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Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

The effect of the Y108V mutation of human glutathione S-transferase P1-1 (hGST P1-1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 fi Val resulted in a 3D-structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H-site) and glutathione binding site (G-site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H-site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K d~0 .5 lM) when compared with those of the parent compounds, K EA d~1 3 lM, K GSH d~2 5 lM. The EA moiety of the conjugate binds in the H-site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the DC p values of binding can also be correlated with the potential stacking interactions between Additional Supporting Information may be found in the online version of this article.Abbreviations: DMSO, dimethylsulfoxide; DSC, differential scanning calorimetry; DTT, dithiothreitol; EA, ethacrynic acid; EACys, ethacrynic-cysteine conjugate; EAME, ethacrynic-mercaptoethanol conjugate; EASG, ethacrynic-glutathione conjugate; GST, glutathione transferase; HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; hGST P1-1, human glutathione transferase P1-1; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; MPD, 2-methyl-2,4-pentanediol; wt, wild type.Indalecio Quesada-Soriano and Lorien J. Parker contributed equally to this work.The coordinates for the structures of the Y108V apo, EA and EASG complex are deposited with the Protein Databank (http:// rcsg.org/pdb/) with the entry codes 3HJM, 3HKR, and 3HJO, respectively. ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.