Human Hepatoma Cell Line HepG2 Research Articles

Study on chemical constituents of seeds of Croton tiglium and their cytotoxicities

Seven compounds were isolated from the seeds of Croton tiglium by preparative TLC, semi-preparative HPLC, and column chromatography over silica gel, MCI, and Sephadex LH-20, etc. Their structures were elucidated by spectroscopic data analysis as bis(2,3-dihydroxypropyl) nonanedioate (1), 12-O-(α-methyl)butyrylphorbol-13-decanoate (2), 12-O-tiglylphorbol-13-decanoate (3), (9S,10R,11E,13R)-9,10,13-trihydroxyoctadec-11-enoic acid (4), methyl (9S,10R,11E,13R)-9,10,13-trihydroxyoctadec-11-enoate (5), 4(1H)-quinolinone (6), and 5-hydroxy-2-pyridinemethanol (7). Compound 1 was a new compound and compounds 4-7 were isolated from family Euphorbiaceae for the first time. Compounds 2 and 3 showed cytotoxic activities against human lung cancer cell line A549 with IC₅₀ values of 47.8 and 7.0 μmol•L ⁻¹, respectively, and against human hepatocarcinoma cell line HepG2 with IC₅₀ values of 71.4 and 44.0 μmol•L ⁻¹, respectively.

Insulin-Mediated Downregulation of Apolipoprotein A-I Gene in Human Hepatoma Cell Line HepG2: The Role of Interaction Between FOXO1 and LXRβ Transcription Factors.

Apolipoprotein A-I (ApoA-I) is a key component of high density lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα, and LXRβ transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 h, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRβ, or LXRα abrogated this effect. FOXO1 forms a complex with LXRβ and insulin treatment impairs FOXO1/LXRβ complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα, and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRβ complex, FOXA2, and LXRα on hepatic enhancer of apoA-I gene. J. Cell. Biochem. 118: 382-396, 2017. © 2016 Wiley Periodicals, Inc.

MicroRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line.

BackgroundIt is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation.MethodsTo determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes.ResultsThe hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress.ConclusionThese results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2762-7) contains supplementary material, which is available to authorized users.

Enhanced pro-protein convertase subtilisin/kexin type 9 expression by C-reactive protein through p38MAPK-HNF1α pathway in HepG2 cells.

Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.

Suppression of StarD7 promotes endoplasmic reticulum stress and induces ROS production

StarD7 is an intracellular lipid transport protein identified as up-regulated in the choriocarcinoma JEG-3 cell line. StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria, and StarD7 knockdown causes a reduction in phospholipid synthesis. Since inhibition of PC synthesis may lead to endoplasmic reticulum (ER) stress we hypothesized that StarD7 may be involved in maintaining cell homeostasis. Here, we examined the effect of StarD7 silencing on ER stress response and on the levels of reactive oxygen species (ROS) in the human hepatoma cell line HepG2. StarD7 knockdown induced alterations in mitochondria and ER morphology. These changes were accompanied with an ER stress response as determined by increased expression of inositol-requiring enzyme 1α (IRE1α), calnexin, glucose regulated protein 78/immunoglobulin heavy chain-binding protein (Grp78/BiP), protein kinase-like ER kinase (PERK) as well as the phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α). Additionally, a downregulation of the tumor suppressor p53 by a degradation mechanism was observed in StarD7 siRNA cells. Furthermore, StarD7 silencing induced ROS generation and reduced cell viability after H2O2 exposure. Decreased expression of StarD7 was associated to increased levels of the heme oxygenase-1 (HO-1) and catalase enzymes as well as in catalase enzymatic activity. Finally, no changes in levels of autophagy and apoptosis markers were observed in StarD7 siRNA treated cells respect to control cells. Taken together, these results indicate that StarD7 contributes to modulate cellular redox homeostasis.

Anchoring PEG-oleate to cell membranes stimulates reactive oxygen species production

Polyethylene glycol (PEG) derivatives possessing oleyl and reactive groups for conjugating functional substrates, such as proteins and quantum dots, are useful materials for cell-surface engineering and cell immobilization onto substrates. The reagent is known as a biocompatible anchor for cell membranes (BAM). Here, BAM-anchoring on cell membranes is reported to stimulate reactive oxygen species (ROS) production in those cells. Significant increases in ROS production and release to the surrounding environment were detected in mouse fibroblast cell line 10T1/2 when soaked in a solution containing BAM conjugated with 1/10mol/mol bovine serum albumin at 1.5μM-protein. ROS production stimulation was confirmed to be independent of the protein crosslinked with BAM and of cell type. Similar stimulation was detected for BAMs conjugated with ovalbumin and casein, in human hepatoma cell line HepG2, and human umbilical vein endothelial cells. Considering the effects of ROS on a variety of cellular processes, these results demonstrated the necessity for focusing attention on the effects of generated and released ROS on the behaviors of cells in the studies applying BAM to cells.

