Activity In HepG2 Cells Research Articles

Olive (Olea europaea L.) Seed as New Source of Cholesterol-Lowering Bioactive Peptides: Elucidation of Their Mechanism of Action in HepG2 Cells and Their Trans-Epithelial Transport in Differentiated Caco-2 Cells.

The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

Structural changes and anti-hepatocellular carcinoma activity of interferon-γ after interaction with sinensetin

Exploring the interaction of small molecules with therapeutic proteins can provide useful information about development of ligand-protein complexes as synergistically therapeutic platforms. In this study, the interaction of sinensetin with human interferon gamma (IFNγ) was evaluated experimentally and theoretically. Also, the synergistic effects of IFNγ- sinensetin complex on the inhibition of hepatocellular carcinoma HepG2 cell proliferation were assessed by cell viability and quantitative real time PCR assays. It was realized that sinensetin interacts with IFNγ through a static quenching mechanism and hydrophobic forces mediated by presence of Lys55 and Lys58 amino acid residues in the binding site were the main contributing forces in the spontaneous formation of IFNγ-sinensetin complex. Also, the interaction of sinensetin with IFNγ did not induce a significant change in the secondary and tertiary structure of the protein. Cellular assays revealed a synergistic effect of sinensetin on IFNγ -triggered anticancer action in HepG2 cells through overexpression of caspase-3 mRNA and protein. In conclusion, this study may hold great promise for the development of potential ligand- protein complexes for therapeutic purposes.

Investigation of the intestinal trans-epithelial transport and antioxidant activity of two hempseed peptides WVSPLAGRT (H2) and IGFLIIWV (H3)

A preceding paper has shown that a hempseed peptic hydrolysate displays a cholesterol-lowering activity with a statin-like mechanism of action in HepG2 cells and a potential hypoglycemic activity by the inhibition of dipeptidyl peptidase-IV in Caco-2 cells. In the framework of a research aimed at fostering the multifunctional behavior of hempseed peptides, we present here the identification and evaluation of some antioxidant peptides from the same hydrolysate. After evaluation of its diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, a trans-epithelial transport experiment was performed using differentiated Caco-2 cells that permitted the identification of five transported peptides that were synthesized and evaluated by measuring the oxygen radical absorbance capacity (ORAC), the ferric reducing antioxidant power (FRAP), and the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), and diphenyl-2-picrylhydrazyl radical DPPH assays. The most active peptides, i.e. WVSPLAGRT (H2) and IGFLIIWV (H3), were then tested in cell assays. Both peptides were able to reduce the H2O2-induced reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells, via the modulation of Nrf-2 and iNOS pathways, respectively.

The Klf6 Super-enhancer Determines Klf6 Sensitivity to BRD4 Inhibitors in Human Hepatoma (HepG2) Cells

Background: The Klf6 gene, belonging to Krüppel-like family of C2H2 zinc finger transcription factors, is strongly associated with tumor formation through high somatic mutations in carcinomas of the prostate, liver, colon, stomach, lung, neck, pituitary gland and nervous system. Recently, Klf6 super-enhancer which strongly regulates Klf6 gene expression has been identified, and the function of Klf6 super-enhancer which regulates cell growth was studied. Objective: The development of inhibitors targeting BRD4-binding super-enhancers is a potential target therapeutic strategy for tumor therapy. However, the suppression of Klf6 super-enhancer function by BRD4 inhibitors is not known. Methods: CRISPR-Cas9 editing technique was used for the Klf6 super-enhancer deletion experiment, and the expression levels of several genes for cell clones were detected by qRT-PCR analysis and Western blotting. Cell proliferation assay was applied to evaluate the functional role of Klf6 super-enhancer using several BRD4 inhibitors. The interaction of several BRD4 inhibitors against the target protein was analyzed by molecular docking simulation. Results: JQ-1, a human BRD4 inhibitor, inhibited Klf6 gene expression and its activity in HepG2 cells in a time and dose-dependent manner while simultaneously inhibiting cell growth. Besides, BETd-246, a human BRD4 inhibitor, strongly inhibited Klf6 gene expression, significantly inhibited cell growth, and exhibited higher efficacy than JQ-1. Molecular docking studies revealed that some key residues were critical for ligand-receptor interactions by forming hydrogen bonds with ligands (JQ-1: ASN140, BETd-246: ASN140, TYR106, LYS65, GLN58, MET105, and MET53). Conclusions: Our findings suggest that KLF6 is regulated by Klf6 super-enhancer and the targeting of Klf6 super-enhancer by BRD4 inhibitors may be an effective therapeutic strategy for liver cancer therapy.

