Abstract

One of the most potent cyclopentenone prostaglandins, 15-deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), has been shown to be cytotoxic in some tumor cells and, as a ligand of peroxisome proliferator activated receptor gamma (PPARgamma), to influence the transcriptional regulation of several genes. We examined whether a glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, is formed and if the glutathione conjugate efflux pumps, MRP1 and MRP3, could transport this conjugate, thereby attenuating the cytotoxicity and transactivating activity of 15-d-PGJ(2) in MCF7 breast cancer cells. Formation of 15-d-PGJ(2)-SG was demonstrated both in vitro and in cells, and its structure was determined by ESI/MS and NMR. Expression of MRP1 and MRP3 was achieved by stable transduction of parental MCF7 cells. Membrane vesicles derived from these cells supported efficient, ATP-dependent transport of 15-d-PGJ(2)-SG (K(M) 1.4 and 2.9 microM for MRP1 and MRP3, respectively). When compared with parental, MRP-minus MCF7 cells, expression of MRP1 and MRP3 conferred approximately 2-fold protection from 15-d-PGJ(2) cytotoxicity. 15-d-PGJ(2)-mediated transcriptional activation was evaluated in cells transiently transfected with a reporter gene under the transcriptional control of a PPAR responsive element. Treatment of parental MCF7 cells with 15-d-PGJ(2) resulted in a time-dependent induction of reporter gene activity-induction that was measurable with concentrations of added 15-d-PGJ(2) as low as 100 nM. In contrast, expression of MRP1 or MRP3 abolished 15-d-PGJ(2)-dependent reporter gene induction. Depletion of intracellular glutathione reversed MRP1- and MRP3-mediated attenuation of 15-d-PGJ(2) cytotoxicity and transactivation. These data indicate that MRP1 and MRP3 can modulate the biological effects of 15-d-PGJ(2), and likely other cyclopentenone prostaglandins, in a glutathione-dependent manner. The results are consistent with a mechanism for the attenuation of the biological activities of 15-d-PGJ(2) that involves the formation and active efflux of its glutathione conjugate, 15-d-PGJ(2)-SG.