Oxidative Stress Sensitizes Bladder Cancer Cells to TRAIL Mediated Apoptosis by Down-Regulating Anti-Apoptotic Proteins

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Abstract
Purpose: TRAIL, an endogenous protein involved in immunosurveillance and a novel drug in clinical trials, is of particular interest as cancer therapy because it can induce apoptosis in cancer cells but not in normal cells. Since some cancers develop resistance to TRAIL, safe and effective methods of TRAIL sensitization are of clinical interest. We explored how chemotherapy and oxidative stress affect TRAIL sensitivity and expression of proteins in the apoptotic pathway. Materials and Methods: Sensitivity to TRAIL was assessed in viability assays. Apoptosis was measured by caspase-3/7 activity and/or nuclear condensation using Hoechst staining. Western blotting was used to determine cleavage, phosphorylation or alterations in protein expression. Results: TRAIL decreased the viability of 5637 but not of J82 or T24 bladder carcinoma cells (ATCC®). Chemotherapy with doxorubicin or cisplatin (Ben Venue Laboratories, Bedford, Ohio) decreased the expression of the anti-apoptotic protein cFLIPS and increased caspase-8 cleavage, reversing TRAIL resistance in T24 cells. Specific targeting of cFLIPS by siRNA was insufficient for sensitization to TRAIL in T24 cells. However, chemotherapy mediated TRAIL sensitization was mimicked by low concentrations of H2O2, which resulted in the phosphorylation of translation EF2 and decreased the expression of several short half-life, anti-apoptotic proteins, including FLIPS, XIAP and survivin. Conclusions: Inducing oxidative stress by low H2O2 concentrations may reverse TRAIL resistance. This warrants the further exploration of H2O2 as an adjuvant intravesical treatment to lower the apoptotic threshold of bladder cancer cells.