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Post-translational modification of available cysteine residues by disulfide bond formation with the thiol of free glutathione (GSH; glutathionylation) is an important cellular signaling response to conditions of oxidative and/or nitrosative stress. We have used two novel drugs, a platinum-stabilized form of oxidized glutathione (NOV-002) and a novel glutathione S-transferase activated pro-drug, PABA/NO (O2- [2,4-dinitro-5- (N-methyl-N-4-carboxyphenylamino) phenyl] 1-N, N-dimethylamino) diazen-1-ium-1, 2-diolate) to compare glutathionylation in tumor cells. NOV-002 provides GSSG that in millimolar concentrations can cause direct oxidative stress. PABA/NO liberates nitric oxide (NO) causing nitrosative stress and resulting in the formation of GSNO, the metabolites of which can induce protein glutathionylation. To evaluate the extent of glutathionylation following drug exposure, we utilized monoclonal antibodies directed against glutathionylated cysteine residues. One and two dimensional SDS-PAGE confirmed that treatment of NIH3T3 cells with both NOV-002 and PABA-NO produces a rapid (<5 mins.) time and dose-dependent glutathionylation of a finite number of cellular proteins (between 10 and 30). NIH3T3 cells transfected with MRP1 were less sensitive to the toxicity of PABA/NO and had a dose and time dependent reduction in glutathionylation of target proteins. These data suggested that the transporter-mediated efflux of GSNO minimizes drug effects. PABA/NO has antitumor activity both in vitro and in vivo in nude mice bearing ovarian xenografts. NOV-002 caused tumor cell apoptosis and enhanced bone marrow progenitor cell proliferation. We are using peptide digestion and mass spectral analysis to identify those proteins, the glutathionylation of which are critical to the pharmacological efficacy of each drug.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]