Axis Signaling Pathways Research Articles

Abstract 1791: LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 model

Abstract Objective: Blocking PD-1 with monoclonal antibodies is a highly promising onco-immune therapy and has elicited durable antitumor responses and long-term remissions with a broad spectrum of cancers, However, the response to anti-PD-1 therapy is restricted to only a subset of patients with around 25% response rate in cancer patients. TGF-β, a growth factor, plays a pivotal role in tumor development and progression, by inducing tumor immune evasion, activating cancer associated fibroblasts with induction of migration and invasiveness of tumor cells, and promoting angiogenesis in tumor microenvironment. Therefore, a tetravalent bispecific fusion protein (LBL-015), targeting both PD-1/PD-L1 axis and TGF-β signaling pathway was developed to achieve maximal anti-tumor immunity, while blocking the factors implicated in the tumor microenvironment and overcoming the resistance of tumor cells to anti-PD-1 therapy. Methods: The bispecific fusion protein was designed as 2:2 antibody-cytokine fusion format, using our unique Mab fusion platform. The binding affinity of LBL-015 to human PD-1 and TGF-β1 was determined by Fortebio. The activity of LBL-015 was determined using several in vitro assays, including PD-1/NFAT, TGFβ1/SBE-Luc reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-015 was investigated in the human PD-1 knock-in mouse model (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-015 was measured as 5.24 nM to human PD-1 and 0.14 nM to human TGF-β1, respectively. In vitro LBL-015 could block PD-1-PD-L1 interaction, recovering NFAT reporter gene signal, with EC50 of 0.1565 nM, which was much more potent than M7824 (EC50 of 0.5412 nM). In TGF-β1/SBE-Luc reporter gene assay, LBL-015 inhibited SMAD signaling induced by TGF-β1, with IC50 of 0.00576 nM, which was comparable to M7824. In pre-activated human PBMCs, the release of IFN-γ by PBMCs was completely suppressed with TGF-β1 addition. LBL-015 could relieve the suppression of IFN-γ release induced by PD-1 or TGF-β1, in a dose dependent manner. Surprisingly, M7824 didn't show any effect in this assay system. In a model of huPD-1 knock-in mice (Shanghai Model Organisms) implanted with MC38-OVA cells. LBL-015 was shown significant tumor growth inhibition (TGI) of 84.36% and 55.27% at the dose of 20 mg/kg and 10 mg/kg respectively (p<0.001 compared with vehicle treated mice), indicating its potent anti-tumor activity of this bi-functional fusion protein. Conclusions: LBL-015, an anti-PD-1 and TGF-β bispecific fusion protein was shown a great synergetic anti-tumor efficacy in vitro to antagonize both tumor immune evasion and aberrant microenvironment induced by PD-1 and TGF-β1, also in a mouse tumor model, LBL-015 strongly inhibited tumor growth in a dose dependent manner, hence a promising bispecific fusion protein for further clinical development. Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1791.

Inhibition of PI3K/mTOR/KATP channel blunts sodium thiosulphate preconditioning mediated cardioprotection against ischemia-reperfusion injury.

Recent studies have shown that pre and postconditioning the heart with sodium thiosulfate (STS) attenuate ischemia-reperfusion (IR) injury. However, the underlying mechanism involved in the cardioprotective signaling pathway is not fully explored. This study examined the existing link of STS mediated protection (as pre and post-conditioning agents) with PI3K, mTOR, and mPTP signaling pathways using its respective inhibitors. STS was administered to the isolated perfused rat heart through Kreb's Heinselit buffer before ischemia (precondition: SIPC) and reperfusion (postcondition: SPOC) in the presence and absence of the PI3K, mTOR, and mPTP signaling pathway inhibitors (wortmannin, rapamycin, and glibenclamide respectively). SIPC failed to improve the IR injury-induced altered cardiac hemodynamics, increased infarct size, and the release of cardiac injury markers in the presence of these inhibitors. On the other hand, the SPOC protocol effectively rendered the cardioprotection even in the PI3K/mTOR/KATP inhibitors presence. Interestingly, the SIPC's identified mode of action viz reduction in oxidative stress and the preservation of mitochondrial function were lost in the inhibitors' presence. Based on the above results, we conclude that the underlying mechanism of SIPC mediated cardioprotection works via the PI3K/mTOR/KATP signaling pathway axis activation.

MHTP, a synthetic alkaloid, attenuates combined allergic rhinitis and asthma syndrome through downregulation of the p38/ERK1/2 MAPK signaling pathway in mice

The combined allergic rhinitis and asthma syndrome (CARAS) is a chronic airway inflammation of allergic individuals, with a type 2 immune response. Pharmacotherapy is based on drugs with relevant side effects. Thus, the goal of this study was to evaluate the synthetic alkaloid, MHTP in the experimental model of CARAS. Therefore, BALB/c mice were ovalbumin (OVA) -sensitized and -challenged and treated with MHTP by intranasal or oral routes. Treated animals showed a decrease (p < 0.05) of sneezing, nasal rubbings, and histamine nasal hyperactivity. Besides, MHTP presented binding energy and favorable interaction for adequate anchoring in the histamine H1 receptor. MHTP treatment inhibited the eosinophil migration into the nasal (NALF) and the bronchoalveolar (BALF) fluids. Histological analysis showed that the alkaloid decreased the inflammatory cells in the subepithelial and perivascular regions of nasal tissue and in the peribronchiolar and perivascular regions of lung tissue. The MHTP treatment also reduced the pulmonary hyperactivity by decreasing the smooth muscle layer hypertrophy and the collagen fiber deposition in the extracellular matrix. The immunomodulatory effect of the alkaloid was due to the decrease of cytokines like IL-5 and IL-17A (type 2 and 3), TSLP (epithelial), and the immunoregulatory cytokine, TGF-β. These MHTP effects on granulocytes were dependent on the p38/ERK1/2 MAP kinase signaling pathway axis. Indeed, the synthetic alkaloid reduced the frequency of activation of both kinases independent of the NF-κB (p65) pathway indicating that the molecule shut down the intracellular transduction signals underlie the cytokine gene transcription.

LncRNA DLEU2 is activated by STAT1 and induces gastric cancer development via targeting miR-23b-3p/NOTCH2 axis and Notch signaling pathway

IntroductionGastric cancer (GC) has severely affected the health of patients and caused high mortality around the world. Long non-coding RNAs (lncRNAs) have been validated to play significant roles in biological process of multiple cancers. MethodsQuantitative real-time PCR (RT-qPCR) and western blot analysis were conducted to evaluate the expression levels and protein levels of related genes in GC cells. Functional assays were implemented to explore the effect of deleted in lymphocytic leukemia 2 (DLEU2). The upstream and downstream mechanisms of DLEU2 were verified by mechanism investigations. ResultsThe expression of long non-coding RNA (lncRNA) DLEU2 was observably high in GC cells and tissues. DLEU2 silence depressed the capacities of proliferation, migration and invasion but promoted apoptosis in GC cells. Moreover, DLEU2 was activated by signal transducer and activator of transcription 1 (STAT1) and sequestered microRNA-23b-3p (miR-23b-3p) to modulate the expression of notch receptor 2 (NOTCH2), thereby stimulating Notch signaling pathway. More importantly, DLEU2 contributed to GC progression via targeting miR-23b-3p/NOTCH2 axis. ConclusionsIn summary, our research identified the STAT1/DLEU2/miR-23b-3p/NOTCH2/Notch axis in GC development, indicating that DLEU2 might function as a novel biomarker in GC.

Tumor promoting effects of exosomal microRNA-210 derived from lung cancer cells on lung cancer through the RUNX3/PI3K/AKT signaling pathway axis.

Exosomes are involved in a range of processes in lung cancer such as cell proliferation, metastasis, and angiogenesis. Tumor-derived exosomes participate in the formation and progression of lung cancer by delivering functional biomolecules, including microRNAs (miRNA). The purpose of the present study was to determine the role of lung cancer cell-derived exosomal miR-210 in the proliferation and invasion of lung cancer cells and its underlying mechanism. Initially, exosomes were isolated from A549 cells and characterized by transmission electron microscopy and assessment of exosomal marker expression. RT-qPCR determined that miR-210 expression was elevated in exosomes as well as lung cancer cells. As reflected by dual-luciferase reporter assay, miR-210 negatively regulated RUNX3 expression. Following loss- and gain- function assay, it was found that miR-210 inhibition suppressed biological properties of A549 and H460 cells, which could be reversed by the silencing of RUNX3. miR-210 elevation induced the p-PI3K/PI3K and p-AKT/AKT levels, suggesting the activation of PI3K/AKT signaling pathway. Collectively, exosomal miR-210 targeted and negatively regulated RUNX3 expression to promote malignant properties of lung cancer cells by potentiating PI3K/AKT signaling pathway.

