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© 2007 The American Society for Nutrition J. Nutr. 137:31-36, January 2007

Biochemical, Molecular, and Genetic Mechanisms

Activation of Caspase-8 Contributes to 3,3'-Diindolylmethane-Induced Apoptosis in Colon Cancer Cells1

Eun Ji Kim3, So Young Park2, Hyun-Kyung Shin2,3, Dae Young Kwon4, Young-Joon Surh5 and Jung Han Yoon Park2,3,*

2 Department of Food Science and Nutrition and 3 Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University, Chuncheon, 200-702, Korea; 4 Korea Food Research Institute, Sungnam, 463-746, Korea; and 5 College of Pharmacy, Seoul National University, Seoul, 151-742, Korea

* To whom correspondence should be addressed. E-mail: jyoon{at}hallym.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
3,3'-Diindolylmethane (DIM) is the major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol, which is a promising anticancer agent present in cruciferous vegetables and has itself been reported to have anticarcinogenic properties. This study examined DIM-mediated regulation of apoptosis in the HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer cell lines. DIM (0–30 µmol/L) substantially decreased the number of viable cells and induced apoptosis of HCT116 and HT-29 cells in a concentration-dependent manner. Western-blot analyses of total cell lysates revealed that DIM increased the activation of caspase-3, -7, -8, and -9 and enhanced poly(ADP-ribose) polymerase cleavage in both HCT116 and HT-29 cells. In addition, DIM increased the translocation of cytochrome c and Smac/Diablo from the mitochondria to the cytoplasm. In concert with the caspase-8 activation by DIM, increased levels of Fas and truncated Bid were observed. DIM did not affect the protein levels of p53, Bcl-2, Bax, or Fas ligand (FasL) in HCT116 cells. In HT-29 cells, however, DIM decreased Bcl-2 levels, although the protein levels of Bax or FasL were not affected. The caspase-8 inhibitor Z-IETD-FMK attenuated the DIM-induced apoptosis, indicating that increased activation of this enzyme contributed to the increase in p53-independent apoptosis that was observed in colon cancer cells. We have demonstrated that DIM induces apoptosis in colon cancer cells, providing insights into the mechanisms underlying its antitumorigenic activities.


   Introduction
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 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Colon cancer is one of the leading causes of cancer-related deaths in the United States (1) and the worldwide incidence of this cancer is increasing. It has been estimated that a majority of human cancers can be prevented by modifications of lifestyle, including diet (2). Therefore, substantial efforts have been made to develop dietary chemopreventive agents that could inhibit, delay, or reverse the multistage carcinogenesis (3).

Results from epidemiological studies suggest that cruciferous (Brassica) vegetables, such as broccoli, cabbage, Brussels sprouts, and cauliflower, are effective in reducing the risk of cancers (4,5). Indole-3-carbinol (I3C)6 is an autolysis product of glucobrassicin, a naturally occurring constituent of cruciferous vegetables and is a major bioactive component in these vegetables. Because numerous studies have demonstrated that I3C inhibits carcinogenesis in animal experiments (6–8) and also inhibits the growth of human cancer cells (9–11), I3C has received much attention as a cancer preventive or chemotherapeutic agent in recent years.

I3C is chemically unstable in low pH environments and is rapidly converted in the stomach to a variety of condensation products with distinctive biological activities (12). 3,3'-Diindolylmethane (DIM) is a major acid condensation product of I3C (13). DIM is readily detected in the liver and feces of rodents fed I3C, whereas the parent I3C was not detected in these animals (14). DIM is slowly formed from I3C in cell cultures at a neutral pH over extended incubation periods and during the simple mixture and storage of purified diets used in rodent feeding studies (15,16). These observations suggest that DIM, and not I3C, may exert the observed physiological effects of dietary I3C.

Several studies indicated that DIM exhibits promising cancer preventive effects. A single oral administration of DIM during the initiation stage of tumorigenesis reduced the incidence and multiplicity of dimethylbenazanthracene-induced mammary tumors in rat by 70–80% (17). In addition, repeated oral administrations of DIM during the promotion stage of dimethylbenazanthracene-induced tumorigenesis reduced mammary tumor growth by 95% (18). Recently, Nachshon-Kedmi et al. (19) reported that DIM inhibited tumor growth and induced apoptosis of TRAMP-C2 mouse prostate cancer cells transplanted into C57BL/6 mice. In addition to animal studies, in vitro studies have shown that DIM inhibits the growth of human colon (20,21), pancreas (22), prostate (23–25), and breast (9,16,26,27) cancer cells.

Deregulated proliferation and inhibition of apoptosis lies at the heart of all tumor development, so the control of cell proliferation and apoptosis presents an obvious target for preventive and therapeutic intervention in all cancers (28). Apoptosis can be induced either by an extrinsic pathway mediated via the activation of death receptors or by an intrinsic mitochondria-mediated pathway (reviewed in 29). The death receptor-mediated pathway is initiated by interaction of the ligand with its death receptor, which sequentially recruits receptor-associated death domains, caspase-8, and caspase-3. Caspase-3 then cleaves various substrates leading to apoptosis. In contrast, the mitochondria-mediated pathway involves the alteration of mitochondrial membrane permeability, thereby promoting the release of cytochrome c and Smac/Diablo from mitochondria. Cytosolic cytochrome c, together with apoptosis protease-activating factor-1, activates caspase-9 and the latter then activates caspase-3 (30). Cytosolic Smac/Diablo promotes caspase-9 activation by competing with caspases for binding of the inhibitor of apoptosis protein family, thereby relieving the inhibitory effects of inhibitor of apoptosis protein on caspases (31,32). Mitochondria-mediated apoptosis is regulated by the Bcl-2 family of proteins, which controls mitochondrial membrane permeability (reviewed in 33).

DIM has been shown to induce apoptosis in human breast (9,16,26,27) and prostate (23–25) cancer cells. However, the molecular mechanisms by which DIM inhibits the growth of human colon cancer cells have not been fully elucidated. This study was performed to examine how DIM induces apoptosis in HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer cells. We demonstrate that DIM-induced apoptosis in colon cancer cells occurs at least partly because DIM induces caspase-8 activation, resulting in the induction of the intrinsic (mitochondrial) pathway.


   Materials and Methods
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 LITERATURE CITED
 
    Materials. Reagents used were as follows: DIM (LKT Laboratories); a horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (Amersham); 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and anti-ß-actin (Sigma); DMEM/Ham's F-12 nutrient mixture (DMEM/F-12) (Gibco BRL); fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin (Cambrex Bio Technology); antibodies against cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, cleaved caspase-8, cleaved poly(ADP-ribose) polymerase (PARP), and Bid (Cell Signaling); cytochrome c antibody, phycoerythrin-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) (BD Pharmingen); Z-IETD-FMK and antibodies against Bcl-2, Bax, Fas, Fas ligand (FasL), Smac/Diablo, and heat shock protein (Santa Cruz Biotechnology).

    Cell culture. HCT116 and HT-29, the human colon cancer cell lines, and IEC-6 cells, an intestinal cell line derived from the rat jejunal crypt (34), were obtained from the American Type Culture Collection. Cells were maintained in DMEM/F12 containing 100 mL/L FBS with 100,000 U/L penicillin and 100 mg/L streptomycin. To examine the effect of DIM, we plated the cells in multi-well plates with DMEM/F-12 containing 100 mL/L FBS. Prior to DIM treatment, the cell monolayers were rinsed and serum deprived for 24 h with DMEM/F-12 containing 10 mL/L FBS (serum deprivation medium). After serum deprivation, we replaced the medium with fresh serum deprivation medium with or without various concentrations (10, 20, 30 µmol/L) of DIM. Viable cell numbers were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay as described previously (35). We used the serum deprivation medium containing 10 mL/L FBS to minimize possible effects of various phytochemicals present in FBS. DIM was dissolved in dimethylsufoxide and all cells were treated with dimethylsufoxide at a final concentration of 1 mL/L.

    Hoechst 33258 staining. The characteristic apoptotic morphological changes, chromatin condensation and fragmentation, were assessed by fluorescent microscopy after Hoechst 33258 staining. Briefly, cells were plated on cell culture cover slips in 8-well plates and then treated with 30 µmol/L DIM for 48 h with untreated cells as control. After washing twice with PBS, the treated and nontreated cells were fixed by adding 40 g/L PBS-buffered formaldehyde for 20 min and then stained with 10 mg/L Hoechst 33258 for 30 min. The cells were immediately washed with PBS and then examined using fluorescent microscopy.

    Fluorescence-activated cell sorting analysis. Cells were treated with various concentrations of DIM for 48 h and the number of early apoptotic cells quantified. The cells were trypsinized and then incubated with phycoerythrin-conjugated Annexin V and 7-AAD for 15 min at room temperature in the dark. Apoptotic cells were analyzed by flow cytometry utilizing FACScan (Becton Dickinson). We analyzed the data using ModFit V.1.2. software.

    Western-blot analysis. Total cell lysates were prepared as described previously (36). Cytosolic and mitochondrial proteins were separated as described by Eguchi et al. (37) and the purity of the fractions estimated by Western blotting with an antibody raised against the mitochondrial heat shock protein (38). We determined the protein contents of total cell lysates and cytoplasmic and mitochondrial fractions using the BCA protein assay kit (Pierce). The proteins were resolved on sodium dodecyl sulfate (40–200 mL/L or 100–200 mL/L) polyacrylamide gels and transferred onto a polyvinylidene fluoride membrane (Millipore). The blots were blocked for 1 h in 50 mL/L nonfat dry milk in 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, and 1 mL/L Tween 20 and incubated for 1 h with cleaved PARP (1:1000), cleaved caspase-3 (1:1000), cleaved caspase-7 (1:1000), cleaved caspase-9 (1:1000), cleaved caspase-8 (1:1000), cytochrome c (1:1000), Smac/Diablo (1:1000), p53 (1:1000), Bax (1:1000), Bcl-2 (1:1000), Bid (1:1000), Fas (1:1000), FasL (1:1000), or ß-actin antibody (1:2000). The blots were then incubated with anti-mouse, anti-rabbit, or anti-goat horseradish peroxidase-conjugated antibodies. Signals were detected by means of an enhanced chemiluminescence method using SuperSignal West Dura Extended Duration substrate (Pierce). The relative abundance of each band was quantified using the Bio-profile Bio-1D application (Vilber-Lourmat) and the expression levels were normalized to ß-actin.

    Statistical analysis. The results were expressed as means ± SEM. They were analyzed by 1-way or 2-factor repeated measures of ANOVA. Differences between the treatment groups were analyzed by Duncan's multiple range test. Differences were considered significant at P < 0.05. All statistical analyses were performed with SAS statistical software version 8.12.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    DIM inhibits growth and induces apoptosis of colon cancer cells. To examine whether DIM inhibits colon cancer cell growth, HCT116 and HT-29 cells were treated with various concentrations of DIM in serum deprivation medium containing 10 mL/L FBS. DIM markedly decreased cell viability dose dependently in both HCT116 and HT-29 cells (Table 1) (P < 0.05). The toxicity of DIM against normal small intestinal epithelial cells was assessed with a similar experiment utilizing IEC-6 cells. DIM had no effect on IEC-6 cell growth (data not shown).


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  		TABLE 1 Effect of DIM on growth of HCT116 and HT-29 cells1

 
Hoechst 33258 dye staining revealed chromatin condensation, visualized as an intense pycnotic bluish-white fluorescence in cell nuclei, in both HCT116 (Fig. 1A,B) and HT-29 cells (Fig. 1C,D) treated with 30 µmol/L DIM. The presence of phosphatidylserine on the outer leaflet of apoptotic cell membranes was assessed using Annexin V and 7-AAD staining followed by flow cytometry to quantify the amount of cells in the early stage of apoptosis. The numbers of apoptotic (Annexin V-positive/7-AAD-negative) cells increased in a DIM concentration-dependent manner in both HCT116 and HT-29 cells cultured in serum deprivation medium (Table 2). We also observed that DIM induced apoptosis in HCT116 and HT-29 cells grown in DMEM/F12 supplemented with 100 mL/L FBS (data not shown).


Figure 1
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  		Figure 1  DIM induces apoptosis of HCT116 (A,B) and HT-29 (C,D) cells. Cells were treated with 0 or 30 µmol/L DIM for 48 h. Cells were fixed and stained with a DNA specific dye, Hoechst 33258.

