4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Urinary-Bladder-Neoplasms

The effect of microenvironmental factors on the regulation of intracellular pH (pHi) in MGH U1 cells and EMT-6 cells was studied using the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange were found to be present in both cell types. The activity of both exchangers was dependent on pHi, with low levels of activity at neutral pH and an increase in activity as pHi fell. The level of extracellular pH (pHe) also influenced the operation of the exchangers, with a fall in activity as pHe was reduced over the range 7.4-6.6. This effect was more marked for the Na(+)-dependent Cl-/HCO3- exchanger than for the Na+/H+ antiporter, suggesting that under conditions of reduced pHe the Na+/H+ antiporter is the major mechanism for regulation of pHi. Neither 6 h of radiobiological hypoxia nor variations in the extracellular [Ca2+] over the range 1-6 mM had an effect on the regulation of pHi, while extracellular lactate (5-10 mM) caused a small, concentration-dependent decrease in the combined activity of both exchangers. We conclude that under the microenvironmental conditions found in some regions of tumors, Na+/H+ exchange may be the major method of regulation of pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcium; Carrier Proteins; Cell Hypoxia; Chloride-Bicarbonate Antiporters; Culture Media; Humans; Hydrogen-Ion Concentration; Lactates; Mammary Neoplasms, Animal; Sarcoma; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The environment of cells within solid tumors is known to be acidic relative to that in normal tissue, and the viability of tumor cells may depend on mechanisms which maintain intracellular pH (pHi) above the extracellular pH (pHe). We have assessed therefore the toxicity in vitro of the proton ionophore carbonylcyanide-3-chlorophenylhydrazone (CCCP), since this agent has been reported to be capable of transporting H+ equivalent through artificial lipid bilayers and mitochondrial membranes. CCCP was toxic to the human bladder carcinoma cell line MGHU1 and to the murine mammary sarcoma cell line EMT-6 only at pH, less than 6.5. CCCP transported H+ equivalents through cell membranes at physiological (7.35) and low pHc (6.20). Cell lines were found to have steady-state pHi values approximately 0.1 to 0.2 pH units above pHc at pHc less than 6.50. Addition of CCCP led to a decrease in steady-state pHi values as compared to untreated cells at pHc less than 6.50, whereas there was no apparent effect of CCCP on steady-state pHi values at pHc greater than 6.50. The CCCP-induced reduction in steady-state pHi combined with the uncoupling of oxidative phosphorylation by CCCP appeared to be the major mechanisms leading to cell death at pHc less than 6.50. The toxicity of CCCP under acidic conditions was enhanced by amiloride and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid, agents which are known to inhibit membrane-based ion exchange mechanisms which regulate pHi under acidic conditions. When both agents were combined with CCCP, cell killing was observed at pHc less than 7.30. Our results suggest that mechanisms which regulate pHi under acidic conditions which occur in solid tumors may represent targets for new forms of tumor-specific therapy.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Amiloride; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma; Cell Survival; Drug Synergism; Energy Metabolism; Glycolysis; Humans; Hydrogen-Ion Concentration; Mammary Neoplasms, Experimental; Mice; Nigericin; Nitriles; Sarcoma, Experimental; Time Factors; Tumor Cells, Cultured; Urinary Bladder Neoplasms