Sulforaphane induces colorectal cancer cell proliferation through Nrf2 activation in a p53-dependent manner

Sulforaphane is a well-known phytochemical that stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant cellular response. In this study, we found that sulforaphane promoted cell proliferation in HCT116 human colon cancer cells expressing a normal p53 gene in a dose-dependent but biphasic manner. Since p53 has been reported to contribute to cell survival by regulating various metabolic pathways to adapt to mild stress, we further examined cellular responses in both p53-wild-type (WT) and p53-knockout (KO) HCT116 cells exposed to sulforaphane in vitro and in vivo. Results demonstrated that sulforaphane treatment activated Nrf2-mediated antioxidant enzymes in both p53-WT and p53-KO cells, decreased apoptotic protein expression in WT cells but increased in KO cells in a dose-dependent manner, and increased the expression of a mitochondrial biogenesis marker PGC1α in WT cells but decreased in KO cells. Moreover, a low dose of sulforaphane promoted tumor growth, upregulated the Nrf2 signaling pathway, and decreased apoptotic cell death in p53-WT HCT116 xenografts compared to that in p53-KO HCT116 xenografts in BALB/c nude mice. These findings suggest that sulforaphane can influence colon cancer cell proliferation and mitochondrial function through a crosstalk between the Nrf2 signaling pathway and p53 axis.

The Development of Maillard Reaction, and Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling Inhibitors as Novel Therapeutic Strategies for Patients with AGE-Related Diseases.

Advanced glycation end products (AGEs) are generated by nonenzymatic modifications of macromolecules (proteins, lipids, and nucleic acids) by saccharides (glucose, fructose, and pentose) via Maillard reaction. The formed AGE molecules can be catabolized and cleared by glyoxalase I and II in renal proximal tubular cells. AGE-related diseases include physiological aging, neurodegenerative/neuroinflammatory diseases, diabetes mellitus (DM) and its complications, autoimmune/rheumatic inflammatory diseases, bone-degenerative diseases, and chronic renal diseases. AGEs, by binding to receptors for AGE (RAGEs), alter innate and adaptive immune responses to induce inflammation and immunosuppression via the generation of proinflammatory cytokines, reactive oxygen species (ROS), and reactive nitrogen intermediates (RNI). These pathological molecules cause vascular endothelial/smooth muscular/connective tissue-cell and renal mesangial/endothelial/podocytic-cell damage in AGE-related diseases. In the present review, we first focus on the cellular and molecular bases of AGE–RAGE axis signaling pathways in AGE-related diseases. Then, we discuss in detail the modes of action of newly discovered novel biomolecules and phytochemical compounds, such as Maillard reaction and AGE–RAGE signaling inhibitors. These molecules are expected to become the new therapeutic strategies for patients with AGE-related diseases in addition to the traditional hypoglycemic and anti-hypertensive agents. We particularly emphasize the importance of “metabolic memory”, the “French paradox”, and the pharmacokinetics and therapeutic dosing of the effective natural compounds associated with pharmacogenetics in the treatment of AGE-related diseases. Lastly, we propose prospective investigations for solving the enigmas in AGE-mediated pathological effects.

Exposure to dibutyl phthalate impairs lipid metabolism and causes inflammation via disturbing microbiota-related gut-liver axis.

Dibutyl phthalate (DBP), a kind of typical environmental pollutant, is widely used as plasticizers, and its neurotoxicity and developmental toxicity have been found in recent years. However, whether oral DBP exposure will affect the homeostasis of gut microbiota and its adverse response in liver of mammalians remain unclear. In the present study, 10-week experimental cycles of vehicle or DBP (0.1 and 1 mg/kg) were given to 6-week-old C57BL/6J mice by oral gavage. Our results revealed that the body weight of mice was increased after exposure to both low and high doses of DBP. The serum levels of hepatic triglyceride and total cholesterol were significantly increased in response to both doses of DBP. In addition, some pivotal genes related to lipogenesis were also increased in liver at the mRNA level. Evaluation of gut microbiota by 16S rRNA sequencing technology showed that 0.1 mg/kg DBP exposure significantly affected gut microbiota at the phylum and genus levels. Moreover, DBP exposure decreased mucus secretion and caused inflammation in the gut, leading to the impairment of intestinal barrier function. Exposure to DBP inhibited the expression of peroxisome proliferator-activated receptor-γ and activated the expression of nuclear factor kappa B. In addition, DBP exposure increased the level of lipopolysaccharide in serum, and increased the expression of toll-like receptor 4 and the levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha, in the liver. These results indicated that exposure to DBP disturbed the homeostasis of gut microbiota, induced hepatic lipid metabolism disorder, and caused liver inflammation in mice via the related gut-liver axis signaling pathways.

Inhibitory Effects of the Lactobacillus rhamnosus GG Effector Protein HM0539 on Inflammatory Response Through the TLR4/MyD88/NF-кB Axis.

Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory condition with no effective treatment. Probiotics have gained wide attention because of their outstanding advantages in intestinal health issues. In previous studies, a novel soluble protein, HM0539, which is derived from Lactobacillus rhamnosus GG (LGG), showed significant protective effects against murine colitis, but no clear precise mechanism for this effect was provided. In this study, we hypothesized that the protective function of HM0539 might be derived from its modulation of the TLR4/Myd88/NF-κB axis signaling pathway, which is a critical pathway widely involved in the modulation of inflammatory responses. To test this hypothesis, the underlying anti-inflammatory effects and associated mechanisms of HM0539 were determined both in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in dextran sulfate sodium (DSS)-induced murine colitis. Our results showed that HM0539 inhibited the expression of cyclooxygenase-2 (COX-2) and the expression inducible nitric oxide synthase (iNOS) by down-regulating the activation of their respective promoter, and as a result this inhibited the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Meanwhile, we demonstrated that HM0539 could ultimately modulate the activation of distal NF-κB by reducing the activation of TLR4 and suppressing the transduction of MyD88. However, even though the overexpression of TLR4 or MyD88 obviously reversed the effect of HM0539 on LPS-induced inflammation, HM0539 still retained some anti-inflammatory activity. Consistent with the in vitro findings, we found that HM0539 inhibited to a great extent the production of inflammatory mediators associated with the suppression of the TLR4/Myd88/NF-κB axis activation in colon tissue. In conclusion, HM0539 was shown to be a promising anti-inflammatory agent, at least in part through its down-regulation of the TLR4-MyD88 axis as well as of the downstream MyD88-dependent activated NF-κB signaling, and hence might be considered as a potential therapeutic option for IBD.

MiR-130a/Ndrg2 Axis Inhibits the Proliferation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis.

Studies have found that N-myc downstream-regulated gene 2 (Ndrg2) is involved in the progression of rheumatoid arthritis (RA); however, the specific mechanism still remains unclear. Gene expression profiles in the tibial joints of the collagen-induced rheumatoid arthritis model were obtained using Gene Expression Omnibus database. Western blot and real-time PCR were respectively performed to determine the expression of Ndrg2 and gene messenger RNA. Cell viability was measured by Cell Counting Kit-8 (CCK-8) method, and cell cycle was detected by flow cytometry. Cell scratch assays were carried out to detect migration. The binding ability of miR-130a to Ndrg2-3'-UTR was predicted by TargetScan website and confirmed by dual luciferase assay. A collagen-induced arthritis rat model was constructed to observe the effects of miR-130a on arthritis index, hind limb swelling, volume of rat hind paw, and inflammation. Ndrg2 was found downregulated in RA tissues, and knockdown of Ndrg2 promoted fibroblast-like synoviocytes (FLS) proliferation and inflammation, while overexpressed Ndrg2 produced opposite results. Ndrg2 was predicted as a target gene for miR-130a, and miR-130a mimic promoted FLS proliferation, while miR-130a inhibitor suppressed FLS proliferation. Moreover, we found that miR-130a antagomir could significantly reduce the arthritis index, swelling degree, foot volume, and inflammatory factor levels; inhibit the expression of miR-130a; and promote the expression of Ndrg2. The miR-130a/Ndrg2 axis signaling pathway is involved in the progression of RA. Our findings provide a theoretical basis for the clinical treatment of RA.

