Tumor necrosis factor-alpha sensitizes prostate cancer cells to gamma-irradiation-induced apoptosis

LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by gamma-irradiation and somewhat sensitive to the death-induci ng effects of tumor necrosis factor (TNF)-alpha. Simultaneous exposure of L NCaP cells to TNF-alpha and 8 Gy of irradiation was synergistic and resulte d in a 3-fold increase of apoptotic cells within 72 h compared to TNF-alpha alone. It appeared that TNF-alpha sensitized the cells to irradiation beca use, when cells were irradiated 24 h after exposure to TNF-alpha, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-alpha exposure did not result in more cell death than after TNF-alpha a lone. TNF-alpha induced expression of its own mRNA, but TNF-alpha mRNA indu ction was neither induced nor enhanced by irradiation. Activation of the tr anscription factor nuclear factor kappa B can be induced by TNF-alpha and h as a modulating antiapoptotic effect. But enhancement of TNF-alpha-induced cell death by irradiation did not result from altered activation of nuclear factor kappa B. TNF-alpha treatment of LNCaP cells resulted in partial act ivation 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-alpha and irradiation at 72 h, a time when 60% of the cells were apopto tic, Experiments with peptide inhibitors of cysteine and serine proteases s uggested that caspases were the predominant mediators of apoptosis induced by TNF-alpha alone but that serine proteases contributed significantly to c ell death induced hy TNF-alpha plus irradiation. TNF-alpha increased produc tion of ceramide in LNCaP cells 38 h after exposure. Although irradiation a lone had no effect on ceramide production in LNCaP cells, TNF-alpha plus ir radiation induced significantly more ceramide than TNF-alpha alone. Ceramid e production did not occur immediately after exposure to TNF-alpha, but rat her was delayed such that ceramide levels were increased only 24 h after ex posure to apoptotic stimuli. Moreover, nontoxic levels of exogenous C-2-cer amide sensitized LNCaP cells to irradiation similarly to TNF-alpha, suggest ing that one mechanism by which LNCaP cells were sensitized to irradiation was by increased intracellular ceramide, Hence, ceramide generation is a cr itical component in radiation-induced apoptosis in human prostate cancer ce lls. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer.