Inhibition Of STAT3 Signaling Research Articles

STAT3 signaling pathway is involved in the pathogenesis of miscarriage

IntroductionThe STAT3 signaling pathway plays an important role in the migration and invasion of villous trophoblast cells. In early miscarriage, the activation of STAT3 has been confirmed to decline, but its effect in early pregnancy has not received much attention. MethodsThe number of trophoblast cells were detected by HE staining in 30 cases of earlymiscarriage, 20 cases of recurrent miscarriage and 30 cases of control group.The protein levels of CyclinD1, VEGF, VEGFR1, Ki67, CD34 and phosphorylated STAT3 in three groups weredetected by immunohistochemistry or Western blot.Consistently, the mRNA levels of them weredetected by qPCR. The expression of STAT3 signaling pathway on trophoblast cells were evaluated in HTR-8/SVneo cell treated by AG490. Additionally, we established the situation of AG490-induced STAT3 signaling pathway inhibited in HTR-8/SVneo cell as well. ResultsHE staining showed that the number of trophoblast cells in the two study groups were significantly lower than that in the control group. The expression of STAT3 and its down stream target gene, such as CyclinD1, VEGF were significantly downregulated in abortion tissues (villi and decidua) in patients with early miscarriage. In vitro, AG490 inhibited the growth of trophoblast cells and promoted apoptosis of them by inhibiting STAT3 signaling pathway. DiscussionThe STAT3 signaling pathway might be involved in the pathogenesis of earlymiscarriage.However, further experiments are still needed to verify whether inhibitors of the STAT3 pathway can be used as drug treatment targets for spontaneous abortion.

Inhibition of JNK-Mediated Autophagy Promotes Proscillaridin A- Induced Apoptosis via ROS Generation, Intracellular Ca+2 Oscillation and Inhibiting STAT3 Signaling in Breast Cancer Cells.

Breast cancer is the most heterogenous cancer type among women across the world. Despite concerted efforts, breast cancer management is still unsatisfactory. Interplay between apoptosis and autophagy is an imperative factor in categorizing therapeutics for cancer treatment. Proscillaridin A (PSD-A), a well-known cardiac glycoside used for cardiac arrest and arrythmias, has been unveiled in many cancer types but the underlying mechanism for apoptosis and autophagy in breast cancer is not fully understood. In our study, PSD-A restricted cell growth, inhibited STAT3 activation and induced apoptosis and autophagy in breast cancer cells via ROS generation and Ca+2 oscillation. Pretreatment of NAC and BAPTA-AM restored PSD-A induced cellular events in breast cancer cells. PSD-A induced apoptosis via DNA fragmentation, caspase-cascade activation, PARP cleavage, mitochondrial dysfunction, Bax/Bcl-2 proteins modulation and ER chaperone GRP78 inhibition along with decreased phosphorylation of ERK1/2. Inhibition of STAT3 activation was found to be associated with decreased phosphorylation of SRC. Moreover, PSD-A induced events of autophagy i.e. conversion of LC3-I to LC3-II, and Atg3 expression via JNK activation and decreased mTOR and AKT phosphorylation. In this study, pretreatment of SP600125, a JNK inhibitor, reduced autophagy and enhanced STAT3 inhibition and apoptosis. Additionally, SB203580, a commercial p38 inhibitor, stimulated STAT3 activation and improved autophagic events rate in breast cancer cells, displaying the role of the MAPK signaling pathway in interplay between apoptosis and autophagy. Our data suggest that the rate of apoptotic cell death is improved by blocking JNK-induced autophagy in PSD-A treated MCF-7 and MDA-MB-231 breast cancer cells.

Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity.This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs).Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-β2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.

Abstract CT147: Phase 1 dose escalation of MSC-1, a humanized anti-LIF monoclonal antibody, in patients with advanced solid tumors

Abstract Leukemia Inhibitory Factor (LIF) is a pleiotropic cytokine, which is highly expressed in a subset of tumors and correlates with poor prognosis. LIF is hypothesized to contribute to tumor microenvironment immunosuppression and regulation of cancer stem cells. MSC-1 is a first-in-class humanized IgG1 monoclonal antibody that potently and selectively inhibits LIF. In pre-clinical models, MSC-1 decreases tumor growth through inhibition of STAT3 signaling, promoting immune stimulatory macrophages and increasing tumor infiltration of CD8 T and NK cells. The Phase Ia clinical study employed an accelerated 3 + 3 escalation design to explore safety and tolerability, dose-limiting toxicities (DLTs), preliminary efficacy, define a recommended phase II dose (RP2D), and evaluate exploratory tumor biomarkers. Eligible patients had advanced relapsed/refractory solid tumors and received treatment with MSC-1 intravenously (75mg-1500 mg) once every 3-weeks as a single agent until disease progression. The three highest dose cohorts were expanded to further assess safety, PK, engagement of LIF in the periphery, and assess immuno-regulatory tumor activity in matched pre- and on-treatment tumor biopsies. Forty-one patients (pts) were enrolled (14 in dose escalation; 27 in expanded cohorts) with the last pt completing the study on September 23, 2019. The most common tumor types were pancreatic (13), colorectal (5), head & neck (4) and ovarian (4). Pts had received a median of 3 prior lines of therapy. All pts experienced at least one adverse event (AE). The most common considered drug-related AEs were fatigue (N=8, 20%) and gastrointestinal disorder (N=8, 20%), and there was 1 considered drug-related SAE (Gr 2 osteonecrosis of jaw in a head and neck cancer patient who previously received radiation to the area and denosumab). There were no Dose Limiting Toxicities observed during the first cycle of treatment and 2 pts discontinued treatment due to AEs. The PK profile of MSC-1 was linear with an estimated terminal half-life of ∼13 days and benign anti-drug antibody profile. There was evidence of durable peripheral saturation of LIF binding demonstrating high level of target engagement. Results supported the selection of a RP2D of 1500 mg Q3W. Prolonged stable disease (≥ 16 weeks) was observed in 9 pts. Analysis of paired biopsies collected from matched metastatic lesions supported MSC-1 mediated STAT3 signaling inhibition, stimulatory (M1) to suppressive (M2) macrophage skewing in the majority of paired biopsies evaluated and increased CD8 T-cell infiltration in a subset of samples. Single agent MSC-1 was well tolerated in doses ranged from 75 mg to 1500 mg IV OD in patients with advanced solid tumors, showed promising activity as an anti-cancer therapy, and is Phase 1b/2 ready for combination with other agents. The updated final safety, efficacy, PK, LIF stabilization analyses, and tumor biopsy data will be presented. Citation Format: Alison Schram, Erkut Borazanci, Irene Brana, Maria Vieito Villar, Elena Garralda, Anna Spreafico, Marc Oliva, Nehal Lakhani, Robert Wasserman, Kimberly Hoffmann, Robin Hallett, Judit Anido, Dorotea Maetzel, Patricia Giblin, Enda Moran, Adrianne Kelly, Joan Seoane, Daniel D. Von Hoff, Lillian Siu, Josep Tabernero. Phase 1 dose escalation of MSC-1, a humanized anti-LIF monoclonal antibody, in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT147.

MiR-125b participates in the occurrence of preeclampsia by regulating the migration and invasion of extravillous trophoblastic cells through STAT3 signaling pathway

Preeclampsia (PE) is a major risk factor for maternal and fetal mortality. Studies showed that microRNAs (miRNAs) play important roles in PE, and are closely related to extra-villous trophoblastic proliferation and invasion. The current study determined miR-125b expression in PE patients, and explored the role of miR-125b in the occurrence and development of PE and its possible mechanism, aiming to provide a novel basis for the diagnosis and treatment of PE. The level of miR-125b in serum derived from pregnant women was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, invasion and migration of HTR-8/SVneo were determined by Cell Counting Kit-8 (CCK-8), Transwell and scratch assay, respectively. The target gene of miR-125b was predicted by Targetscan, and verified by luciferase reporter assay. The expressions of related proteins were determined by Western Blotting. The miR-125b level in the serum of PE patients was up-regulated as compared with normal pregnant women, and high level of miR-125b reduced cell proliferation, inhibited invasion and migration of HTR-8/SVneo as well as the expressions of STAT3, p-STAT3 and SOCS3, while low level of miR-125b produced the opposite results. STAT3 was predicted as the target gene of miR-125b, and the high level of miR-125b inhibited STAT3 signaling pathway. High expression of miR-125b may be involved in the occurrence of PE through inhibiting STAT3 pathway to inhibit the migration and invasion of extra-villous trophoblastic cells.

