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Regulation of STAT3 and NF-?B activations by S-nitrosylation in multiple myeloma.

ABSTRACT: Numerous reports suggest that aberrant activations of STAT3 and NF-?B promote survival and proliferation of multiple myeloma (MM) cells. In the present report, we demonstrate that a synthetic S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC), inhibits proliferation and survival of multiple MM cells via S-nitrosylation-dependent inhibition of STAT3 and NF-?B. In human MM cells (e.g. U266, H929, and IM-9 cells), SNAC treatment increased S-nitrosylation of STAT3 and NF-?B and inhibited their activities. Consequently, SNAC treatment resulted in MM cell cycle arrest at G1/S check point and inhibited their proliferation. SNAC also decreased the expression of cell survival factors and increased the activities of caspases, thus increased sensitivity of MM cells to melphalan, a chemotherapeutic agent for MM. In U266 xenografted mice, SNAC treatment decreased the activity of STAT3 and reduced the growth of human CD138 positive cells (U266 cells) in the bone marrow and also reduced their production of human IgE into the serum. Taken together, these data document the S-nitrosylation mediated inhibition of MM cell proliferation and cell survival via inhibition of STAT3 and NF-?B pathways and its efficacy in animal model of MM.

SUBMITTER: Kim J

PROVIDER: S-EPMC5826580 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Regulation of STAT3 and NF-κB activations by S-nitrosylation in multiple myeloma.

Kim Jinsu J Choi Seungho S Saxena Nishant N Singh Avtar K AK Singh Inderjit I Won Je-Seong JS

Free radical biology & medicine 20170221

Numerous reports suggest that aberrant activations of STAT3 and NF-κB promote survival and proliferation of multiple myeloma (MM) cells. In the present report, we demonstrate that a synthetic S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC), inhibits proliferation and survival of multiple MM cells via S-nitrosylation-dependent inhibition of STAT3 and NF-κB. In human MM cells (e.g. U266, H929, and IM-9 cells), SNAC treatment increased S-nitrosylation of STAT3 and NF-κB and inhibited the ...[more]

PMID: 28232202

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Project description:ObjectiveAs an important negative regulatory factor of immunological cells, Tim3 plays a regulating role in tumor immune microenvironment. The purpose of this study was to investigate the expression of Tim3 on MM cells and its effect on the proliferation and apoptosis of MM cells, as well as its potential mechanism.MethodsIn this study, the expression of Tim3 was detected on myeloma cells (CD38+CD138+ cells) of bone marrow by flow cytometry (FCM) from 167 patients with MM and 51 healthy donors as controls and making correlation analysis with related clinical indexes. In vitro, MM cell lines (RPMI-8226 and U266) were treated with Tim3 knock-down alone, bortezomib alone and combination of Tim3 knock-down and bortezomib, then cell proliferation, cell apoptosis and downstream signaling pathway were detected by CCK-8, FCM, RT-PCR and western blot.ResultsThe expression of Tim3 on myeloma cells in MM patients was significantly higher than normal control group and positively correlated with β2 microglobulin, creatine, and plasma cells of bone marrow, negatively correlated with hemoglobin and red blood cells. In vitro, we validated the high expression of Tim3 in RPMI-8226 and U266 cell lines. After Tim3 knock-down, the cell proliferation was inhibited and cell apoptosis was induced, the relative mRNA and protein expression of Tim3 and NF-κB signal pathway (PI3K, AKT, mTOR, NF-κB) were significantly decreased. Also, the cell proliferation was inhibited, cell apoptosis was increased, the relative mRNA and protein expression of NF-κB were decreased significantly in combination group than bortezomib or Tim3 knock-down group.ConclusionsThe high expression of Tim3 on MM cells is associated with progression of MM patients. Tim3 maybe regulate the proliferation of MM cells via NF-κB signal pathway. Down-regulation of Tim3 expression can inhibit proliferation and induce apoptosis of MM cells, also has an additive inhibitory effect of bortezomib on NF-κB signaling pathway, then inhibit proliferation and induce apoptosis. Therefore, Tim3 may be a potential target for the treatment of MM.