 

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  		TABLE 2 Effect of DIM on apoptotic cell numbers in HCT116 and HT-29 cells1

 
    DIM increases the activation of caspases and enhances PARP cleavage. To examine the effect of DIM on caspase cleavage and PARP, the cells were treated with various concentrations of DIM for 36 h and total cell lysates were prepared for Western-blot analysis. DIM increased the amount of the 37-kDa fragment of caspase-9, the 20-kDa fragment of caspase-7, and the 19- and 17-kDa fragments of caspase-3 in both HCT116 and HT-29 cells. The 89-kDa cleavage product of PARP was greatly increased in a DIM concentration-dependent manner in both cell types (Fig. 2).


Figure 2
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  		Figure 2  DIM increases the activation of caspases and PARP cleavage in HCT116 (A) and HT-29 (B) cells. Cells were treated with various concentrations of DIM for 36 h. Total cell lysates were analyzed by Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of 3 independent experiments, are shown. The relative abundance of each band to their own ß-actin was quantified and the control levels were set at 1. The adjusted mean ± SEM, n = 3, of each band is shown above each blot. Means without a common letter differ, P < 0.05.

 
    DIM increases the release of cytochrome c and Smac/Diablo from mitochondria. Because DIM induced the activation of caspase-9, we examined whether it also induces the release of cytochrome c and Smac/Diablo from mitochondria. Immunoblotting was performed with mitochondrial and cytoplasmic fractions prepared from cells that had been treated with various concentrations of DIM. DIM increased the levels of cytochrome c and Smac/Diablo in the cytoplasm and concomitantly decreased the levels of these proteins in the mitochondria of both HCT116 and HT-29 cells (Fig. 3).


Figure 3
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  		Figure 3  DIM increases the release of cytochrome c and Smac/Diablo from mitochondria in HCT116 (A) and HT-29 (B) cells. Cells were treated with various concentrations of DIM for 36 h and subjected to subcellular fractionation. The resulting cytosolic and mitochondrial fractions were analyzed by Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of 3 independent experiments, are shown. The relative abundance of each band was quantified and the control levels were set at 1. The adjusted mean ± SEM, n = 3, of each band is shown above each blot. Means without a common letter differ, P < 0.05.

 
    DIM increases the levels of truncated Bid. As cytochrome c and Smac/Diablo release were increased in cells treated with DIM, we investigated the effect of DIM on the levels of Bcl-2 family proteins, which are known to control mitochondrial membrane permeability (33). DIM did not affect the levels of p53, Bcl-2, and Bax in HCT116 (wild-type p53) cells (Fig. 4A) or Bax levels in HT-29 (mutant p53) cells (Fig. 4B). DIM decreased Bcl-2 levels in HT-29 cells (Fig. 4B) and increased the levels of truncated Bid (t-Bid), the active form of Bid, in both HCT116 and HT-29 cells (Fig. 4A,B).


Figure 4
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  		Figure 4  DIM increases t-Bid levels in HCT116 (A) and HT-29 (B) cells. Cells were treated with various concentrations of DIM for 36 h. Total cell lysates were analyzed by immunoblotting with an antibody raised against Bcl-2, Bax, t-Bid, p53, or ß-actin. Photographs of chemiluminescent detection of the blots, which were representative of 3 independent experiments, are shown. The relative abundance of each band to their own ß-actin was quantified and the control levels were set at 1. The adjusted mean ± SEM, n = 3, of each band is shown above each blot. Means without a common letter differ, P < 0.05.

 
    DIM increases the levels of cleaved caspase-8 and Fas. Because DIM increased the levels of t-Bid (Fig. 4), the effect of DIM on cleaved caspase-8, membrane-bound FasL, and Fas proteins was examined using Western blotting. DIM significantly increased the levels of cleaved caspase-8 in both HCT116 and HT-29 cells. DIM increased the Fas levels in both HCT116 and HT-29 cells but had no effect on the FasL levels (Fig. 5).


Figure 5
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  		Figure 5  DIM increases the levels of cleaved caspase-8 and Fas in HCT116 (A) and HT-29 (B) cells. Cells were treated with various concentrations of DIM for 36 h. Total cell lysates were analyzed by Western blotting with an antibody raised against cleaved caspase-8, Fas, or FasL. Photographs of chemiluminescent detection of the blots, which were representative of 3 independent experiments, are shown. The relative abundance of each band to their own ß-actin was quantified and the control levels were set at 1. The adjusted mean ± SEM, n = 3, of each band is shown above each blot. Means without a common letter differ, P < 0.05.

 
    A caspase-8 inhibitor attenuates DIM-induced apoptosis. As DIM increased the activation of caspase-8 and Bid cleavage, we examined whether the caspase-8 inhibitor Z-IETD-FMK would attenuate DIM-induced apoptosis. In this experiment, HT-29 cells were pretreated with 20 µmol/L Z-IETD-FMK for 4 h prior to treatment with 30 µmol/L DIM for 24 h. The caspase-8 inhibitor decreased DIM-induced apoptosis in HT-29 cells (P < 0.05). The percentage of living cell numbers in control, DIM, and DIM+Z-IETD-FMK was 95.6 ± 0.6, 74.1 ± 0.8, and 86.6 ± 0.8, respectively, and that of apoptotic cell numbers was 1.94 ± 0.7, 21.7 ± 0.8, and 9.48 ± 0.5, respectively.


   Discussion
TOP
 ABSTRACT
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 Materials and Methods
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 Discussion
LITERATURE CITED
 
Cancer development is a complex, multi-step process that seems to progress for an extended period of time and eventually cancer spreads from one area of the body to others during the late metastasic stage. Present clinical cancer therapies include surgery, radiation, and chemotherapy but have limited effect at the late metastasic stage. However, increasing evidence from epidemiological and pathological studies suggest that many human cancers could be prevented or their progression slowed down (39). Recently, chemoprevention of cancer has gained more attention, presumably because it involves the use of dietary bioactive compounds either alone or in combination to prevent, inhibit, or reverse cancer development (40). Therefore, investigations of how dietary bioactive components regulate cancer cell survival could play an important role in the development of new agents with low toxicity to prevent and treat cancer. This study was performed to examine whether and how DIM induces apoptosis in colon cancer cells.

Failure of tumor cells to undergo apoptosis translates into malignant potential and chemotherapeutic resistance (reviewed in 40). Apoptosis has quickly surfaced as a potential target for cancer prevention/treatment at various stages of carcinogenesis. Therefore, induction of apoptosis by dietary bioactive compounds can be an excellent approach to inhibit the promotion and progression of carcinogenesis and to remove genetically damaged, preinitiated, or neoplastic cells from the body. Previously, other investigators have shown that DIM induces apoptosis in breast and prostate cancer cells (9,16,23–27). Using HCT116 and HT-29 cells, we observed the following results: 1) DIM induced apoptosis in both colon cancer cell lines; 2) DIM induced cleavage of caspase-3, -7, -8, and -9; 3) DIM induced the release of cytochrome c and Smac/Diablo from mitochondria; 4) DIM increased the levels of Fas and t-Bid; and 5) a caspase-8 inhibitor mitigated the DIM-induced apoptosis.

The typical executioners of apoptosis are proteolytic enzymes called cysteinyl aspartate-specific proteases (caspases). Our results clearly demonstrate that DIM increased the activation of caspase-3, -7, -8, and -9 and PARP cleavage. The activation of caspases may be one of the major mechanisms by which DIM induces apoptosis.

Caspases are activated via 2 pathways: the extrinsic (death receptor) and the intrinsic (mitochondrial). In the intrinsic pathway, binding of tumor necrosis factor, tumor necrosis factor-related apoptosis-inducing ligand, or FasL to their specific receptors, in association with adaptor molecules such as Fas-associated death domain, leads to cleavage and activation of initiator caspase-8 and -10, which in turn cleave and activate executioner caspase-3, -6, and -7, culminating in apoptosis (41,42). In this study, we found that the levels of Fas and cleaved caspase-8 were increased in DIM-treated colon cancer cells. In addition, the caspase-8 inhibitor Z-IETD-FMK attenuated DIM-induced apoptosis. These results indicate that DIM increases Fas levels, leading to activation of caspase-8, which contributes to activation of caspase-3 and -7.

