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.