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Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201
2 To whom correspondence should be addressed. E-mail: fsarkar{at}med.wayne.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Bax translocation • apoptosis • I3C
Indole-3-carbinol (I3C)3, a compound found in high concentrations in Brassica vegetables, has been known to possess anticarcinogenic effects in experimental animals ( 1, 2). The interest in I3C, as a cancer chemopreventive agent, has increased significantly in the past 10 y. Most in vivo and in vitro studies on I3C have shown the inhibitory effects of I3C on cancer cells ( 1– 4). Administration of I3C to mice has been shown to reduce the incidence of spontaneous mammary tumor formation ( 5) and I3C also has been shown to greatly decrease the incidence of mammary tumor in mice ( 6). One mechanism by which I3C may inhibit carcinogenesis is through the induction of enzymes, such as cytochrome P-450-dependent monooxygenases, glutathione S-transferases (GST) or epoxide hydrolases (EH), which metabolize carcinogens to more polar and excretable forms ( 4, 5, 7, 8). The studies from our laboratories and others have shown that I3C inhibits cell growth, arrests cell cycle at the G1 checkpoint and induces apoptosis in breast, prostate and other cancer cells ( 9– 15). However, the precise molecular mechanism(s) by which I3C exerts its inhibitory effects on cancer cells has not been fully elucidated.
There are several important molecules involved in the apoptotic process. The Bcl-2 family (e.g., Bax, Bcl-2, Bcl-XL, etc.) has been reported to play a major role in determining whether cells will undergo apoptosis ( 16). Bax induces apoptosis, whereas Bcl-2 and Bcl-XL inhibit apoptosis ( 16– 19). It has been reported that the ratio of Bax:Bcl-2 ( 16, 20), rather than Bcl-2 alone, is important for the survival of drug-induced apoptosis in cancer cells. Moreover, the subcellular location of Bax protein has been found to be important in the apoptotic processes. Bax is generally sequestered in the cytosol and removed from the cytosol into mitochondria with large aggregates during apoptosis ( 21– 23). Bax translocation into mitochondria targets the mitochondrial intermembrane contact sites and releases cytochrome c ( 24, 25). Bax is then packaged into large aggregates on mitochondria. The release of cytochrome c from the mitochondria cleaves and activates caspase-3 and caspase-9, resulting in the sequence of apoptotic processes ( 22– 24).
Breast cancer is the most common cancer in women and remains the second leading cause of cancer-related female deaths in the U.S. ( 26). Thus, there is a tremendous need for the development of mechanism-based and targeted preventive and therapeutic strategies for breast cancer. The facts that I3C inhibits the growth of cancer cells and induces apoptosis may make it a potent agent against breast cancer. In the present investigation, we have examined the effect of I3C on breast cancer cells and the molecular changes associated with I3C-induced apoptotic processes. We found that Bax translocation from cytosol into mitochondria, mitochondrial depolarization and release of cytochrome c might be one of the molecular mechanism(s) by which I3C induces apoptosis and inhibits breast cancer growth. However, translocation of Bax into mitochondria is not sufficient for the induction of apoptotic processes as demonstrated by our studies.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The human breast epithelial cell lines, MCF10A (nontumorigenic) and MCF10CA1a (cancer cells) were obtained from the Karmanos Cancer Institute in Detroit, MI. MCF10A is a spontaneously immortalized human breast epithelial cell line that was derived without viral or chemical intervention from immortal diploid human breast epithelial cells containing wild-type p53. MCF10CA1a (CA1a) cells were cancer cells and derived from MCF10AneoT model system. MCF10AneoT is a cell line derived from MCF10A by transfecting mutated H-ras oncogene and has a transformed phenotype. It is a transplantable, xenograft model of human proliferative breast disease with proven neoplastic potential and commonly considered a premalignant human breast epithelial cell line. MCF10CA1a cells were cultured in DMEM/F-12 (1:1) media (Invitrogen, Carlsbad, CA) supplemented with 5% horse serum (Invitrogen), 2 mmol/L L-glutamine, 1% penicillin and streptomycin in a 5% CO2 atmosphere at 37°C. MCF10A cells were cultured in the above media plus 1 mg/L insulin, 0.1 mg/L choleratoxin, 0.5 mg/L hydrocortisone (Sigma, St. Louis, MO), 0.5 mg/L fungizone and 0.02mg/L EGF (Invitrogen).
Cell growth inhibition
The MCF10A and CA1a cells were seeded at a density of 5 x 104/well in a six-well culture dish. After 24 h, the cells were treated with 15, 30, 60 or 100 µmol/L of I3C (Sigma) or 0.1% DMSO (vehicle control). These concentrations were chosen based on previously published reports showing the effect of I3C on breast cancer cells. Cells treated with I3C or DMSO for 1–3 d were harvested by trypsinization, stained with 0.4% trypan blue, and viable cells were counted by using a hemocytometer.
Analysis of poly(ADP-ribose) polymerase (PARP) cleavage
Cells treated with 60 or 100 µmol/L of I3C or with DMSO (as control) for 24, 48 and 72 h were lysed in lysis buffer [10 mmol/L Tris-HCl (pH 7.1), 50 mmol/L sodium chloride, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 100 µmol/L sodium orthovandate, 2 mmol/L iodoacetic acid, 5 µmol/L ZnCl2, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.5% Triton X-100]. The lysates were kept on ice for 30 min and vigorously vortexed before centrifugation at 12,500 x g for 20 min. Fifty µg of total proteins were resolved through 10% SDS-PAGE and then transferred to nitrocellulose membrane. The membrane was incubated with primary monoclonal antihuman PARP antibody (1:5000, Biomol, Plymouth Meeting, PA), washed with TTBS and incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce, Rockford, IL).
Flow cytometry for detecting apoptosis
7-Amino actinomycin D (7AAD) staining and flow cytometry was conducted to detect and quantify apoptosis. Cells treated with 60 or 100 µmol/L I3C for 24, 48, 72 h or with DMSO for 72 h (as control) were subjected to this analysis. Briefly, 7AAD (Calbiochem-Novabiochem, La Jolla, CA) was dissolved in acetone and diluted in PBS to a concentration of 200 mg/L. A total of 100 µL of 7AAD solution was added to 106 cells suspended in 1 mL of PBS and mixed thoroughly. Cells were stained for 20 min at 4°C while protected from light and pelleted by centrifugation. The cells were resuspended in 500 µL of 1% PBS-BSA solution. Unstained fixed cells were used as negative control. Samples were analyzed on a FACscan (Becton, Dickinson, CA) within 30 min of fixation. Data on 10,000 and 20,000 cells were acquired and processed using Lysys II software (Becton). Scattergrams were generated by combining forward light scatter with 7-AAD fluorescence, and regions were drawn around clear-cut populations having negative, dim and bright fluorescence. The frequency of cells with low, medium and high 7-AAD fluorescence was assessed. The purity and enrichment of the sorted populations were then calculated.
Western blot analysis
MCF10A and CA1a cells were plated and cultured in complete medium and allowed to attach for 24 h before the addition of 60 or 100 µmol/L of I3C and incubated for 24, 48 and 72 h. Control cells were incubated in the medium containing 0.1% DMSO for the same time points. After incubation, the cells were lysed in 62.5 mmol/L Tris-HCl and 2% SDS. Protein concentration was then measured using BCA protein assay (Pierce). Cell extracts were subjected to 12% SDS-PAGE, and electrophoretically transferred to nitrocellulose membrane. Membranes were incubated with monoclonal Bcl-2 (1:500, Oncogene, Cambridge, MA), Bax (1:5000, Trevigen, Gaithersburg, MD), and rabbit polyclonal ß-actin (1:5000, Sigma) antibodies, washed with TTBS and incubated with secondary antibodies conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce).
Fluorescence staining for confocal imaging
The 5 x 104 cells were plated on glass cover slips in each well of a six-well plate. The cells were treated with 30, 60 or 100 µmol/L of I3C for 6, 12, 24, 48 and 72 h. After treatment, the cells were fixed in ice-cold 100% methanol for 10 min and washed in PBS three times. The nonspecific binding was blocked with 0.2% BSA in PBS for 45 min. The cells were incubated with Bax antibody (1 mg/L, Trevigen) in PBS-0.1% saponin solution for 2 h and washed with PBS-0.1% saponin solution three times. After washing, the cells were incubated with secondary fluorescent goat anti-mouse antibody (Alexa Fluor 488, 1 mg/L, Molecular Probes, Eugene, OR) in PBS-0.1% saponin and 5% serum for 1 h and washed thereafter with PBS-0.1% saponin. They were next fixed with 100% methanol and washed three times with PBS before being mounted in antifade solution (Molecular Probes) on slides, and the fluorescence images were captured with Zeiss Laser Scanning Inverted Confocal Microscope System 310 with a 63x/1.2 oil immersion objective.
