ABSTRACT
We have previously shown that doxorubicin sensitizes prostate cancer cells to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL). Sensitization correlated with decreased expression of the antiapoptotic cellular FLICE-like inhibitor 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 kinase-independent manner. Treatment with hydrogen peroxide recapitulated the events observed after doxorubicin treatment. In addition, cells treated with hydrogen peroxide expressed less X-linked inhibitor of apoptosis protein (XIAP) and survivin which, like cFLIP(S), are short-half-life proteins with an antiapoptotic function while expression levels of DR5, caspases-8, -9, -3, and Bax are maintained. The doxorubicin-mediated decrease in cFLIP(S) and XIAP and the TRAIL-induced apoptosis were 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.
Subject(s)
Doxorubicin/pharmacology , Peptide Elongation Factor 2/metabolism , Prostatic Neoplasms/drug therapy , Apoptosis/drug effects , Apoptosis/physiology , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , Deferoxamine/pharmacology , Doxorubicin/antagonists & inhibitors , Drug Synergism , Free Radicals/metabolism , Humans , Hydrogen Peroxide/pharmacology , Iron Chelating Agents/pharmacology , Male , Peptide Elongation Factor 2/genetics , Phenotype , Phosphorylation/drug effects , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , Protein Synthesis Inhibitors/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , X-Linked Inhibitor of Apoptosis Protein/biosynthesis , X-Linked Inhibitor of Apoptosis Protein/geneticsABSTRACT
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 cFLIPL in the presence of doxorubicin. Although proteasome inhibitors increased basal levels of short cFLIP isoforms, cFLIPS declined at a similar rate in the absence or presence of proteasome inhibition during doxorubicin treatment. Ectopic expression of a cFLIPSGFP fusion protein protected PC3 cells from TRAIL-induced apoptosis in the absence or presence of doxorubicin, whereas downregulation of cFLIPS by RNA interference resulted in sensitization to TRAIL-induced apoptosis. We conclude that doxorubicin-mediated downregulation of cFLIPS, which occurs at the post-transcriptional level independent of proteasome-mediated pathways, is sufficient to enhance TRAIL sensitivity in PC3 prostate carcinoma cells.
