Inhibitors of intracellular pH regulation induce cisplatin resistance in EMT6 mouse mammary tumor cells.

Previous results from this laboratory indicated that EMT6 cells treated with inhibitors of the intracellular pH (pHi) regulatory mechanisms were resistant to cisplatin (Laurencot, C.M.; Kennedy, K.A. The effect of low pH on the cytotoxicity of cisplatin in EMT6 mouse mammary tumor cells. Oncol. Res. 7:371-380; 1995.) This inhibitor-induced cisplatin resistance was independent of pH. The purpose of the research presented here was to characterize further cisplatin resistance observed in cells cultured with the Na+/H+ antiport inhibitor 5-(N,N hexamethylene) amiloride (NHMA) and the HCO3-/Cl- exchanger inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). Using [195mPt]-DDP, the accumulation of cisplatin into EMT6 mouse mammary tumor cells treated with NHMA and SITS was not changed. Total DNA cross-link formation and DNA-interstrand cross-link formation, however, decreased in NHMA- and SITS-treated cells. Since cisplatin accumulation was unchanged in NHMA- and SITS-treated cells but the amount of DNA cross-links decreased, the intracellular activation of cisplatin appeared to be altered in the cisplatin-resistant cells. Because SITS interferes with chloride transport and chloride has been proposed to be involved in cisplatin activation, cisplatin toxicity in EMT6 cells was evaluated in chloride-deficient medium. EMT6 cells cultured in chloride-deficient medium were less sensitive to cisplatin than cells cultured in chloride-containing medium, but this sensitivity was not altered by NHMA and SITS. Furthermore, the resistance to cisplatin in cells treated with NHMA and SITS was similar in chloride-containing and chloride-deficient medium. These data suggest that the concentration of other ions, in addition to chloride, may be important for cisplatin toxicity.