Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway.

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Zhang, Zhuangwei; Zhang, Huiqin; Chen, Shiyong; Xu, Yan; Yao, Anjun; Liao, Qi; Han, Liyuan; Zou, Zuquan; Zhang, Xiaohong

2017-02-01

The plant flavonol dihydromyricetin (DHM) was reported to induce apoptosis in human hepatocarcinoma HepG2 cells. This study was undertaken to elucidate the underlying molecular mechanism of action of DHM. In the study, DHM down-regulated Akt expression and its phosphorylation at Ser473, up-regulated the levels of mitochondrial proapoptotic proteins Bax and Bad, and inhibited the phosphorylation of Bad at Ser136 and Ser112. It also inhibited the expression of the antiapoptotic protein Bcl-2 and enhanced the cleavage and activation of caspase-3 as well as the degradation of its downstream target poly(ADP-ribose) polymerase. Our results for the first time suggest that DHM-induced apoptosis in HepG2 cells may come about by the inhibition of the Akt/Bad signaling pathway and stimulation of the mitochondrial apoptotic pathway. Dihydromyricetin may be a promising therapeutic medication for hepatocellular carcinoma. Copyright © 2017 Elsevier Inc. All rights reserved.

Nickel oxide nanoparticles exert cytotoxicity via oxidative stress and induce apoptotic response in human liver cells (HepG2).

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Ahamed, Maqusood; Ali, Daoud; Alhadlaq, Hisham A; Akhtar, Mohd Javed

2013-11-01

Increasing use of nickel oxide nanoparticles (NiO NPs) necessitates an improved understanding of their potential impact on human health. Previously, toxic effects of NiO NPs have been investigated, mainly on airway cells. However, information on effect of NiO NPs on human liver cells is largely lacking. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and induction of apoptotic response in human liver cells (HepG2) due to NiO NPs exposure. Prepared NiO NPs were crystalline and spherical shaped with an average diameter of 44 nm. NiO NPs induced cytotoxicity (cell death) and ROS generation in HepG2 cells in dose-dependent manner. Further, ROS scavenger vitamin C reduced cell death drastically caused by NiO NPs exposure indicating that oxidative stress plays an important role in NiO NPs toxicity. Micronuclei induction, chromatin condensation and DNA damage in HepG2 cells treated with NiO NPs suggest that NiO NPs induced cell death via apoptotic pathway. Quantitative real-time PCR analysis showed that following the exposure of HepG2 cells to NiO NPs, the expression level of mRNA of apoptotic genes (bax and caspase-3) were up-regulated whereas the expression level of anti-apoptotic gene bcl-2 was down-regulated. Moreover, activity of caspase-3 enzyme was also higher in NiO NPs treated cells. To the best of our knowledge this is the first report demonstrating that NiO NPs caused cytotoxicity via ROS and induced apoptosis in HepG2 cells, which is likely to be mediated through bax/bcl-2 pathway. This work warrants careful assessment of Ni NPs before their commercial and industrial applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

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Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Radiation induced bystander effect on hepatoma HepG2 cells under hypoxia condition

International Nuclear Information System (INIS)

Zhang Jianghong; Jin Yizun; Shao Chunlin; Prise KM

2009-01-01

Objective: To investigate radiation induced bystander effect and its mechanism on hepatoma HepG2 cells under hypoxia condition. Methods: Non-irradiated bystander hepatoma cells were co-cultured with irradiated cells or treated with the conditioned medium (CM) from irradiated cells, then micronuclei (MN) were measured for both irradiated cells and bystander cells. Results: The MN yield of irradiated HepG2 cells under hypoxic condition was significantly lower than that under normoxia, the oxygen enhancement ratio of HepG2 cells of MN was 1.6. For both hypoxic and normoxic condition, the MN yield of bystander cells were obviously enhanced to a similar high level after co-culturing with irradiated cells or with CM treatment, and it also correlated with the irradiation dose. When the hypoxic HepG2 cells were treated with either DMSO, a scavenger of reactive oxygen species (ROS), or aminoguanidine, an iNOS inhibitor, the yield of bystander MN was partly diminished, and the reducing rate of DMSO was 42.2%-46.7%, the reducing rate of aminoguanidine was 42% . Conclusion: ROS, NO and their downstream signal factors are involved in the radiation induced bystander effect of hypoxic HepG2 cells. (authors)

Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2 through a mitochondrial-mediated pathway

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Ji, Y.; Ji, C.; Yue, L.; Xu, H.

2012-01-01

Objective Many scientific studies have shown that Asparagus officinalis has an antitumour effect and enhances human immunity, but the active components and the antitumour mechanisms are unclear. We investigated the effects of saponins isolated from Asparagus on proliferation and apoptosis in the human hepatoma cell line HepG2. Methods HepG2 cells were treated with varying concentrations of Asparagus saponins at various times. Using mtt and flow cytometry assays, we evaluated the effects of Asparagus saponins on the growth and apoptosis of HepG2 cells. Transmission electron microscopy was used to observe the morphology of cell apoptosis. Confocal laser scanning microscopy was used to analyze intracellular calcium ion concentration, mitochondrial permeability transition pore (mptp), and mitochondrial membrane potential (mmp). Spectrophotometry was applied to quantify the activity of caspase-9 and caspase-3. Flow cytometry was used to investigate the levels of reactive oxygen species (ros) and pH, and the expressions of Bcl2, Bax, CytC, and caspase-3, in HepG2 cells. Results Asparagus saponins inhibited the growth of HepG2 cells in a dose-dependent manner. The median inhibitory concentration (IC50) was 101.15 mg/L at 72 hours. The apoptosis morphology at 72 hours of treatment was obvious, showing cell protuberance, concentrated cytoplasm, and apoptotic bodies. The apoptotic rates at 72 hours were 30.9%, 51.7%, and 62.1% (for saponin concentrations of 50 mg/L, 100 mg/L, 200 mg/L). Treatment with Asparagus saponins for 24 hours increased the intracellular level of ros and Ca2+, lowered the pH, activated intracellular mptp, and decreased mmp in a dose-dependent manner. Treatment also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl2, upregulated the expression of Bax, and induced release of CytC and activation of caspase-3. Conclusions Asparagus saponins induce apoptosis in HepG2 cells through a mitochondrial-mediated and caspase

FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis.

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Wang, Jiao; Liu, Shu; Yin, Yancun; Li, Mingjin; Wang, Bo; Yang, Li; Jiang, Yangfu

2015-03-01

The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2α phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated up-regulation of Bim is a key mechanism underlying rhein-induced cancer cells apoptosis.

Activation of farnesoid X receptor increases the expression of cytokine inducible SH2-containing protein in HepG2 cells.

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Xu, Zhizhen; Huang, Gang; Gong, Wei; Zhao, Yuanyin; Zhou, Peng; Zeng, Yijun; He, Fengtian

2012-11-01

Cytokine inducible SH2-containing protein (CISH), which negatively regulates cytokine signaling by inhibiting JAK2/STAT5 activity, is regarded as a therapeutic target for inflammatory diseases. Farnesoid X receptor (FXR), a ligand-activated transcription factor, has been proposed to play a protective function in the inflammatory responses. However, the role of FXR in modulation of CISH expression is unknown. In the present study, we for the first time identified that in human hepatoma cell line HepG2 the activation of FXR by the natural agonist chenodeoxycholic acid (CDCA) and the synthetic specific agonist GW4064 upregulated CISH at both transcriptional and translational levels, and inhibited interleukin (IL)6-induced STAT5 activation. Moreover, the in vivo experiment demonstrated that gavaging mice with CDCA increased CISH expression and reduced basal STAT5 phosphorylation in liver tissues. Reporter assay showed that FXR agonists enhanced the transcriptional activity of CISH promoter. These data suggest that FXR may serve as a novel molecular target for manipulating CISH expression in hepatocytes. FXR-mediated upregulation of CISH may play an important role in the homeostasis of cytokine signal networks and be beneficial to control cytokine-associated inflammatory diseases.

[Arginase inhibitor nor-NOHA induces apoptosis and inhibits invasion and migration of HepG2 cells].

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Li, Xiangnan; Zhu, Fangyu; He, Yongsong; Luo, Fang

2017-04-01

Objective To investigate the cell inhibitory effect of arginase inhibitor nor-NOHA on HepG2 hepatocellular carcinoma cells and related mechanism. Methods CCK-8 assay was used to detect the cell proliferation and flow cytometry to detect the apoptosis of HepG2 cells treated with (0, 0.5, 1.0, 2.0, 3.0) ng/μL nor-NOHA. The protein levels of arginase 1 (Arg1), P53, matrix metalloproteinase-2 (MMP-2), E-cadherin (ECD) were determined by Western blotting. Real time quantitative PCR was employed to examine the changes in the mRNA level of inducible nitric oxide synthase (iNOS). Griess assay was used to measure the concentration of nitric oxide (NO) in HepG2 cells. Transwell TM assay and wound-healing assay were performed to evaluate the changes of the cell invasion and migration ability, respectively. Results nor-NOHA inhibited the proliferation and induced the apoptosis of HepG2 cells. It also decreased the expression levels of Arg1 and MMP-2, increased the expression levels of P53 and ECD as well as the production of NO; in addition, nor-NOHA inhibited the invasion and migration of HepG2 cells. Conclusion Nor-NOHA can induce cell apoptosis and inhibit the ability of invasion and migration of HepG2 cells by inhibiting Arg1, which is related with the increase of iNOS expression and the high concentration of NO.

Protective effects of flavonoids isolated from Korean milk thistle Cirsium japonicum var. maackii (Maxim.) Matsum on tert-butyl hydroperoxide-induced hepatotoxicity in HepG2 cells.