Preeclampsia Drives Molecular Networks to Shift Toward Greater Vulnerability to the Development of Autism Spectrum Disorder.

Preeclampsia (PE) confers a significant risk for subsequent diagnosis with autism spectrum disorder (ASD), with the mechanisms underlying this observation being largely unknown. To identify molecular networks affected by both PE and ASD, we conducted a large-scale literature data mining and a gene set enrichment analysis (GSEA), followed by an expression mega-analysis in 13 independently profiled ASD datasets. Sets of genes implicated in ASD and in PE significantly overlap (156 common genes; p = 3.14E−67), with many biological pathways shared (94 pathways; p < 1.00E−21). A set of PE-driven molecular triggers possibly contributing to worsening the risk of subsequent ASD was identified, possibly representing a regulatory shift toward greater vulnerability to the development of ASD. Mega-analysis of expression highlighted RPS4Y1, an inhibitor of STAT3 that is expressed in a sexually dimorphic manner, as a contributor to both PE and ASD, which should be evaluated as a possible contributor to male predominance in ASD. A set of PE-driven molecular triggers may shift the developing brain toward a greater risk of ASD. One of these triggers, chromosome Y encoded gene RPS4Y1, an inhibitor of STAT3 signaling, warrants evaluation as a possible contributor to male predominance in ASD.

Th17 cells inhibit CD8+ T cell migration by systematically downregulating CXCR3 expression via IL-17A/STAT3 in advanced-stage colorectal cancer patients

BackgroundCD8+ T cell trafficking to the tumor site is essential for effective colorectal cancer (CRC) immunotherapy. However, the mechanism underlying CD8+ T cell infiltration in colorectal tumor tissues is not fully understood. In the present study, we investigated CD8+ T cell infiltration in CRC tissues and the role of chemokine–chemokine receptor signaling in regulation of T cell recruitment.MethodsWe screened chemokines and cytokines in healthy donor and CRC tissues from early- and advanced-stage patients using multiplex assays and PCR screening. We also utilized transcription factor activation profiling arrays and established a xenograft mouse model.ResultsCompared with tumor tissues of early-stage CRC patients, CD8+ T cell density was lower in advanced-stage tumor tissues. PCR screening showed that CXCL10 levels were significantly increased in advanced-stage tumor tissues. CXCR3 (the receptor of CXCL10) expression on CD8+ T cells was lower in the peripheral blood of advanced-stage patients. The migratory ability of CD8+ T cells to CXCL10 depended on CXCR3 expression. Multiplex arrays showed that IL-17A was increased in advanced-stage patient sera, which markedly downregulated CXCR3 expression via activating STAT3 signaling and reduced CD8+ T cell migration. Similar results were found after CD8+ T cells were treated with Th17 cell supernatant. Adding anti-IL-17A or the STAT3 inhibitor, Stattic, rescued these effects in vitro and in vivo. Moreover, survival analysis showed that patients with low CD8 and CXCR3 expression and high IL-17A levels had significantly worse prognosis.ConclusionsCD8+ T cell infiltration in advanced-stage tumor was systematically inhibited by Th17 cells via IL-17A/STAT3/CXCR3 axis. Our findings indicate that the T cell infiltration in the tumor microenvironment may be improved by inhibiting STAT3 signaling.

Cryptotanshinone prevents muscle wasting in CT26-induced cancer cachexia through inhibiting STAT3 signaling pathway

Ethnopharmacological relevanceSalvia miltiorrhiza bunge (Danshen) has been extensively used to treat a wide variety of diseases including cancers. Cryptotanshinone is a major lipophilic compound extracted from the root of Danshen and has been reported to exert various pharmacological effects, however, its anti-cachectic remains unknown. Aim of the studyThe present study aims to investigate the anti-cachectic efficacy of cryptotanshinone and elucidate the underlying mechanism. Materials and methodsPrevention of muscle wasting by cryptotanshinone in colon adenocarcinoma CT26-induced cachexia and CT26 conditioned medium (TCM)-induced myotubes were investigated. Main features of cancer cachexia were determined after cryptotanshinone administration. The therapeutic effect of cryptotanshinone on myotube atrophy was assessed by morphological observation and myotube fiber width determination. E3 ubiquitin ligases muscle RING-finger containing protein 1 (MuRF1) and muscle atrophy Fbox protein (MAFbx/Atrogin-1) expression and STAT3 activation were examined using western blot, real-time qPCR and dual-luciferase reporter gene assays both in vitro and in vivo. The myotubes were infected with lentiviruses expressing STAT3 or GFP. ResultsIn CT26 tumor-bearing mice, cryptotanshinone (20 and 60 mg/kg) administration drastically prevented systemic cancer cachexia from whole body weight loss and wasting of multiple tissues including heart, fat and skeletal muscle, with a negligible effect on cancer growth at dose of 20 mg/kg cryptotanshinone administration prevented the induction of MuRF1 and MAFbx/Atrogin-1 in cachectic muscles. Moreover, cryptotanshinone (2.5-10 μM) dose-dependently reduced the elevated expression of MuRF1 and MAFbx/Atrogin-1 in C2C12 myotubes, and improved myotube atrophy. We showed that cryptotanshinone significantly suppressed the hyper-activated STAT3 in cachectic muscles and C2C12 myotubes and inhibited STAT3 transcriptional activity, but it did not repress the activation of STAT1. The inhibitory effect of cryptotanshinone on TCM-induced myotube atrophy was blocked by STAT3 overexpression. ConclusionsThese data suggest that cryptotanshinone prevents muscle wasting in cancer cachexia through STAT3 inhibition, and it may be a promising candidate drug for the treatment of cancer cachexia.

Inhibitory Effects of Tangeretin, A Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling.

Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24−) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.

Ellagic acid induces esophageal squamous cell carcinoma cell apoptosis by modulating SHP-1/STAT3 signaling.

Ellagic acid (EA) has been reported to have antiproliferative and antioxidant properties, but its function in esophageal squamous cell carcinoma (ESCC) has not been investigated yet. In the current study, EA was found have a significant anti-tumor activity in ESCC. In specific, EA inhibited ESCC cell survival in both of a concentration- and time-dependent manner. And our results showed that EA promoted ESCC cell apoptosis, including inducing the cleavages of PARP, and inhibiting the expression of anti-apoptotic proteins. In mechanistic, EA markedly suppressed STAT3-driven luciferase activity, and inhibited both of the endogenous and cytokines-induced STAT3 activation in ESCC cells. Further investigations indicated that EA could significantly upregulate SHP-1 expression, a negative modulator of STAT3 signaling. In contrast, knockdown of SHP-1 could attenuate the effects of EA on inhibiting ESCC cell survival. Moreover, we found that EA could inhibit RNF6 expression, an E3 of SHP-1, and overexpressing RNF6 could also significantly attenuate the effects of EA on inhibiting ESCC cell survival, which further revealed that EA could inhibit STAT3 signaling by modulating RNF6/SHP-1 axis. Our present study indicated that EA could be as a novel STAT3 inhibitor for the treatment of ESCC.

IL-37 Represses the Autoimmunity in Myasthenia Gravis via Directly Targeting Follicular Th and B Cells.

IL-37 is a newly identified immune-suppressive factor; however, the function, cellular sources, and mechanism of IL-37 in humoral immunity and Myasthenia gravis (MG) are still unclear. In this study, we found IL-37 were substantially downregulated in the serum and PBMCs of MG patients compared with healthy controls. The lower IL-37 was associated with severer disease (quantitative MG score) and higher follicular Th (Tfh)/Tfh17 and B cell numbers. Flow cytometry analysis revealed that IL-37 was mainly produced by CD4+ T cells without overlapping with Th1, Th17, and Tfh subsets in MG patients. Regulatory IL-37+ T cell rarely expressed Foxp3 and CD25 but produced numerous IL-4. Tfh and B cell expressed high levels of SIGIRR, the receptor of IL-37, in MG patients. Mechanically, IL-37 directly bond to SIGIRR, repressed the proliferation, cytokine production of Tfh and B cells, and the secretion of autoantibody via inhibition of STAT3 signaling in Tfh and B cells.

