Inhibition Of STAT3 Signaling Pathway Research Articles

Progesterone regulates inflammation and receptivity of cells via the NF-κB and LIF/STAT3 pathways

A high concentration of blood ammonia can lead to failure of early embryo implantation in dairy cows. Progesterone (P4) is an important hormone that regulates the changes of uterine receptivity and participates in pregnancy during early implantation. However, whether P4 alleviates the harmful effects of blood ammonia is unknown. Therefore, this experiment treated bovine endometrial epithelial cells (BEEC) with ammonia chloride and P4. The results showed that ammonia decreased the viability of BEEC, upregulated the ROS level and apoptosis rate, and upregulated the expressions of Bax, Bcl-2, P53, Cyt-c, caspase-3, caspase-8 and caspase-9. It also upregulated the expressions of TLR4, NF-κB, and in turn, produced a large amount of IL-6 and downregulated the expressions of EGF and VEGF. After co-treating BEEC with ammonia and P4, P4 had no alleviating effect on cell viability, ROS and apoptosis, but downregulated the expressions of TLR4, NF-κB, IL-6, and upregulated the expressions of LIF, EGF and VEGF. Inhibition of STAT3 signaling pathway, LIF expression was not affected, and VEGF expression was down-regulated. Using LIF treated BEEC, VEGF expression was upregulated, but after the addition of inhibitors, the expression of VEGF was not upregulated by LIF. Therefore, progesterone could not alleviate the oxidative stress and apoptosis induced by ammonia in BEEC. However, progesterone ameliorated the inflammation and receptivity genes of BEEC induced by ammonia via the NF-κB and LIF/STAT3 signaling pathways.

Effect of CHL1 gene on cell viability, invasiveness and apoptosis in neuroblastoma cells

Objective: To investigate the effect and mechanisms of CHL1 gene overexpression on cell viability, invasiveness and apoptosis in neuroblastoma cells. Methods: The empty plasmid (pcDNA3.1 group) and CHL1 recombinant plasmid (pcDNA3.1-CHL1 group) were transfected into SK-N-SH human neuroblastoma cells, and the untransfected cells were used as blank control. Forty-eight hours after transfection, the protein expressions of CHL1, PCNA, MMP-2, Bax, STAT3 and p-STAT3 were detected by western blot. Meanwhile, cell viability, invasion and apoptosis were detected by MTT, transwell and flow cytometry assays, respectively. Results: The expression level of CHL1 protein in pcDNA3.1-CHL1 group was 0.612±0.052, which was higher than that of pcDNA3.1 group 0.122±0.014 and blank control group 0.120±0.013, with statistically significant difference (P<0.05). After 24, 48 and 72 hours of transfection, the absorbance (A) values of SK-N-SH cells in the pcDNA3.1-CHL1 group were 0.328±0.035, 0.502±0.051 and 0.688±0.064, respectively, whereas those in the pcDNA3.1 group were 0.562±0.050, 0.796±0.065 and 0.973±0.077, respectively. The differences were statistically significant (P<0.05). The invaded cells in the pcDNA3.1-CHL1 group were 104.9±3.7, which were lower than that in the pcDNA3.1 group (175.6±4.6), with statistically significant difference (P<0.05). Additionally, the apoptotic rate of pcDNA3.1-CHL1 cells was (23.46±1.22)%, which was higher than that in pcDNA3.1 group (3.45±0.20)%(P<0.05). Furthermore, the levels of PCNA, MMP-2, Bax and p-STAT3 proteins in pcDNA3.1-CHL1 group were 0.156±0.018, 0.122±0.015, 0.285±0.032 and 0.023±0.004, respectively, whereas those in pcDNA3.1 group were 0.542±0.053, 0.196±0.021, 0.073±0.009 and 0.057±0.007, respectively. There were statistically significant differences between two groups (P<0.05). Conclusion: Overexpression of CHL1 inhibits the cell viability and invasion, as well as induces apoptosis of neuroblastoma cells, which is related to the inhibition of STAT3 signaling pathway.

