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

*Children's Cancer Institute Australia for Medical Research, Sydney 2031, Australia;
^†The Heart Research Institute, Sydney 2050, Australia; and
^‡Lady Davis Institute, Jewish General Hospital and Department of Physiology, McGill University, Montreal, QC, Canada H3T 1E2

Communicated by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, March 28, 2006 (received for review March 9, 2006)

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 ⁵⁹Fe 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 ⁵⁹Fe 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 ⁵⁹Fe 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 ⁵⁹Fe 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.

Footnotes

^§To whom correspondence may be addressed. E-mail: prem.ponka{at}mcgill.ca or d.richardson{at}med.usyd.edu.au
Author contributions: D.R.R. designed research; R.N.W. and C.H. performed research; and R.N.W., C.H., P.P., and D.R.R. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

NO,

nitrogen monoxide;

GSH,

glutathione;

MRP1,

multidrug resistance-associated protein 1;

DNIC,

dinitrosyl-dithiol-Fe complex;

NOS,

nitric oxide synthase;

Mφ,

macrophage;

BSO,

l-buthionine-[S,R]-sulfoximine;

Tf,

transferrin;

SperNO,

spermine-NONOate;

VCR,

vincristine.