The Potential Inhibitory Effects of miR-19b on Ocular Inflammation are Mediated Upstream of the JAK/STAT Pathway in a Murine Model of Allergic Conjunctivitis.

PurposeThymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine that initiates allergic inflammatory reaction between epithelial and dendritic cells (DCs). miR-19b was reported to suppress TSLP expression. The present study aimed to examine miR-19b expression, regulation, and function in allergic conjunctivitis (AC).MethodsA murine model of experimental AC was induced in BALB/c mice by short ragweed pollen. The serum, eye balls, conjunctiva, and cervical lymph nodes (CLN) were used for the study. Gene expression was determined by RT-PCR, whereas protein production and activation were evaluated by immunostaining, ELISA, and Western blotting.ResultsIn the murine AC model, miR-19b was aberrantly downregulated, whereas the levels of TSLP and p-STAT3, as well as the number of CD11c+ pSTAT3+ DCs were increased. Moreover, Th2 inflammatory cytokine expression was significantly increased. These severe phenotypes could be counteracted by either applying exogenous miR-19b mimic microRNAs or the JAK/STAT inhibitor CYT387. Moreover, overexpression of miR-19b repressed p-STAT3 expression and the number of CD11c+ cells in AC eye and CLN tissues.ConclusionsThese findings suggested that miR-19b reduced ocular surface inflammation by inhibiting Stat3 signaling via TSLP downregulation in a murine AC model. Moreover, the present study further demonstrated the clinical potential of applying miR-19b and anti-JAK/STAT therapies in the treatment of AC.

B cell-Derived IL35 Drives STAT3-Dependent CD8+ T-cell Exclusion in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell- but not regulatory T cell-specific production of IL35. We also demonstrated that B cell-specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti-PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3-CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell-mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

Igalan from Inula helenium (L.) suppresses the atopic dermatitis-like response in stimulated HaCaT keratinocytes via JAK/STAT3 signaling.

This study aimed to evaluate the protective effect of igalan, a sesquiterpene lactone isolated from Inula helenium (L.), on inhibiting inflammation, regulating the epidermal differentiation gene expression, and reactive oxygen species scavenging in atopic dermatitis (AD)-like inflammatory keratinocytes. HaCaT human keratinocytes were treated with igalan at indicated concentrations before being activated by a combination of TNF-α and IFN-γ or IL-4 representative for T-helper 1 and T-helper 2 cell cytokines, which are associated with AD pathogenesis. By inhibiting the NF-κB pathway as well as the STAT activation, igalan could downregulate several marker inflammatory genes in AD, such as TARC/CCL17, MDC/CCL22, and RANTES/CCL5. In contrast, igalan, acting as JAK inhibitor, could promote the mRNA expression levels of the genes FLG, LOR, KRT10, and DSC1, which encode for essential proteins responsible for keratinocyte differentiation, by inhibiting STAT3 signaling. Furthermore, igalan exerts its antioxidant effect through activating the Nrf2 pathway, triggering the expression of some enzymes that contribute to preventing intracellular ROS generation during inflammation. These findings indicate that igalan, via suppressing JAK/STAT3 signaling, could impair the production of pro-inflammatory chemokines and enhance expression levels of several genes involved in keratinocyte differentiation in AD-like stimulated keratinocytes.

HCT116 colorectal liver metastases exacerbate muscle wasting in a mouse model for the study of colorectal cancer cachexia.

