Signal Transducer And Activator Of Transcription 3 Expression Research Articles

STAT3 in porcine endometrium during early pregnancy induces changes in extracellular matrix components and promotes angiogenesis†.

A molecular interaction between maternal endometrium and implanting conceptus can lead to activation of a variety of transcription factors that regulate expression of several genes necessary for the process of embryo implantation. While, signal transducer and activator of transcription 3 (STAT3) is responsible for decidualization and epithelial remodeling in humans and mice, its role in porcine endometrium has not been explored before. In the present study, we observed a pregnancy dependent increase in gene and protein expression of STAT3. Phosphorylated STAT3 was predominantly present in the endometrium of pregnant animals in luminal and glandular epithelium and in the endothelium of blood vessels with a weak staining in stromal cells. Interleukins, IL-1β and IL-6, and epidermal growth factor (EGF)-induced STAT3 expression and phosphorylation in endometrial explants collected on Day 13 of the estrous cycle. Biological significance of STAT3 was evaluated by blocking its phosphorylation with STAT3-specific inhibitor, Stattic. Using porcine extracellular matrix (ECM) and adhesion molecule array, EGF was shown to induce changes in gene expression of ECM components: MMP1, MMP3, MMP12, LAMA1, SELL, and ICAM1, which was abrogated in the presence of Stattic. Transcriptional activity of STAT3 was observed in promoter regions of MMP3 and MMP12. Additionally, IL-6-induced STAT3 phosphorylation upregulated VEGF and VCAM1 abundances in endometrial-endothelial cells (EEC). Moreover, IL-6 resulted in an increase in EEC proliferation and capillary formation which was reversed in the presence of Stattic. Results of present study reveal a role for STAT3 phosphorylation in regulating extracellular matrix remodeling and angiogenesis in porcine endometrium to facilitate embryo implantation.

Luteolin restricts ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway

African swine fever (ASF) is a devastating infectious disease that causes significant economic losses to the pig industry worldwide. Luteolin is abundant in onion leaves, carrots, broccoli, and apple skin and exerts various biological activities, including anti-cancer and anti-virus effects. Our aim was to demonstrate the mechanism of action and potent antiviral activity of luteolin against ASF virus (ASFV) in porcine alveolar macrophages. We performed cell viability, hemadsorption, indirect immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction assays to investigate the effect of luteolin on ASFV. Notably, luteolin restricted ASFV replication in a dose-dependent manner. The anti-ASFV activity of luteolin was maintained for 24–72 h. Subsequent experiments revealed that luteolin could block multiple stages of the ASFV replication cycle, including those at 6–9 h and 12–15 h after infection, instead of directly interacting with ASFV. Moreover, ASFV infection stimulated the expression of phosphorylated nuclear factor (NF)-κB, interleukin (IL)− 6, and phosphorylated signal transducer and activator of transcription 3 (STAT3). However, luteolin downregulated ASFV-induced NF-κB, IL-6, and STAT3 expression. Importantly, NF-κB agonist CU-T12–9 weakened the inhibitory effects of luteolin on NF-κB and STAT3. Moreover, CU-T12–9 partially restored the inhibitory effect of luteolin on ASFV. Similarly, luteolin reduced ASFV-induced activating transcription factor 6 (ATF6) expression, and CU-T12–9 weakened the inhibitory effect of luteolin on ATF6. Our findings suggested that luteolin inhibited ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway and might provide a rationale for anti-ASFV drug development.