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Jung, Hyun Ah; Abdul, Qudeer Ahmed; Byun, Jeong Su; Joung, Eun-Ji; Gwon, Wi-Gyeong; Lee, Min-Sup; Kim, Hyeung-Rak; Choi, Jae Sue

2017-09-14

Milk thistle leaves and flowers have been traditionally used as herbal remedy to alleviate liver diseases for decades. Korean milk thistle, Cirsium japonicum var. maackii (Maxim.) Matsum has been employed in traditional folk medicine as diuretic, antiphlogistic, hemostatic, and detoxifying agents. The aim of current investigation was to evaluate hepatoprotective properties of the MeOH extract of the roots, stems, leaves and flowers of Korean milk thistle as well as four isolated flavonoids, luteolin, luteolin 5-O-glucoside, apigenin and apigenin 7-O-glucuronide during t-BHP-induced oxidative stress in HepG2 cells. Hepatoprotective potential of the MeOH extracts and flavonoids derived from Korean milk thistle against t-BHP-induced oxidative stress in HepG2 cells were evaluated following MTT method. Incubating HepG2 cells with t-BHP markedly decreased the cell viability and increased the intracellular ROS generation accompanied by depleted GSH levels. Protein expression of heme oxygenase (HO-1) and nuclear factor-E2-related factor 2 (Nrf-2) was determined by Western blot. Our findings revealed that pretreating HepG2 cells with MeOH extracts and bioactive flavonoids significantly attenuated the t-BHP-induced oxidative damage, followed by increased cell viability in a dose-dependent manner. The results illustrate that excess ROS generation was reduced and GSH levels increased dose-dependently when HepG2 cells were pretreated with four flavonoids. Moreover, Western blotting analysis demonstrated that protein expressions of Nrf-2 and HO-1 were also up-regulated by flavonoids treatment. These results clearly demonstrate that the MeOH extracts and flavonoids from Korean milk thistle protected HepG2 cells against oxidative damage triggered by t-BHP principally by modulating ROS generation and restoring depleted GSH levels in addition to the increased Nrf-2/HO-1 signaling cascade. These flavonoids are potential natural antioxidative biomarkers against oxidative stress-induced

Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

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Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

2016-04-01

Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

Influence of TiO{sub 2} nanoparticles on cellular antioxidant defense and its involvement in genotoxicity in HepG2 cells

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Petkovic, Jana; Zegura, Bojana; Filipic, Metka, E-mail: metka.filipic@nib.si [Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, SI-1000 Ljubljana (Slovenia)

2011-07-06

We investigated the effects of two types of TiO{sub 2} nanoparticles (<25 nm anatase, TiO{sub 2}-An; <100 nm rutile, TiO{sub 2}-Ru) on cellular antioxidant defense in HepG2 cells. We previously showed that in HepG2 cells, TiO{sub 2} nanoparticles are not toxic, although they induce oxidative DNA damage, production of intracellular reactive oxygen species, and up-regulation of mRNA expression of DNA-damage-responsive genes (p53, p21, gadd45{alpha} and mdm2). In the present study, we measured changes in mRNA expression of several antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, nitric oxide synthase, glutathione reductase and glutamate-cysteine ligase. As reduced glutathione has a central role in cellular antioxidant defense, we determined the effects of TiO{sub 2} nanoparticles on changes in the intracellular glutathione content. To confirm a role for glutathione in protection against TiO{sub 2}-nanoparticle-induced DNA damage, we compared the extent of TiO{sub 2}-nanoparticle-induced DNA damage in HepG2 cells that were glutathione depleted with buthionine-(S,R)-sulfoximine pretreatment and in nonglutathione-depleted cells. Our data show that both types of TiO{sub 2} nanoparticles up-regulate mRNA expression of oxidative-stress-related genes, with TiO{sub 2}-Ru being a stronger inducer than TiO{sub 2}-An. Both types of TiO{sub 2} nanoparticles also induce dose-dependent increases in intracellular glutathione levels, and in glutathione-depleted cells, TiO{sub 2}-nanoparticle-induced DNA damage was significantly greater than in nonglutathione-depleted cells. Interestingly, the glutathione content and the extent of DNA damage were significantly higher in TiO{sub 2}-An- than TiO{sub 2}-Ru-exposed cells. Thus, we show that TiO{sub 2} nanoparticles cause activation of cellular antioxidant processes, and that intracellular glutathione has a critical role in defense against this TiO{sub 2}-nanoparticle-induced DNA damage.

Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

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Sun, Hengwen [Department of Radiation, Cancer Center of Guangdong General Hospital (Guangdong Academy of Medical Science), Guangzhou, 510080, Guangdong (China); Yang, Shana; Li, Jianhua [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Zhang, Yajie [Department of Pathology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Gao, Dongsheng [Department of Oncology, Guangdong Medical College Affiliated Pengpai Memorial Hospital, Hai Feng, 516400, Gungdong (China); Zhao, Shenting, E-mail: zhaoshenting@126.com [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China)

2016-03-25

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

International Nuclear Information System (INIS)

Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

2016-01-01

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

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Guo, Haiqing; Ren, Feng; Zhang, Li; Zhang, Xiangying; Yang, Rongrong; Xie, Bangxiang; Li, Zhuo; Hu, Zhongjie; Duan, Zhongping; Zhang, Jing

2016-03-01

Kaempferol is a flavonoid compound that has gained importance due to its antitumor properties; however, the underlying mechanisms remain to be fully understood. The present study aimed to investigate the molecular mechanisms of the antitumor function of kaempferol in HepG2 hepatocellular carcinoma cells. Kaempferol was determined to reduce cell viability, increase lactate dehydrogenase activity and induce apoptosis in a concentration‑ and time‑dependent manner in HepG2 cells. Additionally, kaempferol‑induced apoptosis possibly acts via the endoplasmic reticulum (ER) stress pathway, due to the significant increase in the protein expression levels of glucose‑regulated protein 78, glucose‑regulated protein 94, protein kinase R‑like ER kinase, inositol‑requiring enzyme 1α, partial activating transcription factor 6 cleavage, caspase‑4, C/EBP homologous protein (CHOP) and cleaved caspase‑3. The pro‑apoptotic activity of kaempferol was determined to be due to induction of the ER stress‑CHOP pathway, as: i) ER stress was blocked by 4‑phenyl butyric acid (4‑PBA) pretreatment and knockdown of CHOP with small interfering RNA, which resulted in alleviation of kaempferol‑induced HepG2 cell apoptosis; and ii) transfection with plasmid overexpressing CHOP reversed the protective effect of 4‑PBA in kaempferol‑induced HepG2 cells and increased the apoptotic rate. Thus, kaempferol promoted HepG2 cell apoptosis via induction of the ER stress‑CHOP signaling pathway. These observations indicate that kaempferol may be used as a potential chemopreventive treatment strategy for patients with hepatocellular carcinoma.

Signaling dynamics of palmitate-induced ER stress responses mediated by ATF4 in HepG2 cells

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Cho Hyunju

2013-01-01

Full Text Available Abstract Background Palmitic acid, the most common saturated free fatty acid, has been implicated in ER (endoplasmic reticulum stress-mediated apoptosis. This lipoapotosis is dependent, in part, on the upregulation of the activating transcription factor-4 (ATF4. To better understand the mechanisms by which palmitate upregulates the expression level of ATF4, we integrated literature information on palmitate-induced ER stress signaling into a discrete dynamic model. The model provides an in silico framework that enables simulations and predictions. The model predictions were confirmed through further experiments in human hepatocellular carcinoma (HepG2 cells and the results were used to update the model and our current understanding of the signaling induced by palmitate. Results The three key things from the in silico simulation and experimental results are: 1 palmitate induces different signaling pathways (PKR (double-stranded RNA-activated protein kinase, PERK (PKR-like ER kinase, PKA (cyclic AMP (cAMP-dependent protein kinase A in a time dependent-manner, 2 both ATF4 and CREB1 (cAMP-responsive element-binding protein 1 interact with the Atf4 promoter to contribute to a prolonged accumulation of ATF4, and 3 CREB1 is involved in ER-stress induced apoptosis upon palmitate treatment, by regulating ATF4 expression and possibly Ca2+ dependent-CaM (calmodulin signaling pathway. Conclusion The in silico model helped to delineate the essential signaling pathways in palmitate-mediated apoptosis.

Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

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Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

2018-04-25

Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide

International Nuclear Information System (INIS)

Alia, Mario; Ramos, Sonia; Mateos, Raquel; Granado-Serrano, Ana Belen; Bravo, Laura; Goya, Luis

2006-01-01

Flavonols such as quercetin, have been reported to exhibit a wide range of biological activities related to their antioxidant capacity. The objective of the present study was to investigate the protective effect of quercetin on cell viability and redox status of cultured HepG2 cells submitted to oxidative stress induced by tert-butyl hydroperoxide. Concentrations of reduced glutathione and malondialdehyde, generation of reactive oxygen species and activity and gene expression of antioxidant enzymes were used as markers of cellular oxidative status. Pretreatment of HepG2 with 10 μM quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of quercetin (0.1-10 μM) prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butyl hydroperoxide in HepG2 cells. Reactive oxygen species generation induced by tert-butyl hydroperoxide was significantly reduced when cells were pretreated for 2 or 20 h with 10 μM and for 20 h with 5 μM quercetin. Finally, some of the quercetin treatments prevented the significant increase of glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase activities induced by tert-butyl hydroperoxide. Gene expression of antioxidant enzymes was also affected by the treatment with the polyphenol. The results of the biomarkers analyzed clearly show that treatment of HepG2 cells in culture with the natural dietary antioxidant quercetin strongly protects the cells against an oxidative insult

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells.

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Yang, Eun Sun; Lee, Su-Min; Park, Jeen-Woo

2010-07-01

It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells' vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: Role of nonoxidative metabolism

International Nuclear Information System (INIS)

Wu Hai; Cai Ping; Clemens, Dahn L.; Jerrells, Thomas R.; Ansari, G.A. Shakeel; Kaphalia, Bhupendra S.

2006-01-01

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs

Ethanol Extract of Dianthus chinensis L. Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells In Vitro

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Nho, Kyoung Jin; Chun, Jin Mi; Kim, Ho Kyoung

2012-01-01

Dianthus chinensis L. is used to treat various diseases including cancer; however, the molecular mechanism by which the ethanol extract of Dianthus chinensis L. (EDCL) induces apoptosis is unknown. In this study, the apoptotic effects of EDCL were investigated in human HepG2 hepatocellular carcinoma cells. Treatment with EDCL significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. This induction was associated with chromatin condensation, activation of caspases, and cleavage of poly (ADP-ribose) polymerase protein. However, apoptosis induced by EDCL was attenuated by caspase inhibitor, indicating an important role for caspases in EDCL responses. Furthermore, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl. These results support a mechanism whereby EDCL induces apoptosis through the mitochondrial pathway and caspase activation in HepG2 cells. PMID:22645629

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

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Zhang, Yuanyuan; Han, Lirong [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Qi, Wentao [Academy of State Administration of Grain, No.11 Baiwanzhuang Avenue, Xicheng District, Beijing, 100037 (China); Cheng, Dai; Ma, Xiaolei; Hou, Lihua [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Cao, Xiaohong, E-mail: caoxh@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Wang, Chunling, E-mail: wangchunling@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China)

2015-01-24

Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

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Rafael Simó

Full Text Available Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes and in vivo (C57BL6/mice experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

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Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

2014-01-01

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

Protective effects of quercetin on nicotine induced oxidative stress in 'HepG2 cells'.

