Activation Of JNK Signaling Pathway Research Articles

CTRP9 induces macrophages polarization into M1 phenotype through activating JNK pathway and enhances VSMCs apoptosis in macrophages and VSMCs co-culture system

Inflammation plays a critical role in the development of atherosclerosis (AS), which has been identified as a major predisposing factor for stroke. Macrophages and VSMCs are associated with plaque formation and progression. Macrophages can dynamically change into two main functional phenotypes, namely M1 and M2, they can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. As a member of CTRPs family, CTRP9 has been reported play important protective roles in the cardiovascular system. However, whether CTRP9 can regulate macrophage activation status in inflammatory responses and have effect on VSMCs behaviors in co-culture system have not been fully investigated. In the present study, using peritoneal macrophages treated with CTRP9, we found that CTRP9 facilitated macrophages towards M1 phenotype, promoted TNF-α secretion and MMPs expression. CTRP9 showed synergistic effect with LPS in inducing M1 macrophages. In macrophages-VSMCs co-culture system, apoptosis and down-regulated proliferation of VSMCs were accelerated with CTRP9-treated macrophages. Then we attempted to explore the underlying molecular mechanisms of CTRP9 resulting in M1 activation. The c-Jun NH2-terminal kinases (JNK) are members of the mitogen activated protein kinases (MAPK) family, plays a central role in the cell stress response, with outcomes ranging from cell death to cell proliferation and survival. We found JNK expression was upregulated following CTRP9 stimulation, and inhibiting JNK phosphorylation level was associated with decreased expression of M1 markers and TNF-α concentration. Moreover, VSMCs apoptosis were ameliorated after inhibition of JNK. These results suggested that CTRP9 may promote macrophage towards M1 activation status through JNK signaling pathway activation.

C6orf120 gene deficiency may be vulnerable to carbon tetrachloride induced acute hepatic injury in rats.

The function of the C6orf120 gene, which encodes an N-glycosylated protein, remains unknown. The study was performed to characterize the utility of the C6orf120 gene in carbon tetrachloride-induced acute liver injury and to elucidate the potential underlying mechanisms by establishing a C6orf120 gene-knockout (C6orf120-/-) rat model. C6orf120-/- and wild-type (WT) rats were intraperitoneally administered with CCl4 (1 : 1 v/v in olive oil, 2 µl/g). Rats were sacrificed 24 h after CCl4 administration. Liver tissues were collected for H&E, IHC, qRT-PCR, and Western blot analysis. C6orf120 gene deficiency may be vulnerable to CCl4-induced acute liver injury in rats as indicated by the high levels of alanine aminotransferase (WT: 388.7 ±55.96 vs. C6orf120-/-: 915.9 ±118.8, p < 0.001) and greater degree of pathological damage. Quantitative reverse transcription polymerase chain reaction showed that the mRNA levels of inflammation-associated cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, in liver tissues were increased in C6orf120-/- rats compared with those in WT rats. Moreover, western blot showed that the protein expression of cytokines nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing 3 (NLRP3), caspase-1, IL-1β, nuclear factor-κB, c-Jun N-terminal kinases, and Bax were increased in C6orf120-/- rats compared with those in WT rats. C6orf120-/- rats were susceptible to CCl4-induced liver injury, which may be related to NLRP3 inflammasome and JNK signaling pathway activation.

Electroacupuncture Ameliorates Cognitive Impairment by Inhibiting the JNK Signaling Pathway in a Mouse Model of Alzheimer's Disease.

Electroacupuncture (EA) has become popular for its adjustable strength and frequency and easy quantification in the clinic and has demonstrated therapeutic potential for Alzheimer’s disease (AD). However, the mechanism remains unknown. Abnormally activated c-Jun N-terminal kinase (JNK) has been closely related to the pathological process of AD. The aim of this study was to investigate the effect of EA on cognitive impairment and the role of the JNK signaling pathway in AD model amyloid precursor protein (APP)/presenilin 1 (PS1) mice. The memory and learning ability of each group was assessed using the Morris Water Maze (MWM). Immunofluorescence, immunohistochemistry and Western blot were performed to measure the expression of APP, JNK, phosphorylated (P-)JNK, mitogen-activated protein kinase 4 (MKK4), MKK7, c-Jun and caspase-3 in hippocampal tissue samples in APP/PS1 mice after EA intervention. Obvious cognitive deficits were observed in the AD model APP/PS1 mice in the MWM test and were associated with JNK signaling pathway activation and APP upregulation. Four weeks of EA significantly ameliorated the cognitive impairments and inhibited JNK signaling pathway activation and APP upregulation. Taken together, the findings demonstrated that EA can reverse cognitive deficits and substantially lower the burden of APP in AD model APP/PS1 mice, at least partially through inhibiting the JNK signaling pathway and regulating apoptosis signals. Therefore, EA may offer an effective alternative therapeutic approach for AD.

