Human Liver Carcinoma HepG2 Cells Research Articles

Preparation and characterization of stearic acid-pullulan nanoparticles

For a new anticancer drug carrier, we synthesized 4 compositions of amphiphilic stearic acidconjugated pullulan (SAP) and characterized them with FT-IR spectroscopy. Crystalline changes were verified by x-ray diffraction patterns before and after synthesis of the SAP conjugate. SAP nanoparticles were prepared by a diafiltration method, and the fluorescence spectroscopy using pyrene showed particle self-assembly in water. SAP nanoparticles were spherical in TEM photos, and particle size ranged between 200 approximately 500 nm in photon correlation spectroscopy. Release of all-trans-retinoic acid from the SAP nanoparticles was maintained over 5 weeks. For further study in vivo, we tested the cytotoxicity of SAP nanoparticles using an MTT assay, and cytotoxicity was augmented as the molar mass of stearic acid increased in human liver carcinoma HepG2 cells. Therefore, SAP nanoparticles might be a promising longterm delivery carrier for hydrophobic therapeutic molecules with the appropriate composition.

Malathion‐induced oxidative stress, cytotoxicity, and genotoxicity in human liver carcinoma (HepG2) cells

Malathion is an organophosphate pesticide that is known for its high toxicity to insects and low to moderate potency to humans and other mammals. Its toxicity has been associated with the inhibition of acetylcholinesterase activity, leading to the interference with the transmission of nerve impulse, accumulation of acetylcholine at synaptic junctions, and subsequent induction of adverse health effects including headache, dizziness, nausea, vomiting, bradycardia, and miosis. Oxidative stress (OS) has been reported as a possible mechanism of malathion toxicity in humans. Hence, the aim of this study was to examine the role of OS in malathion-induced cytotoxicity and genotoxicity. To achieve this goal, MTT, lipid peroxidation, and single cell gel electrophoresis (Comet) assays were performed, respectively, to evaluate the levels of cell viability, malondialdehyde (MDA) production, and DNA damage in human liver carcinoma (HepG(2)) cells. Study results indicated that malathion is mitogenic at lower levels of exposure, and cytotoxic at higher levels of exposure. Upon 48 h of exposure, the average percentages of cell viability were 100% +/- 11%, 117% +/- 15%, 86% +/- 15%, 35% +/- 9%, and 27% +/- 7% for 0, 6, 12, 18, and 24 mM, respectively. In the lipid peroxidation assay, the concentrations of MDA produced were 12.55 +/- 0.16, 20.65 +/- 0.27, 31.1 +/- 0.40, 34.75 +/- 0.45, and 15.1 +/- 0.20 muM in 0, 6, 12, 18, and 24 mM malathion, respectively. The Comet assay showed a significant increase in DNA damage at the 24 mM malathion exposure. Taken together, our results indicate that malathion exposure at higher concentrations induces cytotoxic and genotoxic effects in HepG(2) cells, and its toxicity may be mediated through OS as evidenced by a significant production of MDA, an end product of lipid peroxidation.

Abstract 4990: Nuclear Factor-Y (NF-Y) regulates the transcription of the human aldo-keto reductase 1C1 (AKR1C1) gene*

Abstract Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Here, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene. The 5′ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5′-flanking, ∼3.0 kb region of the human AKR1C1 gene, identified the region between −128 to −88 as the minimal proximal promoter that is essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Based on computational analysis, a set of potential putative transcription factor binding sites for Oct1, Sp1, NF-Y, C/EBP, p40X, USF, NF1 and AP-2 were identified in this region. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were essential for controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at −109ATTGG−105 of the AKR1C1 gene. These results indicate that the NF-Y transcription factor regulates the basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4990.

