Departments of Pediatrics, Pathology and Laboratory Medicine and Radiation Oncology. Medical University of South Carolina, Charleston, SC, USA.
Redox Biol. 2015 Aug;5:416-417. doi: 10.1016/j.redox.2015.09.021. Epub 2015 Dec 30.
In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. 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 (Tyr705). 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 Cys259 was the target Cys residue of GSNO-mediated S-nitrosylation of STAT3. The replacement of Cys259 residue with Ala abolished the inhibitory role of GSNO in IL-6-induced STAT3 phosphorylation and transactivation, suggesting the role of Cys259S-nitrosylation in STAT3phosphorylation. Since STAT3 activation is involved in tumor progression and metastasis, we investigated the effect of GSNO in cell culture and mouse xenograft model of head and neck squamous cell carcinoma (HNSCC). GSNO treatment of HNSCCN cell lines reversibly decreases the activation (phosphorylation) of STAT3 in a concentration dependent manner. The reduced STAT3/NF-kB activity by GSNO correlated with decreased cell proliferation and increased apoptosis of HNSCC cells. In HNSCC mouse xenograft model, the tumor growth was reduced by systemic treatment with GSNO and was further reduced when the treatment combined with radiation and cisplatin. Accordingly, GSNO treatment also resulted in decreased levels of pSTAT3 and tumor growth regulators (ie. cyclin D2, VEGF and Bcl-2) in tumor tissue. In summary, these findings have implications for the development of new therapeutics targeting of STAT3 for treating diseases associated with inflammatory/immune responses and abnormal cell proliferation, including cancer.
在这项研究中，我们评估了基于 S-亚硝基化的 Janus 激活激酶 2/信号转导和转录激活因子 3（JAK2/STAT3）通路的调节。我们的研究表明，受刺激的小胶质细胞中的 STAT3 经历了两种不同的氧化还原依赖修饰，S-亚硝基化和 S-谷胱甘肽化。STAT3 的 S-亚硝基化与诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）和 S-亚硝基谷胱甘肽（GSNO）有关，而 STAT3 的 S-谷胱甘肽化与细胞氧化应激有关。iNOS 产生的 NO 或用外源性 GSNO 处理小胶质细胞会抑制 STAT3 激活，从而抑制 STAT3 磷酸化（Tyr705）。因此，白细胞介素 6（IL-6）诱导的小胶质细胞增殖和相关基因表达也减少。在使用纯化的 JAK2 和 STAT3 的细胞外激酶测定中，STAT3 的磷酸化通过其与 GSNO 的选择性预孵育而被抑制，但通过 JAK2 与 GSNO 的预孵育未被抑制，表明 GSNO 介导的机制通过 STAT3 的 S-亚硝基化而不是 JAK2 抑制 STAT3 磷酸化。在这项研究中，我们确定 Cys259 是 GSNO 介导的 STAT3 S-亚硝基化的靶 Cys 残基。用丙氨酸取代 Cys259 残基消除了 GSNO 在 IL-6 诱导的 STAT3 磷酸化和转导激活中的抑制作用，表明 Cys259S-亚硝基化在 STAT3 磷酸化中的作用。由于 STAT3 激活参与肿瘤的进展和转移，我们研究了 GSNO 在头颈部鳞状细胞癌（HNSCC）的细胞培养和小鼠异种移植模型中的作用。GSNO 处理 HNSCCN 细胞系以浓度依赖的方式可逆地降低 STAT3 的激活（磷酸化）。GSNO 降低 STAT3/NF-κB 活性与 HNSCC 细胞的增殖减少和凋亡增加相关。在 HNSCC 小鼠异种移植模型中，全身用 GSNO 治疗可减少肿瘤生长，当与放疗和顺铂联合治疗时，肿瘤生长进一步减少。因此，GSNO 治疗还导致肿瘤组织中 pSTAT3 和肿瘤生长调节剂（即细胞周期蛋白 D2、VEGF 和 Bcl-2）水平降低。总之，这些发现为开发针对 STAT3 的新治疗方法提供了依据，用于治疗与炎症/免疫反应和异常细胞增殖相关的疾病，包括癌症。
