Expression In HepG2 Research Articles

A suppressive role of ionizing radiation-responsive miR-29c in the development of liver carcinoma via targeting WIP1.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, and it has been linked to radiation exposure. As a well-defined oncogene, wild-type p53-induced phosphatase 1 (WIP1) plays an inhibitory role in several tumor suppressor pathways, including p53. WIP1 is amplified and overexpressed in many malignancies, including HCC. However, the underlying mechanisms remain largely unknown. Here, we show that low-dose ionizing radiation (IR) induces miR-29c expression in female mouse liver, while inhibiting its expression in HepG2, a human hepatocellular carcinoma cell line which is used as a model system in this study. miR-29c expression is downregulated in human hepatocellular carcinoma cells, which is inversely correlated with WIP1 expression. miR-29c attenuates luciferase activity of a reporter harboring the 3'UTR binding motif of WIP1 mRNA. Ectopic expression of miR-29c significantly represses cell proliferation and induces apoptosis and G1 arrest in HepG2. In contrast, the knockdown of miR-29c greatly enhances HepG2 cell proliferation and suppresses apoptosis. The biological effects of miR-29c may be mediated by its target WIP1 which regulates p53 activity via dephosphorylation at Ser-15. Finally, fluorescence in situ hybridization (FISH) and immunohistochemical analyses indicate that miR-29c is downregulated in 50.6% of liver carcinoma tissues examined, whereas WIP1 is upregulated in 45.4% of these tissues. The expression of miR-29c inversely correlates with that of WIP1 in HCC. Our results suggest that the IR-responsive miR-29c may function as a tumor suppressor that plays a crucial role in the development of liver carcinoma via targeting WIP1, therefore possibly representing a target molecule for therapeutic intervention for this disease.

Mir‐548n Expression in HEPG2 and HEP2 cells Is Affected By The Culture Medium Zinc Concentration

The objective of this research was to study the impact of zinc depletion / supplementation on plasma microRNAs, which may be then used as biomarkers for zinc status. Clinical data from plasma from adolescent females on zinc supplementation (9 mg elemental Zn / wk for 4 weeks) exhibited changes in exosomal microRNAs of which mir-548n, showed a 57% increase. Hepatocyte (HEPG2) and lung epithelium (HEp2) cells were grown in normal (15μM), zinc depleted (Chelex-100 treated) or zinc supplemented (50μM) media for 48 hr to validate the clinical data. HEPG2 cells grown in 15 μM Zn vs. 0 μM Zn showed a 10-fold increase in miR-548n and remained at that level in cells grown in 50 μM Zn. In the HEp2 cells, mir-548n expression decreased 3-fold in cells grown in 15 μM Zn vs. 0 μM Zn, and increased 7-fold in cells grown in 50 μM Zn vs. 15 μM Zn. These data indicate that mir-548n expression is regulated by Zn. Its secretion into plasma exosomes isolated from human subjects may be indicative of its increased expression in tissues in response to Zn supplementation

The role of CD29-ILK-Akt signaling-mediated epithelial-mesenchymal transition of liver epithelial cells and chemoresistance and radioresistance in hepatocellular carcinoma cells.

Hepatocellular carcinomas (HCC) are aggressive cancers, and the prognosis of HCC patients is poor. This study investigated the roles of CD29 in epithelial-mesenchymal transition (EMT) and chemoresistance and radioresistance in HCC tumors. CD29 expression in HCC and peritumoral tissues was measured by immunohistochemistry. CD29 overexpression was established by an adenovirus-carrying CD29 gene expression cassette, while silencing of CD29 expression was established by an adenovirus-carrying shRNA. Western blot was used to measure protein expression, and MTT was used to analyze cell viability. Xenograft HCC mouse model was established by inoculating isolated CD29(+) and CD29(-) HCC tumor cells. Significantly higher percentage of positive CD29 expression was observed in HCC tissues compared to peritumoral tissues. Xenograft CD29(+) tumors grew more quickly than CD29(-) tumors. CD29(+) tumors were more resistant to radiotherapy and cisplatin therapy than CD29(-) tumors. Overexpression of CD29 significantly increased the resistance of CD29(-) tumors to radiation and cisplatin treatment. In contrast, silencing of CD29 expression significantly sensitized CD29(+) tumors to irradiation and cisplatin treatment. Overexpression of CD29 decreased E-cadherin, but increased fibronectin, vimentin, ILK activity, Akt Ser(473) phosphorylation, and mTORC1 protein expression in Hep G2 and THLE-3 cells. Moreover, overexpression of CD29 significantly increased the resistance of Hep G2 and THLE-3 cells to starvation, radiation, and cisplatin treatments. This study suggests that CD29 plays a crucial role in the resistance of HCC to chemo/radiotherapy and EMT of liver epithelial cells.

