Technical Abstract: Gossypol, a natural compound present in cottonseeds, possesses antiproliferative and proapoptotic effects in various cancer cells. The (-)-gossypol enantiomer is a more potent inhibitor of cancer cell growth. Here, we studied the molecular mechanisms of cell viability inhibition and apoptosis induced by (-)-gossypol in human prostate cancer cells. (-)-Gossypol treatment resulted in growth suppression in both primary cultured human prostate cancer epithelial cells and the prostate cancer cell line, DU-145. Inhibition of cancer cell growth was associated with down-regulation of cyclin-D1, Rb, CDK4 and CDK6, and with up-regulation of p21 and TGF-beta1 mRNA and proteins levels, as determined by RT-PCR and Western Blot analyses, respectively. Treatment of cells with 5-10 uM (-)-gossypol significantly enhanced apoptosis measured by DNA fragmentation. We determined that the apoptotic mechanisms might be correlated with down-regulation of Bcl-2 and Bcl-xL and with up-regulation of Bax. Moreover, (-)-gossypol activated caspases-3, -8, and -9 and increased PARP (poly (ADP-ribose) polymerase) cleavage. Furthermore, (-)-gossypol-induced apoptosis might be due to an increase of CAD (caspase-activated deoxyribonuclease) proteins and a decrease of ICAD (inhibitor of CAD) proteins. To further investigate the apoptotic pathways induced by (-)-gossypol, different caspase inhibitors were used to block caspase activities. The data demonstrated that (-)-gossypol resulted in apoptosis via the caspase-dependent pathways. These observations indicate that the apoptotic processes caused by (-)-gossypol in mediated by the regulation of the Bcl-2 and caspase families in human prostate cancer cells. Our data suggest that (-)-gossypol will have chemopreventive benefits in prostate cancer patients as well as in healthy individuals.