NATURAL COMPOUNDS AND THEIR ROLE IN APOPTOTIC CELL SIGNALING PATHWAYS

Mechanism of β-Carotene-Induced Apoptosisof Gastric Cancer Cells: Involvement of

Ataxia-Telangiectasia-MutatedSung Hee Jang,a Joo Weon Lim,b and Hyeyoung Kima

aDepartment of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology,Yonsei University, Seoul, Korea

bInstitute of Gastroenterology, College of Medicine, Yonsei University, Seoul, Korea

β-Carotene acts as an antioxidant or a pro-oxidant depending on the concentrations thatcells are treated with. Oxidative DNA damage is related to apoptosis of various cells.Ataxia-telangiectasia-mutated (ATM), a sensor for DNA-damaging agents, activates avariety of effectors in multiple signaling pathways, such as DNA repair and apoptosis.In the present study, we investigated whether a high concentration of β-carotene inducesapoptosis of gastric adenocarcinoma (AGS) cells and whether ATM is involved in β-carotene-induced apoptosis of AGS cells. We found that β-carotene (100 μmol/L) inducedapoptosis (determined by cell viability), DNA fragmentation, and the protein levels ofp53 and Bcl-2 in AGS cells. ATM levels in the nucleus decreased from β-carotene in AGScells. β-Carotene-induced alterations, including an increase in DNA fragmentation andp53 levels and a decrease in nuclear ATM and cellular Bcl-2 levels, were inhibited in thecells transfected with full-length ATM cDNA compared to wild-type cells or the cellstransfected with control vector plasmid control DNA vector (pcDNA). In conclusion,β-carotene induces apoptosis by increasing apoptotic protein p53 and decreasing anti-apoptotic Bcl-2 as well as nuclear ATM in AGS cells. Nuclear loss of ATM may be theunderlying mechanism of β-carotene-induced apoptosis of gastric cancer cells.

Key words: β-carotene; ataxia-telangiectasia-mutated; apoptosis

Introduction

β-Carotene shows antioxidant and anti-inflammatory activities in various cells and tis-sues.1 β-Carotene effectively scavenges certainreactive oxygen species, especially peroxyl rad-ical and singlet oxygen in the cells exposedto oxidative stress. Antioxidant activity of β-carotene has been suggested for prevention ofinflammatory diseases, such as atherosclero-sis and rheumatoid arthritis.2–4 However, re-cent studies demonstrate that a high concen-tration of β-carotene behaves as a pro-oxidantby propagating free radical-induced reactions,

Address for correspondence: Hyeyoung Kim, Department of Food andNutrition, College of Human Ecology, Yonsei University, Seoul 120-749,Korea. Voice: 82-2-2123-3125; fax: 82-2-364-5781. kim626@yonsei.ac.kr

consuming endogenous antioxidants, and in-ducing oxidative DNA damage of the cells.5,6

β-Carotene induces apoptosis of colon cancercells and melanoma cancer cells and leukemiacells.5,7,8

Apoptosis, also called programmed celldeath, is an energy-driven process by which acell actively destroys itself in response to extra-cellular signals or developmental cues. Apop-tosis is characterized by cell shrinkage, chro-matin condensation, DNA fragmentation, andthe presence of apoptotic bodies. Bcl-2, Bax,and p53 could be the central executioners ofthe apoptotic pathway because they inducemost of the visible changes that characterizeapoptotic cell death. Bcl-2 is an intracellularsuppressor of apoptosis by heterodimerizingwith proapoptotic Bax. p53 controls the cellcycle and apoptosis in response to abnormal

Natural Compounds and Their Role in Apoptotic Cell Signaling Pathways: Ann. N.Y. Acad. Sci. 1171: 156–162 (2009).doi: 10.1111/j.1749-6632.2009.04711.x c© 2009 New York Academy of Sciences.

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Jang et al.: β-Carotene-Induced Apoptosis of Gastric Cancer Cells 157

proliferative signals and stress, including DNAdamage.8

DNA repair is a crucial physiological mech-anism maintaining genomic DNA integrity ineach nuclear cell. The ataxia-telangiectasia-mutated (ATM), a sensor for DNA-damagingagents, activates a variety of effectors in multi-ple signaling pathways, such as cell cycle check-points, DNA repair, and apoptosis. FollowingDNA damage, ATM undergoes autophospho-rylation at S1981, leading to the dissociation ofinactive complex and subsequent triggering ofa signaling cascade involving phosphorylationof several substrates, which in turn results in twocrucial responses to DNA damage: the activa-tion of cell cycle checkpoints and the initiationof DNA repair. DNA repair failure may resultin the activation of apoptosis. In other words,active p53 by ATM induces cell cycle arrestand the cell is then repaired by DNA repairprotein, such as ATM and Ku. However, con-tinuous cell cycle arrest by p53 induces apopto-sis. Moreover, ATM is specifically cleaved in thecells during the apoptotic process by a varietyof stimuli. ATM cleavage in vivo is dependent oncaspases, revealing that ATM is an efficient sub-strate for caspase-3 but not caspase-6 in vitro.9

In the present study, we purposed to in-vestigate whether a high concentration ofβ-carotene induces apoptosis of gastric adeno-carcinoma (AGS) cells and whether ATM is in-volved in β-carotene-induced apoptosis of AGScells, using wild-type cells or the transfectedcells with full-length ATM cDNA or plasmidcontrol DNA vector (pcDNA).

Materials and Methods

Cell Culture and β-Carotene Treatment

A human gastric cancer cell line, AGS, waspurchased from the American Type CultureCollection (ATCC CRL 1739, Rockville, MD)and cultured in RPMI 1640 medium supple-mented with 10% fetal bovine serum and an-tibiotics (100 U/mL penicillin and 100 μg/mLstreptomycin; all from GIBCO, Grand Island,

NY). β-Carotene and tetrahydrofuran (THF,inhibited with 250 mg/L butylated hydroxy-toluene) were purchased at Sigma (St. Louis,MO). β-Carotene was dissolved in tetrahydro-furan before each experiment, and all proce-dures using β-carotene were carried out in thedark.

Experimental Protocol

For determination of cell viability and DNAfragmentation, AGS cells were cultured in theabsence or presence of β-carotene (20, 50, and100 μmol/L) for 24 h. For Western blotting ofp53, Bcl-2, and ATM, the cells were culturedin the presence of β-carotene (100 μmol/L) forvarious time points (0, 4, 8, 12 h). To investi-gate the relation of ATM and the expressionof p53 and Bcl-2, AGS cells were transfectedwith control vector pcDNA or full-length ATMcDNA. After transfection of pcDNA or ATMcDNA, the cells were cultured with β-carotene(100 μmol/L) for 12 h (Western blotting ofp53, Bcl-2, and ATM) or 24 h (DNA fragmen-tation).

Cell Viability

Viable cell numbers were determined by thetrypan blue exclusion test (0.2% trypan blue).AGS cells were plated at 5 × 104 cells/well ina 24-well culture plate and treated with vari-ous concentrations of β-carotene (20, 50, and100 μmol/L) for 24 h.

Quantification of DNA Fragmentation

AGS cells (5 × 104 cells/well), either wildtype or transfected, were treated with β-carotene (50 and/or 100 μmol/L) for 24 h.DNA fragmentation was determined byamount of oligonucleosome-bound DNA in thecell lysate. The relative increase of nucleosomesin the cell lysate, determined at 405 nm, wasexpressed as an enrichment factor (cell deathdetection ELISAplus kit; Roche Molecular Bio-chemicals GmbH, Mannheim, Germany).

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Preparation of Nuclear Extractsand Whole-Cell Extracts

AGS cells were seeded at 2.5 × 106 cellsper 10-cm dish and cultured overnight to reach80% confluency. After treatment with 100 μMβ-carotene, the cells were harvested at 0, 4,8, 12, and 24 h by scraping into PBS; cellswere then pelleted by centrifugation at 300 g

for 5 min at 4◦C.For the preparation of nuclear extracts, the

cells were collected and resuspended in 100 μLof the hypotonic buffer [10 mmol/L HEPES,pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2,0.5% Nonidet P-40 (NP-40), 0.5 mmol/Ldithiothreitol (DTT), and 0.5 mmol/L phenyl-methylsulfonylfluoride (PMSF)] and placed onice for 20 min. The extracts were centrifugedat 15,000 g for 10 min at 4◦C. The pel-lets were washed once with hypotonic buffer,resuspended in 50 μL of extraction buffer(20 mmol/L HEPES, pH 7.9, 420 mmol/LNaCl, 0.5 mmol/L EDTA, 1.5 mmol/LMgCl2, 25% glycerol, 0.5 mmol/L DTT, and0.5 mmol/L PMSF) and placed on ice for20 min. The extracts were centrifuged at15,000 g for 10 min at 4◦C, and the super-natants (nuclear extracts) were used for Westernblot analysis of ATM and histone H1 (nuclearcontrol).

For Western blot analysis of p53, Bcl-2, andactin (loading control) in whole-cell extracts,the cells were trypsinized, washed, and ho-mogenized in Tris-HCl (pH 7.4) buffer con-taining 1% NP-40 and protease inhibitor cock-tail (Boehringer Mannheim, Indianapolis, IN).The protein concentration of each sample wasdetermined by Bradford assay (Bio-Rad labo-ratories, Hercules, CA).

Western Blot Analysis

The protein (70–100 μg) was loaded perlane, separated by 6–10% SDS-PAGE underreducing conditions, and transferred onto ni-trocellulose membranes (Amersham Inc., Ar-lington Heights, IL) by electroblotting. The

membranes were blocked using 5% nonfatdry milk in TBS-T (Tris-buffered saline and0.2% Tween 20) for 2 h at room tempera-ture. The proteins were detected with poly-clonal antibodies for ATM, p53, Bcl-2, andactin (all from Santa Cruz Biotechnology, SantaCruz, CA) and histone H1 (Upstate Biotech-nology, Lake Placid, NY) at 1:1000 dilutionin TBS-T containing 5% dry milk and in-cubated at 4◦C overnight. After washing inTBS-T, the immunoreactive proteins were vi-sualized using secondary antibodies conjugatedto horseradish peroxidase, which was followedby enhanced chemiluminescence (Santa CruzBiotechnology) using exposure to Agfa film(Agfa Health Care, Mortsel, Belgium).

Transfection with ATM Gene

To determine the relation of ATM andthe expression of p53 and Bcl-2, AGS cellswere transfected with human full-length ATMcDNA (+ATM) or control vector pcDNA(mock control). ATM cDNA was kindly pro-vided by Dr. Yosef Shiloh (Department ofHuman Molecular Genetics and Biochem-istry, Sackler School of Medicine, Tel AvivUniversity, Tel Aviv, Israel). AGS cells weretransfected with 1 μg/mL of the pcDNAor ATM cDNA, using transfection reagentDOTAP (Boehringer-Mannheim, Pentzberg,Germany), for 20 h to improve stability andintracellular delivery of DNAs. After transfec-tion, the cells were treated with β-carotene(100 μmol/L) for 24 h (DNA fragmentation)and 12 h (Western blotting for ATM, p53, andBcl-2).

Statistical Analysis

Results are expressed as mean ± SE of fourseparate experiments. Statistical analysis wasperformed via one-way ANOVA followed byDuncan’s test. P < 0.05 was considered statis-tically significant.

Jang et al.: β-Carotene-Induced Apoptosis of Gastric Cancer Cells 159

Figure 1. Effect of β-carotene on cell viability (A)and DNA fragmentation (B) of gastric adenocarci-noma (AGS) cells. The cells were seeded in 24-wellculture plates at 5 × 104 cells per well and cul-tured to reach 80% confluency. After treatment ofβ-carotene, cells were incubated for 24 h. (A) Cellviability was determined by the trypan blue exclu-sion test. (B) DNA fragmentation was determined byamount of oligonucleosome-bound DNA in the celllysate. The relative increase of nucleosomes in thecell lysate, determined at 405 nm, was expressed asan enrichment factor. Results are expressed as mean± SE of four separate experiments. ∗, P < 0.05 com-pared to 0 (the cells without treatment of β-carotene).

