Doxorubicin sensitizes human bladder carcinoma cells to Fas-mediated cytotoxicity

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Abstract
The resistance of bladder carcinoma to anticancer chemotherapeutic agents remains a major problem. Hence, several immunotherapeutic approaches have been developed to treat the drug-resistant cancer cells. Fas antigen (Fas) and Fas ligand participate in cytotoxicity mediated by T lymphocytes and natural killer cells. Like Fas ligand, anti-Fas monoclonal antibody (MoAb) induces apoptosis of the cells expressing Fas. This study examined whether bladder carcinoma cells are sensitive to cytotoxicity mediated by anti-Fas MoAb and whether anticancer agents synergize with anti-Fas MoAb in cytotoxicity. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. The T24 human bladder carcinoma cell line constitutively expressed the Fas on the cell surface; however, T24 line was resistant to anti-Fas MoAb. Treatment of T24 cells with anti-Fas MoAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, treatment of T24 cells with a combination of anti-Fas MoAb and doxorubicin resulted in a synergistic cytotoxic effect. In addition, the doxorubicin-resistant T24 cells were sensitive to treatment with a combination of anti-Fas MoAb and doxorubicin. Synergy was also achieved in three other bladder carcinoma cell lines and four freshly derived human bladder carcinoma cells. Treatment with anti-Fas MoAb in combination with epirubicin or pirarubicin also resulted in a synergistic cytotoxic effect on T24 cells. The mechanisms of synergy were examined. Anti-Fas MoAb did not affect the intracellular accumulation of doxorubicin, the expression of P-glycoprotein, or the expression of the antioxidant glutathione S-transferase-π mRNA. However, treatment with doxorubicin enhanced the expression of Fas on T24 cells. This study demonstrated that treatment of bladder carcinoma cells with doxorubicin sensitized the cells to lysis by anti-Fas MoAb. The synergistic effect obtained with established doxorubicin-resistant bladder carcinoma cells and freshly isolated bladder carcinoma cells suggests that drug-resistant bladder carcinoma cells can be sensitized by doxorubicin to Fas- and Fas ligant-mediated cytotoxicity by lymphocytes. Furthermore, the sensitization required low concentrations of doxorubicin, thus supporting the in vivo application of a combination of chemotherapy and immunotherapy in the treatment of drug-resistant and/or immunotherapy-resistant bladder carcinoma. Cancer 1997; 79:1180-9. © 1997 American Cancer Society.