Human Hepatoma Huh7 Cells Research Articles

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

ObjectivesReceptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon (GCG) is important for carbohydrate and lipid metabolism. MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. ResultsGCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from β-arrestin-2 recruitment coupled with increased activation of Gαs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2. ConclusionsBy retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.

Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes.

Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands.

Anti-miR-518d-5p overcomes liver tumor cell death resistance through mitochondrial activity

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient’s overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.

Pooling Strategies to Improve Throughput of High Resolution Mass Spectrometry‐Based Analysis of Xenobiotic Metabolites Generated from Huh7 Human Hepatoma Cell Lines Transduced with P450 Enzymes

As an approach to identify unknown mass spectral features corresponding to xenobiotic metabolites and simultaneously identify the enzyme(s) responsible, we developed a panel of lentivirally transduced human hepatoma cell lines (Huh7) that express xenobiotic metabolizing enzymes. However, screening thousands of chemicals individually against 40-50 different cell lines each would require immense analytical resources. We hypothesized that pooling samples can improve analytical efficiency and throughput without significantly affecting metabolite generation or detection, and designed two pooling strategies to generate and detect metabolites from 36 different chemicals (including a number of drugs whose metabolism is well characterized) and 8 cytochrome P450-expressing HuH7 cell lines. In the pre-pooling strategy, 36 chemicals were grouped into 12 pools of 6 randomized compounds, with each compound present in two separate pools. The 12 pools were each incubated with 8 different cell lines for 0 and 2h resulting in 192 samples for analyses. In the post-pooling strategy, 36 chemicals were incubated individually with 8 different cell lines. After incubation, the 36 chemicals were pooled by the same system as the pre-pooling strategy. 10 μL aliquots of sample extracts were analyzed using liquid chromatography (Dionex Ultimate 3000) and Fourier-transform high-resolution mass spectrometry (Thermo Scientific Fusion) in both HILIC+ and C18- modes. Expression of the P450 proteins was verified by immunoblotting. Results from the pre-pooled samples showed that the anti-cancer drug pazopanib was metabolized by the CYP3A4- and CYP2C8-expressing cell lines. Bupropion and β-naphthoflavone were metabolized mainly by CYP2B6 and CYP1A2, verifying that the cell lines express functional P450s. Next, we compared the metabolomic profiles of the xenobiotics from the two different pooling strategies. The post-pooling strategy generally showed more metabolite generation, although it required 3 times as many biological sample incubations. Hydroxybupropion and β-naphthoflavone dihydrodiol generation were similar between the two pooling strategies. Qualitatively, the generation of hydroxy trifluralin, a possible metabolite of the commonly used herbicide, showed no significant differences between the two pooling strategies, although the signal levels using pre-pooling were 13 times higher than in post-pooling samples. Thus, either pooling strategy can be used as screens to identify enzyme-compound pair reactions and to guide subsequent metabolite generation and characterization. The presence of each xenobiotic in two pools increases stringency for qualifying a feature as a metabolite, while achieving the same confirmation as technical replicates. While the pre-pooling strategy may be vulnerable to confounding effects of potential interactions among xenobiotics in a given pool, the presence of each substrate in two different pools mitigated this concern.

Apoptotic effect of compound K in hepatocellular carcinoma cells via inhibition of glycolysis and Akt/mTOR/c-Myc signaling.

