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1.
10—羟基喜树碱对人肝癌Hep G2细胞的分化诱导作用 总被引:11,自引:0,他引:11
目的:研究羟基喜树碱HCPT对人肝癌Hep G2细胞分化诱导作用的机制。方法:用免疫细胞化学染色检测增殖细胞核抗原表达;流式细胞仪检测细胞周期和野生型p53蛋白表达;端粒重复扩增法检测端粒酶活性。结果:以诱导分化剂量(5-20μg·L~(-1))的HCPT处理6 d,人肝癌Hep G2细胞明显受阻于G_2/M期,PCNA阳性表达率降低。经HCPT 5μg·L~(-1)处理6 d,Hep G2细胞的野生型p53蛋白表达明显增强,但端粒酶活性没有改变。结论:HCPT诱导人肝癌Hep G2细胞分化的作用与细胞阻滞于G_2/M期、PCNA表达降低及野生型p53蛋白表达增强有关。 相似文献
2.
Copper, though essential, is highly toxic when present in excess, as in Wilson disease, a genetic disorder of hepatic copper metabolism. We hypothesized that mitochondria are a major target of copper-induced cytotoxicity in Wilson disease. We used the human hepatoma line Hep G2 to examine copper-mediated cytotoxicity and three different methods to assess organelle damage: MTT assay (mitochondria), neutral red (NR; lysosomes) and Trypan blue exclusion assay (TB; plasma membrane). For all assays, cells at approximately 60% confluence in microtitre plates were incubated with CuCl(2) (concentration range: 50-100-150-200 microM) for 24 or 48 h. Results were expressed as percent of untreated control. At 24 h, cytotoxicity as detected by NR assay was significantly higher at all concentrations of copper than for MTT or TB ( p<0.005 at all concentrations). Cytotoxicity as detected by MTT was higher than that detected by TB at all concentrations except at 200 microM (p<0.05 for 50 microM, p<0.005 for 100 microM, p = 0.001 for 150 microM). Results at 48 h were similar (NR versus others: p <0.001 MTT versus TB: NS except at 150 microM where p<0.01). We investigated reactive oxygen species (ROS) production in copper-associated hepatocytoxicity by incubating sub-confluent cells with 2('),7(')-dichlorodihydrofluorescein diacetate dye plus copper (concentration range: 0-200 microM) for 1-1.5 h. Copper, but not zinc, produced significant increases in ROS (p<0.001). In summary, Hep G2 lysosomes appeared more susceptible to Cu-mediated damage than mitochondria; the cell membrane was highly resistant to damage. 相似文献
3.
Variation in drug-metabolizing enzyme activities during the growth of human Hep G2 hepatoma cells 总被引:2,自引:0,他引:2
H Doostdar A Demoz M D Burke W T Melvin M H Grant 《Xenobiotica; the fate of foreign compounds in biological systems》1990,20(4):435-441
1. The activities of several drug-metabolizing enzymes change during the growth cycle (exponential growth to confluence) of Hep G2 cells in culture. As the rate of cell growth slowed down (days 7 to 10 after passage) the activities of ethoxy- and methoxy-resorufin O-dealkylase and of NADPH cytochrome c- and NADH cytochrome b5-reductase increased. In contrast, the O-dealkylations of pentoxy- and benzyloxy-resorufin did not change significantly during culture. 2. UDP-glucuronyltransferase activities also showed substrate-dependent alterations with time in culture. In contrast, glutathione-S-transferase activity remained constant despite a decline in the intracellular reduced glutathione content. 3. Epoxide hydrolase activity altered throughout time in culture, with an initial decrease in activity followed by a marked increase between days 7 and 10 after passage. 4. These results indicate the importance of standardizing the protocol with regard to the timing of experiments within the growth period of the cells when using hepatoma cell lines for assessing the metabolism and cytotoxicity of chemicals. 相似文献
4.