Project description:S-nitrosylation and S-glutathionylation, redox-based modifications of protein thiols, are recently emerging as important signaling mechanisms. In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway that plays critical roles in immune/inflammatory responses and tumorigenesis.Our studies show that STAT3 in stimulated microglia underwent two distinct redox-dependent modifications, S-nitrosylation and S-glutathionylation. STAT3 S-nitrosylation was associated with inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) and S-nitrosoglutathione (GSNO), whereas S-glutathionylation of STAT3 was associated with cellular oxidative stress. NO produced by iNOS or treatment of microglia with exogenous GSNO inhibited STAT3 activation via inhibiting STAT3 phosphorylation (Tyr(705)). Consequently, the interleukin-6 (IL-6)-induced microglial proliferation and associated gene expressions were also reduced. In cell-free kinase assay using purified JAK2 and STAT3, STAT3 phosphorylation was inhibited by its selective preincubation with GSNO, but not by preincubation of JAK2 with GSNO, indicating that GSNO-mediated mechanisms inhibit STAT3 phosphorylation through S-nitrosylation of STAT3 rather than JAK2. In this study, we identified that Cys(259) was the target Cys residue of GSNO-mediated S-nitrosylation of STAT3. The replacement of Cys(259) residue with Ala abolished the inhibitory role of GSNO in IL-6-induced STAT3 phosphorylation and transactivation, suggesting the role of Cys(259) S-nitrosylation in STAT3 phosphorylation.Microglial proliferation is regulated by NO via S-nitrosylation of STAT3 (Cys(259)) and inhibition of STAT3 (Tyr(705)) phosphorylation.Our results indicate the regulation of STAT3 by NO-based post-translational modification (S-nitrosylation). These findings have important implications for the development of new therapeutics targeting STAT3 for treating diseases associated with inflammatory/immune responses and abnormal cell proliferation, including cancer.

Project description:BackgroundMultiple myeloma (MM) is an incurable hematological malignancy. Although proteasome inhibitors and immunomodulators have significantly improved patient outcomes, some patients respond poorly to treatment and almost all patients will relapse. Mechanisms of proteasome inhibitor resistance in multiple myeloma have not been fully elucidated. ZHX2 is a transcription regulator degraded via proteasome and presents both oncogenic or tumor suppressive effect in different cancers, however, it is still unknown that the role of ZHX2 in myeloma. In this study, we aim to demonstrate the effect and mechanism of ZHX2 on proteasome inhibitor resistance in MM.MethodsGSE24080 gene expression profile datasets from Gene Expression Omnibus (GEO) were analyzed to evaluate the relationship between ZHX2 expression level and survival in MM. Expression of ZHX2 in human MM cell lines at baseline and after bortezomib (BTZ) treatment was determined by Western blotting (WB). The proliferation and apoptosis rate of MM cells treated with BTZ after the knockdown of ZHX2 were analyzed by flow cytometry. Nuclear translocation of NF-κB after the knockdown of ZHX2 was evaluated by WB and immunofluorescence, and the expression of NF-κB target genes was measured by real-time quantitative PCR. Co-immunoprecipitation (Co-IP) and WB were used to detect the interaction of ZHX2 with NF-κB.ResultsWe found that higher ZHX2 expression was correlated with poorer clinical outcomes of patients. In addition, ZHX2 expression was relatively higher in RPMI-8226 and MM.1S cell lines and the level of ZHX2 protein was upregulated after BTZ treatment. Knockdown of ZHX2 significantly enhanced the sensitivity of MM cells to BTZ, inhibited nuclear translocation of NF-κB, and reduced mRNA expression of NF-κB target genes. It was also revealed that ZHX2 directly binds to NF-κB.ConclusionOur study showed that ZHX2 can promote proteasome inhibitor resistance in MM cells by regulating the nuclear translocation of NF-κB.

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Regulation of STAT3 and NF-?B activations by S-nitrosylation in multiple myeloma.

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Regulation of STAT3 and NF-κB activations by S-nitrosylation in multiple myeloma.

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