Since the AKT/mammalian target of rapamycin (mTOR)/c-Myc signaling plays a pivotal role in the modulation of aerobic glycolysis and tumor growth, in the present study, the role of AKT/mTOR/c-Myc signaling in the apoptotic effect of Compound K (CK), an active ginseng saponin metabolite, was explored in HepG2 and Huh7 human hepatocellular carcinoma cells (HCCs). Here, CK exerted significant cytotoxicity, increased sub-G1, and attenuated the expression of pro-Poly (ADP-ribose) polymerase (pro-PARP) and Pro-cysteine aspartyl-specific protease (pro-caspase3) in HepG2 and Huh7 cells. Consistently, CK suppressed AKT/mTOR/c-Myc and their downstreams such as Hexokinase 2 (HK2) and pyruvate kinase isozymes M2 (PKM2) in HepG2 and Huh7 cells. Additionally, CK reduced c-Myc stability in the presence or absence of cycloheximide in HepG2 cells. Furthermore, AKT inhibitor LY294002 blocked the expression of p-AKT, c-Myc, HK2, PKM2, and pro-cas3 in HepG2 cells. Pyruvate blocked the ability of CK to inhibit p-AKT, p-mTOR, HK2, and pro-Cas3 in treated HepG2 cells. Overall, these findings provide evidence that CK induces apoptosis via inhibition of glycolysis and AKT/mTOR/c-Myc signaling in HCC cells as a potent anticancer candidate for liver cancer clinical translation.

Effect of Synchronous Versus Sequential Regimens on the Pharmacokinetics and Biodistribution of Regorafenib with Irradiation.

This study was performed to evaluate the interaction between conventional or high-dose radiotherapy (RT) and the pharmacokinetics (PK) of regorafenib in concurrent or sequential regimens for the treatment of hepatocellular carcinoma. Concurrent and sequential in vitro and in vivo studies of irradiation and regorafenib were designed. The interactions of RT and regorafenib in vitro were examined in the human hepatoma Huh-7, HA22T and Hep G2 cell lines. The RT–PK phenomenon and biodistribution of regorafenib under RT were confirmed in a free-moving rat model. Regorafenib inhibited the viability of Huh-7 cells in a dose-dependent manner. Apoptosis in Huh-7 cells was enhanced by RT followed by regorafenib treatment. In the concurrent regimen, RT decreased the area under the concentration versus time curve (AUC)regorafenib by 74% (p = 0.001) in the RT2 Gy × 3 fraction (f’x) group and by 69% (p = 0.001) in the RT9 Gy × 3 f’x group. The AUCregorafenib was increased by 182.8% (p = 0.011) in the sequential RT2Gy × 1 f’x group and by 213.2% (p = 0.016) in the sequential RT9Gy × 1 f’x group. Both concurrent regimens, RT2Gy × 3 f’x and RT9Gy × 3 f’x, clearly decreased the biodistribution of regorafenib in the heart, liver, lung, spleen and kidneys, compared to the control (regorafenib × 3 d) group. The concurrent regimens, both RT2Gy × 3 f’x and RT9Gy × 3 f’x, significantly decreased the biodistribution of regorafenib, compared with the control group. The PK of regorafenib can be modulated both by off-target irradiation and stereotactic body radiation therapy (SBRT).

Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro

Alpha-amanitin (α-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography–tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Instability of the NS1 Glycoprotein from La Reunion 2018 Dengue 2 Virus (Cosmopolitan-1 Genotype) in Huh7 Cells Is Due to Lysine Residues on Positions 272 and 324.

La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.

Role of lncRNA NR2F1-AS1 and lncRNA H19 Genes in Hepatocellular Carcinoma and Their Effects on Biological Function of Huh-7.

