Influence of Extracellular pH on the Accumulation and Cytotoxicity of N-(4-Methylphenylsulfonyl)-N′-(4-chlorophenyl)urea in Human Cell Lines

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Abstract
The effect of extracellular pH (pHe) on the accumulation and cytotoxicity of the diarylsulfonylureaantitumor agent N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (MPCU) has been examined. In ahuman colon adenocarcinoma cell line, GC3/C1, the initial rate of uptake of [3H]MPCU (2.4 μM) wasincreased by 4.5-fold as pHe was reduced from 7.4 to 6.5. Steady state levels of MPCU were inversely proportionalto pHcand were 5-fold greater at pH 6.0 compared to 7.4. Similar results were obtained using Rh30cells derived from an alveolar rhabdomyosarcoma. MPCU rapidly re-equilibrated after achieving steady statewhen pHe was altered, indicating that MPCU was not tighdy bound within cells. In both cell lines, the uncouplingagent, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), significandy reduced (GC3/C1) orcompletely inhibited (Rh30) accumulation of MPCU at each pHe, examined. Sodium azide had the sameeffect on the accumulation of MPCU as FCCP. The effects of FCCP and azide appeared to be due to collapseof the pH differential across the mitochondrial inner membrane rather than the gradient across the plasmamembrane. As extracellular pH (pHc) decreased, intracellular pH(pHi) also decreased in GC3/C1cells, suchthat the greatest pH differential (pHi - pHe) was 0.2 units at pHe, 6.0. Neither FCCP nor azide significantlyaltered this pH gradient, indicating a minor role, if any, for the plasma membrane pH gradient in accumulationof MPCU in GC3/C1 cells. The effect of pHe(7.4 to 6.0) on cytotoxicity of MPCU was determined afterexposure of cells for 4 hr to various concentrations of MPCU in the presence of 10% fetal bovine serum. Decreasing the pHe from 7.4 to 6.0 increased the potency of MPCU by 4.7- and 4.5-fold in Rh30 and GC3/C1 cells, respectively. In cells exposed to drug/pHccombinations that resulted in 50% reduction in colony formingpotential, the steady state levels of [3H]MPCU were similar (range 8.8 ± 0.9 to 10.56 ± 0.6 nmol/106cells). These results demonstrate that decrease of pHe significantly enhanced the uptake of MPCU accumulationinto an FCCP/azide-sensitive compartment, and cytotoxicity of this agent. These data further support thehypothesis that sequestration of diarylsulfonylureas into the FCCP/azide-sensitive compartment (probablymitochondria) was associated with its cytotoxicity. The role of pHe in determining therapeutic selectivity ofdiarylsulfonylureas is discussed.

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