Proteins In HepG2 Cells Research Articles

Anti-hyperglycemic effects and signaling mechanism of Perilla frutescens sprout extract.

BACKGROUND/OBJECTIVESPerilla frutescens (L.) Britton var. (PF) sprout is a plant of the labiate family. We have previously reported the protective effects of PF sprout extract on cytokine-induced β-cell damage. However, the mechanism of action of the PF sprout extract in type 2 diabetes (T2DM) has not been investigated. The present study was designed to study the effects of PF sprout extract and signaling mechanisms in the T2DM mice model using C57BL/KsJ-db/db (db/db) mice.MATERIALS/METHODSMale db/db mice were orally administered PF sprout extract (100, 300, and 1,000 mg/kg of body weight) or rosiglitazone (RGZ, positive drug, 1 mg/kg of body weight) for 4 weeks. Signaling mechanisms were analyzed using liver tissues and HepG2 cells.RESULTSThe PF sprout extract (300 and 1,000 mg/kg) significantly reduced the fasting blood glucose, serum insulin, triglyceride and total cholesterol levels in db/db mice. PF sprout extract also significantly improved glucose intolerance and insulin sensitivity, decreased hepatic gluconeogenic protein expression, and ameliorated histological alterations of the pancreas and liver. Levels of phosphorylated AMP-activated protein kinase (AMPK) protein expression also increased in the liver after treatment with the extract. In addition, an increase in the phosphorylation of AMPK and decrease in the phosphoenolpyruvate carboxykinase and glucose 6-phosphatase proteins in HepG2 cells were also observed.CONCLUSIONSOur results sugges that PF sprout displays beneficial effects in the prevention and treatment of type 2 diabetes via modulation of the AMPK pathway and inhibition of gluconeogenesis in the liver.

Differentially expressed genes of HepG2 cells treated with gecko polypeptide mixture.

Gecko (Gekko japonicus) extracts have been used in traditional Chinese medicine for many years. It has been proven that the gecko polypeptide mixture (GPM) extracted from gecko can inhibit the growth of multiple types of tumor cells. In order to investigate the possible anti-tumor molecular mechanisms of GPM, we used RNA-seq technology to identify the differentially expressed genes (DEGs) of human hepatocellular carcinoma (HCC) HepG2 cells treated with or without GPM. MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe morphological changes in the nuclei of HepG2 cells. Western blot analysis was applied to observe the expressions of apoptosis-related and endoplasmic reticulum stress (ERS)-related proteins in HepG2 cells. Flow cytometry assay was performed to detect the apoptosis and reactive oxygen species (ROS) in HepG2 cells. Our results showed that GPM inhibited HepG2 cells proliferation and induced the apoptosis of HepG2 cells. RNA-seq analysis suggested that the ER-nucleus signaling pathway involved in the anti-cancer molecular mechanism of GPM. Therefore, GPM may induce apoptosis in HepG2 cells via the ERs pathway.

Hepatitis C virus NS4B protein induces epithelial-mesenchymal transition by upregulation of Snail

BackgroundChronic hepatitis C virus (HCV) infection is an important cause of hepatocellular carcinoma (HCC). Epithelial to mesenchymal transition (EMT) is a key process associated with tumor metastasis and poor prognosis. HCV infection, HCV core and NS5A protein could induce EMT process, but the role of NS4B on EMT remains poorly understood.MethodsWe overexpressed HCV NS4B protein in HepG2 cells or Huh7.5.1 cells infected by HCVcc, the E-cadherin expression, N-cadherin expression and the EMT-associated transcriptional factor Snail were determined. The migration and invasion capabilities of the transfected cells were evaluated using wound-healing assay. Additionally, we used Snail siRNA interference to confirm the relation of HCV NS4B and Snail on EMT promotion.ResultsHCV NS4B increased the expression of EMT related markers and promoted cell migration and invasion. Snail knock-down almost completely eliminated the function of NS4B protein in EMT changes and reversed cell migration capacity to lower level. HCV NS4B protein could reduce the expression of Scribble and Hippo signal pathway were subsequently inactivated, resulting in the activation of PI3K/AKT pathway, which may be the reason for the up-regulation of Snail.ConclusionsThis study demonstrates that HCV NS4B protein induces EMT progression via the upregulation of Snail in HCC, which may be a novel underlying mechanism for HCV-associated HCC development, invasion and metastasis.

Fructose-1,6-bisphosphate decreases IL-8 levels and increases the activity of pro-apoptotic proteins in HepG2 cells.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver tumor that affects the world population. Liver cancer inevitably causes great harms and its treatment is extremely difficult. Its development is related to the existence of chronic liver injury, such as in cirrhosis. Cancer is a disease related to the process of inflammation so, research with anti-inflammatory agents has been performed for the development of anti-tumor drugs. Fructose-1,6-bisphosphate (FBP), a metabolite of the glycolytic route, has shown anti-inflammatory actions. The purpose of this study is to investigate the effect of FBP on HepG2 cells growth and inflammatory parameters. Results showed that FBP decreased the proliferation of HepG2 cells through trypan blue assay, without causing necrosis, shown by the intracellular release of LDH. By flow cytometry, we observed a significant IL-8 decrease which is closely related to the tumoral progression and chemotherapeutic resistance, especially in HCC. Then, we found, by RT-PCR, a high expression level of pro-apoptotic protein, such as Bax and p53, and decreased the expression levels of anti-apoptotic proteins, like Bcl-2 suggesting apoptosis. Finally, our results showed that FBP can be a potential therapeutic agent to slow the progress of HCC.

Anti-tumor effect of evodiamine by inducing Akt-mediated apoptosis in hepatocellular carcinoma

BackgroundEvodiamine is an alkaloid extracted from Euodia rutaecarpa (Juss.) Benth. There is little information about the mechanisms of evodiamine on the apoptosis of hepatocellular carcinoma (HCC). Materials and methodsA xenograft model and CCK8 assay were used to investigate the anti-HCC effect of evodiamine. The effect of evodiamine on apoptosis was evaluated by DAPI staining and flow cytometry. Western blot analyses and immunohistochemistry were processed to assess the protein expressions of Akt and apoptotic proteins. ResultsEvodiamine suppressed tumor growth, improved the expression of cleaved-caspase3 and decreased tumor specific growth factor (TSGF) and alpha fetoprotein (AFP) activities. Furthermore, evodiamine inhibited cell viability and induced cell cycle arrest. DAPI staining revealed nuclear condensation in evodiamine-treated groups. Meanwhile, evodiamine increased the number of apoptotic cells. Furthermore, evodiamine suppressed Akt and regulated apoptotic proteins in HepG2 cells. Evodiamine decreased p-Akt levels activated by SC79, which led to the increase of bax/bcl-2 and cleaved-caspase3. ConclusionsOur findings suggested that evodiamine could exert anti-HCC effect through inducing Akt-mediated apoptosis. Evodiamine has the potential to be a therapeutic medicine for HCCs.

Essential oil from Siegesbeckia pubescens induces apoptosis through the mitochondrial pathway in human HepG2 cells

Siegesbeckia pubescens (SP) has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP (SPEO) on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of HepG2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of HepG2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in HepG2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in HepG2 cells through activating the mitochondrial pathway.

Anti-oxidative Activity of Hydrolysate from Rice Bran Protein in HepG2 Cells.

Glutathione (GSH) is an ubiquitous thiol-containing tripeptide, which plays important roles in cellular protection from oxidative stress. In our search for a dietary source that can increase GSH levels, we discovered that a 24 h treatment of HepG2 cells with rice bran protein hydrolysate (RBPH), prepared by Umamizyme G-catalyzed hydrolysis, increased the GSH content in a dose-dependent manner. RBPH elevated the expression levels of γ-glutamylcysteine synthetase (γ-GCS), which constitutes the rate-limiting enzyme of GSH synthesis, and of another two enzymes, hemeoxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1). This induction was preceded by the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) inside the nucleus, which is a key transcription factor for the expression of the γ-GCS, HO-1, and NQO1. Pre-treatment of cells with RBPH produced a significant protective effect against cytotoxicity caused by H2O2 or ethanol. These results indicate that RBPH exerts a protective effect against oxidative stress by modulating GSH levels and anti-oxidative enzyme expression via the Nrf2 pathway.

