Identification of apoptotic signal transduction

pathway via Bcl-2 and NF-κB in

echinomycin-treated HT-29 colon cancer cells

Ju Youn Park

The Graduate School

Yonsei University

Department of Biomedical Laboratory Science

ii

Identification of apoptotic signal transduction

pathway via Bcl-2 and NF-κB in

echinomycin-treated HT-29 colon cancer cells

A Dissertation

Submitted to the Department of Biomedical Laboratory Science

and the Graduate School of Yonsei University

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

Ju Youn Park

June 2007

iii

This certifies that the dissertation of Ju Youn Park is

approved.

___________________________

Thesis Supervisor: Yong-Suk Ryang

___________________________

Jong-Bae Kim: Thesis Committee Member

___________________________

Hyeyoung Lee: Thesis Committee Member

___________________________

Soo-Ki Kim: Thesis Committee Member

___________________________

Eung Ho Choi: Thesis Committee Member

The Graduate School

Yonsei University

June 2007

iv

Eye Fixed on the Far Horizon

Never look down to est the ground before taking your step; only he who keeps his

eye fixed on the far horizon will find his right road.

- Dag Hammarskjold

-

Believe, Discover and Work

Life is not easy for any of us.

We must work and above all we must believe in ourselves.

We must believe that each one of us is able to do something well, and that, when we

discover what this something is, we must work until we succeed.

- Madame Curie -

For my father and mother

&

Special thanks to Prof. Ryang Y. S., Prof. Kim S. K., Prof. Yoo. K. H., Prof. Lyu H. Y.

i

Table of Contents

Abbreviations ------------------------------------------------------------------------------------------ iv

List of Figures ------------------------------------------------------------------------------------------ vi

Abstract ------------------------------------------------------------------------------------------------ vii

Chapter I. Bcl-2 dependence of echinomycin-induced apoptosis in HT-29 human colon

cancer cells ---------------------------------------------------------------------------------------------- 1

1. Introduction ------------------------------------------------------------------------------------------ 2

2. Materials and Methods ------------------------------------------------------------------------------ 4

Chemotherapeutics ---------------------------------------------------------------------------------- 4

Cell culture ------------------------------------------------------------------------------------------- 4

Treatment of HT-29 cells with echinomycin ----------------------------------------------------- 4

Preparation of cell extracts ------------------------------------------------------------------------- 5

DNA fragmentation analysis ----------------------------------------------------------------------- 5

Apoptosis assay -------------------------------------------------------------------------------------- 6

Quantitative analysis of apoptosis by flow cytometry ------------------------------------------ 6

Western blot analysis -------------------------------------------------------------------------------- 6

Detection of cytochrome c release ---------------------------------------------------------------- 7

Transfection ------------------------------------------------------------------------------------------ 8

Mitochondrial depolarization ---------------------------------------------------------------------- 8

Statistical evaluation -------------------------------------------------------------------------------- 8

3. Results ----------------------------------------------------------------------------------------------- 10

Echinomycin treatment induces apoptosis of HT-29 cells ------------------------------------ 10

Echinomycin induces mitochondrial membrane depolarization ----------------------------- 12

Transiently transfected with Bax-dominant negative fails to protect echinomycin-induced

ii

apoptosis -------------------------------------------------------------------------------------------- 14

Transient overexpression of Bcl-2 inhibits echinomycin-induced apoptosis in a dose-

dependent manner ---------------------------------------------------------------------------------- 16

Echinomycin treatment affects overexpression of Bcl-2 in HT-29 cells ------------------- 19

4. Discussion ------------------------------------------------------------------------------------------- 21

Chapter II. NF-κB activation and regulation of IL-8 expression in HT-29 human colon

cancer cell stimulated by DNA intercalation agent, echinomycin ------------------------------ 24

1. Introduction ----------------------------------------------------------------------------------------- 25

2. Materials and Methods ---------------------------------------------------------------------------- 27

Chemotherapeutics --------------------------------------------------------------------------------- 27

Cell culture ------------------------------------------------------------------------------------------ 27

Treatment of HT-29 cells with echinomycin --------------------------------------------------- 27

Preparation of cell extracts ----------------------------------------------------------------------- 28

Western blot analysis ------------------------------------------------------------------------------ 28

Transient transfection and luciferase reporter gene assay ------------------------------------ 29

Electrophoretic mobility shift assays ------------------------------------------------------------ 29

Reverse transcription polymerase chain reaction ---------------------------------------------- 30

Enzyme-linked immunosorbent assay ---------------------------------------------------------- 31

Quantitative analysis of apoptosis by flow cytometry ---------------------------------------- 32

Statistical evaluation -------------------------------------------------------------------------------32

3. Results ----------------------------------------------------------------------------------------------- 33

Induction of nuclear translocation of NF-κB by echinomycin in HT-29 cells ------------- 33

Echinomycin induces the translocation of p50-p65 heterodimeric subunits of NF-κB -- 35

Expression profiles of IκB and p-IκB in HT-29 cells treated with echinomycin ---------- 37

Echinomycin treatment triggers IL-8 production in HT-29 cells ---------------------------- 39

iii

Proteasome inhibitor blocks NF-κB activation, consequently upregulating IL-8 expression

in echinomycin-treated HT-29 cells ------------------------------------------------------------- 41

IL-8 production at echinomycin treatment in HT-29 cells occurs via both caspase-3 and

NF-κB dependent signal pathway --------------------------------------------------------------- 43

Echinomycin-induced apoptosis in HT-29 cells occurs via only NF-κB activation

independent of caspase-3 activation ------------------------------------------------------------- 45

4. Discussion ------------------------------------------------------------------------------------------- 49

Conclusions -------------------------------------------------------------------------------------------- 53

References --------------------------------------------------------------------------------------------- 55

Abstract in Korean ------------------------------------------------------------------------------------ 63

iv

Abbreviations

Boc-D-FMK : Boc-Asp (OMe)-CH2F

DMSO : dimethyl sulfoxide

DN : dominalt negative

DSB : double-strand breaks

ECL : electrogenerated chemiluminescent

ELAM-1 : endothelial-leukocyte adhesion molecule-1

EMSAs : electrophoretic mobility shift assays

FBS : fetal bovine serum

FITC : fluorescein isothiocyanate

HIF : hypoxia inducible factor

HIV : human immunodeficiency virus

HRP : horseradish peroxidase

ICAM-1 : intercellular adhesion molecule-1

IL-8 : interleukin-8

JC-1 : 5,5’,6,6’-tetrachloro-1,1’.3,3’-tetraethylbenzimidazoly-carbocyanine iodide

MAP kinase : mitogen-activated protein kinase

MGB : minor groove binders

MOMP : mitochondrial outer membrane permeabilization

NF-κB : nuclear factor κB

PBS : phosphate-buffered saline

PI : propidium iodide

PMSF : phenylmethylsulfonyl fluoride

PVDF : polyvinylidene difluoride

v

RT-PCR : reverse transcription polymerase chain reaction

SDS : sodium dodecyl sulfate

TAT : transactivator of transcription

TNF-α : tumor necrosis factor-alpha

VCAM-1 : vascular cell adhesion molecule-1

VRE : vancomycin-resistant enterococci

Z-DEVD-FMK : Z-Asp (OMe)-Glu (OMe)-Val-Asp (OMe)-CH2F

vi

List of Figures

Figure I-1. Echinomycin-induced apoptosis in HT-29 cells ------------------------------------- 11

Figure I-2. Mitochondrial membrane potential in echinomycin-treated cells ---------------- 13

Figure I-3. Treansfection of Bax-DN did not protect echinomycin-induced apoptosis ----- 15

Figure I-4. Overexpression of Bcl-2 blocks echinomycin-induced apoptosis ---------------- 18

Figure I-5. Overexpression of Bcl-2 blocks echinomycin-induced cytochrome c release and

caspase-3 activation ------------------------------------------------------------------- 20

Figure II-1. NF-κB activation was induced in echinomycin-treated HT-29 cells ------------ 34

Figure II-2. Echinomycin induces the translocation of p50-p65 heterodimeric subunits of

NF-κB ----------------------------------------------------------------------------------- 36

Figure II-3. Degradation of IκB in echinomycin treated cells ---------------------------------- 38

Figure II-4. Echinomycin inhibited the IL-8 production in HT-29 cells ----------------------- 40

Figure II-5. Apoptotic cell death inhibition by proteasome inhibitor in echinomycin-treated

cells -------------------------------------------------------------------------------------- 42

Figure II-6. Regulation of IL-8 production was caspase-3 and NF-κB dependent by

Echinomycin ---------------------------------------------------------------------------- 44

Figure II-7. Involvement of NF-κB and caspase pathways in the regulation of apoptosis by

echinomycin in HT-29 cells ---------------------------------------------------------- 46

Figure II-8. Relationship of NF-κB activation and caspase3 signalig pathway in HT-29 by

echinomycin ---------------------------------------------------------------------------- 47

Figure II-9. Schematic of the signaling pathway by echinomycin treatment in HT-29 cells - 48

vii

ABSTRACT

Identification of apoptotic signal transduction pathway via Bcl-2 and NF-κκκκB in

echinomycin-treated HT-29 colon cancer cells

Ju Youn Park

Dept. of Biomedical Laboratory Science

The Graduate School

Yonsei University

The present study was aimed to identify specific signal pathway via Bcl2 and NF-κB in

echinomycin-mediated apoptosis of HT-29 colon cancer cells. In first experiment to verify

Bcl-2 pathway, inactive form Bax-DNA failed to prevent echinomycin-induced apoptosis in

HT-29 cells. In contrast, combined analyses of DNA fragmentation and flow cytometric

analysis clearly verified that echinomycin-induced apoptosis was drastically attenuated by

Bcl-2 overexpression, whereas a control vector rarely affected echinomycin-induced

apoptosis. Given these, these first part of data verify that Bcl-2 regulates echinomycin-

induced apoptosis in HT-29 cells. Next, in this study was investigated whether echinomycin-

induced apoptosis would be NF-κB-dependent and if so, echinomycin would activate or

inhibit NF-κB as well as resultant chemokine, IL-8 expression. As sequential findings of

NF-κB-dependency in echinomycin-induced apoptosis, EMSA employing NF-κB specific

probe and transfection with luciferase reporter plasmids containing, NF-κB binding

elements showed that echinomycin-induced DNA-protein complex formation was inhibited

and echinomycin induced the binding activity of NF-κB promoter. In line, EMSA in the

viii

presence of antibodies specific for p50 and p65 subunits indicated that echinomycin induces

the translocation of p50-p65 heterodimeric subunits of NF-κB. Using Western blots, levels

of IκB were detected at initial echinomycin treatment, and thereafter decreased, faintly seen

after a 6 h treatment. In contrast, p-IκB levels were clearly detected throughout 6 to 24 h of

echinomycin treatment, albeit initially fainted. Finally, in experiment to clarify the role of

NF-κB on IL-8 expression in echinomycin-mediated apoptosis of HT-29 cells. ELISA plus

RT-PCR clearly showed that IL-8 production is inducible and manipulative by echinomycin

treatment. Using a specific inhibitor approach, it was clear that IL-8 production at

echinomycin treatment in HT-29 cells occurs via both caspase-3 and NF-κB dependent

signal pathway. To confirm whether two different pathways (NF-κB and caspase) would be

coupled, only both NF-κB inhibitor (PDTC) and caspase-3 specific inhibitor (Z-DEVD-

FMK) significantly attenuated echinomycin-initiated apoptosis of HT-29 cells, whereas

pretreatment of HT-29 cells with NF-κB inhibitor (PDTC) rarely affected echinomycin-

induced procaspase-3 activation. Collectively, second part of data indicate that echinomycin-

induced apoptosis in HT-29 cells occurs via NF-κB activation independent of caspase-3

activation, finally modulating the resultant-linked key chemokine IL-8 expression.

