title: D,L-sulforaphane-induced apoptosis in human breast cancer cells is regulated by the adapter protein p66Shc.
abstract: Cancer chemopreventive response to D,L-sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, is partly attributable to apoptosis induction, but the mechanism of cell death is not fully understood. The present study demonstrates a critical role for adapter protein p66(Shc) in SFN-induced apoptosis. Immortalized mouse embryonic fibroblasts (MEF) derived from p66(shc) knockout mice were significantly more resistant to SFN-induced apoptosis, collapse of mitochondrial membrane potential, and reactive oxygen species (ROS) production compared with MEF obtained from the wild-type mice. Notably, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to SFN-induced ROS production and apoptosis. Stable overexpression of manganese superoxide dismutase in MCF-7 and MDA-MB-231 human breast cancer cells conferred near complete protection against SFN-induced apoptosis and mitochondrial membrane potential collapse. SFN treatment resulted in increased S36 phosphorylation and mitochondrial translocation of p66(shc) in MDA-MB-231 and MCF-7 cells, and SFN-induced apoptosis was significantly attenuated by RNA interference of p66(shc) in both cells. SFN-treated MDA-MB-231 and MCF-7 cells also exhibited a marked decrease in protein level of peptidyl prolyl isomerase (Pin1), which is implicated in mitochondrial translocation of p66(shc) . However, stable overexpression of Pin1 failed to alter proapoptotic response to SFN at least in MCF-7 cells. Finally, SFN-induced S36 phosphorylation of p66(Shc) was mediated by protein kinase Cβ (PKCβ), and pharmacological inhibition of PKCβ significantly inhibited apoptotic cell death resulting from SFN exposure. In conclusion, the present study provides new insight into the mechanism of SFN-induced apoptosis involving PKCβ -mediated S36 phosphorylation of p66(shc).
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Kozue
authors.initials: K
authors.lastname: Sakao
authors.forename: Shivendra V
authors.initials: SV
authors.lastname: Singh
mesh_terms: D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D055786:Gene Knockout Techniques; D006801:Humans; D017879:Isothiocyanates; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013454:Sulfoxides; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology*
chemical_list: D016588:Anticarcinogenic Agents; D017879:Isothiocyanates; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D017382:Reactive Oxygen Species; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013454:Sulfoxides; D013861:Thiocyanates; D013482:Superoxide Dismutase; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase; C000606850:Pin1 protein, mouse; C016766:sulforaphane