Effect and Mechanism of Running on Mouse Emphysema Model Induced by Cigarette Extract

<bold>Objective</bold> To observe the effect of running on mouse emphysema model induced by cigarette extract, and to explore its mechanism based on signal transducer and activator of transcription 3 (STAT3). <bold>Methods</bold> A total of 40 male C57BL/6 mice were randomly divided into control group, exercise group, emphysema group and emphysema + exercise group, with 10 mice in each group. The control group and the exercise group were intraperitoneally injected with 0.3 mL phosphate buffer saline (PBS) at the 0, 11th and 22th day in the experiment; the emphysema group and the emphysema + exercise group were intraperitoneally injected with 0.3 mL cigarette extract at the 0, 11th and 22th day in the experiment. The control group and the emphysema group were free to move in the cage from the 23rd to 29th day; the exercise group and the emphysema + exercise group received aerobic running training from the 23rd day to the 29th day (15 m/min, 30 minutes a day). Hematoxylin eosin (HE) staining was used to observe the pathological changes of lung tissue and evaluate the severity of emphysema in the four groups; the expression levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid (BAL) and serum were analyzed to evaluate the pulmonary and systemic inflammation of the four groups; Western blot method was used to analyze the expression of STAT3 in lung tissues. <bold>Results</bold> 1) Severity of emphysema: compared with the control group, the alveolar chord length of mice in the emphysema group and the emphysema + exercise group significantly increased, the difference was statistically significant (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the alveolar chord length in the emphysema + exercise group significantly decreased，the difference was statistically significant (<italic>P</italic>&lt;0.05). 2) Lung inflammation: compared with the control group, the contents of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-6, and IL-17 in BAL of the emphysema group and the emphysema + exercise group significantly increased, while the IL-10 level significantly decreased (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the counts of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-6, and IL-17 in BAL of the emphysema + exercise group were statistically lower, and the IL-10 level was statistically higher, the difference was statistically significant (<italic>P</italic>&lt;0.05). 3) Systemic inflammation: compared with the control group, the counts of white blood cells, neutrophils and lymphocytes in the emphysema group statistically increased (<italic>P</italic>&lt;0.05), the expression levels of IL-1β, IL-17, and TNF-α in the emphysema group and the emphysema + exercise group statistically increased, and the IL-10 level statistically decreased (<italic>P</italic>&lt;0.05). Compared with emphysema group, the counts of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-17, and TNF-α in the emphysema + exercise group were lower, and the IL-10 level was statistically higher, the difference was statistically significant (<italic>P</italic>&lt;0.05). 4) STAT3 expression level in lung tissues: compared with the control group, p-STAT3 expression level in lung tissues of the emphysema group and the emphysema + exercise group statistically increased (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the expression level of p-STAT3 in lung tissues of the emphysema + exercise group statistically decreased, the difference was statistically significant (<italic>P</italic>&lt;0.05). <bold>Conclusion</bold> Running could reduce the pulmonary and systemic inflammation, and alleviate the pathological manifestations of emphysema in the mice model of emphysema, and its mechanism may be related to the inhibition of STAT 3 activation.

Anti-inflammatory Effect of (-)-Epigallocatechin-3-O-gallate on Human Umbilical Vein Endothelial Cells Grown on 316L Stainless Steel via STAT3/NF-κB Signaling

Stainless steel (316L SS) is commonly used to build coronary artery stents for the treatment of occluded arteries. However, tissues in contact with the stent may develop inflammation that can lead to restenosis. The natural substances derived from normal diet provide a pool of candidates that have potential to treat cardiovascular diseases. (-)-Epigallocatechin-3- O-gallate (EGCG), a polyphenolic flavonoid present in green tea, has antioxidant, antithrombogenic, and anti-inflammatory effects, and may reduce the risk of cardiovascular diseases. This study aimed to investigate whether EGCG has an anti-inflammatory effect on human umbilical vein endothelial cells (HUVECs) attached to the surface of 316L SS. We evaluated cell proliferation using the dimethyl thiazolyl tetrazolium bromide method in HUVECs after treatment with EGCG. Enzyme-linked immunosorbent assay (ELISA) assessed the level of inflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor (TNF) in HUVECs. We further investigated the regulatory mechanisms of the signal transducer and activator of transcription 3 (STAT3)/NF-κB signaling pathway in HUVECs by Western-blot analysis. We found that HUVECs cultured on 316L SS had increased cell proliferation and inflammation, and these can be inhibited by treatment with EGCG. EGCG reduced the secretion of IL-6 and TNF and decreased the expression of STAT3 and NF-κB in HUVECs cultured on 316L SS. Consequently, our study demonstrated that EGCG treatment ameliorates the proliferation of HUVEC when cultured with 316L SS, potentially by modulating the inflammation responses via the STAT3/NF-κB signaling pathways.

