6 research outputs found
S-Glutathionylation at Cys328 and Cys542 Impairs STAT3 Phosphorylation.
STAT3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in cancers. Although many reports provide evidence that STAT3 is a direct target of oxidative stress, its redox regulation is poorly understood. Under oxidative conditions STAT3 activity can be modulated by S-glutathionylation, a reversible redox modification of cysteine residues. This suggests the possible cross-talk between phosphorylation and glutathionylation and points out that STAT3 is susceptible to redox regulation. Recently, we reported that decreasing the GSH content in different cell lines induces inhibition of STAT3 activity through the reversible oxidation of thiol groups. In the present work, we demonstrate that GSH/diamide treatment induces S-glutathionylation of STAT3 in the recombinant purified form. This effect was completely reversed by treatment with the reducing agent dithiothreitol, indicating that S-glutathionylation of STAT3 was related to formation of protein-mixed disulfides. Moreover, addition of the bulky negatively charged GSH moiety impairs JAK2-mediated STAT3 phosphorylation, very likely interfering with tyrosine accessibility and thus affecting protein structure and function. Mass mapping analysis identifies two glutathionylated cysteine residues, Cys328 and Cys542, within the DNA-binding domain and the linker domain, respectively. 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, identified the more redox-sensitive cysteines within STAT3 structure, and may have important implications for development of new drugs
Mild oxidative stress induces S-glutathionylation of STAT3 and enhances chemosensitivity of tumoural cells to chemotherapeutic drugs.
STAT3 is a transcription factor constitutively activated in a variety of cancers that has a critical role in the inhibition of apoptosis and induction of chemoresistance. Inhibition of the STAT3 signaling pathway suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting that direct inhibition of STAT3 function is a viable therapeutic approach. Herein, we identify the naturally occurring sesquiterpene lactone cynaropicrin as a potent inhibitor of both IL-6-inducible and constitutive STAT3 activation (IC50=12\u3bcM). Cynaropicrin, which contains an \u3b1-\u3b2-unsaturated carbonyl moiety and acts as potent Michael reaction acceptor, induces a rapid drop in intracellular glutathione (GSH) concentration, thereby triggering S-glutathionylation of STAT3. Furthermore, glutathione ethylene ester, the cell permeable form of GSH, reverts the inhibitory action of cynaropicrin on STAT3 tyrosine phosphorylation. These findings suggest that this sesquiterpene lactone is able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein to regulate its function. STAT3 inhibition led 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 for 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. Finally, cynaropicrin displayed a slight to strong synergism with two well-established chemotherapeutic drugs, cisplatin and docetaxel. Taken together our studies suggest that cynaropicrin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumour cells to chemotherap
S‑Glutathionylation at Cys328 and Cys542 Impairs STAT3 Phosphorylation
STAT3 is a latent transcription factor
that promotes cell survival
and proliferation and is often constitutively active in cancers. Although
many reports provide evidence that STAT3 is a direct target of oxidative
stress, its redox regulation is poorly understood. Under oxidative
conditions STAT3 activity can be modulated by S-glutathionylation,
a reversible redox modification of cysteine residues. This suggests
the possible cross-talk between phosphorylation and glutathionylation
and points out that STAT3 is susceptible to redox regulation. Recently,
we reported that decreasing the GSH content in different cell lines
induces inhibition of STAT3 activity through the reversible oxidation
of thiol groups. In the present work, we demonstrate that GSH/diamide
treatment induces S-glutathionylation of STAT3 in the recombinant
purified form. This effect was completely reversed by treatment with
the reducing agent dithiothreitol, indicating that S-glutathionylation
of STAT3 was related to formation of protein-mixed disulfides. Moreover,
addition of the bulky negatively charged GSH moiety impairs JAK2-mediated
STAT3 phosphorylation, very likely interfering with tyrosine accessibility
and thus affecting protein structure and function. Mass mapping analysis
identifies two glutathionylated cysteine residues, Cys328 and Cys542,
within the DNA-binding domain and the linker domain, respectively.
Site direct mutagenesis and <i>in vitro</i> 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, identified the more redox-sensitive
cysteines within STAT3 structure, and may have important implications
for development of new drugs