ABSTRACTColorectal cancer (CRC) is often accompanied by formation of liver metastases (LM) and skeletal muscle wasting, i.e. cachexia. Despite affecting the majority of CRC patients, cachexia remains underserved, understudied and uncured. Animal models for the study of CRC-induced cachexia, in particular models containing LM, are sparse; therefore, we aimed to characterize two new models of CRC cachexia. Male NSG mice were injected subcutaneously (HCT116) or intrasplenically (mHCT116) with human HCT116 CRC tumor cells to disseminate LM, whereas experimental controls received saline (n=5-8/group). Tumor growth was accompanied by loss of skeletal muscle mass (HCT116: −20%; mHCT116: −31%; quadriceps muscle) and strength (HCT116: −20%; mHCT116: −27%), with worsened loss of skeletal muscle mass in mHCT116 compared with HCT116 (gastrocnemius: −19%; tibialis anterior: −22%; quadriceps: −21%). Molecular analyses revealed elevated protein ubiquitination in HCT116, whereas mHCT116 also displayed elevated Murf1 and atrogin-1 expression, along with reduced mitochondrial proteins PGC1α, OPA1, mitofusin 2 and cytochrome C. Further, elevated IL6 levels were found in the blood of mHCT116 hosts, which was associated with higher phosphorylation of STAT3 in skeletal muscle. To clarify whether STAT3 was a main player in muscle wasting in this model, HCT116 cells were co-cultured with C2C12 myotubes. Marked myotube atrophy (–53%) was observed, along with elevated phospho-STAT3 levels (+149%). Conversely, inhibition of STAT3 signaling by means of a JAK/STAT3 inhibitor was sufficient to rescue myotube atrophy induced by HCT116 cells (+55%). Overall, our results indicate that the formation of LM exacerbates cachectic phenotype and associated skeletal muscle molecular alterations in HCT116 tumor hosts.

The IκB Kinase Inhibitor ACHP Targets the STAT3 Signaling Pathway in Human Non-Small Cell Lung Carcinoma Cells.

STAT3 is an oncogenic transcription factor that regulates the expression of genes which are involved in malignant transformation. Aberrant activation of STAT3 has been observed in a wide range of human malignancies and its role in negative prognosis is well-documented. In this report, we performed high-throughput virtual screening in search of STAT3 signaling inhibitors using a cheminformatics platform and identified 2-Amino-6-[2-(Cyclopropylmethoxy)-6-Hydroxyphenyl]-4-Piperidin-4-yl Nicotinonitrile (ACHP) as the inhibitor of the STAT3 signaling pathway. The predicted hit was evaluated in non-small cell lung cancer (NSCLC) cell lines for its STAT3 inhibitory activity. In vitro experiments suggested that ACHP decreased the cell viability and inhibited the phosphorylation of STAT3 on Tyr705 of NSCLC cells. In addition, ACHP imparted inhibitory activity on the constitutive activation of upstream protein tyrosine kinases, including JAK1, JAK2, and Src. ACHP decreased the nuclear translocation of STAT3 and downregulated its DNA binding ability. Apoptosis was evidenced by cleavage of caspase-3 and PARP with the subsequent decline in antiapoptotic proteins, including Bcl-2, Bcl-xl, and survivin. Overall, we report that ACHP can act as a potent STAT3 signaling inhibitor in NSCLC cell lines.

Sphingosine 1-Phosphate Signaling Is Involved in Impaired Blood-Brain Barrier Function in Ischemia-Reperfusion Injury.

Sphingosine 1-phosphate (S1P) is a major bioactive lipid mediator in the vascular and immune system. Here, we have shown that inhibition of S1P signaling prevents blood-brain barrier (BBB) dysfunction after ischemia both in vitro and in vivo. In the in vitro BBB models, oxygen-glucose deprivation and reoxygenation (OGD/R) enhanced the expression of an S1P synthesizing enzyme (Sphk1) and S1P transporters (Abca1, Spns2), increasing S1P in culture media. Inhibitors of Sphk1 (SKI-II) or Abca1 (probucol) attenuated the decrease in transendothelial electrical resistance and the increase in permeability caused by OGD/R. In the middle cerebral artery occlusion and reperfusion (MCAO/R) model of mice, probucol administration after MCAO operation reduced the infarction area and vascular leakage, preserving the integrity of tight junction proteins. Furthermore, MCAO/R caused activation of STAT3, a downstream mediator of S1P signaling, which was suppressed by postoperative probucol administration. Accordingly, S1P activated STAT3, both in cultured vascular endothelial cells and pericytes, and STAT3 signaling inhibitor (Stattic) protected BBB dysfunction in OGD/R-treated in vitro BBB models. These results suggest that inhibition of S1P signaling is a strategy to treat BBB impairment after cerebral ischemia and highlight the potential alternative use of probucol, a classical anti-hyperlipidemic drug, for emergency treatment of stroke.