Abstract 4380: Cadmium-induced toxicity in human liver carcinoma (HepG2) cells: Involvement of oxidative stress, phosphatidylserine externalization, and nucleosomal DNA fragmentation

Abstract Human liver carcinoma (HepG2) cells were used as a test model to evaluate the in vitro cytotoxicity of cadmium and to determine whether oxidative stress plays a key role in cadmium induced apoptosis. To achieve this goal, we performed the MTT assay to measure cell viability and lipid peroxidation to determine the level of oxidative cell/tissue damage. The annexin V assay and DNA fragmentation analysis were performed for early and late apoptosis, respectively. The results of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a dose-dependent manner, showing a 24 h-LD50 of 3.6 µg/mL. We detected a significant increase in lipid peroxidation (malondialdehyde) concentrations in cadmium chloride-treated HepG2 cells compared to the control. Similarly, a strong dose-response relationship (p < 0.05) was obtained in connection with cadmium chloride-induced apoptosis. Cadmium chloride-induced apoptosis was characterized by a significant increase in the percentage of annexin-V positive cells (apoptotic cells), as well by the occurrence of nucleosomal DNA fragmentation. In summary, this study indicated cadmium chloride is highly cytotoxic to HepG2 cells. This cytotoxic effect may be mediated at least in part through oxidative cell/tissue damage, phosphatidylserine externalization and occurrence of nucleosomal DNA fragmentation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4380.

Thioamide derivative modulates annexin v expression in human liver carcinoma (HepG2) cells

Thioamide is one of a group of antithyroid drugs prescribed in the treatment of hyperthyroidism. Thioamide derivative is currently used for the treatment of thyroid disease, tuberculosis, and leprosy. Recent in vitro experiment in our laboratory demonstrated thioamide is highly cytotoxic to human liver carcinoma (HepG2) cells. Although published studies indicate that thioamide derivative has medicinal properties effective against many diseases other than liver cancer, the molecular mechanisms under which this compound induces apoptosis of cancer cells remain largely unknown. In the present study, we proposed to use human liver carcinoma (HepG2) cells as a test model to evaluate the basis apoptotic mechanism of thioamide derivative. To achieve this goal, the expression of annexin V was measured by flow cytometric analysis. Human liver carcinoma (HepG2) cells were treated with different doses of thioamide derivative for 48 hours. Results from the flow cytometric assessment (Annexin V FITC/PI) showed a strong dose‐response relationship between thioamide derivative exposure and annexin V positive cells undergoing early stage apoptosis in HepG2 cells. Taken together, our results indicate that phosphatidylserine externalization was involved in thioamide derivative induced toxicity to HepG2 cells. This grant is supported in part by NIH‐NIGMS Grant R25GM50117 and in part by NIH Grant 2G12RR013459‐11.

Thioamide derivative‐induced cytotoxic effect in human liver carcinoma (HepG2) cells

Thioamide is an antithyroid drug that has been shown to cross the placental barrier. Use of antithyroid medications in pregnancy may result in fetal hypothyroidism, goiter, and cretinism. The literature review indicated that about 10% of patients taking thioamide drug have skin eruptions, maculespapules, urticaria, dermatitis, fever, and arthralgia. However, the precise mechanisms involved in thioamide toxicity are not well elucidated. In the present study, we use human liver carcinoma (HepG2) cells as a test model to evaluate the cytotoxicity of three thioamide derivatives. To achieve this goal, cell survival was determined by the means of MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐ diphenyltetrazolium bromide, a tetrazole) assay. Human liver carcinoma (HepG2) cells were treated with different doses of thioamide derivative for 48 hours. Results from MTT assay indicate that thioamide derivative gradually reduce the viability of HepG2 cells in a dose‐dependent manner, showing LD50 value of about 13.58, 32, and 38μM, upon 48 h of exposure, respectively. Based on this in vitro, our thioamide derivative appears to be highly cytotoxic to HepG2 cells. This research was supported in part by NIH‐NIGMS Grant R 25 GM 50117, and in part by a grant from the National Institutes of Health, Grant No. 2G12RR013459‐11, through the RCMI‐Center for Environmental Health at Jackson State University.

The CCAAT box binding transcription factor, nuclear factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1C1 (AKR1C1) gene

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene. The 5′ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5′ flanking, ∼ 3.0 kb region of the human AKR1C1 gene identified the region between − 128 and − 88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 and 2008/C13*), lung (H23 and A549) and liver carcinoma (HepG2) cells. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel-supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at − 109 ATTGG − 105 of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Ectopic expression of NF-Ys increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or suppression of NF-YA decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that were CCAAT box-dependent. These results indicate that the NF-Y regulates the basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and the cisplatin-induced transcription in human ovarian carcinoma cells.