The STAT3 inhibitor GPB730 enhances the sensitivity to enzalutamide in prostate cancer cells

Enzalutamide is a second-generation anti-androgen which has shown increased survival in patients with metastatic prostate cancer. However, some patients do not respond to this therapy or will develop resistance to treatment over time. Signal Transducer and Activator of Transcription 3 (STAT3) is known to be involved in castration-resistant prostate cancer and to interact with androgen receptor (AR)-signaling. This study aims to investigate the combination enzalutamide and the small molecule STAT3 inhibitor GPB730 for enhanced therapeutic effect in advanced prostate cancer in vitro. The prostate cancer cell lines LNCaP (androgen dependent) and C4–2 (androgen insensitive) were used. The effect of enzalutamide and GPB730, alone and in combination, was investigated on viability and IC50 values calculated. Enzalutamide and GPB730 treated LNCaP and C4–2 cells were subjected to western blot and QPCR analyses in order to investigate the expression of AR, STAT3 and down-stream targets. C4–2 were less sensitive to growth inhibition by enzalutamide than LNCaP cells. GPB730 enhanced the growth inhibitory effect of enzalutamide in LNCaP and C4–2 cells. The addition of GPB730 to enzalutamide decreased the IC50 values for enzalutamide by 3.3-fold for LNCaP and by 12-fold for C4–2. In C4–2 cells, GPB730 alone decreased PSA expression and enhanced the enzalutamide induced decrease in NKX3.1 expression. GPB730 and enzalutamide in combination enhanced inhibition of c-myc and survivin expression. This study suggests that enzalutamide may be combined with the STAT3 inhibitor GPB730 in order to enhance the efficacy of enzalutamide, offering a new therapeutic approach in advanced prostate cancer.

JMJD3 Promotes Porphyromonas gingivalis Lipopolysaccharide-Induced Th17-Cell Differentiation by Modulating the STAT3-RORc Signaling Pathway.

The immune response mediated by Th17 cells is essential in the pathogenesis of periodontitis. Emerging evidence has demonstrated that lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) could promote Th17-cell differentiation directly, while the downstream signaling remains elusive. This study was aimed to explore the role of JMJD3 (a JmjC family histone demethylase) and signal transducers and activators of transcription 3 (STAT3) in Th17-cell differentiation triggered by Pg-LPS and clarify the interaction between them. We found that the expression of JMJD3 and STAT3 was significantly increased under Th17-polarizing conditions. Pg-LPS could promote Th17-cell differentiation from CD4+ T cells, with an increased expression of JMJD3 and STAT3 compared to the culture without Pg-LPS. The coimmunoprecipitation results showed that the interactions of JMJD3 and STAT3, STAT3 and retinoid-related orphan nuclear receptor γt (RORγt) were enhanced following Pg-LPS stimulation during Th17-cell differentiation. Further blocking assays were performed and the results showed that inhibition of STAT3 or JMJD3 both suppressed the Th17-cell differentiation, JMJD3 inhibitor could reduce the expression of STAT3 and p-STAT3, while JMJD3 expression was not affected when STAT3 was inhibited. Taken together, this study found that JMJD3 could promote Pg-LPS induced Th17-cell differentiation by modulating the STAT3-RORc signaling pathway.

