and breast cancer risk
Animal Dosage Feeding Period Main Findings Mechanism Ref
Sprague-
Dawley
female rats
5 or 10%
(w/w)
flaxseed
1) Gestation and
lactation; 2) after
weaning (PND 21-
50); 3) lifetime
(gestation to PND 50)
Gestation, lactation and lifetime exposure
Both 5 and 10% flaxseed ↓TEB density ↑AB density
5% flaxseed ↓puberty onset ↓estrous cycle
10% flaxseed ↑puberty onset ↓estrous cycle length
5% flaxseed anti-estrogenic effects
↑mammary gland atrophy
10%flaxseed estrogenic effects
↑mammary gland differentiation
(173)
Sprague-
Dawley
female rats
10%
(w/w)
flaxseed
1) Lactation only;
2) From lactation to
PND 50
During lactation only or continuously
10% flaxseed ↓TEB density ↑AB density
10% flaxseed
↑differentiation of TEB to AB (174)
Sprague-
Dawley
female rats
(DMBA)
10%
(w/w)
flaxseed
During suckling
(from birth to PND 21
day)
At PND 21
10% flaxseed ↑number of TEBs
↑cell proliferation of terminal ductal epithelium
At PND 49-51
10% flaxseed ↓number of TEBs
At PND 21
10% flaxseed ↓epithelial ER-β ↑EFGR
At PND 49-51
10% flaxseed ↓ EGFR and EGF
(175)
Sprague-
Dawley
female rats
(DMBA)
10%
(w/w)
flaxseed
During suckling
(from birth to PND 21
day)
10% flaxseed ↓tumor incidence
↓average tumor size and tumor number
↓total tumor load and final tumor weights
N/A (176)
Sprague-
Dawley
female rats
(DMBA)
5 or 10%
(w/w)
flaxseed
1) In utero; 2) during
lactation (PND 5-25)
5% flaxseed ↓ mammary tumor latency
10% flaxseed ↓ mammary tumor latency
↑ tumor multiplicity
Flaxseed exposure didn’t affect number of TEBs or the
cell proliferation within the epithelial structures.
10% flaxseed ↑lobular ER-α
↓ER-β in lobules and ducts (177)
Sprague-
Dawley
female rats
(DMBA)
15%
(w/w)
defatted
flax flour
In utero
15% defatted flax
No significant changes in mammary gland morphology
↑apoptotic cells in mammary gland at PND week 8
↑tumor incidence ↑tumor multiplicity
15% defatted flax ↓mRNA of BRCA1
(at PND 8-week) ↑mRNA of p53 (178)
58
Table 2.2 Late life exposure to flaxseed and breast cancer risk (Carcinogen -induced rodent models)
Animal Dosage Feeding Period Main Findings Mechanism Ref
Sprague-
Dawley
female rats
5 and 10%
(w/w) flaxseed
flour
5 and 10%
(w/w) defatted
flaxseed meal
Start at 5-week-old, feed
for 4 weeks, before
DMBA administration
Flaxseed flour is more potent than defatted
flaxseed meal
↓epithelial cell proliferation
↓nuclear aberration in TEB
5% flaxseed flour exerted optimum effects
N/A (180)
Sprague-
Dawley
female rats
5% (w/w)
flaxseed flour
1) Initiation: post-
weaning to DMBA
administration
2) Promotion: after
DMBA administration
3) Initiation and
Promotion: after weaning
until end of 25 week
No significant difference in tumor incidence
5%FS during initiation or throughout the study
↓tumor multiplicity
5% FS during promotion ↓tumor size
↑ALA
↓DPA (n-6 PUFA)
No significant changes in
EPA and DHA content in
tumor or mammary
tissues
(180,
181)
Sprague-
Dawley
female rats
2.5 and 5%
flaxseed
13 weeks after DMBA
administration
Treat for 7 weeks
2.5% FS ↓new tumor incidence
2.5 and 5% FS ↓established tumor volume >50%
↓new tumor number and volume
2.5 and 5% FS ↓volume of established + new
tumor
N/A (182)
Sprague-
Dawley
female rats
2.5 and 5%
flaxseed
2 days after MNU
injection
Treat for 22 weeks
No significantly difference on tumor incidence,
volume, multiplicity and weight
↓tumor invasiveness and grade
↓plasma IGF-I in rats +/-
MNU (183)
59
Table 2.2 Late life exposure to flaxseed and breast cancer risk (Xenograft rodent models, continue)
Animal Dosage Feeding Period Main Findings Mechanism Ref
Balb/c nu/nu mice
MCF-7
+/- estrogen
10% (w/w)
flaxseed +/- 5mg
tamoxifen
after tumor was
established
Treat for 6 weeks
Under both low and high estrogen level
↑tumor regression ↓tumor size
↑effectiveness tumor inhibitory effect of
TAM
Under both low and high estrogen
↓Ki-67 index in tumor
↑ tumor cell apoptosis
(3)
Ncr nu/nu mice
MDA-MB-435
10% (w/w)
flaxseed
after tumor was
established
Treat for 7 weeks
↓tumor growth rate, final tumor volume
and weight
↓lymph node metastasis incidence
↓number of lung metastatic tumor
↓ Ki67, IGF-I, EGFR in primary tumors (184)
Balb/c nu/nu mice
MCF-7
Low estrogen
10% (w/w)
flaxseed
after tumor was
established
Treat for 25 weeks
↓ tumor growth stimulating effect of soy
protein
↓ tumor volume and weight
↓ Ki67 cell proliferation index
↑ number of apoptotic tumor cells
↓pMAPK, IGF-1 and EGFR
↓ERα and cyclin D1
↑ERβ and HER2
(185,
186)
Balb/c nu/nu mice
MCF-7
Low estrogen
5 and 10% (w/w)
flaxseed +/- 5mg
tamoxifen
after tumor was
established
Treat for 16 weeks
5 and 10% FS ↑tumor regression
↓tumor volume and weight
10% FS ↑effectiveness of TAM in
tumor regression
5 or 10% FS combined with TAM
↓Ki-67, cyclin D1, ERα, HER2, IGF-1R (189)
Balb/c nu/nu mice
MCF-7
High estrogen
5 and 10% (w/w)
flaxseed +/- 5mg
tamoxifen
after tumor was
established
Treat for 8 weeks
5 and 10% FS alone ↓tumor growth (dose-
dependent)
FS combined with TAM ↓tumor growth
(5% FS is most effective)
FS alone ↓Ki-67, IGF-I, HER2, PgR
↑apoptotic cell
FS/TAM ↑apoptotic cell, ERα
↓Ki-67, IGF-I, PgR
(188)
Balb/c nu/nu mice
MCF-7
Low estrogen
10% (w/w)
flaxseed
after tumor was
established
Treat for 8 weeks
↓tumor size ↑tumor regression rate
↓Ki-67 index ↑apoptotic cells
↓Bcl-2, cyclin D1, ERα, ERβ, EGFR,
HER2, IGF-1R
(210)
60
Table 2.3 Late life exposure to flaxseed oil and breast cancer risk
Animal Dose of FSO Feeding Period Main Findings Mechanism Ref
Carcinogen -induced rodent models
Sprague-
Dawley rats
DMBA
1.82% FSO:
18.2g/kg diet
(11.4g ALA/kg
diet)
7-week
(13 weeks after
DMBA
administration)
↓ established tumor volume (>50%)
no inhibitory effect on newly formed
tumor
↑ALA, EPA, DHA and total n-3
PUFA in tumors
↑ n-3: n-6PUFA
(182)
C3H/Heston
mice
DMBA
6% FSO: 60g/kg
diet
(28.2g ALA/kg
diet)
48-week
(1 week before
DMBA
administration)
↓ reduce tumor incidence (201)
Transgenic rodent models
TG.NK mice
with
MMTV/c-neu
0.05-0.2ml FSO
(59.4% ALA) 30-week
0.2ml FSO (when n3:n6 PUFA →1)
↓tumor incidence, overall tumor weight (208)
61
Table 2.3 Late life exposure to flaxseed oil and breast cancer risk (Xenograft rodent models, continue)
Animal Dose of FSO Feeding Period Main Findings Mechanism Ref
Ovariectomized
anthymic mice with
high E2
MCF-7
4% FSO: 40g/kg diet
(22.8g ALA/kg diet)
8-week
(fed after tumor
established)
↓ tumor size and cell
proliferation
↑ tumor cell apoptosis
↑ serum ALA, EPA and DHA
↓ EGFR, HER-2 and Akt
protein
(78)
Ovariectomized
anthymic mice with
low E2
MCF-7
4% FSO: 38.5g/kg
diet + TAM
(21.2g ALA/kg diet)
8-week
(fed after tumor
established)
↓tumor size, cell proliferation
↑tumor cell apoptosis
↑effectiveness of TAM
↓HER-2, IGF-1R, Bcl-2 and
pMAPK
(105)
BALB/c mice
410 and 410.4 mouse
tumor cell
10% FSO: 100g/kg
diet
(55.9g ALA/kg diet)
3-8week
(fed before tumor
established)
no significant effect on 410
tumor growth
↓410.4 tumor growth and
metastasis
↑ALA, EPA and DHA in
tumors
↓tumor prostaglandin
production
(135)
Athymic mice
BT-474
(overexpressing HER-
2)
High estrogen
4% FSO: 40g/kg diet
+/- 2.5 mg/kg bw
TRAS
(22.8g ALA/kg diet)
4-week
(fed after tumor
established)
4% FSO alone has no effect on
tumor growth
FSO enhances the effectiveness
of TRAS
FSO+TRAS ↓tumor cell
proliferation
↑ tumor cell apoptosis
4% FSO ↑ALA, EPA and
DHA
↓n-6:n-3 PUFA ratio
FSO+TRAS ↓Ki-67
↓HER-2, pHER-2, pAkt,
MAPK protein expression
↓ mRNA of EFGR
(159)
62
Table 2.3 Late life exposure to flaxseed oil and breast cancer risk (Xenograft rodent models, continue)
Animal Dose of FSO Feeding Period Main Findings Mechanism Ref
Athymic nude mice
MDA-MB-435
4% FSO: 36.53g/kg diet
(20.82g ALA/kg diet)
7-week
(fed after tumor
established)
↓tumor growth rate
↓distant lymph node metastasis (52%)
↓ Ki-67
↑ apoptosis index (202)
Athymic nude mice
MDA-MB-435
4% FSO: 36.53g/kg diet
(20.82g ALA/kg diet)
7-week
(fed after tumor
established)
↓lung and total metastases
No significant effect in tumor
recurrence
(203)
BALB/c athymic
nude mice with
MCF-7 with low
estrogen
8.2% FS cotyledon +/-
5mg TAM
= 33.37 FSO g/kg diet
(19g ALA/kg diet)
8-week
(fed after tumor
established)
↓ tumor size, tumor growth, cell
proliferation
↑ the effectiveness of TAM
↓Ki67
↓HER-2, pHER-2,
ERα, pMAPK, pAkt
↓mRNA of IGF-1R
(205)
Athymic mice
BT-474
(overexpressing
HER-2)
High estrogen
8% FSO: 80g/kg diet +
2.5 or 5mg/kg bw TRAS
(45.6g ALA/kg diet)
6-week
(fed after tumor
established)
↓ tumor size, tumor cell proliferation
↑ tumor cell apoptosis
↑ the effectiveness of TRAS
↓ Ki-67
(207)
63
Chapter Three
Rationale, Hypothesis, Objective and Experimental Design
64
3.1 Rationale
Breast cancer (BC) remains a major health concern as it is the leading cancer
killer of women worldwide (1, 2, 211). Growing evidence suggests that fish consumption
or intake of marine-derived long-chain n-3 polyunsaturated fatty acids (PUFA), such as
eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), may
influence mammary gland (MG) development at early life stages and potentially reduce
the risk of BC (8, 11, 34, 164). However, substantive evidence for a biological role for
the precursor, alpha-linolenic acid (ALA, 18:3n-3), remains equivocal. ALA is a plant-
based n-3 PUFA, but the conversion of ALA to EPA and DHA is highly inefficient in
humans. Meanwhile, ALA is known as the principle source of n-3 PUFA in the Western
countries, where the intake of EPA and DHA are typically low (32, 44). Therefore, the
clarification of ALA’s involvement in BC is essential. To the best of our knowledge, no
study has directly compared the effects of plant- and marine-based n-3 PUFA on
mammary tumor development. This fundamental knowledge is lacking, and is highly
important for the development of dietary strategies to prevent BC in the Western
countries. Thus, the research objective was to 1) determine the relative inhibitory effects
of lifelong exposure to plant-derived versus marine-based n-3 PUFA on mammary tumor
development; and to 2) identify potential mechanisms by which they exert anticancer
effects.
3.2 Mouse Model: MMTV-neu (ndl) YD5
The creation of transgenic mouse models has provided a direct method of
observing tumor growth and greatly enhanced our understanding of molecular
mechanisms underlying BC development and progression. For instance, the mouse
mammary tumor virus (MMTV) mouse models have been informative models for human
BC despite morphological, hormonal and lifestyle differences between mice and humans.
MMTV is an oncoRNA virus of the Retroviridae family, which causes breast tumors
once activated (212). Efficient replication of MMTV occurs predominantly in the
alveolar epithelial cells of the MG, and the expression of MMTV is under the influence
65
of steroid hormones (212). The MMTV induce premalignant lesions and malignant
tumors of the MG by acting as an insertional mutagen or by activating transcription of
nearby oncogenes (117, 213). ErbB2 (Neu/HER-2) is an epidermal growth factor receptor;
the term neu is used when referring to the murine gene or cDNA, whereas the human
homolog are designated as HER-2 (214). Overexpression of HER-2 is responsible for 25-
30% invasive human BC cases, which correlates with poor patient prognosis (73, 215,
216). In this study, transgenic mice used were engineered to overexpress the neu
protooncogene under the transcriptional control of MMTV enhancer. Oncogenic
activation of neu occurs as the result of a single point mutation in the transmembrane
domain, converting a valine residue to glutamic acid (217). Although comparable levels
of neu protein were detected in both normal and tumor tissues, catalytically active neu
was detected solely in tumors (214). Additionally, MMTV-neu breast tumors exhibit
sporadic mutations in neu resulting in its constitutive activation. Mutations commonly
encountered include an in-frame deletion within the extracellular domain of the neu
protein (212, 214). The neu deletion mutants (ndl) are able to promote the transforming
activity of neu transgene and lead to the development of mammary tumors faster than the
observed in the original MMTV-neu mice (212, 214). Furthermore, biochemical
characterization of these mammary tumors revealed the presence of neu receptor dimers
that were constitutively tyrosine phosphorylated (117). YD (tyrosine residue 1227) is one
of the neu autophosphorylation sites that can independently mediate the transforming
signals. Female MMTV transgenic mice expressing activated neu alleles of YD5 were
capable of inducing mammary tumors at an average age of 102 days (117).