Anticancer activity and mediation of apoptosis in hepatoma carcinoma cells induced by djulis and its bioactive compounds

• Betanin, rutin, kaempferol, quercetin and other compounds were present in EECF. • EECF and its bioactive compounds induced HepG2 cell apoptosis. • EECF and quercetin significantly reduced tumor growth in nude mice. • EECF could act as a potential source of liver cancer chemoprevention. Anticancer activity and mediation of apoptosis in hepatoma carcinoma cells induced by ethanolic extracts of djulis (EECF) and its bioactive compounds were investigated. EECF (50–500 μg/ml) exhibited significant cytotoxicity in HepG2 cells. AnnexinV-FITC/PI and DAPI staining assay showed that EECF induced apoptosis, accompanied with Sub-G0 phase arrest, ROS generation, loss of mitochondrial membrane potential (ΔΨm), increase of Bax/Bcl-2 ratio, caspase-3 activation and PARP cleavage. According to the HPLC-DAD and HPLC-MS/MS analysis, betanin, rutin, kaempferol, and quercetin were present in EECF, and showed antiproliferative action in HepG2 cells. Among the four bioactive compounds, quercetin showed the highest apoptotic effect toward HepG2 cells. In vivo , EECF and quercetin significantly reduced tumor growth in nude mice in tumor xenografts of HepG2 cells. Overall, the presence of bioactive compounds in EECF may account for the anticancer activity of EECF, and EECF could act as a potential chemopreventive agent against growth of hepatoma carcinoma cells.

Assessment of a panel of cellular biomarkers and the kinetics of their induction in comparing genotoxic modes of action in HepG2 cells.

One major challenge for in vitro genotoxicology is the determination of the genotoxic mode of action of tested compounds. The quantification of the phosphorylation of the histones H3 (pH3) and H2AX (γH2AX) allows an efficient discrimination between aneugenic and clastogenic compounds. However, these two biomarkers do not permit to deduct the specific mechanisms involved in the action of clastogenic compounds. The aim of this study was to investigate other possible cellular biomarkers allowing differentiating clastogenic properties. For this purpose, we analyzed γH2AX and pH3 plus six other biomarkers involved in the DNA damage signaling pathway in HepG2 cells treated with nine clastogens exhibiting different mechanisms of action, as well as one aneugen. All compounds were tested at various concentrations and with kinetics of 2, 6, 24 and 48 hr. Our results demonstrate the activation of the investigated biomarkers by the tested compounds in a time and concentration dependent manner. Notably, we observed for some nondirect genotoxic clastogens, notably dNTPs pool imbalance inducers, a different kinetic of DNA damage induction compared with direct genotoxins (oxidative stress). However, no specific biomarker signature of mechanisms of clastogenic action could be specified. Multiparametric analysis demonstrates a strong correlation between γH2AX and p-p53(S15) for clastogen compounds. Environ. Mol. Mutagen. 59:516-528, 2018. © 2018 Wiley Periodicals, Inc.

Zanthoxylum ailanthoides Suppresses Oleic Acid-Induced Lipid Accumulation through an Activation of LKB1/AMPK Pathway in HepG2 Cells.