The Limonoids TS3 and Rubescin E Induce Apoptosis in Human Hepatoma Cell Lines and Interfere with NF-κB Signaling.

Hepatocellular carcinoma (HCC) is extremely resistant towards pharmacological therapy. To date, the multi-kinase inhibitor Sorafenib is the only available therapeutic agent with the potential to prolong patient survival. Using the human hepatoma cell lines HepG2 and Huh7, we analyzed anti-cancer activities of 6 purified havanensin type limonoids isolated from the traditional African medicinal plant Trichilia rubescens Oliv. Our results show that two of the compounds, TR4 (TS3) and TR9 (Rubescin E) reduced hepatoma cell viability, but not primary hepatocyte viability, at TC50s of 5 to 10 μM. These were significantly lower than the TC50s for Sorafenib, the histone deacetylase inhibitor SAHA or 5-Fluoruracil. In comparison, TR3 (Rubescin D), a limonoid isolated in parallel and structurally highly similar to TR4 and TR9, did not interfere with hepatoma cell viability. Both, TR4 and TR9, but not TR3, induced apoptosis in hepatoma cells and interfered with NF-κB activation. TR4 as well as TR9 significantly supported anti-cancer activities of Sorafenib. In summary, the limonoids TR4 and TR9 exhibit anti-cancer activities and support Sorafenib effects in vitro, having the potential to support future HCC therapy.

In vitro toxicity of reuterin, a potential food biopreservative

Reuterin has a high potential as a food preservative due to both its chemical characteristics and its antimicrobial activity against food-borne pathogens and spoilage bacteria. However, there is a lack of information about its toxicity and its capacity to interfere with the metabolism of drugs by inhibiting cytochrome P450 (CYP) activity. The results of this study indicated that reuterin exhibited a moderate cytotoxicity in the human hepatoma cell line HepG2 according to assays measuring three different endpoints in the same set of cells. Reuterin was much less toxic than acrolein and only four times more toxic than diacetyl, a generally recognized as safe flavoring compound. In vitro experiments utilizing human liver microsomes showed that reuterin presents low possibility of displaying in vivo drug interactions by inhibition of CYP3A4, CYP2D6, and CYP2C9. Therefore, reuterin can be considered a promising food biopreservative, although additional toxicology research is needed before permission for use can be granted.

Effect of Fanbaicao (Herba Potentillae Discoloris) oil on the expression of p21 and CDK4 in HepG2 cells

ObjectiveTo research the anti-cancer mechanism of the Traditional Chinese Medicine Fanbaicao (Herba Potentillae Discoloris) oil in the human hepatoma cell line HepG2. MethodsGas chromatography was used to analyze the components of Fanbaicao (Herba Potentillae Discoloris). We tested the inhibitory effect of Fanbaicao (Herba Potentillae Discoloris) oil on the human hepatoma cell line HepG2 in vitro using 3-(4, 5-Dimet hylt hiazol-2-yl)-2,5-dip henyltetrazolium bromide assays. Fluorescence activating cell sorter analysis was used to examine the levels of apoptosis, and western blot and immunofluorescence were used to detect the expression of p21, p-p21 and CDK4 proteins. ResultsFanbaicao (Herba Potentillae Discoloris) oil contains 45 ingredients, and L-ascorbic acid 2, 6-bispalmitate was the main component and accounted for 44.96% of total drive-off peak area. Other components included (Z)−14-met hyl-8-exadecenal-acetal (8.56%), phytol (7.74%) and lauric acid (6.31%). Fanbaicao (Herba Potentillae Discoloris) oil treatment reduced the proliferation of HepG2 cells and the half growth inhibition concentration (IC50) was 2.03 mg/mL. Furthermore, we also observed significantly increased HepG2 cell apoptosis in a dose-dependent manner (P < 0.05). Fanbaicao (Herba Potentillae Discoloris) oil significantly increased the expression of p21 and p-p21 and significantly decreased the expression of CDK4 in HepG2 cells compared with controls (P < 0.01). ConclusionOur results showed that Fanbaicao (Herba Potentillae Discoloris) oil has anti-cancer activities in HepG2 cells, which is probably related to the upregulation of p21 and p-p21 and downregulation of CDK4 expression.

Spacer Length: A Determining Factor in the Design of Galactosyl Ligands for Hepatoma Cell-Specific Liposomal Gene Delivery.