Altogether, the MMTV-neu (ndl) YD5 is a highly aggressive BC model, which
can cause 50% of the MMTV transgenic mice to develop mammary tumors within 102
days of age (21, 116). Since the neu-oncogene corresponds to HER2 gene in humans, this
mouse model will be useful in detailed research and analysis of the signaling cascades
involved in HER-2 positive BC.
66
3.3 Hypothesis
The overall hypothesis is that lifelong exposure to both marine- and plant-derived
n-3 PUFA can prevent mammary tumor development. Specifically, it is hypothesized that:
1) Lifelong exposure to n-3 PUFA can influence MG development early in life by
reducing the number of terminal end buds (TEB) in MMTV-neu (ndl)-YD5 mice
2) Lifelong exposure to n-3 PUFA have long-term preventive impact on
mammary tumor development by delaying tumor latency, reducing tumor volume and
multiplicity in MMTV-neu (ndl)-YD5 mice
3) Plant-derived n-3 PUFA can inhibit mammary tumor development in a dose-
dependent manner
3.4 Objective
To determine the effects of lifelong n-3 PUFA exposure on:
1) The number and density of TEBs in mammary glands of mice at 6 weeks
2) Mammary tumor development including tumor latency, tumor volume and tumor
multiplicity of mice over 20 weeks
3) Fatty acid composition of tumor tissues and adjacent MGs, as well as serum
4) Expression of proteins that involved in tumor cell proliferation and apoptosis
3.5 Experimental Design
Heterozygous MMTV-neu (ndl)-YD5 males were bred with wild-type female
FVB mice. From this cross, two genetically distinct offspring were obtained: wild-type
mice (WT) and heterozygous MMTV-neu (ndl)-YD5 (MMTV). The harem mice were
67
fed one of the four diets, containing 1) 10% safflower oil, 2) 3% flaxseed oil plus 7%
safflower oil, 3) 10% flaxseed oil alone, or 4) 3% menhaden oil plus 7% safflower oil. 10%
safflower oil is an n-6 PUFA rich diet, which served as a positive control; while 3%
menhaden oil is rich in marine-derived n-3 PUFA and used as a negative control. The
amount of EPA and DHA present in 3% menhaden oil diet is equivalent to the level
present in traditional Japanese diets (1-2% of daily energy as marine-derived n-3 PUFA).
3% and 10% flaxseed oil contain different levels of ALA that are designed to test the
dose-response relationship between plant-derived n-3 PUFA exposure and BC outcome.
Female transgenic MMTV offspring were maintained on the same diets as their parents
for 6 or 20 weeks. At 6 weeks, mice were randomly chosen for euthanization, the number
and density of TEBs in the mouse mammary gland were measured. Starting at week 12,
the MGs of mice were palpated for new tumors; the tumor volume and multiplicity were
tracked for the duration of the 20-week-study. Mice were euthanized at 20 weeks to
collect tissues and organs. To determine the effect of diet on tissue fatty acid composition,
fatty acid analysis was conducted on tumor tissue, MGs and serum samples from 6- and
20-week-mice. Finally, the expressions of relevant protein targets in mouse tumors were
assessed to determine the impact of n-3 PUFA on cell proliferation and apoptosis.
68
Chapter Four
Plant- and Marine-derived N-3 Polyunsaturated Fatty Acids Prevent
Mammary Tumor Development
69
4.1 Abstract
Marine-derived n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA), have been shown to inhibit mammary
carcinogenesis. However, evidence regarding α-linolenic acid (ALA), a plant-based and
major n-3 PUFA in the Western diet, remains equivocal. Therefore, this study examined
the relative inhibitory potency of plant versus marine based n-3 PUFA on mammary
tumor development in a relevant model of human breast cancer over expressing human
epidermal growth factor receptor 2 (HER-2/neu). Heterozygous MMTV-FVB males were
bred with FVB female mice and fed one of four diets: 1) 10% safflower oil, 2) 3%
flaxseed oil plus 7% safflower oil, 3) 10% flaxseed oil alone, or 4) 3% menhaden oil plus
7% safflower oil. Transgenic female offspring were maintained on the parental diet for 6
or 20 weeks. Compared to the 10% safflower oil, intake of 10% flaxseed and 3%
menhaden oil significantly decreased the number and density of terminal end buds in the
mammary glands of mice at 6 weeks of age (p <0.05). At 20 weeks, a significant (p<0.05)
dose-dependent reduction of tumor volume (24% and 78%) and multiplicity (15% and
43%) was observed in mice fed 3% or 10% flaxseed oil, respectively. Relative to 3%
menhaden oil, 10% flaxseed oil resulted in a similar reduction in tumor volume (78% and
72%) but led to a greater reduction in multiplicity (27% vs. 43%). However, 3% flaxseed
oil had weaker inhibitory potency compared to 3% menhaden oil (p<0.05). Based on the
fatty acid composition of these n-3 PUFA diets, plant-derived ALA was only 1/8 as
potent as marine-based EPA and DHA in inhibiting mammary development. 3%
menhaden and 10% flaxseed oil down-regulated tumor expression of proteins involved in
cell proliferation (i.e. HER-2, pHER-2, pAkt and Ki67; p<0.05); and increased the
expression of the cell apoptotic protein, cleaved-caspase-3 (p<0.05). However, 3%
flaxseed oil was less potent than 3% menhaden or 10% flaxseed oil. The dose-dependent
effect of flaxseed oil clearly demonstrates a role for ALA in cancer prevention. However,
it is not possible to determine whether this effect is directly due to ALA or the conversion
to EPA and DHA. Overall, this study demonstrates that while marine-based n-3 PUFA
are more potent than plant-derived ALA, lifelong exposure to n-3 PUFA, whether from
marine or plant source, can mitigate tumor outcomes.
70
4.2 Introduction
Breast cancer (BC) is the most common cancer among women worldwide (1, 2).
It is the second leading cause of death for women in Canada and the United States (1, 2).
As estimated in 2014, 24,400 Canadian women will be diagnosed and 5,000 will die from
BC (211). This represents 26% of all newly diagnosed cancer and 14% of all cancer
deaths in Canadian women (211). BC is described based on receptor status, such as
human epidermal growth factor receptor 2 (HER-2) (73). Overexpression of HER-2
occurs in 25-30% of invasive human BC cases, and is associated with aggressive tumor
growth and poor prognoses (215, 216, 218). Though it has yet to be determined what
initially causes the onset of BC, research has found links between dietary habits and BC
risk. Dietary fatty acids in particular have garnered attention for their potential role in
modifying BC risk (32, 159, 164). Epidemiological studies have found significant
differences in BC incidence between populations consuming Asian diets and those
consuming Western diets (8, 25). Asian diets typically include a high intake of fish,
which are enriched in n-3 polyunsaturated fatty acids (n-3 PUFA), and associated with
lower incidence of BC compared to Westerners (8). The marine-derived n-3 PUFA, such
as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are
the downstream metabolites of α-linolenic acid (ALA, 18:3n-3) (164). In contrast,
Westerners typically consume more n-6 PUFA, such as linoleic acid (LA, 18:2n-6) and
arachidonic acid (AA, 20:4n-6), and often lack recommended doses of n-3 PUFA (0.6%-
1.2% of total fat intake as ALA and 500 mg EPA+DHA per day) (17, 25). Furthermore,
Asians emigrating to Western cultures and adopting local dietary habits have reported
rises in BC incidence reaching rates similar to that of Western countries (24). Thus,
dietary fatty acids may play a critical role in the prevention of BC development.
Though a number of human observational studies have associated n-3 PUFA
intake with a reduction in BC risk, the totality of evidence and the specific mechanisms
responsible for the protective effects of n-3 PUFA remain inconclusive. Emerging
research suggests that early life exposure at the critical period when the mammary gland
(MG) is undergoing extensive modeling and remodeling, may alter the susceptibility to
develop BC during adulthood (29, 168, 219). MG development is unique in that it
71
develops throughout life; the gland starts as a blank fat pad with an initial primary duct
that starts at the nipple and branches posteriorly throughout the fat pad over the course of
development (168, 220). In rodents, these ducts are led by club-shaped structures called
terminal end buds (TEB), and have been identified as the sites of tumor initiation in MGs,
likely due to the presence of a cap of highly proliferative stem cells on each bud (171,
221, 222). Therefore, MG development is a time of increased vulnerability to
carcinogenic exposures owing to the presence of TEBs. Similar structure in a human
breast is called terminal ductal lobular unit 1(TDLU 1), which appear to be the sites of
BC initiation in most women (169, 172). Exposure to n-3 PUFA during the early life
periods have been shown to decrease the TEB structures in mouse MG and reduce the
risk mammary tumorigenesis later in life (168, 173, 174, 177).
Experimental studies in rodent models have provided more consistent evidence
supporting an anticancer role of n-3 PUFA. The comparative nature of MG development
between humans and murine animals has allowed for numerous transgenic mouse models
of BC to be developed in order to recapitulate molecular pathways activated during
human mammary tumor development (223, 224). The mouse mammary tumor virus
(MMTV)-neu (ndl)-YD5 model is a highly aggressive BC model that overexpress HER-2
via the muring-equivalent neu-oncogene, and results in 50% of the MMTV transgenic
mice to develop mammary tumors within 100 days of age (117, 213, 225). Based on its
highly aggressive phenotype, the MMTV-neu (ndl)-YD5 mouse represents a relevant
model system for elucidating potential strategies aimed at prevention and/or treatment of
HER-2 positive BC (21, 116). Previously, we have demonstrated that lifelong exposure to
n-3 PUFA can mitigate mammary tumor development in mice expressing MMTV-neu
(ndl)-YD5 (116). Intake of a 3% (w/w) menhaden fish oil based diet reduced tumor
volume and multiplicity compared to mice receiving a 10% (w/w) n-6 PUFA diet (116).
Furthermore, a dose-response relationship of 0%, 3% and 9% (w/w) menhaden oil on
tumor size reduction was observed in MMTV-neu (ndl)-YD5 mice, and it was negatively
correlated with a dose-dependent incorporation of EPA and DHA into MG and tumor
phospholipid classes (21). As a result, the incorporation of n-3 PUFA into cellular and
tumor lipids is hypothesized to be an important mechanism by which n-3 PUFA elicit
their anti-tumorigenic effects, although other mechanisms may also be included.
72
Previous published studies have mainly focused on the protective role of fish
consumption or intake of marine-derived n-3 PUFA, such as EPA and DHA in BC
development (11, 31, 164); whereas the evidence regarding the biological role of the
precursor, ALA, a plant-based n-3 PUFA, remains equivocal. Flaxseed oil (FSO) is one
of the richest plant sources of ALA, where ALA comprises approximately 57% of total
fatty acids (155, 156). Research studies have demonstrated the anti-tumorigenic
properties of flaxseed were mainly attributed to its oil components (78, 159, 182, 205,
209). More importantly, ALA is known as the principal source of n-3 PUFA in Western
diets, but its endogenous conversion to EPA and DHA is not efficient in humans (58,
129). Therefore, the clarification of ALA’s involvement in BC is essential. To the best of
our knowledge no experimental study has directly compared the effects of plant- and
marine-based n-3 PUFA on mammary tumor development. This would help to determine
the relative effectiveness of ALA versus EPA/DHA. Also, it is the fundamental
knowledge for the development of dietary strategies to prevent BC in Western countries.
Therefore, the present study was designed to determine the relative inhibitory potency
and potential mechanisms of lifelong exposure to plant-derived versus marine-based n-3
PUFA on mammary tumor development in female MMTV-neu (ndl)-YD5 mice. The
results from this study may provide guidance regarding the potential use of ALA-rich
foods, such as flaxseed, in the management of BC, and whether FSO can be used as an
alternative dietary supplement in the Western diet when intakes of fish oil, or EPA and
DHA are typically low.
4.3 Material and Methods
4.3.1 Animals and diets
MMTV-neu (ndl)-YD5 mice, on an FVB background, were obtained from an in-
house breeding colony derived from animals originally obtained as a generous gift from
Dr. William Muller (McGill University). Mice were housed in ventilated cages in a
temperature- and humidity- controlled environment and were exposed to a 12 hour light-
dark cycle. Harems consisted of one male heterozygous MMTV-neu (ndl)-YD5 mouse
73
and three female FVB mice yielding progeny with a wild type or a heterozygous (MMTV)
genotype. All mice had ad libitum access to food and double-distilled water. Harems
were randomly assigned to one of four modified AIN93G diets (Research Diet Inc.): 1)
10% safflower oil (enrich in n-6 PUFA, 0% n-3 PUFA diet), or 2) 3% flaxseed oil+7%
safflower oil (3% plant-based n-3 PUFA diet), or 3) 10% flaxseed oil alone (10% plant-
based n-3 PUFA diet), or 4) 3% menhaden oil+7% safflower oil (3% marine-based n-3
PUFA diet). All diets were isocaloric and provided for 20 kcal% protein, 58 kcal%
carbohydrate and 22 kcal% fat. Diet ingredients and fatty acid composition of the diets
are listed in Tables 4.1 and 4.2, respectively. The mice consuming the 10% safflower oil
(n-6 PUFA diet) were used as a positive control to reflect an n-6 PUFA enriched Western
style diet. The mice fed 3% menhaden fish oil served as a negative control as it is
supplemented with a similar level of EPA and DHA as consumed by Asian countries. 3%
and 10% plant-based n-3 PUFA diets containing different amounts (%w/w) of flaxseed
oil are used to examine the dose-dependent relationship between ALA and BC.
All offspring were weaned and genotyped at 3 weeks of age as described
previously (116). Female transgenic (MMTV) offspring were kept and maintained on
their parental diet while all the males were euthanized post-weaning. Therefore, female
transgenic offspring received the same experimental diet throughout life, from in utero
until termination at 6 or 20 weeks of age. All experimental procedures were approved by
the institutional animal care committee (University of Guelph).
4.3.2 Food intake, body weights, puberty onset
Starting at 3 weeks of age, female mice were checked daily for vaginal opening, a
marker of puberty onset. Food intake and body weights were measured weekly.