Results

Cell Viability and DNA Fragmentationof AGS Cells Treated with β-Carotene

To determine whether β-carotene inducescell death and DNA fragmentation, the cellswere treated with various concentrations ofβ-carotene for 24 h. The number of viable cells,determined by the trypan blue exclusion test,was reduced by treatment with β-carotene ina concentration-dependent manner (Fig. 1A).It paralleled with the increase in nucleosome-bound DNA, an index of DNA fragmentation(Fig. 1B).

Figure 2. Effect of β-carotene on protein levels ofataxia-telangiectasia-mutated (ATM), p53, and Bcl-2in AGS cells. AGS cells were seeded at 2.5 × 106

cells per 10-cm dish and cultured overnight toreach 80% confluency. After treatment of β-carotene(100 μmol/L), the cells were harvested at 0, 4, 8,and 12 h. Western blotting was performed for ATMin nuclear extracts (A) and p53 and Bcl-2 in whole-cell extracts (B). Histone H1 was used for nuclearcontrol, while actin served as a loading control.

Levels of ATM, p53, and Bcl-2 in AGSCells Treated with β-Carotene

As shown in Figure 2A, β-carotene (100μmol/L) decreased the nuclear level of ATMtime dependently. The level of histone H1,as a nuclear marker, was not changed by β-carotene. The protein level of apoptotic p53was increased by β-carotene, while that ofantiapoptotic Bcl-2 was decreased by β-carotene in AGS cells in a time-dependentmanner. The level of actin, a loading control,was not altered by treatment of β-carotene inAGS cells (Fig. 2B).

DNA Fragmentation of Wild-Type Cellsor Transfected Cells Treated

with β-Carotene

To determine the relation of ATM and DNAfragmentation of the cells, wild-type cells or thetransfected cells with pcDNA or ATM cDNAwere treated with β-carotene (100 μmol/L) for

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Figure 3. DNA fragmentation of wild-type cellsor the transfected cells, which were treated with β-carotene. The cells were seeded in 24-well cultureplates at 2 × 104 cells per well and transfectedwith 1 μg/mL of pcDNA or ATM cDNA, using thetransfection reagent DOTAP, for 20 h. After treatmentof β-carotene (100 μmol/L), the cells were culturedfor 24 h. DNA fragmentation was detected by theamount of oligonucleosome-bound DNA in the celllysate. The relative increase of nucleosomes in thecell lysate, determined at 405 nm, was expressedas an enrichment factor. Results are expressed asmean ± SE of four separate experiments. ∗, P < 0.05compared to none; +, P < 0.05 compared to mock;none, wild-type cells without treatment of β-carotene;control, wild-type cells with treatment of β-carotene;mock, the cells transfected with control vector pcDNAwith treatment of β-carotene; +ATM, the cells trans-fected with ATM cDNA with treatment of β-carotene.

24 h. β-Carotene-induced DNA fragmentationwas inhibited in the cells transfected with ATMcDNA (+ATM) compared to control vectorpcDNA (mock control) (Fig. 3).

Levels of ATM, p53, and Bcl-2 inWild-Type Cells or Transfected Cells

Treated with β-Carotene

To investigate the relation between ATMdegradation and the expression of apoptosis-related proteins, wild-type cells or the trans-fected cells with pcDNA or ATM cDNA weretreated with β-carotene (100 μmol/L) for 12 h.The β-carotene-induced decrease in ATM inthe nucleus and Bcl-2 in the whole cells was

Figure 4. Levels of ATM, p53, and Bcl-2 in wild-type cells or transfected cells, which were treated withβ-carotene. The cells were seeded in a 10-cm dishat 2 × 106 cells and transfected with 1 μg/mL ofpcDNA or ATM cDNA, using DOTAP, for 20 h. Aftertreatment of β-carotene (100 μmol/L), the cells wereharvested at 12 h. Western blotting was performedfor ATM in nuclear extracts (A) and p53 and Bcl-2in whole-cell extracts (B). Histone H1 was used forthe nuclear control, while actin served as a loadingcontrol.

