Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane.

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.

Apoptosis in cervical cancer cells: implications for adjunct anti-estrogen therapy for cervical cancer.

BACKGROUND: Many tumors show dependence on estrogen for growth and establishment of drug resistance. We examined the effects of estrogen on cervical cancer cells exposed to apoptotic agents including drugs used for treatment. MATERIALS AND METHODS: We tested the effect of estradiol on apoptosis in three cervical cancer cell lines. Apoptosis was measured by endonucleolytic degradation of DNA. Bcl-2 was measured by Western analysis. RESULTS: Estradiol reduced the percentage of cells undergoing apoptosis after exposure to the DNA-damaging agents UVB, mitomycin-C and cisplatin. Protection against taxol-induced apoptosis was marginal. Protection was independent of HPV gene expression, and not specific to apoptosis induced by DNA damage, since estradiol significantly reduced the number of apoptotic cells produced after exposure to indole-3-carbinol (I3C), a non-genotoxic phytochemical effective in preventing HPV-induced tumors. Higher concentrations of I3C overcame the anti-apoptotic effect of estradiol. Treatment with I3C resulted in loss of the survival protein Bcl-2, and estradiol partially reversed this effect. CONCLUSION: Estrogen protects cervical cancer cells treated with DNA-damaging agents; UVB, mitomycin-C and cisplatin, from apoptotic death. For I3C, which induces apoptosis and is anti-estrogenic, the amount of apoptosis versus survival and the level of Bcl-2 depend on the I3C/estradiol ratio.

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells exposed to diindolylmethane in vitro.

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical cancer in animal model studies and in human clinical trials. I3C and its physiologic condensation product diindolylmethane (DIM) also induce apoptosis of tumor cells in vitro and in vivo, suggesting that these phytochemicals might be useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic acid microarray studies on transformed keratinocytes and tumor cell lines exposed to pharmacologic concentrations of DIM in vitro are consistent with a cellular response to nutritional deprivation or disruptions in protein homeostasis such as endoplasmic reticulum (ER) stress. In this report we investigate whether specific stress response pathways are activated in tumor cells exposed to DIM and whether the ER stress response might contribute to DIM's cytotoxicity. Induction of the stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78, GRP94, and asparagine synthase was documented by Western blot and real-time reverse transcriptase-polymerase chain reaction in C33A cervical cancer cells, and induction of a subset of these was also observed in cancer cell lines from breast (MCF-7) and prostate (DU145). The results are consistent with activation of more than 1 stress response pathway in C33A cells exposed to 75 microM DIM. Phosphorylation elF2alpha was rapidly and transiently increased, followed by elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional activation dependent on an ATF6-XBP-1 binding site was detected by transient expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT); induction of the GADD153 (CHOP) promoter was also confirmed by transient expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved at later times, coinciding with the onset of apoptosis. The results support the hypothesis that cytotoxic concentrations of DIM can activate cellular stress response pathways in vitro, including the ER stress response. Conversely, DIM was especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing degrees; nutrient limitation had a similar, but even more pronounced, effect. Because DIM toxicity in vitro is enhanced in cells undergoing nutritional deprivation and ER stress, it is possible that stressed cells in vivo, such as those within developing solid tumors, also have increased sensitivity to killing by DIM.

Indole-3-carbinol is a negative regulator of estrogen.

Studies increasingly indicate that dietary indole-3-carbinol (I3C) prevents the development of estrogen-enhanced cancers including breast, endometrial and cervical cancers. Epidemiological, laboratory, animal and translational studies support the efficacy of I3C. Whereas estrogen increases the growth and survival of tumors, I3C causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. Our long-range goal is to best use I3C together with other nutrients to achieve maximum benefits for cancer prevention. This study examines the possibility that induction of growth arrest in response to DNA damage (GADD) in genes by diindolylmethane (DIM), which is the acid-catalyzed condensation product of I3C, promotes metabolically stressed cancer cells to undergo apoptosis. We evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of I3C/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-alpha. Expression of GADD was evaluated by real-time reverse transcription-polymerase chain reaction. Proliferation and apoptosis were measured by a mitochondrial function assay and by fluorescence-activated cell sorting analysis. The luciferase reporter assay was used to specifically evaluate expression driven by ER-alpha. The estrogen-sensitive MCF-7 breast cancer cell line was used for these studies. We show a synergistic effect of I3C and genistein for induction of GADD expression, thus increasing apoptosis, and for decrease of expression driven by ER-alpha. Because of the synergistic effect of I3C and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phytochemical when used in combination.

Diindolylmethane alters gene expression in human keratinocytes in vitro.

Indole-3-carbinol (I3C) and its dimer 3,3'-diindolylmethane (DIM), obtained from dietary consumption of cruciferous vegetables, have multiple biochemical activities. Both compounds have been effective clinically in treating precancerous lesions of the cervix and laryngeal papillomas, pathologies with a human papillomavirus (HPV) component. Using cDNA microarrays, we examined early changes in gene expression after treatment with 100 micro mol/L DIM in C33A and CaSki cervical cancer cells and in an immortalized human epithelial cell line (HaCat), as well as in normal human foreskin keratinocytes (HFK). Multiple analyses were done after treating C33A cells for 6 h; other analyses included 4- and 12-h treatments of C33A and 6-h treatments of CaSki, HaCat and HFK cells. DIM consistently altered the expression of >100 genes at least twofold. Many of the stimulated genes encode transcription factors and proteins involved in signaling, stress response and growth. Results were comparable between transformed cells with and without integrated HPV sequences, and many of the same genes were induced in these cancer-derived cells and in noncancer cells. Eight genes encoding bZip proteins were among the most consistently and robustly induced, including the stress-associated immediate early gene GADD153 (>50 fold in C33A) and nuclear factor-interleukin 6 (NF-IL6), also known as c/EBPbeta, (>5 fold in C33A), which has been shown to reduce expression of HPV oncogenes. Induction of GADD153, NF-IL6 and ATF3 was confirmed by Western analysis. In functional analyses, DIM not only suppressed transcription of a luciferase gene driven by the HPV11 upstream regulatory region (URR) in C33A, CaSki, HaCat and HFK cells from >2-fold to 37-fold depending on the type of cells, but also reduced endogenous transcription of HPV16 oncogenes to undetectable levels in CaSki cells as determined by an RNase protection assay. Ectopic expression of GADD153 or NF-IL6 suppressed transcription in a dose-dependent manner driven by the HPV11 URR in C33A, CaSki, HaCat and HFK cells. These results identify unexpected ways in which dietary I3C and DIM invoke cellular responses and are consistent with a potential antiviral effect of DIM on keratinocytes, but they do not explain the differential sensitivity of transformed keratinocytes to apoptosis by DIM.