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Yarahmadi, Amir; Zal, Fatemeh; Bolouki, Ayeh

2017-10-01

Nicotine is a natural component of tobacco plants and is responsible for the addictive properties of tobacco. Nicotine has been recognized to result in oxidative stress by inducing the generation of reactive oxygen species (ROS). The purpose of this work was to estimate the hepatotoxicity effect of nicotine on viability and on antioxidant defense system in cultures of HepG2 cell line and the other hand, ameliorative effect of quercetin (Q) as an antioxidant was analyzed. Nicotine induced concentration dependent loss in HepG2 cell line viability. The results indicated that nicotine decreased activity of superoxide dismutase (SOD) and glutathione reductase (GR) and increased activities of catalase (CAT) and glutathione peroxidase (GPx) and glutathione (GSH) content in the HepG2 cells. Q significantly increased activity of SOD, GR and GSH content and decreased activity of GPX in nicotine + Q groups. Our data demonstrate that Q plays a protective role against the imbalance elicited by nicotine between the production of free radicals and antioxidant defense systems, and suggest that administration of this antioxidant may find clinical application where cellular damage is a consequence of ROS.

Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

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DU Senrong

2015-06-01

Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P＜0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

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Bosquet, Alba; Guaita-Esteruelas, Sandra; Saavedra, Paula; Rodríguez-Calvo, Ricardo; Heras, Mercedes; Girona, Josefa; Masana, Lluís

2016-06-01

Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 μM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. eFABP4, per se, induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Campomanesia adamantium (Myrtaceae fruits protect HEPG2 cells against carbon tetrachloride-induced toxicity

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Thaís de Oliveira Fernandes

2015-01-01

Full Text Available Campomanesia adamantium (Myrtaceae is an antioxidant compounds-rich Brazilian fruit popularly known as gabiroba. In view of this, it was evaluated the hepatoprotective effects of pulp (GPE or peel/seed (GPSE hydroalcoholic extracts of gabiroba on injured liver-derived HepG2 cells by CCl4 (4 mM. The results showed the presence of total phenolic in GPSE was (60% higher when compared to GPE, associated with interesting antioxidant activity using DPPH·− assay. Additionally, HPLC chromatograms and thin layer chromatography of GPE and GPSE showed the presence of flavonoids. Pretreatment of HepG2 cells with GPE or GPSE (both at 800–1000 μg/mL significantly (p < 0.0001 protected against cytotoxicity induced by CCl4. Additionally, the cells treated with both extracts (both at 1000 μg/mL showed normal morphology (general and nuclear contrasting with apoptotic characteristics in the cells only exposed to CCl4. In these experiments, GPSE also was more effective than GPE. In addition, CCl4 induced a marked increase in AST (p < 0.05 and ALT (p < 0.0001 levels, while GPE or GPSE significantly (p < 0.0001 reduced these levels, reaching values found in the control group. In conclusion, the results suggest that gabiroba fruits exert hepatoprotective effects on HepG2 cells against the CCl4-induced toxicity, probably, at least in part, associated with the presence of antioxidant compounds, especially flavonoids.

Copper(ii) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response

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Piret, Jean-Pascal; Jacques, Diane; Audinot, Jean-Nicolas; Mejia, Jorge; Boilan, Emmanuelle; Noël, Florence; Fransolet, Maude; Demazy, Catherine; Lucas, Stéphane; Saout, Christelle; Toussaint, Olivier

2012-10-01

The potential toxic effects of two types of copper(ii) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu2+ released in cell culture medium suggested that Cu2+ cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.The potential toxic effects of two types of copper(ii) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major

Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway

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Dae Sung Kim

2012-01-01

Full Text Available Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

Garcinia dulcis Fruit Extract Induced Cytotoxicity and Apoptosis in HepG2 Liver Cancer Cell Line

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Mohd Fadzelly Abu Bakar

2015-01-01

Full Text Available Garcinia dulcis or locally known in Malaysia as “mundu” belongs to the family of Clusiaceae. The study was conducted to investigate the anticancer potential of different parts of G. dulcis fruit extracts and their possible mechanism of action in HepG2 liver cancer cell line. MTT assay showed that the peel, flesh, and seed extracts of G. dulcis induced cytotoxicity in HepG2 cell line with IC50 values of 46.33 ± 4.51, 38.33 ± 3.51, and 7.5 ± 2.52 µg/mL, respectively. The flesh extract of G. dulcis induced cell cycle arrest at sub-G1 (apoptosis phase in a time-dependent manner. Staining with Annexin V-FITC and propidium iodide showed that 41.2% of the cell population underwent apoptosis after 72 hours of exposure of the HepG2 cell line to G. dulcis flesh extract. Caspase-3 has been shown to be activated which finally leads to the death of HepG2 cell (apoptosis. GC-MS analysis showed that the highest percentage of compound identified in the extract of G. dulcis flesh was hydroxymethylfurfural and 3-methyl-2,5-furandione, together with xanthones and flavonoids (based on literature, could synergistically contribute to the observed effects. This finding suggested that the flesh extract of G. dulcis has its own potential as cancer chemotherapeutic agent against liver cancer cell.

Casein Glycomacropeptide Hydrolysates Exert Cytoprotective Effect against Cellular Oxidative Stress by Up-Regulating HO-1 Expression in HepG2 Cells

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Tiange Li

2017-01-01

Full Text Available Oxidative stress is considered as an important mediator in the progression of metabolic disorders. The objective of this study was to investigate the potential hepatoprotective effects and mechanisms of bovine casein glycomacropeptide hydrolysates (GHP on hydrogen peroxide (H2O2-induced oxidative damage in HepG2 cells. Results showed that GHP significantly blocked H2O2-induced intracellular reactive oxygen species (ROS generation and cell viability reduction in a dose-dependent manner. Further, GHP concentration-dependently induced heme oxygenase-1 (HO-1 expression and increased nuclear factor-erythroid 2-related factor 2 (Nrf2 nuclear translocation. Moreover, pretreatment of GHP increased the activation of p38 mitogen-activated protein kinase (p38 MAPK and extracellular signal-regulated protein kinase 1/2 (ERK1/2, which were shown to contribute to Nrf2-mediated HO-1 expression. Taken together, GHP protected HepG2 cells from oxidative stress by activation of Nrf2 and HO-1 via p38 MAPK and ERK1/2 signaling pathways. Our findings indicate that bovine casein glycomacropeptide hydrolysates might be a potential ingredient in the treatment of oxidative stress-related disorders and further studies are needed to investigate the protective effects in vivo.

Inflammation response at the transcriptional level of HepG2 cells induced by multi-walled carbon nanotubes

International Nuclear Information System (INIS)

Piret, Jean-Pascal; Vankoningsloo, Sebastien; Noel, Florence; Saout, Christelle; Toussaint, Olivier; Mendoza, Jorge Mejia; Lucas, Stephane

2011-01-01

Poor information are currently available about the biological effects of multi-walled carbon nanotubes (MWCNT) on the liver. In this study, we evaluated the effects of MWCNT at the transcriptional level on the classical in vitro model of HepG2 hepatocarcinoma cells. The expression levels of 96 transcript species implicated in the inflammatory and immune responses was studied after a 24h incubation of HepG2 cells in presence of raw MWCNT dispersed in water by stirring. Among the 46 transcript species detected, only a few transcripts including mRNA coding for interleukine-7, chemokines receptor of the C-C families CCR7, as well as Endothelin-1, were statistically more abundant after treatment with MWCNT. Altogether, these data indicate that MWCNT can only induce a weak inflammatory response in HepG2 cells.

Inflammation response at the transcriptional level of HepG2 cells induced by multi-walled carbon nanotubes

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Piret, Jean-Pascal; Vankoningsloo, Sébastien; Noël, Florence; Mejia Mendoza, Jorge; Lucas, Stéphane; Saout, Christelle; Toussaint, Olivier

2011-07-01

Poor information are currently available about the biological effects of multi-walled carbon nanotubes (MWCNT) on the liver. In this study, we evaluated the effects of MWCNT at the transcriptional level on the classical in vitro model of HepG2 hepatocarcinoma cells. The expression levels of 96 transcript species implicated in the inflammatory and immune responses was studied after a 24h incubation of HepG2 cells in presence of raw MWCNT dispersed in water by stirring. Among the 46 transcript species detected, only a few transcripts including mRNA coding for interleukine-7, chemokines receptor of the C-C families CCR7, as well as Endothelin-1, were statistically more abundant after treatment with MWCNT. Altogether, these data indicate that MWCNT can only induce a weak inflammatory response in HepG2 cells.

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

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Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-04-12

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Polyethylenimine-functionalized silver nanoparticle-based co-delivery of paclitaxel to induce HepG2 cell apoptosis

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Li Y

2016-12-01

Full Text Available Yinghua Li,1,* Min Guo,1,* Zhengfang Lin,1 Mingqi Zhao,1 Misi Xiao,1 Changbing Wang,1 Tiantian Xu,1 Tianfeng Chen,2 Bing Zhu1 1Center Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, 2Department of Chemistry, Jinan University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Hepatocarcinoma is the third leading cause of cancer-related deaths around the world. Recently, a novel emerging nanosystem as anticancer therapeutic agents with intrinsic therapeutic properties has been widely used in various medical applications. In this study, surface decoration of functionalized silver nanoparticles (AgNPs by polyethylenimine (PEI and paclitaxel (PTX was synthesized. The purpose of this study was to evaluate the effect of Ag@PEI@PTX on cytotoxic and anticancer mechanism on HepG2 cells. The transmission electron microscope image and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay showed that Ag@PEI@PTX had satisfactory size distribution and high stability and selectivity between cancer and normal cells. Ag@PEI@PTX-induced HepG2 cell apoptosis was confirmed by accumulation of the sub-G1 cells population, translocation of phosphatidylserine, depletion of mitochondrial membrane potential, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose polymerase cleavage. Furthermore, Ag@PEI@PTX enhanced cytotoxic effects on HepG2 cells and triggered intracellular reactive oxygen species; the signaling pathways of AKT, p53, and MAPK were activated to advance cell apoptosis. In conclusion, the results reveal that Ag@PEI@PTX may provide useful information on Ag@PEI@PTX-induced HepG2 cell apoptosis and as appropriate candidate for chemotherapy of cancer. Keywords: silver nanoparticles, polyethylenimine, paclitaxel, reactive oxygen species, apoptosis

Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid, Induces Cytotoxicity, Oxidative Stress, and Apoptosis in HepG2 Cells.