Synthesizing both data, these coupled data indicate that echinomycin-mediated apoptosis in

HT-29 colon cancer cells occurs via Bcl-2 and NF-κB dependent pathway. Accordingly, the

present study laid a ground for understanding the intracellular signal transduction

mechanism by echinomycin in combination with apoptosis mechanism in HT-29 colon

cancer cells and applying the mechanism.

---------------------------------------------------------------------------------------------------------------

Key words: Echinomycin, Apoptosis, HT-29 colon cancer cell, Bcl-2, Caspase-3, NF-κB,

IL-8

１

CHAPTER I

Bcl-2 dependence of echinomycin-induced apoptosis

in HT-29 human colon cancer cells

２

1. Introduction

Echinomycin was classically known as DNA bis-intercalating or DNA damaging-

quinoxaline molecule. Emerging evidences have shown that echinomyin or DNA bis-

intercalator owns novel medico-biologic activities such as hypoxia inducible factor (HIF)

suppression, transactivator of transcription (TAT) (of human immunodeficiency virus

(HIV)) binding, anti-vancomycin-resistant enterococci (VRE) activity, and anti-thrombotic

activity (Lee et al., 2007; Jayasuriya et al., 2005; Kong et al., 2005; Kim et al., 2004). Such

diversified activities are unexplainable only by simple notion of echinomycin’s DNA

targeting, suggesting that echinomycin might modulate cellular machinery involving

apoptosis and differentiation in prokaryotes or eukaryotes. Hypothesizing this, it was

presented evidences that echinomycin triggers intracellular signaling pathways such as the

mitogen-activated protein (MAP) kinase, mitochondrial, and caspase pathways,

consequently leading to apoptosis of HT-29 cells (Park et al., 2006; Park et al., 2004).

However, previous experiment had clear limitations in terms of demonstrating the

engagement of Bcl-2 in echinomycin-mediated apoptosis (Park et al., 2006).

It was widely accepted that apoptosis via the mitochondrial pathway induces

mitochondrial outer membrane permeabilization (MOMP), then leading to the release of

cytochrome c. This key step toward apoptosis is mediated by proteins of the Bcl-2 family

(Spierings et al., 2005).

Compelling data have shown that Bcl-2 overexpression inhibits apoptosis induced by

diverse anticancer drug (Panaretakis et al., 2003). Despite a partial identification of

prototypic signal path triggered by echinomycin, the role of Bcl-2 in this signaling pathway

is unclear.

３

To address this, it was explored whether echinomycin would overcome the anti-apoptotic

impact of Bcl-2 in HT-29 cells by exploiting the controlled Bcl-2 overexpression. This study

shows that Bcl-2 regulates echinomycin-induced apoptosis in HT-29 cells.

４

2. Materials and Methods

Chemotherapeutics

Echinomycin (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO)

(Sigma, St. Louis, MO, USA) and added to the culture medium at the indicated

concentration. The concentration of DMSO in the medium was less than 1% (v/v). Cells

were incubated 37℃ for the indicated times and harvested.

Cell culture

Human colon cancer cell line HT-29, others were purchased from American Type Culture

Collection (ATCC) (Rockville, MD, USA). HT-29 cells were cultured in RMPI1640 (Gibco

BRL, Hercules, CA, USA) and supplemented with 10% fetal bovine serum (FBS), penicillin

(100 U/ml), and streptomycin (100 U/ml) in an atmosphere of 5% CO2 in air at 37℃.

Treatment of HT-29 cells with echinomycin

Echinomycin (Sigma, St. Louis, MO, USA) was dissolved in DMSO (Sigma, St. Louis,

MO, USA), and added to the culture medium at the indicated concentration. The

concentration of DMSO in the medium was less than 0.1% (v/v). Cells were incubated at

37 for the indicated times℃ , and harvested.

５

Preparation of cell extracts

Cells from a dish were harvested, pelleted, and washed in phosphate-buffered-saline

(PBS). The cell pellet was then resuspended in an equal volume of lysis buffer (100 mM Tris,

150 mM NaCl, 10% glycerol, 0.6% Triton-X 100, 5 mM EDTA, 1 mM sodium

orthovanadate, 10 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride (PMSF)). The

cells were incubated for 60 min on ice, and centrifuged at 14,000 rpm for 30 min at 4 . ℃

The soluble fraction was transferred to a new tube, and the preparation was stored at -70 .℃

DNA fragmentation analysis

Approximately 2 × 106 cells were lysed with 0.2 ml of lysis buffer containing 10 mM Tris-

HCl, 20 mM EDTA, and 0.5 % Triton X-100 (pH 8.0), and placed on ice for 30 min. Cell

extracts were clarified by centrifugation at 14,000 rpm for 10 min. The supernatant

containing DNA cleavage products was precipitated overnight using isopropyl alcohol

(Merck, Clarkston, MI, USA). The lysates was incubated with 0.3 mg/ml proteinase K

(Boehringer Mannheim, Mannheim, Germany) at 37℃ for 1 h. Dry DNA pellets were then

resuspended in TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA), containing 0.5

mg/ml RNase A (Boehringer Mannheim, Manneheim, Germany). DNA fragments were

separated on a 1.8 % agarose gel, and visualized by ethidium bromide staining (Bio-Rad,

Hercules, CA, USA), and photographed.

６

Apoptosis assay

HT-29 cells were seeded in 35-mm plastic dishes (3× 105 cells per dish) and treated with

echinomycin for 24 h. Cells were prepared and analyzed using APOPercentageTM apoptosis

assay according to the manufacturer’s instructions (Biocolor, Newtonabbey, Northern

Ireland).

Quantitative analysis of apoptosis by flow cytometry

Cells were harvested, washed with PBS, and resuspended in a binding buffer (10 mM

HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After 15 min of incubation with Annexin

V-fluorescein isothiocyanate (Sigma, St. Louis, MO, USA) and propidium iodide (PI)

(Pharmingen, San Diego, CA, USA) at room temperature, the fluorescence emitted by cells

(10,000 cells/sample) was analyzed on a flow cytometer (Becton-Dickinson, Franklin Lakes,

NJ, USA).

Western blot analysis

HT-29 cells were seeded in 35-mm plastic dishes (3 × 105 cells per dish) and incubated

with echinomycin for different time periods. Cells were lysed in the lysis buffer (50 mM

Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 µg/ml

aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM sodium orthovanadate). After

centrifugation at 15,000 rpm at 4℃ for 30 min, supernatant was collected, 20 µg of lysates

７

from each sample was run on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and

then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. PVDF

membranes were rinsed in TBST (10 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.05% Tween 20,

and 1 mM EDTA) and blocked in blocking buffer (TBST containing 5% bovine serum

albumin) overnight at 4℃. PVDF membranes were incubated with primary antibodies

overnight at 4℃, washed, and incubated with goat anti-rabbit IgG conjugated with

horseradish peroxidase (HRP) or goat anti-mouse IgG conjugated with HRP for 1 h at room

temperature. The membrane was developed with electrogenerated chemiluminescent (ECL)

substrate (Amersham Life Sciences, Arlington Heights, IL, USA), and exposed to Biomax

MS autoradiography x-ray film (Kodak, Rochester, NY, USA).

Detection of cytochrome c release

1 × 107 cells were trypsinized and collected at the indicated times. The pellets were

washed with ice-cold PBS and resuspended in 150 µl of cytosol extraction buffer (250 mM

sucrose, 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM

EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride). The cell pellets

were then homogenized with 30 strokes of a homogenizer (Wheaton, Millville, NJ, USA).

The mitochondria-enriched fraction was pelleted by centrifugation at 14,000 rpm for 30 min.

The supernatant was subjected to Western blot analysis with a monoclonal antibody to

cytochrome c.

８

Transfection

Transfection was performed using LipofectAMINE2000 (Invitrogen, Carlsbad, CA, USA),

according to the supplier’s protocol. HT-29 cells were plated in 6-well plates (1 × 106

cells/well) and transfected with each expression vector (pcDNA3-Bcl-2, or pcDNA3-Bax-

dominalt negative (DN)) or with a control vector (pcDNA3, empty vector). The plasmids

used in this experiment were kindly provided by Dr. Kim, H. S. (University of Catholic,

Republic of Korea). Twenty-four hours after transfection, the cells were treated with

echinomycin for different time periods as indicated for Western blot analysis, DNA

fragmentation and quantitative analysis of apoptosis by flow cytometry (Becton-Dickinson,

Franklin Lakes, NJ, USA).

Mitochondrial depolarization

To measure mitochondrial depolarization, drug-treated or untreated cells (5× 106 cells/ml)

were incubated with 5 µg/ml JC-1 (5,5’,6,6’-tetrachloro-1,1’.3,3’-tetraethylbenzimidazoly-

carbocyanine iodide) as described in HT-29 cells. After incubation for 20 min at room

temperature in the dark, cells were washed once with PBS. Flow cytometry was performed

on a FACScan (Becton-Dickinson, Franklin Lakes, NJ, USA).

Statistical evaluation

All experiments were performed at least three times. Results are presented as means ± SD.

９

Data were analyzed by the Student’s t-test.