S3I-201 derivative incorporating naphthoquinone unit as effective STAT3 inhibitors: Design, synthesis and anti-gastric cancer evaluation

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of many human cancers and has been widely recognized as a promising target for cancer therapy. A variety of small-molecule inhibitors have been developed for targeting STAT3, and some of them are now undergoing clinical trials. S3I-201, a known STAT3 inhibitor, may block STAT3 function in cancer cells by binding to the STAT3 SH2 domain to disrupt STAT3 protein complex formation. Using S3I-201 as a starting point for drug development, we synthesized a series of new STAT3 inhibitors 9a-x in this study by introducing naphthoquinone unit, a privileged fragment in STAT3 inhibitors. Most of the compounds exhibited strong anti-proliferation activity of gastric cancer cells (MGC803, MKN28, MNK1, and AGS). The representative compound 9n (SIL-14) could effectively inhibit the colony formation and migration of gastric cancer cells MGC803, arrest the cell cycle and induce MGC803 cell apoptosis at low micromolar concentrations in vitro. In addition, SIL-14 can also inhibit the phosphorylation of STAT3 protein and significantly decrease the expression of total STAT3, suggesting that it may exert anticancer effects by blocking the STAT3 signaling pathway. These results support that SIL-14 may be a promising STAT3 inhibitor for the further development of potential anti-gastric cancer candidates.

Prognostic significance of STAT3 gene expression in patients with glioblastoma tumors: a study from Western India

ObjectiveGlioblastoma Multiforme (GBM), a devastating the most common primary malignant intracranial brain tumors. In India, the incidence of this malignancy is escalating, however, there are very few studies on this tumor entity from Indian population. The present study sought to investigate the prevalence and prognostic significance of Signal Transducer and Activator of Transcription 3 (STAT3) gene expression in GBM patients from Western India.MethodSTAT3 gene expression using real-time PCR was detected in total 55 GBM patients. The impact of STAT3 aberrant expression on progression-free survival (PFS) and overall (OS) was analysed using univariate and multivariate survival analysis. The data were analysed using SPSS statistical software and p value ≤0.05 was considered as significant.ResultsThe aberrant STAT3 expression was found in 85% (47/55) of patients with -1.12 fold change down-regulation in 49% (23/47) and 3.36 fold change up-regulation was noted in 51% (24/47) of patients. In wild type IDH tumors (n=30), down regulation and up regulation of STAT3 was noted in 63% and 27% of patients, respectively, whereas, for IDH mutant GBM tumors (n=25), the incidence of low expression and high expression of STAT3 was noted in 16% and 68% of patients, respectively. Thus, we found that incidence of STAT3 down regulation was significantly high in patients with IDH wild type tumors, whereas, in IDH mutant GBM tumors, the incidence of up-regulated STAT3 was significantly high (P=0.021, χ2=12.81, r=+0.310). In Kaplan-Meier univariate survival analysis, a part from age (P=0.006), tumor location (P=0.025), and KPS score (P=0.002), co-detection of STAT3 up regulation and presence of IDH mutation (P=0.030) remained significant prognostic factors for PFS and OS. In multivariate survival analysis also, co-detection of STAT3 high expression and presence of IDH mutation remained independent prognosticators for PFS (HR=6.45, 95% CI=1.32-31.40, P=0.021) and OS (HR=8.69, 95% CI=1.66-45.51, P=0.010).ConclusionFor GBM tumors, STAT3 up-regulation and presence of IDH mutations together predicts better survival. This reflects unique molecular etiology for GBM patients. Therefore, they would be useful in the future for targeted therapy and for clinicians they would be useful for better patient management. However, study on a larger sample size is required for validation.

Altered Expression of STAT3, RORγt, and IL-17A Proteins and Th17 Cell in Intrahepatic Cholestasis of Pregnancy

