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Targeted Therapies and Clinical Translational Research: Drug Sensitivity and Resistance

TRAIL sensitization of PC3 prostate cancer cells by doxorubicin can be blocked by the anti-oxidant N-acetyl-cysteine or by the iron chelator deferoxamine

Medical University of South Carolina, Charleston, SC

Abstract

B59

The purpose of this study was to investigate the mechanism whereby doxorubicin sensitizes cancer cells to TNF-Related Apoptosis Inducing Ligand (TRAIL). We used PC3 prostate cancer cells which are partially resistant to TRAIL but can be more fully sensitized with doxorubicin treatment. Doxorubicin downregulates the short form of the anti-apoptotic protein Flice-Like Inhibitory Protein (FLIPs), which correlates with sensitization to TRAIL. The mechanism of FLIPs downregulation is not transcriptional or mediated by ubiquitination. Rather, doxorubicin globally inhibits translation suggesting that short half-life proteins such as FLIPs would disappear quickly. Polysome and Western blot analysis indicate that translation initiation is not inhibited. Elongation factor 2 (EF2), which mediates translocation in peptide chain elongation, is inactivated by phosphorylation. We found that doxorubicin treatment results in strong EF2 phosphorylation and therefore translational elongation became the focus of our mechanism study. EF2 is normally phosphorylated by EF2 kinase. However, EF2k is not the likely cause of doxorubicin-mediated phosphorylation because, as a short half life protein, it is also quickly downregulated by doxorubicin. As an alternative, we explored whether EF2 phosphorylation is the result of oxidative stress. When doxorubicin is administered, it binds to cellular iron, generating free radicals through Fenton chemistry. We demonstrate with hydrogen peroxide treatments that free radicals can both phosphorylate EF2 and downregulate FLIPs. Further, these effects of doxorubicin can be abrogated through the pretreatment of cells with an anti-oxidant to scavenge free radicals, N-Acetyl-Cysteine (NAC), or an iron chelator to preclude the doxorubicin/iron complex, deferoxamine (DFO). Upon pretreatment with NAC or DFO, doxorubicin treatment fails to downregulate FLIPs, phosphorylate EF2, or sensitize cells to TRAIL as shown by Western blot. However, NAC and DFO do not abrogate the G2 cell-cycle arrest that results from doxorubicin treatment. This result indicates that the arrest is likely due to the DNA damaging aspect of doxorubicin activity rather than the iron-chelation aspect. This observation may help explain why patients have a wide range of responses to doxorubicin treatment. Anti-oxidants may inadvertently protect tumors from the effectiveness of doxorubicin while a patient would still suffer the side effects of cell cycle arrest such as anemia, hair loss, and immune suppression. This potential clinical outcome merits further study. Tumors may require initial protein profiling before treatment decisions are made in order to best tailor treatment and adjunct therapy to oncogenes specific to the tumor.