Zanthoxylum ailanthoides (ZA) has been used as folk medicines in East Asian and recently reported to have several bioactivity; however, the studies of ZA on the regulation of triacylglycerol (TG) biosynthesis have not been elucidated yet. In this study, we examined whether the methanol extract of ZA (ZA-M) could reduce oleic acid- (OA-) induced intracellular lipid accumulation and confirmed its mode of action in HepG2 cells. ZA-M was shown to promote the phosphorylation of AMPK and its upstream LKB1, followed by reduction of lipogenic gene expressions. As a result, treatment of ZA-M blocked de novo TG biosynthesis and subsequently mitigated intracellular neutral lipid accumulation in HepG2 cells. ZA-M also inhibited OA-induced production of reactive oxygen species (ROS) and TNF-α, suggesting that ZA-M possess the anti-inflammatory feature in fatty acid over accumulated condition. Taken together, these results suggest that ZA-M attenuates OA-induced lipid accumulation and inflammation through the activation of LKB1/AMPK signaling pathway in HepG2 cells.

Inhibition of ERK1/2 Signaling Impairs the Promoting Effects of TGF-β1 on Hepatocellular Carcinoma Cell Invasion and Epithelial‐Mesenchymal Transition

Transforming growth factor-β (TGF-β) and ERK signaling have been implicated in various human cancers including hepatocellular carcinoma, but the underlying mechanism remains largely unclear. In this study, we aimed to explore the role of ERK1/2 in the regulation of TGF-β’s promoting and suppressive activities in HCC cells. Our data showed that treatment with TGF-β1 enhanced invasion and epithelial–mesenchymal transition (EMT) in HCC HepG2 cells, accompanied with increased MMP9 production and activation of Smad2/3 and ERK1/2, but inhibited tumor cell proliferation. These effects were eliminated by treatment with SB431542, a TGF-β inhibitor. Afterward, treatment with the MEK1/2 inhibitor U0126 reduced the TGF-β1-induced invasion and vimentin and MMP9 secretion in HepG2 cells, without affecting the inhibitory effects of TGF-β1 on HepG2 cell proliferation. Moreover, inhibition of Smad2/3 expression attenuated TGF-β1-induced cell invasion, ERK1/2 phosphorylation, and MMP9 production in HepG2 cells. However, knockdown of Slug only reduced cell invasion but did not affect ERK1/2 activation and MMP9 secretion in HepG2 cells. These data indicate that TGF-β1 activates ERK1/2 in HepG2 cells through the Smad2/3 pathway but not the Slug pathway. In summary, our study demonstrates that inhibition of ERK1/2 signaling attenuates the promoting effects of TGF-β1 on the metastatic phenotypes of HCC cells without affecting its suppressive effects on HCC cell proliferation. Therefore, we suggest that ERK1/2 may be used as a molecular target for the treatment of TGF-β-responsive HCC.

Free Fatty Acids Impair FGF21 Action in HepG2 Cells

Background/Aims: Fibroblast growth factor 21 (FGF21) is a key mediator of glucose and lipid metabolism. However, the beneficial effects of exogenous FGF21 administration are attenuated in obese animals and humans with elevated levels of circulating free fatty acids (FFA). Methods: We investigated in vitro how FFA impact FGF21 effects on hepatic lipid metabolism. Results: In the absence of FFA, FGF21 reduced lipogenesis and increased lipid oxidation in HepG2 cells. Inhibition of lipogenesis was associated with a down regulation of SREBP-1c, FAS and SCD1. The lipid-lowering effect was associated with AMPK and ACC phosphorylation, and up regulation of CPT-1α expression. Further, FGF21 treatment reduced TNFα gene expression, suggesting a beneficial action of FGF21 on inflammation. In contrast, the addition of FFA abolished the positive effects of FGF21 on lipid metabolism. Conclusion: In the absence of FFA, FGF21 improves lipid metabolism in HepG2 cells and reduces the inflammatory cytokine TNFα. However, under high levels of FFA, FGF21 action on lipid metabolism and TNFα gene expression is impaired. Therefore, FFA impair FGF21 action in HepG2 cells potentially through TNFα.

