STAT3 DNA-binding Activity Research Articles

Linker Variation and Structure-Activity Relationship Analyses of Carboxylic Acid-based Small Molecule STAT3 Inhibitors.

The molecular determinants for the activities of the reported benzoic acid (SH4-54), salicylic acid (BP-1-102), and benzohydroxamic acid (SH5-07)-based STAT3 inhibitors were investigated to design optimized analogues. All three leads are based on an N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities and the CH2 group of the glycinamide scaffold were separately modified. The replacement of the pentafluorobenzene or cyclohexylbenzene, or replacing the benzene ring of the aromatic carboxylic or hydroxamic acid motif with heterocyclic components (containing nitrogen and oxygen elements) all decreased potency. Notably, the Ala-linker analogues, 1a and 2v, and the Pro-based derivative 5d, all with (R)-configuration at the chiral center, had improved inhibitory activity and selectivity against STAT3 DNA-binding activity in vitro, with IC50 of 3.0 ± 0.9, 1.80 ± 0.94, and 2.4 ± 0.2 μM, respectively. Compounds 1a, 2v, 5d, and other analogues inhibited constitutive STAT3 phosphorylation and activation in human breast cancer and melanoma lines, and blocked tumor cell viability, growth, colony formation, and migration in vitro. Pro-based analogue, 5h, with a relatively polar tetrahydropyranyl (THP) ring, instead of the cyclohexyl, showed improved permeability. In general, the (R)-configuration Pro-based analogs showed the overall best profile, including physicochemical properties (e.g., microsomal metabolic stability, Caco-2 permeability), and in particular, 5d showed improved tumor-cell specificity.

Abstract B137: Novel potent salicylic, benzoic, and benzohydroxamic acid-based small-molecule STAT3 inhibitors induce antitumor effects in breast cancer xenografts

Abstract We investigated the molecular determinants for the activity of the previously reported salicylic BP-1-102 (IC50 6.8 ± 2.5 μM), benzoic SH4-54 (IC50 4.4 ± 0.3 μM), and benzohydroxamic acid SH5-07 (IC50 3.9 ± 0.6 μM) small-molecule STAT3 inhibitors in order to derive structurally optimum analogs with improved potency and pharmacokinetic parameters. All three leads are based on N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities were separately modified to generate various series of new analogs. The inhibitory activities of the new analogs against STAT3 DNA-binding in vitro were determined by electrophoretic motility shift assay. Some new analogs show dramatically improved potency compared to the corresponding lead compounds. Notably, analogs H098, H127, H142, H145, H152, and H155 showed more than 10-fold improved potency in inhibiting STAT3 DNA-binding activity in vitro, with IC50 of 300 - 600 nM. Using analogs H105 and H120, we show that the new analogs decreased STAT3 DNA-binding activity and phosphorylation at Tyr705 in MDA-MB-231 and MDA-MB-468 cells, two human triple-negative breast cancer lines harboring constitutively active STAT3, within as early as 30 min. Furthermore, the new analogs potently inhibited the growth and viability of the human cancer cells with persistently active STAT3, with IC50 of 1.2 - 2.0 µM. The potent analogs specifically disrupted STAT3 DNA-binding activity in vitro over that of STAT1 and STAT5, and they further showed minimum inhibitory effect on the proliferation of human cancer cells that do not harbor persistently active STAT3. Initial in vivo efficacy study in nude mouse model showed that oral administration of H105 and H120 suppressed the growth of tumor xenografts generated from human breast cancer line harboring constitutively active STAT3. Thus, we have made substantial progress and broken the nanomolar barrier in developing STAT3 inhibitors, and these compounds are the first nanomolar STAT3 inhibitors reported to date that are direct disruptors of STAT3 activation, which show in vivo activity. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Christine Brotherton-Pleiss, Marcus Tius, James Turkson. Novel potent salicylic, benzoic, and benzohydroxamic acid-based small-molecule STAT3 inhibitors induce antitumor effects in breast cancer xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B137.

Proscillaridin A Promotes Oxidative Stress and ER Stress, Inhibits STAT3 Activation, and Induces Apoptosis in A549 Lung Adenocarcinoma Cells.