１０

3. Results

Echinomycin treatment induces apoptosis of HT-29 cells

Echinomycin treatment results in apoptosis of many different cell types (Kim et al.,

2004). To examine the ability of echinomycin to induce apoptosis in HT-29 cells, cultures

were treated with a condition of various does and time, after which they were examined

APOPercentageTM apoptosis assay. As shown in Figure I-1, echinomycin caused apoptosis

of HT-29 cells in a time-dependent manner, from a concentration of 0.2 µg/ml echinomycin.

Figure I-1 clearly confirmed that echinomycin drives apoptosis of HT-29 cells.

１１

0

0.5

1

1.5

2

2.5

N 6h 12h 24h 48h

O.D

(55

0 nm

)

Figure I-1. Echinomycin-induced apoptosis in HT-29 cells. Echinomycin was incubated with

HT-29 cells induce apoptosis for indicated time. After the echinomycin treatment (2 µg/ml),

cells undergoing apoptosis were detected by the uptake of a purple dye (APOPercentageTM

Apoptotic Assay, Biocolor).

１２

Echinomycin induces mitochondrial membrane depolarization

To determine the depolarization of mitochondria in response to echinomycin, the

membrane potential-sensitive dye JC-1 was added to cultures of control and echinomycin-

treated cells. Thereafter, the fraction of cells with the depolarized mitochondria was

quantified by flow cytometry. The mitochondrial depolarization was seen in as early as 6 h

(Figure I-2). This loss of mitochondrial potential was increased time dependently.

１３

0

20

40

60

80

0 6 12 18 24

Time (h)

% l

oss

of m

itoc

hond

rial

mem

bran

ce p

oten

tial

Figure I-2. Mitochondrial membrane potential in echinomycin-treated cells. HT-29 cells

were untreated (control, circles) or treated with 2 µg/ml echinomycin (squares) for the times

indicated. The cells were then stained with JC-1, and the cells with intact mitochondrial

membrane potential were scored. The percentage of cells with intact membrane potential is

plotted as a function of time.

１４

Transiently transfected with Bax-dominant negative fails to protect echinomycin-

induced apoptosis

The simple correlation between Bcl-2 family expression and echinomycin-induced

apoptosis was examined in previous study (Park et al., 2006). In order to examine the effect

of Bax expression to prevent echinomycin-induced apoptosis in HT-29 cells, HT-29 cells

were transiently transfected with Bax-DN prior to echinomycin treatment. Immunoblotting

confirmed that the transfectants expressed lowly levels of Bax in echinomycin treatment at

24 h (Figure I-3A) and the Bax-DN in HT-29 cells was not changed throughout the

apoptosis process after echinomycin treatment (Figure I-3B).

１５

A.

Mock + - - -Bax-DN (µµµµg) - 1 2 4Echinomycin + + + +

Bax

Actin

B.

Mock + - - - -Bax-DN (µµµµg) - - 1 2 4Echinomycin - + + + +

Figure I-3. Treanfection of Bax-DN did not protect echinomycin-induced apoptosis. HT-29

cells were transiently transfected with an empty vector as a control or pcDNA3/Bax-DN.

Then, 2 µg/ml echinomycin was added to HT-29 cells for 24 h. (A) Bax-DN was determined

by immunoblotting in a dose-dependent manner. For immunoblotting, anti-actin antibody

was used as a loading control. (B) Apoptotic DNA fragmentation was determined by agarose

gel electrophoresis.

１６

Transient overexpression of Bcl-2 inhibits echinomycin-induced apoptosis in a dose-

dependent manner

While mitochondrial dysfunction triggered by echinomycin was partially elucidated, the

role of Bcl-2 in this signaling pathway has been still unclear. Thus, HT-29 cells were

transiently transfected with Bcl-2 DNA prior to echinomycin treatment. Immunoblotting

confirmed that the transfectants expressed high levels of Bcl-2 (Figure I-4A). To further

confirm this, HT-29 cells were overexpressed by using Bcl-2 and then treated with

echinomycin for 24 h. It is clear that echinomycin-induced apoptosis in HT-29 cells was

drastically attenuated by Bcl-2 transfection, whereas a control vector rarely affected it. This

clear proof was fully evidenced by DNA fragmentation (Figure I-4B) and flow cytometric

analysis (Figure I-4C). These data shows that echinomycin–induced apoptosis is indeed

sensitive to the overexpression of Bcl-2.

１７

A.

Bcl-2

Actin

N Mock Bcl-2

Mock + - - -Bcl-2 (µµµµg) - - 2 4

Echinomycin - + + +M

B.

１８

0

20

40

60

80

100A

popt

osis

(%

)

Vector - + - - -Bcl-2 (µµµµg) - - - 2 4

Echinomycin - - + + +

C.

Figure I-4. Overexpression of Bcl-2 blocks echinomycin-induced apoptosis. (A)

Overexpressed Bcl-2 expression was determined by immunoblotting. Immunoblotting with

anti-actin antibody was used as a loading control. HT-29 cells were transiently transfected

with an empty vector or pcDNA3/Bcl-2. Then, 2 µg/ml echinomycin was added to HT-29

cells for 24 h. (B) Apoptotic DNA fragmentation was determined by agarose gel

electrophoresis (M: marker). (C) Cells were stained with Annexin V-FITC and propidium

iodide (PI). Apoptotic cells are determined by counting the % of Annexin V-FITC(+), PI(-)

cells and the % of Annexin V-FITC(+), PI(+) cells.

１９

Echinomycin treatment affects overexpression of Bcl-2 in HT-29 cells

Echinomycin-induced apoptosis through sequential activation of ERK-casapase pathway

was shown in previous study (Park et al., 2006). But, the relationship between Bcl-2 and

caspase activation in echinomycin-induced apoptosis was unknown. To clarify this issue,

Bcl-2 overexpresion would abrogate the cytochrome c release as well as the activation of

caspase-3. Figure I-5 clearly shows that Bcl-2 overexpression abrogates cytochrome c

release as well as the cleavage of procaspase-3 in a transfected dose-dependent fashion.

２０

Cytochrome C

Mock - + - - -Bcl-2 (µµµµg) - - - 2 4

Echinomycin - - + + +

Procaspase 3

Actin

Figure I-5. Overexpression of Bcl-2 blocks echinomycin-induced cytochrome c release and

caspase-3 activation. HT-29 cells were transiently transfected with an empty vector or

pcDNA3/Bcl-2. Then, 2 µg/ml echinomycin was added to HT-29 cells for 24 h. Cytochrome

c and procaspase-3 were detected by immunoblotting with corresponding antibodies. Similar

results were achieved in three separate experiments with comparable outcomes. Actin was

used as a loading control.

２１

4. Discussion

This study verifies that Bcl-2 regulates echinomycin-induced apoptosis in HT-29 cells.

This verifying evidence was that using combined analyses of DNA fragmentation plus flow

cytometric analysis, echinomycin-induced apoptosis was drastically attenuated by Bcl-2

overexpression, whereas a control vector rarely affected echinomycin-induced apoptosis.

Indirect evidence was that transfection of Bax-donimant negative DNA failed to prevent

echinomycin-induced apoptosis.

The Bcl-2 was known to play a pivotal role in promoting tumor cell survival through

inhibition of apoptotic cell death following a variety of stimuli (Reed et al., 1995). The Bcl-2

family as apoptosis regulators holds anti- (Bcl-2 and Bcl-xL) and pro-apoptotic (Bax, Bad,

Bid, Bik, Bak, and Bcl-xS) effect (Shankar et al., 2007).

To explore the involvement of Bcl-2 family in echinomycin-mediated apoptosis,

preliminary experiment using immunoblotting has shown that the down-regulation of anti-

apoptotic protein Bcl-2 as well as the up-regulation of pro-apoptotic protein Bax could

trigger mitochondria-mediated apoptosis on HT-29 cells (Park et al., 2006). This clue

provides two plausible hypotheses: 1) Bcl-2 dependent, not Bad-dependent or 2) Bcl-2

independent, not Bad-dependent. However, previous experiment had some defects in terms

of failure to prove the regulatory machinery between Bcl-2 and Bad at upstream level of

mitochondria-MAP kinase-capsase pathway in echinomycin-mediated apoptosis. To resolve

this, functional opposite expression system: 1) Bcl-2 overexpresson 2) transfection of Bax-

DN. Major hypothesis was that Bcl-2 might regulate echinomycin-mediated apoptosis in

HT-29 cells. In initial experiment to rule out the possible engagement of Bax, it was found

that overexpression of Bax-DN failed to block echinomycin-induced apoptosis in HT-29

２２

cells (Figure I-3), indicating that echinomycin-induced apoptosis of HT-29 cells may be

independent of Bax expression. In this care, data coupled with previous data (down-

regulation of anti-apoptotic protein Bcl-2) indirectly verify this study hypothetical path (Bcl-

2 dependent, not Bad-dependent). It was well documented that Bcl-2 overexpression

protects apoptosis induced by anticancer agents (Nuessler et al., 1999; Takahashi et al.,

1999). In particular, DNA-damaging agent was known to cause apoptosis via direct or

indirect inhibition of Bcl-2 expression (Walton et al., 1993). However, this research was the

first suggestion that of diverse, structured minor groove binders (MGB), the prototypic

echinomycin might control the apoptotic signaling via Bcl-2-mitochondria pathway.

Previous study showed that echinomycin-induced apoptosis through sequential activation

of ERK-caspase pathway (Park et al., 2006). But, the direct linkage between Bcl-2 and

caspase activation in echinomycin-induced apoptosis was elusive. On this question, these

data (Figure I-5) clearly were shown that Bcl-2 overexpression abrogates cytochrome c

release as well as the activation of procaspase-3 in a transfected dose-dependent fashion.

These results were not clearly compatible with apoptosis triggered by DNA damaging or

intercalating agents (Marquis et al., 2005; Roaten et al., 2002).

Several line of evidences were suggested that mitochondrial damage might cause

degradation of Bcl-2 (Zhou et al., 2006; Panaretakis et al., 2003). Consistently, it was

demonstrated that caspase-3 and cytochrome c release were directly involved in the

execution of echinomycin-induced apoptosis in HT-29 (Park et al., 2006).

It was generally accepted that the cytochrome c release and depolarization are essential to

mitochondrial apoptosis (Liao et al., 2006). Given this, echinomycin triggers the

depolarization of mitochondria in the presence of cytochrome c release, followed by the

activation of caspase-3. Current data plus a previous report clearly reconfirm that the

mitochondrial dysfunction was requisite to echinomycin-mediated apoptosis.

