Studies on the cellular pharmacology of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)-urea.

The cellular pharmacology of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)-urea (MPCU) has been examined in a cloned line of GC3 human colon adenocarcinoma cells. There was a rapid concentrative accumulation of drug, which could be separated into energy-independent and -dependent phases. Accumulation over 15 sec was linear and temperature dependent, but not energy dependent (azide insensitive). The rate of uptake was a linear function of concentration over a wide range (0.0026 to 5 mM). No saturation kinetics were demonstrated. Steady state was achieved within 10 min, and drug levels associated with GC3/C1 cells exceeded the extracellular concentration by 4- to 6-fold. This second phase "concentrative accumulation" of drug was azide sensitive. When cells were incubated to steady state in the presence of azide, removal of azide (with addition of glucose) resulted in a further uptake of sulfonylurea to a higher steady state. When [3H]MPCU was removed from the medium after achieving steady state, loss of drug from cells was rapid (T1/2 approximately 130 sec), and no tight-binding component was apparent. After achieving steady state, cell-associated drug was lost into drug-containing medium reaching a lower steady state if 10 mM azide (+/- glucose) was added. These data indicate that MPCU may enter cells by a non-saturable energy-independent process (passive diffusion) and bind weakly to some intracellular component or become sequestered to some compartment in an energy-dependent manner.

Evidence for mitochondrial localization of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea in human colon adenocarcinoma cells.

N-(4-Methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) is a new agent that exhibits high therapeutic activity against human and rodent tumor models. Initial studies indicated that in vitro [3H]MPCU was concentrated 4- to 6-fold in GC3/c1 human colon adenocarcinoma cells in an azide-sensitive manner. In this study the dependence of uptake and concentrative accumulation of MPCU upon temperature, plasma membrane potential, and the electrochemical potential of mitochondria has been examined. Accumulation and efflux of MPCU were temperature dependent. At 3.6 microM MPCU, initial rates of uptake (15 s) were 1.4, 38.0, and 84.2 pmol/min/10(6) cells at 2 degrees C, 23 degrees C, and 37 degrees C, respectively. The rate of uptake and concentrative accumulation within GC3/c1 cells was not altered in high K+ buffer or by 1 mM ouabain, indicating that plasma membrane potential was not significant in these processes. Concentrative accumulation, but not initial uptake, was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol, and sodium azide. Glucose partially antagonized the inhibition of these agents which uncouple oxidative phosphorylation. Oligomycin, an inhibitor of mitochondrial ATP synthase, did not inhibit uptake or concentrative accumulation of MPCU. However, oligomycin in the presence of 2-deoxyglucose significantly inhibited concentrative accumulation of MPCU. These results suggested that concentrative accumulation of MPCU was dependent upon the mitochondrial transmembrane gradient rather than ATP, although direct implication of ATP could not be excluded. To examine which component of this gradient was predominant in causing MPCU sequestration, the ionophores valinomycin and nigericin were used. Valinomycin, which collapses the charge gradient across the mitochondrial matrix membrane, caused only slight inhibition of MPCU accumulation, and the effect was similar at 2 or 10 mumol. In contrast, nigericin (which collapses the pH gradient and increases mitochondrial membrane potential) inhibited by approximately 90% concentrative accumulation of MPCU. These data suggested that MPCU was being concentrated in mitochondria and that this was dependent upon the pH gradient across mitochondrial membrane. In cells exposed to MPCU or the analogue N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, enlargement of mitochondria was observed within 24 h and appeared to be the initial morphological change associated with drug treatment. These results implicate mitochondria as a site of sequestration of diarylsulfonylureas and as a potential site of action.

N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, a novel agent equally cytotoxic to nonproliferating human colon adenocarcinoma cells.

Diarylsulfonylureas have been shown to have therapeutic activity against rodent and human tumor models, notably causing regressions in some lines of human colon adenocarcinomas in mice. At present the mechanism of cytotoxicity is unknown, although preliminary data implicate mitochondria as a potential site of action. In this study, the cytotoxicity of the diarylsulfonylurea N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) has been examined in GC3/c1 human colon adenocarcinoma cells. At cytotoxic concentrations of ISCU, in the presence of albumin as a drug binding species, there was only slight inhibition of [3H]thymidine and [3H]uridine incorporation at concentrations of ISCU up to 140 micrograms/ml and no inhibition of synthesis of protein as determined by incorporation of L-leucine. In the absence of albumin, incorporation of [3H]thymidine into DNA or [3H]uridine into RNA was inhibited at greater than 70 micrograms/ml and 140 micrograms/ml, respectively. As ISCU is highly bound to serum albumin (greater than 99%), it would appear that inhibition of nucleic acid synthesis occurs only at supralethal concentrations of ISCU. The cytotoxicity of ISCU in proliferating or quiescent cell populations was examined. GC3/c1 cells grown in medium containing 0.5% fetal calf serum (FCS) had a 60% reduction in rate of growth, but were more sensitive than cells exposed for 24 h in 10% FCS-medium (IC50 1.9 and 31 micrograms/ml, respectively). When the albumin concentration was adjusted (240 mg/100 ml) to allow equivalent drug binding, IC50 values were similar. In cultures of GC3/c1 cells growth rate was related to the concentration of FCS. In the absence of serum, growth rate was 2.5 to 3.2% that of exponentially growing control cultures in the presence of 10% FCS. Addition of FCS to quiescent cultures after 1 to 6 days in serum-free conditions resulted in immediate growth of cells. Clonogenic potential was also unchanged for at least 6 days under serum-free conditions. Under these conditions, where albumin concentration was adjusted to be equivalent to medium containing 10% FCS, sensitivity of proliferatively quiescent GC3/c1 cells was similar to that in exponentially growing control cultures in which the population doubling time was approximately 22 h. Further, there was no recovery of clonogenic potential when cells were exposed for 24 h to ISCU and maintained in a quiescent state for up to 4 days prior to serum stimulation. These data suggest that the cytotoxic effects of ISCU are independent of the proliferative state of the cell population.(ABSTRACT TRUNCATED AT 400 WORDS)