Cardiac glycosides are natural compounds used for the treatment of cardiovascular disorders. Although originally prescribed for cardiovascular diseases, more recently, they have been rediscovered for their potential use in the treatment of cancer. Proscillaridin A (PSD-A), a cardiac glycoside component of Urginea maritima, has been reported to exhibit anticancer activity. However, the cellular targets and anticancer mechanism of PSD-A in various cancers including lung cancer remain largely unexplored. In the present study, we found that PSD-A inhibits growth and induces apoptosis in A549 lung adenocarcinoma cells. The anticancer activity of PSD-A was found to be associated with the activation of JNK, induction of ER stress, mitochondrial dysfunction, and inhibition of STAT3 activation. PSD-A induces oxidative stress as evidenced from ROS generation, GSH depletion, and decreased activity of TrxR1. PSD-A-mediated ER stress was verified by increased phosphorylation of eIF2α and expression of its downstream effector proteins ATF4, CHOP, and caspases-4. PSD-A triggered apoptosis by inducing JNK (1/2) activation, increasing bax/bcl-2 ratio, dissipating mitochondrial membrane potential, and inducing cleavage of caspases and PARP. Further study revealed that PSD-A inhibits both constitutive and inducible STAT3 activations and decreases STAT3 DNA-binding activity. Moreover, PSD-A-mediated inhibition of STAT3 activation was found to be associated with increased SHP-1 expression, decreased phosphorylation of Src, and binding of PSD-A with STAT3 SH2 domain. Finally, STAT3 knockdown by shRNA inhibited growth and enhanced apoptotic efficacy of PSD-A. Taken together, the data suggest that PSD-A could be developed into a potential therapeutic agent against lung adenocarcinoma.

(E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol suppresses ovarian cancer cell growth via inhibition of ERK and STAT3.

In the present study, we synthesized several non-aldehyde analogues of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal which showed anti-cancer effect. Interestingly, among the 16 compounds, we found that (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) showed the most significant anti-proliferative effect on PA-1 and SK-OV-3 ovarian epithelial cancer cells. MMPP treatment (0-15 µg/mL) induced apoptotic cell death, enhanced the expression of cleaved caspase-3, and cleaved caspase-9 in a concentration dependent manner. Notably, DNA binding activity of STAT3, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 was significantly decreased by MMPP treatment. However, ERK siRNA augmented MMPP-induced inhibitory effect on cell growth rather than p38 siRNA or JNK siRNA. Moreover, combination treatment of MMPP with ERK inhibitor U0126 (10 µM) augmented MMPP-induced inhibitory effect on cell growth and DNA binding activity of STAT3, and enhanced expression of cleaved caspase-3 and cleaved caspase-9. In addition, STAT3 siRNA transfection augmented MMPP-induced cell growth inhibition. In PA-1 bearing xenograft mice model, MMPP (5 mg/kg) suppressed tumor growth significantly. Immunohistochemistry staining showed that the expression levels of p-ERK, PCNA, p-STAT3 were decreased while the expression level of caspase-3 was increased by MMPP treatment. Thus, MMPP may be a promising anti-cancer agent in ovarian epithelial cancer treatment.

The mechanism of CIRP in inhibition of keratinocytes growth arrest and apoptosis following low dose UVB radiation.

UV induces CIRP expression and subsequent Stat3 activation, but the biological function and mechanism of CIRP and Stat3 in mediating UVB-induced skin carcinogenesis have not been fully elucidated. In this study, we demonstrate that CIRP is elevated in all tested melanoma and non-melanoma skin cancer cell lines; and the expression of CIRP is upregulated in keratinocytes after being irradiated with relatively low dose (<5 mJ/cm2 ), but not high dose (50 mJ/cm2 ), UVB acutely and chronically. The increased expression of CIRP, either induced by UVB or through overexpression, leads to resistance of keratinocytes to UVB-induced growth arrest and death; and reduced expression of CIRP by RNA knockdown sensitizes keratinocyte cells to the low dose UVB radiation. We also demonstrated that CIRP expression is required for the low dose UVB-induced Tyr705-phosphorylation, but not total amount, of Stat3. The p-Stat3 level is correlated with the expression levels of cyclin D1 and VEGF, two known downstream cell growth regulators of Stat3, as well as Bag-1/S, an apoptosis regulator. Inhibition of Stat3 DNA-binding activity by S3I-201 leads to a reduction of the p-Stat3 and Bag-1/S along with growth and survival of keratinocytes post-UVB; and the effect of S3I-201 on the UVB-irradiated cells can be partially inhibited by overexpression of CIRP or Bag-1/S. Furthermore, the overexpression of Bag-1/S can totally inhibit UVB-induced PARP cleavage and caspase 3 activation. The results presented above led us to propose that CIRP-p(705)Stat3 cascade promotes cell proliferation and survival post-UVB via upregulating the expression of cyclin D1 and Bag-1/S, respectively. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Myricitrin Modulates NADPH Oxidase‐Dependent ROS Production to Inhibit Endotoxin‐Mediated Inflammation by Blocking the JAK/STAT1 and NOX2/p47phox Pathways

Myricitrin, a naturally occurring polyphenol hydroxy flavonoid, has been reported to possess anti-inflammatory properties. However, the precise molecular mechanism of myricitrin's effects on LPS-induced inflammation is unclear. In the present study, myricitrin significantly alleviated acute lung injury in mice. Myricitrin also markedly suppressed the production of NO, TNF-α, IL-6, and MCP-1 in RAW264.7 macrophage cells. The inhibition of NO was concomitant with a decrease in the protein and mRNA levels of iNOS. The phosphorylation of JAKs and STAT-1 was abrogated by myricitrin. Furthermore, myricitrin inhibited the nuclear transfer and DNA binding activity of STAT1. The JAK-specific inhibitor ruxolitinib simulated the anti-inflammatory effect of myricitrin. However, myricitrin had no impact on the MAPK signalling pathway. Myricitrin attenuated the generation of intracellular ROS by inhibiting the assembly of components of the gp91phox and p47phox. Suppression of ROS generation using NAC or apocynin or by silencing gp91phox and p47phox all demonstrated that decreasing the level of ROS inhibited the LPS-induced inflammatory response. Collectively, these results confirmed that myricitrin exhibited anti-inflammatory activity by blocking the activation of JAKs and the downstream transcription factor STAT1, which may result from the downregulation of NOX2-dependent ROS production mediated by myricitrin.

Natural product pectolinarigenin inhibits osteosarcoma growth and metastasis via SHP-1-mediated STAT3 signaling inhibition.

Signal transducer and activator of transcription 3 (STAT3) has important roles in cancer aggressiveness and has been confirmed as an attractive target for cancer therapy. In this study, we used a dual-luciferase assay to identify that pectolinarigenin inhibited STAT3 activity. Further studies showed pectolinarigenin inhibited constitutive and interleukin-6-induced STAT3 signaling, diminished the accumulation of STAT3 in the nucleus and blocked STAT3 DNA-binding activity in osteosarcoma cells. Mechanism investigations indicated that pectolinarigenin disturbed the STAT3/DNA methyltransferase 1/HDAC1 histone deacetylase 1 complex formation in the promoter region of SHP-1, which reversely mediates STAT3 signaling, leading to the upregulation of SHP-1 expression in osteosarcoma. We also found pectolinarigenin significantly suppressed osteosarcoma cell proliferation, induced apoptosis and reduced the level of STAT3 downstream proteins cyclin D1, Survivin, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma extra-large (Bcl-xl) and myeloid cell leukemia 1 (Mcl-1). In addition, pectolinarigenin inhibited migration, invasion and reserved epithelial–mesenchymal transition (EMT) phenotype in osteosarcoma cells. In spontaneous and patient-derived xenograft models of osteosarcoma, we identified administration (intraperitoneal) of pectolinarigenin (20 mg/kg/2 days and 50 mg/kg/2 days) blocked STAT3 activation and impaired tumor growth and metastasis with superior pharmacodynamic properties. Taken together, our findings demonstrate that pectolinarigenin may be a candidate for osteosarcoma intervention linked to its STAT3 signaling inhibitory activity.

Abstract LB-058: Linker variation and structure-activity relationship analyses of salicylic, benzoic and hydroxamic acid-based small molecule Stat3 inhibitors

Abstract Benzoic acid, salicylic acid and hydroxamic acid-based small molecules have been reported that inhibit Stat3 activation. However, the structural basis for their Stat3-inhibitory activities are not clearly defined. To investigate the molecular determinants for activity and to derive structurally optimized analogs of improved pharmacokinetic parameters and potency, we conducted a computer-aided molecule optimization program and synthesized new analogs of the previously reported lead Stat3 inhibitors, BP-1-102 (IC50, 6.8 ± 2.5), SH4-54 (IC50 4.4 ± 0.3), and SH5-07 (IC50 3.9 ± 0.6). All three leads are based on N-methylglycinamide scaffold with its two amine moieties condensed with three different functionalities. The three functionalities or the CH2 group of the glycolamide scaffold were separately modified to generate a series of compounds, some of which show improved potency compared to the corresponding parental compounds. Specifically, H012, H070 and H072 had improved inhibitory activity against Stat3 DNA-binding activity in vitro, with IC50 of 3.0 ± 0.9, 2.4 ± 0.4, and 2.2 ± 0.9 μM, respectively. The replacement of the pentafluorobenzene or the cyclohexylbenzene with other components was detrimental to activity and led to decreased potency of the new analogues, as did the substitution of the benzene of the pTyr-mimetic moiety with heterocyclic components containing nitrogen and oxygen elements. Pharmacokinetic study showed that analog H058 has improved permeability in Caco-2 bidirectional permeability studies, compared to the lower relative permeability of the lead agents. Ala-based analogs, such as H048 and H058, were low in microsomal stability, presumably due to the labile methyl groups of the Ala. Select active analogs inhibited constitutive Stat3 activation and the DNA-binding activity of Stat3 in cancer cells, and they further decreased the growth, colony formation, and migration of human cancer cells harboring persistently active Stat3. Studies together demonstrate a significant progress towards developing a more suitable Stat3 inhibitor with drug-like features. Additional efforts are focused on refining the physicochemical properties to further improve the activity and efficacy and ultimately furnish suitable Stat3-inhibiting candidates for clinical application. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Christine Brotherton-Pleiss, Marcus Tius, James Turkson. Linker variation and structure-activity relationship analyses of salicylic, benzoic and hydroxamic acid-based small molecule Stat3 inhibitors. [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 LB-058.

Abstract LB-028: CIRP promotes keratinocytes survival following low-dose UVB irradiation through Stat3-mediated Bag-1/S expression

Abstract UV induces CIRP expression and subsequent Stat3 activation, but the biological function and mechanism of CIRP and Stat3 in mediating UVB-induced skin carcinogenesis have not been fully elucidated. In this study, we demonstrate that CIRP is elevated in all tested melanoma and non-melanoma skin cancer cell lines; and the expression of CIRP is upregulated in keratinocytes after being irradiated with relatively low dose (&amp;lt;5 mJ/cm2), but not high dose (50 mJ/cm2), UVB acutely and chronically. The increased expression of CIRP, either induced by UVB or through overexpression, leads to resistance of keratinocytes to UVB-induced growth arrest and death; and reduced expression of CIRP by RNA knockdown sensitizes keratinocyte cells to the low dose UVB irradiation. We also demonstrated that CIRP expression is required for the low dose UVB-induced Tyr705-phosphorylation, but not total amount, of Stat3. The p-Stat3 level is correlated with the expression levels of cyclin D1 and VEGF, two known downstream cell growth regulators of Stat3, as well as Bag-1/S, an apoptosis regulator. Inhibition of Stat3 DNA-binding activity by S3I-201 leads to a reduction of the p-Stat3 and Bag-1/S along with growth and survival of keratinocytes post-UVB; and the effect of S3I-201 on the UVB-irradiated cells can be partially inhibited by overexpression of CIRP or Bag-1/S. Furthermore, the overexpression of Bag-1/S can totally inhibit UVB-induced PARP cleavage and caspase 3 activation. The results presented above led us to propose that CIRP-p(705)-Stat3 cascade promotes cell proliferation and survival post-UVB via upregulating the expression of cyclin D1 and Bag-1/S respectively. Citation Format: Yi Liao, Liling Tang, Shiyong Wu. CIRP promotes keratinocytes survival following low-dose UVB irradiation through Stat3-mediated Bag-1/S expression. [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 LB-028.

Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer

ABSTRACTWe have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.

10 Oncostatin M regulates trophoblast invasion through STAT1 expression

Introduction Trophoblast cell invasion is one of key steps for successful pregnancy, which is controlled by several factors including cytokines that activate intracellular signaling cascades including the Janus kinase / signal transducer and activator of transcription (JAK-STAT) system. Objectives We aimed to analyze the effect of cytokines on STAT1 expression and trophoblast cell functions. Methods Immortalized HTR-8/SVneo cells were treated with interleukin-6 (IL-6) IL-11, granulocyte macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF) and oncostatin M (OSM). STAT1 phosphorylation (tyr-701) was analyzed by Western blotting and immunofluorescence staining. Simultaneously, cells were incubated with 50 μ M Fludarabine (specific STAT1 inhibitor) or small interfering RNA (siRNA) to inhibit the expression of STAT1 and followed by cytokine stimulation. STAT1 DNA-binding activity, cell viability and invasion were analyzed by DNA-binding, MTS and Matrigel invasion assays. Results Western blot analysis and immunofluorescence staining of STAT1 phosphorylation revealed that OSM induced phosphorylation of STAT-1 stronger than LIF in HTR-8/SVneo. IL-6, IL-11 and GM-CSF had no effect on STAT1 activation. STAT1 phosphorylation was inhibited by STAT1 siRNA and Fludarabine. STAT1 inhibition reduced STAT1 DNA binding activity and OSM could not rescue this effect. OSM stimulated HTR-8/SVneo cell viability and invasion, whereas STAT1 inhibition decreased trophoblast cell invasion. This reduction could not be prevented by OSM. Conclusions These findings demonstrate that the OSM-STAT1 axis plays an important role in trophoblastic cells and STAT1 activation may induce a variety of cell functions

(-)-Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma.

We explored the anti-cancer capacity of (−)-oleocanthal in human hepatocellular carcinoma (HCC). (−)-Oleocanthal inhibited proliferation and cell cycle progression and induced apoptosis in HCC cells in vitro and suppressed tumor growth in an orthotopic HCC model. (−)-Oleocanthal also inhibited HCC cell migration and invasion in vitro and impeded HCC metastasis in an in vivo lung metastasis model. ( )-Oleocanthal acted by inhibiting epithelial-mesenchymal transition (EMT) through downregulation Twist, which is a direct target of STAT3. (−)-Oleocanthal also reduced STAT3 nuclear translocation and DNA binding activity, ultimately downregulating its downstream effectors, including the cell cycle protein Cyclin D1, the anti-apoptotic proteins Bcl-2 and survivin, and the invasion-related protein MMP 2. Overexpression of constitutively active STAT3 partly reversed the anti cancer effects of (−)-oleocanthal, which inhibited STAT3 activation by decreasing the activities of JAK1 and JAK2 and increasing the activity of SHP-1. These data suggest that (−)-oleocanthal may be a promising candidate for HCC treatment.

Chikusetsusaponin IVa Butyl Ester (CS-IVa-Be), a Novel IL6R Antagonist, Inhibits IL6/STAT3 Signaling Pathway and Induces Cancer Cell Apoptosis.

The activation of IL6/STAT3 signaling is associated with the pathogenesis of many cancers. Agents that suppress IL6/STAT3 signaling have cancer-therapeutic potential. In this study, we found that chikusetsusaponin IVa butyl ester (CS-IVa-Be), a triterpenoid saponin extracted from Acanthopanas gracilistylus W.W.Smith, induced cancer cell apoptosis. CS-IVa-Be inhibited constitutive and IL6-induced STAT3 activation, repressed STAT3 DNA-binding activity, STAT3 nuclear translocation, IL6-induced STAT3 luciferase reporter activity, IL6-induced STAT3-regulated antiapoptosis gene expression in MDA-MB-231 cells, and IL6-induced TF-1 cell proliferation. Surprisingly, CS-IVa-Be inhibited IL6 family cytokines rather than other cytokines induced STAT3 activation. Further studies indicated that CS-IVa-Be is an antagonist of IL6 receptor via directly binding to the IL6Rα with a Kd of 663 ± 74 nmol/L and the GP130 (IL6Rβ) with a Kd of 1,660 ± 243 nmol/L, interfering with the binding of IL6 to IL6R (IL6Rα and GP130) in vitro and in cancer cells. The inhibitory effect of CS-IVa-Be on the IL6-IL6Rα-GP130 interaction was relatively specific as CS-IVa-Be showed higher affinity to IL6Rα than to LIFR (Kd: 4,910 ± 1,240 nmol/L) and LeptinR (Kd: 4,990 ± 915 nmol/L). We next demonstrated that CS-IVa-Be not only directly induced cancer cell apoptosis but also sensitized MDA-MB-231 cells to TRAIL-induced apoptosis via upregulating DR5. Our findings suggest that CS-IVa-Be as a novel IL6R antagonist inhibits IL6/STAT3 signaling pathway and sensitizes the MDA-MB-231 cells to TRAIL-induced cell death. Mol Cancer Ther; 15(6); 1190-200. ©2016 AACR.

Argon mediates protection by interleukin-8 suppression via a TLR2/TLR4/STAT3/NF-κB pathway in a model of apoptosis in neuroblastoma cells invitro and following ischemia-reperfusion injury in rat retina invivo.

Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and anti-inflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's invivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-light-chain-enhancer' of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) but not STAT5 or cAMP-response element-binding protein (CREB) phosphorylation and DNA-binding activity. Argon treatment attenuated apoptosis by preservation of mitochondrial membrane potential and decline in reactive oxygen species (ROS) generation. NF-κB and STAT3 inhibition, as well as TLR2 and TLR4 inhibition reversed argon's effects on IL-8 mRNA expression. Argon attenuated rotenone-induced IL-8 protein and mRNA expression invitro. Inhibition of TLR2 and 4 attenuated argon's protective effect invivo reducing IRI driven retinal IL-8 expression. IL-8 expression was found in the retina in co-localization with Müller cells and retinal ganglion cells. Argon mediates its neuroprotective effects by TLR-mediated regulation of transcription factors NF-κB and STAT3, thus decreasing interleukin-8 expression invitro and invivo. These findings may open up new opportunities to effectively treat cerebral ischemia and reperfusion injury through the inhalation of argon. Argon exerts its protective effects invitro and in vivo via toll-like receptors TLR2 and TLR4 signaling, followed by alteration of downstream enzymes. In conclusion, argon mediates its beneficial effects by suppression of STAT3 and NF-κB phosphorylation and subsequent suppression of interleukin IL-8 protein expression. These novel findings may open up opportunities for argon as a therapeutic agent, particularly in the treatment of neuronal injury. Cover image for this issue: doi: 10.1111/jnc.13334.

Tumor growth suppressive effect of IL-4 through p21-mediated activation of STAT6 in IL-4Rα overexpressed melanoma models.

To evaluate the significance of interleukin 4 (IL-4) in tumor development, we compared B16F10 melanoma growth in IL-4-overespressing transgenic mice (IL-4 mice) and non-transgenic mice. In IL-4 mice, reduced tumor volume and weight were observed when compared with those of non-transgenic mice. Significant activation of DNA binding activity of STAT6, phosphorylation of STAT6 as well as IL-4, IL-4Rα and p21 expression were found in the tumor tissues of IL-4 mice compared to non-transgenic mice. Higher expression of IL-4, STAT6 and p21 in human melanoma tissue compared to normal human skin tissue was also found. Higher expression of apoptotic protein such as cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, p53 and p21, but lower expression levels of survival protein such as Bcl-2 were found in the tumor of IL-4 mice. In vitro study, we found that overexpression of IL-4 significantly inhibited SK-MEL-28 human melanoma cell and B16F10 murine melanoma cell growth via p21-mediated activation of STAT6 pathway as well as increased expression of apoptotic cell death proteins. Moreover, p21 knockdown with siRNA abolished IL-4 induced activation of STAT6 and expression of p53 and p21 accompanied with reduced IL-4 expression as well as melanoma cell growth inhibition. Therefore, these results showed that IL-4 overexpression suppressed tumor development through p21-mediated activation of STAT6 pathways in melanoma models.

Hydroxamic Acid and Benzoic Acid-Based STAT3 Inhibitors Suppress Human Glioma and Breast Cancer Phenotypes In Vitro and In Vivo.

STAT3 offers an attractive target for cancer therapy, but small-molecule inhibitors with appealing pharmacologic properties have been elusive. Here, we report hydroxamic acid-based and benzoic acid-based inhibitors (SH5-07 and SH4-54, respectively) with robust bioactivity. Both inhibitors blocked STAT3 DNA-binding activity in vitro and in human glioma, breast, and prostate cancer cells and in v-Src-transformed murine fibroblasts. STAT3-dependent gene transcription was blocked along with Bcl-2, Bcl-xL, Mcl-1, cyclin D1, c-Myc, and survivin expression. Nuclear magnetic resonance analysis of STAT3-inhibitor complexes defined interactions with the SH2 and DNA-binding domains of STAT3. Ectopic expression of the SH2 domain in cells was sufficient to counter the STAT3-inhibitory effects of SH4-54. Neither compound appreciably affected STAT1 or STAT5 DNA-binding activities, STAT3-independent gene transcription, or activation of a panel of oncogenic kinases in malignant cells. Each compound decreased the proliferation and viability of glioma, breast, and prostate cancer cells and v-Src-transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates for further development as cancer therapeutics.

Berberine and Curcumin Target Survivin and STAT3 in Gastric Cancer Cells and Synergize Actions of Standard Chemotherapeutic 5-Fluorouracil

Aberrantly expressed survivin and STAT3 signaling have emerged as major determinants of chemoresistance in gastric cancer. We evaluated effects of potent herbal derivatives curcumin, berberine, and quercetin on STAT3 signaling, survivin expression, and response to 5-fluorouracil (5-FU) treatment in gastric cancer cells (AGS). Cytotoxic and inhibitory effects of berberine, curcumin, and quercetin alone or in combination with 5-FU were examined by MTT assay, and their effect on survivin, STAT3, and the phosphorylated active STAT3 (pSTAT3) expression was examined by western blotting. Effect of these herbal derivatives on STAT3 DNA binding activity was measured by electrophoretic mobility shift assay. Curcumin, berberine, and quercetin effectively downregulated pSTAT3 levels, survivin expression, and gastric cancer cells viability in a dose-dependent manner (with corresponding IC50 values of 40.3μM, 29.2μM and 37.5μM, respectively). Berberine was more effective in inhibiting survivin expression as compared to other herbal agents. 5-FU in combination with berberine or curcumin showed a synergistic inhibition of survivin and STAT3 level resulting in enhanced cell death in gastric cancer cells. Overall, our data suggest use of berberine and curcumin as adjunct therapeutics to overcome chemoresistance during treatment of gastric malignancies.

Abstract 3807: Capsazepine attenuates JAK/STAT3 signaling pathway, proliferation, survival of prostate carcinoma DU145 cells through induction of the protein tyrosine phosphatase epsilon (PTP-epsilon)

Abstract Constitutive activation of signal transducers and activators of transcription (STAT) 3 is a major distinguishing feature of various human cancer cells and its activation leads to survival, proliferation, and metastasis of tumor cells. Whether the anti-proliferative, pro-apoptotic, and anti-invasive effects of capsazepine (Capz), a synthetic analog of capsaicin, are linked to its capability to inhibit STAT3 activation was investigated. We found that Capz suppressed both constitutive and IL-6-inducible STAT3 activation in human prostate carcinoma cells. Capz also suppressed nuclear translocation and DNA binding activity of STAT3. The suppression was mediated through the inhibition of activation of the upstream kinases JAK1/2 and c-Src. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate treatment reversed the Capz-induced down-regulation of STAT3, thereby suggesting the involvement of a PTP. Capz induced the expression of PTPϵ protein and mRNA. Moreover, knockdown of PTPϵ by small interfering RNA suppressed the induction of PTPϵ, reversed the inhibition of STAT3 activation, suggesting the critical role of PTPϵ in its possible mechanism of action. Capz downregulated the expression of STAT3-regulated proliferative, antiapoptotic, angiogenetic, and metastatic gene products; and this correlated with suppression of cell proliferation and invasion, the accumulation of cells in sub-G1 phase of cell cycle, and induction of apoptosis. Overexpression of phosphorylated STAT3 led to the attenuation of Capz-mediated cleavage of PARP as compared to the control. Thus, overall, our results suggest that Capz is a novel inhibitor of STAT3 activation and thus may have a potential in negative regulation of growth, metastasis, and angiogenesis of tumor cells. Citation Format: Jong Hyun Lee, Chulwon Kim, Seok-Geun Lee, Junhee Lee, Jung-woo Lee, Kwang Seok Ahn. Capsazepine attenuates JAK/STAT3 signaling pathway, proliferation, survival of prostate carcinoma DU145 cells through induction of the protein tyrosine phosphatase epsilon (PTP-epsilon). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2015-3807

Abstract 2050: Persistent STAT3 signaling contributes to the resistance of anti cancer drugs doxorubicin and cisplatin, and MEK inhibitor AZD6244 in human sarcoma cells

Abstract Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway is activated in response to cytokines and growth factors. Persistently activated STAT3 are frequently detected in many types of human cancers and are believed to play an important role in their oncogenesis. Stat3 has been classified as a proto-oncogene because activated STAT3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. We previously demonstrated that persistently activated STAT3 is frequently detected in human sarcoma such as rhabdomyosarcoma. To further examine the role of persistent TAT3 signaling plays in rhabdomyosarcoma, we used genetic approaches to either knock-down the expression of STAT3 using short hairpin RNA (ShRNA) or expressed constitutive active STAT3 protein. Knock-down the expression of STAT3 in rhabdomyosarcoma cells that express persistent STAT3 signaling, reduced the cell viability and sensitized cells to anti-cancer drugs doxorubicin and cisplatin. On the other hand, expression of constitutive active STAT3 protein in rhabdomyosarcoma cell lines lacking persistent STAT3 signaling decreased the sensitivity of those cells to doxorubicin and cisplatin. Furthermore, a MEK inhibitor, AZD6244 that is being tested in human cancer clinical trials unexpectedly induced STAT3 phosphorylation suggesting inducing of STAT3 may induce intrinsic resistance to MEK inhibitor. Accordingly, knock down of STAT3 sensitize sarcoma cells to MEK inhibitor treatments. To further translate our finding to potential cancer therapy, we tested a small molecular STAT3-selective inhibitor LY5 in sarcoma cells expressing persistent STAT3 signaling. Our data demonstrated that LY5 inhibited STAT3 phosphorylation, STAT3 DNA binding activity, and the migration of sarcoma cells. In addition, the combination of LY5 with anti-cancer drugs doxorubicin and cisplatin and the MEK inhibitor, AZD6244 also shown stronger inhibitory effects than single agent alone. In summary, our results using both genetic and small molecule approaches demonstrated that persistent STAT3 signaling contributes to the resistance of anti-cancer drugs doxorubicin and cisplatin, and MEK inhibitor in human sarcoma cells. They also implicated a potential cancer therapy strategy with the combination of STAT3 inhibitor with doxorubicin, cisplatin, and MEK inhibitor in sarcoma cells expressing persistent STAT3 signaling. Citation Format: Xiaojuan Wu, Hui Xiao, Chenglong Li, Jiayuh Lin. Persistent STAT3 signaling contributes to the resistance of anti cancer drugs doxorubicin and cisplatin, and MEK inhibitor AZD6244 in human sarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2015-2050

Obesity Drives Th17 Cell Differentiation by Inducing the Lipid Metabolic Kinase, ACC1

Chronic inflammation due to obesity contributes to the development of metabolic diseases, autoimmune diseases, and cancer. Reciprocal interactions between metabolic systems and immune cells have pivotal roles in the pathogenesis of obesity-associated diseases, although the mechanisms regulating obesity-associated inflammatory diseases are still unclear. In the present study, we performed transcriptional profiling of memory phenotype CD4 T cells in high-fat-fed mice and identified acetyl-CoA carboxylase 1 (ACC1, the gene product of Acaca) as an essential regulator of Th17 cell differentiation in vitro and of the pathogenicity of Th17 cells in vivo. ACC1 modulates the DNA binding of RORγt to target genes in differentiating Th17 cells. In addition, we found a strong correlation between IL-17A-producing CD45RO(+)CD4 T cells and the expression of ACACA in obese subjects. Thus, ACC1 confers the appropriate function of RORγt through fatty acid synthesis and regulates the obesity-related pathology of Th17 cells.