4-Carvomenthenol ameliorates the murine combined allergic rhinitis and asthma syndrome by inhibiting IL-13 and mucus production via p38MAPK/NF-κB signaling pathway axis

The aim of this study was to analyze the 4-carvomenthenol (carvo) oral treatment on the experimental model of the combined allergic rhinitis and asthma syndrome (CARAS). BALB/c mice were OVA-sensitized on day zero and 7th (50 μg/mL OVA in 10 mg/mL Al (OH)3) and OVA-challenged (5 mg/mL, 20 μL/animal) for three weeks. In the last week, the animals were dally challenged with aerosol of OVA and the carvo treatment (12.5, 25 or 50 mg/kg) occurred one hour before each OVA-challenge. Data were analyzed and p < 0.05 was considered significant. Carvo (12.5–50 mg/kg) decreased significantly the eosinophil migration into the nasal (NALF) and bronchoalveolar (BALF) cavities as well as on the nasal and lung tissues of sick animals. The treatment also decreased mucus production on both tissue sections stained with PAS (periodic acid-Schiff satin). In addition, the histological analyzes demonstrated that sick mice presented hyperplasia and hypertrophy of the lung smooth muscle layer followed by increasing of extracellular matrix and carvo (50 mg/kg) inhibited these asthmatic parameters. We analyzed the allergic rhinitis signals as nasal frictions and sneezing and observed that carvo decreased these two signals as well as serum OVA-specific IgE titer, type 2 cytokine synthesis, mainly IL-13, with increasing of IL-10 production. Decreasing of IL-13 production corroborated with decreasing of mucus production and these effects were dependent on p38MAPK/NF-κB(p65) signaling pathway inhibition. Therefore, these data demonstrated that a monoterpene of essential oils presents anti-allergic property on an experimental model of CARAS suggesting a new drug prototype to treat this allergic syndrome.

LINC00675 activates androgen receptor axis signaling pathway to promote castration-resistant prostate cancer progression

The development of prostate cancer (PCa) from androgen-deprivation therapy (ADT) sensitive to castration resistant (CRPC) seriously impacts life quality and survival of PCa patients. Emerging evidence shows that long noncoding RNAs (lncRNAs) play vital roles in cancer initiation and progression. However, the inherited mechanisms of how lncRNAs participate in PCa progression and treatment resistance remain unclear. Here, we found that a long noncoding RNA LINC00675 was upregulated in androgen-insensitive PCa cell lines and CRPC patients, which promoted PCa progression both in vitro and in vivo. Knockdown of LINC00675 markedly suppressed tumor formation and attenuated enzalutamide resistance of PCa cells. Mechanistically, LINC00675 could directly modulate androgen receptor’s (AR) interaction with mouse double minute-2 (MDM2) and block AR’s ubiquitination by binding to it. Meanwhile, LINC00675 could bind to GATA2 mRNA and stabilize its expression level, in which GATA2 could act as a co-activator in the AR signaling pathway. Notably, we treated subcutaneous xenografts models with enzalutamide and antisense oligonucleotides (ASO) targeting LINC00675 in vivo and found that targeting LINC00675 would benefit androgen-deprivation-insensitive models. Our findings disclose that the LINC00675/MDM2/GATA2/AR signaling axis is a potential therapeutic target for CRPC patients.

Peptides from Euphausia superba Promote Longitudinal Bone Growth by Accelerating Growth Plate Chondrocyte Proliferation and Hypertrophy.

With the improvements in living standards, height is getting more attention. Malnutrition is one of the main causes of children's short stature; therefore, nutritional intervention in adolescence is the key to prevent short stature. The peptides from Antarctic Krill (AKPs), the ideal protein model, act in bone formation and anti-osteoporosis. However, the studies on promoting longitudinal bone growth by AKPs have not been reported. Three-week-old male ICR mice, to construct the adolescent mice model, randomly divided into three groups: normal group, casein group (casein, 300 mg/kg·BW), and AKPs group (AKPs, 300 mg/kg·BW). After 21 days of drug administration, the effects of AKPs on serum biochemical indexes and femur histomorphology of mice, and the mechanism of AKPs promoting longitudinal bone growth was discussed. AKPs significantly increased longitudinal bone growth and improved bone strength. In addition, AKPs remarkably promoted proliferation and hypertrophy of chondrocytes in the growth plate. The further mechanism revealed that AKPs increased serum Growth Hormone (GH) and Insulin-Like Growth Factors-1(IGF-1) contents, which activated the downstream GH/IGF-1 axis signaling pathways. Moreover, AKPs induced the secretion and expression of bone morphogenetic protein 2 (BMP- 2) and triggered the activation of BMP2-dependent Smads signaling. AKPs also activated Wnt/ β-catenin signaling, and synergistically activated the expression of Runt-related transcription factor 2 (Runx 2) and Osterix (OSX). AKPs promoted longitudinal bone growth by activating GH/IGF-1 axis, BMP-2/Smads and Wnt/β-catenin pathways, suggesting AKPs to be a potential nutrient fortifier for longitudinal bone growth.