Silencing of microRNA-517a induces oxidative stress injury in melanoma cells via inactivation of the JNK signaling pathway by upregulating CDKN1C

BackgroundMelanoma is notoriously resistant to current treatments, and less than 25% of metastatic melanoma cases respond to existing therapies. Growing evidence has shown that microRNAs (miRNAs) play a vital role in the prognosis of melanoma. MiR-517a has been implicated in many types of cancer; however, its expressional features and potential biological functions in melanoma remain unclear. The present study aimed to investigate the possible effects of miR-517a on oxidative stress (OS) in melanoma cells.MethodsmiR-517a expression in melanoma was determined using RT-qPCR. After treatment with different concentrations of H2O2, cell viability was determined in order to identify the most appropriate H2O2 concentration. Through loss and gain of function experiments, the interactions between miR-517a, the cyclin dependent kinase inhibitor 1C (CDKN1C) and the c-Jun NH2-terminal kinase (JNK) signaling pathway, as well as their roles in OS of melanoma cells were identified. Moreover, the expression of Cleaved Caspase-3, extent of ERK1/2 phosphorylation, Bax/Bcl-2 ratio, levels of T-AOC, ROS and MDA, and SOD activity were also tested. Finally, melanoma cell viability and apoptosis were detected.ResultsMiR-517a was upregulated, while CDKN1C was downregulated in melanoma tissues and cells. MiR-517a targets CDKN1C and consequently reduced its expression. Inhibition of miR-517a was shown to increase Cleaved Caspase-3 expression, Bax/Bcl-2 ratio, levels of ROS and MDA, as well as cell apoptosis but decrease extent of ERK1/2 phosphorylation, T-AOC levels, SOD activity, along with cell proliferation and mitochondrial membrane potential.ConclusionsOverall, silencing miR-517a results in upregulated CDKN1C expression, and inhibited JNK signaling pathway activation, consequently promoting OS in melanoma cells.

TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.

Piperlongumine potentiates the antitumor efficacy of oxaliplatin through ROS induction in gastric cancer cells.

Oxaliplatin is one of the most commonly used chemotherapeutic agents in the treatment of various cancers, including gastric cancer. It has, however, a narrow therapeutic index due to its toxicity and the occurrence of drug resistance. Therefore, there is a pressing need to develop novel therapies to potentiate the efficacy and reduce the toxicity of oxaliplatin. Piperlongumine (PL), an alkaloid isolated from Piper longum L., has recently been identified as a potent agent against cancer cells in vitro and in vivo. In the present study, we investigated whether PL can potentiate the antitumor effect of oxaliplatin in gastric cancer cells. Cellular apoptosis and ROS levels were analyzed by flow cytometry. Thioredoxin reductase 1 (TrxR1) activity in gastric cancer cells or tumor tissues was determined using an endpoint insulin reduction assay. Western blotting was used to analyze the expression levels of the indicated proteins. Nude mice xenograft models were used to test the effects of PL and oxaliplatin combinations on gastric cancer cell growth in vivo. We found that PL significantly enhanced oxaliplatin-induced growth inhibition in both gastric and colon cancer cells. Moreover, we found that PL potentiated the antitumor effect of oxaliplatin by inhibiting TrxR1 activity. PL combined with oxaliplatin markedly suppressed the activity of TrxR1, resulting in the accumulation of ROS and, thereby, DNA damage induction and p38 and JNK signaling pathway activation. Pretreatment with antioxidant N-acetyl-L-cysteine (NAC) significantly abrogated the combined treatment-induced ROS generation, DNA damage and apoptosis. Importantly, we found that activation of the p38 and JNK signaling pathways prompted by PL and oxaliplatin was also reversed by NAC pretreatment. In vivo, we found that PL combined with oxaliplatin significantly suppressed tumor growth in a gastric cancer xenograft model, and effectively reduced the activity of TrxR1 in tumor tissues. Remarkably, we found that PL attenuated body weight loss evoked by oxaliplatin treatment. Our data support a synergistic effect of PL and oxaliplatin and suggest that application of its combination may be more effective for the treatment of gastric cancer than oxaliplatin alone.