２３

These cumulative results clearly indicated that echinomycin-mediated apoptosis of HT-29

cells occurs via Bcl-2-mitochondria path. Another important implication was to unravel the

hidden apoptotic signal path regarding MGB or DNA bis-intercalating agent. Collectively,

these data verify was that Bcl-2 regulates echinomycin induced apoptosis in HT-29 cells.

This discovery will not suffice to explain the full scenario of echinomycin-induced apoptosis.

Every MGB might take a unique signal path. In that context, further discovery of a novel

signal path taken by echinomycin or other MGB should shed light on the creation of novel

therapeutics against aberrant signal-transducing disease, such as cancer, hyper-proliferative

disease, infections.

２４

CHAPTER II

NF-κκκκB activation and regulation of IL-8 expression in

HT-29 human colon cancer cell stimulated by DNA

intercalation agent, echinomycin

２５

1. Introduction

DNA has served as one of most favored targets for cancer chemotherapy (July et al.,

2004; Chen et al, 2002). Of DNA damaging anticancer agents, double-strand breaks (DSB)

inducer such as topoisomerase inhibitors (Jacob et al., 2005), anthracycline (Capranico et al.,

1987) and DNA intercalating agents such as echinomycin have been preclinically or

clinically used for treatment of a variety of solid cancer (Ryu et al., 2000). These compounds

not only directly attack random or specified site of cancer cell DNA but also trigger a wide

array of intracellular signaling pathways. Both direct and delicate, complex network of

genotoxic signals synergistically induce efficient apoptosis of targeted cancer cells (Janssens

et al., 2006; D’Aqostini et al., 2005). While the apoptotic signal path of DSB inducer has

been well documented, how DNA intercalating agents initiate or elicit apoptotic signaling

has been in mystery.

Previous reports have presented clear evidences regarding the existence of mitochondrial

dependent, MAP kinase pathway in echinomycin, the prototypic DNA bis-intercalator

treated HT-29 colon cancer cells. This discovery might confer overall insight into unraveling

the exact, specified intracellular signal path taken by each DNA intercalator or minor groove

binder (MGB) because these compounds would be a good candidate in view of owning

unique cytotoxic profiles against chemo-resistant or intractable type of cancer (Geroni et al.,

2002; Baraldi et al., 2001). Recently, previous study results have demonstrated that

echinomycin can induce cell apoptosis via Bcl-2 dependent and Bax independent pathway.

These uncovering signal paths in echinomycin-induced apoptosis still mainly stayed in

cytoplasmic level. This apoptotic signal might converge in a nucleus, consequently

modulating nuclear factor κB (NF-κB) transcription factor. Emerging data have suggested

２６

that DSB inducer would directly trigger NF-κB pathway, thereby leading to apoptosis via

activating or suppressing NF-κB. Despite partial knowledge of DSB-initiated NF-κB,

however, the relaying molecular events between cytoplasmic signaling a path and an intra

nuclear path in DNA intercalating agents-initiated apoptosis remain to be unsettled.

Transcription factor, the inhibitory protein of NF-κB and IκB can be phosphorylated,

ubiquitinated, and then degraded by proteosome, which results in upregulation of NF-κB

mediated gene expression of inflammation and cancer. NF-κB is involved not only in

inflammatory diseases and in oncogenesis but also in apoptotic processes induced by

cytokine, chemokine and antitumor drugs (Mayo et al., 2000; Barkett et al., 1999).

Cytokines, such as tumor necrosis factor (TNF)-α, interferon-β, interleukin-1 (IL-1), IL-6,

and IL-8, and the adhesion molecules endothelial-leukocyte adhesion molecule (ELAM)-1,

intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1

are products of some of the genes that are regulated by NF-κB-dependent mechanisms

(Baeuerle et al., 1996). In an intestinal epithelial cell such as HT-29, NF-κB can be a central

regulator of chemokine gene expression (Sougioultzis et al., 2006; Jijon et al., 2005). This

chemokine may play some role in survival or death of cancer cells. Of chemokines,

interleukin (IL)-8 has been known to be intimately linked with NF-κB pathway in HT-29

cells (Mormina et al., 2006). Still, the connective link between NF-κB and IL-8 expression

in echinomycin-mediated apoptosis of HT-29 cells was unknown.

To clarify these complicated issues, it was investigated whether echinomycin-induced

apoptosis would be NF-κB-dependent and if so, echinomycin would activate or inhibit NF-

κB as well as resultant chemokine, expression of IL-8.

２７

2. Materials and Methods

Chemotherapeutics

Echinomycin (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO)

(Sigma, St. Louis, MO, USA) and added to the culture medium at the indicated

concentration. The concentration of DMSO in the medium was less than 1% (v/v). Cells

were incubated 37℃ for the indicated times and harvested.

Cell culture

Human colon cancer cell line HT-29, others were purchased from American Type Culture

Collection (ATCC) (Rockville, MD, USA). HT-29 cells were cultured in RMPI1640 (Gibco

BRL, Hercules, CA, USA) and supplemented with 10% fetal bovine serum (FBS), penicillin

(100 U/ml), and streptomycin (100 U/ml) in an atmosphere of 5% CO2 in air at 37℃.

Treatment of HT-29 cells with echinomycin

Echinomycin (Sigma, St. Louis, MO. USA) was dissolved in DMSO (Sigma, St. Louis,

MO, USA), and added to the culture medium at the indicated concentration. The

concentration of DMSO in the medium was less than 0.1% (v/v). Cells were incubated at

37℃ for the indicated times, and harvested. In some experiments, HT-29 cells were treated

２８

with MG-132 (Z-Leu-Leu-Leu-aldehyde), PDTC (pyrrolidine dithiocarbamate), Boc-D-

FMK (Boc-Asp (OMe)-CH2F and Z-DEVD-FMK (Z-Asp (OMe)-Glu (OMe)-Val-Asp

(OMe)-CH2F) were from Calbiochem (San Diego, CA, USA)) for 1 h before exposure to

echinomycin and during the incubation period of the experiment.

Preparation of cell extracts

Cells from a dish were harvested, pelleted, and washed in phosphate-buffered-saline

(PBS). The cell pellet was then resuspended in an equal volume of lysis buffer (100 mM Tris,

150 mM NaCl, 10% glycerol, 0.6% Triton-X 100, 5 mM EDTA, 1 mM sodium

orthovanadate, 10 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride (PMSF)). The

cells were incubated for 60 min on ice, and centrifuged at 14,000 rpm for 30 min at 4 ℃.

The soluble fraction was transferred to a new tube, and the preparation was stored at -70 ℃.

Western blot analysis

HT-29 cells were seeded in 35-mm plastic dishes (3 × 105 cells per dish) and incubated

with echinomycin for different time periods. Cells were lysed in the lysis buffer (50 mM

Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 µg/ml

aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM sodium orthovanadate). After

centrifugation at 15,000 rpm at 4℃ for 30 min, supernatant was collected, 20 µg of lysates

from each sample was run on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and

then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. PVDF

２９

membranes were rinsed in TBST (10 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.05% Tween 20,

and 1 mM EDTA) and blocked in blocking buffer (TBST containing 5% bovine serum

albumin) overnight at 4℃. PVDF membranes were incubated with primary antibodies

overnight at 4℃, washed, and incubated with goat anti-rabbit IgG conjugated with

horseradish peroxidase (HRP) or goat anti-mouse IgG conjugated with HRP for 1 h at room

temperature. The membrane was developed with electrogenerated chemiluminescent (ECL)

substrate (Amersham Life Sciences, Arlington Heights, IL, USA), and exposed to Biomax

MS autoradiography x-ray film (Kodak, Rochester, NY, USA).

Transient transfection and luciferase reporter gene assay

The pGL3-NF-κB promoter constructs were transfected into HT-29 cells by

LipofectAmine 2000 (Invitrogen, California, CA, USA). Luciferase activity was assayed by

using a luciferase assay kit according to the manufacturer's instructions (Promega, Madison,

WI, USA). Cell extracts were prepared in 500 µl 1× reporter lysis buffer. The lysates were

centrifuged at 13,000g for 5 min and supernatant was used for detection of luciferase

activity in a Microlumat LB96P Luminometer (Perkin Elmer, Fremont, CA, USA). The

luciferase reporter assay was repeated at least three times.

Electrophoretic mobility shift assays

Assays were performed with the gel shift assay system (Promega, Madison, WI, USA)

according to the manufacturer’s protocol, with 5 to 10 µg of nuclear protein. Cells (2 × 107)

３０

were lysed in 200 µl lysis buffer (20 mM HEPES (pH 7.9), 10 mM NaCl, 3 mM MgCl2,

0.1% Nonidet P-40, 10% glycerol, 0.2 mM EDTA, 1 mM DTT (dithiothreitol), 0.4 mM

PMSF, and 1 µg/ml leupeptin). The lysates were incubated on ice for 15 min and then

centrifuged at 2000 rpm for 5 min. The pellets were washed once in 20 mM HEPES (pH7.9)

containing 20% glycerol, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 1 µg/ml leupeptin.

The pellets were resuspended in 200 µl wash buffer containing 400 mM NaCl and incubated

on ice for 45 min. Finally, suspensions were centrifuged (13,000 rpm, 15 min, 4℃) and

supernatants were collected. Protein concentration was estimated according to the Bradford

method (Stoscheck, 1990), and the aliquots of the supernatants were stored at -70℃.

Sequences of double stranded consensus oligonuclotides for NF-κB (Promega, Madison, WI,

USA) used in gel shift reactions were as follows 5’-AGT TGA GGG GAC TTT CCC AGG

C-3’. Probe labeling was carried out as specified by the manufacturer with [γ-32P] ATP

(3,000 Ci/mmol; 10 mCi/ml) (Amersham Life Sciences, Arlington Heights, IL, USA).

Specificity studies were performed with a 50-fold molar excess of unlabeled oligonucleotide

added to the reaction mixtures prior to the addition of radiolabeled oligonucleotides.

Reaction mixtures were analyzed on 5% nondenatured polyacrylamide gels with 0.5x TBE

(tris-borate-EDTA) (89 mM Tris-HCl [pH 8.0], 89 mM boric acid, 2 mM EDTA) as the

running buffer. The gels were electrophoresed at 100 V for 3 h, dried in gel dryer, and

subjected to autoradiographic exposure for 12 to 48 h.

