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Background: Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance., Methods: We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II., Results: In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment., Conclusions: Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC.

Febrile hyperthermia enhanced TNF-stimulated apoptosis of MCF-7 cells and overcame resistance in a TNF-resistant, MCF-7 variant (3E9), increasing their TNF-sensitivity by 10- and 100-fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF-7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase-8 activation, as the specific inhibitor, Z-IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase-8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF-dependent loss of mitochondrial membrane potential and activation of capspase-9 that was initiated and dependent on upstream caspases. MCF-7 and 3E9 cells were equally sensitive to exogenous C(6)-ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro-palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF-7 cells and overcomes TNF-resistance by sustained activation of caspase-8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase-dependent apoptosis by febrile hyperthermia., (Copyright 2006 Wiley-Liss, Inc.)