Perfluorinated compounds (PFCs) are emerging compounds of concern. They are widely distributed in the environment, wildlife
and human. Concern has been raised over their possible adverse effects on human health. This study was designed to determine
cytotoxic effects of two important PFCs, perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), in a single and a
mixture of them exposure to Hep G2 cells. The results showed that PFOA and PFOS (50–200 μmol/l) induced production of reactive
oxygen species (ROS), dissipation of mitochondria membrane potential and apoptosis of Hep G2 cells. Moreover, activities of
superoxide dismutase, catalase and glutathione reductase were increased, whereas activities of glutathione-S-transferase and glutathione peroxidase were decreased. Glutathione content was reduced. Differential expression of genes,
such as p53, Bcl-2, caspase-9, was evident in PFOA or PFOS exposure groups. The possible mechanism was that they could overwhelm
homeostasis of antioxidative systems, boost ROS generation, impact mitochondria, and affect genes expression of apoptotic
regulators, which resulted in start-ups of apoptosis program. Cells exposed to mixture of PFOA and PFOS and each of them showed
non-apoptotic rate significant difference, which indicated that the combined effect of two compounds was summation effect,
but neither synergistic nor antagonistic effect. 相似文献
5.
Metabolism of propranolol by the human hepatoma cell line Hep G2 was studied. Although metabolism qualitatively was similar to that in-vivo, the P450-mediated N-desisopropylation clearly predominated. Pretreatment of cells with 3-methylcholanthrene increased the activity of this pathway 14-fold, whereas phenobarbitone had no effect. This is similar to the pathway-selective inductive response observed for cigarette smoking in-vivo. As in-vivo, secondary metabolism of N-desisopropylpropranolol was extensive. This could, however, be completely blocked by 0.1 microM clorgyline, a potent MAO type A inhibitor. As in human liver microsomes, the stereochemistry of propranolol metabolism demonstrated a preference for the R(+)-enantiomer. These observations emphasize the usefulness of the Hep G2 cell line as a model of man. 相似文献
6.
Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209) 总被引:1,自引:0,他引:1
Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 micromol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 micromol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. 相似文献
7.
Epirubicin HCl is a new anthracycline analog and derivative of doxorubicin. Doxorubicin is a potent anticancer agent, the use of which is limited by its cumulative dose-dependent cardiotoxicity. Epirubicin HCl has more favorable therapeutic index than doxorubicin and possesses less hematologic and cardiac toxicity at comparable doses. Hepatoma G2 cells are a valuable model to study hepatocellular carcinoma and the liver, where drugs are metabolized. The goal of our study was to evaluate the cytotoxic effect of epirubicin HCl on viability of Hep G2 cells measured using the MTT cytotoxicity test. Epirubicin HCl produced a concentration- and time-dependent cytotoxicity to Hep G2 cells. The mechanism of cytotoxicity of epirubicin HCl (IC(50) value of 1.6 mug/ml within 24 h) appeared to involve a production of free radical species since activities of free radical scavenging enzymes (SOD, catalase, Se-dependent GPx) were increased. Addition of SOD prevented cytotoxicity of epirubicin HCl, and also counteracted the apoptosis. DNA fragmentation was determined to evaluate apoptosis. Western blot analysis indicated a decrease in GST-pi expression and increased activity of NADPH-dependent cytochrome P450 reductase which is a major enzyme in the conversion of epirubicin HCl to a free radical. It is proposed that production of reactive oxygen species increased by the treatment with epirubicin HCl can cause lipid peroxidation, which subsequently promotes apoptosis and reduces cell viability. Superoxide dismutase, catalase and glutathione peroxidase must be considered as a part of the intracellular antioxidant defense mechanism of Hep G2 cells against single electron reducing quinone-containing anticancer antibiotics. 相似文献
8.
Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line 总被引:4,自引:0,他引:4
In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 microM benz(a)anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O-deethylation (EROD) was increased 15-fold by BA but not by PB, while the O-dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by alpha-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O-dealkylation and elevated microsomal epoxide hydrolase activities. 相似文献
9.
10.