An Unbiased Mass Spectrometry Approach Identifies Glypican-3 as an Interactor of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR) in Hepatocellular Carcinoma Cells

The mechanism of LDL receptor (LDLR) degradation mediated by the proprotein convertase subtilisin/kexin type 9 (PCSK9) has been extensively studied; however, many steps within this process remain unclear and still require characterization. Recent studies have shown that PCSK9 lacking its Cys/His-rich domain can still promote LDLR internalization, but the complex does not reach the lysosome suggesting the presence of an additional interaction partner(s). In this study we carried out an unbiased screening approach to identify PCSK9-interacting proteins in the HepG2 cells' secretome using co-immunoprecipitation combined with mass spectrometry analyses. Several interacting proteins were identified, including glypican-3 (GPC3), phospholipid transfer protein, matrilin-3, tissue factor pathway inhibitor, fibrinogen-like 1, and plasminogen activator inhibitor-1. We then validated these interactions by co-immunoprecipitation and Western blotting. Furthermore, functional validation was examined by silencing each candidate protein in HepG2 cells using short hairpin RNAs to determine their effect on LDL uptake and LDLR levels. Only GPC3 and phospholipid transfer protein silencing in HepG2 cells significantly increased LDL uptake in these cells and displayed higher total LDLR protein levels compared with control cells. Moreover, our study provides the first evidence that GPC3 can modulate the PCSK9 extracellular activity as a competitive binding partner to the LDLR in HepG2 cells.

Ergosterol peroxide activates Foxo3-mediated cell death signaling by inhibiting AKT and c-Myc in human hepatocellular carcinoma cells.

Sterols are the important active ingredients of fungal secondary metabolites to induce death of tumor cells. In our previous study, we found that ergosterol peroxide (5α, 8α-epidioxiergosta-6, 22-dien-3β-ol), purified from Ganoderma lucidum, induced human cancer cell death. Since the amount of purified ergosterol peroxide is not sufficient to perform in vivo experiments or apply clinically, we developed an approach to synthesize ergosterol peroxide chemically. After confirming the production of ergosterol peroxide, we examined the biological functions of the synthetic ergosterol peroxide. The results showed that ergosterol peroxide induced cell death and inhibited cell migration, cell cycle progression, and colony growth of human hepatocellular carcinoma cells. We further examined the mechanism associated with this effect and found that treatment with ergosterol peroxide increased the expression of Foxo3 mRNA and protein in HepG2 cells. The upstream signal proteins pAKT and c-Myc, which can inhibit Foxo3 functions, were clearly decreased in HepG2 cells treated with ergosterol peroxide. The levels of Puma and Bax, pro-apoptotic proteins, were effectively enhanced. Our results suggest that ergosterol peroxide stimulated Foxo3 activity by inhibiting pAKT and c-Myc and activating pro-apoptotic protein Puma and Bax to induce cancer cell death.

Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability.

Quantitative proteomics analysis reveals glutamine deprivation activates fatty acid β-oxidation pathway in HepG2 cells.

Glutamine, a multifunctional amino acid, functions in nutrient metabolism, energy balance, apoptosis, and cell proliferation. Lipid is an important nutrient and controls a broad range of physiological processes. Previous studies have demonstrated that glutamine can affect lipolysis and lipogenesis, but the effect of glutamine on the detailed lipid metabolism remains incompletely understood. Here, we applied the quantitative proteomics approach to estimate the relative abundance of proteins in HepG2 cells treated by glutamine deprivation. The results showed that there were 212 differentially abundant proteins in response to glutamine deprivation, including 150 significantly increased proteins and 62 significantly decreased proteins. Interestingly, functional classification showed that 43 differentially abundant proteins were related to lipid metabolism. Further bioinformatics analysis and western blotting validation revealed that lipid accumulation may be affected by β-oxidation of fatty acid induced by glutamine deprivation in HepG2 cells. Together, our results may provide the potential for regulating lipid metabolism by glutamine in animal production and human nutrition. The MS data have been deposited to the ProteomeXchange Consortium with identifier PXD003387.

