• The Journal of Internal Korean Medicine
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• v.27 no.1
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• pp.27-39
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• 2006

Objectives : Induction of CYP2E1 by ethanol is believed to be one of the major mechanism by which ethanol generate a state of oxidative stress. Previous studies showed that treatment with Chungganhaeju-tang prevents hepatic inflammation and apoptosis in alcoholic liver disease. The purpose of our study is to determine if Chungganhaeju-tang can also protect against alcohol-induced cytotoxicity in CYP2E1-transfected HepG2 cells. Materials and Methods : CYP2E1-transfected HepG2 cells and control vector-transfected HepG2 cells were exposed for isx hours to Chungganhaeju-tang, and then 50 mM of ethanol was added and left for two days. Results : Ethanol significantly decreased cell viability in CYP2E1-transfected HepG2 cells and increased apoptosis. These alterations were attenuated by Chungganhaeju-tang. This was accompanied by an improvement of NF-${\kappa}B$ and Akt activation. Conclusion : These results suggest that Chungganhaeju-tang exerts inhibitory effect against the cytotoxicity induced by alcohol in CYP2E1-transfected HepG2 cells, and that this is a protective action due, at least in part, to an activation of NF-${\kappa}B$ that plays a key role in the protection mechanism, and in reducing hepatotoxic cytokine gene expression.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1310-1316
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• 2005

To evaluate the toxicological properties of human cytochrome P450 2E1 (CYP2E1) induced by ethanol and possible protective effects of various green tea catechins on alcohol-induced toxicity, transfected HepG2 cells that stably and constitutively express human CYP2E1 were established using the recombinant retroviral expression vector. Exposure of the CYP2E1-expressing HepG2 cells to high concentration of ethanol (200 mM) for 5 days resulted in a more than $50\%$ increase of cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) production, and loss of normal morphology, in comparison with HepG2 cells containing control vector. Treatment of the cells with various catechins increased cell viability by more than 2-fold. (-)-Epicatechin gallate and(-)-catechin gallate at the lowest concentration ($5\;{\mu}M$) attenuated cell death induced CYP2E1 by $60-65\%$. Therefore, the results showed that the catechins, including epimerized catechins, have strong protective effects against alcohol-induced CYP2E1 toxicity, and it is correlated with antioxidant effect.

• The Journal of Internal Korean Medicine
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• v.28 no.4
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• pp.694-708
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• 2007

Objectives : Jageum-Jung is used as an anti-cancer agent in oriental medicine, but the mechanism by which it induces cell death in cancer cells is still unclear. The purpose of this study was to investigate the effects of Jageum-Jung on apoptosis and cell cycle arrest in HepG2 hepatoma cells. Methods : Various cancer cell lines including HepG2, C6 glioma, SH-SY5Y, PANC-1, and MCF-7 cells, were used. Apoptosis was determined by DAPI nuclei staining and flow cytometry in HepG2 cells treated with various concentrations (from 25 to 200 ${\mu}g/ml$) of $H_2O$ extract of Jageum-Jung (JGJ) for 48 hrs. Expression of cell cycle arrest mediators including Rb, p53, p21, cyclin B1, cdk4, and cyclin E proteins were measured by Western blot analysis. To estimate intracellular hydrogen peroxide levels and intracellular nitric oxide levels, HepG2 cells were stained with DCFH-DA dye and DAF dye, subjected on flow cytometric analysis. Results : 1. Jageum-Jung decreased the viability of HepG2 cells in a dose-dependent manner. 2. Jageum-Jung induced the catalytic activation of caspase-3 in HepG2 cells. 3. Jageum-Jung increased the intracellular hydrogen peroxide and NO in HepG2 cells. 4. Jageum-Jung increased the expression of Rb, p53 and p21 in HepG2 cells. 5. Jageum-Jung induced the expression of cyclin B1, cdk4, and cyclin E in HepG2 cells. Conclusions : Taken together, we suggest that Jageum-Jung exhibits cytotoxic effects on HepG2 cells, causing apoptosis and cell cycle arrest. The results showed that Jageum-Jung may do so by regulating the expression of specific target molecules that promote efficient apoptotic cell death following $G_2$/M phase arrest in a dose-dependent manner.

