Signal Transducer And Activator Of Transcription 3 Inhibitor S3I-201 Research Articles

Melatonin attenuates cellular senescence and apoptosis in diabetic nephropathy by regulating STAT3 phosphorylation

AimsMelatonin is an endogenous hormone related to the regulation of biorhythm. Previous researchers have found that melatonin can ameliorate diabetic nephropathy (DN), but the mechanism remains to be elucidated. To discover the possible mechanism by which melatonin prevents DN, we investigated the potential effects of melatonin on signal transducer and activator of transcription 3 (STAT3) on the progression of cellular senescence and apoptosis. Main methodsCellular senescence, apoptosis and the underlying mechanism of melatonin were investigated both in vivo and in vitro. C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ) to establish DN. For an in vitro model of DN, human renal cortex proximal epithelial tubule (HK-2) cells were exposed to high glucose conditions. Key findingsMelatonin inhibited the phosphorylation of STAT3, decreased the expression of senescence proteins p53, p21 and p16INK4A. Melatonin also downregulated the expression of apoptotic proteins, including cleaved PARP1, cleaved caspase-9 and -3. Melatonin treatment decreased the positive area of senescence-associated galactosidase (SA-β-gal) staining and the number of TUNEL-positive cells in kidneys of DN mice. In vitro, melatonin inhibited STAT3 phosphorylation and lowered cellular senescence and apoptosis markers, in a manner similar to the STAT3 inhibitor S3I-201. In addition, the inhibition effect of melatonin on cellular senescence and apoptosis in HK-2 cells was reversed by the usage of recombinant IL-6 (rIL-6), which can induce STAT3 phosphorylation. SignificanceWe, for the first time, demonstrate that melatonin inhibits STAT3 phosphorylation, which is involved in alleviating the cellular senescence and apoptosis in DN.

Design, synthesis, and biological characterization of a potent STAT3 degrader for the treatment of gastric cancer.

Gastric cancer is a common malignant tumor that threatens human health, and its occurrence and development mechanism is a complex process involving multiple genes and multiple signals. Signal transducer and activator of transcription 3 (STAT3) has been elucidated as a promising target for developing anticancer drugs in gastric cancer. However, there is no FDA-approved STAT3 inhibitor yet. Herein, we report the design and synthesis of a class of STAT3 degraders based on proteolysis-targeting chimeras (PROTACs). We first synthesized an analog of the STAT3 inhibitor S3I-201 as a ligand, using the cereblon (CRBN)/cullin 4A E3 ligase ligand pomalidomide to synthesize a series of PROTACs. Among them, the SDL-1 achieves the degradation of STAT3 protein in vitro, and exhibits good anti-gastric cancer cell proliferation activity, inhibits invasion and metastasis of MKN1 cell, and induces MKN1 cell apoptosis and arrests cell cycle at the same time. Our study shows that SDL-1 is a potent STAT3 degrader and may serve as a potential anti-gastric cancer drug, providing ideas for further development of drugs for clinical use.

Hepatocyte growth factor protects pulmonary endothelial barrier against oxidative stress and mitochondria-dependent apoptosis.

Background:Pulmonary microvascular endothelial cells (PMVECs) were not complex, and the endothelial barrier was destroyed in the pathogenesis progress of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF), which was secreted by bone marrow mesenchymal stem cells, could decrease endothelial apoptosis. We investigated whether mTOR/STAT3 signaling acted in HGF protective effects against oxidative stress and mitochondria-dependent apoptosis in lipopolysaccharide (LPS)-induced endothelial barrier dysfunction and ALI mice.Methods:In our current study, we introduced LPS-induced PMEVCs with HGF treatment. To investigate the effects of mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 were, respectively, used to inhibit mTOR/STAT3 signaling. Moreover, lentivirus vector-mediated mTORC1 (Raptor) and mTORC2 (Rictor) gene knockdown modifications were introduced to evaluate mTORC1 and mTORC1 pathways. Calcium measurement, reactive oxygen species (ROS) production, mitochondrial membrane potential and protein, cell proliferation, apoptosis, and endothelial junction protein were detected to evaluate HGF effects. Moreover, we used the ALI mouse model to observe the mitochondria pathological changes with an electron microscope in vivo.Results:Our study demonstrated that HGF protected the endothelium via the suppression of ROS production and intracellular calcium uptake, which lead to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection) and specific proteins (complex I), raised anti-apoptosis Messenger Ribonucleic Acid level (B-cell lymphoma 2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2 have the same protective effects in mitochondria damage and apoptosis. In in vivo experiments of ALI mouse, HGF also increased mitochondria structural integrity via the mTOR/STAT3 pathway.Conclusion:In all, these reveal that mTOR/STAT3 signaling mediates the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis, and endothelial junction protein in ARDS, contributing to the pulmonary endothelial survival and barrier integrity.

STAT3/HIF-1\u03b1 signaling activation mediates peritoneal fibrosis induced by high glucose

BackgroundEpithelial-mesenchymal transition (EMT) of mesothelial cells is a key step in the peritoneal fibrosis (PF). Recent evidence indicates that signal transducer and activator of transcription 3 (STAT3) might mediate the process of renal fibrosis, which could induce the expression of hypoxia-inducible factor-1α (HIF-1α). Here, we investigated the effect of STAT3 activation on HIF-1α expression and the EMT of mesothelial cells, furthermore the role of pharmacological blockade of STAT3 in the process of PF during peritoneal dialysis (PD) treatment.MethodsFirstly, we investigated the STAT3 signaling in human peritoneal mesothelial cells (HPMCs) from drained PD effluent. Secondly, we explored the effect of STAT3 signaling activation on the EMT and the expression of HIF-1α in human mesothelial cells (Met-5A) induced by high glucose. Finally, peritoneal fibrosis was induced by daily intraperitoneal injection with peritoneal dialysis fluid (PDF) so as to explore the role of pharmacological blockade of STAT3 in this process.ResultsCompared with the new PD patient, the level of phosphorylated STAT3 was up-regulated in peritoneal mesothelial cells from long-term PD patients. High glucose (60 mmol/L) induced over-expression of Collagen I, Fibronectin, α-SMA and reduced the expression of E-cadherin in Met-5A cells, which could be abrogated by STAT3 inhibitor S3I-201 pretreatment as well as by siRNA for STAT3. Furthermore, high glucose-mediated STAT3 activation in mesothelial cells induced the expression of HIF-1α and the profibrotic effect of STAT3 signaling was alleviated by siRNA for HIF-1α. Daily intraperitoneal injection of high-glucose based dialysis fluid (HG-PDF) induced peritoneal fibrosis in the mice, accompanied by the phosphorylation of STAT3. Immunostaining showed that phosphorylated STAT3 was expressed mostly in α-SMA positive cells in the peritoneal membrane induced by HG-PDF. Administration of S3I-201 prevented the progression of peritoneal fibrosis, angiogenesis, macrophage infiltration as well as the expression of HIF-1α in the peritoneal membrane induced by high glucose.ConclusionsTaken together, these findings identified a novel mechanism linking STAT3/HIF-1α signaling to peritoneal fibrosis during long-term PD treatment. It provided the first evidence that pharmacological inhibition of STAT3 signaling attenuated high glucose-mediated mesothelial cells EMT as well as peritoneal fibrosis.

Leukemia inhibitory factor protects photoreceptor cone cells against oxidative damage through activating JAK/STAT3 signaling.

BackgroundThe present study aimed to investigate the protective role of leukemia inhibitory factor (LIF) against oxidative damage in photoreceptor cone cells.MethodsIn vivo, dark-adapted mice were injected with LIF or phosphate-buffered saline (PBS) intravitreously prior to being exposed to 5,000 lux bright light to determine the protective effect of LIF against light damage in cone cells. Oxidative damage to cone cells was analyzed using electroretinograms, immunostaining, Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In vitro, 661W cells were pretreated with 5 ng/mL of LIF with or without 50 µM of signal transducer and activator of transcription 3 (STAT3) inhibitor S3I201 for 1 h prior to treatment with 1 mM H2O2; cell survival, apoptosis, the oxidative stress index, and the activation of STAT3, extracellular signal-regulated kinase (ERK1/2), and AKT were subsequently determined.ResultsIn vivo, light induction damaged the function and morphology of cone cells, and LIF was observed to protect cone cells from this light damage. Moreover, the activation of the Janus tyrosine kinase (JAK)/STAT3 signaling pathway and the subsequent changes in apoptosis and proliferation-related genes were found to be involved in the protective effect of LIF against light-induced retinal damage. In the H2O2-induced 661W cell model, H2O2 increased cellular apoptosis rates, the expression levels of Bcl-2–associated X-protein (BAX) and cleaved caspase 3, reactive oxygen species (ROS) production, and malondialdehyde content, while decreasing the cell viability, and Bcl-2, superoxide dismutase, catalase, and glutathione peroxidase activity. LIF was observed to block these events; however, the administration of the STAT3 inhibitor S3I201 reversed the beneficial effects of LIF on H2O2-triggered apoptosis and ROS production.ConclusionsIn conclusion, the present study suggested that LIF may relieve oxidative damage in cone cells through suppressing apoptosis and oxidative stress by targeting the STAT3 signaling pathway.

Eucannabinolide, a novel sesquiterpene lactone, suppresses the growth, metastasis and BCSCS-like traits of TNBC via inactivation of STAT3

Signal transducer and activator of transcription 3 (STAT3) is an important therapeutic target to triple negative breast cancer (TNBC) treatment. In the present study, we aim to investigate the potential activity of Eucannabinolide (Euc), a novel sesquiterpene lactone separated from Eupatorium cannabinum Linn. against TNBC by targeting STAT3 and expect that Euc will be developed as an inhibitor of STAT3 in the treatment of TNBC. We found that Euc effectively suppressed STAT3 activation at tyrosine 705, inhibited its translocation to nucleus, and decreased its DNA binding capacity. Moreover, introduction of STAT3-short hairpin RNAs or STAT3 inhibitor S3I-201 attenuates the Euc-induced inhibition of cell viability. And, Euc inhibited cell viability, proliferation, metastasis and breast cancer stem cell-like traits but did not induce cytotoxicity in human mammary epithelial cells. The in vivo study similarly demonstrated that administration of Euc inhibited the growth of xenograft tumors and impaired tumor metastasis of a lung metastasis model. The above phenomena were associated with STAT3 dysfunction induced by Euc. In conclusion, Euc elicits the effects of anti-proliferation, anti-metastasis and anti-breast cancer stem cell-like traits in TNBC via targeting STAT3. These data highlight that development of Euc as a STAT3 inhibitor may offer a promising therapeutic strategy for TNBC.

Differentiation-inducing factor-1 suppresses cyclin D1-induced cell proliferation of MCF-7 breast cancer cells by inhibiting S6K-mediated signal transducer and activator of transcription 3 synthesis.

Differentiation‐inducing factor‐1 (DIF‐1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase‐3 (GSK‐3). Here, we report an alternative mechanism underlying the action of DIF‐1 in human breast cancer cell line MCF‐7, on which the effects of DIF‐1 have not been examined previously. Intragastric administration of DIF‐1 reduced the tumor growth from MCF‐7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF‐7, DIF‐1 arrested the cell cycle in G0/G1 phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF‐1 did not inhibit the phosphorylation of GSK‐3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF‐1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I‐201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation‐inducing factor‐1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF‐1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF‐1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70S6K/p85S6K). Inhibition of p70S6K/p85S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF‐1 suppresses MCF‐7 proliferation by inhibiting p70S6K/p85S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression.

