Effects Of Suppressor Of Cytokine Signaling 3 Research Articles

Effects of SOCS3 on the development of colon cancer via regulation of HIF-1α

Purpose: To investigate the influence of suppressor of cytokine signaling 3 (SOCS3) on rats with colon cancer (CC).
 Methods: Sprague-Dawley (SD) rats were randomly divided into CC group and control group, and then CC rat model was constructed. The expression of SOCS3 in CC tissues was determined by quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin-eosin staining (H&E) was used to examine colon tissue morphology. Immunohistochemistry (IHC) staining assay was performed to determine the expression of SOCS3 protein in colon tissues. The contents of HIF-1α, phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) proteins were determined by Western blotting (WB).
 Results: Compared with that in the control group, the number of tumors in CC group was significantly increased (p < 0.05). 2). On the other hand, protein and message ribonucleic acid (mRNA) expressions of SOCS3 were down-regulated in CC group (p < 0.05). 3), while protein expressions of p-PI3K, p-AKT and HIF-1α were raised in CC group (p < 0.05).
 Conclusion: SOCS3 is lowly expressed in CC rats, and promotes the expression of HIF-1α by activating PI3K/AKT signaling pathway. Thus, SOCS3 provides a therapeutic strategy for the management of colon cancer.
 Keywords: Colon cancer; Suppressor of cytokine signaling protein 3 (SOCS3); Hypoxia inducible factor-1α

Gene therapy with SOCS1 induces potent preclinical antitumor activities in oral squamous cell carcinoma.

Constitutive activation of STAT3 promotes oncogenesis and growth of oral squamous cell carcinoma (OSCC). We investigated the mechanism of action of suppressor of cytokine signaling 1 (SOCS1), an endogenous inhibitor of JAK, as gene therapy for OSCC. Antitumor effect of SOCS1 was compared to JAK inhibitor I by cell proliferation assay, cell cycle analysis, and apoptosis analysis in vitro. In addition, antitumor effect was evaluated using xenograft mouse models in vivo. JAK inhibitor I inhibited the proliferation of KOSC2 cl3-43 or T3M-1 clone2 OSCC cell lines in vitro. While JAK inhibitor I arrested both cell lines at the G2/M phase, induction of apoptosis was observed in T3M-1 clone2 cells, but not KOSC2-cl3-43 cells. An adenoviral vector expressing SOCS1 (AdSOCS1) significantly decreased the proliferation of both OSCC cell lines and induced G2/M phase cell cycle arrest and apoptosis, suggesting that induction of apoptosis of KOSC2 cl3-43 cells by AdSOCS1 is regulated by the JAK/STAT independent pathway. Overexpression of SOCS1 inhibited activation of the JAK/STAT and p44/42 MAPK pathways, while JAK inhibitor I inhibited activation of the JAK/STAT pathway only. Consistently, expression of Mcl-1 was decreased by overexpression of SOCS1, but not JAK inhibitor I. Additionally, KOSC2 cl3-43 or T3M-1 clone2 OSCC cells were subcutaneously implanted in the flanks of two xenograft mouse models. As compared to a control adenovirus vector (AdLacZ), intratumor injection of AdSOCS1 significantly decreased the tumor volume and induced apoptosis in vivo. SOCS1 gene therapy may be a beneficial approach for the treatment of OSCC.

Effect of SOCS3 on lung injury in rats with severe acute pancreatitis through regulating JAK2/STAT3 signaling pathway.

To explore the effect of suppressor of cytokine signaling 3 (SOCS3) on the lung injury in rats with severe acute pancreatitis (SAP) by regulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. Sprague-Dawley rats were divided into control group (n=20) and SAP model group (established via injection of 5% sodium taurocholate, n=40). Then, SOCS3 was overexpressed using the adenovirus in 20 rats in SAP model group. The serum amylase (AMY) was detected, whether the transfection was successful was verified via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the hepatic function indexes were detected, the pathological changes were observed using hematoxylin-eosin (HE) staining, and the wet/dry weight ratio (W/D) was calculated. Moreover, the content of serum inflammatory factors was detected via enzyme-linked immunosorbent assay (ELISA) and the expression levels of JAK2/STAT3 signaling pathway genes and proteins were detected through RT-PCR and Western blotting. The content of AMY in SAP model group was significantly increased, indicating the successful modeling. SOCS3 was significantly increased in transfection group, suggesting that the transfection efficiency was significant. The content of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in SOCS3 transfection group was significantly lower than in model group. According to the histopathological observation, there were lung injury, pulmonary edema, hemorrhage, severe inflammatory response, and alveolar congestion in SAP model group. There were almost no pathological changes in SOCS3 transfection group. In SOCS3 transfection group, the content of serum interleukin-6 (IL-6), IL-18 and tumor necrosis factor-α (TNF-α), the mRNA and protein expressions of IL-6, JAK2, and STAT3 were all remarkably declined. SOCS3 inhibits the activation of the JAK2/STAT3 pathway and the increase of inflammatory factors, promoting the repair of lung injury in SAP rats.

Effect of SOCS1 on diabetic renal injury through regulating TLR signaling pathway.

To clarify the effect of suppressor of cytokine signaling 1 (SOCS1) on diabetic nephropathy (DN)-induced renal injury by regulating the Toll-like receptor (TLR) signaling pathway. The Sprague-Dawley rats were divided into control group (n=10) and DN group (established with streptozotocin injection, n=20). The DN rats were administrated with SOCS1 lentivirus to upregulate the in vivo expression. The rat blood glucose was detected to confirm the successful preparation of the DN model. The hepatic and renal function indexes, including blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and creatinine (CR) were detected. The pathological lesions in the kidney were observed via hematoxylin-eosin (HE) staining. Besides, the serum levels of the inflammatory factors in rats were detected via enzyme-linked immunosorbent assay (ELISA). The relative levels of genes in the TLR signaling pathway were detected via RT-PCR and Western blotting. The blood glucose level in rats of the DN group was significantly enhanced, indicating the successful modeling. The expression of SOCS1 was significantly upregulated in rats administrated with SOCS1 lentivirus. The contents of BUN, ALP, ALT, and CR in rats of SOCS1 overexpression group were significantly lower than those in the DN group. The inflammatory infiltration in the kidney and the glomerular injury were pronounced in the DN group. The serum levels of interleukin-1 (IL-1), interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α) were significantly declined in SOCS1 overexpression group. Besides, the mRNA expressions of myeloid differential protein-88 (MyD88), TLR2, and INF-γ, and the protein expression of TLR2 were all remarkably downregulated in SOCS1 overexpression group. SOCS1 can promote renal injury repair in DN rats by inhibiting the TLR pathway. Therefore, SOCS1 is expected to be a new target for the repair of DN renal injury.

Protective Role of SOCS3 Modified Bone Marrow Mesenchymal Stem Cells in Hypoxia-Induced Injury of PC12 Cells

We attempted to explore the possible effects of SOCS3 (suppressor of cytokine signaling 3)-modified bone marrow mesenchymal stem cells (BMSCs) on the hypoxic injury of rat adrenal gland pheochromocytoma (PC-12) cells. PC12 cells were cultured with EGFP (enhanced green fluorescent protein)-BMSCs and SOCS3-BMSCs respectively under hypoxia in vitro and classified into control, hypoxia, EGFP-BMSCs, and SOCS3-BMSC groups. CCK-8, Hoechst 33258 staining, and Annexin V-FITC/PI staining were assessed to measure the viability and apoptosis of hypoxia-induced PC12 cells. The JAK/STAT3 pathway- and apoptosis-related proteins were identified by Western blot. Finally, rat models of permanent middle cerebral artery occlusion (pMCAO) were established to verify the potential influences of SOCS3-BMSCs in vivo. SOCS3-modified BMSCs can stably express SOCS3 protein. EGFP-BMSCs, especially SOCS3-BMSCs, can improve cell viability and SOD content, and reduce cell apoptosis, LDH viability, and MDA content in hypoxia-induced PC12 cells (all P < 0.05). Besides, EGFP-BMSCs and SOCS3-BMSCs decreased cleaved caspase-3 level and increased Bcl-2/Bax of hypoxia-induced PC12 cells, while SOCS3-BMSCs could also elevate SOCS3 protein and reduce p-STAT3 protein level in hypoxia-induced PC12 cells (all P < 0.05). In vivo experiments confirmed that EGFP-BMSCs, particularly SOCS3-BMSCs, could ameliorate infarct size and inhibit neuronal apoptosis of different degrees in pMACO rats (all P < 0.05). SOCS3-modified BMSCs can alleviate oxidative stress, improve cell viability, and reduce neuronal apoptosis by downregulation of JAK/STAT3 pathway, thereby exerting the neuroprotective role in ischemic brain injury.

Cardiac Ablation of SOCS3 Aggravates DOCA-Salt-Induced Hypertrophic Remodeling by Activation of Gp130-Dependent Signaling in Mice

Background/Aims: Cardiac remodeling is a critical pathogenetic process leading to heart failure. Suppressor of cytokine signaling-3 (SOCS3) is demonstrated as a key negative regulator of the gp130 receptor to inhibit cardiac hypertrophy. However, the role of SOCS3 in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. Methods: Cardiac-specific SOCS3 knockout (SOCS3cKO) and wild-type (WT) C57BL/6J mice were subjected to uninephrectomy and DOCA-salt for 3 weeks. The effect of SOCS3 on cardiac remodeling and inflammation was evaluated by histological analysis. Gene and protein levels were measured by real-time PCR and immunoblotting analysis. Results: After DOCA-salt treatment, the expression of SOCS3, activation of gp130/JAK/STAT3, cardiac dysfunction and fibrosis in DOCA-salt mice were significantly elevated, which were markedly attenuated by eplerenone, a specific mineralocorticoid receptor (MR) blocker. Moreover, DOCA-salt-induced cardiac dysfunction, hypertrophy, fibrosis and inflammation were aggravated in SOCS3cKO mice, but were significantly reduced in AAV9-SOCS3-injected mice. These effects were mostly associated with activation of gp130/STAT3/AKT/ERK1/2, TGF-β/Smad2/3 and NF-κB signaling pathways. Conclusions: Our data demonstrate that loss of SOCS3 in cardiomyocytes promotes DOCA-salt-induced cardiac remodeling and inflammation, and it may be a novel potential therapeutic target for hypertensive heart disease.

SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes.

CAAT/enhancer-binding protein-beta (C/EBPβ) is a transcription factor that regulates interleukin-1β (IL-1β)-induced catabolic pathways, including the expression of matrix metalloproteinases (MMPs), in chondrocytes. We previously reported that suppressor of cytokine signaling 1 (SOCS1) inhibits IL-1β signaling in chondrocytes. However, the effect of SOCS1 on C/EBPβ has not been explored. To investigate the interaction between SOCS1 and C/EBPβ, we established human SW1353 cells with overexpression or knockdown of SOCS1 or C/EBPβ. Both SOCS1 and C/EBPβ were involved in transcription of MMP-3 and MMP-13. When stimulated with IL-1β, C/EBPβ levels were significantly increased by SOCS1 knockdown and decreased by SOCS1 overexpression. A similar change in IL-1β-induced C/EBPβ expression was observed in SOCS1-transfected human articular chondrocytes. However, C/EBPβ overexpression or knockdown did not change the levels of IL-1β-induced SOCS1. SOCS1 regulated the levels of C/EBPβ mRNA by ubiquitination of C/EBPβ as well as transcriptional regulation. Furthermore, it suppressed the phosphorylation of cAMP response element-binding protein (CREB), an active transcription factor of C/EBPβ. In addition, p38 mitogen-activated protein kinases, a target of SOCS1, was involved in CREB phosphorylation. The chromatin immunoprecipitation assay confirmed that SOCS1 overexpression led to reduced binding of C/EBPβ to the MMP-13 promoter. Taken together, our results demonstrate that SOCS1 downregulates the p38-CREB-C/EBPβ pathway resulting in increased expression of MMPs in chondrocytes.

Transcriptional repression of SOCS3 mediated by IL-6/STAT3 signaling via DNMT1 promotes pancreatic cancer growth and metastasis.

BackgroundPrevious studies have investigated the sustained aberrantly activated Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is crucial for pancreatic cancer growth and metastasis. Suppressor of cytokine signaling 3 (SOCS3), as a key negative feedback regulator of this signaling pathway, is usually down-regulated in various cancers. In the present study, we aim at exploring the biological function and the underlying molecular regulation mechanisms of SOCS3 in pancreatic cancer.MethodsThe expression of SOCS3 and other genes in pancreatic cancer was examined by Quantitative real-time PCR, western blotting and immunohistochemical staining. The interaction between pSTAT3 and DNA Methyltransferase 1 (DNMT1) was investigated by co-immunoprecipitation assay. Luciferase reporter assay was used to investigate the transcriptional regulation of pSTAT3 and DNMT1 on the SOCS3 gene. The effects of SOCS3 on the biological behavior of pancreatic cancer cells were assessed both in vitro and vivo. Furthermore, we performed a comprehensive analysis of the expression of SOCS3 in a pancreatic cancer tissue microarray (TMA) and correlated our findings with pathological parameters and outcomes of the patients.ResultsWe showed that SOCS3 expression was decreased in phosphorylated STAT3 (pSTAT3)-positive tumors and was negatively correlated with pSTAT3 in pancreatic cancer cells. We also found that IL-6/STAT3 promoted SOCS3 promoter hypermethylation by increasing DNMT1 activity; silencing DNMT1 or 5-aza-2-deoxycytidine (5-AZA) treatment could reverse the down-regulation of SOCS3 mediated by IL-6. Using co-immunoprecipitation and luciferase reporter assays, we found that STAT3 recruited DNMT1 to the promoter region of SOCS3 and inhibited its transcriptional activity. Overexpression of SOCS3 significantly inhibited cell proliferation, which may be due to the increase in G1-S phase arrest; overexpression of SOCS3 also inhibited cell migration and invasion as well as tumorigenicity in nude mice. Pancreatic cancer tissue microarray analysis showed that high SOCS3 expression was a good prognostic factor and negatively correlated with tumor volume and metastasis.ConclusionWe demonstrated that activated IL-6/STAT3 signaling could induce SOCS3 methylation via DNMT1, which led to pancreatic cancer growth and metastasis. These data also provided a mechanistic link between sustained aberrantly activated IL-6/STAT3 signaling and SOCS3 down-regulation in pancreatic cancer. Thus, inhibitors of STAT3 or DNMT1 may become novel strategies for treating pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0301-7) contains supplementary material, which is available to authorized users.

Abstract 722: Gene therapy for peritoneal dissemination model of gastric cancer using SOCS-1 by Adenoviral Vector

Abstract Objective: The peritoneal metastasis is one of the most common recurrence style of gastric cancer (GC) and an effective treatment for their patients has not been established. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of various cytokine signaling and has been recently focused on as a therapeutic target in treatment for cancer. The aim of this study was to evaluate the effect with the enforced SOCS-1 expression by adenoviral vector (AdSOCS-1). METHODS: We evaluated the therapeutic effect of SOCS-1 in 6 GC cell lines with proliferation assay. Moreover, we examined the mechanism of antitumor effect of SOCS-1 focusing on the cell signalling and cell cycle in vitro. In vivo study, we evaluated the therapeutic effect in mice model. MKN45-Luc cell line was intraperitoneally injected into ICR nu/nu mice. And, we evaluated the peritoneal dissemination by the in vivo imaging system using Xenogen IVIS ® Imaging System. We treated these mice using the intraperitoneal injection of AdSOCS-1, twice a week for 4weeks. We evaluated the peritoneum dissemination by the photon intensity with time course repeatedly. Finally, we examined these tumor specimen pathologically and the overall survival (N=22). RESULTS: In 3 GC cell lines among 6 GC cell lines, AdSOCS-1 suppressed the proliferation. SOCS-1 showed an anti-proliferative potential in the GC cell proliferation via the inhibition of JAK/STAT and p38 MAPK signaling pathways and also induced apoptosis in vitro. Additionally, SOCS-1 is proven to influence the cell cycle in G2/M phase activating the Chk2 in a JAK/STAT independent manner. In Xenograft peritoneal dissemination model, the total count of photon after therapy in AdSOCS-1 group was significantly lower than that in control group. By TUNEL immunohistochemical analysis, tumor specimen in AdSOCS-1 group included more apoptosis area comparing with that in the control group. To assess whether overexpression of SOCS-1 also reduced proliferation of MKN-45 cells in vivo, Ki-67 expression was determined by immunohistochemistry. AdSOCS-1 group showed decreased Ki-67 positive cells compared to control group. We compared the overall survival of the peritoneal dissemination mice in two groups. AdSOCS-1 group showed better outcome than control group significantly. CONCLUSIONS: Our results indicated that SOCS-1 inhibited the progression of gastric cancer in Xenograft peritoneal dissemination model. SOCS-1 can be a novel therapeutic application for peritoneal metastasis in gastric cancer. Citation Format: Rie Nakatsuka, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yoshihito Souma, Yasuhiro Miyazaki, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Yuichiro Doki, Tetsuji Naka. Gene therapy for peritoneal dissemination model of gastric cancer using SOCS-1 by Adenoviral Vector. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 722. doi:10.1158/1538-7445.AM2014-722

Hydrodynamic tail vein injection of SOCS3 eukaryotic expression vector in vivo promoted liver lipid metabolism and hepatocyte apoptosis in mouse

Suppressor of cytokine signaling 3 (SOCS3), a signal transduction cytokine, is involved in lipid metabolism as well as in cell proliferation, differentiation, apoptosis, and so on. To explore the effects of SOCS3 on apoptosis and lipid metabolism in liver, we used a simple effective method named hydrodynamic tail vein injection to overexpress SOCS3. Then orbital blood was obtained for the assessment of blood lipid after injection. Lipid metabolism related genes were detected by Western blot after the determination of serum lipids. Meanwhile, liver cell apoptosis was observed by Hoechst and TUNEL staining and the expression of apoptosis related proteins Bax, Bcl-2, and Caspase3 were detected as well as the JAK2/STAT3 signaling pathway. In addition, we also demonstrated the effect of SOCS3 in prime hepatocyte by overexpression or interference of SOCS3 along with SD1008, which is a specific inhibitor of the JAK2/STAT3 signaling pathway. Taken together, all the results indicated that SOCS3 promoted lipid synthesis in mice liver and promoted hepatocyte apoptosis by inhibiting the activation of the JAK2/STAT3 signaling pathway, however the detailed regulation mechanism had not yet been fully understood and needs further study.