Short- and long-term effects on reproductive parameters of female Wistar rats after exposure to rosuvastatin starting in pre-puberty

Statins are a class of drugs that act lowering lipid levels by inhibiting cholesterol biosynthesis. Additionally, statins can act by “pleiotropic effects”, related to the inhibition of synthesis of the other mevalonate pathway products. Rosuvastatin is a third-generation statin and has shown better results in reducing cholesterol concentrations when compared to other statins. Recent studies suggest that rosuvastatin may act as an endocrine disruptor that potentially damages the hormonal axis and, consequently reproductive development and function of male rats. However, the effects of rosuvastatin exposure on rat female reproductive parameters remain unknown. In this study female rats were exposed to rosuvastatin at the doses of 0 (control), 3, or 10 mg/Kg.bw−1/day from pre-puberty to adulthood. No alterations in the female reproductive parameters were observed at a dose of 3 mg/Kg.bw−1. However, females exposed to 10 mg/Kg.bw−1 exhibited shorter estrous cycles, altered copulatory behavior, decreased serum prolactin level, and alterations in the liver, pituitary and placental weights, parameters to some extent influenced by the reproductive hormonal axis signaling pathway. On the other hand, pubertal onset, reproductive hormone levels, fertility, and histological parameters of the ovary, uterus, and placenta were unaltered by exposure to both doses of this statin. Thus, rosuvastatin exposure, at the higher dose, altered the reproductive function of female rats, probably due to the pleiotropic effects of this statin. Additional studies on the effects of this statin on female reproductive function and development are encouraged to better characterize its mode of action.

Long non‑coding RNA NNT‑AS1 knockdown represses the progression of gastric cancer via modulating the miR‑142‑5p/SOX4/Wnt/β‑catenin signaling pathway.

Patients with advanced gastric cancer (GC) have a poor prognosis with a median overall survival of 10–12 months. Long non-coding RNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) and sex-determining region Y-related high mobility group box 4 (SOX4) have been reported to be associated with the progression of various types of cancer; however, the regulatory mechanism between NNT-AS1 and SOX4 in GC is not completely understood. Reverse transcription-quantitative PCR was used to detect the expression levels of NNT-AS1, microRNA (miR)-142-5p and SOX4. Western blotting was performed to assess the protein expression levels of SOX4, β-catenin, c-Myc, Bcl-2 and E-cadherin. The proliferation, apoptosis, migration and invasion of GC cells were determined using MTT, flow cytometry and Transwell assays. The relationship between miR-142-5p and NNT-AS1 or SOX4 was investigated using a dual-luciferase reporter assay. NNT-AS1 and SOX4 were upregulated, whereas miR-142-5p was downregulated in GC tissues and cells compared with normal tissues and cells. Both NNT-AS1 and SOX4 knockdown inhibited GC cell proliferation, migration and invasion, and enhanced GC cell apoptosis. Moreover, the results indicated that NNT-AS1 modulated SOX4 expression by sponging miR-142-5p. In addition, SOX4 overexpression reversed NNT-AS1 knockdown-mediated effects on GC cell proliferation, apoptosis, migration and invasion. NNT-AS1 knockdown blocked the Wnt/β-catenin signaling pathway via the miR-142-5p/SOX4 axis. Collectively, the present study indicated that NNT-AS1 knockdown decreased GC cell proliferation, migration and invasion, and induced GC cell apoptosis by regulating the miR-142-5p/SOX4/Wnt/β-catenin signaling pathway axis.