Synthesis and antiproliferative activity of indolizine derivatives incorporating a cyclopropylcarbonyl group against Hep-G2 cancer cell line

Indolizine and annulated indolizine derivatives incorporating a cyclopropylcarbonyl group were synthesized in a one pot procedure by the tanden reactions of [3 + 2] cycloaddition of the corresponding N-ylide with electron deficient alkene. Seventeen indolizine derivatives were reported for the first time. All the compounds were examined for their antiproliferative activity against the human hepatocellular liver carcinoma ( Hep-G2) cell line by MTT method. Among the compounds tested, 5a, 5d, 5g and 5j showed the most favorable activities with IC 50 values of 0.39, 0.48, 0.29 and 0.20 μg/mL. Especially, compound 5j displayed potent antiproliferative activities with IC 50 value of 0.20 μg/mL, and showed significant EGFR kinase inhibitory activity with IC 50 value of 0.085 μM. Docking simulations of 5j were carried out to illustrate the binding mode of the molecular into the EGFR active site.

Abstract A39: In vitro mechanisms of cadmium-induced apoptosis in human liver carcinoma (HEPG2) cells

Abstract Cadmium is a heavy metal that has been shown to manifest its toxicity in humans and animals. Many documented in vitro studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Diseases like autoimmune thyroidism are thought to be caused, in large part, by overexposure to cadmium. Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, the mechanisms of the carcinogenic activity of cadmium is not clearly defined. Therefore, the purpose of this study was to elucidate some of the molecular mechanisms that are involved in cadmium-induced toxicity in human liver carcinoma (HepG2) cells. To achieve this goal, we performed the MTT assay to measure cell viability. Annexin V assay and DNA fragmentation analysis were performed for early and late apoptosis, respectively. The result of MTT assay indicated that cadmium induces toxicity to HepG2 cells in a dose-dependent manner, showing a 24 h-LD50 of 3.6 µg/mL cadmium chloride. Similarly, a strong dose-response relationship (p < 0.05) was obtained in connection with cadmium chloride-induced apoptosis. Cadmium chloride-induced apoptosis was characterized by a significant increase in the percentages of annexin-V positive cells, as well by the occurrence of nucleosomal DNA fragmentation. In summary, this study indicated cadmium chloride is highly cytotoxic to HepG2 cells. This cytotoxic effect may be mediated at least in part through phosphatidylserine externalization and occurrence of nucleosomal DNA fragmentation. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A39.

Polyhydroxy Steroids and Saponins from China Sea Starfish Asterina pectinifera and Their Biological Activities

A new polyhydroxy sterol ester, (25S)-5alpha-cholestane-3beta,6alpha,7alpha,8,15alpha,16beta-hexahydroxyl-26-O-14'Z-eicosenoate (1), together with seven known steroid derivatives (2-8), were isolated from the EtOH extract of the whole body of China Sea starfish Asterina pectinifera. The structure of 1 was determined by using extensive spectra analysis (IR, 1D and 2D NMR, and MS), chemical degradation, and comparison with the known compound (25S)-5alpha-cholestane-3beta,6alpha,7alpha,8,15alpha,16beta,26-heptol (2). All the isolates were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) and their cytotoxicity against human liver carcinoma HepG2 cell line in vitro. Compounds 3-6, and 8 exhibited antiviral activity against HSV-1 virus with the minimal inhibitory concentration (MIC) values of 0.2, 0.05, 0.2, 0.22, and 0.07 microM, respectively. While compounds 4 and 5 exhibited cytotoxicity against HepG2 cells with IC(50) values of 0.2 and 1.6 microM, respectively.