TLR4 promotes liver inflammation by activating the JNK pathway.

Drug-induced liver injury has become a serious public health problem that cannot be ignored. Although the mechanism of acetaminophen (APAP)-induced liver injury has been investigated for several decades, there are still many deficiencies. However, only a deeper study of its mechanism can provide more effective measures of prevention and treatment for APAP-induced liver injury. The aim of this study was to investigate whether toll-like receptor 4 (TLR4) participates in and regulates APAP-induced liver injury, which may provide a new direction for the prevention and treatment of clinical drug-induced hepatitis. WT mice were treated with APAP (300 mg/kg) or equivalent PBS. The livers of mice were taken at 1 h, 3 h, 6 h and 12 h after treatment. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect TLR4 mRNA expression level in the liver. After TLR4 involving in APAP-induced liver injury was confirmed, we investigated the relationship between TLR4 expression and hepatic inflammation. WT and TLR4-/- mice received APAP (3000 mg/kg) intraperitoneal injection after 16 h of fasting; serum was collected after 8 h and 24 h, and serum alanine aminotransferase (ALT) and reduced glutathione (GSH) activity were measured. Rat liver tissue was observed for histological changes by hematoxylin and eosin (H&E) staining. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) assay were performed to analyze proinflammatory cytokines expression (such as TNF-α, IL-1β, MCP-1, IL-6). After isolating mononuclear cells (MNCs) in the liver of mice, flow cytometry was used to detect cell activation level and infiltration of macrophages and neutrophils. Western blotting was used to analyze the activation of phosphorylated JNK and p38 signaling pathways in livers of WT and TLR4-/- mice. In addition, after stimulated with APAP, the silence of TLR4 in RAW264.7 cells could activate phosphorylated JNK and p38 signaling pathways. After APAP stimulation, WT mice exhibited more severe liver injury than TLR4-/- mice, with higher ALT levels, lower GSH levels, and more necrotic or apoptotic cells. TLR4-/- mice have lower levels of inflammatory cytokines including MCP-1 and IL-6; at the same time, the number of infiltrating macrophages and neutrophils in liver tissue of TLR4-/- mice was significantly lower than that of WT mice. The activation of JNK signaling pathway was strikingly enhanced in WT mice treated with APAP, but no significant difference was observed in the activation of JNK phosphorylation in TLR4-/- mice after the same dose of APAP stimulation. Similarly, in RAW264.7 cells, the activation of phosphorylated JNK and p38 was remarkably inhibited by TLR4-siRNA, but was activated in the control group, which was consistent in vivo. APAP-treated TLR4-/- mice showed milder liver injury compared to WT mice. It was confirmed that TLR4 could activate the JNK signaling pathway to induce the secretion of inflammatory factors and the infiltration of macrophages to promote APAP-induced liver injury. This finding might provide a new prevention and treatment idea for clinical drug-induced hepatitis.

Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology, tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer’s disease

