Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1

Abstract

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron (Fe) release. We have shown that NO-mediated Fe efflux from cells required glutathione (GSH), and we have hypothesized that a GS–Fe–NO complex was released. Hence, we studied the role of the GSH-conjugate transporter multidrug resistance-associated protein 1 (MRP1) in NO-mediated Fe efflux. MCF7-VP cells overexpressing MRP1 exhibited a 3- to 4-fold increase in NO-mediated 59 Fe and GSH efflux compared with WT cells (MCF7-WT) over 4 h. Similar results were found for other MRP1-overexpressing cell types but not those expressing another drug efflux pump, P-glycoprotein. NO-mediated 59 Fe and GSH efflux were temperature- and energy-dependent and were significantly decreased by the GSH-depleting agent and MRP1 transport inhibitor l -buthionine-[ S , R ]-sulfoximine. Other MRP1 inhibitors, MK571, probenecid, and difloxacin, significantly inhibited NO-mediated 59 Fe release. EPR spectroscopy demonstrated the dinitrosyl-dithiol-Fe complex (DNIC) peak in NO-treated cells was increased by MRP1 inhibitors, indicating inhibited DNIC transport from cells. The extent of DNIC accumulation correlated with the ability of MRP1 inhibitors to prevent NO-mediated 59 Fe efflux. MCF7-VP cells were more sensitive than MCF7-WT cells to growth inhibition by effects of NO, which was potentiated by l -buthionine-[ S , R ]-sulfoximine. These data indicate the importance of GSH in NO-mediated inhibition of proliferation. Collectively, NO stimulates Fe and GSH efflux from cells via MRP1. Active transport of NO by MRP1 overcomes diffusion that is inefficient and nontargeted, which has broad ramifications for understanding NO biology.

Nitric Oxide Storage and Transport in Cells Are Mediated by Glutathione S-Transferase P1-1 and Multidrug Resistance Protein 1 via Dinitrosyl Iron Complexes

“…Protein Labeling-Apo-transferrin (Apo-Tf; Sigma-Aldrich) was labeled with 59 Fe (PerkinElmer Life Sciences) to generate diferric 59 Fe-Tf using established methods (12). 59 Fe was monitored using a ␥-counter (PerkinElmer Life Sciences).…”

“…Efflux of 59 Fe from MEFs-Cells were prelabeled for 24 h with 59 Fe-Tf (0.75 M), washed, and then preincubated for 30 min at 37°C with medium containing the MRP1 inhibitor, MK571 (20 M) (12). The cells were then reincubated for 6 h at 37°C with MK571 (20 M) in the presence or absence of the NO generator, S-nitroso-glutathione (GSNO; 0.5 mM).…”

“…Subsequently, we discovered that the glutathione (GSH) transporter, multidrug resistance protein 1 (MRP1), mediates NO-stimulated iron and GSH efflux from cells and that this process could be inhibited by L-buthionine-(S,R)-sulfoximine (BSO) (12), an inhibitor of GSH synthesis (13). Several recent investigations have suggested that DNICs such as dinitrosyl-diglutathionyl iron complexes can bind to glutathione S-transferase (GST) isoenzymes with high affinity (K d ϭ 10 Ϫ7 -10 Ϫ10 M) (14,15).…”

“…Considering the coordinated role of GSTs and MRP1 in detoxification processes (16 -18) and our studies showing that MRP1 is involved in NO-mediated iron and GSH efflux from cells (12), we have for the first time examined the hypothesis that GST isoenzymes ␣ (GST A1-1), (GST M1-1), and (GST P1-1) and MRP1 act as coordinate partners involved in the storage and transport of DNICs, respectively. In the current study, we show that GST P1-1, but not GST A1-1 or GST M1-1, decreased NO-mediated 59 Fe release from cells via MRP1, demonstrating a relationship between the proteins in terms of NO storage and transport and the role of DNICs in these processes.…”

“…Cell Proliferation-Proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and validated by viable cell counts (12).…”