74
Table 4.1 Composition of purified oil diets
Macronutrient
10% safflower 3% flaxseed 10% flaxseed 3% menhaden
g% kcal% g% kcal% g% kcal% g% kcal%
Protein 21 20 21 20 21 20 21 20
Carbohydrate 60 58 60 58 60 58 60 58
Fat 10 22 10 22 10 22 10 22
Total 100 100 100 100
Kcal/g 4.1 4.1 4.1 4.1
Ingredient
gm/Kg
Diet Kcal
gm/Kg
Diet Kcal
gm/Kg
Diet Kcal
gm/Kg
Diet Kcal
Casein 200 800 200 800 200 800 200 800
L-Cystine 3 12 3 12 3 12 3 12
Corn starch 336.7 1347 336.7 1347 336.7 1347 336.7 1347
Maltodextrin 10 132 528 131 528 131 528 132 528
Sucrose 100 400 100 400 100 400 100 400
Cellulose, BW200 50 0 50 0 50 0 50 0
Soybean oil 0 0 0 0 0 0 0 0
Safflower oil 97 873 67.9 611 0 0 67.9 611
Flaxseed oil 0 0 29.1 262 97 873 0 0
Menhaden oil 0 0 0 0 0 0 29.1 262
t-Butylhyfroquinone 0.02 0 0.02 0 0.02 0 0.02 0
Mineral mix 35 0 35 0 35 0 35 0
Vitamin mix 10 40 10 40 10 40 10 40
Choline Bitatrate 2.5 0 2.5 0 2.5 0 2.5 0
Total 966.3 4000 966.3 4000 966.3 4000 966.3 4000
Composition of AIN-93G modified diets with 10% safflower oil, 7% safflower oil+3% flaxseed
oil, 10% flaxseed oil, and 7% safflower oil+3% menhaden oil as provided by manufacture,
Research Diets.
75
Table 4.2 Fatty acid composition of diets
Fatty acid 10% safflower oil 3% flaxseed oil 10% flaxseed oil 3% menhaden oil
12:0 0.0 0.0 0.0 0.1
14:0 0.2 0.2 0.1 2.7
15:0 0.0 0.0 0.0 0.2
16:0 6.5 6.3 5.8 10.2
16:1c9 0.1 0.1 0.1 0.1
18:0 2.7 2.8 3.2 3.1
18:1c9 16.3 16.0 15.0 14.3
18:1c11 0.7 0.7 0.8 1.4
18:2n6 71.5 55.4 16.5 56.1
18:3n6 0.3 0.2 0.0 0.1
18:3n3 0.2 17.4 57.6 0.6
18:4n3 0.1 0.0 0.0 0.8
20:0 0.4 0.4 0.1 0.4
20:1c11 0.0 0.0 0.1 0.4
20:2n6 0.0 0.0 0.0 0.1
20:3n6 0.0 0.0 0.0 0.1
20:4n6 0.0 0.0 0.0 0.5
20:3n3 0.0 0.0 0.1 0.1
20:5n3 0.0 0.0 0.0 3.9
22:0 0.3 0.0 0.1 0.0
22:1n9 0.0 0.0 0.0 0.1
22:2n6 0.0 0.0 0.0 0.1
22:4n6 0.0 0.0 0.0 0.1
22:3n3 0.0 0.0 0.0 0.0
22:5n6 0.0 0.0 0.0 0.2
22:5n3 0.0 0.0 0.0 0.8
24:0 0.2 0.0 0.0 0.0
22:6n3 0.0 0.0 0.0 3.4
24:1 0.0 0.0 0.0 0.0
Total n-6 71.8 55.7 16.7 57.3
Total n-3 0.4 17.6 57.8 9.5
Total SFA 10.5 9.8 9.5 16.8
Total MUFA 17.3 16.9 16.0 16.3
Total PUFA 72.2 73.3 74.4 66.9
Fatty acid composition (%) of n-6 and n-3 PUFA diets. Lipids were extracted from three
individual pellets from each diet and analyzed by gas chromatography. SFA: saturated fatty acids;
MUFA: monosaturated fatty acids; PUFA: polyunsaturated fatty acid.
76
4.3.3 Early mammary gland developmental period (6-week-timepoint)
4.3.3.1 Euthanization and tissue collection
At 42 days postnatal, mice were randomly chosen from each diet group (n=12-16)
to assess the effect of dietary n-3 PUFA exposure on MG development. Mice at 6 weeks
of age were selected as previously reported that the number of TEBs in the mouse MGs
typically reaches their maximum during this period. A vaginal smear was taken by
flushing the vagina with 30 µL of a phosphate buffer saline solution. The solution was
then placed on a glass slide and viewed under a Nikon Eclipse TS100 microscope for
estrus cycle classification. Mice were classified as being in one of four estrous cycle
stages including proestrus, estrus, metaestrus and diestrus. If in proestrus, estrus or
metaestrus, mice were euthanized via carbon dioxide asphyxiation followed by cervical
dislocation. If the mice were in diestrus, estrous check was then repeated the next day,
and euthanization was delayed in order to control for the impact of hormone fluctuations
on cell proliferation profiles. This was done for a maximum of two days past the set
euthanization date, at which point estrous stage was checked and recorded, and mice
were euthanized regardless of stage, in order to maintain consistency in MG
measurements.
At necropsy, blood was collected by cardiac puncture, allowed to sit for 30 min
before separating into cells and serum by centrifuge (10000 xg for 5 min), and stored in
an -80˚C freezer. The mouse pelt with MGs attached was removed for wholemounting
analysis. The 5th
MGs were excised, weighed and snap-frozen in liquid nitrogen for fatty
acid analysis.
4.3.3.2 Mammary gland whole mounts
The skin pelt was stretched on corkboard and fixed in 10% formaldehyde. After
48-hour fixation, the 4th
left abdominal MG was dissected from the skin pelt and de-fatted
in acetone for two days (refreshed daily). This was followed by a series of rehydration
steps in decreasing concentrations of 99%, 95% and 70% ethanol, and distilled water for
77
1 h each and then stained in carmine alum overnight. The following day the glands were
destained in a 2% hydrochloric acid/70% ethanol solution for 1 h, then stepwise
dehydration through an increasing series of graded ethanol (70%, 95%, and 99% each for
1 h) and transferred into xylene overnight. The whole mount MGs were preserved in
heat-sealed polyethylene packages containing 2ml methyl salicylate.
4.3.3.3 Counting of TEB structures
The whole mount mammary glands were coded so that the investigator was
blinded to the identity of the glands. The most undifferentiated TEBs are mostly
concentrated in the periphery of the MGs. The distal portions of MGs were viewed under
a stereoscope at a 10× magnification (10× ocular lens and 1.0× objective lens, Zeiss
Stemi 2000-c) to enumerate number of TEBs and other measures including total fat pad
area, ductal tree area, and length of ductal infiltration. Density of TEBs per MG was
calculated using the formula (number of TEBs) / (ductal tree area). Mammary gland
morphology measures were evaluated blindly by 2 investigators.
4.3.4 Mammary tumor developmental period (20-week-timepoint)
4.3.4.1 Tumor palpation
The remaining female mice continued on the same experimental diet until the 20
weeks of age (n=12 per genotype per diet group). Starting at 10 weeks of age, mice were
palpated for mammary tumor formation. When a new tumor was detected, measurements
of tumor size and multiplicity were recorded 3 times per week for the duration of the
study. Tumors were measured along the sagittal (length) and transverse (width) planes
with the use of digital calipers, and the tumor volume was calculated as follows: [(length)
× (width)2]/2.
78
4.3.4.2 Euthanization and tissue collection
At 20 weeks of age, all the mice were checked for estrous cycle and euthanized by
CO2 asphyxiation as described above. For ethical reasons mice that lost more than 20% of
their body weight or possessed tumors exceeded either 17mm in length/width or more
than 5000mm3 in volume were euthanized prior to the 20-week time point.
At necropsy, blood and serum samples were first collected as described above.
For tumor-bearing mice, MG and tumors were photographed, and final tumor weight and
volume measurements were recorded. One of the excised primary tumors (per mouse)
was immediately preserved in 10% formaldehyde for immunohistochemistry analysis.
The remainder of the tumors tissues, the 4th
and 5th
MGs were cleaned of surrounding
tissue, removed, weighed and snap frozen in liquid nitrogen for fatty acid and molecular
analysis.
4.3.4.3 Fatty acid analysis
Lipid extraction. Lipids were extracted from serum, MGs and tumor tissues via
the Foch Method (226). In brief, 50ul serum sample from either 6- or 20-week-old mice
was first vortexed with 0.1M KCl and 4ml of CHCl3: MeOH (2:1), and then incubated
overnight at 4˚C to facilitate lipid exaction. Lipids from MGs and tumor tissue were
extracted in a similar manner. The entire 4th
MG from 6-week old mice or the tumor
adjacent MG from 20-week-old mice, was homogenized in 0.1M KCl. For tumor samples,
only 0.1g of the tissue was homogenized in 0.1M KCl. Homogenates were then added to
10ml of CHCl3: MeOH (2:1), vortexed and chilled overnight.
The following day, samples were centrifuged (1460 rpm, 10 min) and the
chloroform layer was then collected, dried down under a gentle stream of nitrogen and
reconstituted to 10mg/ml. Serum samples were examined by total lipid analysis, while
MGs and tumor tissues were analyzed by phospholipid-class TLC in the following
manner. Phospholipid class analysis of MGs and tumors provides more detailed
information regarding potential mechanisms of action.
79
Total lipid analysis (saponification). 0.5 M KOH prepared in methanol was added
into the serum sample. A C17:0 FFA standard was transferred to each sample using a
Hamilton syringe. Samples were vortexed and saponified at 100˚C for 1 hour. Samples
were checked every 10 min to avoid evaporation.
Phospholipid-class TLC. Lipid samples extracted from MGs and tumor tissues
were spotted on an activated H-plate (EMD Chemicals, #5721-7). The TLC solvent was
made fresh by combining 30ml chloroform, 9ml methanol, 25ml 2-propanol, 6ml of
0.25M KCl and 18ml trimethylamine. The TLC plate was lightly sprayed with 0.1% (w/v)
ANSA (Fluka, #GA12046) and visualized under UV light. Phosphatifylcholine (PC) and
phosphatidylethanolamine (PE) were collected for analysis. A C17:0 FFA standard was
included using a Hamilton syringe.
Methylation. Methylation for serum, MGs and tumor samples was the same.
Hexane and 14% BF3-MeOH (Sigma, N1252) were added to each sample and incubated
for 90 min at 100˚C. Following methylation, 2ml of double distilled H2O was added to
stop methylation. Samples were centrifuged (1460rpm, 10 min), then the hexane layer
was collected and dried down under nitrogen before reconstitution in 2ml of hexane.
Fatty acid methyl esters were analyzed using a gas chromatography system (Agilent
Technologies, 7890B). Fatty acid composition was expressed as a percentage of total
fatty acids.
Sample size from each diet group was as follows: for both 6-and 20-week mice,
serum sample size (n) was equal to 5 per diet; for 6-week mice, 5th
MG tissue was taken
from mice in 10% safflower oil (n=5), 3% flaxseed oil (n=6), 10% flaxseed oil (n=6), and
3% menhaden oil (n=5); for 20-week mice, 4th MG tissue was taken from mice fed with
10% safflower oil (n=5), 3% flaxseed oil (n=4), 10% flaxseed oil (n=6), and 3%
menhaden oil (n=5); tumor tissues were taken from mice consumed 10% safflower oil
(n=6), 3% flaxseed oil (n=5), 10% flaxseed oil (n=5), and 3% menhaden oil (n=5).
80
4.3.4.4 Western blot analysis
Western blotting was performed on the mouse mammary tumor tissues to analyze
the protein expression of HER2 and phospho-HER2. For each MMTV mice (n=6 per diet
group), a portion (about 20mg) of tumor tissues was homogenized in 1.5ml RIPA buffer
(cell signaling technology, #9806S) containing 15ul protease inhibitors (cell signaling
technology, #5817S). The homogenate was mixed gently on a rotator at 4˚C for 2 hours.
Next, the lysates were clarified by centrifugation at 10000xg at 4˚C for 10 min and
quantified for total protein concentration by using Coomassie Plus Protein Assay kit
(Thermo Sicentific, #1856210). Equal amount of tumor protein extract (20µg) from each
mouse were made up to 25µl with PBS and 5µl of 5×SDS PAGE loading dye. Then the
mixed tumor protein samples were separated by electrophoresis on 7.5% polyacrylamide
gels under denaturing conditions, and transferred to polyvinylidene difluoride membranes.
Membranes were incubated overnight at 4˚C in blocking buffer (5% skim milk in Tris-
buffered saline/0.1% Tween-20, TBST). After blocking, membranes were incubated for 2
hours under room temperature with primary rabbit anti-human antibodies, including
HER2/ErbB2 (1:2000, cell signaling technology, #4290) and Phospho-HER2
(Tyr1221/1222) (1:1000, cell signaling technology, #2243). The primary antibodies were
diluted in TBST containing 3% (v/w) bovine serum albumin (Sigma-Aldrich Canada Ltd).
The membranes were also probed under the same conditions with primary goat anti-
human antibody for β-actin (1:500, Santa Cruz Biotechnology, sc1616) to confirm even
protein loading. After extensive washing in TBST, membranes were probed with
secondary anti-rabbit IgG HRP-linked antibody (1:5000, cell signaling technology #7074)
for 1 hour. For β-actin, secondary donkey anti-goat IgG-HRP (1:2000, Santa Cruz
Biotechnology, sc2020) was used. The bound antibodies were visualized using an
enhanced chemiluminescence kit (PerkinElmer, Inc) and detected in FluorChem Imaging
System (Alpha Innotech). The intensity of bands was quantified using Alpha View SA
(Alpha Innotech), and the relative intensity unit of each tumor protein bands was
normalized with the average intensity of β-actin bands.
81
4.3.4.5 ELISA
Mouse mammary tumor Akt and phosphor-Akt protein expression was quantified
by an ELISA kit (ebioscience). Tumor proteins were extracted as described previously
and equal amounts of tumor protein extract (40 µg) from each MMTV mouse (n=6 per
diet group) was used for the ELISA measurement. The relative abundance of total Akt
(ebioscience, #85-86047-11) and phospho-Akt 1/2/3 (Ser473) (ebioscience, #85-86042-
11) were measured in all the tumor protein samples according to the manufacturer’s
instructions.