inhibited in the cells transfected with ATMcDNA (+ATM) compared to control vectorpcDNA (mock control) or control (wild-typecells treated with β-carotene). An increase inp53 by β-carotene was suppressed in the cellstransfected with ATM cDNA (+ATM) com-pared to control vector pcDNA (mock control)or control (Fig. 4) cells.

Discussion

The present study provides evidence for apossible mechanism by which β-carotene in-duces apoptosis of gastric cancer cells. Wefound that high concentration of β-carotene(100 μmol/L) induced apoptosis of AGS cellsin parallel with nuclear loss of ATM protein.

Jang et al.: β-Carotene-Induced Apoptosis of Gastric Cancer Cells 161

β-Carotene-induced alterations (increase inDNA fragmentation and p53 level and decreasein Bcl-2 level) were suppressed in the cells trans-fected with ATM cDNA.

The effect of β-carotene and othercarotenoids at a high concentration to in-hibit tumor cell growth has been reported.10

β-Carotene reduces the growth of tumor celllines, including colon cancer cells and leukemiccells,5,7,8 while it induces apoptotic protein ex-pression, such as p53 and Bax, and suppressesBcl-2 expression.5,8

The bcl-2 family plays a critical role in de-termining cell fate in the apoptotic pathway. Itinfluences mitochondrial membrane polariza-tion and the cleavage-mediated caspase activa-tion, which are the ultimate effectors in apop-tosis signaling. Low-expression level of bcl-2has been observed in cells undergoing apopto-sis.5,8 Naturally occurring compounds, such asresveratrol and curcumin, have been reportedto induce apoptosis in carcinoma cell lines bydecreasing bcl-2 expression.11,12 In the presentstudy, we show that β-carotene decreases theexpression of bcl-2 in gastric cancer cells.

The protein p53 shows an essential role incancer prevention and apoptosis. The level ofp53 is consistent in cells without stress. Dur-ing cellular stress, p53 protein is phosphory-lated and interacts with mouse double minute 2(MDM 2), resulting in apoptosis. In the presentstudy, p53 was induced in the cells treated withβ-carotene, which indicates that p53 may me-diate β-carotene-induced apoptosis of gastriccancer AGS cells.

In response to DNA damage, ATM activatesmultiple signaling pathways, such as cell cy-cle checkpoints, DNA repair, and apoptosis. Inthe present study, nuclear level of ATM de-creased in the cells treated with β-carotene. Inaddition, the cells that overexpress the ATMgene showed protective effect for the cells fromapoptotic changes, such as DNA fragmenta-tion, increase in apoptotic p53, and decrease inBcl-2 of the cells. Because ATM is specificallycleaved in cells undergoing apoptosis by a vari-ety of stimuli, 9 ATM level in the nucleus may

be important for cell survival besides its role inthe DNA repair process.

In conclusion, β-carotene induces apoptosisby increasing apoptotic protein p53 and de-creasing anti-apoptotic protein Bcl-2 as wellas nuclear ATM in gastric cancer AGS cells.Nuclear loss of ATM may be the underlyingmechanism of β-carotene-induced apoptosis ofgastric cancer cells.

Acknowledgments

This study was supported by a grant (F01-2006-000-10063-0, Joint Research Project un-der the Korea-Japan Basic Scientific Cooper-ation Program) from the Korea Science andEngineering Foundation made in the programyear of 2006 to H.K., and KOSEF grant fundedby the Korea Government (Ministry of Sci-ence and Technology) (R11-2007-040-01002-0). The authors are grateful to Dr. T. Morio(Tokyo Medical and Dental University, Tokyo,Japan) for valuable discussion and the BrainKorea 21 Project, College of Human Ecology,Yonsei University.

Conflicts of Interest

The authors declare no conflicts of interest.

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