Berbamine Enhances the Efficacy of Gefitinib by Suppressing STAT3 Signaling in Pancreatic Cancer Cells.

BackgroundSmall molecular inhibitors such as gefitinib (Gefi), which target EGF receptor (EGFR), are considered to be a viable pathway for the selective inhibition of pancreatic cancer (PC) development. However, the large difference in Gefi response between PC patient individuals and PC cell lines severely limits the clinical efficacy of Gefi. Berbamine (BBM) is a well-known natural-derived antitumor agent. However, no study yet exists on whether BBM can enhance the sensitivity of PC cells to Gefi or its underlying mechanisms.MethodsMTS assay and clonogenic assay were used to determine whether BBM could enhance the anti-PC activity of Gefi by. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after combined treatment with BBM and Gefi. Surface plasmon resonance (SPR) and Western blot experiments were carried out to detect the STAT3 binding affinity and the STAT3 inhibitory effect of BBM. Molecular docking and Molecular dynamic simulation were used to predicting the dominant interaction between BBM and STAT3.ResultsThis study found that BBM synergizes with Gefi to inhibit cell growth and induce cell cycle arrest and PC cell apoptosis. Mechanistically, our results showed that BBM and Gefi have synergistic inhibitory effects on STAT3 phosphorylation, but have little effect on other EGFR downstream pathways, suggesting that BBM may exert sensitization through the inhibition of STAT3. Besides, BBM has a high affinity for STAT3 and a good inhibitory effect on STAT3 activation, further indicating that BBM was a potent direct STAT3 inhibitor. Molecular modeling between STAT3 and BBM suggested that BBM formed several key hydrophilic interactions with STAT3.ConclusionOur findings suggest that the combination of BBM and Gefi could be further developed as a potential PC therapy.

Bioactivation of Napabucasin Triggers Reactive Oxygen Species-Mediated Cancer Cell Death.

Napabucasin (2-acetylfuro-1,4-naphthoquinone or BBI-608) is a small molecule currently being clinically evaluated in various cancer types. It has mostly been recognized for its ability to inhibit STAT3 signaling. However, based on its chemical structure, we hypothesized that napabucasin is a substrate for intracellular oxidoreductases and therefore may exert its anticancer effect through redox cycling, resulting in reactive oxygen species (ROS) production and cell death. Binding of napabucasin to NAD(P)H:quinone oxidoreductase-1 (NQO1), and other oxidoreductases, was measured. Pancreatic cancer cell lines were treated with napabucasin, and cell survival, ROS generation, DNA damage, transcriptomic changes, and alterations in STAT3 activation were assayed in vitro and in vivo. Genetic knockout or pharmacologic inhibition with dicoumarol was used to evaluate the dependency on NQO1. Napabucasin was found to bind with high affinity to NQO1 and to a lesser degree to cytochrome P450 oxidoreductase (POR). Treatment resulted in marked induction of ROS and DNA damage with an NQO1- and ROS-dependent decrease in STAT3 phosphorylation. Differential cytotoxic effects were observed, where NQO1-expressing cells generating cytotoxic levels of ROS at low napabucasin concentrations were more sensitive. Cells with low or no baseline NQO1 expression also produced ROS in response to napabucasin, albeit to a lesser extent, through the one-electron reductase POR. Napabucasin is bioactivated by NQO1, and to a lesser degree by POR, resulting in futile redox cycling and ROS generation. The increased ROS levels result in DNA damage and multiple intracellular changes, one of which is a reduction in STAT3 phosphorylation.