4.3.4.6 Immunohistochemistry analysis (IHC)
To evaluate the dietary effect of n-3 PUFA on tumor cell proliferation and
apoptosis, Ki-67 and cleaved-caspases-3 protein expression levels in mouse mammary
tumor tissues were assessed by IHC. Briefly, paraffin tumor sections (n=6 per diet group)
were deparaffinized in xylene, rehydrated through graded ethanol solutions to distilled
water, and washed in phosphate-buffered saline (PBS). Endogenous peroxidase activity
was quenched by incubating in a dual endogenous enzyme-blocking reagent (Dako,
#S2003) for 5 min. Next, tumor sections were subject to heat-induced antigen retrieval in
a decloaking chamberTM
N×Gen (Biocare Medical) containing 0.01 M citrate buffer
(pH=6.0) According to the tested protein targets, the tumor sections were treated
differentially on boiling temperature and time during the antigen retrieval (i.e. Ki-67,
90°C for 25 min; cleaved-caspase 3, 95°C for 20 min). Nonspecific binding sites were
blocked using 3% (w/v) BSA for 1 hour. Tumor sections were then incubated overnight
at 4°C with 1:200 dilutions of rabbit monoclonal antibody against Ki67 (Abcam,
#ab16667) or 1:1000 dilutions of rabbit polyclonal antibody against cleaved-caspase-3
(Asp 175) (Cell Signaling, #9661). Instead of primary antibody, the tumor section
incubated with PBS was used as a negative control. In the following day, after several
washes with PBS, the tumor sections were incubated with secondary anti-rabbit
EnVision+ System-HRP-labelled polymer (Dako, #K400211-2) for 1 hour at room
temperature. To show antigens, tumor sections were treated with 3, 3’-diaminobenzidine
82
chromogen solution and its specific substrate buffer (Dako, #K4010) according to the
manufacturer’s instructions for 10 min. The tumor sections were then counterstained with
hematoxylin, dehydrated and mounted. The tumor slides (n=6 per diet group) were read
blindly with a light microscopy (Olympus Bx46, ZK62809) at 400× magnification to
identity the samples. For each slide, 5-10 fields were randomly chosen to photograph by
Q-capture pro-7 system; the number of total cells and of the positive cells of Ki-67 and
cleaved-caspase-3 per field were scored blindly using image J software. Approximately
over 1000 cells were counted per field. Ki-67 and cleaved-caspase-3 labeling index was
expressed as a percentage of total tumor cells, and calculated as follows: (number of
positive cells / total cells counted) ×100.
4.3.5 Statistical analysis
The predetermined upper limit of probability for statistical significance was p ≤
0.05 for all analyses using SASv9.1. A one-way analysis of variance (ANOVA) was
conducted to determine differences in daily food intake, puberty onset, number and
density of TEB structures, tumor latency, final overall tumor weight, fatty acid
composition and protein expression between diet groups, and followed, if justified by
statistical significance, by Tukey’s Studentized Range test. When the data was not
normally distributed, variables were log or square root transformed to adjust for
nonnormality. Log-Rank test was used to analyze the difference in proportion of mice
free of tumors in each diet group throughout the 20-week study period. The Kruskal-
Wallis procedure was performed followed, if justified by statistical significance, by
Wilcoxon two-sample rank sums. A repeated-measures test was conducted for total tumor
volume and tumor multiplicity over the 20-week time course to determine differences
between diet groups over time, as well as for mouse body weight between diet groups
(116). Values are reported as mean SD.
For the purpose of this study, the following definitions for tumor parameters were
used. Tumor latency values were determined as time when the first tumor was palpated.
Tumor multiplicity values were determined by the total number of tumor palpated in each
83
mouse. Total tumor volume values were calculated as the sum of all tumor volumes for
each mouse. Final tumor volume and multiplicity values were taken when mice reached
20 weeks of age, except the ones required enthanization prior to 20 weeks of age. Due to
ethical reasons, seven of 48 tumor-carrying mice were terminated before the 20 week
time point (4 mice from 10% safflower oil diet and 3 mice from 3% flaxseed oil diet
group were terminated between 18-19 weeks of age).
4.4 Results
4.4.1 Food intake, body weight gain and puberty onset
There were no significant differences between diet groups in daily food intake
(data not shown), body weight change from 3 to 20 weeks of age (Figure 4.1) or tissue
weights measured at euthanization (data not shown). The average daily intake for mouse
is ~2g per day. Timing of vaginal opening is a marker of puberty onset. The onset of
puberty was significantly (p<0.05) delayed in mice fed with n-3 PUFA diets compared to
mice that consumed an n-6 PUFA diet (10% safflower oil, 25.9 ± 0.8 days). A significant
delay (p<0.05) was also observed in mice fed 10% flaxseed oil (29.2 ± 1.6 days)
compared to mice fed 3% flaxseed oil (27.9 ± 1.4 days), 3% menhaden oil group were at
intermediate level (28.2 ± 1.6 days) (Figure 4.2).
84
Figure 4.1 Mice body weight changes throughout 3 to 20 weeks of age. No significant
difference existed between diet groups determined by repeated measures analysis.
10
15
20
25
30
35
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Aver
age
bod
y w
eigh
t (g
)
Time (Week)
10% safflower 3% flaxseed 10% flaxseed 3% menhaden
85
Figure 4.2 Average pubertal onset as measured by vaginal opening in mice fed 10%
safflower oil (n-6 PUFA diet, n=27), 3% flaxseed oil (n-3 PUFA, n= 24), 10% flaxseed
oil (n-3 PUFA, n=24) or 3% menhaden oil (n-3 PUFA, n=26). A one-way ANOVA was
performed; different letters denote significant difference (p<0.05) between diet groups.
4.4.2 TEB structure in 6-week-old mice
At 6 weeks of age, significant differences in the number of TEB structures were
observed in the MG of mice between diet groups. See Figure 4.3 for representative
images of MG taken at 6 weeks of age from each diet group. When compared with mice
consuming 10% safflower oil (an n-6 PUFA diet), lifelong exposure to 10% flaxseed and
3% menhaden oil significantly (p<0.05) reduced the number and density of TEBs;
however, the mice exposed to 3% flaxseed oil did not have any significant effect on
numbers of TEB structures (Figure 4.4). There were no significant differences between
diet groups in ductal tree area and length of ductal infiltration (data not shown).
20
24
28
32
10% safflower (n-6)
3% flaxseed (n-3)
10% flaxseed (n-3)
3% menhaden (n-3)
Days
Post
nata
l
c
b
a
ab
86
Figure 4.3 Representative stereoscopic wholemount images (20×) of the left fourth MG
of MMTV mice at 6 weeks of age fed a) 10% safflower oil, b) 3% flaxseed oil, c) 10%
flaxseed oil, and d) 3% menhaden oil. Arrows (→) show representative terminal end buds
(TEB) enumerated. Lymph node (LN).
a. 10% safflower
oil
b. 3% flaxseed oil
d. 3% menhaden oil c. 10% flaxseed oil
LN LN
LN LN
87
Figure 4.4 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on the
average total number and density of terminal end buds (TEB) in the MG of mice at 6
weeks of age (n=12-15 mice per diet group). The density of TEBs was calculated using
the following equation: (number of TEBs) / (ductal tree area). Bars not sharing a letter
are significantly different (p < 0.05) based on a one-way ANOVA test.
0
5
10
15
20
25
10% safflower (n-6)
3% flaxseed (n-3)
10% flaxseed (n-3)
3% menhaden (n=3)
Aver
age
nu
mb
er o
f T
EB
s
a
a
bb
(n-3)(n-3)
88
65
75
85
95
105
115
125
10% safflower (n-6)
3% flaxseed (n-3)
10% flaxseed (n-3)
3% menhaden (n-3)
Days
Post
nata
l
a a
b
b
4.4.3 Tumor latency and tumor free status
Time to palpation of first tumor was tracked in the mice from four different diet
groups. Average tumor latency was delayed (p<0.05) in mice fed 10% flaxseed (105.3 ±
8.2 days) or 3% menhaden oil (103.9 ± 10.8 days) relative to 10% safflower oil (86.7 ±
7.7 days). However, there was no significant difference between mice fed 3% flaxseed
(93.9 ± 7.6 days) and 10% safflower oil diets (Figure 4.5). Similarly, tumor free status
followed the same trend (Figure 4.6). At the T50 threshold, the median age when
mammary tumor was induced in 50% of MMTV mice, n-3 PUFA diets delayed tumor
latency compared to mice fed 10% safflower oil. However, only T50 for mice fed with
either 10% flaxseed (107 days) or 3% menhaden oil (102 days) was significantly (p<0.05)
higher than mice fed 3% flaxseed and 10% safflower oil diets (93 and 86 days,
respectively).
Figure 4.5 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on the
average tumor latency in mice (n=12 mice per diet group). Different letters denote
significant (p < 0.05) differences between means.
89
Figure 4.6 The proportion of mice tumor free throughout the duration of the study fed 10% safflower (n-6 PUFA), 3% flaxseed (n-3
PUFA), 10% flaxseed (n-3 PUFA) or 3%menhaden oil (n-3 PUFA) (n=12 mice per diet group). Significant differences between diet
groups were analyzed by Log-Rank test (p < 0.05).
0
10
20
30
40
50
60
70
80
90
100
110
1 74 79 84 89 94 99 104 109 114 119 124 129 134 139
Pro
po
rtio
n o
f m
ice
tum
or
free
Age of Tumor Onset (day)
10% safflower (n-6)
3% flaxseed (n-3)
3% menhaden (n-3)
10% flaxseed (n-3)
TD 50
b
b
a
a
90
Figure 4.7 Tumor volume. Average total tumor volume of mice within each diet group was tracked over 20 weeks (n=12 mice per diet
group). Length and width of palpated tumors were recorded, and tumor volume was calculated using the following equation: [length ×
(width) 2
]/2. Measurements were taken three times per week. Different letters denote significant differences (p<0.05) between diet
groups determined by repeated measures analysis.
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
10 11 12 13 14 15 16 17 18 19 20
Av
era
ge
tota
l tu
mo
r v
olu
me (
mm
3)
per
mo
use
Time (week)
10% safflower (n-6)a
3% menhaden (n-3)c
10% flaxseed (n-3)c
3% flaxseed (n-3)b
91
Figure 4.8 Tumor multiplicity. Average total numbers of palpated tumors per mouse within each diet group were tracked over 20
weeks (n=12 mice per diet group). Mice were palpated for tumors three times per week. Different letters denote significant
differences (p<0.05) between diet groups determined by repeated measures analysis.
0
1
2
3
4
5
6
7
10 11 12 13 14 15 16 17 18 19 20
Av
era
ge
tota
l n
um
ber
of
palp
tate
d t
um
ors
per
mou
se
Time (Week)
10% safflower (n-6)
3% Flaxseed (n-3)
3% Menhaden (n-3)
10% Flaxseed (n-3)
a
b
c
d
92
4.4.4 Tumor volume
Total tumor volume accumulated over time was compared between diet groups by
repeated measures. Total tumor volume in mice fed any of the n-3 PUFA diets was
significantly (p<0.05) less than that carried by mice fed 10% safflower oil over the 20-
week time course (Figure 4.7). In comparison to the 10% safflower oil group, the total
tumor volume began to diverge significantly (p<0.05) at 15 weeks of age for mice fed
either 10% flaxseed or 3% menhaden oil, and at 17 weeks of age for mice fed 3%
flaxseed oil. There was a significant (p<0.05) dose-dependent reduction of total tumor
volume in mice fed with 3 and 10% flaxseed oil. At 20 weeks of age, the final tumor
volumes carried by mice were 24% and 78% lowered in 3% and 10% flaxseed oil groups
relative to 10% safflower oil, respectively.
When compared with 3% menhaden oil, total tumor volume significantly different
starting at week 16 relative to mice fed 3% flaxseed oil; while total tumor volume was
not different between 3% menhaden and 10% flaxseed oil diets. Moreover, 10% flaxseed
oil resulted in a similar reduction in final tumor volume as 3% menhaden oil (78% and
72%, respectively), but 3% flaxseed oil had a much weaker inhibitory effect (p<0.05) on
final tumor volume compare to 3% menhaden oil (24% vs.72%).
4.4.5 Tumor multiplicity
Total tumor multiplicity over time was compared between diet groups by repeated
measures. Feeding any of the n-3 PUFA diets (p<0.05) reduced the number of tumors
palpated in mice compare to 10% safflower oil. This reduction started at week 13 and
maintained until the end of the study at 20 weeks (Figure 4.8). A significant (p<0.05)
dose-dependent reduction was also observed in total tumor multiplicity in mice fed 3%
and 10% flaxseed oil. This was accompanied by a dose-dependent reduction (15% and
42%, respectively) in the final tumor multiplicity of 20-week-old mice on 3% and 10%
flaxseed oil diets relative to 10% safflower oil.
93
When compared with 3% menhaden oil diet, feeding 10% flaxseed oil was more
potent in reducing total tumor multiplicity (p<0.05), which began to diverge significantly
at week 18 and resulted in a greater reduction in final tumor multiplicity (27% vs. 42%).
However, 3% flaxseed oil was significantly (p<0.05) weaker in reducing total tumor
multiplicity relative to 3% menhaden oil. This significant diverged starting at week 14
and led to a significant differences in final tumor multiplicity (15% vs. 27%) at the end of
20-week time course.
4.4.6 Final total tumor weight
At euthanization, the final total tumor weight carried by mice fed either 10%
flaxseed or 3% menhaden oil was significantly (p<0.05) less than that carried by 10%
safflower oil treated mice (Figure 4.9). However, feeding 3% flaxseed oil did not result in
a reduction in the final total tumor weight compared to 10% safflower oil.
Figure 4.9 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on the
final total tumor weight carried by mice at 20 weeks of age (n=12 mice per diet group).
Different letters denote significant (p < 0.05) differences between means.
94
4.4.7 Fatty acid composition
4.4.7.1 Fatty acid composition in serum
Fatty acid analysis of serum from mice at 6 or 20 weeks of age was conducted to
verify the experimental diet intake (Table 4.3). Serum from 10% safflower oil fed mice
exhibited fatty acid profiles high in LA, AA and n-6 DPA (Docosapentaenoic acid, 22:5
n-6). In comparison, feeding n-3 PUFA diets significantly (p<0.05) increased the serum
level of these fatty acids in both 6-and 20-week-old mice. As expected, there was a
significant decrease in n-6/n-3 PUFA ratio (p<0.05) in the serum of mice exposed to n-3
PUFA diets relative to 10% safflower oil. In comparison to 3% menhaden oil, diets
containing flaxseed oil resulted in a significant (p<0.05) accumulation of ALA into serum,
and further 10% flaxseed oil significantly (p<0.05) increased serum level of EPA.
However, mice fed 3% menhaden oil had the greatest DHA in serum than any other
dietary treatments (p<0.05). Overall, there was no significant difference in serum fatty
acid composition in 6- and 20-week-old mice.
4.4.7.2 Fatty acid composition in mammary glands
Phospholipid analysis of MG from 6- or 20-week mice were assessed to
determine if the observed effects on tumor outcomes were related to the incorporation of
n-3 PUFA into the target tissue. Selected n-6 and n-3 PUFA in phosphatidylethanolamine
(PE) fraction shown in Figure 4.10, and complete details of phosphatidylcholine (PC) and
PE are shown in Table 4.4. Feeding 10% safflower oil increased n-6 PUFA incorporation
into MGs of mice. For both 6- and 20-week mice, there was a significant (p<0.05) dose-
dependent increase in ALA, EPA and n-3 DPA (22:5 n-3) in MGs of mice fed increasing
levels of flaxseed oil in comparison to mice fed 10% safflower oil. Correspondingly, a
significant (p<0.05) dose-dependent decrease in AA was observed. Moreover, exposure
to 3% menhaden oil markedly (p<0.05) increased EPA, DPA (n-3) and DHA content of
MG, with a corresponding decrease in AA and n-6 DPA, when compared with safflower
oil fed mice.