7-O-Methylaromadendrin Stimulates Glucose Uptake and Improves Insulin Resistance in Vitro

The stimulation of glucose uptake into peripheral tissues is an important mechanism for the removal of glucose in blood and for the management of diabetes mellitus (DM). Since recent results have demonstrated the beneficial effects of flavonoids in relation to DM, this study was designed to examine the effects of 7-O-methylaromadendrin (7-O-MA), a flavonoid isolated from Inula viscosa, on glucose uptake into liver and fat tissue, and investigate the molecular mechanisms involved. 7-O-MA at 10 microM significantly stimulated insulin-induced glucose uptake measured by 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) in both human hepatocellular liver carcinoma (HepG2) cells and differentiated 3T3-L1 adipocytes. Adipocyte-specific fatty acid binding protein (aP2) gene expression was increased by 7-O-MA in adipocytes, and both gene and protein level of peroxisome proliferator-activated receptor gamma2 (PPARgamma2) was also increased. Moreover, 7-O-MA stimulated the reactivation of insulin-mediated phosphorylation of phosphatidylinositol 3-kinase (PI3K)-linked protein kinase B (Akt/PKB) and adenosine 5'-monophosphate-activated protein kinase (AMPK) in high glucose-induced, insulin-resistant HepG2 cells, and this effect was blocked by either LY294002, a PI3K inhibitor, or compound C, an AMPK inhibitor. Therefore, these results suggest that 7-O-MA might stimulate glucose uptake via PPARgamma2 activation and improve insulin resistance via PI3K and AMPK-dependent pathways, and be a potential candidate for the management of type 2 DM.

Porphyrin and Galactosyl Conjugated Micelles for Targeting Photodynamic Therapy

To study the targeting and photodynamic therapy efficiency of porphyrin and galactosyl conjugated micelles based on amphiphilic copolymer galactosyl and mono-aminoporphyrin (APP) incoporated poly(2-aminoethyl methacrylate)-polycaprolactone (Gal-APP-PAEMA-PCL). Poly(2-aminoethyl methacrylate)-polycaprolactone (PAEMA-PCL) was synthesized by the combination of ring opening polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization, and then Gal-APP-PAEMA-PCL was obtained after conjugation of lactobionic acid and 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (APP) to PAEMA-PCL. The chemical structures of the copolymers were characterized, and their biological properties were evaluated in human laryngeal carcinoma (HEp2) and human hepatocellular liver carcinoma (HepG2) cells. Both APP-PAEMA-PCL and Gal-APP-PAEMA-PCL did not exhibit dark cytotoxicity to HEp2 cells and HepG2 cells. However, Gal-APP-PAEMA-PCL was taken up selectively by HepG2 cells and had the higher phototoxicity effect. Both polymers preferentially localized within cellular vesicles that correlated to the lysosomes. The results indicated that porphyrin and galactosyl conjugated polymer micelles exhibited higher targeting and photodynamic therapy efficacy in HepG2 cells than in HEp2 cells.

Potassium Dichromate Induced Cytotoxicity, Genotoxicity and Oxidative Stress in Human Liver Carcinoma (HepG2) Cells

Chromium is a widespread industrial waste. The soluble hexavalent chromium Cr (VI) is an environmental contaminant widely recognized to act as a carcinogen, mutagen and teratogen towards humans and animals. The fate of chromium in the environment is dependent on its oxidation state. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction to trivalent chromium, resulting in the formation of reactive oxygen species together with oxidative tissue damage and a cascade of cellular events. However, the results from in vitro studies are often conflicting. The aim of this study was to develop a model to establish relationships between cytotoxicity, genotoxicity and oxidative stress, in human liver carcinoma [HepG2] cells exposed to potassium dichromate. HepG2 cells were cultured following standard protocols and exposed to various concentrations [0–50 μM] of potassium dichromate [K2Cr2O7]. Following exposure to the toxic metal, the MTT assay was performed to assess the cytotoxicity, the thiobarbituric acid test to evaluate the degree of lipid peroxidation as an indicator of oxidative stress and the alkaline comet assay was used to assess DNA damage to study genotoxicity. The results of the study indicated that potassium dichromate was cytotoxic to HepG2 cells. The LD50 values of 8.83 ± 0.89 μg/ml, 6.76 ± 0.99 μg/ml, respectively, for cell mortality at 24 and 48 hrs were observed, indicating a dose- and time-dependent response with regard to the cytotoxic effects of potassium dichromate. A statistically significant increase in the concentration of malondialdehyde [MDA], an indicator of lipid peroxidation, was recorded in exposed cells [15.9 – 69.9 μM] compared to control [13 μM]. Similarly, a strong dose-response relationship (p<0.05) was also obtained with respect to potassium dichromate induced DNA damage (comet assay) in HepG2 cells exposed [3.16 ± 0.70 – 24.84 ± 1.86 microns – mean comet tail length]; [12.4 ± 1.45% – 76 ± 1.49% – % tail DNA] to potassium dichromate than control [3.07 ± 0.26 microns – mean comet tail length]; [2.69 + 0.19% – % Tail DNA], respectively. The results demonstrated that potassium dichromate was highly cytotoxic to HepG2 cells, and its cytotoxicity seems to be mediated by oxidative stress and DNA damage.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor gamma (PPARgamma), NF-kappaB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC(50) was measured as 8.45 micromol/L and 191.55 micromol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC(50) was 9.27 micromol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 micromol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 micromol/L ADFMChR than when treated with 30.0 micromol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 micromol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 micromol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 micromol/L GW9662, a blocker of PPARgamma. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 micromol/L ADFMChR, PPARgamma and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-kappaB expression decreased; however, pre-incubation with 10.0 micromol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 micromol/L ADFMChR on PPARgamma and NF-kappaB protein expression in HepG2 cells. ADFMChR induces apoptosis of HepG2 cell lines by activating PPARgamma, inhibiting protein expression of Bcl-2 and NF-kappaB, and increasing Bax expression.