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Liu, Ying; Cheng, Yajun; Li, Jinwei; Wang, Yuanpeng; Liu, Yuanfa

2018-05-23

In the present study, effects of cis-9,10-epoxy stearic acid (ESA) generated by the thermal oxidation of oleic acid on HepG2 cells, including cytotoxicity, apoptosis, and oxidative stress, were investigated. Our results revealed that ESA decreased the cell viability and induced cell death. Cell cycle analysis with propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1 phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and time-dependent manner; the apoptosis of cells after treated with 500 μM ESA for 12, 24, and 48 h was 32.16, 38.70, and 65.80%, respectively. Furthermore, ESA treatment to HepG2 cells resulted in an increase in reactive oxygen species and malondialdehyde (from 0.84 ± 0.02 to 8.90 ± 0.50 nmol/mg of protein) levels and a reduction in antioxidant enzyme activity, including superoxide dismutase (from 1.34 ± 0.27 to 0.10 ± 0.007 units/mg of protein), catalase (from 100.04 ± 5.05 to 20.09 ± 3.00 units/mg of protein), and glutathione peroxidase (from 120.44 ± 7.62 to 35.84 ± 5.99 milliunits/mg of protein). These findings provide critical information on the effects of ESA on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for the evaluation of the biosafety of the oxidative product of oleic acid.

SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150

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Jung, T.W.; Lee, K.T.; Lee, M.W.; Ka, K.H.

2012-01-01

Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

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Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.

Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells

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Marino, Maria; Acconcia, Filippo; Trentalance, Anna

2003-01-01

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1–S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1–S phase transition. PMID:12808053

Hepatoprotective potential of Lavandula coronopifolia extracts against ethanol induced oxidative stress-mediated cytotoxicity in HepG2 cells.

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Farshori, Nida Nayyar; Al-Sheddi, Ebtsam S; Al-Oqail, Mai M; Hassan, Wafaa H B; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Siddiqui, Maqsood A

2015-08-01

The present investigations were carried out to study the protective potential of four extracts (namely petroleum ether extract (LCR), chloroform extract (LCM), ethyl acetate extract (LCE), and alcoholic extract (LCL)) of Lavandula coronopifolia on oxidative stress-mediated cell death induced by ethanol, a known hepatotoxin in human hapatocellular carcinoma (HepG2) cells. Cells were pretreated with LCR, LCM, LCE, and LCL extracts (10-50 μg/ml) of L. coronopifolia for 24 h and then ethanol was added and incubated further for 24 h. After the exposure, cell viability using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red uptake assays and morphological changes in HepG2 cells were studied. Pretreatment with various extracts of L. coronpifolia was found to be significantly effective in countering the cytotoxic responses of ethanol. Antioxidant properties of these L. coronopifolia extracts against reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and glutathione (GSH) levels induced by ethanol were investigated. Results show that pretreatment with these extracts for 24 h significantly inhibited ROS generation and LPO induced and increased the GSH levels reduced by ethanol. The data from the study suggests that LCR, LCM, LCE, and LCL extracts of L. coronopifolia showed hepatoprotective activity against ethanol-induced damage in HepG2 cells. However, a comparative study revealed that the LCE extract was found to be the most effective and LCL the least effective. The hepatoprotective effects observed in the study could be associated with the antioxidant properties of these extracts of L. coronopifolia. © The Author(s) 2013.

AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin

Science.gov (United States)

Hua, Hui; Yin, Yancun; Wang, Jiao; Luo, Ting; Jiang, Yangfu

2016-01-01

AMP-activated protein kinase (AMPK) is an important energy sensor that may inhibit cell proliferation or promote cell survival during stresses. Besides cyclooxygenase, AMPK is another target of the nonsteroid anti-inflammatory agent aspirin. Preclinical and clinical investigations demonstrate that aspirin can inhibit several types of cancer such as colorectal adenomas and hepatocellular carcinoma (HCC). However, little is known about the cellular response to aspirin that may lead to aspirin resistance. Here, we show that aspirin induces the expression of MCL-1 in HepG2 and SW480 cells through AMPK-mTOR-Akt/ERK axis. Treatment of HepG2 and SW480 cells with aspirin leads to increased MCL-1 expression, Akt and ERK1/2 phosphorylation. Inhibition of Akt/MEK abrogates the induction of MCL-1 by aspirin. Aspirin activates AMPK, which in turn up-regulates mTORC2 activity, Akt, ERK1/2 phosphorylation and MCL-1 expression. MCL-1 knockdown sensitizes cancer cells to aspirin-induced apoptosis. Combination of aspirin and AMPK, Akt or MEK inhibitor results in more significant inhibition of cell proliferation and induction of apoptosis than single agent. Moreover, sorafenib blocks aspirin-induced MCL-1 up-regulation. Combination of aspirin and sorafenib leads to much more cell death and less cell proliferation than each drug alone. Treatment of HCC and colon cancer xenografts with both aspirin and sorafenib results in more significant tumor suppression than single agent. These data demonstrate that AMPK-mediated up-regulation of mTORC2 and MCL-1 may compromise the anticancer effects of aspirin. Combination of aspirin and sorafenib may be an effective regimen to treat HCC and colon cancer. PMID:26918349

Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

International Nuclear Information System (INIS)

Yuan, Li; Wang, Jing; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

2012-01-01

Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer. Highlights: �

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

Directory of Open Access Journals (Sweden)

Paul B. Tchounwou

2007-06-01

Full Text Available 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 dosedependent 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 ≤ 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.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

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Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

2015-05-04

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Selective killing of hepatocellular carcinoma HepG2 cells by three-dimensional nanographene nanoparticles based on triptycene

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Xiong, Xiaoqin; Gan, Lu; Liu, Ying; Zhang, Chun; Yong, Tuying; Wang, Ziyi; Xu, Huibi; Yang, Xiangliang

2015-03-01

Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents.Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702

Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

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Gorgani-Firuzjaee, Sattar [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Adeli, Khosrow [Division of Clinical Biochemistry, The Hospital for Sick Children, University of Toronto, Toronto (Canada); Meshkani, Reza, E-mail: rmeshkani@tums.ac.ir [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of)

2015-08-21

The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway.

Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

International Nuclear Information System (INIS)

Gorgani-Firuzjaee, Sattar; Adeli, Khosrow; Meshkani, Reza

2015-01-01

The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway

Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Luo, Yi, E-mail: yi.luo@pfizer.com; Rana, Payal; Will, Yvonne

2012-06-01

Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients. -- Highlights: ► Palmitic acid and cyclosporine (CsA) synergistically increased cytotoxicity. ► The impairment of mitochondrial functions may contribute to the enhanced toxicity. ► Inhibition of JNK activity attenuated

Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

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Wang Mu [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Ruan Yuxia [Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Xing Xiaobo; Chen Qian; Peng, Yuan [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Cai Jiye, E-mail: tjycai@jnu.edu.cn [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China)

2011-07-04

Graphical abstract: Highlights: > In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. > We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. > Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. > The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 {+-} 4.62 nm to 129.70 {+-} 43.72 nm) and the expression of CD44 decreased (99.79 {+-} 0.16% to 75.14 {+-} 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 {mu}M curcumin-treated) and 50-120 pN (20 {mu}M curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

International Nuclear Information System (INIS)

Wang Mu; Ruan Yuxia; Xing Xiaobo; Chen Qian; Peng, Yuan; Cai Jiye

2011-01-01

Graphical abstract: Highlights: → In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. → We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. → Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. → The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

Oxidative stress induced lipid accumulation via SREBP1c activation in HepG2 cells

International Nuclear Information System (INIS)

Sekiya, Mika; Hiraishi, Ako; Touyama, Maiko; Sakamoto, Kazuichi

2008-01-01

SREBP1c (sterol regulatory element-binding protein 1c) is a metabolic-syndrome-associated transcription factor that controls fatty acid biosynthesis under glucose/insulin stimulation. Oxidative stress increases lipid accumulation, which promotes the generation of reactive oxygen species (ROS). However, we know little about the role of oxidative stress in fatty acid biosynthesis. To clarify the action of oxidative stress in lipid accumulation via SREBP1c, we examined SREBP1c activity in H 2 O 2 -treated mammalian cells. We introduced a luciferase reporter plasmid carrying the SREBP1c-binding site into HepG2 or COS-7 cells. With increasing H 2 O 2 dose, SREBP1c transcriptional activity increased in HepG2 cells but declined in COS-7 cells. RT-PCR analysis revealed that mRNA expression of SREBP1c gene or of SREBP1c-regulated genes rose H 2 O 2 dose-dependently in HepG2 cells but dropped in COS-7 cells. Lipid accumulation and levels of the nuclear form of SREBP1c increased in H 2 O 2 -stimulated HepG2 cells. ROS may stimulate lipid accumulation in HepG2 cells via SREBP1c activation

Cytoprotective effect exerted by geraniin in HepG2 cells is through microRNA mediated regulation of BACH-1 and HO-1.

Science.gov (United States)

Aayadi, Hoda; Mittal, Smriti P K; Deshpande, Anjali; Gore, Makarand; Ghaskadbi, Saroj S

2017-11-01

Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3β). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].

HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

Science.gov (United States)

Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

1995-03-01

We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

Science.gov (United States)

Yoon, Jaemin; Ham, Hyeonmi; Sung, Jeehye; Kim, Younghwa; Choi, Youngmin; Lee, Jeom-Sig; Jeong, Heon-Sang; Lee, Junsoo

2014-01-01

BACKGROUND/OBJECTIVES The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress. PMID:24741394

Investigating free radical generation in HepG2 cells using immuno-spin trapping.

