Doxorubicin generates a proapoptotic phenotype by phosphorylation of elongation factor 2

Abstract

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.

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

“…This important observation may explain why eEF-2K activation is proapoptotic ( Figure 5) during ER stress, because a persistent block in all translational elongation would be expected to promote cell death. Consistent with this model, inactivating eEF-2 in other ways, such as doxorubicin-induced phosphorylation 55 or diphtheria toxin-mediated ADP ribosylation 56,57 of eEF-2, also causes cell death, whereas potentiating eEF-2 activity can protect cells from some apoptotic stimuli. 58 Therefore, eEF-2K may promote cell death during ER stress via its effect on translation elongation.…”

ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

“…Because doxorubicin also causes RNA damage 10 and inhibits DNA and RNA synthesis, 11,12 it is not unexpected that doxorubicin would also inhibit the synthesis of proteins. 13,14 In addition to inhibition of protein translation, doxorubicin induces the activation of SAPKs in a number of normal cell types, including hepatocytes, 6 primary mouse macrophages 7 and cardiomyocytes. 8,9 Our work presented here demonstrates that doxorubicin inhibits protein synthesis (Fig.…”

Doxorubicin Exerts Cytotoxic Effects through Cell Cycle Arrest and Fas-Mediated Cell Death

“…Thus, Fas proteins, which are increased by DOX, interact with FasL on T cells and result in Fas-mediated death of T cells. Furthermore, DOX changes the expression pattern of a multitude of genes involved in cell cycle regulation (p53, CDK10, and Gadd45) and apoptosis and cell damage (Fas and Bax) [13][14][15][16] . The p53 tumor suppressor gene, which is an essential transcription factor involved in cell cycle checkpoints, upregulates cell cycle modulators such as the cyclin-dependent kinase inhibitor p21 and GADD45, which is rapidly induced by genotoxic reagents [17] .…”