95
Table 4.3 Serum fatty acid composition of mice at 6 or 20 weeks of age.
-Major n-6 and n-3 PUFA were displayed in the table. Other measured fatty acids are including 12:0, 14:0, 15:0, 16:0, 16:1n-7, 18:0,
18:1n-7, 18:1n-9, 18:3n-6, 20:0, 20:1n-9, 20:2n-6, 20:3n-6, 20:3n-3, 22:0, 22:1n-9, 22:2n-6, 22:4n-6. 22:3n-3, 24:0 and 24:1.
-One-way ANOVA was performed on serum fatty acids of 6- and 20-week-mice independently; different letters denote significant
difference (p < 0.05) between dietary treatments in each age group.
Serum
Fatty acid
6-week-old mice 20-week-old mice
10%
safflower
(n-6)
3% flaxseed
(n-3)
10%
flaxseed
(n-3)
3%
menhaden
(n-3)
10%
safflower
(n-6)
3% flaxseed
(n-3)
10%
flaxseed
(n-3)
3%
menhaden
(n-3)
LA (18:2n-6) 30.8 1.2b 33.6 1.9
a 20.4 2.0
c 33.2 1.0
a 34.6 0.8
a 33.1 0.8
a 21.6 1.1
c 29.6 2.3
b
AA (20:4n-6) 18.9 1.2a 9.1 0.8
b 2.3 0.3
d 6.8 0.5
c 18.4 4.3
a 10.8 1.3
b 3.0 0.4
c 10.6 1.8
b
DPA (22:5n-6) 1.8 0.1a 0.1 0.1
b 0.0 0.0
bc 0.1 0.1
b 1.6 0.2
a 0.0 0.0
b 0.0 0.0
b 0.1 0.0
b
ALA (18:3n-3) 0.1 0.0c 4.1 0.8
b 18.2 1.1
a 0.2 0.1
c 0.1 0.0
c 3.8 0.6
b 16.6 3.6
a 0.1 0.0
c
EPA (20:5n-3) 0.0 0.0d 1.0 0.2
c 6.7 0.6
a 4.2 0.7
b 0.0 0.0
d 0.7 0.1
c 6.2 1.4
a 2.5 0.9
b
DPA (22:5n-3) 0.1 0.0c 0.5 0.1
b 0.8 0.1
a 0.9 0.0
a 0.1 0.1
d 0.4 0.0
c 0.9 0.1
a 0.6 0.1
b
DHA (22:6n-3) 1.0 0.1c 4.1 0.7
b 3.8 0.4
b 6.7 0.3
a 1.1 0.1
c 4.4 0.3
b 4.2 0.7
b 6.8 0.6
a
Total n-6 53.6 1.5a 44.7 1.7
b 23.6 2.4
d 41.8 1.5
c 56.9 3.5
a 45.8 1.4
b 25.6 1.4
c 43.3 2.6
b
Total n-3 1.2 0.2c 10.0 0.8
d 30.4 0.8
a 12.3 0.7
b 1.3 0.2
c 9.7 0.7
b 28.7 2.2
a 10.3 1.0
b
n-6/n-3 Ratio 46.3 7.3a 4.5 0.3
b 0.8 0.1
d 3.4 0.3
c 45.4 5.9
a 4.7 0.4
b 0.9 0.1
c 4.3 0.7
b
96
Figure 4.10 MG fatty acid composition. Upper panel: PE fraction in 6-week-old mice;
lower panel: PE fraction in 20-week-old mice. The fatty acid composition of MG
phospholipid differs markedly between dietary treatments (n=4-6 per diet). Bars not
sharing a letter are significantly different (p<0.05) based on a one-way ANOVA test. Full
fatty acid profile is displayed in Table 4.4.
97
When compared with 3% menhaden oil, feeding low (3%) and high (10%)
flaxseed oil diets significantly (p<0.05) increased ALA incorporation into MG tissue.
Notably, mice fed 10% flaxseed oil had significantly (p<0.05) higher level of EPA than
mice fed 3% menhaden oil at 20 weeks, and displayed a significant (p<0.05) reduction of
AA at both age groups. Interestingly, DHA accumulation was significantly (p<0.05)
higher in the mice fed 3% menhaden oil than any flaxseed oil diets. Similar effects were
observed in the PC fraction (Table 4.4, Appendix).
4.4.7.3 Fatty acid composition in mammary tumors
There were significant differences in the fatty acid composition of phospholipid
from mammary tumors in the four different diet groups (n=5-6 per diet), which varied
between PC and PE phospholipid fractions. The PUFA composition in PC and PE are
shown in Figure 4.11 and complete details of all fatty acids are reported in Table 4.5.
Compared to mice fed 10% safflower oil, a significant (PC and PE, P<0.05) dose-
dependent decrease in AA and a significant dose-dependent increase in ALA, EPA and n-
3 DPA were observed in the tumors of mice fed increasing levels of flaxseed oil. In
contrast, a dose-dependent reduction of LA was only observed in tumor PE (p<0.05).
Likewise, tumors from mice receiving a 3% menhaden oil diet had significantly lower
levels of AA and n-6 DPA (PC and PE, p<0.05), higher levels of EPA, n-3 DPA and
DHA (PC and PE, p<0.05), and a significant reduction of LA in tumor PE (p<0.05),
when compared with mice on 10% safflower oil.
98
Table 4.4 Fatty acid composition of MG phospholipids.
Fatty Acids
Mammary Gland of 20-week-old Mice
PC PE
10%
ssafflower
3%
flaxseed
10%
flaxseed
3%
menhaden
10%
safflower
3%
flaxseed
10%
flaxseed
3%
menhaden
12:0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
14:0 0.3±0.4 0.2±0.0 0.4±03 0.5±0.2 0.8±0.6 0.8±0.2 1.0±0.6 0.6±0.7
15:0 0.1±0.1b 0.1±0.1
ab 0.1±0.1
a 0.2±0.0
ab 0.5±0.3 0.1±0.1 0.1±0.1 0.1±0.1
16:0 24.5±3.3 21.7±2.1 23.3±2.0 23.8±1.3 10.4±2.2 10.2±1.3 8.1±2.0 11.1±4.6
18:0 16.7±4.2 19.3±1.9 16.5±3.2 19.2±1.6 18.6±1.9 17.5±1.6 15.0±2.5 16.6±4.8
20:0 0.1±0.1 0.2±0.0 0.2±0.0 0.1±0.1 0.2±0.1 0.2±0.0 0.1±0.1 0.1±0.1
22:0 0.2±0.2 0.2±0.0 0.2±0.0 0.2±0.1 0.3±0.1 0.3±0.1 0.1±0.1 0.3±0.2
24:0 0.1±0.2 0.1±0.1 0.0±0.0 0.0±0.0 0.1±0.1 0.0±0.0 0.0±0.0 0.0±0.0
Total SFA 42.0±3.3 41.7±0.3 40.7±2.1 44.0±1.8 30.8±3.4 29.2±2.2 27.0±3.0 29.4±3.9
16:1n-7 1.5±0.6b 1.4±0.2
b 3.4±1.3
a 2.2±0.4
ab 1.6±0.3
b 2.1±0.5
ab 3.0±0.6
a 2.5±0.4
a
18:1n-7 10.4±1.6b 11.1±1.0
b 15.5±1.9
a 9.8±1.2
b 14.3±1.3
bc 15.5±2.0
b 20.1±1.9
a 11.1±2.8
c
18:1n-9 3.0±0.9 2.0±1.2 3.9±1.4 2.7±1.5 1.9±0.5b 2.5±0.4
ab 3.5±1.5
a 2.6±0.4
a
20:1n-9 0.1±0.2 0.2±0.1 0.1±0.2 0.1±0.1 0.4±0.3 0.3±0.3 0.5±0.3 0.4±0.3
22:1n-9 0.3±0.2 0.3±0.1 0.3±0.2 0.2±0.2 0.8±0.7 1.1±0.7 0.5±0.3 1.4±1.0
24:1 0.0±0.0 0.1±0.1 0.1±0.1 0.1±0.1 0.1±0.1ab
0.2±0.2ab
0.1±0.1b 0.4±0.2
a
Total
MUFA 15.3±2.9
b 15.1±1.5
b 23.4±4.4
a 15.1±2.2
b 19.2±1.3
b 21.7±2.2
ab 27.2±3.2
a 18.9±3.9
b
18:2n-6 26.9±5.3ab
30.7±1.3a 22.1±4.4
b 28.2±1.7
ab 18.9±1.3
a 19.8±1.9
a 12.3±3.3
b 13.3±3.3
b
18:3n-6 0.1±0.1 0.1±0.1 0.0±0.0 0.1±0.1 0.0±0.0 0.0±0.0 0.0±0.0 0.1±0.1
18:3n-3 0.0±0.0c 0.8±0.0
b 4.7±1.9
a 0.1±0.0
c 0.1±0.1
c 1.4±0.6
b 5.1±2.6
a 0.0±0.0
c
20:2n-6 1.0±0.2a 0.7±0.1
b 0.3±0.0
d 0.5±0.0
c 0.5±0.0
a 0.4±0.1
a 0.1±0.1
b 0.2±0.2
b
20:3n-6 1.1±0.2 1.0±0.1 09±0.4 1.1±0.2 0.7±0.3 0.9±0.2 1.2±0.8 0.8±0.3
20:4n-6 11.1 2.6a 6.1 0.6
b 2.4 1.0
c 4.9 0.7
b 19.7±1.9
a 14.6±2.0
b 8.0±1.8
c 12.6±4.3
bc
20:3n-3 0.0±0.0c 0.1±0.1
b 0.4±0.1
a 0.2±0.1
b 0.0±0.0
b 0.0±0.0
b 0.3±0.1
a 0.0±0.0
b
20:5n-3 0.0±0.0c 0.2±0.0
c 2.0±0.4
a 1.2±0.5
b 0.1±0.1
d 0.7±0.1
c 6.0±0.9
a 2.1±0.5
b
22:2n-6 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
22:4n-6 1.0±0.4a 0.4±0.0
b 0.0±0.0
c 0.2±0.1
bc 3.9±0.5
a 1.5±0.4
b 0.3±0.1
c 0.7±0.3
c
22:3n-3 0.0±0.0c 0.1±0.1
b 0.4±0.1
a 0.2±0.1
b 0.0±0.0 0.0±0.0 0.0±0.0 0.1±0.1
22:5n-6 0.7±0.4a 0.1±0.1
b 0.0±0.0
b 0.0±0.0
b 3.0±0.8
a 0.0±0.0
c 0.1±0.1
bc 0.2±0.2
b
22:5n-3 0.0±0.0c 0.6±0.2
b 1.0±0.1
a 0.9±0.1
a 0.4±0.2
d 2.0±0.2
c 4.3±0.7
a 2.7±0.4
b
22:6n-3 0.2±0.2c 1.9±0.5
b 1.4±0.2
b 2.9±0.7
a 2.6±1.0
c 7.8±1.0
b 6.7±0.3
b 11.1±1.5
a
Total n-6 41.9±3.2a 39.0±1.2
ab 25.7±3.3
c 35.0±2.0
b 46.6±4.8
a 37.3±0.9
b 23.2±1.7
c 34.3±1.2
b
Total n-3 0.2±0.2c 3.6±0.8
b 9.8±1.8
a 5.3±1.2
b 3.3±1.5
d 11.8±0.8
c 22.7±2.2
a 17.5±4.0
b
n-6/n-3
ratio 209 16.1
a 11.1 2.0
b 2.7 0.8
d 6.8 1.5
c 17.1 7.7
a 3.2 0.3
b 1.0 0.1
d 2.0 0.3
c
Total PUFA 42.1±3.0a 42.6±1.5
a 35.5±3.2
b 40.3±2.4
a 49.9±3.2
ab 49.1±1.1
ab 46.0±2.6
a 51.8±2.3
b
99
In comparison to 3% menhaden oil, there was greater incorporation of ALA in
tumors of mice fed increasing levels of flaxseed oil (p<0.05). However, tumors from
mice on low flaxseed oil (3%) diet had a significantly (p<0.05) lower EPA and DHA than
tumors from mice on 3% menhaden oil. Additionally, a significant increase in AA and
decrease in n-3 DPA were observed in the tumor PC of mice fed 3% flaxseed oil
compared with 3% menhaden oil (p<0.05). In contrast, feeding high dose of flaxseed oil
(10%) significantly reduced LA and AA and increased EPA and n-3 DPA incorporation
into tumors compared to mice fed 3% menhaden oil. Notably, DHA accumulation was
the highest (p<0.05) in tumors of mice exposed to 3% menhaden oil. Furthermore, mice
on n-3 PUFA diets also displayed a significant reduction of n-6/n-3 PUFA ratio in both
tumor and adjacent MG tissue compared to n-6 PUFA fed mice (Table 4.4 and 4.5,
p<0.05). In comparison to tumor PC, there was a larger reduction in n-6/n-3 PUFA ratio
in tumor PE, which declined from 16:1 in 10% safflower oil to 3:1 and 2:1 in 3%
flaxseed and 3% menhaden oil, respectively, as well as finally to 1:1 in mice exposed to
10% flaxseed oil (Table 4.5). In addition, the majority of n-3 PUFA in the form of EPA,
n-3 DPA and DHA were accumulated to a greater extent in PE relative to PC.
100
0
5
10
15
20
25
30
35
18:2n-6 20:4n-6 22:5n-6 18:3n-3 20:5n-3 22:5n-3 22:6n-3
% C
om
po
siti
on
Tumor-PE
10% safflower 3% flaxseed
10% flaxseed 3% menhaden
ab
c b
a
ab
c
b
a
bb bc
b ac d bc
a
c ba
b
a
bb
c
0
5
10
15
20
25
18:2n-6 20:4n-6 22:5n-6 18:3n-3 20:5n-3 22:5n-3 22:6n-3
% C
om
posi
tion
Tumor-PC
10% safflower 3% flaxseed10% flaxseed 3% menhaden
bb
b
a
a
d
c
a cc ab bd
ac bd
ac
b
b b b c
ab b
Figure 4.11 Mammary tumor fatty acid composition. Percentage composition of major n-6 and n-3 PUFA in the representative PC
and PE phospholipid fraction of mouse mammary tumor tissue are shown in the bar graph. Upper panel: PC fraction; lower panel: PE
fraction. The fatty acid composition of tumor phospholipid classes differs markedly between dietary treatments (n=5-6 per diet). Bars
not sharing a letter are significantly different (p<0.05) based on a one-way ANOVA test. Full fatty acid profile is displayed in Table
4.5.
101
Table 4.5 Fatty acid composition of mammary tumor phospholipids.
