The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects

Abstract

Mean values of extracellular pH (pHe) in tumours tend to be about 0.5 pH units lower than in normal tissues, whereas values of intracellular pH (pHi) in tumours and normal tissues are similar. Previous studies have shown that drugs that acidify cells at lower pHe such as nigericin, used alone or with agents that inhibit the regulation of pHi, have toxicity to cultured cells at pHe < 6.5 in short-term exposure; these agents also lead to modest anti-tumour effects in mice when given acutely. To evaluate the long-term effects of these drugs at levels of pHe that might occur commonly in tumours, we exposed cells for up to 72h at pHe 6.8 or 7.2 in vitro. Nigericin (0.033 microM) caused time-dependent cell killing of murine KHT and EMT-6 cells at pHe 6.8 (but not at pHe 7.2) with a surviving fraction approximately 5 x 10(-3) after 72 h exposure. Cell killing was increased in the presence of 4,4-diisothiocyanstilbene 2,2-disulphonic acid (DIDS), an inhibitor of Na+-dependent HCO3-/CI- exchange, and to a lesser extent in the presence of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of Na+/H+ exchange. Cell killing was exquisitely sensitive to the level of pHe. Osmotic pumps were used to obtain a 72 h continuous infusion of nigericin in mice; this led to dose-dependent killing of cells in KHT tumours with surviving fraction of approximately 0.1 at maximum tolerated doses. Hydralazine, which may cause tumour hypoxia and lower pHi as well as pHe, caused cytotoxity when given alone by chronic infusion, and enhanced the cytotoxicity due to nigericin. The addition of DIDS and/or EIPA (using two pumps) further enhanced anti-tumour toxicity, with a surviving fraction of approximately 0.002 at tolerated doses of the four drugs used to treat KHT tumours. The experiments demonstrate the activity of drugs that inhibit the regulation of pHi against murine tumours when delivered by chronic infusion.