Molecular mechanisms of gossypol-mediated regulation of apoptosis in human prostate cancer cells

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Prostate cancer is the most malignant cancer in men and is the second leading cause of male cancer death in the U. S. Gossypol (GP), a natural compound present in cottonseeds, exists as (±)enantiomers and has been shown to possess antiproliferative and pro-apoptotic effects on various cancer cells. The (−)GP is a more potent inhibitor for cancer cell growth. Our preliminary results showed that (−)GP inhibited prostate cancer cell proliferation and down-regulated Bcl-2 mRNA expression. Our current study examined the molecular mechanisms of anti-proliferation and induction of apoptosis by (−)GP in the human prostate cancer cell line, DU 145. After a 24 hour treatment with (−)GP at 0 to 5μM, apoptosis was detected by DNA fragmentation. (−)GP mediated apoptotic mechanisms were measured by immunoblot analysis. Different caspase inhibitors were used to investigate the apoptotic pathways induced by (−)GP. Our results show that (−)GP significantly induced DNA fragmentation in the DU 145 cells and appeared a dose-dependent manner. (−)GP at 5μM inhibited cell proliferation involved in cyclin D1, Rb and CDK4/6 down-regulation at least 50% compared with control. (−)GP-induced apoptosis was mediated by the down-regulation of the anti-apoptotic Bcl-2 members Bcl-2 and Bcl-xL by 70% and 50%, respectively and by up-regulation of the pro-apoptotic Bcl-2 member Bax more than 2 folds. Moreover, (−)GP activated caspase-3, 7, 8 and 9 about 40% compared with control and increased PARP, DFF40 and DFF45 levels at least 50% when treated with (−)GP at 5μM. Finally, experimental data generated by using caspase inhibitors demonstrated that (−)GP induce apoptosis through the mitochondrial pathway as well as the death receptor pathway. These observations imply that (−)GP induces the apoptotic process through regulation of Bcl-2 and caspase families in human prostate cancer cells. Recently, a clinical trial on advanced, metastatic breast cancer patients showed GP affected the cell cycle regulatory proteins, cyclin D1 and Rb. Researchers found that (−)GP inhibited the function of Bcl-2/xL in human prostate tumors implanted in mice and made the cancer more sensitive to radiation therapy and chemotherapy. Our previous data reported that the milk collected from GP-administrated dairy cows demonstrated antitumor activity. Furthermore, human epidemiological data show that states, like Uzbekistan, that consume large amounts of cottonseed oil have lower cancer incidence than low-cottonseed oil consumptive states such as Armenia and U.S.A. These evidences support that consumption of dietary (−)GP cottonseed oil from daily dietary intake will have chemopreventive benefits through its mechanisms mediated by inhibition of the cell cycle and by enhancing cancer cell apoptosis in the healthy individuals and in prostate cancer patients. (Supported by NIH Grants CA94718 & CA95915 and DOD Grants DAMD 8140, 0319 & 9341)