Measurement of loss of mitochondrial potential by JC-1 staining
The 4 x 105 cells were cultured in six-well plates. After being treated with I3C for 6 h, the cells were collected by trypsinization and washed with PBS followed by resuspending in JC-1 (10 µmol/L, Molecular Probes) and incubated at 37°C for 15 min. Cells were rinsed three times with DMEM high glucose-containing medium without phenol red and suspended in prewarmed medium. The cells were then subjected to flow cytometric analysis on a FACstar Plus Flow Cytometer (Becton) in the FL1, FL2/FL3 channel to detect mitochondrial potential.
Measurement of cytochrome c release
Cells were fractionated into cytosolic and mitochondrial fraction using the Apo Alert Fractionation kit (Clontech, Palo Alto, CA). The 30 µg cytosolic fraction (for cytochrome c) and 20 µg mitochondrial fraction (for Bax) isolated from control and I3C treated cells were loaded on a 12% SDS-PAGE gel and then, following a standard Western blot procedure, the membranes were probed with cytochrome c antibody (1:100, Clontech), Bax antibody (1 µg/mL, Trevigen) or COX-4 antibody (1:500, Clontech).
Densitometric analysis
Autoradiograms of Western blots were scanned with Gel Doc 1000 image scanner (Bio-Rad, Hercules, CA). The bidimensional optical density (O.D.) of Bcl-2, Bax, cytochrome c and actin proteins on the films were quantified and analyzed with Molecular Analyst software (Bio-Rad). The ratio of Bax/Bcl-2 (total), cytochrome c/ß-actin (cytosolic) and Bax/COX-4 (mitochondrial) were calculated by standardizing the ratios of each control to the unit value.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The effect of I3C on the growth of MCF10A and CA1a cells are depicted in Figure 1. The treatment of MCF10A and CA1a cells with 15, 30, 60 and 100 µmol/L of I3C for 24–72 h resulted in dose- and time-dependent inhibition of cell proliferation. There appeared to be more pronounced growth inhibition by low concentration of I3C in CA1a cells than in MCF10A cells. The degree of cell growth inhibition was lower in MCF10A nontumorigenic cell lines with 60 and 100 µmol/L of I3C as compared to CA1a cells ( Fig. 1). The proliferation of CA1a cells was significantly inhibited by 100 µmol/L of I3C treatment for 48 and 72 h as compared to MCF10A cells. This inhibition of cell proliferation could be due to cell cycle arrest or induction of apoptotic cell death induced by I3C. Therefore, we investigated whether I3C could induce apoptosis in these cells.
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The apoptosis was measured in both cell lines treated with 60 and 100 µmol/L of I3C by Western blot analysis of PARP. The PARP cleavage analysis showed that the full size PARP (116 KD) was cleaved to yield an 85 KD fragment after treatment with I3C in both cell lines (
Fig. 2). However, the cleaved fragment was much more pronounced in CA1a cells as compared to MCF10A cells in accordance with the data of cell growth inhibition. To quantify the apoptotic cells induced by I3C, flow cytometric analysis with 7-amino actinomycin D (7-AAD) staining was conducted. The results showed an increased number of apoptotic cells (
84% apoptotic cells,
Fig. 3) in CA1a cells treated with 60 µmol/L I3C for 72 h as compared to untreated control (16% apoptotic cells). In contrast, apoptotic cells were increased only twofold in MCF10A cells with 72 h of I3C treatment (
Fig. 3), suggesting that the degree of apoptotic cell death is much more pronounced in breast cancer cells as compared to nontumorigenic breast epithelial cells. To explore the molecular mechanism(s) by which I3C induces apoptosis, we further investigated the alterations in the expression of selected genes, which are involved in the complex processes of apoptotic cell death.
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The effects of I3C on Bcl-2 and Bax expression in MCF10A and CA1a cells were studied by Western blot analysis. The expression of Bcl-2 in I3C treated CA1a cells was found to be significantly downregulated ( Fig. 4). No significant changes in the expression of Bcl-2 were found in I3C treated MCF10A cells. Optical density measurement showed a significant increase in the ratio of Bax to Bcl-2 protein expression in CA1a cells treated with 60 or 100 µmol/L I3C for 48 h ( Fig. 4), and no such induction was observed in MCF10A cells, which is likely due to no changes in Bcl-2 protein expression. These results suggest that I3C can inhibit the anti-apoptotic activity of Bcl-2, and that increased ratio of Bax/Bcl-2 may contribute to the induction of apoptotic processes in I3C-treated breast cancer cells. Therefore, we focused our investigation on mitochondrial translocation of Bax, which is known to play important roles in the regulation of apoptotic processes.
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Treatment of cells with 30, 60 and 100 µmol/L of I3C for 6, 12, 24, 48 and 72 h resulted in a cellular redistribution of Bax as shown by immunostaining and confocal imaging. Bax had a diffuse pattern in untreated cells, and became a more punctate pattern in MCF10A and CA1a cells within 6 and 12 h after the treatment with 60 µmol/L of I3C ( Fig. 5). The punctate staining of Bax clearly suggested its mitochondrial translocation after I3C treatment, providing strong evidence that the translocation of Bax to mitochondria in I3C treated cells may lead to the induction of apoptotic processes. However, the question remains why I3C treated MCF10A cells do not show significant apoptosis, even though Bax appears to be translocated to the mitochondria. It is well known that the mitochondrial translocation of Bax leads to the loss of mitochondrial potential. Hence, we sought to investigate whether I3C treatment could result in the loss of mitochondrial potential in these cells.
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JC-1 staining was conducted to demonstrate changes in the mitochondrial potential. The dye, JC-1, is taken up by hyperpolarized mitochondria within the cell, but in depolarized mitochondria it remains within the cytosol. The mitochondrial population with varying potential could be detected by flow cytometric analysis of JC-1 stained cells due to different energetic states of mitochondria. There was a significant (p = 0.01) loss of mitochondrial potential that occurred at 6 h after 60 or 100 µmol/L of I3C treatment of CA1a cells ( Fig. 6). In contrast, there was no significant loss of mitochondrial potential when MCF10A cells were treated with 60 or 100 µmol/L of I3C for 6 h ( Fig. 6), suggesting differential effects of I3C on the loss of mitochondrial potential in breast cancer cells and nontumorigenic breast epithelial cells. The loss of mitochondrial potential also is known to be associated with the subsequent release of cytochrome c from the mitochondria to the cytoplasm of the cells. Hence, we investigated whether I3C treatment could lead to the release of cytochrome c in our experimental system.
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Cytochrome c release was significantly (p = 0.04) increased in CA1a cells within 6 h of treatment with I3C ( Fig. 7). In contrast, MCF10A cells treated with I3C failed to release cytochrome c in the cytoplasm ( Fig. 7), providing further direct evidence for the differential effects of I3C on apoptotic processes in tumorigenic and nontumorigenic breast epithelial cells. In addition, we found significantly increased levels of mitochondria-associated Bax (p < 0.05) in CA1a cells treated with 60 or 100 µmol/L of I3C for 6 h as compared to MCF10A cells when corrected for cytochrome c oxidase (COX-4) levels ( Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bax translocation from cytosol into mitochondria has been known as a critical event that occurs during apoptotic processes ( 21, 22). In this study, we observed translocation of Bax from cytosol into mitochondria in both MCF10A nontumorigenic and MCF10CA1a cancer cells treated with I3C. However, no significant apoptosis was observed in MCF10A nontumorigenic cells treated with I3C, suggesting that Bax translocation from cytosol into mitochondria alone is not sufficient to induce apoptotic cell death. It has been reported that the translocation of Bax from cytosol into mitochondria targets the mitochondrial intermembrane contact sites, causing the mitochondrial permeability transition, loss of mitochondrial potential, release of cytochrome c, subsequent activation of caspases and DNA fragmentation, resulting in apoptotic cell death ( 22– 25). We found that I3C failed to induce the loss of mitochondrial potential and the release of cytochrome c in MCF10A nontumorigenic cells, even though Bax was translocated from cytosol into mitochondria upon I3C treatment, suggesting that I3C-induced loss of mitochondrial potential is a more important and direct event for the release of cytochrome c and induction of apoptosis in cancer cells. The fact that I3C selectively induces the loss of mitochondrial potential, the release of cytochrome c, and apoptosis in CA1a breast cancer cells rather than MCF10A nontumorigenic cells, makes I3C an ideal agent for preventive and/or therapeutic purposes against breast cancer.
