C-Jun N-terminal kinase is required for phorbol ester- and thapsigargin-induced apoptosis in the androgen responsive prostate cancer cell line LNCaP

In early, androgen dependent stages of prostate cancer, androgen withdrawal, the major course of therapy in prostate cancer, leads to a rapid regression of the tumor as a result of apoptosis. However, prostate cancer invariably progresses to an androgen independent and apoptosis resistant stage for which no curative treatment is available. The molecular details of regression upon androgen withdrawal and progression to a resistant state are largely unknown. Here we show that c-Jun N-terminal Kinase (JNK) is activated strongly and in a sustained fashion by 12-O-tetradecanoylphorbol 13-acetate (TPA) and thapsigargin (TG), two agents which were previously shown to lead to apoptosis in the androgen responsive prostate cancer cell line LNCaP. The time course of JNK induction by both compounds correlated very well with the onset and progression of apoptosis in LNCaP cells. Inhibition of either ERK or p38 pathways did not affect TPA-induced cell death. In the androgen-independent prostate cancer cell lines DU-145 and PC-3, and in the cervical carcinoma cell line HeLaS3, TPA did not lead to apoptosis and there were no significant changes in JNK activity upon TPA treatment. The failure of TPA to induce JNK activity in PC-3, DU-145, and HelaS3 cells was not due to a general defect in JNK signaling since ultraviolet (UV) irradiation dramatically increased JNK activity in all four cell lines. Specific inhibition of JNK by expression of the JNK Inhibitory Protein (JIP) dramatically inhibited both TPA- and TG-induced apoptosis. Furthermore, apoptosis induced by both agents was completely blocked by ectopic expression of the baculovirus caspase-inhibitor P35. Surprisingly, zVAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases, had no effect on TPA-induced apoptosis, whereas it completely inhibited TG-induced cell death; JNK activity was not affected in either case. This indicates that zVAD-fmk does not inhibit some of the caspases involved in TPA-induced apoptosis, and that despite the common requirement of JNK activation, TPA- and TG-induced cell death are mechanistically different. Furthermore, it also suggests that JNK is either upstream or independent of caspases in LNCaP cells. Collectively, these results indicate that apoptosis in LNCaP cells requires a sustained increase in JNK activity and caspase activation; components of these signaling pathways may be defective in the androgen independent prostate cancer cell lines.