Bruceine D inhibits tumor growth and stem cell-like traits of osteosarcoma through inhibition of STAT3 signaling pathway.

Patients with osteosarcoma exhibiting resistance to chemotherapy or presenting with metastasis usually have a poor prognosis. Osteosarcoma stem cells (OSCs) are a potential cause of tumor metastasis, relapse, and chemotherapy resistance. Therefore, it is necessary to develop novel therapeutic drugs, which not only kill osteosarcoma cells but also target OSCs. This study aims to explore the anti‐osteosarcoma effects of Bruceine D (BD), a natural compound derived from Brucea javanica, and investigate its underlying mechanisms. Results demonstrated that BD could significantly inhibit cell proliferation and migration, induce cell cycle arrest, and promote apoptosis in osteosarcoma cells. Besides, BD could also suppress the sphere‐forming and self‐renewal ability of OSCs. Mechanistically, the inhibitory role of BD on osteosarcoma cell growth and migration including OSC stemness was partially executed through the inhibition of STAT3 signaling pathway. More importantly, BD showed significant anti‐osteosarcoma activity without obvious side effects in vivo. Collectively, the results of this study demonstrated that BD exerts a strong inhibitory effect on tumor growth and stem cell like traits of osteosarcoma which may be partially due to STAT3 inhibition, suggesting that BD maybe a promising therapeutic candidate against osteosarcoma.

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells.

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.

Inhibition of Stat3 signaling pathway decreases TNF-α-induced autophagy in cementoblasts

Autophagy is a self-digestive process that eliminates impaired or aged proteins and potentially toxic intracellular components to maintain homeostasis. We previously demonstrated that TNF-α played a critical role in cementoblast differentiation, mineralization and apoptosis; however, the effect of TNF-α on cementoblast autophagy has remained unclear. In this study, an elevated immunofluorescence signal of LC3B and autophagic vacuoles, autophagosomes and autolysosomes were detected under TNF-α stimulation in OCCM-30 cells. Autophagy-related genes and proteins, Beclin-1, LC3A and Atg-5, were significantly upregulated by TNF-α in a time- and concentration-dependent manner. During this process, the activity of Stat3 was dramatically enhanced and when the activity of Stat3 was blocked by either a specific chemical inhibitor or siRNA transfection before TNF-α stimulation, the TNF-α-induced upregulation of autophagy-related genes and proteins was strongly inhibited. Our results suggest that TNF-α induced autophagy in cementoblasts was dependent, or partially dependent on the activity of Stat3 signaling pathway.

Discovery of new benzensulfonamide derivatives as tripedal STAT3 inhibitors

Persistent activated STAT3 has a striking correlation with cancer development and inhibition of STAT3 signaling pathway is a novel therapeutic way for human cancers. Among STAT family, STAT1 and STAT3 play opposite roles in tumorigenesis. However, the discovery of selective STAT3 inhibitors is still challenging to date. In this study, a series of small-molecular (MW < 500) benzensulfanilamide derivatives were designed to selectively suppress STAT3 activation for anti-cancer treatment. The most potent compound 11 inhibited both overexpressed and IL-6 induced STAT3 phosphorylation, whereas 11 displayed little effect on the phosphorylation of other STAT isoforms STAT1, STAT5, demonstrating 11 was a selective STAT3 inhibitor. Meanwhile, 11 dismissed STAT3 DNA binding activity and colony formation. In addition, 11 elevated the ROS level and induced apoptosis of cancer cells. Furthermore, 11 effectively suppressed tumor growth in an in vivo mouse-xenograft model.