Reverse transcription polymerase chain reaction

HT-29 cells were used for reverse transcription polymerase chain reaction (RT-PCR)

analysis to examine IL-8 mRNA expression levels HT-29 cells samples during echinomycin

３１

treatment. These samples were snap-frozen in liquid nitrogen and stored at -80℃ until use.

For RNA extraction, the HT-29 cells were treated with total RNA isolation reagent, TRIzol

reagent (Gibco BRL, Hercules, CA, USA), as specified by the manufacturer. After isolation,

total RNA was reverse transcriptase (Gibco BRL, Hercules, CA, USA) following

measurement of the total RNA concentration, and agarose gel electrophoresis was performed.

PCR was performed with gene-specific primer sets for IL-8 and GAPDH genes. PCR for IL-

8 gene were subjected to 30 cycles of denaturation (94℃ for 1 min), annealing (65℃ for 1

min), and extension (72℃ for 2 min). PCR for GAPDH gene was subjected to 25 cycles of

denaturation (94℃ for 30 sec), annealing (60℃ for 30 sec), and extension (72℃ for 1

min). The primer sequences and PCR product sizes were as follows: for IL-8, 5'-ATG ACT

TCC AAG CTG GCC GTG GCT-3' (sense) and 5'-TCT CAG CCC TCT TCA AAA ACT

TCT C-3' (anti-sense), 292 bp; for GAPDH, 5'-ACC ACA GTC CAT GCA TCA C-3' (sense)

and 5'-TCC ACC ACC CTG TTG CTG TA-3' (anti-sense), 451 bp. Amplification was

carried out with a thermal cycler (model 480, Perkin-Elmer Cetus). 10 µl of each PCR

product was used for electrophosesis in a 1.5% agarose gel and visualized using ethidium

bromide staining.

Enzyme-linked immunosorbent assay

In order to quantify specific cytokine concentrations in the culture supernatants,

commercial available enzyme-linked immunosorbent assay (ELISA) kits were used. Culture

supernatants was harvested after echinomycin treatment and frozen at -70℃ until use. The

amounts of released human IL-8 proteins were analyzed using ELISA-kits purchased from

R&D Systems (Morrisville, NC, USA). All assays were performed in accordance to

３２

manufacturer's specifications.

Quantitative analysis of apoptosis by flow cytometry

Cells were harvested, washed with PBS, and resuspended in a binding buffer (10 mM

HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After 15 min of incubation with Annexin

V-fluorescein isothiocyanate (Sigma, St. Louis, MO, USA) and propidium iodide (PI)

(Pharmingen, San Diego, CA, USA) at room temperature, the fluorescence emitted by cells

(10,000 cells/sample) was analyzed on a flow cytometer (Becton-Dickinson, Franklin Lakes,

NJ, USA).

Statistical evaluation

All experiments were performed at least three times. Results are presented as means ± SD.

Data were analyzed by the Student’s t-test.

３３

3. Results

Induction of nuclear translocation of NF-κκκκB by echinomycin in HT-29 cells

A major presumption in this study was that echinomycin-initiated apoptosis might be NF-

κB dependent. Specifically, nuclear apoptotic signaling elicited by DNA bis-intercalators

has been poorly documented. In initial experiment to delineate this, time course of nuclear

localization of NF-κB upon echinomycin treatment was determined in HT-29 cells. HT-29

cells were treated with 2 µg/ml echinomycin for over a period of 48 h. Nuclear extracts were

collected, and electrophoretic mobility shift assays (EMSAs) were performed with a 32P-

labeled double-stranded oligonucleotide probe containing the NF-κB binding element. As

shown in Figure II-1A, echinomycin treatment finally formed DNA-protein complexes; NF-

κB activation was incremental in a time-dependent manner. Competition reactions showed

that inducible DNA-protein complexes detected following echinomycin treatment were

specifically NF-κB complexes. In the presence of an excess unlabeled NF-κB specific probe,

echinomycin-induced DNA-protein complex formation was inhibited (Figure II-1A). In

addition, HT-29 cells were transiently transfected with luciferase reporter plasmids

containing, pGL3-NF -κB promoter NF-κB binding elements. Twenty-four hours after

transfection, cells were lysed and assessed for luciferase activity. This study was examined

NF-κB promoter activity whether echinomycin treatment influences NF-κB activation in

HT-29 cells. Herein, echinomycin induced the binding activity of NF-κB promoter (Figure

II-1B), suggesting that echinomycin renders NF-κB bindable complexes to translocate into

nucleus of HT-29 cells.

３４

Cold NF-kBCompetitor

N 1 12 24 48 1 12 24 48 (h)

A.

0

10

20

30

40

50

N 1h 6h 12h 24h

Luc

ifer

ase

Act

ivit

y(f

old)

B.

Figure II-1. NF-κB activation was induced in echinomycin-treated HT-29 cells. (A) Nuclear

extracts were prepared from untreated cells (N), or cells treated with 2 µg/ml echinomycin.

Controls included EMSAs reactions with 50-fold excess cold NF-κB competitor. (B) HT-29

cells were transfected with a NF-κB luciferase reporter plasmid (0.5 µg) for 48 h. After

treatment with the echinomycin for 1, 6, 12, and 24 h, luciferase activity was determined.

The diagram is depicted as fold induction at the basal level. Each column represents an

average (± S.E.) of three individual experiments.

３５

Echinomycin induces the translocation of p50-p65 heterodimeric subunits of NF-κκκκB

NF-κB probe was then added, and EMSAs were performed. A supershift was seen in

echinomycin-treated nuclear extracts, primarily in the presence of antibodies specific for

p50 and p65 subunits. This supershifting strongly suggested the translocated NF-κB

complexes were p50 and p65 heterodimer. Since the transcription factor NF-κB consists of a

variety of homo- and heterodimers, supershift analysis was carried out by incubation of

nuclear extracts with antibodies raised against p50 or p65 NF-κB subunits prior to EMSAs

(Figure II-2). Both antibodies were clearly reacted to supershifted nuclear extracts. This

reaction verifies that echinomycin induces the translocation of p50-p65 heterodimeric

subunits of NF-κB.

３６

1 12 24 1 12 24 (h)

Anti-p50Antibody

Anti-p65Antibody

Figure II-2. Echinomycin induces the translocation of p50-p65 heterodimeric subunits of

NF-κB. Nuclear extracts were prepared from treated cells with 2 µg/ml echinomycin.

Polyclonal antibodies (0.5 µg/ml) against the p50 or p65 subunits of NF-κB were included

in the EMSAs reactions. Supershifted complexes are indicated by the arrow.

３７

Expression profiles of IκκκκB and p-IκκκκB in HT-29 cells treated with echinomycin

Since the expression of IκB and p-IκB precedes the appearance of NF-κB DNA binding

activity, the cytoplasmic IκB level was examined whether IκB degradation correlates with

NF-κB nuclear translocation by echinomycin treatment in HT-29 cells. The presence of IκB

and p-IκB in the whole cell lysate was analyzed by Western blots. Levels of IκB were

detected at initial echinomycin treatment, and thereafter decreased, faintly seen after a 6 h

treatment. In contrast, p-IκB levels were clearly detected throughout 6 to 24 h of

echinomycin treatment, albeit initially fainted (Figure II-3). These results show that IκB

degradation precede the appearance of NF-κB binding activity at echinomycin treatment.

Viewed together, these coupled data suggest that echinomycin induces prolonged activation

of NF-κB in HT-29 cells by triggering simultaneous phosphorylation and degradation of the

nuclear IκB.

３８

IkB

p-IkB

Actin

N 1 6 12 24 (h)

Figure II-3. Degradation of IκB in echinomycin treated cells. At the indicated time points,

cells were lysed and subjected to immunoblot analysis using anti-IκB antibody and anti-

phospho-IκB antibody. Actin is shown as the loading control. Similar results were obtained

in three independent experiments.

３９

Echinomycin treatment triggers IL-8 production in HT-29 cells

NF-κB might control the transcriptional regulation of IL-8 expression (Mukaida et al.,

1994). In experiment to clarify the role of NF-κB on IL-8 expression in echinomycin-

mediated apoptosis of HT-29 cells, it was examined if echinomycin-initiated NF-κB

activation in HT-29 cells would induce IL-8 production. It was found that echinomycin

initially upregulates IL-8 expression followed by downregulation of the surged IL-8

expression (Figure II-4A). In order to confirm the IL-8 suppression following echinomycin

treatment, the amount of IL-8 from supernatants of HT-29 cells were quantitated by ELISA:

In accordance with RT-PCR results, IL-8 production was increased up to maximal level post

1 h echinmycin treatment, and decreased afterward, until post 24 h treatment (Figure II-4B).

These data show that echinomycin treatment triggers IL-8 production in HT-29 cells.

４０

IL-8

N 1 6 12 24 (h)

A.

GAPDH

0

1

2

3

4

5

6

N 1 6 12 24

IL-8

(ng

/ml)

B.

(h)

Figure II-4. Echinomycin inhibited the IL-8 production in HT-29 cells. Cells were treated

with 2 µg/ml echinomycin for the indicated period of time. (A) Effect of echinomycin on

expression on expression mRNA expression of IL-8 was determined by RT-PCR. (B) At the

indicated time points, IL-8 production was determined using ELISA kit.

４１

Proteasome inhibitor blocks NF-κκκκB activation, consequently upregulating IL-8

expression in echinomycin-treated HT-29 cells

Still, whether NF-κB directly affects IL-8 expression at echinomycin treatment in HT-29

cells was unclear. To the end, EMSAs was performed to determine the NF-κB DNA binding

activity using proteosome inhibitor, MG-132. While NF-κB binding activity was detected in

HT-29 cells after 24 h for echinomycin treatment, pretreatment with MG-132 inhibited the

echinomycin-induced NF-κB binding activity (Figure II-5A). In parallel, pretreatment with

MG-132 sustained the level of IL-8 release throughout 6 to 24 hr of echinomycin treatment,

albeit initially fainted (Figure II-5B). In summary, proteasome inhibitor blocks NF-κB

activation, consequently upregulating IL-8 expression in echinomycin-treated HT-29 cells.

These coupled data suggest that IL-8 production is regulated by NF-κB activation via

proteosome pathway.

４２

Echinomycin - + +MG132 + - +

A.

B.

N 1 6 12 24 (h)

MG132+Echinomycin

IL-8

GAPDH

Figure II-5. Apoptotic cell death inhibition by proteasome inhibitor in echinomycin-treated

cells. (A) After the echinomycin (2 µg/ml)-treatment for 24 h in the presence of proteasome

inhibitor MG-132 (50 µM) or without MG-132 , nuclear extracts were subjected to EMSAs.