A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

More than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV) vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

Impurities contained in antifungal drug ketoconazole are potent activators of human aryl hydrocarbon receptor

Antifungal drug ketoconazole is a mixture of (+)/(-) cis-enantiomers, which also contains several impurities. Ketoconazole was identified as an activator of aryl hydrocarbon receptor AhR by three independent research teams. In the current paper we demonstrate that impurities contained in ketoconazole preparations are strong activators of human AhR and inducers of CYP1A1. Impurity IMP-C had similar potency (EC50), but 10-15 times higher efficacy (magnitude of induction) towards AhR, comparing to (+)-ketoconazole, as revealed by gene reporter assay in AZ-AHR stably transfected cells. Impurities IMP-B and IMP-C, and in lesser extent IMP-E, induced a formation of AhR-DNA complex, as demonstrated by electromobility shift assay EMSA. Impurities IMP-C and IMP-E dose-dependently induced CYP1A1 mRNA after 24 h, and their effects were comparable to those by (+)-ketoconazole. The level of CYP1A1 protein in HepG2 cells was strongly increased by IMP-C after 48h. In conclusion, our data further elucidated molecular effects of ketoconazole towards AhR signaling pathway, with possible implications in ketoconazole role in skin chemoprevention and/or damage, involving AhR.

Abstract 1083: Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase, ACOX2, in breast cancer

Abstract Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. In this study we show that an intronic start variant of ACOX2, peroxisomal Acyl-CoA oxidase 2 (ACOX2-i9), is expressed and translated into a 25kDa protein in HepG2 cells as well as in several breast cancer cell lines, where it is required for optimal cell proliferation. shRNA knock down of the ACOX2-i9 variant results in decreased cell viability as assessed by colony formation assay of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 is estrogen regulated, being induced by estradiol and inhibited by tamoxifen in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript is present predominantly in breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA Breast Cancer dataset. It is specifically enriched in ER+ breast cancers where it's expression is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast cancer. Citation Format: Sunniva Bjørklund, Vessela N. Kristensen, Michael Seiler, Grethe I. Grenaker Alnaes, Yao Ming, John Kerrigan, Bjørn Naume, Ravi Sachidanandam, Gyan Bhanot, Anne-Lise Børresen-Dale, Shridar Ganesan. Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase, ACOX2, in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2015-1083

Preliminary study on hepatotoxicity induced by dioscin and its possible mechanism

Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 µmol · L(-1) exposed to HepG2 cells for 12 h, cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope, and the effect on production of reactive oxygen species (ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 µmol · L(-1). Compared with the control, the LDH release rate and ROS were significantly increased. The expression of CYPlA and AhR mRNA was increased. The expression of CYP1Al protein was increased after dioscin treatment, and resveratrol, an AhR antagonist, could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.

Mechanism of the effect of glycosyltransferase GLT8D2 on fatty liver.

BackgroundRecent studies have shown that some glycosyltransferases are involved in the development of nonalcoholic fatty liver disease (NAFLD). The objective of this study was to explore the effect and mechanism of glycosyltransferase GLT8D2 on fatty liver.MethodsRat model of NAFLD was established by induction with high-fat-diet. The GLT8D2 expression in rat liver was examined using immunohistochemistry. Oil Red O staining and triglyceride assay were used to measure the effect of abnormal GLT8D2 expression on lipid accumulation in HepG2 cells. The expression levels of lipid metabolism-related key molecules, namely sterol regulatory element-binding protein-1c (SREBP-1c), stearoyl-coA desaturase (SCD), carnitine palmitoyltransferase-1 (CPT1) and microsomal triglyceride transfer protein (MTP), in HepG2 cells with abnormal GLT8D2 expression were determined by western blot analyses.ResultsThe expression of GLT8D2 was higher in the liver of rats with NAFLD than in the control rats, and GLT8D2 was mainly located around lipid droplets in hepatocytes. GLT8D2 expression increased in steatosis HepG2 cells compared with that in normal HepG2 cells. GLT8D2 positively regulated lipid droplet accumulation and triglyceride content in HepG2 cells. Upregulation or knockdown of GLT8D2 had no effect on the expressions of SREBP-1c, SCD or CPT-1 proteins in HepG2 cells. However, GLT8D2 expression negatively regulated the expression of MTP protein in HepG2 cells.ConclusionGLT8D2 participated in NAFLD pathogenesis possibly by negatively regulating MTP expression. Specific inhibition of GLT8D2 via an antagonistic strategy could provide a potential candidate approach for treatment of NAFLD.