ObjectiveThis research was designed to probe into the expression and related mechanism of lncRNA NR2F1-AS1 and H19 in hepatocellular carcinoma (HCC).MethodsForty-two HCC patients who came to our hospital from February 2018 to August 2019 were included into a research group (RG). Meanwhile, 46 healthy controls were regarded as a control group (CG). BEL-7402, Huh-7 human hepatoma cells and HL-7702 human normal liver cells were purchased, and the NR2F1-AS1 and H19 levels in serum and tissues of HCC patients were detected. PcDNA3.1-NR2F1-AS1, si-NR2F1-AS1, NC, pcDNA3.1-H19 and si-H19 were transfected into BEL-7402 and Huh-7 cells. The NR2F1-AS1 and H19 levels in samples were detected via qRT-PCR, and the expression of apoptosis-related proteins in cells was tested through WB. Cell proliferation, invasion, or apoptosis was detected by CCK8, Transwell or flow cytometry, respectively.ResultsThe NR2F1-AS1 and H19 levels were high in human hepatoma cells, and AUCs of lncRNA NR2F1-AS1 and lncRNA H19 were both >0.8. The lncRNA NR2F1-AS1 and lncRNA H19 were associated with HCC staging. After transfection of pcDNA3.1-NR2F1-AS1, si-NR2F1-AS1, NC, pcDNA3.1-H19, si-H19 BEL-7402 and Huh-7 cells, silencing NR2F1-AS1 and H19 expression can promote apoptosis and inhibit cell growth, while silencing their over-expression can inhibit the EMT process of Huh-7 cells.ConclusionlncRNA NR2F1-AS1 and lncRNA H19 genes are abnormally expressed in HCC. Furthermore, the two can suppress the EMT process of Huh-7 cells and promote apoptosis effectively.

Icaritin ameliorates hepatic steatosis via promoting fatty acid β-oxidation and insulin sensitivity

AimThis study aimed to reveal the effects of icaritin (ICT) on lipotoxicity induced by palmitate (PA) in hepatic cells and steatosis in high-fat diet (HFD)-fed mice as well as exploring the potential mechanisms. Main methodsPrimary mouse hepatocytes and human hepatoma Huh7 cells were used to evaluate ICT effect in vitro. HFD-fed mice were used to evaluate the ICT effect in vivo. ResultsIn vitro study indicated that ICT significantly rescued PA-induced steatosis, mainly through a combination of robust increased mitochondrial respiration, fatty acid oxidation and mildly decreased synthesis of fatty acid. An HFD-fed mouse model with 8 weeks HFD-fed showed metabolic disorders, while ICT application significantly reduced the weight, serum glucose levels, insulin resistance, hepatic steatosis level and adipose contents. In consistent with the observations in cell lines, ICT rescued the HFD-impaired functions and contents of key factors related to fatty acid β-oxidation through elevated expression of peroxisome proliferator-activated receptor α (PPARα). Meanwhile, it also reversed the decreased phosphoryl levels of AKT and glucogen synthase kinase 3 (GSK3β), leading to the improvement of insulin resistance. SignificanceICT administration had a therapeutic effect on PA- or HFD-induced hepatic steatosis and metabolic disorders. It may provide a novel strategy to construct preventive and therapeutic means for hepatic steatosis.

(-)-Epigallocatechin-3-O-gallate at a high concentration may induce lipolysis via ATP consumption by activation of stress defense mechanisms.

Green tea catechins have thus far been demonstrated to have antiobesity effects in a variety of experimental models. However, upstream molecular events triggering those phenomena remain to be identified. In this study, we found that (-)-epigallocatechin-3-O-gallate (EGCG) promoted lipolysis in lipid-loaded Huh7 human hepatoma cells. Notably, EGCG at a high concentration induced both oxidative stress and protein stress (proteo-stress), leading to activation of stress defense mechanisms, such as mRNA expressions of antioxidant and phase-2 detoxifying enzymes, and heat shock proteins. Furthermore, EGCG decreased the level of intracellular ATP, while glucose uptake from culture media was promoted possibly for energy homeostasis. EGCG also upregulated the expression of adipose triglyceride lipase, and activated AMP-activated protein kinase. Collectively, these results suggest that EGCG induces lipolysis to compensate for ATP reduction derived from activation of stress defense systems against its oxidative and proteo-stress properties.

Phytofabrication of cobalt oxide nanoparticles from Rhamnus virgata leaves extract and investigation of different bioactivities.