Therapeutic Effects of STAT3 Inhibition on Experimental Murine Dry Eye

To investigate the therapeutic effects of targeting signal transducer and activator of transcription-3 (STAT3) activation on the ocular surface damage of dry eye in mice. Adult Balb/C and C57BL/6 mice with benzalkonium chloride (BAC) treatment, lacrimal gland excision, and meibomian gland dysfunction were used as dry eye models. The levels of phosphorylated STAT3 (p-STAT3) were detected with immunofluorescence staining and Western blotting. STAT3 inhibition was performed by topical application of STAT3 inhibitor S3I-201. Corneal epithelial barrier function, tear production, and conjunctival goblet cell density were quantified with fluorescein sodium staining, phenol red cotton test, and histochemical staining. The expressions of matrix metalloproteinase (MMP)-3/9, TUNEL, and inflammation cytokines were assessed with immunofluorescence staining, qPCR, and ELISA assays. The therapeutic effect of S3I-201 was further compared with the Janus kinase inhibitor tofacitinib and ruxolitinib. Elevated levels of nuclear p-STAT3 were detected in the corneal and conjunctival epithelium of three dry eye models. Topical application of S3I-201 improved corneal epithelial barrier function, increased tear production and conjunctival goblet cell density in BAC-induced dry eye mice. Moreover, S3I-201 decreased the expression of MMP-3/9, suppressed the apoptosis of corneal and conjunctival epithelial cells, and reduced the levels of IL-1β, IL-6, IL-17A, and IFN-γ. Compared with tofacitinib and ruxolitinib, the STAT3 inhibitor S3I-201 showed superior improvement of tear production and inflammatory cytokine expression in lacrimal gland. Elevated STAT3 activation is involved in the pathogenesis of dry eye, while targeting STAT3 effectively alleviates BAC-induced ocular surface damage.

Abstract 844: β IV -spectrin Regulates Cardiac Fibroblast Phenotype, Fibrosis, and Cardiac Function

Increased fibrosis is associated with cardiac dysfunction and arrhythmias. Activation of quiescent cardiac fibroblasts (CFs) occurs in response to injury although the underlying mechanistic pathways remain unclear. The cytoskeletal protein β IV -spectrin has been shown to control targeting and activity of the multifunctional transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) for maladaptive remodeling. The mechanism linking spectrin dysfunction to altered STAT3 signaling, fibrosis, and function remains unknown. The objective of this study was to investigate the role of stress-induced disruption of β IV -spectrin/STAT3 complex in modulating CF phenotype, fibrosis, and cardiac function. Cardiac function (echocardiography) and fibrosis (Masson’s trichrome) were evaluated in adult wildtype (WT) and qv 4J mice expressing truncated β IV -spectrin lacking STAT3 interaction. A subset of WT mice were subjected to 6 wks of transaortic constriction (TAC) to induce heart failure. From these groups, CFs were isolated from ventricles and analyzed for STAT3 localization, gene expression, and activity. CFs were treated with selective STAT3 inhibitor S3I-201 (100μM) or transfected for 72 hrs with β IV -spectrin fragment from repeat 10 to C-terminus (β IV,10-C , contains STAT3 binding motif). Ejection fraction was decreased in qv 4J and WT TAC relative to WT baseline (49.5±0.85% vs 34.9±2.2% vs 63.2±0.60% respectively, p<0.05, N=5. Ventricular fibrosis was augmented in qv 4J and WT TAC compared to WT baseline (2.4±0.09% vs 3.2±0.25% vs 0.48±0.01% respectively, p<0.05, N=5). qv 4J CFs showed STAT3 mislocalization (49.7±10.7% nuclear accumulation vs 5.3±.1.8% in WT, p<0.05, N=5) with higher STAT3 transcriptional activity (2.6±0.17-fold increase over WT, p<0.05, N=9). In parallel, qv 4J CFs displayed elevated expression of fibrotic genes associated with CF activation. At the functional level, qv 4J CFs showed increased proliferation (1.9±0.19-fold increase over WT at 24 hrs, p<0.05, N=4). STAT3 inhibition with S31-201 or transfection with β IV,10-C normalized gene expression and proliferation in qv 4J CFs compared to WT. These studies identify a requirement of β IV -spectrin for normal STAT3 signaling and quiescent phenotype in CFs.

