Potentiation by vitamin D analogs of TNF alpha and ceramide-induced apoptosis in MCF-7 cells is associated with activation of cytosolic phospholipaseA(2)

Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer c ells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions be tween two vitamin D analogs (CB1093 and EB1089) and known mediators of apop tosis, TNF alpha and ceramide, Pretreatment of MCF-7 breast cancer cells wi th CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFa as assessed by cell viability assay, DNA fragmentation and videomicroscopy, N o significant chan ges in the levels of TNF alpha or TNF-RI transcripts wer e detected. CB1093 primed cells demonstrated enhanced responsiveness to cel l permeable C-2-ceramide in terms of increased DNA fragmentation and loss o f cell viability. Activation of cytosolic phospholipase A(2) (cPLA(2)) has been implicated in TNF alpha-mediated apoptosis, As assessed by [H-3]-arach idonic acid release, cells primed for 48 h with CB1093 (50 nM)showed enhanc ed cPLA(2) activation in response to TNF alpha or ceramide. CB1093 treatmen t alone led to cPLA(2) activation and loss of cell viability which was inhi bited by the specific inhibitor AACOCF(3), These results suggest that TNF a lpha and vitamin D analogs share a common pathway leading to apoptosis invo lving cPLA(2) activation and/or ceramide generation.