Tumor necrosis factor-α sensitizes prostate cancer cells to γ- irradiation-induced apoptosis

LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by γ-irradiation and somewhat sensitive to the death- inducing effects of tumor necrosis factor (TNF)-α. Simultaneous exposure of LNCaP cells to TNF-α and 8 Gy of irradiation was synergistic and resulted in a 3-fold increase of apoptotic cells within 72 h compared to TNF-α alone. It appeared that TNF-α sensitized the cells to irradiation because, when cells were irradiated 24 h after exposure to TNF-α, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-α exposure did not result in more cell death than after TNF-α alone. TNF-α induced expression of its own mRNA, but TNF-α mRNA induction was neither induced nor enhanced by irradiation. Activation of the transcription factor nuclear factor κB can be induced by TNF-α and has a modulating antiapoptotic effect. But enhancement of TNF-α-induced cell death by irradiation did not result from altered activation of nuclear factor κB. TNF-α 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-α 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-α alone but that serine proteases contributed significantly to cell death induced by TNF-α plus irradiation. TNF-α increased production of ceramide in LNCaP cells 48 h after exposure. Although irradiation alone had no effect on ceramide production in LNCaP cells, TNF-α plus irradiation induced significantly more ceramide than TNF- α alone. Ceramide production did not occur immediately after exposure to TNF-α, 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-α, 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 radiation-induced apoptosis in human prostate cancer cells. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer.