STAT3 Nuclear Translocation Research Articles

Trillin prevents proliferation and induces apoptosis through inhibiting STAT3 nucleartranslocation in hepatoma carcinoma cells.

Trillin is a constituent of total Trillium Tschonoskii Maxim (TTM), which is extracted from TTM and displayed anti-tumor effect in many tumor cell lines. However, the anti-tumor mechanism of trillin is still unclear. This study demonstrated that trillin could dramatically inhibit hepatoma carcinoma cell proliferation, induce apoptosis and decrease migration and invasion through suppressing phosphorylated STAT3 translocated to nucleus. Trillin could down-regulate Bcl-2 and Survivin, up-regulate cleaved PRAP, leading to dramatically apoptosis; trillin could also down-regulate MMP1, MMP2, MucI and VEGF, which displayed an inhibition effect on hepatocellular tumor cells invasion and development. The results of this study indicated the potential utility of trillin as a STAT3 inhibitor for the treatment of cancers.

Diallyl Trisulfide Inhibits Leptin-induced Oncogenic Signaling in Human Breast Cancer Cells but Fails to Prevent Chemically-induced Luminal-type Cancer in Rats.

Previous studies have demonstrated inhibitory effect of garlic component diallyl trisulfide (DATS) on growth of breast cancer cells in vitro and in vivo. This study investigated the effect of DATS on oncogenic signaling regulated by leptin, which plays an important role in breast carcinogenesis. Leptin-induced phosphorylation and nuclear translocation of STAT3 was inhibited significantly in the presence of DATS in MCF-7 (a luminal-type human breast cancer cell line) and MDA-MB-231 (a basal-like human breast cancer cell line). Leptin-stimulated cell proliferation, clonogenic cell survival, and migration and/or invasion ability in MCF-7 and/or MDA-MB-231 cells were also suppressed by DATS treatment. DATS exposure resulted in inhibition of leptin-stimulated expression of protein and/or mRNA levels of Bcl-2, Bcl-xL, Cyclin D1, vascular endothelial growth factor, and matrix metalloproteinase-2. Western blotting revealed a decrease in protein levels of phosphorylated STAT3 in breast cancer xenografts from DATS-treated mice when compared to controls in vivo. However, the incidence of N-methyl-N-nitrosourea-induced luminal-type breast cancer development in rats was not affected by oral administration of 5 mg/kg or 25 mg/kg DATS. The present study reveals that oncogenic signaling induced by leptin is inhibited in the presence of DATS but higher doses of this phytochemical may be required to achieve chemopreventive activity.

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production.

The lipopolysaccharide (LPS)-induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701 Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of ARID5A and IL12B The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr749 Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

Xanthoplanine attenuates macrophage polarization towards M1 and inflammation response via disruption of CrkL-STAT5 complex

Monocyte infiltration and macrophage polarization are widely considered as pivotal steps for the initiation and progression of atherosclerosis. Previous studies suggested that zanthoxylum piperitum had strong analgesic and anti-inflammatory effects. However, it remains unclear whether zanthoxylum piperitum inhibits inflammation via macrophage function. In the present study, we investigated the effects of xanthoplanine (the total alkaloid extract of zanthoxylum piperitum) on macrophage function. CCK-8 kit was performed to determine cell viability and the preferred concentration of xanthoplanine. We assayed the effects of xanthoplanine on markers of macrophage polarization and inflammation via quantitative PCR and enzyme-linked immunosorbent assay, and measured the production of reactive oxygen species (ROS) by flow cytometry. Immunoblots, co-immunoprecipitation, immunofluorescence and Luciferase activity were performed to investigate the molecular mechanism of STAT signaling pathway in response to xanthoplanine. We found that xanthoplanine (50 and 100 μM) significantly reduced M1 polarization and promoted M2 polarization. The contents of inflammatory cytokines measured by ELISA were markedly decreased in macrophages pretreated with xanthoplanine, compared with those induced by LPS and IFN-γ. In parallel, xanthoplanine alleviated the production of ROS in macrophages induced by LPS and IFN-γ. Moreover, xanthoplanine alleviated STAT5 phosphorylation and blocked STAT5 nuclear translocation without alterations in CrkL expression, subsequently interrupting the interaction between p-STAT5 and CrkL. Likewise, xanthoplanine prominently attenuated the transcription activity of STAT5 induced by LPS and IFN-γ but did not affect the transcription activity of STAT1 and STAT3. Xanthoplanine attenuated M1 phenotypic switch and macrophage inflammation via blocking the formation of CrkL-STAT5 complex.

The Anticancer Effects of Atractylenolide III Associate With the Downregulation of Jak3/Stat3-Dependent IDO Expression.

Objective: Indoleamin-2,3-dioxygenase-1 (IDO) has been identified as a checkpoint protein involved in generating the immunosuppressive tumor microenvironment that supports tumor growth. It has been reported that atractylenolide III (ATLIII) has anticancer and immune modulatory effects. This study is to determine the anticancer effects of ATLIII with the Jak3/Stat3-dependent IDO inactivation.Methods: We assessed the cytotoxicity of ATLIII and IFN-γ on lung cancer cells by MTT. We determined the efficacy of ATLIII on IFN-γ-induced IDO expression by RT-PCR and Western blot. We also determined the efficacy of ATLIII on Jak3/Stat3 pathway expression induced by IFN-γ and Jak3/Stat3-dependent IDO activation. Further molecular docking assay predicted the binding activity and site of ATLIII to Jak3 protein. Additional immunofluorescence staining was used to measure the Stat3 intracellular localization. Finally, we performed mouse animal experiments to observe changes in the expression of IDO, p-Jak3, p-Stat3, and tryptophan/kynurenine after ATLIII administration.Results: ATLIII showed no cytotoxicity at a wide of dosage range. ATLIII reduced the phosphorylation level of Jak3 and Stat3 in response to IFN-γ stimulation, then remarkably reduced the nuclear translocation of p-Stat3 by IFN-γ. Lastly, ATLIII significantly downregulated the expression level of IDO at a wide dosage range. Molecular docking assay showed that the oxygen atom on the five-membered ring of ATLIII was capable of forming a hydrogen bond with Leu905-NH2 site of Jak3 protein. Further evidence showed that though IFN-γ had normal capacity to trigger Stat3 phosphorylation, nuclear translocation, and promoter luciferase activity, ATLIII failed to trigger efficacy on reducing these changes under forced Jak3–Leu905 mutant expression condition. Finally, we confirmed this view in in vivo experiments.Conclusion: ATLIII has shown significant efficacy to inhibit IFN-γ-triggered Jak3/Stat3 pathway-dependent IDO activation, and do so through a direct binding to Jak3 protein. This study elucidated a new mechanism for the anticancer effect of ATLIII, which may provide a feasible target for the clinical immunotherapy of malignant tumors.

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

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

Targeting STAT3 and STAT5 in Tumor-Associated Immune Cells to Improve Immunotherapy.

Oncogene-induced STAT3-activation is central to tumor progression by promoting cancer cell expression of pro-angiogenic and immunosuppressive factors. STAT3 is also activated in infiltrating immune cells including tumor-associated macrophages (TAM) amplifying immune suppression. Consequently, STAT3 is considered as a target for cancer therapy. However, its interplay with other STAT-family members or transcription factors such as NF-κB has to be considered in light of their concerted regulation of immune-related genes. Here, we discuss new attempts at re-educating immune suppressive tumor-associated macrophages towards a CD8 T cell supporting profile, with an emphasis on the role of STAT transcription factors on TAM functional programs. Recent clinical trials using JAK/STAT inhibitors highlighted the negative effects of these molecules on the maintenance and function of effector/memory T cells. Concerted regulation of STAT3 and STAT5 activation in CD8 T effector and memory cells has been shown to impact their tumor-specific responses including intra-tumor accumulation, long-term survival, cytotoxic activity and resistance toward tumor-derived immune suppression. Interestingly, as an escape mechanism, melanoma cells were reported to impede STAT5 nuclear translocation in both CD8 T cells and NK cells. Ours and others results will be discussed in the perspective of new developments in engineered T cell-based adoptive therapies to treat cancer patients.

NO-releasing STAT3 inhibitors suppress BRAF-mutant melanoma growth

Constitutive activation of STAT3 can play a vital role in the development of melanoma. STAT3-targeted therapeutics are reported to show efficacy in melanomas harboring the BRAFV600E mutant and also in vemurafenib-resistant melanomas. We designed and synthesized a series of substituted nitric oxide (NO)-releasing quinolone-1,2,4-triazole/oxime hybrids, hypothesizing that the introduction of a STAT3 binding scaffold would augment their cytotoxicity. All the hybrids tested showed a comparable level of in vitro NO production. 7b and 7c exhibited direct binding to the STAT3-SH domain with IC50 of ∼ 0.5 μM. Also, they abrogated STAT3 tyrosine phosphorylation in several cancer cell lines, including the A375 melanoma cell line that carries the BRAFV600E mutation. At the same time, they did not affect the phosphorylation of upstream kinases or other STAT isoforms. 7c inhibited STAT3 nuclear translocation in mouse embryonic fibroblast while 7b and 7c inhibited STAT3 DNA-binding activity in the A375 cell line. Their anti-proliferating activity is attributed to their ability to trigger the production of reactive oxygen species and induce G1 cell cycle arrest in the A375 cell line. Interestingly, 7b and 7c showed robust cell growth suppression and apoptosis induction in two pairs of BRAF inhibitor-naïve (-S) and resistant (-R) melanoma cell lines containing a BRAF V600E mutation. Surprisingly, MEL1617-R cells that are known to be more resistance to MEK inhibition by GSK1120212 than MEL1617-S cells exhibit a similar response to 7b and 7c.

STAT1-NFκB crosstalk triggered by interferon gamma regulates noradrenaline-induced pineal hormonal production.

Melatonin production by pineal glands is modulated by several immune signals. The nuclear translocation of nuclear factor kappa-B (NFκB) homodimers, lacking transactivation domains, once induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), inhibits the expression of Aanat gene and the synthesis of noradrenaline (NA)-induced melatonin. Interferon gamma (IFN-γ), on the other hand, increases melatonin synthesis. Furthermore, this cytokine activates the signal transducer as well as the activator of transcription 1 (STAT1) pathway, which was never evaluated as a melatonin synthesis modulator before. Reports demonstrated that IFN-γ might also activate NFκB. The present study evaluated the role of STAT1-NFκB crosstalk triggered by IFN-γ regarding the regulation of NA-induced pineal glands' hormonal production. Moreover, IFN-γ treatment increased NA-induced Aanat transcription, in addition to the synthesis of N-acetylserotonin (NAS) and melatonin. These effects were associated with STAT1 nuclear translocation, confirmed by the co-immunoprecipitation of STAT1 and Aanat promoter. Pharmacological STAT1 enhancement augmented NA-induced Aanat transcription as well as NAS and melatonin production. Additionally, IFN-γ induced the nuclear translocation of RelA-NFκB subunits. The blockade of this pathway prevented IFN-γ effects on the pineal function. The present data show that STAT1 and NFκB crosstalk controls melatonin production through a synergistic mechanism, disclosing a new integrative mechanism regarding pineal hormonal activity control.

Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells

To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 μg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 μg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. Calenduloside E below 20 μg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1β, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

DYRK1A inhibition suppresses STAT3/EGFR/Met signalling and sensitizes EGFR wild-type NSCLC cells to AZD9291.

DYRK1A is considered a potential cancer therapeutic target, but the role of DYRK1A in NSCLC oncogenesis and treatment requires further investigation. In our study, high DYRK1A expression was observed in tumour samples from patients with lung cancer compared with normal lung tissues, and the high levels of DYRK1A were related to a reduced survival time in patients with lung cancer. Meanwhile, the DYRK1A inhibitor harmine could suppress the proliferation of NSCLC cells compared to that of the control. As DYRK1A suppression might be effective in treating NSCLC, we next explored the possible specific molecular mechanisms that were involved. We showed that DYRK1A suppression by siRNA could suppress the levels of EGFR and Met in NSCLC cells. Furthermore, DYRK1A siRNA could inhibit the expression and nuclear translocation of STAT3. Meanwhile, harmine could also regulate the STAT3/EGFR/Met signalling pathway in human NSCLC cells. AZD9291 is effective to treat NSCLC patients with EGFR‐sensitivity mutation and T790 M resistance mutation, but the clinical efficacy in patients with wild‐type EGFR remains modest. We showed that DYRK1A repression could enhance the anti‐cancer effect of AZD9291 by inducing apoptosis and suppressing cell proliferation in EGFR wild‐type NSCLC cells. In addition, harmine could enhance the anti‐NSCLC activity of AZD9291 by modulating STAT3 pathway. Finally, harmine could enhance the anti‐cancer activity of AZD9291 in primary NSCLC cells. Collectively, targeting DYRK1A might be an attractive target for AZD9291 sensitization in EGFR wild‐type NSCLC patients.

Targeting the PI3K/STAT3 axis modulates age-related differences in macrophage phenotype in rats with myocardial infarction.

Ageing is associated with impaired repair mechanisms in cardiovascular diseases. Macrophages contribute to cardiac fibrosis after myocardial infarction (MI). The phosphatidyl‐inositol‐3‐kinase (PI3K) pathway has been shown to play a role in cardiac remodelling after MI. It remained unclear whether n‐butylidenephthalide, a major component of Angelica sinensis, can attenuate cardiac fibrosis by regulating the PI3K/signal transducer and activator of transcription 3 (STAT3)‐mediated macrophage phenotypes in ageing rats after MI. Twenty‐four hours after ligation of the left anterior descending artery, young (2‐month‐old) and ageing (18‐month‐old) male Wistar rats were treated with either vehicle or n‐butylidenephthalide for 4 weeks. There were similar infarct sizes in both age groups. Compared with young rats, ageing rats exhibited significant increased cardiac fibrosis after MI, which can be attenuated after administering n‐butylidenephthalide. MI was associated with decreased activities of PI3K and STAT3 in ageing rats compared with young rats. In both age groups, n‐butylidenephthalide effectively provided a significant increase of STAT3 phosphorylation, STAT3 activity, STAT3 nuclear translocation, myocardial IL‐10 levels and the percentage of M2c macrophage and a decrease of myofibroblast infiltration. The effects of n‐butylidenephthalide on increased IL‐10 levels were reversed by LY294002 or S3I‐201. Furthermore, LY294002 abolished the STAT3 phosphorylation, whereas PI3K activity was not affected following the inhibition of STAT3. In conclusions, the host environment is responsible for ageing‐related myofibroblast dysregulation in response to MI which can be improved by administering n‐butylidenephthalide via macrophage differentiation towards M2 phenotype by targeting the PI3K/STAT3 axis.

Signaling mechanisms inducing hyporesponsiveness of phagocytes during systemic inflammation

AbstractThe inflammatory responsiveness of phagocytes to exogenous and endogenous stimuli is tightly regulated. This regulation plays an important role in systemic inflammatory response syndromes (SIRSs). In SIRSs, phagocytes initially develop a hyperinflammatory response, followed by a secondary state of hyporesponsiveness, a so-called “tolerance.” This hyporesponsiveness can be induced by endotoxin stimulation of Toll-like receptor 4 (TLR4), resulting in an ameliorated response after subsequent restimulation. This modification of inflammatory response patterns has been described as innate immune memory. Interestingly, tolerance can also be triggered by endogenous TLR4 ligands, such as the alarmins myeloid-related protein 8 (MRP8, S100A8) and MRP14 (S100A9), under sterile conditions. However, signaling pathways that trigger hyporesponsiveness of phagocytes in clinically relevant diseases are only barely understood. Through our work, we have now identified 2 main signaling cascades that are activated during MRP-induced tolerance of phagocytes. We demonstrate that the phosphatidylinositol 3-kinase/AKT/GSK-3β pathway interferes with NF-κB–driven gene expression and that inhibition of GSK-3β mimics tolerance in vivo. Moreover, we identified interleukin-10–triggered activation of transcription factors STAT3 and BCL-3 as master regulators of MRP-induced tolerance. Accordingly, patients with dominant-negative STAT3 mutations show no tolerance development. In a clinically relevant condition of systemic sterile stress, cardiopulmonary bypass surgery, we confirmed the initial induction of MRP expression and the tolerance induction of monocytes associated with nuclear translocation of STAT3 and BCL-3 as relevant mechanisms. Our data indicate that the use of pharmacological JAK-STAT inhibitors may be promising targets for future therapeutic approaches to prevent complications associated with secondary hyporesponsiveness during SIRS.

Flubendazole demonstrates valid antitumor effects by inhibiting STAT3 and activating autophagy

BackgroundSignal transducer and activator of transcription 3 (STAT3) is an oncogene, which upregulates in approximately 70% of human cancers. Autophagy is an evolutionarily conserved process which maintains cellular homeostasis and eliminates damaged cellular components. Moreover, the STAT3 signaling pathway, which may be triggered by cancer cells, has been implicated in the autophagic process.MethodsIn this study, we found that the anthelmintic flubendazole exerts potent antitumor activity in three human colorectal cancer (CRC) cell lines and in the nude mouse model. The inhibition of cell proliferation in vitro by flubendazole was evaluated using a clonogenic assay and the MTT assay. Western blot analysis, flow cytometry analysis, siRNA growth experiment and cytoplasmic and nuclear protein extraction were used to investigate the mechanisms of inhibiting STAT3 signaling and activation of autophagy induced by flubendazole. Additionally, the expression of STAT3 and mTOR was analyzed in paired colorectal cancer and normal tissues collected from clinical patients.ResultsFlubendazole blocked the IL6-induced nuclear translocation of STAT3, which led to inhibition of the transcription of STAT3 target genes, such as MCL1, VEGF and BIRC5. In addition, flubendazole also reduced the expression of P-mTOR, P62, BCL2, and upregulated Beclin1 and LC3-I/II, which are major autophagy-related genes. These processes induced potent cell apoptosis in CRC cells. In addition, flubendazole displayed a synergistic effect with the chemotherapeutic agent 5-fluorouracil in the treatment of CRC.ConclusionsTaken together, these results indicate that flubendazole exerts antitumor activities by blocking STAT3 signaling and inevitably affects the autophagy pathway. Flubendazole maybe a novel anticancer drug and offers a distinctive therapeutic strategy in neoadjuvant chemotherapy of CRC.

Abstract 387: Pyrimethamine inhibits STAT3 transcriptional activity via dihydrofolate reductase

Abstract Cancer is often characterized by aberrant gene expression patterns that alter cellular function. Such alterations are commonly caused by the inappropriate activation of transcription factors. STAT3, in particular, is a key transcriptional regulator of many pro-tumorigenic processes, including inflammation, proliferation, and survival, and is persistently phosphorylated and activated in more than 70% of breast tumors. Accordingly, it is a promising therapeutic target for the treatment of breast cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we performed an unbiased, high-throughput screen of the Prestwick collection, a library of 1,120 small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity at concentrations known to be safely achieved in humans. Intriguingly, unlike STAT3 phosphorylation inhibitors and STAT3 SH2 domain inhibitors, pyrimethamine does not affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of STAT3 inhibition. To elucidate this mechanism and identify the direct molecular target of pyrimethamine, we performed thermal proteomic profiling. We detected 4,462 proteins, four of which demonstrated a statistically significant shift in thermal stability in the presence of pyrimethamine. We further characterized these hits by assessing the effect of siRNA-mediated knockdown on STAT3 transcriptional activity. Of the four hits, only knockdown of dihydrofolate reductase (DHFR) consistently reduced STAT3 transcriptional activity. Likewise, treatment with methotrexate, a known inhibitor of DHFR, also reduced STAT3 activity. The combination of pyrimethamine and methotrexate had no additive effect on STAT3 inhibition, suggesting that both compounds were acting through a common mechanism. Finally, we tested whether folinic acid, a reduced form of folate, could rescue pyrimethamine-dependent loss of STAT3 activity. Strikingly, folinic acid, but not folic acid, eliminated the effect of pyrimethamine on STAT3 transcriptional activity in a dose-dependent manner. Together, these results suggest that DHFR is the molecular target responsible for the STAT3-inhibitory effects of pyrimethamine. Moreover, these findings implicate folate metabolism in the regulation of STAT3 transcriptional activity, revealing a previously unknown node within the STAT3 pathway that may be important for the development and treatment of STAT3-driven breast cancer. Citation Format: Lisa N. Heppler, Sarah R. Walker, Sanaz Attarha, Brent D. Page, David A. Frank. Pyrimethamine inhibits STAT3 transcriptional activity via dihydrofolate reductase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 387.

Efficacy and safety of intraperitoneally administered resveratrol against rat orthotopic ovarian cancers.

BackgroundResveratrol (Res) inhibits ovarian cancer (OC) cell growth but its in vivo anti-OC effects are unclear due to the low bioavailability of systemically administered Res. Intraperitoneal administration may overcome this therapeutic dilemma because it makes Res directly affect the abdominal tumors. Ethanol and DMSO are common Res solvents, while their reliability and safety for long-term in vivo treatment remain unknown.MethodsA rat orthotopic OC model was established using the rat NUTU-19 OC cell line. Res dissolved in 10% ethanol or 0.2% DMSO was injected intraperitoneally (20 mg/kg/day) into tumor-free and tumor-bearing rats for 2 weeks. The tumors were collected for gross, morphological and molecular examinations, and blood and ascitic samples were obtained for a CA125 ELISA. Res concentration in ovarian tissues was determined by high performance liquid chromatography (HPLC).ResultsThe average tumor weight (0.187±0.065 g) of the Res-in-DMSO group was lower than that of untreated (0.426±0.091 g; P<0.01) and Res-in-ethanol (0.238±0.073 g; P<0.05) group. The average bloody ascitic volumes collected from untreated, Res-in-ethanol, and Res-in-DMSO groups were 5.65±0.27, 2.75±0.14, and 2.09±0.11 ml, respectively. Abundant TUNEL-positive cells, ARHI and PIAS3 upregulation, CA125 reduction, and decreased STAT3 nuclear translocation were found in the Res-in-ethanol and, especially, the Res-in-DMSO group. Widespread plaques of Res deposits were found on the abdominal serosa of the Res-in-ethanol group, but not in the Res-in-DMSO group. HPLC revealed a higher Res concentration in Res-in-DMSO-treated tumor tissues than in those treated by Res-in-ethanol (P<0.01). Fertility was maintained after long-term Res treatment.ConclusionIntraperitoneal administration of Res effectively inhibited rat orthotopic ovarian cancer growth without affecting normal tissues. The Res-in-DMSO group had the highest drug bioavailability and therefore stronger tumor-suppressive effects on ovarian cancer tissues.

Biliary tumorigenic effect on hypopharyngeal cells is significantly enhanced by pH reduction.

Biliary reflux has been considered a potential risk factor in upper aerodigestive tract malignancies. It is not yet clearly known how pH affects the bile‐induced activation of NF‐κB and its related oncogenic pathway previously linked to hypopharyngeal carcinogenesis. In this study, repetitive applications of conjugated primary bile acids with unconjugated secondary bile acid, deoxycholic acid (DCA), on human hypopharyngeal primary cells reveal that strongly acidic pH (4.0) optimally enhances the tumorigenic effect of bile, by inducing activation of NF‐κB, STAT3 nuclear translocation, bcl‐2 overexpression and significant overexpression of the oncogenic mRNA phenotype, compared to weakly acidic pH (5.5) or neutral pH (7.0). As the pH becomes less acidic the partially activated primary bile acids and activated DCA begin to exert their influence; however, with significantly less intensity compared to bile acids at strongly acidic pH. Our findings suggest that biliary tumorigenic effect is strongly pH dependent. Controlling pH during reflux events may be therapeutically effective in reducing the potential risk of bile‐induced hypopharyngeal cancer.

Luteolin-7-O-β-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes.

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-β-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

Retinoic Acid-Related Orphan Receptor C Regulates Proliferation, Glycolysis, and Chemoresistance via the PD-L1/ITGB6/STAT3 Signaling Axis in Bladder Cancer.

Retinoic acid-related orphan receptor C (RORC) is a member of the nuclear orphan receptor family and performs critical regulatory functions in cell proliferation, metastasis, and chemoresistance in various types of malignant tumors. Here we showed that expression of RORC is lost in tumor tissues of bladder cancer patients. Enhanced expression of RORC suppressed cell proliferation and glucose metabolism and increased cisplatin-induced apoptosis in vitro and in vivo. RORC bound the promoter region of programmed death ligand-1 (PD-L1) and negatively regulated PD-L1 expression. PD-L1 directly interacted with integrin β6 (ITGB6) and activated the ITGB6/FAK signaling pathway. RORC prevented the nuclear translocation of STAT3 via suppression of the PD-L1/ITGB6 signaling pathway, which further inhibited bladder cell proliferation and glucose metabolism and increased cisplatin-induced apoptosis. These findings reveal that RORC regulates bladder cancer cell proliferation, glucose metabolism, and chemoresistance by participating in the PD-L1/ITGB6/STAT3 signaling axis. Moreover, this new understanding of PD-L1 signaling may guide the selection of therapeutic targets to prevent tumor recurrence. SIGNIFICANCE: These findings suggest that RORC-mediated regulation of a PD-L1/ITGB6/FAK/STAT3 signaling axis in bladder cancer provides several potential therapeutic targets to prevent tumor progression.

In vitro inhibitory effects of cirsiliol on IL-6-induced STAT3 activation through anti-inflammatory activity.

Many studies have identified and described various medicinal effects of cirsiliol. Here, we investigated the signaling pathway involved in the anti-inflammatory effects of cirsiliol on IL-6-induced activity. Cirsiliol showed no cytotoxicity and inhibited pSTAT3-induced luciferase activity. At the molecular level, cirsiliol suppressed the expression of IL-6-induced inflammatory marker genes such as CRP, IL-1β, ICAM-1 and SOCS3, IL-6-induced activation of Jak2, gp130, STAT3 and ERK and nuclear translocation of STAT3, as measured by PCR, immunofluorescence staining and western blot analysis. However, the interaction between IL-6 and its receptor was not affected by cirsiliol treatment. These results indicate that cirsiliol attenuates IL-6-induced cellular signaling by regulating Jak2 phosphorylation. Therefore, cirsiliol could be a therapeutic agent for IL-6-related inflammatory diseases.