Use of nucleic acids to treat acquired or inherited hepatic diseases has considerable potential. Although recombinant viruses are popular vectors, interest in cheaper, often less immunogenic, non-viral modalities, is increasing. Thus hepatotropic, galactosylated lipoplexes directed to the hepatic asialoglycoprotein receptor (ASGP-R) are promising candidates. Here we examine the effect that galactosyl ligand spacer length has on the transfection activity of ASGP-Rtargeted lipoplexes in the human hepatoma cell line HepG2. Galactosyl ligands with spacer lengths in the range 2.4-24.1 Å were prepared and formulated into lipoplexes that were characterized by cryo-TEM, band shift, dye displacement and nuclease digestion assays. Cytotoxicity and transfection profiles were determined in liver-derived HepG2 cells and the renal ASGP-R-negative HEK293 line. Lipoplexes, which formed at endpoint +/- charge ratios in the range 1:1-3:1, accorded cargo DNA good protection from serum nuclease digestion and were well-tolerated by both cell lines. Transfection activities in the hepatoma cell line decreased markedly in the presence of a competing ASGP-R cognate ligand and also as the ligand spacer length increased, while activities in HEK293 cells were significantly lower (P &lt;0.05-0.001). Targeted lipoplexes enter HepG2 cells by receptor mediation and the uptake of transfecting complexes and those displaying more rigid short and medium length spacers is more efficient. This observation will inform the design of hepatotropic lipoplexes that are suitable for applications in vivo.

Diameter size and aspect ratio as critical determinants of uptake, stress response, global metabolomics and epigenetic alterations in multi-wall carbon nanotubes

The present study aimed to refurbish the fact of size and aspect ratio specific biological response of MWCNT in different cell systems. The bioactivities of three MWCNTs, differing in length/diameter [10–30 μm/20–30 nm (LW); 10–30 μm/8–15 nm (LN); 0.5–2.0 μm/8–15 nm (SN)], were then evaluated in terms of uptake and various toxicological end points (anti-proliferative, oxidative stress, inflammatory, DNA damage-repair and differential mode of marker gene expressions) in two different cell types - human bronchial epithelial (BEAS-2B) and human hepatoma (HepG2) cell lines. The surface enhanced Raman scattering (SERS) with dark-field microscopy attested the intracellular uptake of SN, but not the LW/LN, supports length dependency. The MWCNTs showed size specific stress responses (greatest in short sized one, SN) in both type of cells and aspect ratio dependent pro-fibrogenic potentiality, the LN with highest aspect ratio exhibited highest fibrogenicity in lung cells. Furthermore, alterations in global metabolomics and global DNA methylation status (DNMT3B dependent hypo-methylation in BEAS-2B cells and hyper-methylation in HepG2 cells) were also evident. Taken together, our findings provide evidence of variations in stress response, epigenetic modifications and global metabolomics alterations in association with differential size and aspect ratio of MWCNT which might be translated to the safe-by-design concept as well as risk assessment.

Abstract 4742: Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists

Abstract We have already found that a nuclear receptor PXR can modulate the cytotoxicity of CDDP for cancer cells, and ketoconazole, a PXT antagonist, can be an agent enhancing the antitumor activity of CDDP due to increase of the intracellular platinum content as the mechanism. However, nuclear receptor ligands have a variety of functions, and there is a possibility that PXR antagonist may enhance the cytotoxicity of CDDP through the mechanism other than the inhibition of transporter-mediated efflux of CDDP. The goal of this study was to compare the enhancing ability of three PXR antagonists, ketoconazole (KTZ), phenethyl isothiocyanate (PEITC) and metformin (MFM), for the antitumor effect of CDDP and clarify the contribution of inhibition of the efflux transporter as the mechanism of PXR antagonist. Cell lines used in this study were human hepatoma cell line HepG2. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Total intracellular platinum contents were determined by ICP-AES. When HepG2 cells were treated for 24 hr with 10 μM of CDDP, no change of caspase-3 activity was observed when compared to control cells. Also, 30 μM of KTZ, 30 μM of PEITC or 5 mM of MFM had no affect on the caspase-3 activity in treated cells compared with control cells. When KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 24-hr treatment with 10 μM of CDDP, the caspase-3 activity was significantly increased to 461%, 355% and 326% of the control, respectively, in comparison with that in CDDP alone. When HepG2 cells were treated for 8 hr with 25 μM of CDDP, the intracellular platinum content was 110 ng/mg protein. On the other hand when KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 8-hr treatment with 25 μM of CDDP, the intracellular platinum content was apparently increased to 227%, 491% and 161% of CDDP alone, respectively. Three PXR antagonists resulted in an increase in the intracellular platinum content that seems to be due to inhibition of the platinum efflux transporter, but there is no correlation between increase in intracellular platinum content and enhancement of antitumor activity by PXR antagonists. These results concluded that PXR antagonist can be an agent enhancing the antitumor activity of CDDP, but the ability of PXR antagonist for enhancing antitumor activity of CDDP can not be determined from only the increase in the intracellular platinum content. Citation Format: Shuichi Kishimoto, Megumi Yasuda, Megumi Tomita, Yuki Yamane, Ryosuke Suzuki, Shoji Fukushima. Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4742.

