Fibroblast Growth Factor 11 Inhibits Hepatitis B Virus Gene Expression Through FXRα Suppression.

Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF family, which shows different signal transmission compared with other FGF superfamily members. The molecular function of FGF11 is not clearly understood. In this study, we identified the inhibitory effect of FGF11 on hepatitis B virus (HBV) gene expression through transcriptional suppression. FGF11 decreased the mRNA and protein expression of HBV genes in liver cells. While the nuclear receptor FXRα1 increased HBV promoter transactivation, FGF11 decreased the FXRα-mediated gene induction of the HBV promoter by the FXRα agonist. Reduced endogenous levels of FXRα by siRNA and the dominant negative mutant protein (aa 1-187 without ligand binding domain) of FXRα expression indicated that HBV gene suppression by FGF11 is dependent on FXRα inhibition. In addition, FGF11 interacts with FXRα protein and reduces FXRα protein stability. These results indicate that FGF11 inhibits HBV replicative expression through the liver cell-specific transcription factor, FXRα, and suppresses HBV promoter activity. Our findings may contribute to the establishment of better regimens for the treatment of chronic HBV infections by including FGF11 to alter the bile acid mediated FXR pathway.

Urban aerosol particulate matter promotes mitochondrial oxidative stress-induced cellular senescence in human retinal pigment epithelial ARPE-19 cells.

Environmental exposure to urban particulate matter (UPM) is a serious health concern worldwide. Although several studies have linked UPM to ocular diseases, no study has reported effects of UPM exposure on senescence in retinal cells. Therefore, this study aimed to investigate the effects of UPM on senescence and regulatory signaling in human retinal pigment epithelial ARPE-19 cells. Our study demonstrated that UPM significantly promoted senescence, with increased senescence-associated ß-galactosidase activity. Moreover, both mRNA and protein levels of senescence markers (p16 and p21) and the senescence-associated secretory phenotype, including IL-1ß, matrix metalloproteinase-1, and -3 were upregulated. Notably, UPM increased mitochondrial reactive oxygen species-dependent nuclear factor-kappa B (NF-κB) activation during senescence. In contrast, use of NF-κB inhibitor Bay 11-7082 reduced the level of senescence markers. Taken together, our results provide the first in vitro preliminary evidence that UPM induces senescence by promoting mitochondrial oxidative stress-mediated NF-κB activation in ARPE-19 cells.

Fisetin Protects C2C12 Mouse Myoblasts from Oxidative Stress-Induced Cytotoxicity through Regulation of the Nrf2/HO-1 Signaling.

Fisetin is a bioactive flavonol molecule and has been shown to have antioxidant potential, but its efficacy has not been fully validated. The aim of the present study was to investigate the protective efficacy of fisetin on C2C12 murine myoblastjdusts under hydrogen peroxide (H2O2)-induced oxidative damage. The results revealed that fisetin significantly weakened H2O2-induced cell viability inhibition and DNA damage while blocking reactive oxygen species (ROS) generation. Fisetin also significantly alleviated cell cycle arrest by H2O2 treatment through by reversing the upregulation of p21WAF1/CIP1 expression and the downregulation of cyclin A and B levels. In addition, fisetin significantly blocked apoptosis induced by H2O2 through increasing the Bcl-2/Bax ratio and attenuating mitochondrial damage, which was accompanied by inactivation of caspase-3 and suppression of poly(ADP-ribose) polymerase cleavage. Furthermore, fisetin-induced nuclear translocation and phosphorylation of Nrf2 were related to the increased expression and activation of heme oxygenase-1 (HO-1) in H2O2-stimulated C2C12 myoblasts. However, the protective efficacy of fisetin on H2O2-mediated cytotoxicity, including cell cycle arrest, apoptosis and mitochondrial dysfunction, were greatly offset when HO-1 activity was artificially inhibited. Therefore, our results indicate that fisetin as an Nrf2 activator effectively abrogated oxidative stress-mediated damage in C2C12 myoblasts.

Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells.

PURPOSE: Numerous studies have linked particulate matter2.5 (PM2.5) to ocular surface diseases, but few studies have been conducted on the biological effect of PM2.5 on the cornea. The objective of this study was to evaluate the harmful effect of PM2.5 on primary rat corneal epithelial cells (RCECs) in vitro and identify the toxic mechanism involved. MATERIALS AND METHODS: Primary cultured RCECs were characterized by pan-cytokeratin (CK) staining. In PM2.5-exposed RCECs, cell viability, microarray gene expression, inflammatory cytokine levels, mitochondrial damage, DNA double-strand break, and signalling pathway were investigated. RESULTS: Exposure to PM2.5 induced cytotoxicity and morphological changes in RCECs. In addition, PM2.5 markedly up-regulated pro-inflammatory mediators but down-regulated the wound healing-related transforming growth factor-ß. Furthermore, PM2.5 promoted mitochondrial reactive oxygen species (ROS) production and mediated cellular damage to mitochondria and DNA, whereas these cellular alterations induced by PM2.5 were markedly suppressed by a potential ROS scavenger. Noteworthy, removal of ROS selectively down-regulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the activation of the nuclear factor-κB (NF-κB) p65 in PM2.5-stimulated cells. Additionally, SB203580, a p38 MAPK inhibitor, markedly suppressed these PM2.5-mediated cellular dysfunctions. CONCLUSIONS: Taken together, our findings show that PM2.5 can promote the ROS/p38 MAPK/NF-κB signalling pathway and lead to mitochondrial damage and DNA double-strand break, which is ultimately caused inflammation and cytotoxicity in RCECs. These findings indicate that the ROS/p38 MAPK/NF-κB signalling pathway is one mechanism involved in PM2.5-induced ocular surface disorders.

Core promoter mutation of nucleotides A1762T and G1764A of hepatitis B virus increases core promoter transactivation by hepatocyte nuclear factor 1.

Hepatitis B virus (HBV) infection highly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). The clinical manifestation of HBV infection is determined by the mutual interplay of the viral genotype, host genetic factors, mode of transmission, adaptive mutations, and environmental factors. Core promoter activation plays a critical role in the pre-genomic RNA transcription of HBV for HBV replication. The mutations of core promoter have been implicated in HCC development. We had obtained HBV genes from Myanmar HBV infectants and identified gene variations at the core promoter region. For measuring the relative transactivation activity on core promoter, we prepared the core-promoter reporter construct. Both of A1762T and G1764A mutation were consistently found in the HBV genes with hepatocellular carcinoma. The A1762T/G1764A mutation was corresponding to K130M/V131I of HBx protein. We prepared the core promoter-luciferase reporter construct containing the double A1762T/G1764A mutation and the K130M/V131I HBx protein expression construct. The A1762T/G1764A mutation highly was responsive to core promoter transactivation by HBx, regardless of HBx mutation. The A1762T/G1764A mutation newly created hepatocyte nuclear factor 1 (HNF1) responsive element. Ectopic expression of HNF1 largely increased the HBV core promoter containing A1762T/G1764A mutation. In addition, hepatic rich fatty acid, palmitic acid and oleic acid, increased K130M/V131I HBx level by core promoter activation. These results provide biological properties and clinical significance of specific HBV core promoter mutants related with HCC development.

Fibroblast growth factor 11 inhibits foot-and-mouth disease virus gene expression and replication in vitro.