Gamma-tocotrienol treatment increased peroxiredoxin-4 expression in HepG2 liver cancer cell line.

BackgroundTo determine the antiproliferative effect of gamma-tocotrienol (GTT) treatment on differential protein expression in HepG2 cells.MethodsHepG2 cells were treated with 70 μM GTT for 48 hours and differentially expressed protein spots were determined by two-dimensional electrophoresis (2DE), identified by MALDI-TOF mass spectrometer (MS) and validated by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsGTT treatment on HepG2 cells showed a total of five differentially expressed proteins when compared to their respective untreated cells where three proteins were down-regulated and two proteins were up-regulated. One of these upregulated proteins was identified as peroxiredoxin-4 (Prx4). Validation by qRT-PCR however showed decreased expression of Prx4 mRNA in HepG2 cells following GTT treatment.ConclusionsGTT might directly influence the expression dynamics of peroxiredoxin-4 to control proliferation in liver cancer.

Repression of the Nuclear Receptor Small Heterodimer Partner by Steatotic Drugs and in Advanced Nonalcoholic Fatty Liver Disease

The small heterodimer partner (SHP) (NR0B2) is an atypical nuclear receptor that lacks a DNA-binding domain. It interacts with and inhibits many transcription factors, affecting key metabolic processes, including bile acid, cholesterol, fatty acid, and drug metabolism. Our aim was to determine the influence of steatotic drugs and nonalcoholic fatty liver disease (NAFLD) on SHP expression and investigate the potential mechanisms. SHP was found to be repressed by steatotic drugs (valproate, doxycycline, tetracycline, and cyclosporin A) in cultured hepatic cells and the livers of different animal models of NAFLD: iatrogenic (tetracycline-treated rats), genetic (glycine N-methyltransferase-deficient mice), and nutritional (mice fed a methionine- and choline-deficient diet). Among the different transcription factors investigated, CCAAT-enhancer-binding protein α (C/EBPα) showed the strongest dominant-repressive effect on SHP expression in HepG2 and human hepatocytes. Reporter assays revealed that the inhibitory effect of C/EBPα and steatotic drugs colocalize between -340 and -509 base pair of the SHP promoter, and mutation of a predicted C/EBPα response element at -473 base pair abolished SHP repression by both C/EBPα and drugs. Moreover, inhibition of major stress signaling pathways demonstrated that the mitogen-activated protein kinase kinase 1/2 pathway activates, while the phosphatidylinositol 3 kinase pathway represses SHP in a C/EBP-dependent manner. We conclude that SHP is downregulated by several steatotic drugs and in advanced NAFLD. These conditions can activate signals that target C/EBPα and consequently repress SHP, thus favoring the progression and severity of NAFLD.

Investigation of Astragalus honey and propolis extract's cytotoxic effect on two human cancer cell lines and their oncogen and proapoptotic gene expression profiles.