Cryptotanshinone sensitizes antitumor effect of paclitaxel on tongue squamous cell carcinoma growth by inhibiting the JAK/STAT3 signaling pathway

Cryptotanshinone, a natural compound isolated from the roots of Salvia miltiorrhiza Bge (Danshen), has been found to induce cancer cells apoptosis and impair cell migration and invasion in various malignancies, but its antiproliferation and chemosensitization effects of Cryptotanshinone on tongue squamous cell carcinoma(TSCC)still remain fully elucidated. In this study, the effects of Cryptotanshinone on the proliferation, apoptosis and cell cycle of human TSCC cells, including CAL 27 and SCC 9 cells, were measured. The results demonstrated that Cryptotanshinone dose-dependently inhibited cell migration and proliferation, and induced apoptosis in TSCC cells. Mechanistic study revealed that Cryptotanshinone suppressed the expression of p-STAT3, Bcl-2, CDK2, Snail and MMP2, and induced the expression of E-cadherin, P53, P21 and β-catenin. Furthermore, we found that the combination treatment of Cryptotanshinone and paclitaxel more effectively inhibited TSCC cell proliferation and migration, and induced apoptosis via the inhibition of STAT3 signaling pathway. In brief, we provided the new evidence that Cryptotanshinone could enhance the efficacy of paclitaxel on TSCC cells in vitro and demonstrated that STAT3 signaling pathway played an important role in Cryptotanshinone-induced anticancer effects in human TSCC.

Abstract 5813: Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway

Abstract RAB GTPases is involved in membrane trafficking vesicle formation and cell motility. Recently, exocytotic RABs received growing attention in cancer research. However, the molecular mechanism of exocytotic RABs in colorectal tumorigenicity remains unclear. In this study, we found RAB GTPase 3C (RAB3C) has the most significant association with higher pathological stage, tumor recurrence, frequent distant metastasis, and poor prognosis in 215 colorectal adenocarcinoma cohort using tissue microarray (TMA) analysis. In multivariate analyses, high RAB3C expression retained its independently prognostic significance for both overall survival (p=0.001) and disease-free survival (p &amp;lt; 0.001). Further experimental results showed increased migration and invasion ability in RAB3C overexpressing colon cancer cells compared with control group in vitro and in vivo. The cell culture medium collected from RAB3C overexpressing model could promote parental colon cancer cells further indicated that the metastasis-promoting role of RAB3C is exocytosis dependent. We then integrated the results from previously microarray and proteomics datasets and discovered that increased production of multiple cytokines in RAB3C overexpressing cell lines. Of these cytokines, IL-6 pathway is the top pathway corresponding to gene expression changes after RAB3C overexpression. Blocking of IL-6 function with IL-6 antibody or IL-6 knockdown significantly reduced the migration ability in RAB3C overexpressing colon cancer cells. Furthermore, secretion of IL-6 after RAB3C overexpression promoted the JAK2-STAT3 signaling with increased STAT3 phosphorylation levels to promote migration. In addition, Ruxolitinib, a JAK2 inhibitor, was found to significantly inhibited RAB3C induced colon cancer cell migration through blocking STAT3 phosphorylation. In conclusion, our study showed that RAB3C overexpression promoted tumor metastasis and is associated with poor prognosis of colon cancer patients through modulating the IL6 exocytosis of cancer cells to activate JAK2-STAT3 pathway. These findings suggest a new therapeutic strategy through the inhibition of STAT3 signaling pathway in RAB3C-IL-6 axis by Ruxolitinib to prevent metastasis of colon cancer. Note: This abstract was not presented at the meeting. Citation Format: Chi-Long Chen. Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5813. doi:10.1158/1538-7445.AM2017-5813

Synergistic Cytotoxicity of β-Elemene and Cisplatin in Gingival Squamous Cell Carcinoma by Inhibition of STAT3 Signaling Pathway.

BackgroundCisplatin remains one of the most active agents and is the mainstay of combination chemotherapy regimens against gingival squamous cell carcinoma. However, the efficacy of cisplatin is limited by its high toxicity and the development of drug resistance. β-elemene, isolated from the Chinese herb Rhizoma zedoariahas, is highly effective against malignancies and has low toxicity, but the development of β-elemene sensitizing chemotherapy in targeting the STAT3 signaling pathway remains unexplored in gingival squamous cell carcinoma. The present study was conducted to assess the chemosensitizing effects of β-elemene for enhancing the cytotoxicity of cisplatin in gingival squamous cell carcinoma.Material/MethodsThe gingival squamous cell carcinoma YD-38 cell line was used. MTT assay, clonogenic assay, annexin V/PI apoptosis assay, Western blot analysis, and xenograft model treatment were carried out in vitro and in vivo.Resultsβ-elemene significantly enhanced proliferative inhibition and cisplatin induced apoptosis in gingival squamous cell carcinoma. Cisplatin combined with β-elemene decreased the expressions of p-STAT3, p-JAK2, and Bcl-2, and increased the expressions of Bax and caspase-3 significantly compared to cisplatin only treatment, as well as in the xenograft model.ConclusionsThe results indicated that β-elemene promoted the anti-proliferative and apoptotic effect of cisplatin by inhibiting STAT3 and blocking the JAK2-STAT3 signaling pathway in GSCC in vitro and in vivo.

Inhibition of Stat3 signaling pathway by nifuroxazide improves antitumor immunity and impairs colorectal carcinoma metastasis

Colorectal carcinoma (CRC) is the one of the most common cancers with considerable metastatic potential, explaining the need for new drug candidates that inhibit tumor metastasis. The signal transducers and activators of the transcription 3 (Stat3) signaling pathway has an important role in CRC and has been validated as a promising anticancer target for CRC therapy. In the present study, we report our findings on nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3. Our studies showed that nifuroxazide decreased the viability of three CRC cell lines and induced apoptosis of cancer cells in a concentration-dependent manner. Moreover, western blot analysis demonstrated that the occurrence of its apoptosis was correlated with the activation of Bax and cleaved caspase-3, and decreased the expression of Bcl-2. In addition, nifuroxazide markedly impaired CRC cell migration and invasion by downregulating phosphorylated-Stat3Tyr705, and also impaired the expression of matrix metalloproteinases (MMP-2 and MMP-9). Furthermore, our studies showed that nifuroxazide also significantly inhibited the tumor metastasis in lung and abdomen metastasis models of colon cancer. Meanwhile, nifuroxazide functionally reduced the proliferation index, induced tumor apoptosis and impaired metastasis. Notably, nifuroxazide reduced the number of myeloid-derived suppressor cells in the blood, spleens and tumors, accompanied by the increased infiltration of CD8+ T cells in the tumors. Importantly, a marked decrease in the number of M2-type macrophages in tumor in the abdomen metastasis model was also observed. Taken together, our results indicated that nifuroxazide could effectively inhibit tumor metastasis by mediating Stat3 pathway and it might have a therapeutic potential for the treatment of CRC.

4-Carbonyl-2,6-dibenzylidenecyclohexanone derivatives as small molecule inhibitors of STAT3 signaling pathway

Inhibition of STAT3 signaling pathway is proposed to be a promising strategy for cancer treatment. In this study, a series of 4-carbonyl-2,6-dibenzylidenecyclohexanone derivatives were prepared and evaluated as anticancer agents. The most potent compound 13r was discovered to exhibit antiproliferative activity against a broad rang of cancer cell lines and relatively low cytotoxicity against normal human cells. Besides, 13r effectively suppressed STAT3 expression as well as phosphorylation, and surface plasmon resonance analysis confirmed the direct interaction of 13r with STAT3. Docking simulation showed that 13r could inhibit STAT3 by targeting SH2 domain. This study provided evidence for these compounds to be further developed as antitumor agents through inhibition of the STAT3 pathway.

