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Fig. 6. Iron (Fe) and glutathione (GSH) efflux increase concurrently after incubation with the NO generator, spermine-NONOate (SperNO), and are more marked in MRP1 hyper-expressing MCF7-VP cells than MCF7-WT or SK-N-MC (childhood neuroblastoma). Both NO-mediated 59Fe and GSH efflux were inhibited by the GSH synthesis inhibitor, buthionine sulfoximine (BSO). NO-mediated 59Fe and GSH efflux was examined by preincubating cells with or without 0.1 mM BSO for 20 h at 37°C before labelling for 3 h at 37°C with 59Fe—transferrin (59Fe-Tf) (0.75 ^M), washing, and subsequent incubation with the NO generator, SperNO (0.5 mM) for 3 h at 37°C. (A) NO-mediated 59Fe efflux results are expressed as a percentage of the control. (B) Results calculated as a percentage of total cell 59Fe. These data are expressed as net 59Fe efflux over that found for the control. (C) Total cellular 59Fe (i.e. cell 59Fe + efflux 59Fe). (D) NO-mediated GSH efflux expressed as a percentage of total GSH. (E) GSH efflux data expressed as net GSH efflux over that found for the control. (F) Total GSH measured showing the GSH depletion in the presence of BSO. Results are mean ± SD (4 determinations) in a typical experiment of 4. "Denotes p < 0.0001.

sera, activated macrophages and a range of different cell types [26,27,29,31-34,93]. A recent study has suggested that the dinitrosyl-diglutathionyl-Fe complex can associate with glutathione ^-transferase enzymes to stabilize NO for many hours (fx/2 = 4.5-8 h; [95]) which markedly exceeds the f1/2 of "free NO" which is 2 ms-2 s [98]. Furthermore, DNICs (e.g. (NO)2(GS)2Fe complexes) can transverse cell membranes to donate Fe to tissues [97] and can transnitrosylate acceptor targets in vitro and in vivo [96,99,100] demonstrating their bioavailability and potential role as NO-carrier molecules.

The efficient efflux of DNICs by an active transport mechanism could be crucial at sites where NO is produced in small physiological quantities as a messenger molecule [89]. For example, in blood vessels where the small quantities of DNICs released from endothelial cells could be important for regulating smooth muscle tone [15] (Fig. 10A). The ability of cells to actively transport and traffic NO overcomes the random process of diffusion that would be inefficient and non-targeted.

In converse to the situation when NO acts as a messenger molecule, under conditions where NO is used as a cytotoxic effector, the substantial quantities generated by iNOS of activated macrophages could lead to the efflux of a relatively large proportion of Fe and

Fig. 7. NO-mediated 59Fe and GSH efflux from parental (MCF7-WT) and MRP1 hyper-expressing MCF7-VP cells are temperature dependent (A,B) and NO-mediated 59Fe efflux is decreased by metabolic inhibitors (C). (A,B) Cells were prelabelled for 3 h at 37°C with 59Fe-transferrin (59Fe—Tf; 0.75 ^M), washed 4 times, and reincubated with or without SperNO (0.5 mM) at 4, 20 or 37°C. (C) Cells were prelabelled with 59Fe-Tf as described above, and then pre-treated for 30 min with or without cyanide (CN; 5 mM), azide (30 mM), oligomycin (15 ^M) or rotenone (20 ^M). Cells were then incubated for 3 h at 37°C with or without SperNO (0.5 mM) in the presence or absence of the inhibitors. Results are mean ± SD (4 determinations) from a typical experiment of 4. (Taken with permission from Ref. [89].)

Fig. 7. NO-mediated 59Fe and GSH efflux from parental (MCF7-WT) and MRP1 hyper-expressing MCF7-VP cells are temperature dependent (A,B) and NO-mediated 59Fe efflux is decreased by metabolic inhibitors (C). (A,B) Cells were prelabelled for 3 h at 37°C with 59Fe-transferrin (59Fe—Tf; 0.75 ^M), washed 4 times, and reincubated with or without SperNO (0.5 mM) at 4, 20 or 37°C. (C) Cells were prelabelled with 59Fe-Tf as described above, and then pre-treated for 30 min with or without cyanide (CN; 5 mM), azide (30 mM), oligomycin (15 ^M) or rotenone (20 ^M). Cells were then incubated for 3 h at 37°C with or without SperNO (0.5 mM) in the presence or absence of the inhibitors. Results are mean ± SD (4 determinations) from a typical experiment of 4. (Taken with permission from Ref. [89].)

GSH from tumor target cells (Fig. 10B) [89]. Since Fe and GSH are critical for proliferation [11,84], their release from tumor cells in large amounts would be cytotoxic. In addition to our current results, this hypothesis is strongly supported by previous studies where cyto-toxic macrophages induced the release of a large proportion (64%) of intracellular Fe from tumor target cells [3], an effect described to be mediated by NO [4]. Previous investigations have shown that increased GSH efflux from cells is a key signal that mediates apopto-sis [101], and it is well-known that Fe mobilization from cells using chelators results in marked anti-tumor activity [102]. Hence, the dual action of NO resulting in both Fe and GSH mobilization may play a vital role in the cytotoxic activity of activated macrophages against tumor cells. We also showed that under conditions that lead to Fe and GSH efflux, proliferation of MCF7-VP cells hyper-expressing MRP1 was more sensitive to the effects of NO than their wild-type counterparts (Fig. 11) [89]. This supports the hypothesis that enhanced GSH and Fe efflux from cells hyper-expressing MRP1 leads to greater anti-proliferative activity. In addition, GSH depletion increased the cytotoxicity of NO particularly in the MRP1 hyper-expressing MCF7-VP cell type, suggesting the critical role played by GSH in the anti-proliferative activity of NO (Fig. 11).

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