Doxorubicin generates a proapoptotic phenotype by phosphorylation of elongation factor 2.
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.
White
Shai J
SJ
Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA.
Kasman
Laura M
LM
Kelly
Margaret M
MM
Lu
Ping
P
Spruill
Laura
L
McDermott
Paul J
PJ
Voelkel-Johnson
Christina
C
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000096:biosynthesis / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D004317:Doxorubicin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004357:Drug Synergism; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D008297:Male; D020652:Peptide Elongation Factor 2 / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000096:biosynthesis / Q000235:genetics
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D005609:Free Radicals; D007502:Iron Chelating Agents; D020652:Peptide Elongation Factor 2; D011500:Protein Synthesis Inhibitors; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D003676:Deferoxamine