MiR-29a levels are elevated in the db/db mice liver and its overexpression leads to attenuation of insulin action on PEPCK gene expression in HepG2 cells

MicroRNAs comprise a class of small (∼22 nucleotide) non-coding RNA species and they bind to their complementary sequence on the 3′UTR of target genes and cause translational repression. In the present study, we report that miR-29a levels are significantly elevated in the diabetic db/db mice liver. Further, we report the effects of such elevation on insulin action in HepG2 cells. Overexpression of miR-29a narrowed down insulin mediated Akt phosphorylation without altering the total Akt levels presumably due to another upstream mediator being directly targeted by miR-29a. This hunt led us to the discovery that the p85α subunit of PI3K (phosphoionositide-3-kinase), the upstream molecule in the insulin signaling cascade harbors the miR-29a binding site on its 3′UTR and a marked inhibition of PI3Kp85α was observed by this microRNA. This was consequently accompanied by attenuation of insulin inhibition of PEPCK gene expression. All these events could be significantly prevented in the presence of the miR-29a inhibitor. Our results, for the first time, show the effect of miR-29a in counteracting insulin action on PEPCK gene expression by primarily targeting PI3K and abrogating downstream insulin signaling in HepG2 cells.

Autophagy Inhibition Enhances Apoptosis Induced by Ginsenoside Rk1 in Hepatocellular Carcinoma Cells

Our previous study indicated that ginsenoside Rk1 has anti-tumor activity and that its mode of action in HepG2 cells treated for 48 h involves coordinated inhibition of telomerase and induction of apoptosis. In the present study, we found that Rk1 induces both G(1) phase arrest and autophagy, but not apoptosis, at an earlier stage of treatment. A 24-h incubation of HepG2cells with Rk1 induced G(1) phase arrest. Rk1-induced autophagy was documented by the conversion of microtubule associated protein light chain 3 (LC3)-I to LC3-II, an autophagosome marker, and monodansylcadaverine (MDC) incorporation into autolysosomes. Combination of Rk1 with an autophagy inhibitor, such as bafilomycin A1 or beclin 1 siRNA, enhanced the anti-tumor effect of Rk1. These results imply that autophagy functions as a survival mechanism in HepG2 cells against Rk1-induced apoptosis. Taken together, our results support the use of autophagy inhibitors in combination with Rk1 as an effective anti-cancer regimen in HepG2 cells.

Coptis chinensis inhibits hepatocellular carcinoma cell growth through nonsteroidal anti-inflammatory drug-activated gene activation

Conventional chemotherapy of liver cancer fails to provide satisfactory remission and may cause serious side effects, thus it is crucial to derive alternative treatments that effectively inhibit cancer cell growth with known mechanisms of action. In the present study, we investigated the anti-carcinogenic effects of Coptis chinensis and its major constituent, berberine, in HepG2 hepatocellular carcinoma (HCC) cells and attempted to elucidate the underlying mechanism, including involvement of the nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1). Inhibition of cell proliferation, induction of apoptosis and cell cycle arrest at the G2/M phase were observed in HepG2 cells treated with Coptis chinensis or berberine. The pro-apoptotic effects were associated with corresponding down-regulation of Bcl-2, activation of procaspase-3 and -9 as well as cleavage of poly (ADP-ribose) polymerase. We further demonstrated the involvement of NAG-1 in the pro-apoptotic events following prior activation of its upstream transcriptional factor early growth response gene (Egr-1). This was confirmed by increased NAG-1 promoter activity preceded by the elevation of Egr-1/ DNA binding activity. Our results suggest that both Coptis chinensis and berberine are potential anti-carcinogenic agents in treating HCC by inducing cell cycle arrest and promotion of apoptosis, while NAG-1 is a molecular target during the drug-induced pro-apoptotic action in HepG2 cells.