MTOR/STAT-3 pathway mediates mesenchymal stem cell-secreted hepatocyte growth factor protective effects against lipopolysaccharide-induced vascular endothelial barrier dysfunction and apoptosis.

Mesenchymal stem cells (MSCs) protect the endothelial barrier complex and survival, implicated in the pathogenesis of acute lung injury (ALI) via paracrine hepatocyte growth factor (HGF). However, the mechanism of HGF in endothelial regulation remains unclear. Here, we introduced a coculture protocol of pulmonary microvascular endothelial cells (PMVECs) and overexpression of the HGF gene of MSCs (MSC-HGF). Immunofluorescence and endothelial permeability analysis revealed that MSC-HGF protected endothelial tight junction protein occludin expression and attenuated cellular permeability as well as endothelial apoptosis. To investigate the novel mechanism mammalian TOR (mTOR)/ signal transducer and activator of transcription 3 (STAT-3) signaling in HGF protective effects against endothelial barrier and apoptosis, we used recombinant mouse HGF in endothelial cells. In addition, we used mTOR inhibitor rapamycin to inhibit the mTOR pathway. Our study demonstrated that rapamycin decreased the protective effects of HGF on the endothelium by decreasing tight junction protein occludin expression and cell proliferation, and raising lipopolysaccharide (LPS)-induced endothelial permeability, endothelial cell injury factors ET-1 and vWF. Similarly, the protective effects of HGF on reducing endothelial barrier and apoptosis were weakened when PMVECs were treated with the STAT-3 inhibitor S3I-201. Moreover, mTOR/STAT-3 were activated by HGF demonstrated as raising mTOR (Ser2448) and STAT3 (Ser727) phosphorylation proteins, leading to endothelial barrier improvement and survival. Reversely, rapamycin or S3I-201 inhibited mTOR/STAT-3 activation. Taken together, our findings highlight that the activation of the mTOR/STAT-3 pathway provides novel mechanistic insights into MSC-secreted HGF protection against LPS-induced vascular endothelial permeability dysfunction and apoptosis, which contributes to decreasing microvascular loss and lung injury.

Advanced glycation end products induced IL-6 and VEGF-A production and apoptosis in osteocyte-like MLO-Y4 cells by activating RAGE and ERK1/2, P38 and STAT3 signalling pathways

Advanced glycation end products (AGEs) are involved in osteopenia in people with diabetes and the elderly. Interleukin-6 (IL-6) and vascular endothelial growth factor-A (VEGF-A) are potent regulators of bone metabolism, and in bone tissue, osteocytes are an important source of these regulators. However, whether AGEs can directly regulate IL-6 and VEGF-A secretion by osteocytes is unknown. In this study, we evaluated the effect of AGEs on IL-6 and VEGF- A production as well as apoptosis in osteocyte-like MLO-Y4 cells. We also studied the involvement of receptor for advanced glycation end products (RAGE) and the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2), P38 and signal transducer and activator of transcription 3 (STAT3) signalling pathways. We found that 100μg/ml AGEs significantly induced apoptosis and up-regulated the expression of IL-6 and VEGF-A in MLO-Y4 cells. Additionally, AGEs significantly activated the ERK1/2, P38 and STAT3 signalling pathways. The ERK1/2 inhibitor U0126, the P38 inhibitor SB239063 and the STAT3 inhibitor S3I-201 all attenuated the effects of AGEs on MLO-Y4 cell apoptosis and IL-6 and VEGF-A secretion. Moreover, activation of the three signalling pathways was abolished by their respective inhibitors. Additionally, the AGEs-induced effects, including increased apoptosis, up-regulated expression of IL-6 and VEGF-A and activation of the three signalling pathways, were all abolished by pre-treating the osteocytes with the RAGE antagonist FPS-ZM1. Together, these data convince us that AGEs can activate the ERK1/2, P38 and STAT3 signalling pathways via RAGE and that their activation involves the AGEs-induced up-regulation of IL-6 and VEGF-A production as well as apoptosis in osteocytes. These results highlight the role of osteocytes in the regulation of bone metabolism by AGEs.

Targeting STAT3 to Suppress Systemic Pro-Oncogenic Effects from Hepatic Radiofrequency Ablation.

Purpose To (a) identify key expressed genes in the periablational rim after radiofrequency ablation (RFA) and their role in driving the stimulation of distant tumor growth and (b) use adjuvant drug therapies to block key identified mediator(s) to suppress off-target tumorigenic effects of hepatic RFA. Materials and Methods This institutional animal care and use committee-approved study was performed in C57BL6 mice (n = 20) and F344 rats (n = 124). First, gene expression analysis was performed in mice after hepatic RFA or sham procedure; mice were sacrificed 24 hours to 7 days after treatment. Data were analyzed for differentially expressed genes (greater than twofold change) and their functional annotations. Next, animals were allocated to hepatic RFA or sham treatment with or without STAT3 (signal transducer and activator of transcription 3) inhibitor S3I-201 for periablational phosphorylated STAT3 immunohistochemistry analysis at 24 hours. Finally, animals with subcutaneous R3230 adenocarcinoma tumors were allocated to RFA or sham treatment with or without a STAT3 inhibitor (S3I-201 or micellar curcumin, eight arms). Outcomes included distant tumor growth, proliferation (Ki-67 percentage), and microvascular density. Results At 24 hours, 217 genes had altered expression (107 upregulated and 110 downregulated), decreasing to 55 genes (27 upregulated and 28 downregulated) and 18 genes (four upregulated, 14 downregulated) at 72 hours and 7 days, respectively. At 24 hours, STAT3 occurred in four of seven activated pathways associated with pro-oncogenic genes at network analysis. Immunohistochemistry analysis confirmed elevated periablational phosphorylated STAT3 24 hours after RFA, which was suppressed with S3I-201 (percentage of positive cells per field: 31.7% ± 3.4 vs 3.8% ± 1.7; P < .001). Combined RFA plus S3I-201 reduced systemic distant tumor growth at 7 days (end diameter: 11.8 mm ± 0.5 with RFA plus S3I-201, 19.8 mm ± 0.7 with RFA alone, and 15 mm ± 0.7 with sham procedure; P < .001). STAT3 inhibition with micellar curcumin also suppressed postablation stimulation of distant tumor growth, proliferation, and microvascular density (P < .01). Conclusion Gene expression analysis identified multiple pathways upregulated in the periablational rim after hepatic RFA, of which STAT3 was active in four of seven. Postablation STAT3 activation is linked to increased distant tumor stimulation and can be suppressed with adjuvant STAT3 inhibitors. © RSNA, 2017.

Epigenetic upregulation of CXCL12 expression mediates antitubulin chemotherapeutics-induced neuropathic pain.