Background:Cancer is one of the major fatal human diseases. Natural products have been used in the treatment of cancer for long time. Bee products including honey and propolis have been introduced for malignancy treatment in recent decades. In this study cytotoxicity of bee products and their effects on the expression of proapoptotic genes have been investigated.Materials and Methods:Cytotoxic effects of Astragalus honey, ethanol extract of propolis and a sugar solution (as control) against HepG2, 5637 and L929 cell lines have been evaluated by the MTT assay. Total RNAs of treated cells were isolated and p53 and Bcl-2 gene expression were evaluated, using real-time PCR.Results:Propolis IC50 values were 58, 30 and 15 μg/ml against L929, HepG2 and 5637, respectively. These values for honey were 3.1%, 2.4% and 1.9%, respectively. Propolis extract has increased the expression of the Bcl-2 gene in all cell lines whereas the honey decreased that significantly (P < 0.05). Also, we found that honey and propolis decreased p53 gene expression in HepG2 and 5637 significantly but not in L929 cells. The sugar solution increased the expression of p53 in two cancer cell lines but no significant changes were observed in the expression of this gene in L929 as normal mouse cell.Conclusion:By downregulation of Bcl-2 expression it could be concluded that the cytotoxicity of honey was more than two fold against tested cancer cells compared with the sugar solution. No significant changes were observed in the expression of p53 in honey-treated cells. Propolis had no significant effect on Bcl-2 and p53 gene expressions (P > 0.05).

Evaluation of the selenotranscriptome expression in two hepatocellular carcinoma cell lines.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. Since the selenium is able to fight the oxidative damage which is one of the major origins of cell damage as well as cancer, we have recently focused our attention on selenoprotein family and their involvement in HCC. In the present paper we have carried out a global analysis of the selenotranscriptome expression in HepG2 and Huh7 cells compared to the normal human hepatocytes by reverse transcription-qPCR (RT-qPCR). Our data showed that in both cells there are three downregulated (DIO1, DIO2, and SELO) and ten upregulated (GPX4, GPX7, SELK, SELM, SELN, SELT, SELV, SEP15, SEPW1, and TrxR1) genes. Additionally, interactomic studies were carried out to evaluate the ability of these down- and upregulated genes to interact between them as well as to identify putative HUB nodes representing the centers of correlation able to exercise a direct control over the coordinated genes.

IFN-CSP inhibiting hepatitis B virus in HepG2.2.15 cells involves JAK-STAT signal pathway.

Frequent and high-dose administration of interferon to patients with viral hepatitis results in various side effects. In our previous study, a novel liver-targeting interferon (IFN-CSP) combining Plasmodium region I peptide with IFNα2b was successfully designed and expressed in the Escherichia coli expression systems. This targeting would target the IFNα2b specifically to the liver, thus reducing the adverse events. In the present study, we further investigated the anti-HBV effects and molecular mechanisms of recombinant IFN-CSP in HepG2.2.15 cell line. Hepatitis B surface antigen (HBsAg) and HBe antigen (HBeAg) in the culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). HBV-DNA was measured by real-time quantitative PCR. HBV core protein was assayed by immunofluorescent and western blot analysis. The expressions of signal transducers and transactivator 1 (STAT1), STAT2, IFN regulatory factor 9 (IRF-9), and 2′-5′-oligoadenylate synthetase 1 (OAS1) were investigated by the reverse transcription PCR and western blot analysis. Results indicate IFN-CSP efficiently inhibited HBsAg and HBeAg secretion, HBV-DNA replication, and HBV core protein expression in HepG2.2.15 cells. The anti-HBV mechanisms involve activation of JAK-STAT signaling and increase of the anti-HBV protein OAS expression. IFN-CSP could be a good substitute for IFNα2b for anti-HBV treatment.

Bioreducible dextran-polyethylenimine conjugates regulate transgene expression distribution in vivo.