Effect of ethyl acetate extract from Marchantia convoluta on inhibition of human liver carcinoma HepG2 cells

The effect of the dichloromethane fraction (DF) of the ethyl acetate extract from Marchantia convoluta on the inhibition of human liver carcinoma cell line HepG2 was investigated. DF can remarkably induce HepG2 cell apoptotic, accompanied by a decrease of the cell viability and an increase of the fraction of S-phase cells by 62.5% in a concentration-dependent manner.

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. In this study, we designed a secretory protein by adding a secretory signal peptide (SP) to the N terminus of Transactivating Transcription (TAT)-apoptin (SP-TAT-apoptin), to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. In human liver carcinoma HepG2 cells, SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h, however, it translocated into the nuclear compartment and caused massive apoptotic cell death, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not, however, cause any cell death in non-malignant human umbilical vein endothelial cells (HUVECs). Most importantly, the conditioned medium from Chinese hamster ovary (CHO) cells transfected with SP-TAT-apoptin also induced significant cell death in HepG2 cells, but not in HUVECs. The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

N-Acetyl-L-Cysteine Affords Protection against Lead-Induced Cytotoxicity and Oxidative Stress in Human Liver Carcinoma (HepG2) Cells

Although lead exposure has declined in recent years as a result of change to lead-free gasoline, several epidemiological have pointed out that it represents a medical and public health emergency, especially in young children consuming high amounts of lead-contaminated flake paints. A previous study in our laboratory indicated that lead exposure induces cytotoxicity in human liver carcinoma cells. In the present study, we evaluated the role of oxidative stress in lead-induced toxicity, and the protective effect of the anti-oxidant n-acetyl-l-cysteine (NAC). We hypothesized that oxidative stress plays a role in lead-induced cytotoxicity, and that NAC affords protection against this adverse effect. To test this hypothesis, we performed the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay and the trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test for lipid peroxidation. Data obtained from the MTT assay indicated that NAC significantly increased the viability of HepG2 cells in a dose-dependent manner upon 48 hours of exposure. Similar trend was obtained with the trypan blue exclusion test. Data generated from the thiobarbituric acid test showed a significant (p <or= 0.05) increase of MDA levels in lead nitrate-treated HepG2 cells compared to control cells. Interestingly, the addition of NAC to lead nitrate-treated HepG2 cells significantly decreased cellular content of reactive oxygen species (ROS), as evidenced by the decrease in lipid peroxidation byproducts. Overall, findings from this study suggest that NAC inhibits lead nitrate-induced cytotoxicity and oxidative stress in HepG2 cells. Hence, NAC may be used as a salvage therapy for lead-induced toxicity in exposed persons.