Coating independent cytotoxicity of citrate- and PEG-coated silver nanoparticles on a human hepatoma cell line

The antibacterial potential of silver nanoparticles (AgNPs) resulted in their increasing incorporation into consumer, industrial and biomedical products. Therefore, human and environmental exposure to AgNPs (either as an engineered product or a contaminant) supports the emergent research on the features conferring them different toxicity profiles. In this study, 30nm AgNPs coated with citrate or poly(ethylene glycol) (PEG) were used to assess the influence of coating on the effects produced on a human hepatoma cell line (HepG2), namely in terms of viability, apoptosis, apoptotic related genes, cell cycle and cyclins gene expression. Both types of coated AgNPs decreased cell proliferation and viability with a similar toxicity profile. At the concentrations used (11 and 5μg/mL corresponding to IC50 and ~IC10 levels, respectively) the amount of cells undergoing apoptosis was not significant and the apoptotic related genes BCL2 (anti-apoptotic gene) and BAX (pro-apoptotic gene) were both downregulated. Moreover, both AgNPs affected HepG2 cell cycle progression at the higher concentration (11μg/mL) by increasing the percentage of cells in S (synthesis phase) and G2 (Gap 2 phase) phases. Considering the cell-cycle related genes, the expression of cyclin B1 and cyclin E1 genes were decreased. Thus, this work has shown that citrate- and PEG-coated AgNPs impact on HepG2 apoptotic gene expression, cell cycle dynamics and cyclin regulation in a similar way. More research is needed to determine the properties that confer AgNPs at lower toxicity, since their use has proved helpful in several industrial and biomedical contexts.

Arsenic Metabolism and Toxicity Influenced by Ferric Iron in Simulated Gastrointestinal Tract and the Roles of Gut Microbiota.

Iron (Fe) is a common trace element in drinking water. However, little is known about how environmental concentrations of Fe affect the metabolism and toxicity of arsenic (As) in drinking water. In this study, influence of Fe at drinking water-related concentrations (0.1, 0.3, and 3 mg Fe (total)/L) on As metabolism and toxicity, and the roles of gut microbiota during this process were investigated by using in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME). Results showed that Fe had ability to decrease bioaccessible As by coflocculation in small intestine. 0.1 and 0.3 mg/L Fe significantly increased As methylation in simulated transverse and descending colon. Gut microbiota played an important role in alteration of As species, and Fe could affect As metabolism by changing the gut microbiota. Bacteroides, Clostridium, Alistipes, and Bilophila had As resistance and potential ability to methylate As. Cytotoxicity assays of effluents from simulated colons showed that the low levels of Fe decreased As toxicity on human hepatoma cell line HepG2, which might be due to the increase of methylated As. When assessing the health risk of As in drinking water, the residual Fe should be considered.

Chronic ethanol treatment depletes glutathione regeneration capacity in hepatoma cell line HepG2

Chronic alcohol abuse is the leading cause of liver cirrhosis in western countries. Ethanol, even at lower concentrations, can cause pleiotropic effects at the cellular level such as the formation of reactive oxygen species (ROS) and DNA damage. Ethanol is oxidized to acetaldehyde by liver alcohol dehydrogenase and by a microsomal ethanol-oxidizing system which is dependent on CYP2E1. There are numerous reports on CYP2E1-mediated formation of ROS. These lower the levels of intracellular glutathione, an efficient antioxidant. We were interested to investigate ethanol effects on glutathione levels independent of CYP2E1. We chose human hepatoma cell line HepG2 as a model known to lack physiological CYP2E1 expression. We found that 40 mM ethanol, a dosage comparable to blood ethanol concentration after heavy alcohol drinking, reduced intracellular HepG2 glutathione levels only by 18%. Within 24 hours, this effect could be normalized by the glutathione regeneration system. When HepG2 cells were exposed to 40 mM ethanol for one week, the cells gradually lost their ability to regenerate intracellular glutathione stores. We conclude that chronic ethanol exposure has a substantial effect on the glutathione regeneration capacity in liver cells which might contribute to alcohol-induced liver disease.

Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα.

ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H2S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H2S regulates ABCA1 expression. The effect of H2S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE−/− mice with a high-cholesterol diet. NaHS (an exogenous H2S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H2S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE−/− mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H2S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H2S. H2S may be a promising potential drug candidate for the treatment of atherosclerosis.

Chemopreventive effect of 18β-glycyrrhetinic acid via modulation of inflammatory markers and induction of apoptosis in human hepatoma cell line (HepG2).

Hepatocellular carcinoma is one of the most common lethal diseases worldwide and there is no effective treatment till date. Natural products derived from the plants play an important role in chemoprevention and act as therapeutic antitumor agents. Licorice is a plant that has been used in food and medicine for the treatment of various diseases. 18β-Glycyrrhetinic acid (18β-GA), a pentacyclic triterpenoid obtained from the roots of licorice plant, is reported to possess various pharmacological properties such as antitumor and antiinflammatory activities. The present study was designed to elucidate the chemopreventive effect of 18β-GA through antiinflammation, antiproliferation, and induction of apoptosis in human hepatoma cell line HepG2. 18β-GA significantly inhibits the proliferation of HepG2 cell without affecting the normal liver cell line (Chang's). In the present study, 18β-GA increased the formation of reactive oxygen species, nitric oxide production, and loss of mitochondrial membrane potential, suggesting the involvement of 18β-GA in apoptosis which was also confirmed by assessing the markers involved in apoptosis like caspase-3, caspase-9, Bax:Bcl-2 ratio, and cleaved PARP. 18β-GA also downregulated the expression of inflammatory proteins such as NF-κB, iNOS, and COX-2. Keeping these data into consideration, our results suggest that 18β-GA may be used as a chemopreventive agent in liver cancer.

Induction of micronuclei and alteration of gene expression by an organomodified clay in HepG2 cells

Clay2 is an organomodified montmorillonite developed by the Technological Institute of Packaging, Transport and Logistic (ITENE) in order to improve polymeric materials used in food packaging. There is not much known on Clay2 toxic potential, particularly at DNA level, therefore it is mandatory to assess its toxicity prior to its commercialization. In the present study the human hepatoma cell line (HepG2) was exposed to non-cytotoxic concentrations of Clay2 and the genomic stability was studied with the Cytokinesis block micronucleus cytome assay, by determining the formation of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Moreover, the expression of various genes involved in the mechanisms of its action using the real-time quantitative PCR was studied. The results obtained provide the evidence that Clay2 is potentially genotoxic as it increased the frequency of micronuclei. In addition it deregulated genes involved in the metabolism, immediate-early response/signaling, DNA damage and oxidative stress showing new valuable information on the cellular response to Clay2. Nonetheless, further studies are highly needed to elucidate the molecular mechanisms of clays toxicity.

Atg5 siRNA inhibits autophagy and enhances norcantharidin-induced apoptosis in hepatocellular carcinoma

Background: Cantharidin is a terpenoid isolated from Chinese blister beetles, and norcantharidin (NCTD) is a demethylated analog of cantharidin. It has been reported that cantharidin and norcantharidin have anticancer activities. Growing evidence suggests that inhibiting autophagy can induce apoptosis in the human hepatoma cell line HepG2. The objective of the present study was to determine whether inhibition of autophagy enhances NCTD-induced apoptosis in HepG2 cells. Methods: HepG2 cells were cultured in DMEM containing NCTD. Autophagy was upregulated in the presence of HBSS media supplemented with Ca2+ and Mg2+ and 10 mM HEPES and downregulated in the presence of 3-methyladenine (3-MA) and Atg5 siRNA. Autophagy, cell viability, and the expression of apoptotic proteins were assessed in HepG2 cells. Results: Our data showed that cell apoptosis generally increased after norcantharidin treatment in HepG2 cells. Expression of LC3-II, an autophagosomemarker, increased when cells were treated with HBSS media. It also increased cell viability. However, in the presence of 3-MA and Atg5 siRNA, autophagy was inhibited, LC3-II expression decreased and cell apoptosis increased. There was increased expression of Bax, cytochrome c, cleaved caspase-3, caspase-9 and PARP and the mitochondrial membrane potential was disrupted. Additionally, increased apoptosis was accompanied by increased reactive oxygen species (ROS) production. Conclusion: NCTD has anticancer activity, and Atg5 siRNA-mediated downregulation of autophagy enhanced its anticancer actions due to ROS generation and activation of the mitochondrial apoptosis pathway.

3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation

3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model.