Science.gov (United States)

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

2014-10-01

Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

International Nuclear Information System (INIS)

Vidyashankar, Satyakumar; Nandakumar, Krishna S.; Patki, Pralhad S.

2012-01-01

Highlights: ► Ethanol induced cytotoxicity in HepG2 cells in absence of lipogenesis. ► Ethanol inhibited HMG-CoA reductase activity. ► Ethanol induced HMG-CoA reductase inhibition is due to decreased cell viability. ► Incubation with mevalonate could not increase the cholesterol. ► Cytotoxicity brought about by CoQ10 depletion and increased TNF-alpha. -- Abstract: Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP 450 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP 450 2E1. The results showed that ethanol at 100 mM concentration caused 40% cytotoxicity at 72 h as determined by MTT assay. The incorporation of labeled [2- 14 C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24 h incubation, whereas incorporation of labeled [2- 14 C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q 10 which is detrimental for cell viability. But vitamin E (10 mM) could partially restore coenzyme-Q 10 and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

Science.gov (United States)

Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

2012-07-01

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

Energy Technology Data Exchange (ETDEWEB)

Yu, Yongbo [Capital Medical University, Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital (China); Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [Capital Medical University, School of Public Health (China)

2016-11-15

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Polyethyleneimine-coated quantum dots for miRNA delivery and its enhanced suppression in HepG2 cells

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Liang G

2016-11-01

Full Text Available Gaofeng Liang,1 Yang Li,1 Wenpo Feng,1 Xinshuai Wang,2 Aihua Jing,1 Jinghua Li,1 Kaiwang Ma1 1Department of Biomedical Engineering, School of Medical Technology & Engineering, 2Department of Oncology, The First Affiliated Hospital, Henan University of Science & Technology, Luoyang, People’s Republic of China Abstract: Quantum dots (QDs have been intensively investigated for bioimaging, drug delivery, and labeling probes because of their unique optical properties. In this study, CdSe/ZnS QDs-based nonviral vectors with the dual functions of delivering miR-26a plasmid and bioimaging were formulated by capping the surface of CdSe/ZnS QDs with polyethyleneimine (PEI. The PEI-coated QDs were capable of condensing miR-26a expression vector into nanocomplexes that can emit strong red luminescence when loaded with CdSe/ZnS QDs. Further results showed that PEI-modified nanoparticles (NPs could transfect miR-26a plasmid into HepG2 cells in vitro. Meanwhile, imaging of living cells could be achieved based on the CdSe/ZnS QDs. Further study suggested that miR-26a transfection up-regulated miR-26a expression, induced cycle arrest, and triggered proliferation inhibition in HepG2 cells. The results indicated that PEI-coated QD NPs possess the capability of bioimaging and gene delivery and could be a promising vehicle with the engineering of QD NPs for gene therapy in the future. Keywords: miR-26a, PEI/QDs, HepG2, gene delivery, bioimaging

Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxorubicin in HepG2 cells.

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Komori, Yuki; Arisawa, Sakiko; Takai, Miho; Yokoyama, Kunihiro; Honda, Minako; Hayashi, Kazuhiko; Ishigami, Masatoshi; Katano, Yoshiaki; Goto, Hidemi; Ueyama, Jun; Ishikawa, Tetsuya; Wakusawa, Shinya

2014-02-05

The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

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Zheng, Juanjuan; Zhang, Yu [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Xu, Wentao, E-mail: xuwentaoboy@sina.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Luo, YunBo [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Hao, Junran [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Shen, Xiao Li [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Yang, Xuan [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Li, Xiaohong [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Huang, Kunlun, E-mail: hkl009@163.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China)

2013-04-15

Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by

Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

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Zheng, Juanjuan; Zhang, Yu; Xu, Wentao; Luo, YunBo; Hao, Junran; Shen, Xiao Li; Yang, Xuan; Li, Xiaohong; Huang, Kunlun

2013-01-01

Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ m ). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in

Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis.

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Yun-Lan Li

Full Text Available Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01 at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V--fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001, indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01 while mitochondrial membrane potential reduced significantly (p<0.001 compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01, while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01. The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001 while that of Bcl-2 decreased (p<0.001. Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the

Potentiation of LPS-Induced Apoptotic Cell Death in Human Hepatoma HepG2 Cells by Aspirin via ROS and Mitochondrial Dysfunction: Protection by N-Acetyl Cysteine.

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Haider Raza

Full Text Available Cytotoxicity and inflammation-associated toxic responses have been observed to be induced by bacterial lipopolysaccharides (LPS in vitro and in vivo respectively. Use of nonsteroidal anti-inflammatory drugs (NSAIDs, such as aspirin, has been reported to be beneficial in inflammation-associated diseases like cancer, diabetes and cardiovascular disorders. Their precise molecular mechanisms, however, are not clearly understood. Our previous studies on aspirin treated HepG2 cells strongly suggest cell cycle arrest and induction of apoptosis associated with mitochondrial dysfunction. In the present study, we have further demonstrated that HepG2 cells treated with LPS alone or in combination with aspirin induces subcellular toxic responses which are accompanied by increase in reactive oxygen species (ROS production, oxidative stress, mitochondrial respiratory dysfunction and apoptosis. The LPS/Aspirin induced toxicity was attenuated by pre-treatment of cells with N-acetyl cysteine (NAC. Alterations in oxidative stress and glutathione-dependent redox-homeostasis were more pronounced in mitochondria compared to extra- mitochondrial cellular compartments. Pre-treatment of HepG2 cells with NAC exhibited a selective protection in redox homeostasis and mitochondrial dysfunction. Our results suggest that the altered redox metabolism, oxidative stress and mitochondrial function in HepG2 cells play a critical role in LPS/aspirin-induced cytotoxicity. These results may help in better understanding the pharmacological, toxicological and therapeutic properties of NSAIDs in cancer cells exposed to bacterial endotoxins.

Bitter melon extract ameliorates palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in HepG2 cells and high-fat/high-fructose-diet-induced fatty liver

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Hwa Joung Lee

2018-03-01

Full Text Available Background: Bitter melon (BM improves glucose level, lipid homeostasis, and insulin resistance in vivo. However, the preventive mechanism of BM in nonalcoholic fatty liver disease (NAFLD has not been elucidated yet. Aim & Design: To determine the protective mechanism of bitter melon extract (BME, we performed experiments in vitro and in vivo. BME were treated palmitate (PA-administrated HepG2 cells. C57BL/6J mice were divided into two groups: high-fat/high-fructose (HF/HFr without or with BME supplementation (100 mg/kg body weight. Endoplasmic reticulum (ER stress, apoptosis, and biochemical markers were then examined by western blot and real-time PCR analyses. Results: BME significantly decreased expression levels of ER-stress markers (including phospho-eIF2α, CHOP, and phospho-JNK [Jun N-terminal kinases] in PA-treated HepG2 cells. BME also significantly decreased the activity of cleaved caspase-3 (a well known apoptotic-induced molecule and DNA fragmentation. The effect of BME on ER stress–mediated apoptosis in vitro was similarly observed in HF/HFr-fed mice in vivo. BME significantly reduced HF/HFr-induced hepatic triglyceride (TG and serum alanine aminotransferase (ALT as markers of hepatic damage in mice. In addition, BME ameliorated HF/HFr-induced serum TG and serum-free fatty acids. Conclusion: These data indicate that BME has protective effects against ER stress mediated apoptosis in HepG2 cells as well as in HF/HFr-induced fatty liver of mouse. Therefore, BME might be useful for preventing and treating NAFLD.

BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

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Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

2013-06-01

Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

Cholesterol-lowing effect of taurine in HepG2 cell.

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Guo, Junxia; Gao, Ya; Cao, Xuelian; Zhang, Jing; Chen, Wen

2017-03-16

A number of studies indicate that taurine promotes cholesterol conversion to bile acids by upregulating CYP7A1 gene expression. Few in vitro studies are concerned the concentration change of cholesterol and its product of bile acids, and the molecular mechanism of CYP7A1 induction by taurine. The levels of intracellular total cholesterol (TC), free cholesterol (FC), cholesterol ester (EC), total bile acids (TBA) and medium TBA were determined after HepG2 cells were cultured for 24/48 h in DMEM supplemented with taurine at the final concentrations of 1/10/20 mM respectively. The protein expressions of CYP7A1, MEK1/2, c-Jun, p-c-Jun and HNF-4α were detected. Taurine significantly reduced cellular TC and FC in dose -and time-dependent ways, and obviously increased intracellular/medium TBA and CYP7A1 expressions. There was no change in c-Jun expression, but the protein expressions of MEK1/2 and p-c-Jun were increased at 24 h and inhibited at 48 h by 20 mM taurine while HNF4α was induced after both of the 24 h and 48 h treatment. Taurine could enhance CYP7A1 expression by inducing HNF4α and inhibiting MEK1/2 and p-c-Jun expressions to promote intracellular cholesterol metabolism.

Lignans from Opuntia ficus-indica seeds protect rat primary hepatocytes and HepG2 cells against ethanol-induced oxidative stress.

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Kim, Jung Wha; Yang, Heejung; Kim, Hyeon Woo; Kim, Hong Pyo; Sung, Sang Hyun

2017-01-01

Bioactivity-guided isolation of Opuntia ficus-indica (Cactaceae) seeds against ethanol-treated primary rat hepatocytes yielded six lignan compounds. Among the isolates, furofuran lignans 4-6, significantly protected rat hepatocytes against ethanol-induced oxidative stress by reducing intracellular reactive oxygen species levels, preserving antioxidative defense enzyme activities, and maintaining the glutathione content. Moreover, 4 dose-dependently induced the heme oxygenase-1 expression in HepG2 cells.