The tunable cobalt oxide nanoparticles (CoONPs) are produced due to the phytochemicals present in Rhamnus virgata (RhV) leaf extract which functions as reducing and stabilization agents. The synthesis of CoONPs was confirmed using different analytical techniques: UV-Vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), dynamics light scatterings (DLS), Fourier-transform infrared spectroscopy (FTIR), energy dispersive X-ray, and Raman spectroscopy analyses. Furthermore, multiple biological activities were performed. Significant antifungal and antibacterial potentials have been reported. The in vitro cytotoxic assays of CoONPs revealed strong anticancer activity against human hepatoma HUH-7 (IC50 : 33.25 μg/ml) and hepatocellular carcinoma HepG2 (IC50 : 11.62 μg/ml) cancer cells. Dose-dependent cytotoxicity potency was confirmed against Leishmania tropica (KMH23 ); amastigotes (IC50 : 58.63 μg/ml) and promastigotes (IC50 : 32.64 μg/ml). The biocompatibility assay using red blood cells (RBCs; IC50 : 4,636 μg/ml) has confirmed the bio-safe nature of CoONPs. On the whole, results revealed nontoxic nature of RhV-CoONPs with promising biological potentials.

Nano-formulation of herbo-mineral alternative medicine from linga chenduram and evaluation of antiviral efficacy.

Traditional medicine is becoming a primary source of health care in many countries in recent years. The current study proposes a new dimension of understanding a traditional origin treatment, using herbo-mineral preparations in nanoform. The herbo-mineral preparation, Linga chenduram [HMLC], was prepared according to the ancient palm script protocol dates back to 1000 years. In search of alternative therapy for the coronavirus, an attempt was made to determine this ethnic medicine formulation's therapeutic potential for viral hepatitis infection. The Hepatitis C virus [HCV] has several genomic similarities with SARS-CoV-2 viruses. The herbo-mineral formulation (HMLC) were analyzed using UV–vis, EDAX, FTIR, XRD, SEM, and TEM studies. SEM images confirmed the ' presence of nanoparticles with agglomerated conditions having an average grain size of 18 to 25 nm. EDAX studies showed the presence of metallic components in oxide or sulfide form in HMLC. The HCV inhibitory effects of HMLC indicated a good response. The cytotoxicity of this preparation against the Huh-7 human hepatoma cell line was significant. The HMLC showed a strong inhibitory effect on HCV replication in a dose-dependent manner. The genomic component of HCV is similar to COVID −19 virus. The Hepatitis C virus (HCV) NS3/4A protease has a striking three-dimensional structural similarity to the SARS-CoV2 Mpro protease, particularly in the arrangement of key active site residues. So HMLC can be tried to treat coronavirus infection. At higher concentrations, HMLC exhibited over 100-fold inhibition. In the MTT assay, HMLC did not show any apparent cytotoxic effect on cell viability at the concentrations 1–100 µg. Histological studies indicated that the liver and kidney did not experience any toxicity by 7 and 15 consecutive days of administration of HMLC on experimental Wistar rats. Hence, the HMLC can be tried as a therapy for COVID −19 infections using the preparations strictly according to ethnopharmacological protocol and optimum doses.

Integrin αvβ3-dependent thyroid hormone effects on tumour proliferation and vascularisation

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvβ3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvβ3, in two murine xenograft tumour models with and without integrin αvβ3 expression. Integrin αvβ3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvβ3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvβ3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvβ3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Molecular Dynamic Study and Synthesis of 1H-benzo[d]imidazole-5-carboxamide Derivatives as Inhibitors for Yellow Fever and Zika Virus Replication

Flaviviridae family comprises the flavivirus genotype that represents a significant world health problem as it includes the Yellow fever virus (YFV) and Zika virus (ZIKV) which are responsible for large outbreaks and for which novel therapies are in urgent demand. The benzimidazole scaffold has been widely reported for its antiviral activity, and hence a new series of 1H-benzo[d]imidazole-5-carboxamide derivatives (VIIa-x, VIIIa-h & IXa, b) was designed, synthesized, and biologically evaluated for their antiviral activity. 5 Compounds (VIId, VIIe, VIIh, VIIn and VIIt) showed antiviral activity against YFV in the low micromolar range using the human hepatoma Huh-7 cells and Vero cells. One compound (VIId) exhibited activity on both YFV (EC50=1.7 ± 0.8µM) and ZIKV (EC50=4.5 ± 2.1µM). Molecular docking and molecular dynamics simulation studies were conducted to understand the SAR of newly synthesized compounds, to explore the potential target of compound VIId and to investigate the possible binding mode to its target.