Fatty Acids
Mammary Tumors
PC PE
10%
ssafflower
3%
flaxseed
10%
flaxseed
3%
menhaden
10%
safflower
3%
flaxseed
10%
flaxseed
3%
menhaden
12:0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
14:0 1.3±0.3 1.3±0.2 1.3±0.2 1.0±0.5 0.3±0.1 0.2±0.1 0.2±0.1 0.2±0.1
15:0 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
16:0 31.7±1.2 30.9±0.9 29.6±0.9 27.9±4.6 6.8±1.1 6.7±0.8 6.5±0.5 6.9±0.8
18:0 8.6±1.3 8.6±0.9 7.7±1.4 10.6±3.8 14.1±1.7 13.7±1.9 13.1±2.3 14.8±1.1
20:0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0
22:0 0.0±0.0b 0.1±0.0
ab 0.1±0.0
ab 0.1±0.1
a 0.1±0.1 0.1±0.0 0.1±0.0 0.1±0.0
24:0 0.0±0.0 0.1±0.0 0.0±0.0 0.1±0.1 0.1±0.1 0.0±0.0 0.1±0.0 0.0±0.0
Total SFA 42.0±1.3 41.3±1.2 38.9±1.7 40.0±3.8 21.5±2.7 20.8±2.6 19.9±2.0 22.2±1.1
16:1n-7 3.0±0.4b 3.3±0.7
b 5.2±1.0
a 3.3±0.9
b 1.6±0.3
b 1.7±0.3
b 2.7±0.7
a 1.9±0.4
b
18:1n-7 12.7±0.7b 14.1±1.9
b 18.7±1.5
a 12.7±2.7
b 17.8±2.8
b 20.5±3.0
ab 23.9±1.3
a 18.2±1.8
b
18:1n-9 5.0±0.6b 5.4±0.6
ab 7.0±1.2
a 5.1±1.4
b 4.0±1.3
b 4.4±1.2
ab 6.9±2.2
a 4.8±1.1
ab
20:1n-9 0.3±0.1b 0.5±0.2
ab 0.7±0.3
a 0.4±0.2
b 0.5±0.4 0.8±0.2 0.6±0.2 0.5±0.2
22:1n-9 0.1±0.0 0.2±0.1 0.2±0.1 0.1±0.1 0.3±0.3 0.3±0.2 0.2±0.0 0.3±0.1
24:1 0.1±0.0 0.2±0.1 0.3±0.1 0.2±0.2 0.2±0.0 0.2±0.1 0.2±0.1 0.1±0.0
Total MUFA 21.4±1.5b 23.7±1.3
b 32.1±2.0
a 21.9±4.9
b 24.4±4.2
b 27.8±3.9
ab 34.6±3.5
a 25.8±2.8
b
18:2n-6 11.6±1.3b 14.0±0.8
b 11.7±0.2
b 19.3±1.4
a 12.1±4.1
a 10.3±2.1
b 8.0±0.8
c 9.7±0.4
b
18:3n-6 0.2±0.1a 0.1±0.1
ab 0.1±0.0
b 0.1±0.0
b 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0
18:3n-3 0.0±0.0c 0.3±0.0
b 1.6±0.4
a 0.1±0.0
c 0.1±0.1
c 0.4±0.2
b 1.6±0.2
a 0.1±0.0
c
20:2n-6 1.5±0.5a 1.4±0.4
a 0.7±0.2
b 1.1±0.4
ab 1.4±0.3
a 1.0±0.2
ab 0.5±0.1
c 0.9±0.3
bc
20:3n-6 2.9±0.6 3.0±1.2 2.5±0.5 2.8±1.1 3.5±1.7 4.0±0.7 3.0±0.6 3.5±0.5
20:4n-6 17.1±1.0a 12.1±0.9
b 4.1±1.1
d 8.4±2.1
c 25.3±2.6
a 22.2±1.6
ab 10.0±1.5
c 18.1±3.8
b
20:3n-3 0.0±0.0c 0.1±0.0
b 0.8±0.3
a 0.1±0.0
c 0.0±0.0
b 0.1±0.0
b 0.4±0.2
a 0.1±0.0
b
20:5n-3 0.0±0.0d 0.3±0.1
c 2.8±0.4
a 1.2±0.6
b 0.1±0.1
d 0.8±0.1
c 6.4±0.9
a 1.4±0.2
b
22:2n-6 0.1±0.1a 0.1±0.0
a 0.0±0.0
b 0.1±0.1
ab 0.1±0.0
a 0.1±0.0
a 0.0±0.0
b 0.1±0.0
a
22:4n-6 0.9±0.1a 0.4±0.1
b 0.1±0.0
d 0.2±0.0
c 3.9±1.1
a 1.3±0.4b 0.2±0.1
c 0.8±0.2
b
22:3n-3 0.0±0.0b 0.1±0.1
b 0.8±0.3
a 0.1±0.0
b 0.0±0.0
b 0.0±0.0
b 0.1±0.0
a 0.0±0.0
b
22:5n-6 0.7±0.3a 0.1±0.0
b 0.1±0.1
b 0.1±0.0
b 4.2±1.7
a 0.2±0.0
b 0.1±0.0
b 0.3±0.0
b
22:5n-3 0.1±0.0d 0.5±0.1
c 1.4±0.2
a 0.9±0.2
b 0.5±0.3
c 2.7±0.6
b 6.2±0.6
a 3.3±0.4
b
22:6n-3 0.8±0.1c 1.7±0.3
b 1.8±0.2
b 3.0±0.6
a 2.9±1.5
c 8.4±1.0
b 9.0±0.7
b 13.7±1.8
a
Total n-6 35.0±1.7a 31.2±1.7
b 19.3±1.0
c 32.1±2.8
ab 50.5±2.2
a 39.1±1.8
b 21.8±2.1
d 33.4±3.1
c
Total n-3 1.0±0.1d 3.0±0.4
c 9.1±1.1
a 5.3±1.2
b 3.6±1.3
d 12.3±0.8
c 23.7±1.8
a 18.6±1.9
b
n-6/n-3
ratio 36.1 5.3
a 10.5 1.7
b 2.14 0.3
d 6.4 1.8
c 15.8 6.5
a 3.2 0.3
b 0.9 0.14
d 1.8 0.3
c
Total PUFA 36.0±1.6a 34.2±1.6
a 28.5±0.7
b 37.4±3.3
a 54.1±3.0
a 51.4±1.6
a 45.5±1.7
b 52.0±2.5
a
102
0
5
10
15
20
25
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
Rela
tive in
ten
sity
un
it
HER2
185kDa
β-actin
10%saf 3%flax 10%flax 3%men
a ab
bb
0
5
10
15
20
25
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
Rela
tive in
ten
sity
un
it
a
pHER 2
185kDa
β-actin
10%saf 3%flax 10%flax 3%men
bb
a
a. b.
4.4.8 Protein analysis
4.4.8.1 HER-2 and pHER-2
HER-2 (human epidermal growth factor receptor 2) is a 185-kD transmembrane
receptor tyrosine kinase that is involved in human mammary oncogenesis. The term neu
is homologous to HER-2 and used when referring to the murine gene or cDNA (73, 214).
Protein expression of HER-2 and its activated form phosphorylated HER-2 (pHER-2) in
tumors were analyzed by Western Blotting as shown in Figure 4.12. Compared to 10%
safflower oil, exposure to 10% flaxseed or 3% menhaden oil significantly lowered the
protein expression of HER-2 in mouse mammary tumors, and simultaneously down-
regulated the tumor protein expression of pHER-2 (p<0.05). However, HER-2 and
pHER-2 expression was not different between 3% flaxseed oil when compared to mice
fed 10% safflower oil.
Figure 4.12 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on
protein expression of (a) HER-2 and (b) pHER-2 in mouse mammary tumors (n=6 per
diet). Different letters denote significant difference (p<0.05) between diet groups was
based on a one-way ANOVA test.
103
0.00
0.20
0.40
0.60
0.80
1.00
1.20
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
Rela
tive a
bso
rb
an
ce o
f to
tal A
kt
a. Total Akt
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
Rela
tive a
bso
rb
an
ce o
f p
Ak
t(S
er 4
73)
b. Phospho-Akt (Ser 473)
a
b
bb
4.4.8.2 Akt and pAkt
Akt is a downstream serine/threonine kinase involved in HER-2 signaling
pathways and functions as an anti-apoptotic signaling molecule (94). Protein expression
of total Akt and its activated form pAkt (phosphorylation at Ser473 site) were assessed by
ELISA as shown in Figure 4.13. Although total Akt was not significantly different
between the four dietary treatments, a significant effect was observed in pAktSer 473
. All n-
3 PUFA diets significantly reduced the pAktSer 473
compared to 10% safflower oil fed
mice.
Figure 4.13 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on
protein expression of (a) total Akt and (b) pAkt
Ser 473in mouse mammary tumors (n=6 per
diet). Different letters denote significant difference (p<0.05) between diet groups was
based on a one-way ANOVA test.
4.4.8.3 Ki67 and Cleaved-caspase-3
Ki67 protein is expressed by proliferating cells and serves as a biomarker of
tumor cell replication and progression (103). While caspase-3 is a cytoplasmic protein
104
0
5
10
15
20
25
30
35
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
KI-
67 l
ab
elli
ng i
nd
ex (
%)
a. Ki67
0
2
4
6
8
10
12
10% safflower
(n-6)
3% flaxseed
(n-3)
10% flaxseed
(n-3)
3% menhaden
(n-3)
Cle
aved
-casp
ase
-3 lab
ellin
g I
nd
ex (
%)
b. Cleaved-caspase-3
a
b
c c cc
a
b
that involved in the activation cascade of caspases responsible for cell apoptosis (227,
228). The tumor protein expression of Ki67 and cleaved caspase-3 (activated caspase-3
by a cleavage adjacent to Asp175) were assessed by immunohistochemistry. The results
are presented as labeling index, which is the percentage of labeled cells relative to total
cells (Figure 4.14 a, b). The photomicrographs of representative immunohistochemical
stained mammary tumor section of MMTV mice are shown in Figures 4.15 and 4.16.
When compared to 10% safflower oil, fed mice any of the n-3 PUFA diets can
significantly reduce the expression of Ki67. Moreover, mice exposed to10% flaxseed and
3% menhaden oil had significant lower levels of Ki67 relative to 3% flaxseed oil.
Exposure to 10% flaxseed and 3% menhaden oil resulted in a significant (p<0.05)
increase in cleaved-caspase-3 compared to 10% safflower oil. Mice fed 3% menhaden oil
diet had significantly (p<0.05) higher cleaved-caspase-3 than mice fed 10% flaxseed oil.
However, no significant difference on cleaved-caspase-3 was observed between mice on
3% flaxseed and 10% safflower oil diets.
Figure 4.14 Effect of lifelong exposure to plant- versus marine-derived n-3 PUFA on
protein expression of (a) Ki-67 and (b) cleaved-caspase-3 in mouse mammary tumors
(n=6 per diet). Different letters denote significant difference (p<0.05) between diet
groups was based on a one-way ANOVA test.
105
Figure 4.15 Immuno-localization of Ki67 in the mammary tumor section of MMTV-neu
(ndl)-YD5 mice fed four different dietary treatments a) 10% safflower oil, b) 3% flaxseed
oil, c) 3% menhaden oil and d) 10% flaxseed oil. Immunohistochemical staining of
paraffinembedded mouse mammary tumor sections stained with Ki-67 antibody (1:200).
Ki67 (dark brown cells) displays a nuclear localization pattern which correlates with it
function in cell cycle progression (magnification: 400×, counterstained with hematoxilin).
Arrows (→) indicate Ki67 immunopositive cells in mouse mammary tumors.
a. 10% safflower
oil
b. 3% flaxseed oil
d. 3% menhaden oil c. 10% flaxseed oil
106
Figure 4.16 Immuno-localization of cleaved-caspase-3 in the mammary tumor section of
MMTV-neu (ndl)-YD5 mice fed four different dietary treatments a) 10% safflower oil, b)
3% flaxseed oil, c) 3% menhaden oil and d) 10% flaxseed oil. Immunohistochemical
staining of paraffinembedded mouse mammary tumor sections stained with cleaved-case-
ase-3 antibody (1:1000). Cleaved-caspase-3 (cells with dark brown outer layers) displays
a cytoplasmic localization pattern which correlates with it function in cell cycle
progression (magnification: 400×, counterstained with hematoxylin). Arrows (→)
indicate cleaved-caspase-3 immunopositive cells in mouse mammary tumors.
a. 10% safflower oil b. 3% flaxseed oil
d. 3% menhaden oil c. 10% flaxseed oil
107
4.5 Discussion
The present study is the first to compare the relative inhibitory potency of plant-
versus marine-derived n-3 PUFA on mammary tumor development. Epidemiological and
experimental studies to date provide evidence in support of a beneficial effect of marine-
based n-3 PUFA in reducing the risk of developing BC. However, a major gap remaining
is substantiating the anti-cancer effect of plant-derived ALA, the principle source of n-3
PUFA consumed in North American. Using the MMTV-neu (ndl)YD5 transgenic mouse
model, the present study demonstrated that lifelong exposure to n-3 PUFA, whether from
plant or marine sources, can prevent breast carcinogenesis and mitigate tumor outcomes
through modulating MG morphology, and signaling protein molecules involved in cell
proliferation and apoptosis. Conservatively, marine-based n-3 PUFA (EPA and DHA)
were 8 times more potent than plant-derived ALA in inhibiting mammary tumorigenesis.
4.5.1 Role of plant- and marine-derived n-3 PUFA in MG development and BC risk
First, the present study provides evidence that the onset of puberty was
significantly (p<0.05) delayed in mice fed either flaxseed or menhaden oil diet as
compared to mice fed 10% safflower oil. Pubertal onset has been linked to BC as earlier
menarches place females at a greater risk of BC (229-231). Therefore, lifelong exposure
to both plant- and marine-derived n-3 PUFA may reduce the risk of BC. Moreover,
estrogen is known as a key regulator of puberty, given that elevated estrogen is typically
associated with accelerated pubertal onset (168, 219, 230). This suggests that n-3 PUFA
may influence circulating estrogen levels. Hilakivi-Clarke et al. has shown that maternal
intake of n-3 PUFA diets significantly increased pregnancy 17β-estradiol levels, but
decreased mammary tumor incidence among the female rat offspring (232).
Unfortunately, serum estrodiol level was not assessed in the present study; future
investigation is needed to verify the role of n-3 PUFA in regulating hormonal effects on
puberty and risk of BC.
