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<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i>) is a popular fruit worldwide with natural antioxidant properties. This study examined how pineapple modified the expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and SULT1A1) and a drug transporter (OATP1B1) in human hepatocarcinoma (HepG2) cells. <b>Materials and Methods:</b> HepG2 cells (2.5×10<sup>5</sup> cells/well in a 24-well plate) were incubated with pineapple juice extract (125-1,000 μg mL<sup>1</sup>) for 48 hrs in phenol red-free medium. Resazurin reduction, ROS, AST and ALT assays were performed. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple juice slightly reduced HepG2 cell viability to 80% of the control, while ROS, AST and ALT levels were not changed. Pineapple juice did not alter the expression of CYP1A2, CYP2C9 and UGT1A6 mRNA. All tested concentrations of pineapple juice suppressed CYP3A4, NAT2 and OATP1B1 expression, while SULT1A1 expression was induced. <b>Conclusion:</b> Though pineapple juice slightly decreased the viability of HepG2 cells, cell morphology and cell function remained normal. Pineapple juice disturbed the expression of phase I (CYP3A4) and phase II (NAT2 and SULT1A1) metabolizing genes and the drug transporter OATP1B1. Therefore, the consumption of excessive amounts of pineapple juice poses a risk for drug interactions.

Background and Aims: Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis., Approach and Results: In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels., Conclusions: LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD., (© 2020 by the American Association for the Study of Liver Diseases.)

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involves the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.

To build a microfluidic device with various morphological features of the tumor vasculature for study of the effects of tumor vascular structures on the flow field and tumor cellular flow behaviors. The designed microfluidic device was able to approximatively simulate the in vivo structures of tumor vessels and the flow within it. In this models, the influences of the angle of bifurcation, the number of branches, and the narrow channels on the flow field and the influence of vorticity on the retention of HepG2 cells were significant. Additionally, shear stress below physiological conditions of blood circulation has considerable effect on the formation of the lumen-like structures (LLSs) of HepG2 cells. These results can provide some data and reference in the understanding of the interaction between hemorheological properties and tumor vascular structures in solid tumors., (Copyright © 2018. Published by Elsevier Inc.)

Background &aims:Low levels of adiponectin (APN), an adipose-derived adipokine, are associated with obesity and non-alcoholic steatohepatitis although its role in high-fat diet-induced hepatic injury and steatosis remains unclear. Here we hypothesized that APN deficiency alters fat diet-induced hepatic function. To this end, we examined the effect of APN deficiency on high-fat diet-induced hepatic injury, apoptosis and steatosis., Methods: Adult wild type and APN knockout mice were fed a low- or high-fat diet for 20 weeks. Serum levels of liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, hepatic triglycerides, steatosis, pro-inflammatory cytokines, apoptosis and autophagy were examined., Results: High-fat feeding led to elevated body (48.2%) and liver weights (18.8%), increased levels of ALT (87.8%), serum cholesterol (104.4%), hepatic triglycerides (305.6%) and hepatic fat deposition as evidenced by Oil Red O staining, along with a reduced AST/ALT ratio and unchanged AST. Although APN knockout itself did not affect hepatic function and morphology, it reconciled fat diet-induced hepatic injury (P<0.05 vs WT-HF group) without reversing changes in body and liver weights, serum cholesterol and hepatic steatosis. In addition, fat diet intake promoted AMPK phosphorylation, p62 accumulation and apoptosis, including elevated Bax and cleaved Caspase-3 and downregulated Bcl-2, along with suppressed phosphorylation of Akt, STAT3 and JNK, and the autophagy makers Atg7, Beclin-1 and LC3B (P<0.05 vs WT-LF group) without affecting hepatic interlelukin-6 and tumor necrosis factor-α levels, the effects were reversed or significantly attenuated by APN knockout (P<0.05 vs WT-HF group). In vitro study using HepG2 cells revealed that STAT3 activation rescued palmitic acid-induced cell injury whereas STAT3 inhibition nullified APN knockdown-offered beneficial effects., Conclusions: Our results revealed that high-fat diet intake promotes hepatic steatosis, apoptosis and interrupted autophagy. APN knockout elicits protective effect against hepatic injury possibly associated with autophagy regulation despite persistent hepatic steatosis.

Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC 50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.

Objective: To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates. Methods: AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC). Results: On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all P <0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all P <0.01). Conclusion: Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.