Background: Intrahepatic cholestasis of pregnancy (ICP) is a disorder specifically associated with pregnancy. Recent evidence suggests that the T helper 17 (Th17) cell population is related to a maternal and foetal immune imbalance associated with ICP. However, there has been insufficient attention paid to the potential roles of signal transducer and activator of transcription 3 (STAT3) and RAR-related orphan receptor gamma (RORγt) in modulations of Th17 cell in ICP. Accordingly, the purpose of our study was to investigate the alterations of Th17 cell in placenta and peripheral blood of patients with ICP and correlations between Th17 cell and STAT3, RORγt, interleukin (IL)-17A in ICP. Methods: Nine pregnant women with ICP and nine women with normal pregnancy served as the ICP and control groups, respectively. STAT3, RORγt, and IL-17A expression were examined by immunohistochemistry and western blotting in placental tissue. Flow cytometry was used to quantify Th17 cell in blood of peripheral circulation. We compared data between groups using Chi-square tests or paired t tests. Pearson or Spearman coefficients were used to measure correlations. Results: STAT3, RORγt, and IL-17A were mainly expressed in the trophoblasts of the two groups of patients. Comparatively to the control group, placental levels of STAT3, RORγt, and IL-17A proteins were significantly elevated in ICP group, as was maternal levels of Th17 cell in peripheral blood. Moreover, placental IL-17A protein level showed significantly positive relationships with placental STAT3 (r = 0.97, p = 2e-05) and RORγt (r = 0.91, p = 0.01) protein in control group, however, not in ICP group (STAT3, r = 0.5, p = 0.17; RORγt, r = 0.62, p = 0.07). Conclusions: Women with ICP showed an increase in Th17 cells in comparison to women with normal pregnancies. STAT3 and RORγt may increase Th17 cell proliferation and differentiation, appears to be altered in ICP. ICP may be adversely affected by excessive accumulation of Th17 cell.

Characterizing the metabolic role of STAT3 in canine osteosarcoma.

Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.

Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3).

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.

The effect of electroacupuncture on the expression of Sirt1 and STAT3 in hippocampus and amygdala of vascular dementia rats.

Inflammation has long been considered a key factor in learning and memory impairment in patients with vascular dementia (VaD). Studies have confirmed that electroacupuncture can improve the learning and memory impairment of patients with VaD by reducing inflammation, but the specific mechanism of this effect is still unclear. The aim of this study was to explore the underlying mechanism of electroacupuncture in the treatment of VaD. The vascular dementia animal model was established by bilateral occlusion of common carotid arteries, and electroacupuncture treatment was given at Baihui (DU20) and Zusanli (ST36). The morris water maze (MWM) was used to test the spatial learning and memory ability of rats in each group. To evaluate the expression of Sirtuin1 (Sirt1), Signal transducer and activator of transcription 3 (STAT3) and inflammatory cytokine (IL-17) in the hippocampus and amygdala, immunohistochemistry and western blot were performed. The MWM test and Nissl staining results show that electroacupuncture can significantly improve the learning and memory impairment of VaD rats, and can repair damaged neurons. Immunohistochemistry and western blot results showed that electroacupuncture could enhance the expression of sirt1 in VaD rats, on the contrary, the expression of STAT3 and IL-17 was reduced due to electroacupuncture. The result suggests that electroacupuncture can suppress inflammation through the Sirt1/STAT3 pathway and improve spatial learning and memory in VaD rats.

Effect of moxibustion combined with acupoint catgut embedding on IL-6/JAK/STAT3 signaling pathway in colonic mucosa of ulcerative colitis rats

To observe the effect of moxibustion combined with acupoint catgut embedding on the content of interleukin 6 (IL-6), and the expressions of janus activated kinase (JAK), signal transducer and activator of transcription 3 (STAT3) in colonic mucosa of rats with ulcerative colitis (UC), so as to explore its mechanism underlying improvement of UC. Thirty SD rats were randomly divided into normal, model, acupoint catgut embedding (ACE), moxibustion and acupoint catgut embedding combined with moxibustion (combination) groups (n=6 rats in each group). The UC model was established by enema of trinitro-benzene-sulfonic acid and ethanol. Moxibustion was applied to bilateral "Tianshu" (ST25), "Dachangshu" (BL25) and "Shangjuxu" (ST37) for 10 min, once daily for 14 days, and ACE applied to the same 3 acupoints, once a week for two weeks. After the treatment, colonic mucosal pathological changes were observed by H.E. staining, the level of IL-6 in colonic mucosa was assayed by ELISA, and the expressions of JAK and STAT3 in colonic mucosa were detected by immunohistochemistry and Western blot. H.E. staining showed severe defect of the colonic mucosal epithelium with infiltration of a large number of inflammatory cells in the model group, which was milder in moxibustion, ACE and combination groups. After modeling, the content of colonic IL-6, and the expression levels of JAK and STAT3 were obviously increased (P<0.01) in the model group relevant to the normal group. Following the intervention, the increase of IL-6 contents, and JAK and STAT3 expressions were reversed (P<0.05, P<0.01) in moxibustion, ACE and combination groups. The therapeutic effects of moxibustion combined with ACE were considerably superior to those of simple ACE and simple moxibustion in down-regulating the levels of JAK and STAT3 expression (P<0.01). Acupoint catgut embedding combined with moxibustion can repair the injured colonic mucosa of UC rats, which may be related with its effect in suppressing the activation of IL-6/JAK/STAT3 signaling pathway.

Abstract CT565: Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer

Abstract Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Effects of STAT3 on aging-dependent neovascularization impairment following limb ischemia: from bedside to bench.

Aging is a major risk factor for ischemic hypoxia-related diseases, including peripheral artery diseases (PADs). Signal transducer and activator of transcription 3 (STAT3) is a critical transcription activator in angiogenesis. Nevertheless, the effect of aging on endothelial cells and their responses to hypoxia are not well studied. Using a hindlimb hypoxic/ischemic model of aged mice, we found that aged mice (80-100-week-old) expressed significantly lower levels of angiogenesis than young mice (10-week-old). In our in vitro study, aged endothelial cells (≥30 passage) showed a significant accumulation of β-galactosidase and a high expression of aging-associated genes, including p16, p21, and hTERT compared with young cells (<10 passage). After 24 hours of hypoxia exposure, proliferation, migration and tube formation were significantly impaired in aged cells compared with young cells. Notably, STAT3 and angiogenesis-associated proteins such as PI3K/AKT were significantly downregulated in aged mouse limb tissues and aged cells. Further, using STAT3 siRNA, we found that suppressing STAT3 expression in endothelial cells impaired proliferation, migration and tube formation under hypoxia. Correspondingly, in patients with limb ischemia we also observed a higher expression of circulating STAT3, associated with a lower rate of major adverse limb events (MALEs). Collectively, STAT3 could be a biomarker reflecting the development of MALE in patients and also a regulator of age-dependent angiogenesis post limb ischemia. Additional studies are required to elucidate the clinical applications of STAT3.

Pirfenidone inhibits cell fibrosis in connective tissue disease-associated interstitial lung disease by targeting the TNF-α/STAT3/KL6 pathway.

BackgroundTo explore the effect and mechanism of pirfenidone in inhibiting pulmonary fibrosis in connective tissue disease-associated interstitial lung disease (CTD-ILD).MethodsFrom 2018 to 2020, 50 CTD-ILD patients were enrolled in the clinical study. Based on whether pirfenidone was used during treatment, patients were enrolled into the pirfenidone group and the control group. Pulmonary function tests were compared before and after treatment. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-α (TNF-α), signal transducer and activator of transcription 3 (STAT3), and Krebs Von den Lungen-6 (KL-6) in venous blood before and after treatment. Rat type II (RLE-6TN) lung epithelial cells were cultivated for in vitro experiments, and they were sorted into the control group, bleomycin group, pirfenidone group, TNF-α group, Stattic group, and TNF-α/Stattic combined treatment group. For the in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate cell proliferation, Reverse Transcription-Polymerase Chain Reaction(RT–PCR) was performed to detect STAT3 and KL-6 mRNA expression levels, ELISA was utilized to detect TNF-α and E-cadherin expression levels, and Western blotting (WB) was performed to determine α-smooth muscle actin (α-SMA), vimentin, TNF-α, STAT3, phosphorylated signal transducer and activator of transcription 3 (PSTAT3) and KL-6 expression.ResultsIn the clinical study, the pulmonary function indices including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC, peak expiratory flow (PEF) and partial pressure (PaO2) of the patients in the study group were superior to those in the control group (P<0.05). The serum TNF-α, STAT3 and KL-6 levels in the study group were significantly lower than those in the control group (P<0.05). In the in vitro experiments, the α-SMA, vimentin, STAT3 and KL-6 levels in the treatment group were significantly lower than those in the bleomycin group (P<0.05). Compared with those in the pirfenidone group, the α-SMA, vimentin, STAT3 and KL-6 levels in the TNF-α-treated group were significantly upregulated (P<0.05). Meanwhile, cell viability was further upregulated (P<0.05), and the expression of STAT3 and KL-6 was further decreased in the Stattic-treated group (P<0.05). In the group treated with infliximab combined with Stattic, TNF-α expression was significantly increased (P<0.05), cell activity was significantly restored (P<0.05), and the STAT3, KL-6 and E-cadherin expression levels were inhibited (P<0.05).ConclusionsPirfenidone improved pulmonary function 1 and decreased TNF-α, STAT3, and KL-6 expression in CTD-ILD patients. Moreover, pirfenidone inhibits cell fibrosis through the TNF-α/STAT3/Mucin 1(MUC1) pathway.