A novel small molecule inhibitor targeting gp130 (CCR3P.217)

Abstract Interleukin-6 (IL-6) is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists we screened our in-house chemical library and identified a novel synthetic compound as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular targets of the compound were investigated. A reporter gene assay showed that the compound suppressed activation of signal transducer and activator of transcription-3 (STAT3) induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, the compound downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein in HepG2 cells and substantially inhibited IL-6-dependent TF-1 cell proliferation. The compound antagonized IL-6-induced TNF-α production in vivo, but did not affect IL-2-induced TNF-α production. In pathologic models, oral administration of the compound ameliorated collagen-induced arthritis and acute pancreatitis in mice. We subsequently demonstrated direct and specific interaction between the compound and gp130, which was measured by SPR analysis (KD = 7.4 μM) and was simulated by a docking study. Taken together, our data suggest that the compound is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.

Exendin-4 Inhibits the Expression of SEPP1 and Fetuin-A via Improvement of Palmitic Acid-Induced Endoplasmic Reticulum Stress by AMPK.

BackgroundSelenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown.MethodsThe human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression.ResultsExendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1α, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA.ConclusionThese data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.

A20 Attenuates FFAs-induced Lipid Accumulation in Nonalcoholic Steatohepatitis.

A20 is a ubiquitin-editing enzyme that attenuates the activity of proximal signaling complexes at pro-inflammatory receptors. It has been well documented that A20 protein plays an important role in response to liver injury and hepatocytes apoptosis in pro-inflammatory pathways. However, there was little evidence showing that A20 protein was involving in fatty-acid homeostasis except the up-regulation of two fatty acid metabolism regulatory genes at mRNA level (PPARa and CPT1a) by adenovirus-mediated A20 protein overexpression. In this study we found that: 1) the expression level of A20 protein was significantly higher in the steatotic liver from MCD-fed mice than the controls; 2) Overexpression of A20 protein suppressed FFAs-stimulated triglyceride deposition in HepG2 cells while under expression of A20 protein increased FFAs-stimulated triglyceride deposition; 3) Overexpression of A20 protein in HepG2 cells upregulated genes that promote β-oxidation and decreased the mRNA levels of key lipogenic genes such as fatty acid synthase (FAS), indicating A20 function as anti-steatotic factor by the activation of mitochondrial β-oxidation and attenuation of de novo lipogenesis; 4) Nonalcoholic steatohepatitis (NASH) patients showed significantly higher A20 expression level in liver compared with control individuals. Our results demonstrated that A20 protein plays an important role in fatty-acid homeostasis in human as well as animals. In addition, our data suggested that the pathological function of A20 protein in hepatocyte from lipotoxicity to NASH is by the alleviation of triglyceride accumulation in hepatocytes. Elevated expression of A20 protein could be a potential therapeutic strategy for preventing the progression of nonalcoholic steatohepatitis.

The selective target of capsaicin on FASN expression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in HepG2 cells.

The inhibition of the mammalian de novo synthesis of long-chain saturated fatty acids (LCFAs) by blocking the fatty acid synthase (FASN) enzyme activity in tumor cells that overexpress FASN can promote apoptosis, without apparent cytotoxic to non-tumor cells. The present study aimed to focus on the potent inhibitory effect of capsaicin on the fatty acid synthesis pathway inducing apoptosis of capsaicin in HepG2 cells. The use of capsaicin as a source for a new FASN inhibitor will provide new insight into its possible application as a selective anti-cancer therapy. The present findings showed that capsaicin promoted apoptosis as well as cell cycle arrest in the G0/G1 phase. The onset of apoptosis was correlated with a dissipation of mitochondrial membrane potential (ΔΨm). Apoptotic induction by capsaicin was mediated by inhibition of FASN protein expression which was accompanied by decreasing its activity on the de novo fatty acid synthesis. The expression of FASN was higher in HepG2 cells than in normal hepatocytes that were resistant to undergoing apoptosis following capsaicin administration. Moreover, the inhibitory effect of capsaicin on FASN expression and activity was found to be mediated by an increase of intracellular reactive oxygen species (ROS) generation. Treatment of HepG2 cells with capsaicin failed to alter ACC and ACLY protein expression, suggesting ACC and ACLY might not be the specific targets of capsaicin to induce apoptosis. An accumulation of malonyl-CoA level following FASN inhibition represented a major cause of mitochondrial-dependent apoptotic induction instead of deprivation of fatty acid per se. Here, we also obtained similar results with C75 that exhibited apoptosis induction by reducing the levels of fatty acid without any change in the abundance of FASN expression along with increasing ROS production. Collectively, our results provide novel evidence that capsaicin exhibits a potent anti-cancer property by targeting FASN protein in HepG2 cells.