Tumor Necrosis Factor-a Sensitizes Prostate Cancer Cells to g-Irradiation-induced Apoptosis

LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by g-irradiation and somewhat sensitive to the deathinducing effects of tumor necrosis factor (TNF)-a. Simultaneous exposure of LNCaP cells to TNF-a and 8 Gy of irradiation was synergistic and resulted in a 3-fold increase of apoptotic cells within 72 h compared to TNF-a alone. It appeared that TNF-a sensitized the cells to irradiation because, when cells were irradiated 24 h after exposure to TNF-a, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-a exposure did not result in more cell death than after TNF-a alone. TNF-a induced expression of its own mRNA, but TNF-a mRNA induction was neither induced nor enhanced by irradiation. Activation of the transcription factor nuclear factor kB can be induced by TNF-a and has a modulating antiapoptotic effect. But enhancement of TNF-a-induced cell death by irradiation did not result from altered activation of nuclear factor kB. TNF-a treatment of LNCaP cells resulted in partial activation of caspase-8 and -6 but not caspase-3. There was only minimal poly(ADP-ribose) polymerase cleavage seen in LNCaP cells after exposure to both TNF-a and irradiation at 72 h, a time when 60% of the cells were apoptotic. Experiments with peptide inhibitors of cysteine and serine proteases suggested that caspases were the predominant mediators of apoptosis induced by TNF-a alone but that serine proteases contributed significantly to cell death induced by TNF-a plus irradiation. TNF-a increased production of ceramide in LNCaP cells 48 h after exposure. Although irradiation alone had no effect on ceramide production in LNCaP cells, TNF-a plus irradiation induced significantly more ceramide than TNF-a alone. Ceramide production did not occur immediately after exposure to TNF-a, but rather was delayed such that ceramide levels were increased only 24 h after exposure to apoptotic stimuli. Moreover, nontoxic levels of exogenous C2-ceramide sensitized LNCaP cells to irradiation similarly to TNF-a, suggesting that one mechanism by which LNCaP cells were sensitized to irradiation was by increased intracellular ceramide. Hence, ceramide generation is a critical component in radiationinduced apoptosis in human prostate cancer cells. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer.