Chronic exposure of cells to

Chronic exposure of cells to cytotoxic drugs can result in the selection of multiple phenotypic and genetic changes. Thus, the use of MCF7/VP cells, which were selected for resistance to VP-16, was potentially problematic. However, a thorough characterization of MCF7/VP cells demonstrated that they are similar to, if not indistinguishable from, MCF7/WT cells with respect to doubling times, morphology, very low GST levels, glutathione levels, and undetectable P-glycoprotein (Table 1 and [20]). Indeed, besides high level MRP expression in MCF7/VP cells, the only reported molecular difference between MCF7/WT and MCF7/VP cells is a modest decrease in MCF7/VP topoisomerase II sensitivity to VP-16-induced cleavable complex formation [20]. This phenotypic change in topoisomerase II sensitivity is probably unrelated to ethacrynic acid, cisplatin, or oxazaphosphorine resistance. Thus, high level MRP is very likely the only molecular change in parental MCF7/VP cells germane to altered sensitivities to the drugs tested in the present report. Ethacrynic Doripenem Hydrate was chosen as a model toxin for our studies for several reasons. Ethacrynic acid is a good substitute for some isozymes of GST including GSTP1-1, and increased GST has been associated with resistance to ethacrynic acid cytotoxicity 7, 8, 9, 26, 44. Some cell lines selected for resistance to ethacrynic acid express increased levels of MRP [45], and MRP-containing membrane vesicles have been shown to transport glutathione conjugates of ethacrynic acid in vitro[46]. Finally, the cellular fate and cytotoxicity of ethacrynic acid are of practical interest because this drug has been used clinically as a diuretic and, in cancer chemotherapy trials, as an inhibitor of GST [47]. Our results establish that MRP, alone or in combination with GSTP1-1, can confer resistance to ethacrynic acid. These results are the first documentation that simultaneous expression of GSTP1-1 and MRP can operate to confer high level cellular resistance to ethacrynic acid (Fig. 3). Increased expression of GSTP1-1 alone, while protective, is insufficient to cause this same high level resistance. This observation suggests that the glutathione conjugate of ethacrynic acid formed in GSTP1-1-catalyzed reactions may itself be toxic to the cell. The mechanism of the putative toxicity is unknown but may involve some direct toxicity of EA-SG. Alternatively, EA-SG may serve as an intracellular reservoir of ethacrynic acid that can later be released as free, toxic ethacrynic acid. Indeed, the formation of EA-SG from ethacrynic acid has been shown to be a reversible reaction [48]. Finally, accumulation of intracellular EA-SG may limit, via EA-SG product inhibition, further detoxification by GSTP1-1-mediated catalysis of EA conjugation 49, 50. In this view, MRP-dependent efflux of EA-SG may augment GST-associated detoxification by: (1) eliminating EA-SG, which itself may be toxic, (2) eliminating EA-SG as a reservoir of free EA by the reverse conjugation reaction, or (3) eliminating EA-SG product inhibition of GST. The oxazaphosphorine compounds include the clinically important anticancer drugs cyclophosphamide, mafosfamide, and ifosfamide. They are metabolized to derivatives that can form conjugates with glutathione 24, 25, 43. Several of these metabolites, including acrolein [23], 4-OH-CP [24], and ifosfamide mustard [25], are known to be substrates of GSTP1-1. Heretofore, it was unclear whether GSTP1-1 alone or in combination with MRP could confer cellular resistance to activated oxazaphosphorines. There is considerable evidence that some of these glutathione conjugates, including the glutathione conjugates of acrolein and its metabolites, are toxic to the cell 51, 52. For the glutathione conjugate of acrolein, toxicity may be a direct consequence of conjugate-mediated oxygen radical formation [51] or may involve the re-release of acrolein from its glutathione conjugate via the reverse reaction [52]. Similarly, it has been suggested that the glutathione conjugate of 4-OH-CP, 4-glutathionylcyclophosphamide, can undergo trans-thiolation reactions leading to the re-release of the penultimate toxins, the tautomers 4-OH-CP/aldophosphamide [24]. Therefore, the possibility that these potentially toxic glutathione conjugates of oxazaphosphorine metabolites may be substrates of MRP-mediated export is a particularly important issue.