Abstract 4118: Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway

Abstract Lung cancer is the leading cause of cancer death in the United States, and metastasis to lymph-node (LN) and distal organs especially brain leads to sever complications and are the major cause of death. Preventing lung cancer development and metastases is important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in magnolia bark extracts, has a good bioavailability profile and recently has also been shown to readily cross both the blood-brain barrier (BBB). We previously demonstrated that HNK can prevent and inhibit squamous lung cancer cell carcinoma both in-vitro and in-vivo. In the current study, we evaluated the anti-metastatic effects of HNK in both LN and brain murine models of lung tumor metastasis. The H2030-BrM3 (BR-brain seeking) lung tumor cells stably transfected with luciferase and GFP were orthotopically injected into lung for LN metastases, or directly to the left ventricle of mouse heart for brain metastases, LN or brain metastasis was monitored using a non-invasive IVIS bio-luminescent imaging system. To test the efficacy of HNK in preventing tumor cells migrating to LN or brain, mice were given 10 mg/kg per body weight HNK by aerosol (for lung to LN metastasis model) or oral gavage starting the day after tumor cell injection, our results reveal that HNK significantly prevented the metastasis of lung cancer cells to LN and brain. Our results demonstrate that HNK decreased LN metastases incidence to 30% compare to 100% in control mice, and inhibited brain metastasis near 70% compare to control. Furthermore, analysis of HNK's mechanism of action by utilizing both RNA sequencing and receptor tyrosine kinase assay suggests that its effect is primarily mediated by inhibiting STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation, and knock-down STAT3 totally abrogated the anti-metastasis effects of HNK in brain metastatic lung cancer cell lines, PC9-BrM3 and H2030-BrM3. These finding suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer brain metastasis. Citation Format: Yongik Lee, Jing Pan, Qi Zhang, Yian Wang, Ming You. Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4118.

Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells.

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. Our study revealed that CA treatment significantly reduced the viability of human colon cancer HCT116, SW480, and HT-29 cells. Treatment with CA induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, activation of caspase-9, and -3, and the cleavage of PARP in HCT116 cells. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of STAT3 in HCT116 cells by blocking the phosphorylation of upstream JAK2 and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, D2, and D3. In STAT3-overexpressed HCT116 cells, CA inhibited cell viability and the expression of cyclin D1 and survivin. Furthermore, CA treatment induced the generation of ROS in these colon cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine abrogated the inhibitory effect of CA on the JAK2-STAT3/Src-STAT3 signaling and rescued cells from CA-induced apoptosis by blocking the induction of p53 and the cleavage of caspase-3 and PARP in HCT116 cells. However, L-buthionine-sulfoximine, a pharmacological inhibitor of GSH synthesis, increased CA-induced ROS production, thereby potentiating apoptotic effect of CA. In conclusion, our study provides the first report that CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases, and inhibition of STAT3 signaling pathway. © 2015 Wiley Periodicals, Inc.

Abstract 1099: Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment

Abstract Purpose: Many chemotherapeutic agents are known to have immunomodulatory activity to improve the effectiveness of adoptive T-cell therapy. We hypothesized that sorafenib could reverse the immunosuppression of tumor microenvironment to enhance T-cell mediated tumor regression. Experimental design: OVA-specific CD8+ T cells were co-cultured with sorafenib-pretreated OVA-overexpressing E.G7 mouse lymphoma cells to evaluate their function using flow cytometry, 51Cr-release assay, and transwell assay. The underlying mechanism of sorafenib-mediated immunomodulation was also explored by Western blotting. In animal studies, the activation of OVA-specific CD8+ T cells transduced with a granzyme B promoter-driven imaging reporter was monitored by bioluminescence imaging and the levels of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were assessed by flow cytometry. Results: We found that sorafenib suppressed the expression of critical immunosuppressive components, including TGF-β , IL-10, VEGF, indoleamine 2,3-dioxygenase (IDO) and CCL2/MCP-1, in E.G7 cells through the inhibition of STAT3 signaling pathway and decreased the percentage of Tregs and MDSCs in tumors. Surprisingly, the anti-tumor effect of small numbers of CD8+ T cells was substantially enhanced when combined with sorafenib in vivo. Citation Format: Jeng-Jong Hwang, Hui-Yen Chuang, Ya-Fang Chang. Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1099. doi:10.1158/1538-7445.AM2014-1099

Tu1598 Proton Pump Inhibitor Selectively Suppresses Proliferation and Restores the Chemosensitivity of Gastric Cancer Cells by Inhibition of STAT3 Signaling Pathway

Tu1598 Proton Pump Inhibitor Selectively Suppresses Proliferation and Restores the Chemosensitivity of Gastric Cancer Cells by Inhibition of STAT3 Signaling Pathway

Inhibition of STAT3 Signaling Pathway by Nitidine Chloride Suppressed the Angiogenesis and Growth of Human Gastric Cancer

STAT3 has been strongly implicated in human malignancies, and constitutive activation of STAT3 serves a crucial role in cell survival, angiogenesis, immune evasion, and inflammation. In this study, we showed that nitidine chloride, a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb) DC, exerts potent anticancer activity through STAT3 signaling cascade. Nitidine chloride dose dependently suppressed VEGF-induced endothelial cell proliferation, migration, and tubular structure formation in vitro and dramatically reduced VEGF-triggered neovascularization in mouse cornea and Matrigel plugs in vivo. This angiogenesis inhibition mediated by nitidine chloride was well interpreted by the suppression of Janus kinase 2/STAT3 signaling and STAT3 DNA-binding activity in endothelial cells. Furthermore, nitidine chloride suppressed the constitutively activated STAT3 protein, its DNA-binding activity, and the expression of STAT3-dependent target genes, including cyclin D1, Bcl-xL, and VEGF in human gastric cancer cells. Consistent with the earlier findings, nitidine chloride inhibited gastric tumor cell growth and induced tumor cell apoptosis in vitro and effectively suppressed the volume, weight, and microvessel density of human SGC-7901 gastric solid tumors (n = 8) at a dosage of 7 mg/kg/d (intraperitoneal injection). Immunohistochemistry and Western blot analysis further revealed that the expression of STAT3, CD31, and VEGF protein in xenografts was remarkably decreased by the alkaloid. Taken together, we propose that nitidine chloride is a promising anticancer drug candidate as a potent STAT3 signaling inhibitor.

Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells

To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata. Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus. Treatment of SaOS2 cells with CEP (2.5-20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor. Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.

Abstract 2576: Mechanism of action of a novel Stat3 inhibitor for cancer therapy

Abstract Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that over-express active Stat3 leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2576. doi:10.1158/1538-7445.AM2011-2576

Alkylation of Cysteine 468 in Stat3 Defines a Novel Site for Therapeutic Development

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that overexpress active Stat3, leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.

Decursin chemosensitizes human multiple myeloma cells through inhibition of STAT3 signaling pathway

Recent reports have indicated that decursin can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this experiment, we investigated how decursin could potentiate the cytotoxic effects of bortezomib in human multiple myeloma cells. We found that decursin inhibited cell viability in U266, MM.1S and ARH77 cells, but not in peripheral blood mononuclear cells (PBMC). Decursin-induced apoptosis through the activation of caspase-8, -9, and -3 in U266 cells. This correlated with the down-regulating of cyclin D1, bcl-2, bcl-xL, survivin, and the vascular endothelial growth factor (VEGF), which are all regulated by the activation of signal transducers and the activator of transcription 3 (STAT3). Indeed, decursin inhibited constitutive STAT3 activation through inhibition of the activation of Janus-activated kinase 2 (JAK2) in U266 cells. In addition, decursin inhibited interleukin-6-inducible STAT3 activation in a time-dependent manner in MM.1S cells. Interestingly, decursin significantly potentiated the apoptotic effects of bortezomib in U266 cells. These effects of decursin were correlated with the suppression of constitutive STAT3 activation in U266 cells. Overall, these results suggest that decursin is a novel blocker of STAT3 activation and it may be a potential candidate for overcoming chemo-resistance through suppression of this signaling.