Background: Although hepatocellular carcinoma cells can sometimes undergo differentiation in an embryonic microenvironment, the mechanism is poorly understood., Aim: The developmental stage-specific embryonic induction of tumor cell differentiation was investigated., Methods: Both chick and mouse liver extracts and hepatoblast-enriched cells at different developmental stages were used to treat human hepatoma HepG2 cells, and the effects on the induction of differentiation were evaluated. The nuclear factors controlling differentiation, hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and upstream stimulatory factor-1 (USF-1), and the oncogene Myc and alpha-fetoprotein (AFP) were measured. HNF-4α RNA interference was used to verify the role of HNF-4α. Embryonic induction effects were further tested in vivo by injecting HepG2 tumor cells into immunodeficient nude mice., Results: The 9-11-days chick liver extracts and 13.5-14.5-days mouse hepatoblast-enriched cells could inhibit proliferation and induce differentiation of HepG2 cells, leading to either death or maturation to hepatocytes. The maturation of surviving HepG2 cells was confirmed by increases in the expressions of HNF-4α, HNF-1α, HNF-6, and USF-1, and decreases in Myc and AFP. The embryonic induction of HepG2 cell maturation could be attenuated by HNF-4α RNA interference. Furthermore, the 13.5-days mouse hepatoblast culture completely eliminated HepG2 tumors with inhibited Myc and induced HNF-4α, confirming this embryonic induction effect in vivo., Conclusions: This study demonstrated that developmental stage-specific embryonic induction of HepG2 cell differentiation might help in understanding embryonic differentiation and oncogenesis.

Staphylococcal enterotoxins C2 (SEC2) is a classical model of superantigens (SAg), which has the powerful ability to activate T cells as well as induce massive cytokine production. This property makes SEC2 and its mutants well concerned as a potential new immune-regulatory agent for cancer therapy. We previously constructed a SEC2 mutant named SAM-3, which had prominently antitumor activity in BALB/c mice model. But, the underlying molecular mechanism for stimulation of human peripheral blood mononuclear cells (PBMCs) and antitumor effect on human tumor cells induced by SAM-3 is not clear. Here, we showed that SAM-3 could activate human TCR Vβ 12, 13A, 14, 15, 17, and 20 CD8(+) subgroup T cells, which secreted the cytokines IL-2, IFN-γ, and TNF-α, and exhibit stimulation activity in a dose-dependent manner. TNF-α secreted from activated T cells could induce apoptosis and G1-phase arrest and lead to the antitumor effect in HepG2 cells. Meanwhile, SAM-3 upregulated the expression of tumor necrosis factor receptor 1 (TNFR1) mRNA and activity of caspase-3 and caspase-8. We also found that the antitumor activity and activity of caspase-3 and caspase-8 were decreased when the neutralizing TNF-α monoclonal antibody presented. These data suggest that TNF-α secreted by SAM-3-activated T cells is an important factor in inducing apoptosis in HepG2 cells.

Alcoholic liver disease is a major source of morbidity and mortality worldwide. Twin studies had demonstrated heritability of alcoholic liver disease. Although to date only Adiponutrin (PNPLA3) rs738409 polymorphism (I148M) had been unequivocally proved to be associated with increased risk of alcoholic liver disease across different ethnicities. This protein was previously thought to have a predominant lipolytic role. However, recent investigations have provided evidence of lipogenic activity of this protein. The current hypothesis paper is summarizing the recent evidences gleaned in biological role of Adiponutrin and bioinformatic pointers towards a role in lipid trafficking. A critical appraisal of the utility of murine models and cell based systems in investigating Adiponutrin is also presented. As the HepG2 cell line harbors the I148M mutation in homozygous state it is hypothesized that this should represent an ideal model system for PNPLA3 biology. Thus, as Adiponutrin is proposed as having both lipolytic and lipogenic/lipid trafficking roles it is termed as a Yin-Yang protein in analogy to ancient Chinese wisdom., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as "type II programmed cell death," and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase-9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox-treated HepG2 cells. Olaquindox-induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy-related MAP-LC3 and Beclin 1 proteins. The autophagy inhibitor 3-methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl-2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c-Jun N-terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox-induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox-induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox-induced apoptosis can be enhanced by 3-methyladenine. Olaquindox-induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS-dependent JNK., (Copyright © 2014 John Wiley & Sons, Ltd.)

3D (three-dimensional) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. In the present study, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. Apo (Apolipoprotein)B as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP (cytochrome P450) enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enables high-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs., (© 2015 Authors.)

Background/purpose: Using gradient ionic detergent, we optimized the preparation procedure for the decellularized liver biologic scaffold, and analyzed its immunogenicity and biocompatibility., Methods: EDTA, hypotonic alkaline solution, Triton X-100, and gradient sodium dodecyl sulfate (1%, 0.5%, and 0.1%, respectively) were prepared for continuous perfusion through the hepatic vascular system. The decellularization of the liver tissue was performed with the optimized reagent buffer and washing protocol. In addition, the preservation of the original extracellular matrix was observed. To analyze its biocompatibility, the scaffold was embedded in a heterologous animal and the inflammation features, including the surrounding cell infiltration and changes of the scaffold architecture, were detected. The cell-attachment ability was also validated by the perfusion culture of HepG2 cells with the scaffold., Results: By using gradient ionic detergent, we completed the decellularization process in approximately 5 h, which was shorter than >10 hours in previous experiments (p<0.001). The extracellular matrix was kept relatively intact, with no obvious inflammatory cellular infiltration or structural damage in the grafted tissue. The engraftment efficiencies of HepG2 were 86±5% (n=8). The levels of albumin and urea synthesis were significantly superior to the ones in traditional two-dimensional culture., Conclusion: The current new method can be used efficiently for the decellularization of the liver biologic scaffold with satisfying biocomparability for application both in vivo and in vitro., (Copyright © 2013. Published by Elsevier B.V.)

Much of the difficulty in elucidating the precise function of S100 protein family has been attributed to functional redundancy and compensation by its conserved family members. In this study, we showed that seven S100 family members were almost totally undetectable in HepG2.2.15 cells, while all of them were highly expressed in its parental HepG2 cells. Re-expression of S100 proteins in HepG2.2.15 cells can partially rescue their defects in cell protrusion and migration through the regulation of cytoskeletons and adhesions. Thus, HepG2.2.15 can serve as a useful model for studying cell protrusion and migration regulated by S100 proteins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We report a biocompatible method of selectively retrieving 3-D cell-encapsulating hydrogel microstructures from a culture substrate. First, poly(l-lysine)/hyaluronic acid (PLL/HA) polyelectrolyte multilayers (PEMs) with methacrylated chitosan (GMA-Chi) on top were formed on an ITO substrate. Then, a cell-encapsulating hydrogel micropattern was formed; a HepG2 cell-encapsulating heparin-based hydrogel micropattern was fabricated by thiol-ene photopolymerization. The application of an oxidative potential of 2 V resulted in the detachment of the cell-encapsulating hydrogels by the dissolution of PEMs. The time of complete retrieval of the hydrogels was controllable by modulating the number of PEM layers. The applied potential did not affect the viability or the function of the cells in the entire hydrogels. In contrast, when a reductive electrochemical potential (-1.8 V) was applied to a silane-modified ITO to release cell-encapsulating hydrogels by the desorption of silane [Chem. Commun., 2009, 5865], extensive cell death at the bottom of the hydrogel adjacent to the electrode was observed.

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct "apicolateral" subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)-positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma., Competing Interests: The authors have declared that no competing interests exist.

Human mesenchymal stem cells (hMSCs) accumulate at carcinomas and have a great impact on cancer cell's behavior. Here we demonstrated that hMSCs could display both the promotional and inhibitive effects on growth of HepG2 and Hela cells by using the conditioned media, indirect co-culture, and cell-to-cell co-culture. Cell growth was increased following the addition of lower proportion of hMSCs while decreased by treatment of higher proportion of hMSCs. We also established a novel noninvasive label way by using internalizing quantum dots (i-QDs) for study of cell-cell contact in the co-culture, which was effective and sensitive for both tracking and distinguishing different cells population without the disturbance of cells. Furthermore, we investigated the role of hMSCs in regulation of cell growth and showed that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways were involved in hMSC-mediated cell inhibition and proliferation. Our findings suggested that hMSCs regulated cancer cell function by providing a suitable environment, and the discovery from the study would provide some clues for development of effective strategy for hMSC-based cancer therapies.

Background: Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure. In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells., Data Sources: A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the key words such as liver failure, bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed., Results: Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines, the incorporation of BAL with GS-HepG2 cells or alginate-encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved., Conclusions: Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functional hepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

Aim: To investigate the possible mechanism by which hepatitis B virus X protein (HBx) mediates apoptosis of HepG2 cells., Methods: HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high-expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX (HBx antagonist) were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases (MAPKs), were measured in each group., Results: Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and JNKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and JNKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group., Conclusion: HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.

A new lectin (42 kDa) from Musca domestica pupae (MPL) has been known to inhibit proliferation in tumor cells. In this study, flow cytometry analysis showed that MPL induced HepG2 cells apoptosis significantly and the cells were arrested at S phase. MPL inhibited IκB-α degradation and NF-κB/p65 translocation from cytoplasm into nucleus. Simultaneously, the expressions of FLIP, which is a target gene of NF-κB/p65 were down-regulated and the caspase-8 and caspase-3 were then activated to induce apoptosis. Taken together, these results showed that MPL induced a caspase-dependent apoptosis via NF-κB/p65 pathway in HepG2 cells.

Unlabelled: It is extensively accepted that hepatitis B virus (HBV) escapes from innate immunity by inhibiting type I interferon (IFN) production, but efficient intervention to reverse the immune tolerance is still not achieved. Here, we report that 5'-end triphosphate hepatitis B virus X gene (HBx)-RNAs (3p-HBx-short interfering [si]RNAs) exerted significantly stronger inhibitory effects on HBV replication than regular HBx-siRNAs in stably HBV-expressing hepatoplastoma HepG2.2.15 cells through extremely higher expression of type I IFNs, IFN-induced genes and proinflammatory cytokines, and retinoic acid inducible gene I (RIG-I) activation. Also, 3p-HBx-siRNA were more efficient to stimulate type I IFN response than HBx sequence-unrelated 3p-scramble-siRNA in HepG2.2.15 cells, indicating that a stronger immune-stimulating effect may partly result from the reversal of immune tolerance through decreasing HBV load. In RIG-I-overexpressed HepG2.2.15 cells, 3p-HBx-siRNAs exerted stronger inhibitory effects on HBV replication with greater production of type I IFNs; on the contrary, in RIG-I-silenced HepG2.2.15 cells or after blockade of IFN receptor by monoclonal antibody, inhibitory effect of 3p-HBx-siRNAs on HBV replication was largely attenuated, indicating that immunostimulatory function of 3p-HBx-siRNAs was RIG-I and type I IFN dependent. Moreover, in HBV-carrier mice, 3p-HBx-siRNA more strongly inhibited HBV replication and promoted IFN production than HBx-siRNA in primary HBV(+) hepatocytes and, therefore, significantly decreased serum hepatitis B surface antigen and increased serum IFN-β., Conclusion: 3p-HBx-siRNAs may not only directly inhibit HBV replication, but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV-induced immune tolerance., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized. A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12 percent v/v Me2SO Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis. The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to about 90 percent, 70 percent and 65 percent of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.

Hepatitis B x-interacting protein (HBXIP), a co-factor of survivin, was originally identified by its binding with the C-terminus of the hepatitis B virus x protein (HBx). We have recently shown that HBXIP promotes the growth of both normal liver cells and hepatoma cells in vitro, but the molecular mechanisms of this have not been documented. In this study, we investigated the potential effects of HBXIP on the proliferation of HepG2 cells and the intracellular signaling pathway mediating these changes. Over-expression of the HBXIP gene promoted the proliferation of HepG2 cells, as shown by the MTT assay. We also showed that HBXIP induced cellular accumulation in the S phase concomitantly with up-regulation of cyclinD(1) and down-regulation of p21 and p53 levels. Moreover, HBXIP over-expression cells showed activation of the PI3K/Akt pathway; this activation was accompanied by an increase in phosphorylation of glycogen synthase kinase 3β. LY294002, a specific inhibitor of PI3K, blocked HBXIP-stimulated Akt phosphorylation and suppressed the cell cycle promotion induced by HBXIP in HepG2 cells. The increase in cyclinD(1) protein levels induced by HBXIP was inhibited when cells were incubated with LY294002. In conclusion, our data suggest that the proliferation of HepG2 cells promoted by HBXIP is associated with activation of the PI3K/Akt signaling pathway.

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation., (Copyright 2010 Elsevier Inc. All rights reserved.)