Selenium enrichment improves anti-proliferative effect of oolong tea extract on human hepatoma HuH-7 cells

Selenium (Se)-enriched tea is attracting increasing interests due to its significantly improved health benefits. This study was to investigate the anti-proliferative effects of Se-enriched oolong tea against human hepatoma HuH-7 cells. Compared with regular oolong tea extract (TE, 0.04 μg selenium/g), Se-enriched oolong tea extract (Se-TE, 0.51 μg selenium/g) exhibited more prominent anti-proliferative effect against HuH-7 cells with an IC50 of 203.1 μg/mL, mainly due to the synergistic effects of organic selenium and tea polyphenols. Our results found that Se-TE increased intracellular ROS production, arrested the cell cycle at G2/M phase, and thus induced cell apoptosis. In addition, western blotting assay revealed the increased expressions of the p53, Bax, caspase 3, and a reduction of Bcl-2 and CDK2, resulting in Se-TE-induced apoptosis. The improved anti-proliferative effect makes Se-enriched oolong tea extract a promising health-promoting ingredient in food industry.

Statin therapy reduces plasma angiopoietin-like 3 (ANGPTL3) concentrations in hypercholesterolemic patients via reduced liver X receptor (LXR) activation

Background and aimsStatins suppress hepatic mRNA expression of ANGPTL3 encoding angiopoietin-like 3 in healthy subjects, but it is unknown if plasma ANGPTL3 concentrations are affected by statins prescribed to hypercholesterolemic patients in clinical practice. We therefore investigated the effect of statin treatment on plasma ANGPTL3 concentrations in hypercholesterolemic patients. In addition, we explored the underlying mechanism by which statins regulate ANGPTL3 in vitro. MethodsPlasma ANGPTL3 concentrations were measured in 93 genetically confirmed familial hypercholesterolemia (FH) patients who were using statin therapy and 61 statin naïve FH patients. Moreover, concentrations were measured in 14 hypercholesterolemic patients who discontinued their statin treatment for 4 weeks. In vitro studies were performed with Huh7 human hepatoma cells. ResultsPlasma ANGPTL3 concentrations were 15% lower in statin treated FH patients compared to statin naïve FH patients (145 (120-193) vs. 167 (135-220) ng/ml, p = 0.012). Statin discontinuation resulted in a 21% (p<0.001) increase of plasma ANGPTL3 concentrations. Simvastatin reduced ANGPTL3 mRNA expression and ANGPTL3 secretion of Huh7 cells. Liver X receptor (LXR) activation with T0901317 increased ANGPTL3 mRNA expression and ANGPTL3 secretion by 6- and 3-fold, respectively. Adding simvastatin did not mitigate this effect but adding the LXR antagonist GSK2230 to simvastatin-incubated Huh7 cells diminished simvastatin-induced reductions in ANGPTL3 mRNA expression and ANGPTL3 secretion. Simvastatin reduced intracellular oxysterol concentrations. Oxysterols are endogenous LXR ligands, implying that simvastatin suppresses ANGPTL3 secretion via reduced oxysterol-mediated LXR activation. ConclusionsStatins lower plasma ANGPTL3 concentrations in hypercholesterolemic patients, likely due to decreased oxysterol-mediated LXR activation.

Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-β1

ABSTRACT Aim In this study, we explored the ability of TAMs to affect the malignant phenotype of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway. Methods The TAMs were characterized by flow cytometry and ELISA assays. Huh-7 cells were treated with conditioned medium of TAMs (TAMs-CM), and the proliferation, migration and invasion abilities were measured by CCK-8, Transwell and scratch assays. The levels of TGF-β1, Gli2, IGF-II and related proteins in the ERK1/2 pathway and the epithelial-mesenchymal transition (EMT) process were examined by RT-qPCR and western blot. Huh-7 cells were injected subcutaneously into nude mice with TAMs to explore the role of TAMs in tumor growth. Results The expression levels of TGF-β1, Gli2 and IGF-II and the cell proliferation, migration and invasion abilities were elevated in Huh-7 cells treated with TAMs-CM. TGF-β1 was upregulated in the conditioned medium and was found to be involved in the promotion of migration, invasion and the EMT of Huh-7 cells. The activation of TGF-β1 signaling increased the expression of Gli2. Knockdown of Gli2 decreased the expression of IGF-II and also reversed the promotional effect of the conditioned medium on migration, invasion and the EMT of Huh-7 cells. TGF-β1/Gli2/IGF-II signaling was shown to promote the malignant phenotype of Huh-7 cells by activating the ERK1/2 signaling pathway. Further, TGF-β1 knockdown attenuated the influence of TAMs on tumor growth in mouse model. Conclusion The TGF-β1 secreted by TAMs promotes the migration, invasion and EMT of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway.

Luciferase reporter assay for small-molecule inhibitors of MIR92b-3p function: Screening cyanopeptolins produced by Nostoc from the Baltic Sea

We developed a cell sensor that detects the liver cancer-specific microRNA MIR92b-3p, involved in hepatocellular carcinoma development and hepatitis C virus infection. To validate our small-molecule screen that employs a Huh7 human hepatoma cell line stably transfected with a pmirGLO vector containing dual luciferase reporters, we used i) a MIR92b-3p antisense or a MIR92b-3p mimicking agent (concentrations from 0.1 pM to 100 nM), ii) expression of XIST, a long non-coding RNA that is a cellular target of MIR92b, and iii) ectopic expression of Luc2 luciferase. This reporter system was used to test four cyanopeptolins from a de novo library of peptides that were isolated from the Baltic Sea cyanobacteria Nostoc edaphicum strain CCNP1411. Exposure of the Huh7-pmirGLO-MIR92b-3p cells to increasing concentrations (from 10 nM to 100 μM) of the cyanopeptolins and microcystin-LR (MC-LR; a treatment control) did not lead to a dose-dependent restoration of the luciferase signal. Instead, when the reporter cells were treated with MC-LR, the luciferase signal decreased markedly, most likely due to non-target, toxic effects of MC-LR on the cells. Although the first use of this reporter system to screen selected Nostoc peptides did not identify inhibitors of MIR92b, this method provides a means to identify functional miRNA regulators and could be readily extended to other compounds.

Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection.

Dengue virus infection is associated with the upregulation of metabolic pathways within infected cells. This effect is common to infection by a broad array of viruses. These metabolic changes, including increased glucose metabolism, oxidative phosphorylation and autophagy, support the demands of viral genome replication and infectious particle formation. The mechanisms by which these changes occur are known to be, in part, directed by viral nonstructural proteins that contact and control cellular structures and metabolic enzymes. We investigated the roles of host proteins with overarching control of metabolic processes, the transcriptional regulators, cyclin-dependent kinase 8 (CDK8) and its paralog, CDK19, as mediators of virally induced metabolic changes. Here, we show that expression of CDK8, but not CDK19, is increased during dengue virus infection in Huh7 human hepatocellular carcinoma cells, although both are required for efficient viral replication. Chemical inhibition of CDK8 and CDK19 with Senexin A during infection blocks virus-induced expression of select metabolic and autophagic genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3), and reduces viral genome replication and infectious particle production. The results further define the dependence of virus replication on increased metabolic capacity in target cells and identify CDK8 and CDK19 as master regulators of key metabolic genes. The common inhibition of CDK8 and CDK19 offers a host-directed therapeutic intervention that is unlikely to be overcome by viral evolution.