The purpose of this work is to design a well-defined cationic dextran for intravenous gene delivery into tumor and examine the effect of the dextran on transfection efficacy in vivo. To this end, disulfide-linked dextran-linear polyethylenimine (Dex-SS-LPEI) conjugates were designed as non-viral vectors for intravenous gene delivery into tumor-bearing Balb/c nude mice. By coupling different molecular weights (2 kDa or 5 kDa) of LPEI disulfide pyridine to thiolated dextran (5 kDa or 10 kDa), Dex-SS-LPEI conjugates were prepared which have dextran as the main chain and disulfide-linked LPEI as the side chain. Dex-SS-LPEI conjugates can condense gene into nanosized polyplexes with moderate surface charge. Besides, the polyplexes of the conjugates have an improved colloidal stability under physiological conditions as compared to that of 22 kDa LPEI lacking dextran and can liberate gene by disulfide cleavage in an intracellular reducing environment. In vitro transfection experiments manifest that Dex-SS-LPEI conjugates mediate efficient gene transfection in different cells. The most efficient transfection in vitro was found for the conjugate with 5 kDa dextran and 5 kDa LPEI. Besides, Dex-SS-LPEI conjugates are practical for systemic gene delivery into tumor-bearing Balb/c nude mice by intravenous injection, affording comparable or higher transgene expression in HepG2 or SKOV-3 tumor than that yielded by 22 kDa LPEI as a positive control. Further, the polyplexes of the conjugate with 10 kDa dextran and 5 kDa LPEI can induce comparable transgene expression in the tumor but lower expression in the lung when compared to those of 22 kDa LPEI. The results of this study indicate that dextran plays a critical role in regulating in vivo gene delivery properties of Dex-SS-LPEI conjugates and transfection efficacy in tumor-xenografted nude mice. Dex-SS-LPEI conjugates have low cytotoxicity in vitro and cause no death of the mice, showing great potential as safe and highly efficient gene delivery vectors towards cancer gene therapy.

The role of Moloney leukemia virus 10 in hepatitis B virus expression in hepatoma cells

Recent studies have shown that the Moloney leukemia virus 10 (Mov10), a putative RNA helicase, has very broad and potent antiretroviral activities. Hepatitis B virus (HBV) has a reverse transcription process, but the potential role of Mov10 in HBV replication remains unknown. In this study, Mov10 was demonstrated to affect HBV expression in HepG2 and HepG2.2.15 cell lines. The data showed that the over-expression of exogenous Mov10 resulted in an increase of the HBsAg and HBeAg levels in the culture supernatant and HBV mRNA level in transfected cells at a low dose and resulted in a decrease at a high dose, but HBV DNA in culture supernatant was not affected. The knockdown of endogenous Mov10 expression through siRNA treatment could suppress levels of HBsAg, HBeAg and HBV mRNA, but had no effect on HBV DNA. Above results indicate that an appropriate level of exogenous Mov10 is responsible for HBV replication, that any perturbation in the level of Mov10 could affect HBV replication, while the endogenous Mov10 could promote HBV replication in vitro. The precise mechanisms that underlie the action of Mov10 on HBV still need further investigation.

Knockdown of PPP5C inhibits growth of hepatocellular carcinoma cells in vitro.

Ser/Thr protein phosphatase 5 (PPP5C) has been reported to participate in tumor progression. However, its functional role in hepatocellular carcinoma (HCC) remains unknown yet. In this study, we firstly evaluated the expression levels of PPP5C in six HCC cell lines by real-time PCR and found that PPP5C was widely expressed in HCC cells. To explore the role of PPP5C in HCC cell growth, lentivirus-mediated short hairpin RNA (shRNA) was employed to silence PPP5C expression in HepG2 and Bel-7404 cells. The expression of PPP5C was significantly downregulated in PPP5C knockdown cells. Knockdown of PPP5C markedly suppressed the proliferation and colony formation ability of HCC cells. Moreover, cell cycle analysis showed that PPP5C depletion in HepG2 cells led to G0/G1 phase and G2/M phase arrest. We demonstrate for the first time that PPP5C is essential for growth of HCC cells, which suggests that inhibition of PPP5C by RNAi may be a potential therapeutic strategy for the treatment of HCC.

Modulation of hepatitis B surface antigen secretion by annexin II expressed in hepatitis B virus‑producing hepatoma cells.