Akt and NF-
B pathways are two important cell-signaling pathways that are related to the Bcl-2 family and apoptotic processes. Akt exerts its antiapoptotic effects through several downstream targets, including the proapoptotic Bc1-2 family member Bad, Forkhead transcription factors and the cyclic AMP response element-binding protein (CREB) (
28–
30). It has been shown that Akt inhibits a conformational change in Bax protein and prevents its translocation into mitochondria, thus inhibiting the disruption of the mitochondrial inner membrane potential, caspase-3 activation, and apoptosis (
31,
32), and that the inactivation of Akt will facilitate the translocation of Bax from cytosol to mitochondria. It has been reported that NF-B exerts its antiapoptotic function in these cells through the control of the Bcl-2 family of proteins (
33,
34). The studies from our laboratory have shown that I3C inhibits Akt kinase activity and NF-B activity in I3C treated prostate cancer cells (
9). Thus, the Bax translocation from cytosol to mitochondria in breast cancer cells treated with I3C may be induced through the inhibition of the Akt pathway. The induction of apoptosis in I3C treated breast cancer cells also may be mediated by the inhibition of NF-B pathway and the regulation of the Bcl-2 family of protein. Further investigations are needed to establish the relationship between the Akt pathway and Bax translocation in our system.
In conclusion, our data provide some important evidences in favor of I3C-induced apoptosis in breast cancer cells that are mediated through regulation of the Bcl-2 protein family, induction of Bax translocation, loss of mitochondrial potential and release of cytochrome c. This may be the molecular mechanisms by which I3C inhibits cell growth and induces apoptosis in breast cancer cells. However, more studies are needed to address the differences on the effects of I3C between breast cancer cells and nontumorigenic breast epithelial cells, and to delineate the molecular mechanism of action of I3C against breast cancer. Finally, we hope that the tools of genomics and proteomics will play an important role in facilitating such discoveries.
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FOOTNOTES |
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3 Abbreviations used: EH, epoxide hydrolases; GST, glutathione S-transferases; I3C, indole-3-carbinol; PARP, poly(ADP-ribose)polymerase.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Manson, M. M., Hudson, E. A., Ball, H. W., Barrett, M. C., Clark, H. L., Judah, D. J., Verschoyle, R. D. & Neal, G. E. (1998) Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver—predicting the outcome using early biomarkers. Carcinogenesis 19: 1829–1836.
2. Xu, M., Bailey, A. C., Hernaez, J. F., Taoka, C. R., Schut, H. A. & Dashwood, R. H. (1996) Protection by green tea, black tea, and indole-3-carbinol against 2-amino-3-methylimidazo[4,5-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344 rat. Carcinogenesis 17: 1429–1434.
3. He, Y. H., Smale, M. H. & Schut, H. A. (1997) Chemopreventive properties of indole-3-carbinol (I3C): inhibition of DNA adduct formation of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), in female F344 rats. J. Cell. Biochem. 27(Suppl.): 42–51.
4. He, Y. H., Friesen, M. D., Ruch, R. J. & Schut, H. A. (2000) Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats. Food Chem. Toxicol. 38: 15–23.[Medline]
5. Bradlow, H. L., Michnovicz, J., Telang, N. T. & Osborne, M. P. (1991) Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12: 1571–1574.
6. Malloy, V. L., Bradlow, H. L. & Orentreich, N. (1997) Interaction between a semisynthetic diet and indole-3-carbinol on mammary tumor incidence in Balb/cfC3H mice. Anticancer Res. 17: 4333–4337.[Medline]
7. Vang, O., Jensen, M. B. & Autrup, H. (1990) Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone. Carcinogenesis 11: 1259–1263.
8. Wallig, M. A., Kingston, S., Staack, R. & Jefferey, E. H. (1998) Induction of rat pancreatic glutathione S-transferase and quinone reductase activities by a mixture of glucosinolate breakdown derivatives found in Brussels sprouts. Food Chem. Toxicol. 36: 365–373.[Medline]
9. Chinni, S. R. & Sarkar, F. H. (2002) Akt inactivation is a key event in indole-3-carbinol-induced apoptosis in PC-3 cells. Clin. Cancer Res. 8: 1228–1236.