Clinically, Microtubule-targeted agents-induced neuropathic pain hampers chemotherapeutics for patients with cancer. Here, we found that application of paclitaxel or vincristine increased the protein and mRNA expression of CXCL12 and frequency and amplitude of miniature excitatory post synaptic currents (mEPSCs) in spinal dorsal horn neurons. Spinal local application of CXCL12 induced the long-term potentiation of nociceptive synaptic transmission and increased the amplitude of mEPSCs. Inhibition of CXCL12 using the transgenic mice (CXCL12) or neutralizing antibody or siRNA ameliorated the mEPSC's enhancement and mechanical allodynia. In addition, paclitaxel and vincristine both could increase the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the acetylation of histone H4 in the CXCL12-expressing neurons. Immunoprecipitation and chromatin immunoprecipitation assays demonstrated that antitubulin chemotherapeutics increased the binding of STAT3 to the CXCL12 gene promoter and the interaction between STAT3 and p300, and contributed to the enhanced transcription of CXCL12 by increasing the acetylation of histone H4 in CXCL12 gene promoter. Inhibition of STAT3 by intrathecal injection of adeno-associated virus encoding Cre and green fluorescent protein into STAT3 mice or inhibitor S3I-201 into rats suppressed the CXCL12 upsurge by decreasing the acetylation of histone H4. Finally, blockade of CXCR4 but not CXCR7 ameliorated the paclitaxel- or vincristine-induced mechanical allodynia. Together, these results suggested that enhanced interaction between STAT3 and p300 mediated the epigenetic upregulation of CXCL12 in dorsal horn neurons, which contributed to the antitubulin chemotherapeutics-induced persistent pain.

The Regulation of STAT3 and Its Role in the Adhesion and Migration of Chronic Lymphocytic Leukaemia Cells

The bone marrow and lymph node microenvironments are important in promoting cell proliferation, survival and protection from drug induced apoptosis in chronic lymphocytic leukaemia (CLL). Chemokine networks, such as the CXCR4/CXCL12 axis, in combination with selectins, such as CD62L and integrins allow the migration of CLL cells to these protective niches. The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival. Further, it has been shown to interact with the signal transducer and activator of transcription 3 (STAT3) signalling pathway. The role of the STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells.Here, we investigate the role of STAT3 in CLL cell survival and migration, using pharmacological inhibition and siRNA knockdown. Phospho-tyrosine and phospho-serine STAT3 were assessed by flow cytometry and western blotting. Apoptosis was assessed by Annexin V/Propidium Iodide staining by flow cytometry. The expression of cell surface markers involved in cell adhesion and homing was determined by multicolour flow cytometry.Stimulation of the BCR using immunoglobulin F(ab´)2 fragments induced tyrosine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IgVH) genes (n=7) but not mutated IgVH genes (n=5). This induced tyrosine phosphorylation was abrogated by pre-treatment with the Janus kinase (JAK) inhibitor Ruxolitinib and the BCR inhibitors Ibrutinib and Idelalisib (p<0.05, n=5). Gene expression studies using Taqman Assays showed BCR stimulation resulted in an upregulation of STAT3 regulated genes in CLL cells with unmutated IgVH genes. Interestingly, stimulation of BCR resulted in a significant increase in CD62L expression, which was inhibited by pre-treatment with Ibrutinib and Ruxolitinib (p<0.05, n=5).STAT3 inhibition was shown to have a divergent effect on CLL cell survival: In patient samples with >70% positive serine pSTAT3 cells, the STAT3 inhibitor cucurbitacin I induced apoptosis with a concurrent downregulation in serine phosphorylation (n=3); while in patient samples with <70% positive serine pSTAT3 cells, treatment with cucurbitacin I resulted in a decrease in apoptosis and a concurrent increase in serine phosphorylation (n=3). The STAT3 inhibitor S3I-201 had a similar effect but the upstream JAK inhibitor Ruxolitinib had no effect on serine phosphorylation and no effect on the apoptosis of CLL cells. In addition, siRNA mediated STAT3 knockdown and treatment with cucurbitacin I and S31-201, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=29).The role of STAT3 in CLL cell adhesion under shear flow conditions was investigated using a microfluidics system including a neMESYS Low Pressure syringe pump system and Human Umbilical Vein Endothelial Cells (HUVEC) coated biochips. Treatment of CLL cells with cucurbitacin I resulted in a significant decrease in adhesion to endothelial cells (p<0.001, n=4). The effect of STAT3 inhibition on the chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with cucurbitacin I resulted in a significant decrease in CLL cells migrated in response to the chemokine CXCL12 compared to control (p=0.0001, n=8). In addition, treatment of CLL cells with CXCL12 resulted in an increase in serine pSTAT3 that was downregulated by pretreatment with cucurbitacin I.This study has shown:1. Activation of STAT3 by BCR stimulation occurs in poor prognostic unmutated IgVH genes2. Serine pSTAT3 has a role in cell survival in response to STAT3 inhibition3. A role for STAT3 in CLL cell adhesion and migration, in particular in the regulation of the expression of CD62L.In conclusion, this study shows a role for the STAT3 pathway in cell survival and CLL cell-microenvironment crosstalk, suggesting therapeutic potential by interfering with the migration and homing of CLL cells to the lymph node and bone marrow microenvironments. DisclosuresNo relevant conflicts of interest to declare.

OP0240 Signal Transducer and Activator of Transcription 3 Regulates Fibroblast Activation as A Downstream Mediator of Transforming Growth Factor-Beta

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by uncontrolled activation of fibroblasts with increased deposition of extracellular matrix. Persistent activation of TGFβ signalling is thought to play a key-role...

Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-κB and signal transducer and activator of transcription 3 pathways

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs)-mediated regulation of macrophages is critical for inflammation response and tissue injury repair. However, the underlying mechanism is not well understood. In this study, we investigated the effect of mouse bone marrow-derived MSCs on macrophages under normal and inflammatory conditions. Co-culture with MSCs or treatment with MSC-conditioned medium (MSC-CM) reduced the expression of tumor necrosis factor-α while inducing the expression of interleukin 10 (IL-10) and arginase 1 in lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cells and splenic CD11b(+) cells. MSC-CM treatment increased the expression of CD206, a marker of alternatively activated M2 macrophages, in RAW264.7 cells. In addition, MSC-CM promoted the proliferation and migration of RAW264.7 cells. MSC-CM treatment activated signal transducer and activator of transcription 3 (STAT3) but inhibited nuclear factor-κB (NF-κB) pathways in LPS-stimulated RAW264.7 cells. Moreover, STAT3 inhibitor S3I-201 antagonized the induction of IL-10, arginase 1, and CD206 by MSC-CM in RAW264.7 cells. Conclusively, our findings suggest that mouse MSCs induce macrophage M2 activation through the NF-κB and STAT3 pathways.

STAT3 upregulates miR-92a to inhibit RECK expression and to promote invasiveness of lung cancer cells

Background:Signal transducer and activator of transcription 3 (STAT3) activation is frequently found in human lung cancer and is associated with increased metastasis and reduced survival. How STAT3 enhances invasiveness is unclear.Methods:The expression of microRNAs and target genes was measured by real-time RT–PCR. Protein level was studied by western blotting. Luciferase reporter assay was used to confirm the direct targeting of microRNAs. Gelatin zymography was used to study matrix metalloproteinase (MMP) activity. Transwell assay was used to investigate cell migration and invasion.Results:Enforced expression of STAT3 decreases the endogenous MMP inhibitor RECK protein but not mRNA level in H460 cells. Conversely, STAT3 inhibitor S3I-201 increases RECK protein in STAT3-activating H1299 cells. We demonstrate that STAT3 upregulates miR-92a to repress RECK via post-transcriptional inhibition. The RECK 3′-untranslated region (3′UTR) reporter activity assay suggests that RECK is a direct repression target of miR-92a. Delivery of pre-miR-92a reduces RECK protein level whereas transfection of anti-miR-92a restores STAT3-induced downregulation of RECK. Anti-miR-92a attenuates MMP activity, migration and invasion of H1299 cells and STAT3-overexpressing H460 cells, suggesting miR-92a is critical for STAT3-induced invasiveness.Conclusion:The STAT3-induced miR-92a promotes cancer invasion by suppressing RECK and targeting of the STAT3/miR-92a axis may be helpful for cancer treatment.

A Novel Small Molecule, LLL12, Inhibits STAT3 Phosphorylation and Activities and Exhibits Potent Growth-Suppressive Activity in Human Cancer Cells

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.