The role of annexin II in hepatitis B virus (HBV)‑associated hepatocellular carcinoma (HCC) remains to be elucidated. Intracellular hepatitis B surface antigen (HBsAg)‑retention contributes to the induction of hepatocarcinogenesis. The present study aimed to investigate the regulation of HBsAg secretion by annexin II expressed in HBV‑producing hepatoma cells. The expression of annexin II was analyzed using western blot analysis in SMMC‑7721, HepG2, HepG2.2.15, 293T and Chinese hamster ovary (CHO) cells. CHO cells transfected with an annexin II plasmid were used as a positive control. The localization of annexin II and HBsAg was observed in the HepG2 and HepG2.2.15 cells using indirect immunofluorescence. HepG2.2.15 cells were transfected with a human immunodeficiency virus‑type 1 viral infectivity factor‑hemagglutinin (Vif‑HA) plasmid or a control vector and, 24 h post‑transfection, MG132 was added to the Vif‑complemented HepG2.2.15 cells. Western blot analysis was performed to detect the expression of annexin II and Vif‑HA. HepG2 cells were cotransfected with HBV and annexin II expression vectors. Western blot analysis was performed to examine the expression of annexin II and an Abbott chemiluminescence immunoassay was used to assess the levels of HBsAg. The expression of annexin II was lower in the HepG2.2.15 cells compared with the SMMC‑7721 and HepG2 cells and the fluorescence signal of annexin II in the HepG2 cells was brighter than in the HepG2.2.15 cells. Annexin II colocalized with HBsAg in the cytosol of the HepG2.2.15 cells. MG132 was not able to increase the stability of annexin II expression in HepG2.2.15 cells. Annexin II reduced the secretion of HBsAg when compared with the control‑transfected HepG2 cells. In conclusion, HBV downregulated the expression of annexin II and annexin II decreased the secretion of HBsAg in HBV‑producing hepatoma cells in favor of intracellular HBsAg storage.

Suppressing the CDC37 cochaperone in hepatocellular carcinoma cells inhibits cell cycle progression and cell growth.

The molecular cochaperone CDC37 regulates the activities of multiple protein kinases, and is an attractive broad-spectrum target in many types of cancers in which it is over-expressed. This study investigates the antitumour effects of inhibiting CDC37 in human hepatocellular carcinoma (HCC). A total of 91 patients were enrolled for CDC37 mRNA detection by using quantitative real-time PCR. Cell proliferation, gene expression changes and tumourigenicity were determined by targeting CDC37 using RNA interference in human hepatoma cell lines. We confirmed the significant over-expression of CDC37 transcript and protein in HBV-associated HCC patients. Using a CDC37-specific small oligo-siRNA, we silenced CDC37 expression in HepG2 and Huh7 hepatoma cell lines, and observed inhibition of in vitro cell proliferation, cell cycle arrest at the G1 phase, and enhanced apoptosis. Specifically, we found concomitant down-regulation of Cyclin D1, CDK4, and pRB (S807/811 and S795) upon CDC37 suppression, which could mediate the arrest of cell cycle progression at the G1 phase. Gene expression profiling further identified several genes involved in cell proliferation, cell cycle progression, and apoptosis that are regulated by CDC37 suppression. Huh7 cells with stable knockdown of CDC37 showed decreased in vitro colony formation ability, and significantly slowed xenograft growth in vivo. On the basis of the observed antitumour effects of inhibiting CDC37 expression, we propose that CDC37 is a promising therapeutic target in HCC. Its ability to regulate multiple pathways makes it potentially valuable in treating the heterogeneous subtypes of this malignancy.