10. Chinni, S. R., Li, Y., Upadhyay, S., Koppolu, P. K. & Sarkar, F. H. (2001) Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene 20: 2927–2936.[Medline]
11. Rahman, K. M., Aranha, O., Glazyrin, A., Chinni, S. R. & Sarkar, F. H. (2000) Translocation of Bax to mitochondria induces apoptotic cell death in indole-3-carbinol (I3C) treated breast cancer cells. Oncogene 19: 5764–5771.[Medline]
12. Meng, Q., Goldberg, I. D., Rosen, E. M. & Fan, S. (2000) Inhibitory effects of Indole-3-carbinol on invasion and migration in human breast cancer cells. Breast Cancer Res. Treat. 63: 147–152.[Medline]
13. Rahman, K. M. & Sarkar, F. H. (2002) Steroid hormone mimics: molecular mechanisms of cell growth and apoptosis in normal and malignant mammary epithelial cells. J. Steroid Biochem. Mol. Biol. 80: 191–201.[Medline]
14. Cover, C. M., Hsieh, S. J., Cram, E. J., Hong, C., Riby, J. E., Bjeldanes, L. F. & Firestone, G. L. (1999) Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. Cancer Res. 59: 1244–1251.
15. Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F. & Firestone, G. L. (1998) Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J. Biol. Chem. 273: 3838–3847.
16. Gross, A., McDonnell, J. M. & Korsmeyer, S. J. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13: 1899–1911.
17. Crompton, M. (2000) Bax, Bid and the permeabilization of the mitochondrial outer membrane in apoptosis. Curr. Opin. Cell Biol. 12: 414–419.[Medline]
18. Schmitt, E., Sane, A. T., Steyaert, A., Cimoli, G. & Bertrand, R. (1997) The Bcl-xL and Bax-alpha control points: modulation of apoptosis induced by cancer chemotherapy and relation to TPCK-sensitive protease and caspase activation. Biochem. Cell Biol. 75: 301–314.[Medline]
19. Nunez, G., Merino, R., Simonian, P. L. & Grillot, D. A. (1996) Regulation of lymphoid apoptosis by Bcl-2 and Bcl-XL. Adv. Exp. Med. Biol. 406: 75–82.[Medline]
20. Raisova, M., Hossini, A. M., Eberle, J., Riebeling, C., Wieder, T., Sturm, I., Daniel, P. T., Orfanos, C. E. & Geilen, C. C. (2001) The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J. Invest. Dermatol. 117: 333–340.[Medline]
21. Bedner, E., Li, X., Kunicki, J. & Darzynkiewicz, Z. (2000) Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry. Cytometry 41: 83–88.[Medline]
22. Jia, L., Patwari, Y., Srinivasula, S. M., Newland, A. C., Fernandes-Alnemri, T., Alnemri, E. S. & Kelsey, S. M. (2001) Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis. Oncogene 20: 4817–4826.[Medline]
23. Putcha, G. V., Deshmukh, M. & Johnson, E. M., Jr. (1999) BAX translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, BCL-2, and caspases. J. Neurosci. 19: 7476–7485.
24. Gao, C. F., Ren, S., Zhang, L., Nakajima, T., Ichinose, S., Hara, T., Koike, K. & Tsuchida, N. (2001) Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis. Exp. Cell Res. 265: 145–151.[Medline]
25. De Giorgi, F., Lartigue, L., Bauer, M. K., Schubert, A., Grimm, S., Hanson, G. T., Remington, S. J., Youle, R. J. & Ichas, F. (2002) The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J. 16: 607–609.
26. Howe, H. L., Wingo, P. A., Thun, M. J., Ries, L. A., Rosenberg, H. M., Feigal, E. G. & Edwards, B. K. (2001) Annual report to the nation on the status of cancer (1973 through 1998), featuring cancers with recent increasing trends. J. Natl. Cancer Inst. 93: 824–842.
27. Xu, M., Schut, H. A., Bjeldanes, L. F., Williams, D. E., Bailey, G. S. & Dashwood, R. H. (1997) Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon. Carcinogenesis 18: 2149–2153.
28. Khwaja, A. (1999) Akt is more than just a Bad kinase. Nature 401: 33–34.[Medline]
29. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R. & Nunez, G. (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278: 687–689.
30. Liu, H., Perlman, H., Pagliari, L. J. & Pope, R. M. (2001) Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF)-kappaB, Bad, or caspase activation. J. Exp. Med. 194: 113–126.
31. Tsuruta, F., Masuyama, N. & Gotoh, Y. (2002) The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J. Biol. Chem. 277: 14040–14047.
32. Yamaguchi, H. & Wang, H. G. (2001) The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene 20: 7779–7786.[Medline]
33. Gupta, S., Afaq, F. & Mukhtar, H. (2002) Involvement of nuclear factor-kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells. Oncogene 21: 3727–3738.[Medline]
34. Heckman, C. A., Mehew, J. W. & Boxer, L. M. (2002) NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells. Oncogene 21: 3898–3908.[Medline]
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