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

Abstract

The effect of extracellular pH (pH(e)) on the accumulation and cytotoxicity of the diarylsulfonylurea antitumor agent N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) has been examined. In a human colon adenocarcinoma cell line, GC3/C1, the initial rate of uptake of [3H]MPCU (2.4 microM) was increased by 4.5-fold as pH(e) was reduced from 7.4 to 6.5. Steady state levels of MPCU were inversely proportional to pH(e) and were 5-fold greater at pH 6.0 compared to 7.4. Similar results were obtained using Rh30 cells derived from an alveolar rhabdomyosarcoma. MPCU rapidly re-equilibrated after achieving steady state when pH(e) was altered, indicating that MPCU was not tightly bound within cells. In both cell lines, the uncoupling agent, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced (GC3/C1) or completely inhibited (Rh30) accumulation of MPCU at each pH(e) examined. Sodium azide had the same effect on the accumulation of MPCU as FCCP. The effects of FCCP and azide appeared to be due to collapse of the pH differential across the mitochondrial inner membrane rather than the gradient across the plasma membrane. As extracellular pH (pH(e)) decreased, intracellular pH(pH(i)) also decreased in GC3/C1 cells, such that the greatest pH differential (pH(i) - pH(e)) was 0.2 units at pH(e) 6.0. Neither FCCP nor azide significantly altered this pH gradient, indicating a minor role, if any, for the plasma membrane pH gradient in accumulation of MPCU in GC3/C1 cells. The effect of pH(e) (7.4 to 6.0) on cytotoxicity of MPCU was determined after exposure of cells for 4 hr to various concentrations of MPCU in the presence of 10% fetal bovine serum. Decreasing the pH(e) 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/pH(e) combinations that resulted in 50% reduction in colony forming potential, the steady state levels of [3H]MPCU were similar (range 8.8 +/- 0.9 to 10.56 +/- 0.6 nmol/10(6) cells). These results demonstrate that decrease of pH(e) significantly enhanced the uptake of MPCU accumulation into an FCCP/azide-sensitive compartment, and cytotoxicity of this agent. These data further support the hypothesis that sequestration of diarylsulfonylureas into the FCCP/azide-sensitive compartment (probably mitochondria) was associated with its cytotoxicity. The role of pH(e) in determining therapeutic selectivity of diarylsulfonylureas is discussed.

Glutathione and Mercapturic Acid Conjugates of Sulofenur and Their Activity against a Human Colon Cancer Cell Line

“…These observations were consistent with the inhibitory properties of their corresponding glutathione conjugate analogs derived from the substituted ureas discussed earlier Guan et al, 1994Guan et al, , 1999Jochheim and Baillie, 1994). Furthermore, our study has revealed that both conjugates exhibited inhibitory activity comparable to the parent drug against the human colon adenocarcinoma GC 3 /c1 cell line, a cell line that has been widely employed to explore the anticancer mechanism(s) of sulofenur and other anticancer diarylsulfonylureas (Houghton et al, 1990(Houghton et al, , 1995Sosinski et al, 1991Sosinski et al, , 1993Rush et al, 1992;Phelps et al, 1995).…”

“…The objectives of this study were to determine whether GS-C(O)-NH-R (R ϭ p-chlorophenyl) is a metabolite of sulofenur, whether the conjugate is a GR inhibitor, and further, whether the conjugate exhibits cell growth-inhibitory activity by using human colon adenocarcinoma GC 3 /c1 cells, a sulofenur-sensitive cell line. The GC 3 /c1 cell line has been widely employed to explore the anticancer mechanisms of sulofenur and other anticancer diarylsulfonylureas (Houghton et al, 1990(Houghton et al, , 1995Sosinski et al, 1991Sosinski et al, , 1993Rush et al, 1992;Phelps et al, 1995). In addition, a glutathione conjugate can be enzymatically converted to a mercapturic acid conjugate (Williams, 1995).…”

Antitumor diarylsulfonylureas: novel agents with unfulfilled promise

“…Each agent accumulated approximately 4-fold in cells, and concentrative accumulation was be abrogated by azide, or by ionophores such as nigericin that collapse the pH gradient across the mitochondrial membrane [25]. Accumulation to steady state was increased at lower extra cellular pH (pile), consistent with increased permeability of the uncharged species of these weak acids [26]. Consistent with increased accumulation, DSU were more potent at low pile.…”

“…Consistent with increased accumulation, DSU were more potent at low pile. At low pH~ [26,27], concentrative accumulation was inhibited by uncoupling agents such as carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), which impair mitochondrial energy transduction, or by collapsing the pH gradient across the mitochondrial membrane using nigericin [28]. Thus, cytotoxicity appeared to correlate with sequestration of drug into the FCCP sensitive compartment.…”

Gefitinib Enhances the Antitumor Activity and Oral Bioavailability of Irinotecan in Mice

“…The amount of the fluorescent dye produced was measured on a Cytofluor 2300 (Millipore, Bedford, MA) with an excitation wavelength of 530 nm and emission wavelength of 590 nm. (36). After the appropriate period of incubation at room temperature, medium was rapidly aspirated to terminate drug accumulation.…”