We describe here the purification and characterization of a new Phaseolus vulgaris lectin that exhibits selective toxicity to human hepatoma Hep G2 cells and lacks significant toxicity on normal liver WRL 68 cells. This polygalacturonic acid?Cspecific lectin (termed BTKL) was purified from seeds of P. vulgaris cv. Blue tiger king by liquid chromatography techniques. The 60-kDa dimeric lectin showed strong and broad-spectrum hemagglutinating activity toward human, rabbit, rat, and mouse erythrocytes. Bioinformatic analysis unveils substantial N-terminal sequence similarity of BTKL to other Phaseolus lectins. Among a number of tumor cells tested, BTKL exhibits potent anti-Hep G2 activity which is associated with (1) induction of DNA fragmentation, (2) production of apoptotic bodies and chromatin condensation, (3) triggering of cell apoptosis and necrosis, and (4) depolarization of mitochondrial membrane (low ????m). Furthermore, BTKL could induce inducible nitric oxide synthase (iNOS) expression and subsequent nitric oxide production in vitro in mouse macrophages, which may contribute to its antitumor activity. In addition, BTKL could bring about a significant dose-dependent increase in the production of mRNAs of proinflammatory cytokines including interleukin-1 beta, interleukin-2, tumor necrosis factor alpha, and interferon-gamma. In sum, the antitumor activity and mechanism of BTKL provided here suggest that it has potential therapeutic value for human liver cancer. 相似文献
11.
S Menzel-Soglowek G Geisslinger J Mollenhauer K Brune 《Biochemical pharmacology》1992,43(7):1487-1492
The inversion of 2-arylpropionic acids (2-APAs) has been investigated in vitro using rat H4IIE and human Hep G2 hepatoma cells in continuous culture. The effect of substrate concentration (15-150 micrograms/mL), cell density (1.5-12 x 10(6) cells/dish) and serum content of the culture medium (0-20%) on inversion was examined in rat H4IIE hepatoma cells using R-ibuprofen as model compound. Increasing R-ibuprofen concentrations and decreasing serum content of the medium resulted in increased inversion whereas variation of cell density had no effect. Furthermore, rat H4IIE and human Hep G2 hepatoma cells were incubated with the individual enantiomers of ibuprofen, ketoprofen and flurbiprofen under optimized culture conditions (serum-free culture medium). The elimination rate constants (kel) and fractions inverted (Fi) were determined. Although inversion occurred slowly in the tumor cells and thus long incubation periods (120 hr) were required, the hepatoma cells were nevertheless able to mimic qualitatively the species and substance specificity of inversion of 2-APAs as observed in vivo. 相似文献
12.
目的:观察HepG2细胞表达的FasL蛋白的生理效应,期望能进一步阐明乙型病毒性肝炎的分子发病机制。方法:(1)建立转染HBx基因的HepG2细胞系。(2)HtepC2x细胞表达的FasL生理效应的分析:采用细胞混合培养后,测定LDH释放、DNA Ladder及流式细胞仪方法检测细胞凋亡情况。结果:HepC2x细胞可表达FasL,HepC2x细胞与HepC2o、Jurkat细胞混合培养48小时后,HepG2o及Jurkat细胞特异性LDH释放率均等于零oDNAladder及流式细胞仪检测APO2.7抗体标记细胞结果表明,与HepC2x混合培养后,HepG2o、Jurkat细胞发生凋亡;增强HepG2o细胞表达Fas,细胞凋亡程度也增加。结论:HepC2x细胞表达的FasL具有致表达Fas的肝细胞或Jurkat细胞凋亡的作用。 相似文献
13.
Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC50 values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs. 相似文献
14.