Fucoidan from Fucus vesiculosus Protects against Alcohol-Induced Liver Damage by Modulating Inflammatory Mediators in Mice and HepG2 Cells

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Jung Dae Lim

2015-02-01

Full Text Available Fucoidan is an l-fucose-enriched sulfated polysaccharide isolated from brown algae and marine invertebrates. In this study, we investigated the protective effect of fucoidan from Fucus vesiculosus on alcohol-induced murine liver damage. Liver injury was induced by oral administration of 25% alcohol with or without fucoidan (30 mg/kg or 60 mg/kg for seven days. Alcohol administration increased serum aspartate aminotransferase and alanine aminotransferase levels, but these increases were suppressed by the treatment of fucoidan. Transforming growth factor beta 1 (TGF-β1, a liver fibrosis-inducing factor, was highly expressed in the alcohol-fed group and human hepatoma HepG2 cell; however, the increase in TGF-β1 expression was reduced following fucoidan administration. Treatment with fucoidan was also found to significantly reduce the production of inflammation-promoting cyclooygenase-2 and nitric oxide, while markedly increasing the expression of the hepatoprotective enzyme, hemeoxygenase-1, on murine liver and HepG2 cells. Taken together, the antifibrotic and anti-inflammatory effects of fucoidan on alcohol-induced liver damage may provide valuable insights into developing new therapeutics or interventions.

A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

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Liao, Ningbo; Sun, Liang; Chen, Jiang; Zhong, Jianjun; Zhang, Yanjun; Zhang, Ronghua

2017-03-01

Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata , hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata . It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC 50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

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Ningbo Liao

2017-03-01

Full Text Available Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata, hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata. It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

Antiproliferative Effect of the Isoquinoline Alkaloid Papaverine in Hepatocarcinoma HepG-2 Cells — Inhibition of Telomerase and Induction of Senescence

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Sakineh Kazemi Noureini

2014-08-01

Full Text Available Cancer cells are often immortal through up-regulation of the hTERT gene, which encodes the catalytic subunit of a special reverse transcriptase to overcome end-replication problem of chromosomes. This study demonstrates that papaverine, an isoquinoline alkaloid from the Papaveraceae, can overcome telomerase dependent immortality of HepG-2 cells that was used as a model of hepatocarcinoma. Although this alkaloid does not directly interact with telomeric sequences, papaverine inhibits telomerase through down-regulation of hTERT, which was analysed using thermal FRET and qRT-PCR, respectively. The IC50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC50 for cytotoxicity was 120 µM. Repeated treatments of the cells with very low non-toxic concentrations of papaverine resulted in growth arrest and strong reduction of population doublings after 40 days. This treatment induced senescent morphology in HepG-2 cells, which was evaluated by beta-galactosidase staining. Altogether, papaverine can be regarded as a promising model compound for drug design targeting cancer development.

P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest.

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Shamseddine, A A; Clarke, C J; Carroll, B; Airola, M V; Mohammed, S; Rella, A; Obeid, L M; Hannun, Y A

2015-10-29

Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.

IRE1α links Nck1 deficiency to attenuated PTP1B expression in HepG2 cells.

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Li, Hui; Li, Bing; Larose, Louise

2017-08-01

PTP1B, a prototype of the non-receptor subfamily of the protein tyrosine phosphatase superfamily, plays a key role in regulating intracellular signaling from various receptor and non-receptor protein tyrosine kinases. Previously, we reported that silencing Nck1 in human hepatocellular carcinoma HepG2 cells enhances basal and growth factor-induced activation of the PI3K-Akt pathway through attenuating PTP1B expression. However, the underlying mechanism by which Nck1 depletion represses PTP1B expression remains unclear. In this study, we found that silencing Nck1 attenuates PTP1B expression in HepG2 cells through down-regulation of IRE1α. Indeed, we show that silencing Nck1 in HepG2 cells leads to decreased IRE1α expression and signaling. Accordingly, IRE1α depletion using siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation, reproducing the effects of silencing Nck1 on activation of this pathway. In addition, depletion of IRE1α also leads to reduced PTP1B expression, which was rescued by ectopic expression of IRE1α in Nck1-depleted cells. Mechanistically, we found that silencing either Nck1 or IRE1α in HepG2 cells decreases PTP1B mRNA levels and stability. However, despite miR-122 levels, a miRNA targeting PTP1B 3' UTR and inducing PTP1B mRNA degradation in HepG2 cells, are increased in both Nck1- and IRE1α-depleted HepG2 cells, a miR-122 antagomir did not rescue PTP1B expression in these cells. Overall, this study highlights an important role for Nck1 in fine-tuning IRE1α expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

International Nuclear Information System (INIS)

Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A.; Will, Yvonne

2008-01-01

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction

Activation of human stearoyl-coenzyme A desaturase 1 contributes to the lipogenic effect of PXR in HepG2 cells.

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Jun Zhang

Full Text Available The pregnane X receptor (PXR was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1, long chain free fatty acid elongase (FAE, and lecithin-cholesterol acyltransferase (LCAT, while the expression of acyl:cholesterol acetyltransferase(ACAT1 was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR, the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene.

Active Fragment of Veronica ciliata Fisch. Attenuates t-BHP-Induced Oxidative Stress Injury in HepG2 Cells through Antioxidant and Antiapoptosis Activities

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Yiran Sun

2017-01-01

Full Text Available Excessive amounts of reactive oxygen species (ROS in the body are a key factor in the development of hepatopathies such as hepatitis. The aim of this study was to assess the antioxidation effect in vitro and hepatoprotective activity of the active fragment of Veronica ciliata Fisch. (VCAF. Antioxidant assays (DPPH, superoxide, and hydroxyl radicals scavenging were conducted, and hepatoprotective effects through the application of tert-butyl hydroperoxide- (t-BHP- induced oxidative stress injury in HepG2 cells were evaluated. VCAF had high phenolic and flavonoid contents and strong antioxidant activity. From the perspective of hepatoprotection, VCAF exhibited a significant protective effect on t-BHP-induced HepG2 cell injury, as indicated by reductions in cytotoxicity and the levels of ROS, 8-hydroxydeoxyguanosine (8-OHdG, and protein carbonyls. Further study demonstrated that VCAF attenuated the apoptosis of t-BHP-treated HepG2 cells by suppressing the activation of caspase-3 and caspase-8. Moreover, it significantly decreased the levels of ALT and AST, increased the activities of acetyl cholinesterase (AChE, glutathione (GSH, superoxide dismutase (SOD, and catalase (CAT, and increased total antioxidative capability (T-AOC. Collectively, we concluded that VCAF may be a considerable candidate for protecting against liver injury owing to its excellent antioxidant and antiapoptosis properties.

The Growth Suppressing Effects of Girinimbine on Hepg2 Involve Induction of Apoptosis and Cell Cycle Arrest

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Tang Sook Wah

2011-08-01

Full Text Available Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G0/G1 peak (hypodiploid and caused G0/G1-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.

Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation

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Osabe, Makoto; Sugatani, Junko; Takemura, Akiko; Yamazaki, Yasuhiro; Ikari, Akira; Kitamura, Naomi; Negishi, Masahiko; Miwa, Masao

2008-01-01

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation

Protective Effects of Black Rice Extracts on Oxidative Stress Induced by tert-Butyl Hydroperoxide in HepG2 Cells

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Lee, Seon-Mi; Choi, Youngmin; Sung, Jeehye; Kim, Younghwa; Jeong, Heon-Sang; Lee, Junsoo

2014-01-01

Black rice contains many biologically active compounds. The aim of this study was to investigate the protective effects of black rice extracts (whole grain extract, WGE and rice bran extract, RBE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. Cellular reactive oxygen species (ROS), antioxidant enzyme activities, malondialdehyde (MDA) and glutathione (GSH) concentrations were evaluated as biomarkers of cellular oxidative status. Cells pretreated with 50 and 100 μg/mL of WGE or RBE were more resistant to oxidative stress in a dose-dependent manner. The highest WGE and BRE concentrations enhanced GSH concentrations and modulated antioxidant enzyme activities (glutathione reductase, glutathione-S-transferase, catalase, and superoxide dismutase) compared to TBHP-treated cells. Cells treated with RBE showed higher protective effect compared to cells treated with WGE against oxidative insult. Black rice extracts attenuated oxidative insult by inhibiting cellular ROS and MDA increase and by modulating antioxidant enzyme activities in HepG2 cells. PMID:25580401

Proanthocyanidins modulate microRNA expression in human HepG2 cells.

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Anna Arola-Arnal

Full Text Available Mi(croRNAs are small non-coding RNAs of 18-25 nucleotides in length that modulate gene expression at the post-transcriptional level. These RNAs have been shown to be involved in a several biological processes, human diseases and metabolic disorders. Proanthocyanidins, which are the most abundant polyphenol class in the human diet, have positive health effects on a variety of metabolic disorders such as inflammation, obesity, diabetes and insulin resistance. The present study aimed to evaluate whether proanthocyanidin-rich natural extracts modulate miRNA expression. Using microarray analysis and Q-PCR, we investigated miRNA expression in HepG2 cells treated with proanthocyanidins. Our results showed that when HepG2 cells were treated with grape seed proanthocyanidin extract (GSPE, cocoa proanthocyanidin extract (CPE or pure epigallocatechin gallate isolated from green tea (EGCG, fifteen, six and five differentially expressed miRNAs, respectively, were identified out of 904 mRNAs. Specifically, miR-30b* was downregulated by the three treatments, and treatment with GSPE or CPE upregulated miR-1224-3p, miR-197 and miR-532-3p. Therefore, these results provide evidence of the capacity of dietary proanthocyanidins to influence microRNA expression, suggesting a new mechanism of action of proanthocyanidins.

Comparative cytotoxic response of nickel ferrite nanoparticles in human liver HepG2 and breast MFC-7 cancer cells.

