Oxidative stress induces STAT3 S-glutathionylation and impairs its phosphorylation: in vivo and in vitro study

STAT3 is a transcription factor constitutively activated in highly malignant solid and hematological tumor. Activated STAT3 may participate in oncogenesis by stimulating cell proliferation and inducing resistance to apoptosis, as well as promoting tumor angiogenesis, invasion, and migration. The tight association of STAT3 activation with transformation and tumor progression makes STAT3 a potential therapeutic target. In this work, we identify the naturally occurring sesquiterpene lactone cynaropicrin as a potent inhibitor of both IL-6-inducible and constitutive STAT3 activation (IC50=12 µM). Cynaropicrin, that contains α-β unsatured carbonyl moiety and functions as potent Michael reaction acceptor, induces dose- and time-dependently the drop in intracellular glutathione (GSH) concentration. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, prevents the inhibitory action of cynaropicrin on STAT3 tyrosine phosphorylation. The disturbance in the intracellular redox state induces S-glutathionylation of STAT3. STAT3 inhibition leads to the suppression of two anti-apoptotic genes, Bcl-2 and Survivin, in DU145 cells that constitutively express active STAT3. This event may be responsible of the decline in cell viability after cynaropicrin treatment. As revealed by PI/Annexin-V staining, PARP cleavage and DNA ladder formation, cynaropicrin cytotoxicity is mediated by apoptosis. Moreover, we found that cynaropicrin potentiated cytotoxic effect of two well-establish chemotherapeutic agent, cisplatin and docetaxel. Many reports support the idea that STAT3 S-glutathionylation might represent the key event in redox STAT3 modulation. In order to clarify S-glutathionylation of STAT3 we performed an in vitro study. We demonstrated that the combined treatment with diamide, a thiols specific oxidant, and GSH induces S-glutathionylation of STAT3 in the recombinant purified form. This effect was completely reversed by treatment with dithiothreitol, indicating that S-glutathionylation of STAT3 was related to formation of protein-mixed disulfides. Since STAT3 was glutathionylated in the presence of oxidants, we examined whether this modification might affect STAT3 phosphorylation using in vitro kinase assay. As expected, S-glutathionylation significantly decreased the level of STAT3 tyrosine. The addition of the bulky negatively charged GSH moiety impairs JAK2-mediated STAT3 phosphorylation very likely interfering with tyrosine accessibility thus affecting protein structure and function. Mass mapping analysis identifies two glutathionylated cysteine residues, Cys328 and Cys542. Site direct mutagenesis and in vitro kinase assay confirm the importance of both cysteine residues in the complex redox regulatory mechanism of STAT3. Cells expressing mutant were resistant in this regard. The data presented herein confirmed the occurrence of a redox-dependent regulation of STAT3 and identified the more redox- sensitive cysteines of STAT3 protein to S-glutathionylation.