Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogenindependent) human breast cancer cells in a concentrationdependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WAmediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells. [Cancer Res 2008;68(18):7661-9]

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Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent but the mechanism of its anticancer effect is not fully understood. We now show, for the first time, that PEITC treatment triggers Atg5-dependent autophagic and apoptotic cell death in human prostate cancer cells. Exposure of PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) human prostate cancer cells to PEITC resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. A normal human prostate epithelial cell line (PrEC) was markedly more resistant toward PEITC-mediated cleavage and recruitment of LC3 compared with prostate cancer cells. Although PEITC treatment suppressed activating phosphorylations of Akt and mammalian target of rapamycin (mTOR), which are implicated in regulation of autophagy by different stimuli, processing and recruitment of LC3 was only partially/marginally reversed by ectopic expression of constitutively active Akt or overexpression of mTOR-positive regulator Rheb. The PEITCmediated apoptotic DNA fragmentation was significantly attenuated in the presence of a pharmacologic inhibitor of autophagy (3-methyl adenine). Transient transfection of LNCaP and PC-3 cells with Atg5-specific small interfering RNA conferred significant protection against PEITC-mediated autophagy as well as apoptotic DNA fragmentation. A xenograft model using PC-3 cells and Caenorhabditis elegans expressing a lgg-1:GFP fusion protein provided evidence for occurrence of PEITC-induced autophagy in vivo. In conclusion, the present study indicates that Atg5 plays an important role in regulation of PEITC-induced autophagic and apoptotic cell death.

Background:The tubulin microtubule network remains an attractive anticancer target. Results: The antitumor steroidal lactone withaferin A (WA) down-regulates tubulin and binds to Cys 303 of ␤-tubulin. Conclusion: Tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. Significance: Favorable safety and pharmacokinetic profiles merit clinical investigation of WA for prevention and/or treatment of breast cancer.

Purpose-Present study was undertaken to elucidate the mechanism of cellular responses to D,Lsulforaphane (SFN), a highly promising cancer chemopreventive agent.Methods-Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis-and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry.Results-The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFNinduced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFNinduced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significant inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells.Conclusion-These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells.