Translocation of Bax to mitochondria induces apoptotic cell death in Indole-3-carbinol (I3C) treated breast cancer cells

Abstract

Epidemiological studies have suggested that the consumption of fruits and vegetables that provide several classes of compounds, including Indole-3-carbinol (I3C), may have chemopreventive activity against breast cancer. Several in vitro and in vivo animal studies also provide convincing evidence for the anti-tumor activity of I3C, however, the molecular mechanism(s) by which I3C exerts its biological e ects on breast cancer cells has not been fully elucidated. In this study, we investigated the e ects of I3C in Her-2/neu over-expressing MDA-MB-435 breast cancer cells and compared these results with parental cells transfected with control vector. We focused our investigation in elucidating the molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells. Our data show that I3C inhibits breast cancer cell growth in a dose dependent manner in Her-2/neu overexpressing and in normal Her-2/neu expressing cells. Induction of apoptosis was also observed in these cell lines when treated with I3C, as measured by poly (ADPribose) polymerase (PARP) and caspase-3 activation. In addition, we found that I3C up-regulates Bax, downregulates Bcl-2 and, thereby, increased the ratio of Bax to Bcl-2 favoring apoptosis. These results suggest that the alteration in the expression of these genes may play an important role in mediating the biological e ects of I3C. Moreover, we also show the cellular localization of Bax by confocal microscopy, which showed di use distribution of Bax throughout the cytoplasmic compartment in breast cancer cells in control culture. However, in I3C treated cells, Bax showed a punctate pattern of distribution that was localized in the mitochondria. From these results, we conclude that the over-expression and translocation of Bax to mitochondria causes mitochondrial depolarization and activation of caspases, which may be one of the mechanism(s) by which I3C induces apoptotic processes in I3C treated breast cancer cells. Overall, our present data provide a novel molecular mechanism(s) by which I3C elicits its biological e ects on both Her-2/neu over-expressing and with normal Her-2/neu expressing breast cancer cells, suggesting that I3C could be an e ective agent in inducing apoptosis in breast cancer cells. Oncogene (2000) 19, 5764 ± 5771.

Chemoprevention of Cigarette Smoke–Induced Alterations of MicroRNA Expression in Rat Lungs

“…MiR-125a-prec recognizes as a main target the Erbb2 oncogene, which plays a fundamental role in breast cancer (40) and is also expressed in lung carcinomas (41). I3C exerts antiproliferative and apoptotic effects in breast cancer cells expressing Erbb2 (42). It is noteworthy that I3C was the only chemopreventive agent that had some effect on the ECS-related overexpression of miR-294 , which is involved in gene transcription and recognizes transcriptional repressor genes (e.g., zinc finger protein 697; AT-rich interactive domain 4A) as main targets.…”

Mechanisms and Therapeutic Implications of Cell Death Induction by Indole Compounds

“…As a mechanism of apoptosis induction, I3C has been to shown to down-regulate anti-apoptotic gene products (Bcl-2, Bcl-X L , survivin, inhibitor-of-apoptosis protein; IAP, X chromosome-linked IAP; XIAP, Fas-associated death domain protein-like interleukin-1-beta-converting enzyme inhibitory protein; FLIP), up-regulate pro-apoptotic factors (such as Bax), release mitochondrial cytochrome C as well as activate caspase-9 and caspase-3 [26-37]. In human cancer cell models, indoles (I3C/DIM) have been shown to induce apoptosis in breast [27,28,30-34,38-42], squamous cell carcinoma [43], cholangiocarcinoma [44], colon [45-49], cervical [37,50], ovarian [51], pancreatic [52,53] and prostate [29,54-57] cancer cells.…”

“…Our own investigations with apoptosis-induction by indoles, with focus on modulation of pro-Vs. anti-apoptotic factors have revealed that indoles can inhibit Bcl-2 and Bcl-X L protein expression in prostate and breast cancer cells [30,31]. The ratio of Bax to Bcl-2 was significantly increased after just 24 hours of treatment which corresponded with a jump in the number of apoptotic cells.…”

“…It is believed that the ratio of Bax:Bcl-2, rather than Bcl-2 alone, is important for the survival of drug-induced apoptosis [71]. We also analyzed the intracellular movement and distribution of Bax by confocal microscopy and found Bax translocation to the mitochondria in breast cancer cells, which caused the mitochondrial depolarization and release of cytochrome c [30]. These alterations, in conjunction with alteration in Bid and Bad, contributed to the activation of caspase adapter Apaf-1 and generation of active caspases (caspase-9 and caspase-3) during I3C induced apoptosis.…”

“…These alterations, in conjunction with alteration in Bid and Bad, contributed to the activation of caspase adapter Apaf-1 and generation of active caspases (caspase-9 and caspase-3) during I3C induced apoptosis. No apoptosis was seen in non-tumorigenic cells treated with I3C [30,31] suggesting that translocation of Bax to mitochondria activates the mitochondrial death pathway in I3C-induced apoptosis in breast cancer cells but not in non-tumorigenic breast epithelial cells. In human melanoma cells, I3C has been shown to down-regulate Bcl-2 and activate caspases (caspase-8 and caspase-3) [72].…”