(B) After the echinomycin (2 µg/ml)-treatment for 24 h in the presence of MG-132 (50 µM),

RNA extracts were subjected to RT-PCR analysis. GAPDH mRNA expression was used as

control. Similar results were obtained in three independent experiments.

４３

IL-8 production at echinomycin treatment in HT-29 cells occurs via both caspase-3 and

NF-κκκκB dependent signal pathway

Using a specific inhibitor approach, a pretreatment of HT-29 cells with NF-κB inhibitor

(PDTC), broad caspase inhibitor (Boc-D-FMK) or caspase-3 specific inhibitor (Z-DEVD-

FMK) pathways, this result discretely was explored which pathway in echinomycin-initiated

apoptosis would be involved in IL-8 production. As seen in RT-PCR (Figure II-6A) and

ELISA (Figure II-6B), it was clear that the IL-8 production at echinimycin treatment is

regulated by both caspase-3 and NF-κB signaling.

４４

N PDTC BOC DEVD

IL-8

GAPDH

A.Echinomycin

B.

0

1

2

3

4

5

6

7

N E PDTC BOC DEVD

IL-8

(ng

/ml)

Figure II-6. Regulation of IL-8 production was caspase-3 and NF-κB dependent by

echinomycin. Echinomycin (2 µg/ml)-induced IL-8 production was regulated by

pretreatment with either inhibitor of the caspase pathway, Z-DEVD-FMK (50 µM) or Boc-

D-FMK (50 µM), and inhibitor NF-κB signaling PDTC (100 µM) using RT-PCR(A) and

ELISA kit (B). GAPDH mRNA expression was used as control. Similar results were

obtained in three independent experiments.

４５

Echinomycin-induced apoptosis in HT-29 cells occurs via only NF-κκκκB activation

independent of caspase-3 activation

To confirm whether two different pathway (NF-κB and caspase) would be coupled or

interdependent, apoptosis assay was done in the presence of NF-κB inhibitors (PDTC),

board caspase inhibitor (Boc-D-FMK) and caspase-3 specific inhibitor (Z-DEVD-FMK).

Only both NF-κB inhibitors (PDTC) and caspase-3 specific inhibitor (Z-DEVD-FMK)

significantly attenuated echinomycin-initiated apoptosis of HT-29 cells (Figure II-7). These

data confirm that the NF-κB activation triggers the apoptotic effects of echinomycin on HT-

29 cells. Pretreatment of HT-29 cells with NF-κB inhibitors (PDTC) rarely affected

echinomycin-induced procaspase-3 activation (Figure II-8). Collectively, this data indicate

that echinomycin-induced apoptosis in HT-29 cells occurs via only NF-κB activation

independent of caspase-3 activation, finally modulating the resultant-linked key chemokine

IL-8 expression.

By combining these data (Park et al., 2006) a hypothetical diagram of the signal

transduction cascade which mediates echinomycin-induced mitochondirial and caspase-3

signaling pathway in apoptosis and regulation of IL-8 production and NF-κB activation in

HT-29 cells was made (Figure II-9).

４６

0.0

10.0

20.0

30.0

40.0

50.0

60.0

Control E PDTC BOC DEVD

Apo

ptot

ic c

ells

(%

)

Figure II-7. Involvement of NF-κB and caspase pathways in the regulation of apoptosis by

echinomycin in HT-29 cells. HT-29 cells treated with 2 µg/ml echinomycin, HT-29 cells

treated with Z-DEVD-FMK (50 µM) or PDTC (100 µM) plus 2 µg/ml echinomycin for 24 h,

after which cells were harvested. Cells were stained with Annexin V-FITC and PI subjected

to flow cytometry analysis. Results are expressed as means ±SD and are representative of

three independent experiments (P<0.05).

４７

N DEVD PDTC

procaspase 3

Actin

Echinomycin

Figure II-8. Relationship of NF-κB activation and caspase3 signalig pathway in HT-29 by

echinomycin. HT-29 cells treated with 2 µg/ml echinomycin, HT-29 cells treated with Z-

DEVD-FMK (50 µM) or PDTC (100 µM) plus 2 µg/ml echinomycin for 24 h, after which

cells were harvested. Protein activities were assessed by Western blotting. Hydrodization

with anti-actin served as a loading control.

４８

Bcl-2

Cytochrome C

ERK MAP kinase

Caspase-3

Caspase-1

?

?Caspase-8

NF-kB

Cell deathIL-8

Echinomycin

IkB, p-IkB,

?

Figure II-9. Schematic of the signaling pathway by echinomycin treatment in HT-29 cells.

Dashed lines indicate the signaling not yet confirmed in HT-29 cells

４９

4. Discussion

This study indicates that echinomycin-induced apoptosis occurs via NF-κB activation,

consequently modulating the resultant-linked key chemokine IL-8 release kinetics.

Evidences that echinomycin-induced apoptosis occurs via NF-κB activation have been

demonstrated in three complementary way ; 1) EMSAs using NF-κB specific probe and

transfection with luciferase reporter plasmids containing, pGL3-NF -κB promoter NF-κB

bonding elements showed that echinomycin-induced DNA-protein complex formation was

inhibited (Figure II-1A) and echinomycin induced the binding activity of NF-κB promoter

(Figure II-1B). 2) EMSAs in the presence of antibodies specific for p50 and p65 subunits

indicated that echinomycin induces the translocation of p50-p65 heterodimeric subunits of

NF-κB. 3) Third proof stems from the expression data of IκB and p-I κB in HT-29 cells

treated with echinomycin.

Prior to this proof, this report has shown that echinomycin-initiated apoptosis in HT-29

cell occurs via Bcl2-dependent as well as mitochondrial dependent MAP kinase-caspase

pathway. In that context of convergeable, downstream signaling, it was hypothesized that

echinomycin-initiated apoptosis might be NF-κB dependent. Specifically, intracellular

apoptotic signaling elicited by DNA bis-intercalators has been poorly documented.

Compelling data has demonstrated that DSB causes DNA adducts and DNA intercalation in

nonspecific fashion, thereby leading to apoptosis of cancer cells. Such a DSB-initiated

apoptosis would be implicated with intracellular signaling a pathway such as MAP kinase,

caspase, PI-3K and NF-κB (Golding et al., 2007). However, how echinomycin, a specific

CG nucleotide-targeted bis-intercalating agent, relays apoptotic signal from cell surface-

cytoplasm signal to nucleus interim has been unresolved. To address this concern, EMSA

５０

coupled with proteosome inhibitor MG-132 verified NF-κB dependency in echinomycin-

initiated apoptosis (Figure II-5). Such NF-κB dependency and NF-κB activation revealed in

echinomycin-initiated apoptosis are not generalizable as a model converging on apoptotic

mechanism of DNA intercalators. In fact, some DSB inducer armed with nonspecific DNA

intercalating ability such as doxorubicin would directly activate NF-κB pathway, eventually

leading to apoptosis (Janssens et al., 2006; Strozyk et al., 2006; Janssens et al., 2005).

However, it was widely accepted that NF-κB activation is an anti-apoptotic signal. In this

point, inhibition of NF-κB combined with anticancer chemotherapy may strongly synergize

the cytotoxic effect of anticancer chemotherapy.

As a nuclear transcription factor, NF-κB target genes including several anti-apoptotic

proteins, e.g., the inhibitor of apoptosis protein (IAP) family of caspase inhibitory proteins,

TNFR-associated factor1 (TRAF1) and TNFR-associated factor2 (TRAF2) (which is

suppose to repress caspase-8 activation), Bfl1/A1, Bcl-xL, FLIP, and inducible nitric oxide

synthetics. Interestingly, NF-κB also control promoter activation of certain pro-apoptotic

factors, such as CD95 (Fas) and CD95 ligand (FasL) and TRAIL receptors (TRAIL-R1 and -

R2) (Ivanov et al., 2006; Franco et al., 2001). Given this, whether NF-κB targets pro- or

anti-apoptotic genes depend on the stimulus-specific signaling a pathway activated.

This study noted definite incremental NF-κB activation up to 24 h after echinomycin

treatment. Additionally, translocation of p50-p65 heterodimer into a nucleus as well as IκB

degradation are decisive proof of NF-κB dependency in echinomycin-initiated apoptosis.

Both p50 and p65 heterdimeric translocation for induction of echinomycin-initiated

apoptosis are another new finding. However, whether this NF-κB pathway would be coupled

with Bcl-2-mitochondrial-MAP kinase-caspase pathway is unclear. As corollary, this NF-κB

activation would affect cytokine and chemokine regulation associated with transcription

factors (Ashkenazi et al., 1998; Beg et al., 1993). For example, when human colonic HT-29

５１

cell monolayer were treated with bacterial DNA (Akhtar et al., 2003), inflammatory

cytokine (Sougioultzis et al., 2006) or camptothecin (DNA topoisomerase inhibitor)

(Ikegami et al., 2006, Boland et al., 2000), IL-8 secretion and NF-κB activation were

inducible. In an intestinal epithelial cell such as HT-29, NF-κB can be a central regulator of

chemokine gene expression (Sougioultzis et al., 2006; Jijon et al., 2005; Kim et al., 2005).

This chemokine may play some role in survival or death of cancer cells. Of chemokines,

interleukin (IL)-8 has been known to be intimately linked with NF-κB pathway in HT-29

cells. Still, the connective link between NF-κB and IL-8 expression in echinomycin-

mediated apoptosis of HT-29 cells was elusive. In experiment to clarify these issues, it was

found that echinomycin initially upregulates IL-8 expression followed by downregulation of

the surged IL-8 expression. Surge of IL-8 expression 1 h after echinomycin treatment might

be explained by inherent potent NF-κB activation elicited by echinomycin. Time dependent

decrement of IL-8 expression might be explained by proteasomal regulation. This is clearly

evidenced by reversal of decrement IL-8 expression at pretreatment of proteasome inhibitor

MG132 on HT-29 cells (Figure II-5).

Cytokine or chemokine can trigger two different pathways with opposite effects

simultaneously, the apoptotic pathway mediated by caspase and NF-κB (Ashkenazi et al.,

1998). Figure II-4-6 clearly indicated that IL-8 expression of echinomycin on HT-29 cells is

regulated by the activation of caspase-3 and NF-κB at pre and post-transcriptional level. In

contrast, apoptosis of HT-29 cell was attenuated by a caspase-3 inhibitor (Z-DEVD-FMK)

and NF-κB specific inhibitor (PDTC). Given this, IL-8 expression is conversely linked with

apoptosis triggered by dependent apoptotic pathway such as mitochondria-caspase or NF-κB.

In line, pretreatment of HT-29 cells with NF-κB inhibitors (PDTC) caused no procaspase-3

cleavage (Figure II-8). Collectively, these data indicate that echinomycin-induced apoptosis

in HT-29 cells occurs via only NF-κB activation independent of caspase-3 activation, finally

５２

modulating the resultant-linked key chemokine IL-8 expression.

This is first report that echinomycin-induced apoptosis is NF-κB-dependant and directly

related to NF-κB activation. Although the importance of the echinomycin-induced apoptosis

in vivo is not clear at present, an improved understanding of this pathway triggered by

echinomycin could ultimately lead to identification of new therapeutic targets for an aberrant

signal transducing disease such as colon cancer.

５３

Conclusions

This study demonstrates the core mechanism of Bcl-2 dependent and NF-κB dependent

signaling pathways induced by echinomycin treatment in HT-29 human colon cancer cells.

Eight cored results are summarized in the below.

1. Echinomycin induces apoptosis in HT-29 human colon cancer cells.

2. Bcl-2 family is involved of echinomycin-induced apoptosis, resulting in cytochrome c

release.

3. Echinomycin induces apoptotic cell death in HT-29 human colon cancer cells through the

Bcl-2, but not Bax.

4. The Bcl-2-mediated control of apoptotic cell death via caspase-3 may play a role in

echinomycin-treated HT-29 cells.

5. Activation of NF-κB essentially is triggered by echinomycin in HT-29 cells.

6. Echinomycin treatment triggers IL-8 production in HT-29 cells

7. Echinomycin regulates significantly the levels of IL-8 production of HT-29 cells through

by NF-κB and caspase-3 dependent.

５４

8. Echinomycin-induced apoptosis in HT-29 human colon cancer cells occurs via only NF-

κB activation independent of caspase-3 activation

５５

References

Akhtar, M., Watson, J. L., Nazli, A., and McKay, D. M. (2003). Bacterial DNA evokes

epithelial IL-8 production by a MAPK-dependent, NF-kappaB-independent pathway. The

FASEB Journal, 17, 1319-1321.

Ashkenazi, A., and Dixit, V. M. (1998). Death receptors: signaling and modulation. Science,

281, 1305-1308.

Baeuerle, P. A., and Baltimore, D. (1996). NF-kappa B: ten years after. Cell, 87, 13-20.

Baraldi, P. G., Balboni, G., Pavani, M. G., Spalluto, G., Tabrizi, M. A., Clercq, E. D.,

Balzarini, J., Bando, T., Sugiyama, H., and Romagnoli, R. (2001). Design, synthesis, DNA

binding, and biological evaluation of water-soluble hybrid molecules containing two

pyrazole analogues of the alkylating cyclopropylpyrroloindole (CPI) subunit of the

antitumor agent CC-1065 and polypyrrole minor groove binders. Journal of Medicinal

Chemistry, 44, 2536-2543.

Barkett, M., and Gilmore, T. D. (1999). Control of apoptosis by Rel/NF-kappaB

transcription factors. Oncogene, 18, 6910-6924.

Beg, A. A., and Baldwin, A. S. (1993). The IκB proteins: multifunctional regulators of rel/

NF-κB transcription factors. Genes and Development, 7, 2064–2070.

Boland, M. P., Fitzgerald, K. A., and O'Neill, L. A. (2000). Topoisomerase II is required for

５６

mitoxantrone to signal nuclear factor kappa B activation in HL60 cells. The Journal of

Biological Chemistry, 275, 25231-25238.

Capranico, G., Riva, A., Tinelli, S., Dasdia, T., and Zunino, F. (1987). Markedly reduced

levels of anthracycline-induced DNA strand breaks in resistant P388 leukemia cells and

isolated nuclei. Cancer Research, 47, 3752-3756.

Chen, J. G., and Horwitz, S. B. (2002). Differential mitotic responses to microtubule-

stabilizing and -destabilizing drugs. Cancer Research, 62, 1935-1938.

D'Agostini, F., Izzotti, A., Balansky, R. M., Bennicelli, C., and De Flora, S. (2005).

Modulation of apoptosis by cancer chemopreventive agents. Mutation Research, 591, 173-

186.

Franco, A. V., Zhang, X. D., Van Berkel, E., Sanders, J. E., Zhang, X. Y., Thomas, W. D.,

Nguyen, T., and Hersey, P. (2001). The role of NF-kappa B in TNF-related apoptosis-

inducing ligand (TRAIL)-induced apoptosis of melanoma cells. Journal of Immunology, 166,

5337-5345.

Geroni, C., Marchini, S., Cozzi, P., Galliera, E., Ragg, E., Colombo, T., Battaglia, R.,

Howard, M., D'Incalci, M., and Broggini, M. (2002). Brostallicin, a novel anticancer agent

whose activity is enhanced upon binding to glutathione. Cancer Research, 62, 2332-2336.

Golding, S. E., Rosenberg, E., Neill, S., Dent, P., Povirk, L. F., and Valerie, K. (2007).

Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated,

５７

homologous recombination repair, and the DNA damage response. Cancer Research, 67,

1046-1053.

Ikegami, T., Matsuzaki, Y., Al Rashid, M., Ceryak, S., Zhang, Y., and Bouscarel, B. (2006).

Enhancement of DNA topoisomerase I inhibitor-induced apoptosis by ursodeoxycholic acid.

Molecular Cancer Therapeutics, 5, 68-79.

Ivanov, V. N., and Hei, T. K. (2006). Sodium arsenite accelerates TRAIL-mediated apoptosis

in melanoma cells through upregulation of TRAIL-R1/R2 surface levels and downregulation

of cFLIP expression. Experimental Cell Research, 312, 4120-4138.

Jacob, S., Miquel, C., Sarasin, A., and Praz, F. (2005). Effects of camptothecin on double-

strand break repair by non-homologous end-joining in DNA mismatch repair-deficient

human colorectal cancer cell lines. Nucleic Acids Research, 33, 106-113.

Janssens, S., Tinel, A., Lippens, S., and Tschopp, J. (2005). PIDD mediates NF-kappaB

activation in response to DNA damage. Cell, 123, 1079-1092.

Janssens, S., and Tschopp, J. (2006) Signals from within: the DNA-damage-induced NF-

kappaB response. Cell Death and Differentiation, 13, 773-784.

Jayasuriya, H., Zink, D. L., Polishook, J. D., Bills, G. F., Dombrowski, A. W., Genilloud, O.,

Pelaez, F. F., Herranz, L., Quamina, D., Lingham, R. B., Danzeizen, R., Graham, P. L.,

Tomassini, J. E., and Singh, S. B. (2005). Identification of diverse microbial metabolites as

potent inhibitors of HIV-1 Tat transactivation. Chemistry & Biodiversity, 2, 112-122.

５８

Jijon, H. B., Madsen, K. L., Walker, J. W., Allard, B., and Jobin, C. (2005). Serum amyloid

A activates NF-kappaB and proinflammatory gene expression in human and murine

intestinal epithelial cells. European Journal of Immunology, 35, 718-726.

July, L. V., Beraldi, E., So, A., Fazli, L., Evans, K., English, J. C., and Gleave, M. E. (2004).

Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma

cells both in vitro and in vivo. Molecular Cancer Therapeutics, 3, 223-232.

Kim, J. A., Kim, D. K., Kang, O. H., Choi, Y. A., Park, H. J., Choi, S. C, Kim, T. H., Yun, K.

J., Nah, Y. H., and Lee, Y. M. (2005). Inhibitory effect of luteolin on TNF-alpha-induced IL-

8 production in human colon epithelial cells. International Immunopharmacology, 5, 209-

217.

Kim, J. B., Lee, G. S., Kim, Y. B., Kim, S. K., and Kim, Y. H. (2004). In vitro antibacterial

activity of echinomycin and a novel analogue, YK2000, against vancomycin-resistant

enterococci. International Journal of Antimicrobial Agents, 24, 613-615.

Kim, Y. B., Kim, Y. H., Park, J. Y., and Kim, S. K. (2004). Synthesis and biological activity

of new quinoxaline antibiotics of echinomycin analogues. Bioorganic and Medicinal

Chemistry Letters, 14, 541-544.

Kong, D., Park, E. J., Stephen, A.G., Calvani, M., Cardellina, J. H., Monks, A., Fisher, R. J.,

Shoemaker, R. H., and Melillo, G. (2005). Echinomycin, a small-molecule inhibitor of

hypoxia-inducible factor-1 DNA-binding activity. Cancer Research, 65, 9047-9055.

５９

Lee, Y. K., Park, H. J., Moon, T. H., Lee, Y. D., Yun, H. J., and Byun, Y. (2007). The short-

term effects on restenosis and thrombosis of echinomycin-eluting stents topcoated with a

hydrophobic heparin-containing polymer. Biomaterials, 28, 1523-1530.

Liao, X., Liu, J. M., Du, L., Tang, A., Shang, Y., Wang, S. Q., Chen, L. Y., and Chen, Q.

(2006). Nitric oxide signaling in stretch-induced apoptosis of neonatal rat cardiomyocytes.

The FASEB Journal, 20, 1883-1885.

Mahyar-Roemer, M., Kohler, H., and Roemer, K. (2002). Role of Bax in resveratrol-induced

apoptosis of colorectal carcinoma cells. BMC Cancer, 17, 27.

Marquis, J. C., Hillier, S. M., Dinaut, A. N., Rodrigues, D., Mitra, K., Essigmann, J. M., and

Croy, R. G. (2005). Disruption of gene expression and induction of apoptosis in prostate

cancer cells by a DNA-damaging agent tethered to an androgen receptor ligand. Chemistry

&Biology, 12, 779-787.

Mayo, M. W., and Baldwin, A. S. (2000). The transcription factor NF-kappaB: control of

oncogenesis and cancer therapy resistance. Biochimica et Biophysica acta, 270, 55-62.

Mormina, M. E., Thakur, S., Molleman, A., Whelan, C. J., and Baydoun, A. R. (2006).

Cannabinoid signalling in TNF-alpha induced IL-8 release. European Journal of

Pharmacology, 540, 183-190.

Mukaida, N., Okamoto, S., Ishikawa, Y., and Matsushima, K. (1994). Molecular mechanism

of interleukin-8 gene expression. Journal of Leukocyte Biology, 56, 554-558.