• Toxicological Research
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• v.16 no.2
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• pp.95-100
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• 2000

In previous study, both constitutive expression and 3-methylcholanthrene (3MC)-mediated elevation of CYP1A2 mRNA were demonstrated in human hepatoma HepG2 cells by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that HepG2 cells would be appropriate for the study of human CYP1A2 regulation(Chung and Bresnick, 1994). Further studies were conducted to determine the basis of this induction phenomenon that is observed in HepG2 cells. Since CYP1A1 gene, another polycyclic hydrocarbon(PH)-inducible gene, is regulated by PHs through their interactions via receptors with cis-elements, the 5'-flanking region of human CYP 1A2 gene was analyzed to search such responsive elements. The promoter activity of various lengths of CYP1A2 gene sequence (-3203/+58bp) was measured in transiently-transfected HepG2 cells by fusion constructs containing the CAT, hGH or luciferase genes as a reporter. This region of the CYP1A2 gene, although containing a XRE, was only weakly responsive (less than 2 fold induction) to 10 nM of TCDD or 1 $\mu$M 3 MC treatment. This small enhancement of promoter activity is inconsistent with the previous observation, i.e., 12 to 14 fold-enhanced CYP1A2 mRNA from 1 $\mu$M 3 MC treated HepG2 cells, suggesting that additional mechanisms would exist for PH-mediated induction of CYP1A2 in these cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.4
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• pp.821-828
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• 2008

Onchungeum, a herbal formula, which has been used for treatment of anemia due to bleeding, discharging blood and skin disease. In the present study, it was examined the effects of extract of Onchungeum (OCE) on the growth of human hepatocarcinoma cell lines Hep3B (p53 null type) and HepG2 (p53 wild type) in order to investigate the anti-proliferative mechanism by OCE. Treatment of Hep3B and HepG2 cells to OCE resulted in the growth inhibition in a dose-dependent manner, however Hep3B cell line exhibited a relatively strong anti-proliferative activity to OEC. Flow cytometric analysis revealed that OCE treatment in Hep3B cells caused G1 phase arrest of the cell cycle, which was associated with various morphological changes in a dose-dependent fashion. RT-PCR and immunoblotting data revealed that treatment of OCE caused the down-regulation of cyclin D1 expression, however the levels of cyclin E expression were not changed by OCE. The G1 arrest of the cell cycle was also associated with the induction of Cdk inhibitor p27 by OCE. Because the p53 gene is null in Hep3B cells, it is most likely that the induction of p21 is mediated through a p53-independent pathway. Moreover, p27 detected in anti-Cdk4 and anti-Cdk2 immunoprecipitates from the OCE-treated cells, suggesting that OCE-induced p27 protein blocks Cdk kinase activities by directing binding to the cyclin/Cdk complexes. Furthermore, OCE treatment potently suppresses the phosphorylation of retinoblastoma proteins and the levels of the transcription factor E2F-1 expression. Taken together, these results indicated that the growth inhibitory effect of OCE in Hep3B hepatoma cells was associated with the induction of G1 arrest of the cell cycle through regulation of several major growth regulatory gene products.

• Toxicological Research
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• v.19 no.3
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• pp.235-240
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• 2003

The modification of CYP2E1 activity is of considerable interest because of its role in the metabolic activation of a variety of toxic chemicals. In the present studies, the time-course of changes in human CYP2E1 activities was determined after treatment with ethanol, glycerol, 4-methylpyrazole or isoniazid using intact HepG2 cells transfected by human CYP2E1. Hydroxylation of chlorzoxazone was chosen for the measurement of CYP2E1 activity. CYP2E1 protein levels were increased upon cultivation of cells in the presence of ethanol, glycerol, 4-methylpyrazole or isoniazid for 24 hr. After 24 hr cultivation, ethanol or glycerol increased CYP2E1 activities, whereas 4-methylpyrazole or isoniazid inhibited. This different effect of the chemical inducers on CYP2E1 activi-ties persisted to subsequent 24 hr. Competitive inhibition study suggested that 4-methylpyrazole or isoniazid has stronger binding affinity to CYP2E1 than ethanol or glycerol. These results demonstrate that different binding affinity of the chemical inducers to the active site of CYP2E1 plays important role in determining real CYP2E1 activity in intact cells after treatment with the chemical inducers. Present study would be helpful in precise understanding of human CYP2E1-mediated toxicity.

• Journal of Environmental Health Sciences
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• v.30 no.3
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• pp.230-238
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• 2004

