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cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20–30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20–30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-β-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, “light,” “heavy,” and “extra heavy” fractions we show that no P-gp is found in the “light” fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.

Arsenic trioxide is an effective treatment for acute promyelocytic leukemia, but resistance to metalloïd salts is found in humans. Using atomic absorption spectroscopy, we have measured the rate of uptake of arsenic trioxide and of antimony tartrate in GLC4 and GLC4/ADR cells overexpressing MRP1 and the rate of their MRP1-mediated effluxes as a function of the intracellular GSH concentration. In sensitive cells, after 1 h, a pseudosteady state is reached where intra- and extracellular concentrations of metalloid are the same. This precludes the formation, at short term, of complexes between arsenic or antimony with GSH. In resistant cells reduced intracellular accumulation of arsenic (or antimony), reflecting an increased rate of arsenic (or antimony) efflux from the cells, is observed. No efflux of the metalloid is observed in GSH depleted cells. The two metalloïds and GSH are pumped out by MRP1 with the same efficiency. Moreover for the three compounds 50% of the efflux is inhibited by 2 μM MK571. This led us to suggest that As- and Sb-containing species could be cotransported with GSH.