108
Second, feeding either 10% flaxseed oil or 3% menhaden oil significantly (p<0.05)
decreased the number and density of TEBs in the MG of mice at 6 weeks of age when
compared with 10% safflower oil. TEBs are directly associated with MG development
and BC risk since they are tumor initiation sites that give rise to mammary tumors upon
exposure to a chemical carcinogen (169). It has been proposed that more TEBs at the
time the gland is exposed to a carcinogen, the higher the risk of BC (168). These findings
demonstrate that lifelong exposure to both plant- and marine-derived n-3 PUFA can
influence MG development at an early life stage, and may lower future BC risk. However,
at the same dosage, 3% menhaden oil was more effective in reducing TEBs relative to 3%
flaxseed oil, given that analysis of TEB structures showed no significant difference
between mice fed 3% flaxseed and 10% safflower oil. As a result, marine-based n-3
PUFA was more potent than plant-derived ALA in altering MG structures. These
observations were attributed to the type and level of PUFA present in MGs of mice at 6
weeks of age. An enrichment of n-3 PUFA and a corresponding decrease in AA in mice
fed flaxseed and menhaden oil diets contributed to reduced TEB structures at 6 weeks
(Figure 4.10). More specifically, a significant (p<0.05) increase of ALA and EPA in mice
fed 10% flaxseed oil, as well as higher levels of EPA and DHA in mice fed 3% menhaden
oil was negatively associated with the number of TEBs in MG. Therefore, changes in
fatty acid composition of MG during early periods of rapid growth and development can
influence the long term health of mammary tissue and modify the risk of mammary
tumorigenesis later in life.
The findings from the present study are in agreement with previous rodent studies.
Exposure of female rats to ~3.5% (w/w) menhaden oil or 4% (w/w) flaxseed oil at
discrete developmental stages such as in utero, during suckling to puberty, or throughout
lifetime, significantly (p<0.05) lowered the density of TEBs and subsequently decreased
mammary tumor incidence (110, 173, 174, 176, 220, 233). In contrast, exposure after
weaning had no beneficial effects on TEB structures (173), which suggests timing of
dietary exposure is an important factor to consider in MG development. Moreover, a
recent study showed that female rat offspring exposed to marine n-3 PUFA enriched fish
oil in utero via maternal intake had a greater effect on preventing mammary tumors than
supplementation during puberty or adulthood (29). Therefore, early life stages such as in
109
utero and during suckling are critical periods for inducing structural changes in MG that
reduce BC risk later in life.
4.5.2 Role of plant- and marine-derived n-3 PUFA in mammary tumor development
Multiple parameters in this present study suggest a preventive effect of plant- and
marine-derived n-3 PUFA on BC development. Compared to 10% safflower oil, intake of
10% flaxseed and 3% menhaden oil delayed tumor latency; whereas no significant effect
on tumor latency was found when mice were fed a low (3%) flaxseed oil diet. Likewise,
mice exposed to either 10% flaxseed or 3% menhaden oil diets were tumor free longer
over the whole 20-week-period relative to mice fed 3% flaxseed oil. This demonstrated
that marine-based n-3 PUFA was more effective than plant-derived ALA in delaying
mammary tumor onset.
Mammary tumor development was assessed by measuring tumor volume and
multiplicity which was significant (p<0.05) lower in mice fed either flaxseed or
menhaden oil diet relative to mice fed 10% safflower oil. These results demonstrate that
lifelong exposure to both plant- and marine-derived n-3 PUFA can attenuate aspects of
mammary tumorigenesis in mice expressing MMTV-neu (ndl)YD5, a relevant model of
HER-2 positive BC. In addition, the reduced tumor multiplicity also demonstrated that
both plant- and marine-derived n-3 PUFA can inhibit subsequent tumor initiation.
Interestingly, a significant dose-dependent reduction in both tumor volume and
multiplicity was found in mice fed increasing levels of flaxseed oil. This provides strong
evidence that mammary tumor development was dose-dependently inhibited by plant-
derived ALA in female MMTV-neu (ndl)YD5 mice. Although 3% menhaden oil was
more potent than 3% flaxseed oil in decreasing tumor volume and multiplicity,
supplementation of a high (10%) flaxseed oil diet resulted in a similar reduction of tumor
volume and a greater reduction of tumor multiplicity. Furthermore, overall final tumor
weight was consistent with the observed effects on tumor growth. Exposure to 10%
flaxseed and 3% menhaden oil produced a similar reduction in final tumor weight, which
was significantly (p<0.05) lower than the final tumor weight observed in mice fed 3%
110
flaxseed and 10% safflower oil. Since 3% menhaden oil diet contains 3.9% EPA and 3.4%
DHA, and 10% flaxseed oil diet contains 57.6% ALA, it is concluded that plant-derived
ALA is ~1/8 as potent as marine-based n-3 PUFA (EPA and DHA) in inhibiting
mammary tumorigenesis in MMTV-neu (ndl)YD5 mouse model.
Observations from the present study reinforce our previous findings which have
also shown beneficial outcomes of marine-derived n-3 PUFA in mitigating mammary
tumor development. In one study, the anti-tumorigenic effect of marine-based n-3 PUFA
was examined via both complementary genetic and conventional dietary approaches
(116). Mice carrying the fat-1 transgene, which are capable of endogenously producing n-
3 PUFA from dietary n-6 PUFA, were crossed with MMTV-neu (ndl)YD5 mouse model
to create a novel double-hybrid progeny (116, 234). It was shown that both in the genetic
arm as well as, and more importantly, in the dietary arm of the study, the volume and
number of tumors was significantly decreased in mice fed marine-based n-3 PUFA.
Particularly, final tumor volume and multiplicity carried by MMTV mice that receiving a
3% (w/w) menhaden oil diet were 64% and 40% reduced in comparison to MMTV mice
fed 10% (w/w) safflower oil, respectively (116). Consistent with previous finding, the
same menhaden oil diet was used in the present study and showed a similar reduction
(72%) in final tumor volume and a slightly smaller reduction (27%) in final tumor
multiplicity, when compared with 10% safflower oil. Furthermore, in a recent study, we
examined the dose-response relationship between dietary marine-based n-3 PUFA and
mammary tumor development (21). A significant dose-dependent reduction was observed
in tumor multiplicity in MMTV-neu (ndl)YD5 mice fed increasing levels of menhaden oil
(21). In support of these findings, Yee et al. (2005) examined mammary tumor
development in MMTV-neu mice fed menhaden oil from 7-8 weeks of age onwards and
showed a significant reduction in tumor incidence (30%) and final tumor multiplicity
(55%) by the end of 15 month (115). The MMTV-neu mouse model utilized in this study
is much less aggressive than MMTV-neu (ndl)YD5, and thus requiring a period to induce
mammary tumors (117). More importantly, our study used less amount of menhaden oil
than this study, but the inhibitory effect on tumor multiplicity in our study was greater.
This suggests a potential benefit of lifelong exposure to n-3 PUFA in our study may add
protection against mammary tumor development.
111
A few studies have also investigated the protective effect of plant-derived n-3
PUFA on MMTV-c-neu mouse model. One study looked at the effect of increasing levels
of flaxseed oil (0.05, 0.1 and 0.2 ml) on mammary tumor development, and demonstrated
that daily exposure to 0.2 ml flaxseed oil (equivalent to 186 mg/day) from 4 weeks of age
onwards delayed tumor incident and reduced overall tumor weight (208). This effective
dose (186 mg/day) of flaxseed oil was comparable to the amount provided by a 10%
(w/w) flaxseed oil diet in the present study (average mouse daily intake is 200 mg/day).
However, lifelong exposure to 10% (w/w) flaxseed oil had more diverse anti-tumorigenic
effects, such as reducing tumor volume and multiplicity. This further emphasizes the
importance of investigating the effect of lifelong exposure in the current study. Moreover,
the anti-tumorigenic effect of a low dose of flaxseed oil was also observed. It has been
shown that mice fed a 0.5% (w/w) flaxseed diet, which contain ~0.2 (w/w) flaxseed oil,
significantly reduced the tumor incidence and the numbers of large tumors (> 6mm
diameter) (190). In contrast, 3% (w/w) flaxseed oil in the present study had no significant
effect on tumor latency. The reason for this discrepancy may be due to the lignan
component in the flaxseed diet. Flaxseed is the richest source of mammalian lignan
precursors, namely secoisolariciresinol diglycoside (SDG), which is known as a type of
phytoestrogen that have estrogenic or anti-estrogenic properties (157, 162, 163). Thus,
the anticancer effect of the 0.5% (w/w) flaxseed diet may be partially attributed to SDG.
Altogether, findings from the current study and several rodent studies employing MMTV-
neu related models provide concordant evidence for an anti-cancer effect of both plant-
and marine-derived n-3 PUFA on modulating neu-related mammary tumor development.
This is critical given that nearly one third of invasive human BC cases are HER-2/neu
related (215, 216).
4.5.3 Potential mechanisms of anti-tumorigenic actions
4.5.3.1 Change membrane fatty acid composition
Total lipid analysis of serum from 20-week mice reflected the type and level of
PUFA from the diets. The anti-tumorigenic action of n-3 PUFA may be attributed to
112
changes in membrane fatty acid composition of both tumor tissue and the adjacent MG
tissue. Analysis of tumor and adjacent MG phospholipid fatty acid composition revealed
significant increases in n-3 PUFA, particularly ALA, EPA and DHA, as well as
significant decreases in AA and overall n-6/n-3 PUFA ratio in mice fed flaxseed or
menhaden oil diet compared to 10% safflower oil (p<0.05). A significant (p<0.05)
increase was also found in n-3 DPA content, demonstrating that EPA is incorporated and
further elongated. These changes in membrane fatty acid composition suggest a potential
inhibitory effect of n-3 PUFA on the synthesis of eicosanoid derived from AA.
Overlapping metabolic pathways of n-6 and n-3 PUFA are likely to be one of the most
salient mechanisms by which n-3 PUFA inhibited tumor development. Both AA and EPA
are the substrate for eicosanoid synthesis; while eicosanoids derived from AA are pro-
inflammatory and cancer promoting, in contrast to EPA and DHA, which are anti-
inflammatory and cancer inhibitory (31, 58, 61). Thus, intake of plant- and marine-
derived n-3 PUFA reduced AA incorporation and thus, less AA was available to produce
pro-inflammatory eicosanoids, and hence prevent BC development. In addition, these
changes may also affect plasma membrane properties. N-3 PUFA can significantly affect
membrane fluidity, permeability, and resident protein activity (52, 235).
Analysis of tumor and adjacent MG phospholipids fatty acid composition also
suggests a differential role of individual n-3 PUFA on cancer prevention. Specifically, the
observed dose-dependent reductions on tumor outcomes with increased flaxseed oil
intake were mainly associated with a dose-dependent increase in ALA and EPA. An
earlier study reported in vivo and in vitro evidence that ALA was the likely bioactive
anticancer agent (78). Unfortunately, in the present study we cannot determine whether
the anti-tumorigenic effect was directly attribute to ALA itself, or the downstream
metabolic products, such as EPA and DHA. Although the conversion of ALA to EPA and
DHA is limited, supplementation of the flaxseed oil diets also resulted in a higher
incorporation of EPA and DHA relative to ALA into tumor membranes. This apparent
mismatch between dietary intakes and levels of incorporation may indicate a negative
selection for ALA into tumor cell membrane phospholipids, or preferred in conversion to
EPA and DHA. Thus, it will be important to determine the independent effect of ALA in
the future studies.
113
Moreover, the observed tumor inhibitory effect of 3% menhaden oil was primarily
related to significant increases in EPA and DHA (p<0.05), and more importantly, it is
noted that DHA is considerably more concentrated than EPA in tumor and adjacent MG
tissue. This observation may indicate a preference by tumor cells for DHA over EPA.
Interestingly, evidence has shown that DHA was more predominantly localized to the sn-
2 position on the glycerol backbone of glycerophospholipids compare to EPA (236, 237);
the position was usually linked to an n-6 PUFA, such as AA (52). With the increased
intake of marine-derived n-3 PUFA, more DHA may replace AA at the sn-2 position of
glycerophospholipid relative to EPA. Therefore, we proposed that DHA may be the more
bioactive component of menhaden oil that exerts these anti-tumorigenic effects. This was
in accordance with a recent analysis that demonstrated DHA was more effective than EPA
in suppressing mammary carcinogenesis (120). Furthermore, it is interesting to note there
is a higher level of marine-based n-3 PUFA incorporated in tumor PE relative to PC.
Previous studies also noted a similar observation in brain tissue, which demonstrated the
proportion of PUFA in PE, particularly DHA, was much greater than in PC (238-240). In
addition, a significant (p<0.05) higher incorporation of LA in tumor PE was observed in
mice fed 3% menhaden oil compared to 10% safflower oil. This may indicate a feedback
inhibition of delta-6 desaturase, which is the rate-limiting enzyme responsible for both
the synthesis of AA from LA and the conversion of EPA and DHA from ALA(241). The
presences of DHA or EPA act as a feedback inhibitor of delta-6 desaturase that may
reduce the flow of LA to AA, causing the accumulation of LA in tumor tissue.
Nevertheless, the incorporation of these n-3 PUFA into both the MGs and tumor tissue
demonstrates their direct involvement in modulating mammary tumor development,
which warrants further investigation into mechanisms of action.
4.5.3.2 Modulate cellular signaling molecules
Recent evidence suggest that membrane fatty acid compositional changes may
perturb lipid rafts, which are enriched with many regulatory proteins and growth factor
receptors (52, 235). Lipid rafts are important heterogeneous membrane microdomains
114
that are rich in cholesterol and saturated phospholipids that, when clustered together,
function as operating platforms for signaling molecules (52, 235) . The highly
unsaturated nature of n-3 PUFA make them sterically incompatible with cholesterol,
which disrupt the formation of lipid rafts and thus, suppress raft-mediated cell signaling
transductions (52, 235). The MMTV-neu (ndl)YD5 model used in the current study
represents HER-2 positive BC, therefore, the signaling pathways related to HER-2 over-
expression were considered. HER-2 falls under the family of epidermal growth receptors,
which are involved in BC progression when over-expressed or mutated (214). Once
HER-2 protein is activated, the phsophotyrosine residues serve as docking sites for the
adaptor proteins, such as Shc and Grb2, resulting in the PI3K/Akt and mitogen-activated
protein kinase pathway activation, which promote cell survival and proliferation (242). In
the present study, 3% menhaden and 10% flaxseed oil reduced HER-2 and pHER-2
protein expression in mouse mammary tumors, which then led to down-regulation of
pAkt protein expression downstream. Although the low (3%) flaxseed oil diet did not
affect tumor protein expression of HER-2 or pHER-2, a significant (p<0.05) reduction
was observed in pAkt. These results matched with the observed effects on tumor
outcomes in MMTV-neu (ndl)YD5 mice, which suggest the anti-tumorigenic actions of
plant- and marine-derived n-3 PUFA may in part be due to reduced growth factor
signaling.
