Nigericin enhances mafosfamide cytotoxicity at low extracellular pH

The cytotoxicity of many alkylating anticancer drugs is increased at reduced intracellular pH (pHi). The therapeutic index of such agents could therefore be improved by lowering pHi in the target cells prior to their application. We have previously demonstrated that the formation of lactic acid can be selectively enhanced in malignant tissues via glucose-mediated stimulation of tumor cell glycolysis. However, the resulting reduction in pHi is partly compensated by the extrusion of H+ equivalents into the extracellular space, with pHi remaining closer to the physiological value than extracellular pH (pHe). For full exploitation of the proton-mediated increase in the cytotoxicity of alkylating agents, pHi should therefore be equilibrated with pHe in lactic acid-producing cells. In the present study we investigated the question as to whether nigericin, an H+/K+ antiporter enabling the entry into cells of H+ ions at low pHe, can be used to enhance the cytotoxic effect of mafosfamide (MAFO; a precursor of "activated" cyclophosphamide) on cultured M1R rat mammary carcinoma cells. At pHe 7.4, the cytotoxic effect of combined treatment with MAFO and nigericin was not superior to treatment with MAFO alone. At acidic pHe, however, MAFO cytotoxicity was potentiated by nigericin as indicated by the colony-forming capacity of M1R cells. For example, at pHe 6.2 (corresponding to the approximate mean "aggregated pH" in actively glycolyzing tumors), the colony-forming fraction of cells treated with a combination of MAFO and nigericin was 3 x 10(-5) that of controls, as compared with a value of 5 x 10(-2) found for cells exposed to MAFO alone. These results suggest that agents counteracting cellular mechanisms that control pHi may be candidate compounds for investigations aimed at the enhancement of alkylating drug cytotoxicity following glucose-mediated pH reduction in malignant tumors in vivo.