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Ahamed, Maqusood; Akhtar, Mohd Javed; Alhadlaq, Hisham A; Khan, M A Majeed; Alrokayan, Salman A

2015-09-01

Nickel ferrite nanoparticles (NPs) have received much attention for their potential applications in biomedical fields such as magnetic resonance imaging, drug delivery and cancer hyperthermia. However, little is known about the toxicity of nickel ferrite NPs at the cellular and molecular levels. In this study, we investigated the cytotoxic responses of nickel ferrite NPs in two different types of human cells (i.e., liver HepG2 and breast MCF-7). Nickel ferrite NPs induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) assays. Nickel ferrite NPs were also found to induce oxidative stress, which was evident by the depletion of glutathione and the induction of reactive oxygen species (ROS) and lipid peroxidation. The mitochondrial membrane potential due to nickel ferrite NP exposure was also observed. The mRNA levels for the tumor suppressor gene p53 and the apoptotic genes bax, CASP3 and CASP9 were up-regulated, while the anti-apoptotic gene bcl-2 was down-regulated following nickel ferrite NP exposure. Furthermore, the activities of apoptotic enzymes (caspase-3 and caspase-9) were also higher in both types of cells treated with nickel ferrite NPs. Cytotoxicity induced by nickel ferrite was efficiently prevented by N-acetyl cysteine (ROS scavenger) treatment, which suggested that oxidative stress might be one of the possible mechanisms of nickel ferrite NP toxicity. We also observed that MCF-7 cells were slightly more susceptible to nickel ferrite NP exposure than HepG2 cells. This study warrants further investigation to explore the potential mechanisms of different cytotoxic responses of nickel ferrite NPs in different cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

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Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

2015-01-01

Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

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Lionel Leclere

Full Text Available Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3 protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

Effect of Cudrania tricuspidata and Kaempferol in Endoplasmic Reticulum Stress-Induced Inflammation and Hepatic Insulin Resistance in HepG2 Cells.

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Kim, Ok-Kyung; Jun, Woojin; Lee, Jeongmin

2016-01-21

In this study, we quantitated kaempferol in water extract from Cudrania tricuspidata leaves (CTL) and investigated its effects on endoplasmic reticulum (ER) stress-induced inflammation and insulin resistance in HepG2 cells. The concentration of kaempferol in the CTL was 5.07 ± 0.08 mg/g. The HepG2 cells were treated with 300 µg/mL of CTL, 500 µg/mL of CTL, 1.5 µg/mL of kaempferol or 2.5 µg/mL of kaempferol, followed immediately by stimulation with 100 nM of thapsigargin for ER stress induction for 24 h. There was a marked increase in the activation of the ER stress and inflammation response in the thapsigargin-stimulated control group. The CTL treatment interrupted the ER stress response and ER stress-induced inflammation. Kaempferol partially inhibited the ER stress response and inflammation. There was a significant increase in serine phosphorylation of insulin receptor substrate (IRS)-1 and the expression of C/EBPα and gluconeogenic genes in the thapsigargin-stimulated control group compared to the normal control. Both CTL and kaempferol suppressed serine phosphorylation of IRS-1, and the treatments did not interrupt the C/EBPα/gluconeogenic gene pathway. These results suggest that kaempferol might be the active compound of CTL and that it might protect against ER stress-induced inflammation and hyperglycemia.

Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose simvastatin: A possible role in statin-induced hepatotoxicity?

International Nuclear Information System (INIS)

Tavintharan, S.; Ong, C.N.; Jeyaseelan, K.; Sivakumar, M.; Lim, S.C.; Sum, C.F.

2007-01-01

Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ 10 ). In HepG2 cells, simvastatin decreased mitochondrial CoQ 10 levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ 10 , reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ 10 deficiency plays an important role in statin-induced hepatopathy, and that CoQ 10 supplementation protects HepG2 cells from this complication

Combination Treatment of Deep Sea Water and Fucoidan Attenuates High Glucose-Induced Insulin-Resistance in HepG2 Hepatocytes

OpenAIRE

Shan He; Wei-Bing Peng; Hong-Lei Zhou

2018-01-01

Insulin resistance (IR) plays a central role in the development of several metabolic diseases, which leads to increased morbidity and mortality rates, in addition to soaring health-care costs. Deep sea water (DSW) and fucoidans (FPS) have drawn much attention in recent years because of their potential medical and pharmaceutical applications. This study investigated the effects and mechanisms of combination treatment of DSW and FPS in improving IR in HepG2 hepatocytes induced by a high glucose...

Mercury-Induced Externalization of Phosphatidylserine and Caspase 3 Activation in Human Liver Carcinoma (HepG2 Cells

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Paul B. Tchounwou

2006-03-01

Full Text Available Apoptosis arises from the active initiation and propagation of a series of highly orchestrated specific biochemical events leading to the demise of the cell. It is a normal physiological process, which occurs during embryonic development as well as in the maintenance of tissue homeostasis. Diverse groups of molecules are involved in the apoptosis pathway and it functions as a mechanism to eliminate unwanted or irreparably damaged cells. However, inappropriate induction of apoptosis by environmental agents has broad ranging pathologic implications and has been associated with several diseases including cancer. The toxicity of several heavy metals such as mercury has been attributed to their high affinity to sulfhydryl groups of proteins and enzymes, and their ability to disrupt cell cycle progression and/or apoptosis in various tissues. The aim of this study was to assess the potential for mercury to induce early and late-stage apoptosis in human liver carcinoma (HepG2 cells. The Annexin-V and Caspase 3 assays were performed by flow cytometric analysis to determine the extent of phosphatidylserine externalization and Caspase 3 activation in mercury-treated HepG2 cells. Cells were exposed to mercury for 10 and 48 hours respectively at doses of 0, 1, 2, and 3 μg/mL based on previous cytotoxicity results in our laboratory indicating an LD50 of 3.5 ± 0.6 μg/mL for mercury in HepG2 cells. The study data indicated a dose response relationship between mercury exposure and the degree of early and late-stage apoptosis in HepG2 cells. The percentages of cells undergoing early apoptosis were 0.03 ± 0.03%, 5.19 ± 0.04%, 6.36 ± 0.04%, and 8.84 ± 0.02% for 0, 1, 2, and 3 μg/mL of mercury respectively, indicating a gradual increase in apoptotic cells with increasing doses of mercury. The percentages of Caspase 3 positive cells undergoing late apoptosis were 3.58 ± 0.03%, 17.06 ± 0

Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

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Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Mason, Ronald P

2015-01-01

Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA. Copyright © 2014 Elsevier Inc. All rights reserved.

Egr2 enhances insulin resistance via JAK2/STAT3/SOCS-1 pathway in HepG2 cells treated with palmitate.

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Lu, Lin; Ye, Xinhua; Yao, Qing; Lu, Aijiao; Zhao, Zhen; Ding, Yang; Meng, Chuchen; Yu, Wenlong; Du, Yunfeng; Cheng, JinLuo

2018-05-01

Insulin resistance is generally responsible for the pathogenesis of type 2 diabetes mellitus (T2DM). Early growth response proteins-2 (Egr2) has been reported to be able to increase the expression of the suppressors of cytokine signaling-1 (SOCS-1), and impair insulin signaling pathway through suppression of insulin receptor substrates (IRS), including IRS-1 and IRS-2. However, whether Egr2 is directly involved in the development of insulin resistance, and how its potential contributions to insulin resistance still remain unknown. Here, our present investigation found that the expression levels of Egr2 were up-regulated when insulin resistance occurs, and knockdown of Egr2 abolished the effect of insulin resistance in HepG2 cells induced with palmitate (PA). Importantly, inhibition of Egr2 decreased the expression of SOCS-1 as well as reduced phosphorylation of JAK2 and STAT3. And, our data indicated that silencing of Egr2 accelerated hepatic glucose uptake and reversed the impaired lipid metabolism upon insulin resistance. In summary, the present study confirms that Egr2 could deteriorate insulin resistance via the pathway of JAK2/STAT3/SOCS-1 and may shed light on resolving insulin resistance and further the pathogenesis of T2DM. Copyright © 2017 Elsevier Inc. All rights reserved.

Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

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Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

2014-04-01

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

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Makoto Seo

2008-01-01

Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

miR-203a is involved in HBx-induced inflammation by targeting Rap1a

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Wu, AiRong [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Chen, Huo [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Xu, ChunFang [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhou, Ji; Chen, Si [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Shi, YuQi [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Xu, Jie [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Gan, JianHe, E-mail: j_pzhang@suda.edu.cn [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhang, JinPing, E-mail: ganjianhe@aliyun.com [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China)

2016-11-15

Hepatitis B virus (HBV) causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. Accumulating evidence suggests that inflammation is the key factor for liver cirrhosis and hepatocellular carcinoma. MicroRNAs play important roles in many biological processes. Here, we aim to explore the function of microRNAs in the HBX-induced inflammation. First, microarray experiment showed that HBV{sup +} liver samples expressed higher level of miR-203a compared to HBV{sup -} liver samples. To verify these alterations, HBx-coding plasmid was transfected into HepG2 cells to overexpress HBx protein. The real-time PCR results suggested that over-expression of HBx could induce up-regulation of miR-203a. To define how up-regulation of miR-203a can induce liver cells inflammation, we over-expressed miR-203a in HepG2 cells. Annexin V staining and BrdU staining suggested that overexpression of miR-203a significantly increased the cell apoptosis and proliferation, meanwhile, over-expression of miR-203a could lead to a decrease in G0/G1 phase cells and an increase in G2/M phase cells. Some cytokines production including IL-6 and IL-8 were significantly increased, but TGFβ and IFNγ were decreased in miR-203a over-expressed HepG2 cells. Luciferase reporter assay experiments, protein mass-spectrum assay and real-time PCR all together demonstrated that Rap1a was the target gene of miR-203a. Further experiments showed that these alterations were modulated through PI3K/ERK/p38/NFκB pathways. These data suggested that HBV-infection could up-regulate the expression of miR-203a, thus down regulated the expression of Rap1a and affected the PI3K/ERK/p38/NFκB pathways, finally induced the hepatitis inflammation. - Highlights: • HBX induces the over-expression of miR-203a in HepG2 cells. • miR-203a targets Rap1a to induce the inflammation in HepG2 cells. • miR-203a regulates the apoptosis and cell cycles of HepG2 cells. • miR-203a alters

Protective effects of rambutan (Nephelium lappaceum) peel phenolics on H2O2-induced oxidative damages in HepG2 cells and d-galactose-induced aging mice.

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Zhuang, Yongliang; Ma, Qingyu; Guo, Yan; Sun, Liping

2017-10-01

Rambutan peel phenolic (RPP) extracts were prepared via dynamic separation with macroporous resin. The total phenolic content and individual phenolics in RPP were determined. Results showed that the total phenolic content of RPP was 877.11 mg gallic acid equivalents (GAE)/g extract. The content of geranin (122.18 mg/g extract) was the highest among those of the 39 identified phenolic compounds. RPP protected against oxidative stress in H 2 O 2 -induced HepG2 cells in a dose-response manner. The inhibitory effects of RPP on cell apoptosis might be related to its inhibitory effects on the generation of intracellular reactive oxygen species and increased effects on superoxide dismutase activity. The in vivo anti-aging activity of RPP was evaluated using an aging mice model that was induced by d-galactose (d-gal). The results showed that RPP enhanced the antioxidative status of experimental mice. Moreover, histological analysis indicated that RPP effectively reduced d-gal-induced liver and kidney tissue damage in a dose-dependent manner. Therefore, RPP can be used as a natural antioxidant and anti-aging agent in the pharmaceutical and food industries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide.

