Interactions of vitamin D analogue CB1093, TNF alpha and ceramide on breast cancer cell apoptosis

Mechanisms by which Vitamin D analogues promote apoptosis in tumour cells a re unclear. In this study we have examined possible interactions between th e synthetic vitamin D analogue CB1093 and two other known mediators of apop tosis, TNF alpha and ceramide, in MCF-7, T47D and Hs578T breast cancer cell s. These studies indicated that cytosolic phospholipase A(2) (cPLA(2)) is i nvolved in CB1093 as well as TNF alpha -mediated cell death. CB1093 promote d both TNF alpha and ceramide-induced c-PLA(2) activation, which was invers ely related to loss of cell viability in MCF-7 and Hs578T cells. TNF alpha alone (5-20 ng/ml) failed to induce cytotoxicity and activation of cPLA(2) in T47D cells. However, pretreatment of these cells with CB1093 potentiated C-2-ceramide-induced cPLA(2) activation and cell death. Treatment with CB1 093 alone induced loss of cell viability and DNA fragmentation in all three cell lines by 5 days and these effects were accompanied by activation of c PLA(2). Furthermore, co-treatment with the cPLA(2) inhibitor AACOCF(3) led to partial protection against loss of cell viability induced by CB1093 in H s578T and T47D cells as well as MCF-7 cells. The broad-spectrum caspase inh ibitor z-VAD-fmk prevented TNF alpha but not C-2-ceramide and CB1093-mediat ed release of arachidonic acid and cell death in MCF-7 cells. These results indicate that CB1093 potentiates responsiveness of breast cancer cells to TNF alpha and suggest that ceramide and/or cPLA(2) might be involved as dow nstream effecters in vitamin D-mediated caspase-independent cell death. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.