This paper describes the alginate encapsulation of preformed high-density 3-D HepG2 cell aggregates that guarantees good maintenance of liver-specific biomarker expression. The process involves forming a high-density (> or =7 x 10(4) cells/aggregate) discoid 3-D cell aggregate in an ultrasound trap, which is subsequently recovered and encapsulated in alginate/CaCl(2) hydrogel. Glucose secretion/consumption, lactate release, detoxifying enzyme capacity, cytokeratin-18 expression as well as hypoxia were characterized in encapsulated 3-D HepG2 aggregates over 10 days in culture. Encapsulated 3-D HepG2 aggregates released glucose into the media, although this ability was exhibited only after 1 day in culture and was subsequently lost over the ensuing 9 days. In contrast, lactate was constantly released into the media. Significantly more lactate was secreted after 3 days in culture indicating a more hypoxic environment and hence a higher rate of anaerobic glycolysis. Aggregates consistently expressed cytokeratin-18. Cytochrome P450-1A1 activity reached a maximum on day 1 of culture followed by a progressive reduction to basal levels, while P450-3A4 activity was up-regulated in a time-dependent manner reaching a peak on day 7 in culture. Glutathione-S-transferase activity, on the other hand, was at more physiological levels and remained constant over the 10-day culture period. The ultrasound trap allowed the rapid (within 5 min) generation of uniformly shaped and sized aggregates. The results reported here suggest that ultrasound-formed 3-D HepG2 aggregates can serve as alternative in vitro models providing a quick outlook on toxicity, in a tissue-mimetic manner, thus offering the future option of a cost-effective screening platform for pharmaceutical development.

Saturated free fatty acids (FFAs), e.g. palmitate, have long been shown to induce toxicity and cell death in various types of cells. In this study, we demonstrate that cAMP synergistically amplifies the effect of palmitate on the induction of cell death in human hepatocellular carcinoma cell line, HepG2 cells. Elevation of cAMP level in palmitate-treated cells led to enhanced mitochondrial fragmentation, mitochondrial reactive oxygen species (ROS) generation and mitochondrial biogenesis. Mitochondrial fragmentation precedes mitochondrial ROS generation and mitochondrial biogenesis, and may contribute to mitochondrial ROS overproduction and subsequent mitochondrial biogenesis. Fragmentation of mitochondria also facilitated the release of cytotoxic mitochondrial proteins, such as Smac, from the mitochondria and subsequent activation of caspases. However, cell death induced by palmitate and cAMP was caspase-independent and mainly necrotic., (2009 Elsevier Inc. All rights reserved.)

The expression of genes encoding key hepatic gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), is regulated at the transcriptional level by a network of transcription factors and cofactors, including cAMP response element-binding protein (CREB). It has been suggested that increased endoplasmic reticulum (ER) stress in the liver impairs hepatic glucose metabolism. However, the direct effect of ER stress on hepatic gluconeogenesis is still not clear. Here, we investigated whether ER stress influences hepatic gluconeogenesis and whether this process is mediated by activating transcription factor 6 (ATF6) through the inhibition of cAMP-mediated activation of CREB. A cAMP stimulant, forskolin, and 8-bromoadenosine-cAMP increased PEPCK and G6Pase mRNA expression in H4IIE rat hepatoma cells, and ER stress induced by tunicamycin or thapsigargin decreased the expression of these genes in forskolin or 8-bromoadenosine-cAMP-treated cells. In a transient transfection study, ATF6 inhibited the PEPCK and G6Pase promoters. Also, adenovirus-mediated overexpression of ATF6 in H4IIE cells decreased forskolin-stimulated PEPCK and G6Pase gene expression. Moreover, the inhibition of endogenous ATF6 expression by small interfering RNAs restored the ER stress-induced suppression of PEPCK and G6Pase gene expression. Transient transfection of ATF6 inhibited transactivation by CREB on the PEPCK and G6Pase promoters, and a gel shift assay showed that Ad-ATF6 inhibits forskolin-stimulated CREB DNA-binding activity. Finally, we found that expression of ATF6 decreased fasting-induced PEPCK, G6Pase mRNA expression, and blood glucose levels in mice. Taken together, these data extend our understanding of ER stress and the regulation of liver gluconeogenesis by ATF6.

Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog. Reporter gene assays revealed that the SR ortholog has molecular functions similar to those of the vertebrate ER. Surprisingly, the ER ortholog is an estrogen-insensitive repressor of SR-mediated transcription. Furthermore, we found that the SR ortholog can bind to both estrogen-responsive elements (EREs) and androgen-responsive elements (AREs) and mediates transcriptional activation by estrogens through both types of elements. Our findings suggest that the ancestral SR, but not ER, could bind estrone and induce the ERE- and ARE-dependent transactivation and that it gained the ability to be regulated by ketosteroid and recognize ARE specifically before jawless vertebrates split. These results highlight the importance of comparative experimental approaches for the evolutionary study of endocrine systems.