Truffin D Garçon G Hannothiaux MH Colein P Shirali P Grave-Descampiaux B 《Journal of applied toxicology : JAT》2003,23(2):109-114
Chlorophenols, mainly used as biocides, are compounds with a wide spectrum of toxic effects, including teratogenic and carcinogenic actions. The aim of this study was to examine possible 4-monochlorophenol (4-MCP) toxicity related to metabolic pathways, which may implicate semiquinones and reactive oxygen species (ROS), in human Hep G2 cells. The effects of 4-MCP were performed through cytotoxicity assays (viability, ATP level), metabolic activities (4-MCP intracellular concentration, NADPH cytochrome P-450 reductase (Cyt P-450 red.) and glutathione-S-transferase activities, CYP 3A7 mRNA expression) and oxidative stress (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, glutathione status, malondialdehyde concentration, CYP 2E1 mRNA expression). According to the literature, in this work Hep G2 cells were incubated in the continuous presence of 4-MCP at 350 microM over 24 or 48 h. Results showed statistically significant decreases in ATP levels (24 or 48 h, P < 0.05) versus controls. The 4-MCP intracellular concentrations increased as early as 8-24 h and then decreased (P < 0.01). Decreases in Cyt. P-450 red. (24 h, P < 0.05), catalase (24 h, P < 0.05; 48 h, P < 0.01), glutathione peroxidase activities (48 h, P < 0.05) and reduced glutathione concentrations (48 h, P < 0.05) were observed. In addition, exposure to 4-MCP increased mRNA expressions of CYP 3A7 (24 h, P < 0.05; 48 h, P < 0.01) and CYP 2E1 (24 h, P < 0.01) versus controls. Taken together, these results suggest that 4-MCP metabolites could induce oxidative stress conditions in Hep G2 cells. 相似文献
15.
A Corsini J Beitz A Granata R Fumagalli H J Mest R Paoletti 《Pharmacological research》1989,21(5):521-531
The effect of trapidil (RocornalR) and some of its newly developed derivatives (AR 12456, AR 12463, AR 12465, AR 12464) on the receptor-mediated low density lipoprotein (LDL) binding, uptake and degradation was studied in human skin fibroblasts (HSF) and in human hepatoma cell line Hep G2. Compound AR 12456 influenced this pathway in a selective way: it enhanced the uptake and degradation of 125I-LDL by Hep G2 cells in a dose-dependent manner, but inhibited it in HSF. Scatchard analysis of the saturable LDL binding in Hep G2 indicates that the effect of compound AR 12456 is the result of an increased number of LDL binding sites. Compound AR 12465 was less effective on LDL catabolism. Trapidil and the other derivatives were inactive under the same experimental conditions. When Ar 12456 was preincubated with Hep G2 cells and then the incubation medium was transferred to HSF, a stimulation of specific LDL pathway occurred also in this cell line. These findings suggest that a metabolite(s) of AR 12456 might be responsible for the enhanced expression of LDL receptors in cultured human cells. 相似文献
16.
A. Galijatovic Y. Otake U. K. Walle T. Walle 《Xenobiotica; the fate of foreign compounds in biological systems》2013,43(12):1241-1256
1. Chrysin is one of many bioflavonoids with chemopreventive properties in cardiovascular disease and cancer. In an effort better to understand factors that may affect the oral bioavailability of the bioflavonoids from dietary sources, the metabolism of chrysin by cultured intestinal Caco-2 cells and hepatic Hep G2 cells was studied, together modelling human presystemic metabolism. 2. At concentrations that may be achieved in the diet, chrysin was extensively metabolized to two conjugated metabolites, M1 and M2, with no CYP-mediated oxidation. M1 was identified as a glucuronide, and M2 as a sulphate conjugate by LC/MS and other spectroscopic and biochemical techniques. Sulphate conjugation occurred at a rate twice that of glucuronic acid conjugation in both cell types. 3. M1 was catalyzed by UGT1A6 with a Km=12 muM. M2 was catalyzed both by M- and P-form phenolsulphotransferases (SULT 1A3 and SULT 1A1) with very low Km of 3.1 and 0.05 muM respectively. 4. Pretreatment with 3-methylcholanthrene, interestingly, did not result in oxidation of chrysin but rather in increased glucuronidation. 5. Also, M1 and M2 were the only metabolites formed from chrysin in fresh rat hepatocytes. The metabolism of another flavonoid, apigenin, was very similar to that of chrysin. 6. These observations suggest that both sulphation and glucuronidation are critical determinants of the oral bioavailability of bioflavonoids in humans, although a contribution from CYP-mediated oxidation can not be excluded. 相似文献
17.