６０

Nuessler, V., Stotzer, O., Gullis, E., Pelka-Fleischer, R., Pogrebniak, A., Gieseler, F., and

Wilmanns, W. (1999). Bcl-2, bax and bcl-xL expression in human sensitive and resistant

leukemia cell lines. Leukemia, 13, 1864-1872.

Panaretakis, T., Pokrovskaja, K., Shoshan, M. C., and Grander, D. (2003). Interferon-alpha-

induced apoptosis in U266 cells is associated with activation of the proapoptotic Bcl-2

family members Bak and Bax. Oncogene, 22, 4543-4556.

Park, J. Y., Park, S. J., Shim, K. Y., Lee, K. J., Kim, Y. B., Kim, Y. H., and Kim, S. K. (2004).

Echinomycin and a novel analogue induce apoptosis of HT-29 cells via the activation of

MAP kinases pathway. Pharmacological Research, 50, 201-207.

Park, J. Y., Ryang, Y. S., Shim, K. Y., Lee, J. I., Kim, H. S., Kim, Y. H., and Kim, S. K.

(2006). Molecular signaling cascade in DNA bisintercalator, echinomycin-induced apoptosis

of HT-29 cells: evidence of the apoptotic process via activation of the cytochrome c-ERK-

caspase-3 pathway. The International Journal of Biochemistry & Cell Biology, 38, 244-254.

Roaten, J. B., Kazanietz, M. G., Caloca, M. J., Bertics, P. J., Lothstein, L., Parrill, A. L.,

Israel, M., and Sweatman, T. W. (2002). Interaction of the novel anthracycline antitumor

agent N-benzyladriamycin-14-valerate with the C1-regulatory domain of protein kinase C:

structural requirements, isoform specificity, and correlation with drug cytotoxicity.

Molecular Cancer Therapeutics, 1, 483-492.

Reed, J. C. (1995). Regulation of apoptosis by bcl-2 family proteins and its role in cancer

and chemoresistance. Current Opinion in Oncology, 7, 541-546.

６１

Ryu, C. K., Jeong, H. J., Lee, S. K., Kang, H. Y., Ko, K. M., Sun, Y. J., Song, E. H., Hur, Y.

H., and Lee, C. O. (2000). Modulation of Nad(P)H:quinone oxidoreductase (NQO1) activity

mediated by 5-arylamino-2-methyl-4,7-dioxobenzothiazoles and their cytotoxic potential.

Archives of pharmacal Research, 23, 554-558.

Shankar, S., and Srivastava, R. K. (2007). Involvement of Bcl-2 family members,

phosphatidylinositol 3'-kinase/AKT and mitochondrial p53 in curcumin

(diferulolylmethane)-induced apoptosis in prostate cancer. International Journal of

Oncology, 30, 905-918.

Sougioultzis, S., Simeonidis, S., Bhaskar, K. R., Chen, X., Anton, P. M., Keates, S.,

Pothoulakis, C., Kelly, C. P. (2006). Saccharomyces boulardii produces a soluble anti-

inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression. Biochemical

and Biophysical Research Communication, 343, 69-76.

Spierings, D., McStay, G., Saleh, M., Bender, C., Chipuk, J., and Maurer, U. (2005).

Connected to death: the (unexpurgated) mitochondrial pathway of apoptosis. Science, 310,

66-67.

Stoscheck, C. M. (1990). Quantitation of Protein. Methods in Enzymology, 182, 50-69.

Strozyk, E., Pöppelmann, B., Schwarz, T., and Kulms, D. (2006). Differential effects of NF-

kappaB on apoptosis induced by DNA-damaging agents: the type of DNA damage

determines the final outcome. Oncogene, 25, 6239-6251.

６２

Takahashi, M., Saito, H., Okuyama, T., Miyashita, T., Kosuga, M., Sumisa, F., Yamada, M.,

Ebinuma, H., and Ishii, H. (1999). Overexpression of Bcl-2 protects human hepatoma cells

from Fas-antibody-mediated apoptosis. Journal of Hepatology, 31, 315-322.

Walton, M. I., Whysong, D., O'Connor, P.M., Hockenbery, D., Korsmeyer, S. J., and Kohn,

K. W. (1993). Constitutive expression of human Bcl-2 modulates nitrogen mustard and

camptothecin induced apoptosis. Cancer research, 53, 1853-1861.

Zhou, J., Zhang, S., Ong, C. N., and Shen, H. M. (2006). Critical role of pro-apoptotic Bcl-2

family members in andrographolide-induced apoptosis in human cancer cells. Biochemical

Phamacology, 72, 132-144.

６３

국문요약국문요약국문요약국문요약

Echinomycin 을을을을 처리한처리한처리한처리한 대장암대장암대장암대장암 세포주에서세포주에서세포주에서세포주에서 세포고사세포고사세포고사세포고사 유도유도유도유도 시시시시

Bcl-2와와와와 NF-κκκκB 를를를를 경유하는경유하는경유하는경유하는 신호전달신호전달신호전달신호전달 규명규명규명규명

연세대학교 대학원

임상병리학과

박 주 연

본 연구는 DNA bis-intercalator의 세포고사기전을 규명하기 위한 연구의 일환으

로 두 가지 핵심 연구 주제로 구성되었다. 첫 번째 주제로 대표 화합물인

echinomycin을 HT-29 대장암세포주에 처리하여 세포사를 유발하고 관련 신호전달

과정 중 특히 Bcl-2 신호전달 경로를 규명하였으며, 두 번째 주제로 세포질에서

핵내의 세포고사 과정을 전달 증폭하는 NF-κB 신호전달 경로 및 그 의존성을

탐색하였다. 동시에 NF-κB 활성 변화에 따른 주 표적 케모카인인 IL-8 생성의

변화를 확인하였다. 첫 번째 실험에서 Bax 불활성 유전자를 세포 내로 이입 시

echinomycin에 의해 유도 되는 HT-29대장암세포주의 세포사에 변화가 없었다. 이

현상과 대조적으로 Bcl-2 과발현 유도 시 caspase-3 활성이 억제되었고 세포사도

현저히 감소하였다. 이는 가설인 caspase-3를 경유하는 Bcl-2의 활성신호가 HT-29

세포사와 직접 관련된 신호전달기전임을 규명하였다. 두 번째 실험에서 NF-κB

신호전달 경로 및 그 의존성을 확인하기 위해 동위원소와 항체를 이용한 gel

６４

shift 분석실험에서 NF-κB 활성의 증가와 NF-κB의 이질복합체인 p50과 p65가 핵

내로 이동함을 입증하였다. 이러한 NF-κB 활성의 변화는 세포질내의 IκB 활성

변화 및 인산화된 IκB 활성과 관련됨을 관찰하였다. Echinomycin에 의해 유도된

세포고사와 관련된 NF-κB 활성 변화는 IL-8 생성을 유발하였으며, 각각 caspase-3

활성 및 NF-κB 활성과 관계가 있었다. 그러나, caspase-3 및 NF-κB 활성 억제 실

험에서 이 두 경로는 연관 또는 상호의존적이 아님을 밝혔다. 이 두 가지 실험

을 종합하면 echinomycin 처리 시 HT-29에 유도되는 세포고사는 미토콘드리아와

관련된 Bcl-2 신호전달에 의해서 유도되며, 다양한 세포고사 신호들은 핵내 전사

인사인 NF-κB 의존성 경로로 전달되며 이 과정에서 케모카인인 IL-8 생성의 변

화는 세포고사의 지표인자로 추정된다. 본 연구의 의의는 미지의 DNA bis-

intercalator의 세포사멸기전의 가장 핵심주제에 접근하는 기반을 제공하며 이와

관련된 신호전달기전 이해 및 치료제 개발의 이론적 근거를 마련하였다.

---------------------------------------------------------------------------------------------------------------

핵심되는핵심되는핵심되는핵심되는 말말말말: Echinomycin, 세포사, 대장암세포주, Bcl-2, Capsase-3, NF-κB, 인터루

킨-8

６５

감사의감사의감사의감사의 글글글글

“자전거를 타고 달려와 돌아보면

왜 그리 길이 굽어있는지

분명 반듯하게만 달려왔는데......." 라는 영화 속의 대사가 있습니다.

시간이 빠르게 혹은 더디게 흘러 잠시 뒤를 돌아 볼 시간을 가지게 되었습니다.

그냥 흘러온 시간 같은데 지나고 보니 굽이굽이 굽은 길마다 작은 푯말을

하나씩 붙여 놓고 온 것 같습니다. 하나하나 그 푯말에 먼지를 닦아주며 다시

읽고 싶지만, 시간은 되돌릴 수 없기에 이렇게 지나온 시간을 더듬어 보면

소중하지 않은 날들이 없습니다. 때로는 시간이 빨리 지나갔으면 하며 지루하게

느끼고 가야 할 길을 몰라 서있을 때 그 굽은 길을 잘 돌아 갈 수 있게 하여준

건 그때 만났던 소중하고 고마운 인연이 있었기 때문이었을 것입니다. 휙

불어주는 시원한 바람같이, 길가에 피어있는 들꽃같이, 시원스레 흐르는

물소리처럼 말입니다. 현인들이 말한 “가장 소중한 것은 언제나 우리가 알지

못하는 사이에 가까이 있다” 라는 그 평범한 진리가 맞는 것 같습니다.

이 글을 쓰고 있는 오늘도 또 하나의 소중한 날로 지나가고 앞으로도 많은

날들이 지나갈 것입니다. 지나가고 다시 시작되고 한다는 것은 참으로 감사한

일입니다. 늘 감사하다는 생각을 하고 살수는 없겠지만, 그 마음만큼은 잊지

않고 간직하겠습니다.

마음에 꿈과 비전을 간직하면 행복에 익숙한 사람이 될 수 있다고 생각합니다.

행복은 결코 누군가에 의해서 얻어지는 것은 아니니까요.

이제 다시 멀리 보겠습니다.

６６

하나 하나의 푯말속에 기억되어있지만 제게는 너무 감사한 일들로 남아있는

많은 분들에게 감사의 마음을 전하며, 학위 과정을 잘 이끌어주신 연세대학교

임상병리학과 교수님들과 연세대학교 원주의과대학 미생물학교실 교수님들

그리고 도움을 주신 모든 분들께 감사의 마음을 전합니다. 마지막으로 묵묵히

저를 바라봐 주신 사랑하는 부모님께 감사의 마음을 전합니다.