Isorhynchophylline (IRN) has been demonstrated to have distinct anti-Alzheimer’s disease (AD) activity in several animal models of AD. In this study, we aimed at evaluating the preventive effect of IRN on the cognitive deficits and amyloid pathology in TgCRND8 mice. Male TgCRND8 mice were administered with IRN (20 or 40 mg/kg) by oral gavage daily for 4 months, followed by assessing the spatial learning and memory functions with the Radial Arm Maze (RAM) test. Brain tissues were determined immunohistochemically or biochemically for changes in amyloid pathology, tau hyperphosphorylation and neuroinflammation. Our results revealed that IRN (40 mg/kg) significantly ameliorated cognitive deficits in TgCRND8 mice. In addition, IRN (40 mg/kg) markedly reduced the levels of Aβ40, Aβ42 and tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-1β, and modulated the amyloid precursor protein (APP) processing and phosphorylation by altering the protein expressions of β-site APP cleaving enzyme-1 (BACE-1), phosphorylated APP (Thr668), presenilin-1 (PS-1) and anterior pharynx-defective-1 (APH-1), as well as insulin degrading enzyme (IDE), a major Aβ-degrading enzyme. IRN was also found to inhibit the phosphorylation of tau at the sites of Thr205 and Ser396. Immunofluorescence showed that IRN reduced the Aβ deposition, and suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the cerebral cortex and hippocampus of TgCRND8 mice. Furthermore, IRN was able to attenuate the ratios of p-c-Jun/c-Jun and p-JNK/JNK in the brains of TgCRND8 mice. IRN also showed marked inhibitory effect on JNK signaling pathway in the Aβ-treated rat primary hippocampus neurons. We conclude that IRN improves cognitive impairment in TgCRND8 transgenic mice via reducing Aβ generation and deposition, tau hyperphosphorylation and neuroinflammation through inhibiting the activation of JNK signaling pathway, and has good potential for further development into pharmacological treatment for AD.

GKN2 promotes oxidative stress-induced gastric cancer cell apoptosis via the Hsc70 pathway

BackgroundThe GKN2 is a secretory protein, whose levels decrease in gastric cancer. The present study aimed to investigate the expression, function and mechanism of action of GKN2 in gastric cancer.MethodsMolecular biology assays were performed to elucidate the function and underlying mechanisms of GKN2 in gastric cancer under stress-induced condition in vivo and in vitro. Clinical specimens were used to assess the correlation of GKN2 and prognosis.ResultsWe found that overexpression of GKN2 significantly enhanced apoptosis and growth arrest in vitro. GKN2 expression increased in gastric cancer cells exposed to hydrogen peroxide and promoted reactive oxygen species-induced mitochondrial dysfunction and resulted in increased cell apoptosis via inhibition of NF-κB signaling pathway and activation of JNK signaling pathway through the direct interaction of GKN2 with Hsc70. Trefoil factor 1 might contribute to the tumor suppressing effects of GKN2. MiR-216a downregulated GKN2 expression. GKN2 also inhibited xenograft tumor growth and was an independent and significant prognostic factor for patients with gastric cancer treated with oxaliplatin.ConclusionsTaken together, our data indicate that GKN2 may increase sensitivity of GC cells to the drugs which increase ROS levels in tumors. Inhibition of the interaction between GKN2 and Hsc70 could attenuate the effects induced by GKN2. GKN2 overexpression could be used to determine the subgroup of patients to obtain the more favorable outcome of oxaliplatin treatment and may be used as biomarker of the prognosis of this cancer.

Curcuminoid WZ26, a TrxR1 inhibitor, effectively inhibits colon cancer cell growth and enhances cisplatin-induced cell death through the induction of ROS

Colon cancer is one of the leading causes of cancer-related deaths. Chemotherapy has improved survival in patients with colon cancer, but has a narrow therapeutic window due to its toxicity. Therefore, novel therapies for colon cancer are urgently needed. We previously developed a curcumin analog WZ26 as an anti-cancer agent in pre-clinical evaluation. In the present study, we further explored the mechanism and target of WZ26 in colon cancer cells. Our results show that WZ26 targets thioredoxin reductase 1 (TrxR1) and increases cellular reactive oxygen species (ROS) levels, which results in the activation of JNK signaling pathway in human colon cancer cells. Furthermore, we found that WZ26 significantly enhances cisplatin-induced cell growth inhibition in colon cancer cells. WZ26 combined with cisplatin markedly increases the accumulation of ROS, and thereby induces DNA damage and activation of JNK signaling pathway. Pretreatment with antioxidant N-acetyl-l-cysteine (NAC) significantly abrogates the combined treatment-induced ROS generation, DNA damage and cell death. In addition, the activation of JNK signaling pathway prompted by WZ26 and cisplatin was also reversed by NAC pretreatment. In vivo, WZ26 combined with cisplatin significantly inhibits tumor growth in a colon cancer xenograft model. Remarkably, WZ26 attenuates the body weight loss evoked by cisplatin treatment. This study discloses a previously unrecognized mechanism underlying the biological activity of WZ26, and reveals that WZ26 and cisplatin combinational treatment might potentially become a more effective regimen in colon cancer therapy.

