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We have previously shown that doxorubicin sensitizes prostate cancer cells to TNF-Related Apoptosis Inducing Ligand (TRAIL). Sensitization correlated with decreased expression of the antiapoptotic protein cFLIP S . The decrease in cFLIP S could not be explained by transcriptional regulation or increased degradation, leading us to focus on translational mechanisms. In this study, we found that doxorubicin caused strong and sustained phosphorylation of elongation factor 2 (EF-2), which interferes with protein elongation. Phosphorylation of EF-2 appeared to occur in a kinaseindependent manner. Treatment with hydrogen peroxide recapitulated the events observed after doxorubicin treatment. In addition, cells treated with hydrogen peroxide expressed less XIAP and survivin which, like cFLIP S , are short half-life proteins with an anti-apoptotic function while expression levels of DR5, caspases-8, -9, -3, and Bax are maintained. The doxorubicin-mediated decrease in cFLIP S and XIAP as well as TRAIL-induced apoptosis was prevented by pretreatment with an iron chelator, indicating that expression of these proteins was affected by free radical generation upon interaction of iron with doxorubicin. In conclusion, our data suggest that free radicals can affect the phosphorylation of EF-2 resulting in a net loss of short half-life proteins such as cFLIP S and XIAP, leaving a cell more vulnerable to apoptotic stimuli.

The pro-apoptotic death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received significant attention as a novel cancer therapeutic, since it selectively induces apoptosis in malignant and not normal cells. Unfortunately, prostate cancer cells display little if any susceptibility to TRAIL-induced apoptosis. However, sensitivity to TRAIL is enhanced by doxorubicin, which correlated with a decrease in expression of the caspase-8 inhibitor cFLIP (Kelly et al., Cancer Biol Ther 1:520). In this study we show that doxorubicin induces a time-and dose-dependent loss of cFLIP protein, but does not affect steady-state mRNA levels. Proteasome inhibition stabilized cFLIP L in the presence of doxorubicin. Although proteasome inhibitors increased basal levels of short cFLIP isoforms, cFLIP S declined at a similar rate in the absense or presence of proteasome inhibition during doxorubicin treatment. Ectopic expression of a cFLIP S GFP fusion protein protected PC3 cells from TRAIL-induced apoptosis in the absence or presence of doxorubicin, whereas downregulation of cFLIP S by RNA interference resulted in sensitization to TRAIL-induced apoptosis. We conclude that doxorubicin-mediated downregulation of cFLIP S , which occurs at the post-transcriptional level independent of proteasome-mediated pathways, is sufficient to enhance TRAIL sensitivity in PC3 prostate carcinoma cells.

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