Foot-and-mouth disease virus (FMDV) causes highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. Although FMD vaccine is the traditional way to protect against the disease, the use of FMD vaccines to protect early infection is limited. The alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF. Here, we identified the inhibitory effect of FGF11 on FMDV gene expression through the transcriptional and translational regulation. For the quantitative analysis of FMDV transcription/translation level, we firstly constructed a plasmid reporter system (FMDV five prime untranslated region (5' UTR) -luci) conjugating luciferase encoding gene with FMDV 5' UTR region, which is a non-coding region to control FMDV transcription/translation and includes cis-acting replication element (CRE) and internal ribosome entry site (IRES). FGF11 decreased the gene expression of FMDV 5' UTR-luci reporter in a dose-dependent manner. We further confirmed the inhibitory function of FGF11 on FMDV gene expression a replication in the FMDV-infected pig cells. FGF11 expression inhibited RNA production of FMDV RNA polymerase 3D gene in the FMDV-infected cells. In addition, while FMDV cell infection induced cytopathic effect (CPE) within 24 hr, FGF11 expression dramatically repressed CPE at the basal level. These results indicate that FGF11 inhibits FMDV gene expression and replication in vitro, implicating to provide intervention strategy for FMDV pathogenesis and transmission.

The Analyses of Cetacean Virus-Responsive Genes Reveal Evolutionary Marks in Mucosal Immunity-Associated Genes.

Viruses are the most common and abundant organisms in the marine environment. To better understand how cetaceans have adapted to this virus-rich environment, we compared cetacean virus-responsive genes to those from terrestrial mammals. We identified virus-responsive gene sequences in seven species of cetaceans, which we compared with orthologous sequences in seven terrestrial mammals. As a result of evolution analysis using the branch model and the branch-site model, 21 genes were selected using at least one model. IFN-ε, an antiviral cytokine expressed at mucous membranes, and its receptor IFNAR1 contain cetacean-specific amino acid substitutions that might change the interaction between the two proteins and lead to regulation of the immune system against viruses. Cetacean-specific amino acid substitutions in IL-6, IL-27, and the signal transducer and activator of transcription (STAT)1 are also predicted to alter the mucosal immune response of cetaceans. Since mucosal membranes are the first line of defense against the external environment and are involved in immune tolerance, our analysis of cetacean virus-responsive genes suggests that genes with cetacean-specific mutations in mucosal immunity-related genes play an important role in the protection and/or regulation of immune responses against viruses.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression.

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages.

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1ß, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts.

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.

Betulinic Acid Restricts Human Bladder Cancer Cell Proliferation In Vitro by Inducing Caspase-Dependent Cell Death and Cell Cycle Arrest, and Decreasing Metastatic Potential.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid and generally found in the bark of birch trees (Betula sp.). Although several studies have been reported that BA has diverse biological activities, including anti-tumor effects, the underlying anti-cancer mechanism in bladder cancer cells is still lacking. Therefore, this study aims to investigate the anti-proliferative effect of BA in human bladder cancer cell lines T-24, UMUC-3, and 5637, and identify the underlying mechanism. Our results showed that BA induced cell death in bladder cancer cells and that are accompanied by apoptosis, necrosis, and cell cycle arrest. Furthermore, BA decreased the expression of cell cycle regulators, such as cyclin B1, cyclin A, cyclin-dependent kinase (Cdk) 2, cell division cycle (Cdc) 2, and Cdc25c. In addition, BA-induced apoptosis was associated with mitochondrial dysfunction that is caused by loss of mitochondrial membrane potential, which led to the activation of mitochondrial-mediated intrinsic pathway. BA up-regulated the expression of Bcl-2-accociated X protein (Bax) and cleaved poly-ADP ribose polymerase (PARP), and subsequently activated caspase-3, -8, and -9. However, pre-treatment of pan-caspase inhibitor markedly suppressed BA-induced apoptosis. Meanwhile, BA did not affect the levels of intracellular reactive oxygen species (ROS), indicating BA-mediated apoptosis was ROS-independent. Furthermore, we found that BA suppressed the wound healing and invasion ability, and decreased the expression of Snail and Slug in T24 and 5637 cells, and matrix metalloproteinase (MMP)-9 in UMUC-3 cells. Taken together, this is the first study showing that BA suppresses the proliferation of human bladder cancer cells, which is due to induction of apoptosis, necrosis, and cell cycle arrest, and decrease of migration and invasion. Furthermore, BA-induced apoptosis is regulated by caspase-dependent and ROS-independent pathways, and these results provide the underlying anti-proliferative molecular mechanism of BA in human bladder cancer cells.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Coptisine induces autophagic cell death through down-regulation of PI3K/Akt/mTOR signaling pathway and up-regulation of ROS-mediated mitochondrial dysfunction in hepatocellular carcinoma Hep3B cells.

Coptisine is isoquinoline alkaloid derived from Coptidis Rhizoma and is known to have potential anti-cancer activity toward various carcinomas. Targeting autophagy is one of the main approaches for cancer therapy, but whether the anti-cancer efficacy of coptisine involves autophagy is still unclear. Therefore, this study investigated the effect of coptisine on autophagy in hepatocellular carcinoma (HCC) Hep3B cells, and identified the underlying mechanism. Our results showed that coptisine increased cytotoxicity and autophagic vacuoles in a concentration-dependent manner. Furthermore, the expressions of light chain 3 (LC3)-I/II, Beclin-1 and autophagy genes were markedly increased by coptisine, while the expression of p62 decreased. In addition, we found that pretreatment with bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, markedly reduced coptisine-mediated autophagic cell death, but 3-methyladenine, an inhibitor for autophagosome formation did not. Moreover, our results showed that although coptisine up-regulated AMP-activated protein kinase (AMPK) that partially induced LC3-I/II, coptisine-mediated AMPK signaling did not directly regulate autophagic cell death. Additionally, we found that coptisine suppressed the phosphorylation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR), and this effect was notably enhanced by PI3K inhibitor LY294002. Meanwhile, coptisine significantly increased both the production of mitochondrial reactive oxygen species (ROS) and the recruitment of mitophagy-regulated proteins to mitochondria. Furthermore, N-acetylcysteine, a potential ROS scavenger, substantially suppressed the expression of mitophagy-regulated proteins and LC3 puncta by coptisine. Overall, our results demonstrate that coptisine-mediated autophagic cell death was regulated by PI3K/Akt/mTOR signaling and mitochondrial ROS production associated with mitochondrial dysfunction. Taken together, these findings suggest that coptisine exerts its anti-cancer effects through induction of autophagy in HCC Hep3B cells.

Auranofin Attenuates Non-Alcoholic Fatty Liver Disease by Suppressing Lipid Accumulation and NLRP3 Inflammasome-Mediated Hepatic Inflammation In Vivo and In Vitro.

Non-alcoholic fatty liver disease (NAFLD) causes liver dysfunction and is associated with obesity and type 2 diabetes. Chronic inflammation is associated not only with the development of NAFLD, but also with hepatic diseases, including steatohepatitis, cirrhosis, and hepatocellular carcinoma. Auranofin is a treatment for rheumatoid arthritis and has recently been reported to have potential effects against a variety of diseases, including inflammation, cancer, and viral infection. In this study, auranofin may be considered as a new treatment for the management of metabolic syndrome, as well as in the treatment of NAFLD through immunomodulation. To determine the effect of auranofin on NAFLD, C57BL/6 mice were randomly grouped, fed a regular diet or a high fat diet (HFD), and injected with normal saline or auranofin for 8 weeks. Auranofin significantly decreased the body weight, epididymal fat weight, serum aspartate aminotransferase (AST), and glucose, as well as the serum triglyceride, cholesterol, and low-density lipoprotein cholesterol levels as compared to the HFD group. We also observed that hepatic steatosis was increased in the HFD group and was suppressed by auranofin treatment. In addition, auranofin suppressed the expressions of interleukin (IL)-1ß, IL-18, caspase-1, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) in the liver tissue. Furthermore, the expression of NADPH oxidase 4 and peroxisome proliferator-activated receptor γ (PPARγ), which are a major source of oxidative stress and a regulator of adipogenesis, respectively, were also decreased by auranofin. In addition, primary mouse hepatocytes were incubated with lipopolysaccharide (LPS) and palmitic acid (PA) to induce lipid accumulation and hepatic inflammation for an in vitro model. Auranofin could significantly inhibit LPS- and PA-induced inflammatory activity including nitric oxide and NLRP3 inflammasome-mediated cytokines. The results of this study demonstrate that auranofin treatment inhibits the characteristics of NAFLD through the inhibition of NLRP3 inflammasome. Therefore, auranofin may have potential as a candidate for improving NAFLD symptoms.

Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway.

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

Induction of Apoptosis by Coptisine in Hep3B Hepatocellular Carcinoma Cells through Activation of the ROS-Mediated JNK Signaling Pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and treatment is very limited due to its high recurrence and low diagnosis rate, and therefore there is an increasing need to develop more effective drugs to treat HCC. Coptisine is one of the isoquinoline alkaloids, and it has various pharmacological effects. However, the evidence for the molecular mechanism of the anticancer efficacy is still insufficient. Therefore, this study investigated the antiproliferative effect of coptisine on human HCC Hep3B cells and identified the action mechanism. Our results showed that coptisine markedly increased DNA damage and apoptotic cell death, which was associated with induction of death receptor proteins. Coptisine also significantly upregulated expression of proapoptotic Bax protein, downregulated expression of anti-apoptotic Bcl-2 protein, and activated caspase-3, -8, and -9. In addition, coptisine remarkably increased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and release of cytochrome c into the cytoplasm. However, N-acetylcysteine (NAC), a ROS scavenger, significantly attenuated the apoptosis-inducing effect of coptisine. It is worth noting that coptisine significantly upregulated phosphorylation of ROS-dependent c-Jun N-terminal kinase (JNK), whereas treatment with JNK inhibitor could suppress an apoptosis-related series event. Taken together, our results suggest that coptisine has an anticancer effect in Hep3B cells through ROS-mediated activation of the JNK signaling pathway.

Distribution of hepatitis B virus genotypes in the general population of Myanmar via nationwide study.

BACKGROUND: Hepatitis B virus (HBV) infections are a severe health concern worldwide. HBV is a DNA virus with a rapid rate of mutation. Based on heterogeneity of the nucleotide sequence, the HBV strains are divided into nine genotypes, each with a characteristic geographical distribution. Identifying and tracking alterations of HBV genotypes is important in epidemiological and transmission studies, and contributes to predicting the risk for development of severe liver disease and response to antiviral treatment. The present study was undertaken to detect HBV genotypes and sub-genotypes in the general population of different states and regions in Myanmar. METHODS: In 2015, a total of 5547 adults of the general population, residing in seven states, seven regions and the Nay Pyi Taw Union Territory, were screened for Hepatitis B Surface antigen (HBsAg) by the immunochromatographic test (ICT). Of the 353 HBsAg positive samples, the HBVDNA was identified using polymerase chain reactions (PCR) targeting the DNA sequences encoding the Pre-S region. A total of 153 PCR positive samples were subsequently subjected to genotyping by partial genome sequencing in both directions. The resulting sequences were then edited, aligned, and compared with reference sequences using the National Centre for Biotechnology Information (NCBI) web-based genotyping tool. RESULTS: Three HBV genotypes (HBV genotype B, genotype C and genotype D) were detected in Myanmar, of which genotype HBV genotype C (66.7%) was the most prevalent, followed by HBV genotype D (32%) and HBV genotype B (1.3%). Sub-genotyping revealed a total of 7 variants within the B, C and D genotypes: 2 (B4 and B5) in HBV genotype B, 3 (C1, C5 and C7) in HBV genotype C, and 2 (D3 and D6) in HBV genotype D. CONCLUSION: HBV genotype C, sub-genotype C1 was predominantly distributed in all states and regions of Myanmar. This study is the first report on the nationwide distribution of HBV genotypes and sub-genotypes in Myanmar. We believe our findings will enable huge support for the hepatitis disease surveillance program, since HBV infection is one of the National Priority Diseases in Myanmar.

Mitochondrial dysfunction stimulates HBV gene expression through lipogenic transcription factor activation.