Notoginsenoside R1 alleviates lipopolysaccharide-triggered PC-12 inflammatory damage via elevating microRNA-132

Background Delayed inflammatory response is closely associated with the severity of Spinal cord injury (SCI). Herein, the function and molecular mechanism of notoginsenoside R1 (NGR1) in the in vitro model of SCI inflammation injury were explored. Methods PC-12 neuronal cells were subjected with LPS to construct a cell-based model of SCI inflammatory injury. NGR1 was applied in this cell model. miR-132 was silenced by transfection with miR-132 inhibitor. Cell viability and apoptosis were assessed, respectively. Then, the expression changes of pro-inflammatory cytokines and JNK pathway were examined. Results In this model, LPS was neurotoxic, with inhibiting PC-12 cell viability, inducing apoptosis, and enhancing concentrations of IL-6, IL-8 and TNF-α. However, NGR1 weakened the influence of LPS on PC-12 cells via elevating cell viability, decreasing apoptosis, decreasing pro-inflammatory cytokines expression, and suppressing activation of JNK signalling pathway. miR-132 was up-regulated by NGR1 treatment. Silence of miR-132 eliminated the influence of NGR1 on LPS-stimulated PC-12 cells. Conclusion NGR1 relieved PC-12 cells from LPS-triggered inflammatory damage via elevating miR-132 and hereafter suppressing JNK pathway.

\u03b2-Thujaplicin induces autophagic cell death, apoptosis, and cell cycle arrest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a common liver malignancy worldwide, has high morbidity and mortality. β-Thujaplicin, a tropolone derivative, has been used in some health-care products and clinical adjuvant drugs, but its use for HCC is unknown. In this study, we found that β-Thujaplicin inhibits the growth of HCC cells, but not normal liver cells, with nanomolar potency. Mechanistically, we found that β-Thujaplicin could induce autophagy, as judged by western blot, confocal microscopy, and transmission electron microscopy. Further using β-Thujaplicin combined with an autophagy blocker or agonist treatment HepG2 cells, we found that β-Thujaplicin induced autophagic cell death (ACD) mediated by ROS caused inhibition of the Akt-mTOR signaling pathway. Moreover, β-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and Bax/Bcl-2 ratio, which indicated that β-Thujaplicin induced apoptosis mediated by the mitochondrial-dependent pathway. We also found that increased expression of p21 and decreased expression of CDK7, Cyclin D1, and Cyclin A2 participating in β-Thujaplicin caused the S-phase arrest. It seems that β-Thujaplicin exerts these functions by ROS-mediated p38/ERK MAPK but not by JNK signaling pathway activation. Consistent with in vitro findings, our in vivo study verified that β-Thujaplicin treatment significantly reduced HepG2 tumor xenograft growth. Taken together these findings suggest that β-Thujaplicin have an ability of anti-HCC cells and may conducively promote the development of novel anti-cancer agents.

Retracted: Knockdown of long noncoding RNA GAS5 attenuates H2 O2 -induced damage in retinal ganglion cells through upregulating miR-124: Potential role in traumatic brain injury.

Optic nerve injury is one of the most common and serious complications in traumatic brain injury (TBI). Alleviating degree of optic nerve injury is important to cure of TBI. This study explored the role of long noncoding RNA (lncRNA) GAS5 in mice retinal ganglion cells (RGCs) suffered to H2 O 2 injury. Primary RGC (PRGCs) were treated with H2 O 2 to simulate an in vitro oxidation stress model. LncRNA GAS5 and miR-124 expressions were knocked down by cell transfection with short-hairpin RNA against GAS5 and miR-124 inhibitor, and the transfection efficiency was determined by qRT-PCR. Cell viability, apoptotic cell rate, and production of reactive oxygen species (ROS) was analyzed by CCK-8 assay, PI/FITC-Annexin V method, and DCFH-DA fluorometric assay. Cell apoptosis-associated proteins as well as activations of JAK/STAT3 signaling and JNK signaling were analyzed by Western blot. H2 O 2 treatment-induced cell injury was inhibited by lncRNA GAS5 silence. Specifically, knockdown of GAS5 improved viability of primary PRGCs, inhibited apoptosis, decreased ROS expression, increased antiapoptosis proteins' expressions, and decreased proapoptosis proteins' expressions. It was also found that miR-124 inhibitor treatment impaired the cell protective effect of GAS5 silence, indicating low level of GAS5 protected PRGCs via upregulating miR-124. GAS5 silence might exert cytoprotection effect via activating JAK/STAT3 signaling pathway and inhibiting activation of JNK signaling pathway. Knocking down lncRNA GAS5 alleviated H2 O 2 -induced injury in PRGCs via upregulation of miR-124, which might dependent on activation of JAK/STAT3 signaling pathway and inhibition of JNK signaling pathway.

Casticin inhibits esophageal cancer cell proliferation and promotes apoptosis by regulating mitochondrial apoptotic and JNK signaling pathways.

Casticin, a flavonoid isolated from Vitex species, has been found to have anti-tumor property in multiple human cancers. The present study aimed to investigate the effect of casticin on the proliferation and apoptosis of esophageal cancer (EC) cells, and further illustrate the underlying mechanisms. In in vitro studies, human EC cell lines TE-1 and ECA-109 were treated with various concentrations of casticin (low-, middle-, and high-dose groups). The results showed that casticin dose-dependently inhibited the proliferation and clonogenicity of EC cells and induced cell cycle arrest in sub-G1 and G2 phases. Furthermore, casticin markedly enhanced EC cell apoptosis as detected by flow cytometry and Hoechst 33342 staining. The level of anti-apoptotic Bcl-2 protein was decreased, while the levels of pro-apoptotic Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP were conversely increased in casticin-treated TE-1 and ECA-109 cells. Moreover, casticin decreased the mitochondrial membrane potential and increased the release of mitochondrial cytochrome C into cytoplasm. In addition, the JNK signaling pathway was involved in casticin-medicated anti-proliferation and pro-apoptosis. Cells pretreated with SP600125, a JNK pathway inhibitor, partially abolished the effect of casticin. Finally, the anti-tumor property of casticin was confirmed in in vivo xenograft models. Overall, we provided both in vitro and in vivo evidences that casticin inhibited the proliferation and induced apoptosis of EC cells, and the anti-tumor action of casticin was mediated, in part, by the mitochondrial-dependent apoptosis and the activation of JNK signaling pathway.

Tangeretin protects human brain microvascular endothelial cells against oxygen-glucose deprivation-induced injury.

Tangeretin, a citrus flavonoid extracted from the peel of citrus fruits, was reported to possess antiasthmatic, antioxidant, anti-inflammatory, and neuroprotective properties. However, the effect of tangeretin on human brain microvascular endothelial cells (HBMECs) has not been examined. This study was designed to investigate the protective effects of tangeretin on oxygen-glucose deprivation (OGD)-induced injury of HBMECs, and explore the underlying mechanisms. Our results showed that tangeretin improved HBMECs viability in response to OGD. In addition, tangeretin was able to increase the activity of superoxide dismutase and decrease the levels of reactive oxygen speciesand malondialdehyde (MDA), as well as ameliorate cell apoptosis in OGD-stimulated HBMECs. Mechanistic studies showed that tangeretin prevented the activation of JNK signaling pathway in OGD-stimulated HBMECs. Taken together, our current study demonstrated that tangeretin could ameliorate OGD-induced HBMECs injury through the JNK signaling pathway. Thus, tangeretin might be used as a therapeutic strategy for ischemia-reperfusion brain injury and related diseases.

MiR-326 functions as a tumor suppressor in human prostatic carcinoma by targeting Mucin1.

Accumulating evidence suggests that microRNA-326 (miR-326) serves as a tumor suppressor in the initiation and progression of several human malignancies. However, the biological function and underlying molecular mechanism of miR-326 in prostatic carcinoma (PCa) remains largely unknown. In the present study, we found that miR-326 expression level was significantly downregulated in both primary PCa and castration-resistant PCa (CRPC) tissue samples as detected by qRT-PCR. Downregulation of miR-326 was closely associated with aggressive progression and poor prognosis of primary PCa patients. Gain- and lose- functional experiments revealed that forced expression of miR-326 significantly inhibited cell proliferation, colony formation, migration and invasion, induced G0/G1 cell cycle arrest, and promoted apoptosis in PCa cells in vitro, whereas, knockdown of miR-326 expression showed the opposite results. Overexpression of miR-326 also suppressed tumor growth in xenografted nude mice in vivo. Moreover, Luciferase reporter, qRT-PCR, and western blot assays identified that the 3'-untranslated region (3'-UTR) of Mucin1 (MUC1) was a direct target region of miR-326. Spearman's correlation analysis also confirmed an inverse relationship between miR-326 and MUC1 expressions in primary PCa tissue samples. In addition, restoration of MUC1 expression effectively abrogated the inhibitory effects of miR-326 on PCa proliferation, invasion and migration through the activation of JNK signaling pathway. Therefore, these data indicated that miR-326 functioned as a tumor suppressor in PCa by negatively regulating MUC1, and that miR-326 might serve as a potential therapeutic candidate for PCa treatment.

Pentraxin 3 promotes cardiac differentiation of mouse embryonic stem cells through JNK signaling pathway.

Cardiovascular disease is a leading cause of death worldwide, requiring the development of new therapeutic strategies including stem cell therapy. Pentraxins (PTXs) are a superfamily of proteins highly involved in different myocardial disorders, and thus this study aimed to identify the modulation of long pentraxin 3 (PTX3) in the differentiation of mouse embryonic stem cells (mESCs) toward cardiomyocytes. Cell toxicity of PTX3 was detected by MTT and LDH assays in mESCs. Embryoid bodies (EBs) were differentiated using hanging drop method, and the beating was observed under microscope. Expressional levels of early cardiac progenitor marker genes were assessed by qRT-PCR. Expression of marker proteins in early myocardial development and the activation of JNK signaling pathway was evaluated by Western blot. PTX3 treatment at 50 ng/mL significantly promoted the expression of cardiac-specific marker genes including Nkx2.5, Mef2c, Tbx5, dHand, and αMHC, and increased the expression of cardiac maturity indicative markers including connexin 43 and troponin C1. PTX3 enhanced the phosphorylation of JNK across the incubation duration, whereas the activation of p38 remained the same as control group. Co-treatment of JNK signaling pathway inhibitor SP600125 impaired the PTX3-promoted transcription of Nkx2.5, Mef2c, Tbx5, dHand, and αMHC. This study revealed the promotion of PTX3 in the differentiation of mESCs into cardiomyocytes and the underlying mechanism.

PO-076 HMNQ induces apoptosis and autophagy dependent on reactive oxygen species through activation of JNK signalling pathway

Introduction8-hydroxy-2-methoxy-1,4-naphthoquinone (HMNQ), a natural compound isolated from bark of Juglans sinensis Dode, has been previously reported to possess cytotoxic activity toward various human cancer cells. However, the molecular mechanism of its anticancer effect remains unknown.Material and methodsIn this study, the anticancer activity and molecular mechanism of HMNQ were investigated using cell viability/colony formation assay and wound healing assay. In addition, apoptosis analysis and measerement of mitochondria membrane potential were performed.Results and discussionsOur results showed that HMNQ reduced cell viability, decreased colony formation, and inhibited cell migration in breast, lung, and colon cancer cells. HMNQ effectively induced apoptosis by upregulating the expression of pro-apoptotic protein Bax, cleaved PARP, and downregulating the expression of anti-apoptotic protein Bcl-2 in A549 and MCF7 cells. In addition, HMNQ also induced reactive oxygen species (ROS) production through the decreased mitochondrial-membrane potential and this effect was attenuated by ROS scavengers, N-acetylcysteine (NAC) and l-glutathione (GSH). Furthermore, HMNQ increased the expression of JNK phosphorylation and JNK inhibitor SP600125 suppressed HMNQ-induced decrease in cell viability.ConclusionTaken together, our findings suggest that HMNQ exhibits anti-proliferative activity through induction of ROS-mediated apoptosis in human cancer cells, indicating that HMNQ as a potential anticancer agent.

Dioscorea bulbifera induced apoptosis through inhibition of ERK 1/2 and activation of JNK signaling pathways in HCT116 human colorectal carcinoma cells

Dioscorea bulbifera, also known as air potato, has been cultivated as food crop mainly in tropical countries in Asia and Australia. The tubers are edible and have often been used in Traditional Chinese Medicine (TCM) and Ayurvedic medicine to treat cancer, diabetes, thyroid disease, and inflammation. This study aimed to investigate the effects of D. bulbifera on HCT116 human colorectal carcinoma cells and to unravel the plausible mechanisms underlying its apoptotic effects. The ethanol crude and fractions (hexane, ethyl acetate and water) of D. bulbifera were subjected to cell viability MTT assay against various cancer cell lines. The lowest IC50 of the extract and fractions on selected cancer cells were selected for further apoptosis assay and western blot analysis. HCT116 cancer cells were treated with D. bulbifera and stained with Annexin/PI or Hoechst 33342/PI for preliminary confirmation of apoptosis. The dissipation of mitochondria membrane potential (MMP) was determined by flow cytometry. The protein expressions of apoptosis-related proteins such as Bcl-2 family, caspases, Fas, PARP, ERK1/2 and JNK were detected by western blot analysis. Moreover, the HCT116 cells were treated with UO126 and SP600125 inhibitors to verify the involvement of ERK1/2 and JNK protein expressions in inducing apoptotic cell death. Based on the result, D. bulbifera ethyl acetate fraction (DBEAF) exhibited the most compelling cytotoxicity on HCT116 cells with an IC50 of 37.91 ± 1.30 µg/mL. The induction of apoptosis was confirmed by phosphatidylserine externalization and chromatin condensation. Depolarization of MMP further conferred the induction of apoptosis was through the regulation of Bcl-2 family proteins. Activation of caspase cascades (caspase-3, -9, -8 and -10) was elicited followed by the observation of cleaved PARP accumulation in DBEAF-treated cells. Furthermore, death receptor, Fas was activated upon exposure to DBEAF. Collective apoptotic evidences suggested the involvement of intrinsic and extrinsic pathways by DBEAF in HCT116 cells. Interestingly, the attenuation of ERK1/2 phosphorylation accompanied by the activation of JNK was detected in DBEAF-treated cells. In conclusion, the findings revealed that DBEAF induced apoptosis through intrinsic and extrinsic pathways involving ERK1/2 and JNK.

Exogenous recombinant human thioredoxin-1 prevents acetaminophen-induced liver injury by scavenging oxidative stressors, restoring the thioredoxin-1 system and inhibiting receptor interacting protein-3 overexpression.

Thioredoxin-1 (Trx-1) is a potent therapeutic agent against a variety of diseases because of its actions as an antioxidant and regulator of apoptosis. N-acetyl-p-aminophenol (APAP), commonly known as acetaminophen, generates excessive oxidative stress and triggers hepatocyte cell death, exemplified by regulated necrosis. In the present study, we investigated whether APAP-induced liver injury in a mouse model is associated with "necroptosis," and if pretreatment with recombinant Trx-1 prevents the hepatic injury caused by APAP overdose. We also explored the mechanism underlying the preventive action of Trx-1 against APAP-induced hepatic injury. In a prevention study, C3H/he mice received different doses (0, 10, 50 or 100mgkg-1 body weight) of recombinant human Trx-1 intraperitoneally, followed by a single oral dose of 300mgkg-1 of APAP. In this experimental paradigm, liver injury and lethality were markedly decreased in rhTrx-1-pretreated mice. In survival experiments, mice received rhTrx-1 followed by oral administration of a lethal dose of APAP. APAP overdose caused a series of liver toxicity-associated events, beginning with overexpression of c-fos, excessive production of reactive oxygen species and reactive nitrogen species (RNS) and leading to decreased endogenous Trx-1 expression and activation of JNK signaling pathways. Pretreatment with rhTrx-1 inhibited all of these toxicological manifestations of APAP. In addition, rhTrx-1 significantly reduced the expression of RIP-3, a critical necrosome component. Taken together, our findings indicate that rhTrx-1 prevents APAP-induced liver injury through multiple action mechanisms, including scavenging reactive oxygen species and reactive nitrogen species, restoring endogenous Trx-1 levels and inhibiting RIP-3 overexpression.