Therapeutic effect of Shengjiang Powder on experimental autoimmune encephalomyelitis mice through IL-6/JAK2/STAT3 signaling pathway:based on HPLC

A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.

Signal transducer and activator of transcription 3 cooperates with androgen receptor/cell cycle-related kinase signalling pathway in the progression of hepatitis B virus infection and gender differences.

The study aimed to investigate the role of androgen receptor (AR)/cell cycle-related kinase (CCRK) signalling pathway in chronic hepatitis B virus (HBV) infection and gender differences, and the contribution of AR regulatory factor signal transducer and activator of transcription 3 (STAT3) in it. AR, CCRK, and phosphorylated STAT3 expressions in liver tissues of chronic HBV-infected patients and non-HBV controls were determined by western blot and compared between genders. The relationships of expression levels with serum HBV DNA levels, liver inflammation activity, and fibrosis score were analysed in chronic HBV-infected patients. The relationships between expression levels of three proteins were also analysed. HBV-infected patients had significantly higher expression levels of AR, CCRK, and p-STAT3Tyr705 compared with controls (p < .01). The expression levels of AR, CCRK, and p-STAT3Tyr705 in chronic HBV-infected patients with severe inflammation were significantly higher than those with mild inflammation (p < .05). Expression levels in patients with heavier fibrosis (stage F4) were higher than in those with less fibrosis (stages F0-3) (p < .01). No gender differences were observed in AR, CCRK, and p-STAT3Tyr705 levels in non-HBV controls; higher levels were observed in HBV-infected males than in HBV-infected females (p < .05). AR, CCRK, and p-STAT3Tyr705 levels in liver tissues positively correlated with each other (p < .0001) and with serum HBV DNA levels (p < .0001). In conclusion, in this study, we first found concordant over-expression of AR, CCRK, and STAT3 in liver tissues of chronic HBV-infected patients who have not yet developed HCC, significantly correlated with the severity of the disease and showed gender differences. STAT3 may be a potential therapeutic co-target for chronic HBV infection.

ASC-J9 Blocks Cell Proliferation and Extracellular Matrix Production of Keloid Fibroblasts through Inhibiting STAT3 Signaling.

Keloids are a fibrotic skin disorder caused by abnormal wound healing and featuring the activation and expansion of fibroblasts beyond the original wound margin. Signal transducer and activator of transcription 3 (STAT3) has been found to mediate the biological functions of keloid fibroblasts (KFs). Therefore, we aimed to demonstrate whether ASC-J9, an inhibitor of STAT3 phosphorylation, can suppress the activation of KFs. Western blotting results showed that ASC-J9 inhibited the levels of COL1A1 and FN1 proteins, which were upregulated in KFs, by decreasing the expression of pSTAT3 and STAT3. RNA sequencing and in vitro studies further demonstrated that ASC-J9 treatment of KFs reduced cell division, inflammation, and ROS generation, as well as extracellular matrix (ECM) synthesis. ELISA assays verified that ASC-J9 treatment significantly mitigated IL-6 protein secretion in KFs. Transmission electron microscopy images revealed that ASC-J9 induced the formation of multilamellar bodies in KFs, which is associated with autophagy-related signaling. These results suggested that inhibiting a vicious cycle of the ROS/STAT3/IL-6 axis by ASC-J9 may represent a potential therapeutic approach to suppress cell proliferation and ECM production in KFs.

Effect of fire needling on imiquimod induced psoriasis-like lesion and STAT3 pathway in mice

To observe the effect of fire needling on psoriasis-like lesion and the signal transducer and activator of transcription 3 (STAT3) pathway in mice and compare the therapeutic effect between different interventions of fire needling therapy (surrounding technique of fire needling, fire needling at "Dazhui" [GV 14] and "Zusanli" [ST 36]). Thirty male BALB/c mice were randomized into a blank group, a model group, a dexamthasone group, a surrounding technique group and an acupoint group, 6 mice in each one. Except the blank group, the mice in the rest groups were established as psoriasis-like lesion model by topical application with imiquimod cream, once daily, consecutively for 8 days. From day 4 to day 8, in the dexamthasone group, gastric infusion with 0.2 mL dexamthasone was administered, once daily. On day 4, 6 and 8, in the surrounding technique group, fire needling was exerted around the skin lesion; and fire needling was applied to "Dazhui" (GV 14) and "Zusanli" (ST 36) in the acupoint group, once a day. The changes in skin lesion on the dorsal parts of mice were observed in each group to score the psoriasis area and severity index (PASI). Using HE staining, the dermal morphological changes and epidermal thickness were observed in the mice of each group. The positive expression of proliferating cell-associated antigen Ki-67 was determined by immunofluorescence. Immunohistochemistry method was used to determine the expressions of , and T cells of skin tissue in each group. Using real-time PCR, the expressions of interleukin (IL)-17, IL-22, tumor necrosis factor α(TNF-α) mRNA were determined. Western blot method was adopted to determine the protein expressions of STAT3 and p-STAT3 in skin tissue in each group. Compared with the blank group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all increased in the mice of the model group (P<0.01). Except for the erythema scores of the dexamethasone group and the surrounding technique group, the scores of each item and the total scores of PASI, as well as the epidermal thickness were all decreased in each intervention group as compared with the model group (P<0.01). The infiltration scores and the total scores in the dexamethasone group and the acupoint group were lower than those in the surrounding technique group respectively (P<0.01, P<0.05). In comparison with the blank group, Ki-67 positive cell numbers and the numbers of , and T cells in skin tissue were increased in the mice of the model group (P<0.01). Ki-67 positive cell numbers and the numbers of , and T cells were reduced in each intervention group as compared with the model group (P<0.01), and the numbers of and T cells in the acupoint group were less than the surrounding technique group (P<0.01). Compared with the blank group, the mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all increased in the model group (P<0.01). The mRNA expressions of IL-17, IL-22 and TNF-α and the ratio of p-STAT3 to STAT3 were all decreased in each intervention group as compared with the model group (P<0.01, P<0.05). The mRNA expressions of IL-17, IL-22 and TNF-α in the acupoint group, as well as mRNA expression of IL-17 in the surrounding technique group were all lower than the dexamethasone group (P<0.01), while, the mRNA expression of IL-22 in the acupoint group was lower than the surrounding technique group (P<0.01). Fire needling therapy improves skin lesion severity in imiquimod induced psoriasis-like lesion of the mice, which is probably related to the inhibition of STAT3 pathway activation and the decrease of Th17 inflammatory factors expression. The systemic regulation of fire needling at "Dazhui" (GV 14) and "Zusanli" (ST 36) is superior to the local treatment.