Cellular Responses to Cancer Chemopreventive Agent D,L-Sulforaphane in Human Prostate Cancer Cells Are Initiated by Mitochondrial Reactive Oxygen Species

Purpose  Present study was undertaken to elucidate the mechanism of cellular responses to D,L-sulforaphane (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 SFN-induced 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. SFN-induced 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 significantly 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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This investigation was supported in part by the USPHS grants CA115498 and CA101753 (to S.V.S.), awarded by the National Cancer Institute, and grant 2718/P01/2007/32 (to A.H-A.), awarded by the Polish Ministry of Science and Higher Education. Dong Xiao and Anna A. Powolny contributed equally.