Induction of apurinic endonuclease 1 overexpression by endoplasmic reticulum stress in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Previous studies have noted the induction of endoplasmic reticulum stress or apurinic endonuclease 1 (APE1) expression in many tumors. Therefore, the aim of this study was to investigate the relationship between endoplasmic reticulum (ER stress) and APE1 in hepatocellular carcinoma. Here we investigate the expression of APE1 during ER stress in HepG2 and Huh-7 cell lines. Tunicamycin or brefeldin A, two ER stress inducers, increased APE1 and GRP78, an ER stress marker, expression in HepG2 and Huh-7 cells. Induction of APE1 expression was observed through transcription level in response to ER stress. APE1 nuclear localization during ER stress was determined using immunofluorescence assays in HepG2 cells. Furthermore, expression of Hepatitis B virus pre-S2∆ large mutant surface protein (pre-S2∆), an ER stress-induced protein, also increased GRP78 and APE1 expression in the normal hepatocyte NeHepLxHT cell line. Similarly, tumor samples showed higher expression of APE1 in ER stress-correlated liver cancer tissue in vivo. Our results demonstrate that ER stress and HBV pre-S2∆ increased APE1 expression, which may play an important role in resistance to chemotherapeutic agents or tumor development. Therefore, these data provide an important chemotherapeutic strategy in ER stress and HBV pre-S2∆-associated tumors.

Induction of hepatic apolipoprotein A-I gene expression by the isoflavones quercetin and isoquercetrin

AimsPhytochemicals such as flavonoids, vitamins, and polyphenols have been shown to have beneficial effects in metabolic disease. To determine if select flavonoids regulate hepatic apolipoprotein A-I (apo A-I) and high-density lipoprotein (HDL) synthesis, we examined the effects of quercetin, isoquercetin, and myrescetin on apo A-I gene expression in HepG2 (hepatocytes) and Caco-2 (intestinal) cells. Main methodsApo A-I gene expression was measured by Western blotting, quantitative reverse-transcription polymerase chain reaction, and transient transfection. Estrogen receptor α (ESR1) and estrogen receptor β expression were measured by Western blotting, and ESR1 expression was inhibited using ESR1-specific short inhibitory RNA (siRNA). Key findingsQuercetin and isoquercetin, but not myrecetin, induced apo A-I protein and mRNA synthesis, and induced apo A-I promoter activity. Induction by quercetin required an estrogen-responsive region of the apo A-I promoter. Addition of estrogen receptor blocker ICI-182780 to quercetin-treated cells inhibited the effects of quercetin on apo A-I gene expression. Down-regulation of ESR1 with ESR1 siRNA had no effect on basal apo A-I gene expression; however it prevented quercetin-mediated induction of apo A-I gene expression. SignificanceWe conclude that quercetin induces apo A-I gene expression at least in part through induction of ESR1 and may be useful in treating hypoalphalipoproteinemia.

Hexabromocyclododecane and polychlorinated biphenyls increase resistance of hepatocellular carcinoma cells to cisplatin through the phosphatidylinositol 3-kinase/protein kinase B pathway

Hepatocellular carcinoma (HCC) is one of the most common cancers in China with high mortality, high chemotherapy resistance incidence, and poor prognosis. This study aimed to investigate the influence of polychlorinated biphenyls (PCBs) and hexabromocyclododecane (HBCD) on chemoresistance of HCC cells (HepG2, MHCC97H, and MHCC97L) to cisplatin and to explore the potential molecular mechanism. Cell viability, DNA damage, the expression level and activity of nuclear factor-κB (NF-κB), p53/Mdm4, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway were measured. The results showed that HBCD and PCBs could significantly reduce the chemosensitivity of HCC cells to cisplatin, increasing the cell viability and decreasing DNA damage. Moreover, HBCD and PCBs could induce the transcriptional activity of NF-κb and suppress the p53 expression in HepG2 and MHCC97H cells. In MHCC97L cells, however, opposite changes for NF-κB protein expression, NF-κB transcriptional activity, and p53/Mdm4 expression were observed after HBCD and PCBs exposure. Further investigation revealed that HBCD and PCBs exposure significantly increased the expression level of p-Akt and mammalian target of rapamycin (mTOR) in HepG2 and MHCC97H cells, but reduced that in MHCC97L cells. PI3K inhibitor LY294002 could relieve the influence of HBCD and PCBs on chemoresistance in HepG2 and MHCC97H cells. Taken together, HBCD and PCBs at low concentrations could increase the resistance of HCC cells to cisplatin through modulation on NF-κB pathway activation and p53 function, which is associated with the activity of PI3K/Akt pathway.