Several independent studies have provided consistent data showing that
exogenous supplementation with plant- or marine-derived n-3 PUFA, especially ALA and
DHA, can decrease the expression of HER-2 oncoprotein in BT-474 and SkBr-3 human
BC cells that naturally amplify the HER-2 oncogene (5, 138). Similarly, in athymic mice
implanted with HER-2-overexpressing BT-474 cells, feeding 8% (w/w) flaxseed oil was
found to reduce tumor cell proliferation, and this was likely due to the lowered protein
levels of pHER-2 and pAkt (207, 243). Akt was also shown to be susceptible to EPA,
since the treatment of EPA can decrease protein expression of both total and pAkt in
transfected MCF-7 cells (97). More importantly, treatment of EPA and DHA
independently was shown to disrupt the formation of lipid rafts in BC cell lines and led to
growth arrest (6, 22, 56). These observations were accompanied by the inhibition of
signaling initiated by growth factor receptors, such as HER2 and epidermal growth factor
115
receptor (EFGR) (6, 22, 56). These supportive data may help to explain the differential
effect of plant- and marine-based n-3 PUFA in the current study. Compared to ALA, the
higher degree of unsaturation of EPA and DHA may lead to a greater disruption of lipid
raft, and consequently affect HER-2 mediated signaling pathways. Although little is
known about lipid rafts, evidence from previous studies and the present study support the
idea that n-3 PUFA may be a useful dietary treatment for controlling HER-2-mediated
oncogenesis. Future work will need to examine the effects of plant-derived ALA on lipid
raft disruption.
To further determine the potential mechanism of these anti-tumorigenic actions,
expression of proteins involved in cell proliferation and apoptosis were examined.
Exposure to either flaxseed or menhaden oil diets was shown to reduce tumor protein
expression of Ki67, a nuclear protein widely used as a prognostic or predictive marker of
BC (103). However, at the same dosage, 3% menhaden oil was more potent in reducing
the expression of Ki67 compared to 3% flaxseed oil. These results suggest that marine-
based n-3 PUFA is more effective than plant-derived ALA in inhibiting cell proliferation.
It is also consistent with previous in vitro studies demonstrating that a higher dosage of
ALA (72μM) is required to achieve similar inhibitory effect as EPA (50μM) and DHA
(30μM) at lower levels (86, 124, 146). However, it is not appropriate to compare the
effective dosage of individual n-3 PUFA from different BC cell lines.
In addition to cell proliferation, exposure to flaxseed or menhaden oil diet also
affected tumor cell apoptosis. It has been well-established that apoptosis is regulated and
executed by the activated cysteine-aspartic proteases, called caspases, which comprise
two distinct classes, the initiators and the effectors (228, 244). Caspase-3 is a key
executioner of apoptosis, which is cleaved by initiator caspases at Asp28 or Asp175 to
generate the active large (p17) and small (p12) subunits, forming an active heterotetramer
(228). In the present study, intake of 3% menhaden and 10% flaxseed oil significantly
(p<0.05) increased tumor protein expression of cleaved-caspase-3 compare to 10%
safflower oil, and this elevation was greatest in mice fed a 3% menhaden oil diet than any
other flaxseed oil diets. This demonstrated that dietary exposure to n-3 PUFA can
promote cell apoptosis, but marine-based n-3 PUFA was more potent than plant-derived
116
ALA. Furthermore, it is noted that the actions of n-3 PUFA on modulating protein
expression involved in cell proliferation and apoptosis are consistent with their inhibitory
effect on tumor outcomes. This suggests that plant- and marine-derived n-3 PUFA
manifest their anti-tumorigenic properties by inhibiting cell growth and promoting tumor
cell death.
4.5.4 Physiological relevance to human intake
In regards to physiological relevance, all the n-3 PUFA diets in the present study
provided mice with 22% of total calories from fat. Based on the average mouse daily
intake (2 g/day), the 3% and 10% flaxseed oil diets provided 35 mg/day and 115 mg/day
of ALA, respectively. These amounts corresponding to 3.8% and 12.5% of total calories
per day, respectively. The doses of ALA in the 3% and 10% flaxseed oil diets calculated
on the basis of total caloric intake are equivalent to human doses of 8.4 g and 27.8 g of
ALA per day, respectively, assuming 2000 kcal of caloric intake per person. These
amounts are much higher compared to the typical North American diet that provides
approximately 1.4 g of ALA per day (15). While these dosages of ALA seem unrealistic,
an earlier clinical study showed the effectiveness of daily intake of 25 g flaxseed (5.7
g/day of ALA) in reducing tumor cell proliferation and HER-2 protein expression, and
increasing apoptosis in tumors of postmenopausal BC patients (50). However, it cannot
rule out the beneficial effect of the SDG component in this flaxseed diet.
When considering the marine-based n-3 PUFA diet in the present study,
supplementation of 3% menhaden oil provided mice with 7.8 mg/day of EPA and 6.8
mg/day of DHA. In total, EPA and DHA accounted for 1.6% of the total daily calories
consumed by the mice. In terms of human intake, it is similar to a traditional Japanese
diet which contains 1-2% of daily energy as EPA and DHA, and corresponds to 3.5 g of
EPA and DHA per day (245). Given that 100 g salmon provided approximately 2.2 g of
EPA and DHA, the equivalent quantity of EPA and DHA from the 3% menhaden oil diet
is achievable in humans through the consumption of ~2 servings of 75g salmon steak a
day. A previous cohort study demonstrated that daily doses up to 7.56 g EPA+DHA, for 6
117
month, were well tolerated with excellent compliance in participants at high risk of BC
(118). Further, they found that daily intake of 1.7 g of EPA+DHA either from canned
salmon+albacore or capsule supplements increased EPA and DHA content of breast
adipose tissue over 3 months without significant differences between treatment arms
(246). These clinical data are supportive, and provide evidence that the equivalent dosage
of EPA and DHA from the 3% menhaden oil diet is tolerated by humans, and may
effective in increasing EPA and DHA incorporation into breast adipose tissue of women
to decrease BC risk.
4.6 Conclusions
In conclusion, the present study has shown that lifelong exposure to both plant-
and marine-derived n-3 PUFA can influence MG development and prevent mammary
tumoirgenesis in an aggressive HER-2 positive BC model. These anti-tumorigenic effects
of n-3 PUFA are mediated by altering fatty acid composition in the target tissue and
modulating the expression of tumor proteins involved in cell proliferation and apoptosis.
These findings support the potential importance of plant- and marine derived n-3 PUFA
in maternal and childhood diets throughout the lifecycle as a simple and effective means
of improving overall MG health and mitigating lifelong BC risk. Also, this study has, for
the first time, demonstrated that plant-derived ALA is only 1/8 as potent as marine-based
n-3 PUFA, especially EPA and DHA. This knowledge is very important for the
development of dietary strategies to prevent BC in Western countries.
118
Chapter Five
General Discussion, Limitations, Future Directions and
Concluding Remarks
119
5.1 General Discussion
Studies have shown that fish consumption or intake of marine-derived long-chain
n-3 PUFA, such as EPA and DHA, may prevent mammary carcinogenesis. However, the
anticancer effect of ALA, a plant-based and major n-3 PUFA in the Western diet, remains
equivocal. The present study has for the first time compared the relative potency between
plant- and marine-derived n-3 PUFA on mammary tumor development in MMTV-neu-
(ndl)YD5 mice, an aggressive transgenic mouse model of HER-2 positive BC. First, this
study has clearly shown that lifelong exposure to plant and marine-derived n-3 PUFA can
delay puberty onset, reduce the number of TEB and changed the fatty acid profile in the
MG of mice during the early life. These early changes in MG had a lasting impact on the
long term health of mammary tissue, influencing MG development and subsequently
lowering the risk of mammary tumorigenesis later in life. Second, this study provided
convincing evidence that lifelong exposure to plant and marine-derived n-3 PUFA can
attenuate mammary tumor development by reducing tumor volume and multiplicity, as
well as overall final tumor weight. As phospholipid membranes are the sites for
communication between cells and their environment, it is likely that n-3 PUFA exert their
anti-tumorigenic effect through the alteration in the membrane fatty acid composition in
tumor and MG tissue, and consequently HER-2-mediated signaling and tumor protein
expression related to cell proliferation and apoptosis. Most importantly, this study
demonstrates plant-derived ALA is only 1/8 as potent as marine-based n-3 PUFA in
inhibiting mammary tumorigenesis. This is useful for providing recommendations for
improving the dietary strategies and reducing BC risk in Western countries, where the
intake of EPA/DHA is considerably low (15). The fact that a food nutrient, like n-3 PUFA,
can have a significant effect on mammary tumor development is remarkable and as such
has considerable implications in the field of BC prevention. This study also emphasized
the potential importance of lifetime exposure, from conception onwards, throughout all
critical periods of development. Thus, it is important for pregnant mothers and women
planning to conceive, as well as young girls, to increase their n-3 PUFA intake in order to
mitigate lifelong BC risk.
120
5.2 Limitations
Despite careful consideration of variables and confounding factors, a few
limitations are identified in this study. First, the MMTV-neu-(ndl)YD5 mouse model is
specific to tumors over-expressing HER-2, and though this type of mammary
tumroigenesis occurs in about 25-30% of invasive human BC cases, the nature of the
disease is very complex (215, 216). Indeed, BC can be divided into several molecular
subtypes based on the expression of cell estrogen receptor, progesterone receptor and
HER2 (209). In addition, BC cannot be assumed to have only one pathway of action
initiating and progressing tumor growth. Other mechanisms of mammary tumor
development are likely involved. This study can speculate that lifelong exposure to plant-
and marine-derived n-3 PUFA would likely attenuate the tumor growth of other subtypes
of BC.
The objective of this study was to compare the relative potency between marine-
based n-3 PUFA, mainly EPA and DHA, and plant-derived ALA on mammary tumor
development. Due to the nature of the MMTV-neu (ndl)YD5 mouse model, the
endogenous production of EPA and DHA from ALA obtained from the flaxseed oil diets
still remains. As a result, we cannot rule out the protective effect of EPA and DHA on
tumor development of mice that consumed flaxseed oil diets. Although the dose-
dependent effect of flaxseed oil demonstrates a role for ALA in cancer prevention, it is
not possible to determine whether this effect is directly due to ALA itself or in part from
the conversion of ALA to EPA and DHA.
The present study emphasized the importance of n-3 PUFA exposure throughout
life, however, it was not designed to investigate the protective role of n-3 PUFA exposure
at various stages of life cycle in MMTV-neu (ndl)YD5 mice. Thus, it is difficult to
predict the effect of plant and marine-derived n-3 PUFA exposure during important
developmental stages, such as in utero, during suckling, post-weaning, and in adulthood.
Also, we cannot compare the anti-tumorigenic effect of early life exposure to n-3 PUFA
versus n-3 PUFA exposure during adulthood.
Finally, the positive control diet utilized in the present study contained 10% (w/w)
121
safflower oil which is abundant in LA (~72% LA in total fatty acids) and scarce in other
n-6 PUFA. The n-6 and n-3 PUFA families refer to many structurally similar molecules
which may have different biological effects. Therefore, in this study, we can only
conclude that in comparison to an LA rich n-6 PUFA diet, plant- and marine- derived n-3
PUFA are protective against mammary tumor development. Furthermore, the
interpretation may differ if saturated or monounsaturated fatty acids are used.
5.2 Future Directions
The results from this study demonstrate that dietary exposure to plant- and
marine-derived n-3 PUFA affected tumor protein expression of HER-2, pHER-2, pAkt
and cleaved-casepase-3, and thus it will be of interest to determine the direct effects of n-
3 PUFA on these protein targets at a transcriptional level. Moreover, additional analysis
of expression of genes involved in eicosanoid biosynthesis, inflammatory process, cell
proliferation and apoptosis will also be of interest, to provide a comprehensive
understanding of how plant- and marine-derived n-3 PUFA modulate molecular
mechanisms underlying BC development and progression.
To date, studies examined the protective roles of n-3 PUFA on lipid raft-mediated
signaling pathways remain inconclusive. Future analysis will be of interest to investigate
the effect of plant- and marine-derived n-3 PUFA on the formation of lipid raft in
mammary tumor tissue. More specifically, it will be important to know whether the
anticancer effect of n-3 PUFA was due to decrease the protein level of HER-2 or
inhibition of HER-2 phosphorylation in lipid rafts.
Estrogen is known as an important factor involved in the development and
progression of BC. Future analysis of serum estradiol and other hormones (progesterone,
etc.) will be of interested to assess whether dietary exposure to plant- and marine-derived
n-3 PUFA could modulate the metabolism of these hormones and further modify the risk
of BC.
Trastuzumab (TRAS, Herceptin™) is the most commonly prescribed drugs for
122
HER-2 positive BC (140). It will be of interest to investigate the interactions between
plant- or marine-derived n-3 PUFA and TRAS in future studies. This will help to
determine whether dietary of plant- or marine-derived n-3 PUFA can interfere, enhance,
or have no effect on the effectiveness of TRAS in mitigating mammary tumor
development in MMTV-neu (ndl)-YD5 mice.
The next logical step to take from here would be to develop a novel double-hybrid
mouse model by crossing of a MMTV-neu-(ndl)-YD5 mouse with a delta-6 desaturase
knockout model, which blocking the conversion of ALA to EPA and DHA. This double-
hybrid mouse model will allow us to compare the independent effect of ALA versus that
of EPA and DHA on mammary tumorigenesis. From that, more definitive
recommendations can be made for using plant- or marine-derived n-3 PUFA as nutritional
interventions in the prevention and treatment of BC.
5.3 Concluding Remarks
This study has shown that lifelong exposure to plant- and marine-derived n-3
PUFA can influence MG development and attenuate mammary tumorigenesis in a
relevant model of HER-2 positive BC. However, plant-derived ALA was only 1/8 as
potent compared to marine-based EPA and DHA. The current study provides evidence for
potential dietary modulation of specific BC subtypes and warrants further investigation in
prospective human trials of BC prevention. It also supports the therapeutic potential of
ALA, EPA and DHA supplementation in the treatment of HER-2-positive BC. Nutritional
intervention provides a promising approach to enhancing conventional therapeutics
without negatively affecting a patient's quality of life. In light of these findings,
Westerners that typically consume low levels of n-3 PUFA should be encouraged to
increase their dietary intake of n-3 PUFA enriched food products, including fish or
flaxseed, especially for pregnant mothers and young females for reducing lifelong BC
risk.
123
Chapter Six
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124
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Appendix - MG fatty acid composition (PC fraction)
Upper panel: PC fraction in 6-week-old mice; lower panel: PC fraction in 20-week-old
mice. The fatty acid composition of MG phospholipid differs markedly between dietary
treatments (n=4-6 per diet). Bars not sharing a letter are significantly different (p<0.05)
based on a one-way ANOVA test. Full fatty acid profile is displayed in Table 4.4.
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