Design, synthesis and cytotoxic activity of novel 1-aroyl-4-(2-chloroethyl)semicarbazides

A series of aroyl derivatives of 4-(2-chloroethyl)semicarbazide were designed and synthesized to explore their antiproliferative activity against human brain carcinoma (U251) and human liver carcinoma (Hepg2) cell lines. The synthesized compounds were characterized by elemental analyses and spectroscopic data. It was established that compounds in which semicarbazide fragments are substituted with a (2-indolyl)carbonyl moiety showed a higher cytotoxic activity than the corresponding benzoyl derivatives. 1-[(5-Benzyloxy-1H-indol-2-yl)carbonyl]-4-(2-chloroethyl)semicarbazide (24) showed the highest cytotoxic activity against Hepg2 (IC50= 21 µg/ml), while 4-(2-chloroethyl)-1-[(5-methoxy-1H-indol-2-yl)carbonyl]semicarbazide (23) was the most active compound against U251 (IC50 = 8 µg/ml).

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.

Microarray Analysis of Mercury-Induced Changes in Gene Expression in Human Liver Carcinoma (HepG2) Cells: Importance in Immune Responses

Mercury is widely distributed in the biosphere, and its toxic effects have been associated with human death and several ailments that include cardiovascular diseases, anemia, kidney and liver damage, developmental abnormalities, neurobehavioral disorders, autoimmune diseases, and cancers in experimental animals. At the cellular level, mercury has been shown to interact with sulphydryl groups of proteins and enzymes, to damage DNA, and to modulate cell cycle progression and/or apoptosis. However, the underlying molecular mechanisms of mercury toxicity remain to be elucidated. Our laboratory has demonstrated that mercury exposure induces cytotoxicity and apoptosis, modulates cell cycle, and transcriptionally activates specific stress genes in human liver carcinoma cells. The liver is one of the few organs capable of regeneration from injury. Dormant genes in the liver are therefore capable of reactivation. In this research, we hypothesize that mercury-induced hepatotoxicity is associated with the modulation of specific gene expressions in liver cells that can lead to several disease states involving immune system dysfunctions. In testing this hypothesis, we used an Affymetrix oligonucleotide microarray with probe sets complementary to more than 20,000 genes to determine whether patterns of gene expressions differ between controls and mercury (1-3 microg/mL) treated cells. There was a clear separation in gene expression profiles between controls and mercury-treated cells. Hierarchical cluster analysis identified 2,211 target genes that were affected. One hundred and eighty-eight of these genes were up-regulated, among which forty eight were significant (p = 0.001) with greater than a two-fold change difference in the concentration range (1-3 microg/mL) of mercury-treated cells; twelve genes were moderately over-expressed with an increase of more than one fold (p = 0.004). 2,023 genes were down-regulated with only forty of them reaching statistically significant decline at p = 0.05 according to the Welch's ANOVA/Welch's t-test. Further analyses of affected genes identified genes located on all human chromosomes with higher than normal effects on genes found on chromosomes 1-14, 17-20 (sex-determining region Y)-box18SRY, 21 (splicing factor, arginine/serine-rich 15 and ATP-binding), and X (including BCL6-co-repressor). These genes are categorized as control and regulatory genes for metabolic pathways involving the cell cycle (cyclin-dependent kinases), apoptosis, cytokine expression, Na+/K+ ATPase, stress responses, G-protein signal transduction, transcription factors, DNA repair as well as metal-regulatory transcription factor 1, MTF1 HGNC, chondroitin sulfate proteoglycan 5 (neuroglycan C), ATP-binding cassette, sub-family G (WHITE), cytochrome b-561 family protein, CDC-like kinase 1 (CLK1 HGNC) (protein tyrosine kinase STY), Na+/H+ exchanger regulatory factor (NHERF HGNC), potassium voltage-gated channel subfamily H member 2 (KCNH2), putative MAPK activating protein (PM20, PM21), ras homolog gene family, polymerase (DNA directed), delta regulatory subunit (50 kDa), leptin receptor involved in hematopoietin/interferon-class (D200- domain) cytokine receptor activity and thymidine kinase 2, mitochondrial TK2 HGNC and related genes. Significant alterations in these specific genes provide new directions for deeper mechanistic investigations that would lead to a better understanding of the molecular basis of mercury-induced toxicity and human diseases that may result from disturbances in the immune system.