In the intrinsic (mitochondrial) pathway, cytochrome c released from mitochondria by apoptotic stimulation associates with procaspase-9/apoptosis protease-activating factor-1 to form an apoptosome. The apoptosome processes caspase-9 from inactive proenzyme to its active form (43). This event further triggers caspase-3 activation and eventually leads to apoptosis (44). Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, is a specific substrate of caspase-8 in the Fas apoptotic signaling pathway. Whereas full-length Bid is localized in cytosol as an inactive precursor, t-Bid translocates to the mitochondria and thus transduces apoptotic signals from the cytoplasm to the mitochondria (reviewed in 29). In addition to demonstrating that DIM induced caspase-3 activation, we found that DIM increased the level of t-Bid, which is known to increase mitochondrial membrane permeability and the release of cytochrome c and Smac/Diablo. The up-regulation of t-Bid and down-regulation of Bcl-2 (in HT-29 cells) caused by DIM increased the release of cytochrome c and Smac/Diablo and contributed to the activation of caspase-9. Our results suggest that both the extrinsic and intrinsic pathways are involved in the DIM-mediated regulation of caspase-3 activation in colon cancer cells. In addition to these 2 pathways, DIM could activate the endoplasmic reticulum (ER) stress apoptotic pathway. In pancreatic cancer cells, DIM has been shown to induce apoptosis through ER-dependent upregulation of death receptor 5 (22). It remains to be determined whether DIM induces apoptosis of colon cancer cells via ER-dependent pathways.

p53 levels were not affected by DIM treatment in HCT116 cells, which contain wild-type p53 protein (45). In addition, DIM still induced apoptosis in HT-29 cells, which contain mutant p53. These results indicate that wild-type p53 is not involved in DIM-induced apoptosis in colon cancer cells. Utilizing MCF-7 breast cancer cells that have wild-type p53, Ge et al. (27) have also shown that induction of apoptosis by DIM was independent of the p53 pathway.

In this study, IEC-6 cells, a normal intestinal epithelial cell line, were used to study whether DIM has adverse effects on small intestinal epithelial cell growth. We observed that DIM had no effect on the growth of IEC-6 cells. The observations that DIM induces apoptosis in HCT116 and HT-29 colon cancer cells without affecting normal intestinal epithelial cell growth suggest that DIM has potential as a chemotherapeutic agent that exhibits a low adverse effect on the gastrointestinal tract.

This study used DIM at concentrations of 10–30 µmol/L and other investigators (16,24,25) examined the effects of DIM on prostate and breast cancer cells at higher concentrations (50–100 µmol/L) than those used in our studies. Unfortunately, there is a paucity of information on DIM concentrations in the human blood and colonic lumen. To determine whether the concentrations of DIM used in the cell culture studies are clinically relevant, the concentrations of DIM in human serum and colonic lumen following administration of DIM should be determined in the future.

In conclusion, we demonstrated that DIM decreased growth and induced apoptosis of HCT116 and HT-29 human colon cancer cells, an effect that is mediated by the activation of caspases. Our results indicate that DIM activates caspases via both the extrinsic and intrinsic pathways. Apoptosis plays an important role in the molecular pathogenesis of cancer and can change the outcome of chemotherapy and radiotherapy. Therefore, dietary bioactive compounds such as DIM hold substantial promise for cancer treatment. Our study partly elucidates the molecular basis for using DIM as a potential antitumorigenic agent.


   FOOTNOTES
 
1 Supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-311-C00176) and research grants from the Korea Science and Engineering Foundation (KOSEF) for Biofoods Research Program, Ministry of Science and Technology, Korea. Back

6 Abbreviations used: 7-AAD, 7-amino-actinomycin D; DIM, 3,3'-diindolylmethane; ER, endoplasmic reticulum; FasL, Fas ligand; FBS, fetal bovine serum; IAP, inhibitor of apoptosis protein; I3C, indole-3-carbinol; PARP, poly(ADP-ribose) polymerase; t-Bid, truncated Bid. Back

Manuscript received 25 August 2006. Initial review completed 9 October 2006. Revision accepted 8 November 2006.


   LITERATURE CITED
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 LITERATURE CITED
 

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