The adverse health effects of a number of environment pollutions are related to the formation of free radicals. Induction of antioxidant defensive system in the response to an oxidative attack is an essential element of the cell to survive. CYP2E1 is easily induced by organic solvents and induces continuous formation of reactive oxygen species (ROS). ${\gamma}$-Glutamyltranspeptidase (${\gamma}$GT) plays an important role in glutathione metabolism and xenobiotic detoxification. To evaluate the characteristic of oxidative stress which induces GGT expression and to understand human antioxidant defensive response against oxidative stress induced by CYP2E1, we studied regulation of ${\gamma}$GT enzyme expression in response to various oxidative stresses in human HepG2 cells. The ${\gamma}$GT activity was not modified after exposure of acute oxidative stress inducing agents (ferric nitrilotriacetate, cumene hydroperoxide, ADP-Fe, O-tetradecanoylphorbol-13-acetate, tumor necrosis factor-alpha). To induce continuous exposure of cells to ROS, HepG2 cells were transfected by human CYP2E1 gene transiently. The CYP2E1 activity was verified with chlorzoxazone hydroxylation. Transfection of CYP2E1 showed continuous 60% increase in intracellular ROS and 240 % increase in microsomal ROS. CYP2E1 overexpressing cells showed increased ${\gamma}$GT activity (2.5-fold). The observed enhancement of ${\gamma}$GT activity correlated with a significant increase of ${\gamma}$GT mRNA (2.1-fold). Treatment with antioxidant strongly prevented the increase in ${\gamma}$GT activity. The CYP2E1 overexpression did not modify toxicity index and increased glutathione levels. These results show that continuous exposure of cells to ROS produced by CYP2E1 up-regulates ${\gamma}$GT; This may be one of the adaptive antioxidant responses of cells to oxidative insult. Present study also suggests that the induction of ${\gamma}$GT could be used as a marker of oxidative stress induced by exposure to organic solvents.

• Journal of Environmental Health Sciences
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• v.29 no.3
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• pp.9-15
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• 2003

The modification of CYP2El activity is a matter of considerable interest because of its role in the metabolic activation of a variety of environmental toxicants. In the present study, the time-course of changes in human CYP2El activities was determined following treatment with solvents (acetone, dimethylsulphoxide or pyridine) using intact HepG2 cells transfected by human CYP2El. Hydroxylation of chlorzoxazone was used for the measurement of CYP2El activity. CYP2E1 protein level was increased upon cultivation of cells in the presence of the solvents for 24 hr. Determination of CYP2El activities after 24 ht cultivation with the solvents demonstrated that acetone or dimethylsulphoxide increased, whereas pyridine inhibited the activities. This differential effect of the solvents on CYP2El activities persisted to subsequent 24 ht. Competitive inhibition study suggested that pyridine has stronger binding affinity to CYP2E1 than acetone or dimethylsulphoxide. These results demonstrate that different binding affinity of the solvents to CYP2El plays important role in determining real CYP2El activity in intact cells after exposure to the solvents. Present study would be helpful in precise understanding of human CYP2El-mediated toxicity.

• Journal of Life Science
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• v.28 no.9
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• pp.1007-1015
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• 2018

The E6-associated protein (E6AP) is known to induce the ubiquitination and proteasomal degradation of HCV core protein and thereby directly impair capsid assembly, resulting in a decline in HCV replication. To counteract this anti-viral host defense system, HCV core protein has evolved a strategy to inhibit E6AP expression via DNA methylation. In the present study, we further explored the mechanism by which HCV core protein inhibits E6AP expression. HCV core protein upregulated both the protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b to inhibit E6AP expression via promoter hypermethylation in HepG2 cells but not in Hep3B cells, which do not express p53. Interestingly, p53 overexpression alone in Hep3B cells was sufficient to activate DNMTs in the absence of HCV core protein and thereby inhibit E6AP expression via promoter hypermethylation. In addition, upregulation of p53 was absolutely required for the HCV core protein to inhibit E6AP expression via promoter hypermethylation, as evidenced by both p53 knockdown and ectopic expression experiments. Accordingly, levels of the ubiquitinated forms of HCV core protein were lower in HepG2 cells than in Hep3B cells. Based on these observations, we conclude that HCV core protein evades ubiquitin-dependent proteasomal degradation in a p53-dependent manner.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2565-2570
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• 2014

H19 is an imprinted oncofetal gene, and loss of imprinting at the H19 locus results in over-expression of H19 in cancers. Aflatoxin B1(AFB1) is regarded as one of the most dangerous carcinogens. Exposure to AFB1 would most easily increase susceptibility to diseases such as hepatocellular carcinoma(HCC) but any possible relationship between AFB1 and H19 is not clear. In present study, we found that AFB1 could up-regulate the expression of H19 and promote cell growth and invasion by hepatocellular carcinoma HepG2 cells. Knocking down H19 RNA co ld reverse the effects of AFB1 on cell growth and invasion. In addition, AFB1 induced the expression of E2F1 and its knock-down could down-regulate H19 expression and suppress cell growth and invasion in hepatocellular carcinoma HepG2 cells. Furthermore, E2F1 over-expression could up-regulate H19 expression and promote cell growth and invasion, with binding to the H19 promoter being demonstrated by chromatin immunoprecipitation assays (ChIP). In summary, our results suggested that aflatoxin B1could promote cell growth and invasion in hepatocellular carcinoma HepG2 cells through actions on H19 and E2F1.