In previous studies, we showed two consistent findings regarding the functional relationship between hepatitis B virus (HBV) gene expression and hepatic lipid accumulation. One is that HBV X (HBx) protein expression induces hepatic lipid accumulation via specific transcriptional activation. The other is that hepatic rich lipids increase HBV gene expression. A variety of transcription factors, including nuclear receptors have been defined as regulators of HBV promoters and enhancers. However, the association between these metabolic events and HBV gene expression remains to be clearly elucidated. Here, we showed that lipid accumulation due to mitochondrial dysfunction is associated with an increase in HBV gene expression. Saturated fatty acids increase the expression of lipogenic factors cooperated with C/EBPα and LXRα. In addition, activation of PPARγ and SREBP-1 by fatty acids derived from hepatic lipid accumulation was found to increase HBV gene expression through mitochondrial dysfunction. These results provide that metabolic changes in the hepatic cells play a critical role in the HBV gene induction.

Isorhamnetin Induces Cell Cycle Arrest and Apoptosis Via Reactive Oxygen Species-Mediated AMP-Activated Protein Kinase Signaling Pathway Activation in Human Bladder Cancer Cells.

Isorhamnetin is an O-methylated flavonol that is predominantly found in the fruits and leaves of various plants, which have been used for traditional herbal remedies. Although several previous studies have reported that this flavonol has diverse health-promoting effects, evidence is still lacking for the underlying molecular mechanism of its anti-cancer efficacy. In this study, we examined the anti-proliferative effect of isorhamnetin on human bladder cancer cells and found that isorhamnetin triggered the gap 2/ mitosis (G2/M) phase cell arrest and apoptosis. Our data showed that isorhamnetin decreased the expression of Wee1 and cyclin B1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdk1. In addition, isorhamnetin-induced apoptosis was associated with the increased expression of the Fas/Fas ligand, reduced ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) expression, cytosolic release of cytochrome c, and activation of caspases. Moreover, isorhamnetin inactivated the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway by diminishing the adenosine triphosphate (ATP) production due to impaired mitochondrial function. Furthermore, isorhamnetin stimulated production of intracellular reactive oxygen species (ROS); however, the interruption of ROS generation using a ROS scavenger led to an escape from isorhamnetin-mediated G2/M arrest and apoptosis. Collectively, this is the first report to show that isorhamnetin inhibited the proliferation of human bladder cancer cells by ROS-dependent arrest of the cell cycle at the G2/M phase and induction of apoptosis. Therefore, our results provide an important basis for the interpretation of the anti-cancer mechanism of isorhamnetin in bladder cancer cells and support the rationale for the need to evaluate more precise molecular mechanisms and in vivo anti-cancer properties.

Anti-Proliferative and Pro-Apoptotic Effects of Licochalcone A through ROS-Mediated Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells.

Licochalcone A (LCA) is a chalcone that is predominantly found in the root of Glycyrrhiza species, which is widely used as an herbal medicine. Although previous studies have reported that LCA has a wide range of pharmacological effects, evidence for the underlying molecular mechanism of its anti-cancer efficacy is still lacking. In this study, we investigated the anti-proliferative effect of LCA on human bladder cancer cells, and found that LCA induced cell cycle arrest at G2/M phase and apoptotic cell death. Our data showed that LCA inhibited the expression of cyclin A, cyclin B1, and Wee1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdc2 and Cdk2. LCA activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, LCA increased the Bax/Bcl-2 ratio, and reduced the integrity of mitochondria, which contributed to the discharge of cytochrome c from the mitochondria to the cytoplasm. Moreover, LCA enhanced the intracellular levels of reactive oxygen species (ROS); however, the interruption of ROS generation using ROS scavenger led to escape from LCA-mediated G2/M arrest and apoptosis. Collectively, the present data indicate that LCA can inhibit the proliferation of human bladder cancer cells by inducing ROS-dependent G2/M phase arrest and apoptosis.