Downregulation of galectin-3 causes a decrease in uPAR levels and inhibits the proliferation, migration and invasion of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed and associated with the prognosis of HCC. However, the functions of Gal-3 in HCC cells are not fully understood. To address the function of Gal-3 in HCC cells, we used small interfering RNA (siRNA) to knock down Gal-3 expression in HepG2, an HCC cell line. We found that in vitro the silencing of Gal-3 decreased the proliferative activity, colony formation ability, migratory and invasive potential of HepG2 cells. The silencing of Gal-3 significantly decreased the mRNA and protein levels of urokinase-type plasminogen activator receptor (uPAR) as well as uPAR's downstream signaling transduction pathway, including phosphorylation of AKT. Furthermore, the downregulation of Gal-3 by siRNA resulted in significantly decreased activity of the MEK/ERK signaling pathway, and the treatment of HepG2 cells with MEK/ERK inhibitor U0126 significantly reduced the mRNA and protein levels of uPAR. Taken together, our results suggest that Gal-3 modulates uPAR expression via the MEK/ERK pathway, and that Gal-3 may be a potential therapeutic target for the treatment of HCC.

Reduced cellular Mg2+ content enhances hexose 6-phosphate dehydrogenase activity and expression in HepG2 and HL-60 cells

We have reported that Mg2+ dynamically regulates glucose 6-phosphate entry into the endoplasmic reticulum and its hydrolysis by the glucose 6-phosphatase in liver cells. In the present study, we report that by modulating glucose 6-phosphate entry into the endoplasmic reticulum of HepG2 cells, Mg2+ also regulates the oxidation of this substrate via hexose 6-phosphate dehydrogenase (H6PD). This regulatory effect is dynamic as glucose 6-phosphate entry and oxidation can be rapidly down-regulated by the addition of exogenous Mg2+. In addition, HepG2 cells growing in low Mg2+ show a marked increase in hexose 6-phosphate dehydrogenase mRNA and protein expression. Metabolically, these effects on hexose 6-phosphate dehydrogenase are important as this enzyme increases intra-reticular NADPH production, which favors fatty acid and cholesterol synthesis. Similar effects of Mg2+ were observed in HL-60 cells.These and previously published results suggest that in an hepatocyte culture model changes in cytoplasmic Mg2+ content regulates glucose 6-phosphate utilization via glucose 6 phosphatase and hexose-6 phosphate dehydrogenase in alternative to glycolysis and glycogen synthesis. This alternative regulation might be of relevance in the transition from fed to fasted state.

Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines

To evaluate the effects of the proteasome inhibitor bortezomib (BZB) on E2Fs and related genes in hepatocellular carcinoma (HCC) cells. The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nmol/L) used was chosen to stay well below the maximal amount/cm² recommended for in vivo application, and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Using small interfering RNA's, the effects of E2F8 depletion on cell number was also evaluated. After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F's, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6. The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib's mechanism of action in hepatocellular carcinoma.

PCB-77 disturbs iron homeostasis through regulating hepcidin gene expression

PCBs are a family of persistent environmental toxicants with a wide spectrum of toxic features, such as immunotoxicity, hepatoxicity, endocrine disruption effects, and oncogenic effects. To date, little has been done to investigate the potential influence of PCB exposure on iron metabolism. Deregulated iron would lead to either iron deficiency or iron excess, coupled with various diseases such as anemia or hemochromatosis. Iron metabolism is strictly governed by the hepcidin–ferroportin axis, and hepcidin is the key regulator that is secreted by hepatocytes. Here, we found that PCB-77 could go through plasma membrane and accumulate in hepatocytes. PCB-77 was demonstrated to suppress hepcidin expression in HepG2 and L-02 hepatocytes. Moreover, hepatic hepcidin was observed to be inhibited in mice upon administration of PCB-77. Due to reduced hepcidin concentration, serum iron content was increased, with a significant reduction of splenic iron content. Together, we deciphered the molecular mechanism responsible for PCB-conducted disturbance on iron homeostasis, i.e. through misregulating hepatic hepcidin expression.