Ribozyme-mediated inhibition of PKCalpha sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis.

BACKGROUND: Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Calpha (PKCalpha) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCalpha by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS: An active ribozyme (PKCalphaRZ) targeting codon 4 in human PKCalpha mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCalphamutRZ) was also made by deleting G(12) from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCalpha expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS: Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50%) lower PKCalpha protein level than parental DU145 cells, whereas no reduction in PKCalpha expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS: Results of the study highlight the importance of PKCalpha in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.