Blockade of endothelin receptor A enhances the therapeutic efficacy of gemcitabine in pancreatic cancer cells

Pancreatic adenocarcinoma is currently one of the leading causes of cancer-related death worldwide. The high rate of mortality in pancreatic cancer patients is due to the inability to detect early-stage disease and the disease being highly refractory to therapy. Gemcitabine has been the standard chemotherapy for advanced pancreatic cancer patients for the last two decades. However, gemcitabine resistance develops within a few weeks of treatment, and the associated mechanism remains poorly understood. Therefore, a novel therapeutic strategy is needed to overcome the limited clinical efficacy of gemcitabine in pancreatic adenocarcinoma.In this study, we demonstrated that ET-1/ETAR axis gene expression was upregulated in pancreatic cancer cells after treatment with gemcitabine. Additionally, ETAR expression was significantly higher in tumor tissues than in normal tissues, and patients with high ETAR expression had a notably worse overall survival rate than those with low ETAR expression. Furthermore, our results revealed that bosentan, an ETAR antagonist, enhanced the growth-inhibiting and proapoptotic effects of gemcitabine on pancreatic cancer cells.Thus, our findings indicate that blockade of the ET-1/ETAR axis signaling pathway promotes the antiproliferative effect of gemcitabine on pancreatic cancer. Therefore, combination of ETAR blockade and gemcitabine serves as an effective therapeutic approach to achieve clinical benefits in pancreatic adenocarcinoma patients.

Astragaloside IV protects human cardiomyocytes from hypoxia/reoxygenation injury by regulating miR-101a

Astragaloside IV (AS/IV) is one of the extracted components from the traditional Chinese medicine Astragalus which has been demonstrated to have potential capacity for anti-inflammation activity and for treating cardiovascular disease. Our purpose was to determine the function and underlying molecular mechanism of AS/IV in hypoxia/reoxygenation (H/R) injured in cardiomyocytes. Differentially expressed genes (DEGs) were screened using bioinformatic analysis, and the molecular targeting relationship was verified by the dual-luciferase report system. H/R injured cardiomyocytes were employed to explore the effect of AS/IV. QRT-PCR and Western blot analysis were applied to detect the expression of mRNA and proteins, respectively. Additionally, superoxide dismutase (SOD), lactic dehydrogenase (LDH) and MDA (malondialdehyde) levels were detected to determine the oxidative damage. Cell viability was assessed by CCK-8, and flow cytometry was used to evaluate cell apoptosis ratio. TGFBR1 and TLR2 were selected as DEGs. Additionally, AS/IV could enhance cell proliferation and upregulated miR-101a expression, which suppressed TGFBR1 and TLR2 expression in H/R injured cardiomyocytes. Moreover, the results of Western blot exhibited that the downstream genes (p-ERK and p-p38) in the MAPK signaling pathway were suppressed, which meant AS/IV could inhibit this pathway in H/R injured cardiomyocytes. Overall, this study demonstrated AS/IV could attenuate H/R injury in human cardiomyocytes via the miR-101a/TGFBR1/TLR2/MAPK signaling pathway axis, which means that it could serve as a possible alternate for H/R treatment.

MON-286 Peak Stimulated Growth Hormone Is Lower in Subjects with Nonalcoholic Fatty Liver Disease Than Controls of Similar Sex, Age and BMI

