Synergistic effect of tumor necrosis factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant human ovarian tumor cell lines

Recent studies have demonstrated that diphtheria toxin (DTX) also mediates target cell lysis, and the mechanism of cytotoxicity has many features similar to those of cytotoxicity mediated by TNF-alpha. Thus, we hypothesized that DTX and TNF-alpha, used in combination, may result in either additive or synergistic cytotoxic activity. This was examined on three human ovarian carcinoma cell lines chosen for their differing sensitivities to TNF-alpha and DTX, i.e., 222, which is sensitive to both TNF-alpha and DTX, 222TR, a TNF-alpha-resistant DTX-sensitive variant of 222, and SKOV-3, which is resistant to both DTX and TNF-alpha. The simultaneous use of DTX and TNF-alpha at suboptimal concentrations resulted in synergistic cytotoxic activity against all three lines tested, thus overcoming the TNF-alpha resistance of 222TR and the double resistance of SKOV-3. DNA fragmentation was observed in all three lines treated with DTX and TNF-alpha and occurred as early as 4 h after treatment. Cycloheximide, actinomycin D, or emetine, at concentrations causing greater than 90% protein synthesis inhibition, did not result in cytotoxicity alone or synergy with TNF-alpha, suggesting that synergy by DTX was not due to its ability to inhibit protein synthesis. The use of energy poisons and pH conditions that inhibit DTX-mediated cytotoxicity resulted in the abrogation of synergy. These findings show that the two cytotoxic agents TNF-alpha and DTX, when used at suboptimal concentrations, synergize in their cytotoxic activity against sensitive and resistant cell lines. Because the SKOV-3 cell line used here is also resistant to chemotherapeutic drugs, combination treatment with DTX and TNF-alpha may be beneficial in overcoming drug resistance.