Concerns exist that administration of intravenous (i.v.) iron preparations is associated with oxidative stress. Therefore iron sucrose (CAS 8047-67-4), ferric gluconate (CAS 34098-81-1) and iron dextran (CAS 9004-66-4) were assessed for redox-active iron by a dichlorofluorescein assay and for intracellular reactive oxygen species (ROS) generation and cytotoxicity in HepG2 cells. Examining each i.v. iron preparation at its maximum concentration achieved following clinically frequently used doses in a 70 kg individual in in vitro experiments, redox-active iron was highest with ferric gluconate, followed by iron dextran and iron sucrose. Interestingly, when the i.v. iron preparations were diluted in human serum instead of buffer, redox-active iron was highest with iron dextran, followed by iron sucrose, and practically disappeared with ferric gluconate. ROS production in HepG2 cells was increased by all i.v. iron preparations. However, in the neutral red cytotoxicity assay all i.v. iron preparations were non-toxic. In conclusion, ferric gluconate showed the highest increase in intracellular ROS-production in HepG2 cells and the highest amount of redox-active iron in buffer in the in vitro assays. In contrast to the other i.v. iron preparations, redox-active iron from ferric gluconate was rendered completely redox-inactive by serum, indicating that redox-active iron in the various preparations has different chemical properties.

Objective: Recently, several drugs have been shown to exert beneficial effects for metabolic syndrome through mild regulation of mitochondrial function. Hence, we explored a strategy of targeting mitochondrial function to improve glucose and lipid metabolism., Research Design and Methods: Mitochondrial membrane potential (Deltapsim) is a marker of mitochondrial function; therefore, we set up a high-throughput screening assay of Deltapsim in L6 myotubes. The effects of a selected lead compound were investigated in vitro and in vivo in relation to metabolic syndrome., Results: A novel small-molecule compound, C1, was identified through this high-throughput screening. C1 depolarized Deltapsim in L6 myotubes without cytotoxicity and led to increased cellular AMP-to-ATP ratio, activation of AMP-activated protein kinase (AMPK), and enhanced glucose uptake. It also stimulated the AMPK pathway in HepG2 cells, leading to decreased lipid content. Intriguingly, C1 inhibited respiration in L6 myotubes but did not affect respiration in isolated muscle mitochondria, suggesting that it may depolarize Deltapsim indirectly by affecting the supply of electron donors. Acute administration of C1 in C57BL/6J mice markedly increased fat oxidation and the phosphorylation of AMPK and acetyl-CoA carboxylase in the liver. In diabetic db/db mice, chronic administration of C1 significantly reduced hyperglycemia, plasma fatty acids, glucose intolerance, and the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver., Conclusions: Our results demonstrate a novel small molecule that mildly depolarizes Deltapsim and is able to improve glucose and lipid metabolism to exert beneficial effects for metabolic syndrome. These findings suggest that compounds regulating mitochondrial function may have therapeutic potential for type 2 diabetes.

The effects of ethanol extracts from Thai plants on P-glycoprotein (P-gp) function and cell viability were examined using paclitaxel-resistant HepG2 (PR-HepG2) cells. KP018 from Ellipeiopsis cherrevensis and AT80 from Ancistrocladus tectorius increased both rhodamine 123, a typical P-gp substrate, and [(3)H]paclitaxel uptake in PR-HepG2 cells. However, some extracts such as MT80 from Microcos tomentosa increased rhodamine 123, but not [(3)H]paclitaxel, uptake, while MM80 from Micromelum minutum increased only [(3)H]paclitaxel uptake. Thus, the effects of extracts of Thai plants on rhodamine 123 uptake were not necessarily the same as those on [(3)H]paclitaxel uptake. Purified compounds such as bergapten did not affect the uptake of either substrate. KP018, AT80, and MM80 increased [(3)H]paclitaxel uptake and decreased the cell viability in a concentration-dependent manner. Among these extracts, KP018 showed the most potent cytotoxicity. The cytotoxic potency of KP018 on PR-HepG2 cells was similar to that on wild-type HepG2 cells, and was not potentiated by verapamil. At concentrations resulting in no cytotoxicity, AT80 and MM80 potentiated paclitaxel-induced cytotoxicity in PR-HepG2 cells. These results indicate that K018 may be a useful source to search for a new anticancer drug, while AT80 and MM80 may be useful as modulators of P-gp-mediated multidrug resistance in cancer cells.