Background: Nonalcoholic fatty liver disease (NAFLD), fatty infiltration of the liver in the absence of alcohol use, is a prevalent and serious complication of obesity. Obesity is a state of relative growth hormone (GH) deficiency, and GH has been identified as a candidate disease-modifying target in NAFLD because of its lipolytic and anti-inflammatory properties. However, it is not known whether individuals with NAFLD phenotyped by proton magnetic resonance spectroscopy (1H-MRS), the gold standard imaging modality for assessment of intrahepatic lipid (IHL) content, have lower peak stimulated GH levels as compared to those of similar age, sex and BMI without NAFLD. Methods: We studied 99 generally healthy adults without diabetes or significant alcohol use, ages 19-67 y and BMI >25 kg/m2. All subjects underwent 1H-MRS for assessment of IHL content. Using a cutoff of >5.5%, 65 subjects had NAFLD and 34 did not (controls). GHRH-arginine stimulation testing was performed. GH was measured by immunoassay and IGF-1 by LC/MS/MS (Quest Diagnostics, CA, USA). Visceral and subcutaneous adipose tissue (VAT/SAT) were assessed by cross-sectional CT at L4. Results are reported as mean ±SD. Results: There was no difference between NAFLD vs controls in mean age (48±12 vs 45±14 y, p=0.30), BMI (33±4 vs 33±7 kg/m2, p=0.96), sex (43% vs 44% female, p=0.90) or premenopausal status (50% vs 60%, p=0.50). Mean IHL was 21.8±13.3% (range 5.5-57.8%) and 2.9±1.1% (range 1.0-4.9%) in the NAFLD and control groups, respectively (p<0.0001). NAFLD subjects had higher ALT, total cholesterol, triglycerides, VLDL, LDL and lower HDL than controls. Fasting glucose was statistically but not clinically significantly higher in NAFLD vs controls (90±9 vs 86±7 mg/dL, p=0.03), and mean HbA1c did not differ significantly. There was a trend towards a higher mean VAT in the NAFLD vs controls (157±70 vs 131±67 g, p=0.07) but no difference in SAT. Mean peak stimulated GH was significantly lower in NAFLD vs controls (9.0±6.3 vs 15.4±11.2 ng/mL, p=0.003) which remained significant after controlled for age, BMI, sex and VAT. In a stepwise model including peak stimulated GH, VAT, age, BMI and sex, peak stimulated GH predicted 8% of the variability in IHL (p=0.004); no other variables were significant predictors of IHL. Mean IGF-1 (149±53 vs 151±49 ng/mL, p=0.80) and IGF-1 Z-score (-0.03±0.61 vs -0.03±0.68, p=0.90) were not significantly different between the groups. Conclusion: Subjects with NAFLD have lower peak stimulated GH but similar IGF-1 levels compared to non-NAFLD controls of similar age, BMI and sex. Additionally, lower peak stimulated GH was predictive of higher IHL, independent of age, BMI, sex and VAT. This suggests that the relative GH deficiency of obesity may be an independent contributor to the development of NAFLD and that the GH axis and downstream signaling pathways may be a therapeutic target for this disease where few currently exist.

Vitamin D suppress the production of vascular endothelial growth factor in mast cell by inhibiting PI3K/Akt/p38 MAPK/HIF-1α pathway in chronic spontaneous urticaria

Mast cells play a significant role in urticaria pathogenesis. It's evidenced that vitamin D has positive impact in chronic spontaneous urticaria (CSU) recently, but underlying mechanisms remain unclear. In this study, isobaric tag for relative and absolute quantification-liquid chromatography-mass spectrometer/mass spectrometer was used to detect the expression of proteins in sera of CSU patients and healthy subjects. Thirty-one differentially expressed proteins were identified, in which vitamin D binding protein (VDBP) was higher in CSU patients than that in healthy subjects after verification. Our results indicated that sera of CSU patients induced the production of vascular endothelial growth factor (VEGF) in mast cells through PI3K/Akt/p38 MAPK/HIF-1α axis in an IgE-depended way, and 25(OH)D3 suppressed the expression of VEGF by inhibiting this signaling pathway axis in this process. Collectively, these results suggest VDBP to be a potential biomarker and propose a potential mechanism of benefit for vitamin D therapy in CSU.

Dexmedetomidine protects H9C2 against hypoxia/reoxygenation injury through miR-208b-3p/Med13/Wnt signaling pathway axis

Dexmedetomidine (Dex) has been reported to be cardioprotective. Differential expression of miR-208b-3p is associated with myocardial injury. But it is unknown that aberrant expression of miR-208b-3p is implicated in myocardial protection of Dex. Hypoxia/reoxygenation (HR) model was established in H9C2 cells. qRT-PCR was performed to detect expression levels of miR-208b-3p in H9C2 undergoing HR, Dex preconditioning, overexpression of miR-208b-3p or inhibition, and to assess expression of Med13 in H9C2 following knockdown of Med13 mRNA. CCK8 and, flow cytometry and Western blot were conducted respectively to examine viability, apoptosis rate and protein expressions of H9C2 subjected to a variety of treatments. Dex preconditioning reduced expression of miR-208b-3p and apoptosis of H9C2 cells caused by HR, while Dex preconditioning increased viability of H9C2. Dex preconditioning increased expression of Med13, which was reduced after knockdown of Med13 mRNA in H9C2. Overexpression of miR-208b-3p attenuated Dex exerted protective effects of myocardial cells, which was reversed by inhibition of miR-208b-3p. Increased expression of Med13 or/and decreased expression of miR-208b-3p decreased expression levels of Wnt/β-catenin signaling pathway-related proteins (Wnt3a, Wnt5a and β-catenin), while knockdown of Med13 mRNA or increased expression of miR-208b-3p increased the expression levels of those proteins. Dex protects H9C2 cells against HR injury through miR-208b-3p/Med13/Wnt/β-catenin signaling pathway axis.