Oxidized Metabolites of Oltipraz Exert Cytoprotective Effects against Arachidonic Acid through AMP-Activated Protein Kinase-Dependent Cellular Antioxidant Effect and Mitochondrial Protection

Oltipraz protects cells from chemical-induced carcinogenesis partly because of phase 2 enzyme induction. Certain oltipraz metabolites also induce phase 2 enzymes. This study investigated the cytoprotective effects of the oxidized metabolites of oltipraz against arachidonic acid (AA), a proinflammatory fatty acid that causes cellular reactive oxygen species (ROS) production and mitochondrial impairment, and the mechanistic basis of their action in HepG2 cells. Treatment with 4-methyl-5-(pyrazin-2-yl)-3H-1,2-dithiol-3-one (M1) or 7-methyl-6,8-bis(methylthio)H-pyrrolo[1,2-a]-pyrazine (M2), but not 7-methyl-8-(methylsulfinyl)-6-(methylthio)H-pyrrolo[1,2-a]pyrazine (M3) or 7-methyl-6,8-bis(methylsulfinyl)H-pyrrolo[1,2-a]pyrazine (M4), enabled cells to protect against AA-induced apoptosis. M1 and M2 treatment protected cells from ROS produced by AA and inhibited AA-induced glutathione depletion. Moreover, both M1 and M2 effectively inhibited mitochondrial dysfunction induced by AA, although M2 alone slightly elicited it at a relatively high concentration. M1 and M2 activated AMP-activated protein kinase (AMPK), but M3 and M4 failed to do so. AMPK activation by M1 and M2 contributed to cell survival against AA through a decrease in cellular ROS production and prevention of mitochondrial dysfunction, as shown by the reversal of the metabolites' restoration of mitochondrial membrane potential by compound C treatment or overexpression of a dominant-negative mutant AMPK. Consistently, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, an AMPK activator, also had a cytoprotective and antioxidant effect against AA. Our results demonstrate that, of the major metabolites of oltipraz, M1 and M2 are capable of protecting cells from AA-induced ROS production and mitochondrial dysfunction, which may be associated with AMPK activation.

Histone deacetylase inhibitors induce in human hepatoma HepG2 cells acetylation of p53 and histones in correlation with apoptotic effects

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

Green Tea Extract Enhances the Selective Cytotoxic Activity of Zizyphus jujuba Extracts in HepG2 Cells

Anticarcinogenic effects attributed to phytochemicals may be based on synergistic, additive, or antagonistic interactions of many compounds. In our previous study, we demonstrated that the chloroform fraction (CHCl(3)-F) from Z. jujuba has anticancer activity in HepG2 cells. In China, many people drink jujuba tea and believe in the synergic effects of jujuba and tea for better health. We therefore investigated the effects of CHCl(3)-F and green tea extract (GTE), and their underlying mechanisms of action in HepG2 cells. Our results showed that GTE enhanced the effect of CHCl(3)-F on cell viability in HepG2 cells, without cytotoxicity in rat hepatocytes, which was used as a normal cell model. Furthermore, combination of CHCl(3)-F and GTE caused an effect on G1 phase arrest but not on apoptosis. Interestingly, the mechanism of the G1 arrest was associated, not with an increase in p27(Kip1) levels and the hypohosphorylation of Rb, which are pathways used by CHCl(3)-F on G1 arrest in HepG2 cells, but with increases in p53 and p21(Waf1/Cip1) levels, and a decrease in cyclin E levels. Collectively, our findings suggest that combination of CHCl(3)-F and GTE produces an enhanced cell growth inhibition effect, and that the resultant G1 arrest was caused via a different mechanism as that of CHCl(3)-F treatment alone.

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.