Galijatovic A Otake Y Walle UK Walle T 《Xenobiotica; the fate of foreign compounds in biological systems》1999,29(12):1241-1256
1. Chrysin is one of many bioflavonoids with chemopreventive properties in cardiovascular disease and cancer. In an effort better to understand factors that may affect the oral bioavailability of the bioflavonoids from dietary sources, the metabolism of chrysin by cultured intestinal Caco-2 cells and hepatic Hep G2 cells was studied, together modelling human presystemic metabolism. 2. At concentrations that may be achieved in the diet, chrysin was extensively metabolized to two conjugated metabolites, M1 and M2, with no CYP-mediated oxidation. M1 was identified as a glucuronide, and M2 as a sulphate conjugate by LC/MS and other spectroscopic and biochemical techniques. Sulphate conjugation occurred at a rate twice that of glucuronic acid conjugation in both cell types. 3. M1 was catalyzed by UGT1A6 with a Km = 12 microM. M2 was catalyzed both by M- and P-form phenolsulphotransferases (SULT 1A3 and SULT 1A1) with very low Km of 3.1 and 0.05 microM respectively. 4. Pretreatment with 3-methylcholanthrene, interestingly, did not result in oxidation of chrysin but rather in increased glucuronidation. 5. Also, M1 and M2 were the only metabolites formed from chrysin in fresh rat hepatocytes. The metabolism of another flavonoid, apigenin, was very similar to that of chrysin. 6. These observations suggest that both sulphation and glucuronidation are critical determinants of the oral bioavailability of bioflavonoids in humans, although a contribution from CYP-mediated oxidation can not be excluded. 相似文献
18.
Both (R)- and (S)-3-hydroxy-13-methyltetradecanoic acids were prepared via a lipase-catalyzed enantioselective acylation. The total synthesis of N-4909 and its diastereomer were achieved by a coupling of either (R)- or (S)-3-hydroxy-13-methyltetradecanoic acid moiety with a hexapeptide moiety and by a cyclization with HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HOAt (1-hydroxy-7-azabenzotriazole) in a high dilution condition. The R configuration of 3-hydroxy-13-methyltetradecanoic acid was found to be important for stimulating the activity of apolipoprotein E secretion in human hepatoma Hep G2 cells. 相似文献
19.
Liping Jiang Jun Cao Yu An Chengyan Geng Shuxian Qu Lijie Jiang Laifu Zhong 《Toxicology in vitro》2007,21(8):1486-1492
The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH. 相似文献
20.
Effect of selenium supplementation on the activities of glutathione metabolizing enzymes in human hepatoma Hep G2 cell line 总被引:1,自引:0,他引:1
Cell culture is an important tool for studying injury to cells exposed to oxidative stress. The human hepatoblastoma derived Hep G2 cells retain their morphology and most of their function in culture and are therefore widely used as an in vitro model of human hepatocytes. Conventional cell culture media are deficient in selenium, which is essential for activation of glutathione peroxidase (GPx), a key enzyme in the defense against oxidative stress. Supplementation of the culture media with 1 microM sodium selenite increased the activities of total GPx by threefold and the selenium-dependent GPx by fourfold as compared to cells cultured in control media. The non-selenium-dependent GPx activity was unchanged. The activities of the other glutathione (GSH)-related enzymes were practically unchanged despite a tendency toward elevation. The activities of oxidized glutathione (GSSG) reductase and catalase increased by 22.4 and 27.4%, respectively. These relatively small increases did not carry statistical significance. Supplementation of tissue culture media with selenium may prove important, particularly for cell protection against oxidative stress. 相似文献