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Haider Raza

Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs, including acetaminophen (APAP, have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP causes liver injury in humans and animals. Hepatic glutathione (GSH depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP-induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST and multidrug resistance (MDR1 proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM, a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.

Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

International Nuclear Information System (INIS)

Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

2014-01-01

Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Liang, Jin [Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036 (China); College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Li, Feng [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Fang, Yong; Yang, Wenjian [College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China); An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Hu, Qiuhui, E-mail: qiuhuihu@njau.edu.cn [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China)

2014-03-01

Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

Study on radiation regulation of hypoxia inducible factor-1α expression and its correlation with hepatoma radiosensitivity

International Nuclear Information System (INIS)

Jin Wensen; Kong Zhaolu; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

2008-01-01

Objective: To study the regulation of hypoxia inducible factor-1α (HIF-1α) expression in hepatoma cells after irradiation and the expression of HIF-1α effect on the radiosensitivity of heptoma cells. Methods: HepG2 cells were pretreated by Cobalt chloride (COCl 2 ), a chemical hypoxia agent, to induce and stabilize the expression of HIF-1α, and then exposed to different γ-irradiation doses. Clonogenic assay was used to evaluate HepG2 cell survival fraction (SF) after irradiation under normoxia and chemical hypoxia. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot assay (Western blot) were utilized to detect the changes of intracellular HIF-1α on the level of transcripation and translation. Results: Cell survival level was elevated by chemical hypoxia and there was a statistical difference between chemical hypoxic group and normoxic group. The ratios of SF(SF co /SF o 2 )on two different conditions were increased with irradiation doses. Meanwhile, the irradiation induced up-regulation of HIF-1α in dose-dependent manner. The expression of HIF-1α was correlated with HepG2 cell survival level to some extent. Conclusions: Irradiation could up-regulate the level of HIF-1α expression in HepG2 cells under chemical hypoxic condition. The cells survival level might be influenced by the changes in HIF-1α expression. (authors)

Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2).

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Ahamed, Maqusood; Akhtar, Mohd Javed; Khan, M A Majeed; Alhadlaq, Hisham A; Alshamsan, Aws

2016-12-01

Cobalt iron oxide (CoFe 2 O 4 ) nanoparticles (CIO NPs) have been one of the most widely explored magnetic NPs because of their excellent chemical stability, mechanical hardness and heat generating potential. However, there is limited information concerning the interaction of CIO NPs with biological systems. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and apoptotic response of CIO NPs in human liver cells (HepG2). Diameter of crystalline CIO NPs was found to be 23nm with a band gap of 1.97eV. CIO NPs induced cell viability reduction and membrane damage, and degree of induction was dose- and time-dependent. CIO NPs were also found to induce oxidative stress revealed by induction of ROS, depletion of glutathione and lower activity of superoxide dismutase enzyme. Real-time PCR data has shown that mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were higher, while the expression level of anti-apoptotic gene bcl-2 was lower in cells following exposure to CIO NPs. Activity of caspase-3 and caspase-9 enzymes was also higher in CIO NPs exposed cells. Furthermore, co-exposure of N-acetyl-cysteine (ROS scavenger) efficiently abrogated the modulation of apoptotic genes along with the prevention of cytotoxicity caused by CIO NPs. Overall, we observed that CIO NPs induced cytotoxicity and apoptosis in HepG2 cells through ROS via p53 pathway. This study suggests that toxicity mechanisms of CIO NPs should be further investigated in animal models. Copyright © 2016 Elsevier B.V. All rights reserved.

MicroRNA-122 mimic transfection contributes to apoptosis in HepG2 cells.

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Huang, Hongyan; Zhu, Yueyong; Li, Shaoyang

2015-11-01

There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

Licochalcone A Upregulates Nrf2 Antioxidant Pathway and Thereby Alleviates Acetaminophen-Induced Hepatotoxicity

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Hongming Lv

2018-03-01

Full Text Available Acetaminophen (APAP overdose-induced fatal hepatotoxicity is majorly characterized by overwhelmingly increased oxidative stress while enhanced nuclear factor-erythroid 2-related factor 2 (Nrf2 is involved in prevention of hepatotoxicity. Although Licochalcone A (Lico A upregulates Nrf2 signaling pathway against oxidative stress-triggered cell injury, whether it could protect from APAP-induced hepatotoxicity by directly inducing Nrf2 activation is still poorly elucidated. This study aims to explore the protective effect of Lico A against APAP-induced hepatotoxicity and its underlying molecular mechanisms. Our findings indicated that Lico A effectively decreased tert-butyl hydroperoxide (t-BHP- and APAP-stimulated cell apoptosis, mitochondrial dysfunction and reactive oxygen species generation and increased various anti-oxidative enzymes expression, which is largely dependent on upregulating Nrf2 nuclear translocation, reducing the Keap1 protein expression, and strengthening the antioxidant response element promoter activity. Meanwhile, Lico A dramatically protected against APAP-induced acute liver failure by lessening the lethality; alleviating histopathological liver changes; decreasing the alanine transaminase and aspartate aminotransferase levels, malondialdehyde formation, myeloperoxidase level and superoxide dismutase depletion, and increasing the GSH-to-GSSG ratio. Furthermore, Lico A not only significantly modulated apoptosis-related protein by increasing Bcl-2 expression, and decreasing Bax and caspase-3 cleavage expression, but also efficiently alleviated mitochondrial dysfunction by reducing c-jun N-terminal kinase phosphorylation and translocation, inhibiting Bax mitochondrial translocation, apoptosis-inducing factor and cytochrome c release. However, Lico A-inhibited APAP-induced the lethality, histopathological changes, hepatic apoptosis, and mitochondrial dysfunction in WT mice were evidently abrogated in Nrf2-/- mice. These

microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line

International Nuclear Information System (INIS)

Ueki, Satomi; Murakami, Yuko; Yamada, Shoji; Kimura, Masaki; Saito, Yoshimasa; Saito, Hidetsugu

2016-01-01

It is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation. To determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes. The hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress. These results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs. The online version of this article (doi:10.1186/s12885-016-2762-7) contains supplementary material, which is available to authorized users

Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

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Yu, Jian-Qing; Yin, Yan; Lei, Jia-Chuan; Zhang, Xiu-Qiao; Chen, Wei; Ding, Cheng-Li; Wu, Shan; He, Xiao-Yu; Liu, Yan-Wen; Zou, Guo-Lin

2012-02-01

Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 μg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 μg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 μg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

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Hui Zhang

Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

Wedelolactone Regulates Lipid Metabolism and Improves Hepatic Steatosis Partly by AMPK Activation and Up-Regulation of Expression of PPARα/LPL and LDLR.

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Yun Zhao

Full Text Available Hyperlipidemia is considered one of the greatest risk factors of cardiovascular diseases. We investigated the anti-hyperlipidemic effect and the underlying mechanism of wedelolactone, a plant-derived coumestan, in HepG2 cells and high-fat diet (HFD-induced hyperlipidemic hamsters. We showed that in cultured HepG2 cells, wedelolactone up-regulated protein levels of adenosine monophosphate activated protein kinase (AMPK and peroxisome proliferator-activated receptor-alpha (PPARα as well as the gene expression of AMPK, PPARα, lipoprotein lipase (LPL, and the low-density lipoprotein receptor (LDLR. Meanwhile, administration of wedelolactone for 4 weeks decreased the lipid profiles of plasma and liver in HFD-induced hyperlipidemic hamsters, including total cholesterol (TC, triglycerides (TG, and low-density lipoprotein-cholesterol (LDL-C. The activation of AMPK and up-regulation of PPARα was also observed with wedelolactone treatment. Furthermore, wedelolactone also increased the activities of superoxidase dismutase (SOD and glutathione peroxidase (GSH-Px and decreased the level of the lipid peroxidation product malondialdehyde (MDA in the liver, therefore decreasing the activity of alanine aminotransferase (ALT. In conclusion, we provide novel experimental evidence that wedelolactone possesses lipid-lowering and steatosis-improving effects, and the underlying mechanism is, at least in part, mediated by the activation of AMPK and the up-regulation of PPARα/LPL and LDLR.

Effects of Nano-CeO2 with Different Nanocrystal Morphologies on Cytotoxicity in HepG2 Cells

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Lili Wang

2015-09-01

Full Text Available Cerium oxide nanoparticles (nano-CeO2 have been reported to cause damage and apoptosis in human primary hepatocytes. Here, we compared the toxicity of three types of nano-CeO2 with different nanocrystal morphologies (cube-, octahedron-, and rod-like crystals in human hepatocellular carcinoma cells (HepG2. The cells were treated with the nano-CeO2 at various concentrations (6.25, 12.5, 25, 50, 100 μg/mL. The crystal structure, size and morphology of nano-CeO2 were investigated by X-ray diffractometry and transmission electron microscopy. The specific surface area was detected using the Brunauer, Emmet and Teller method. The cellular morphological and internal structure were observed by microscopy; apoptotic alterations were measured using flow cytometry; nuclear DNA, mitochondrial membrane potential (MMP, reactive oxygen species (ROS and glutathione (GSH in HepG2 cells were measured using high content screening technology. The scavenging ability of hydroxyl free radicals and the redox properties of the nano-CeO2 were measured by square-wave voltammetry and temperature-programmed-reduction methods. All three types of nano-CeO2 entered the HepG2 cells, localized in the lysosome and cytoplasm, altered cellular shape, and caused cytotoxicity. The nano-CeO2 with smaller specific surface areas induced more apoptosis, caused an increase in MMP, ROS and GSH, and lowered the cell’s ability to scavenge hydroxyl free radicals and antioxidants. In this work, our data demonstrated that compared with cube-like and octahedron-like nano-CeO2, the rod-like nano-CeO2 has lowest toxicity to HepG2 cells owing to its larger specific surface areas.

Gymnaster koraiensis and its major components, 3,5-di-O-caffeoylquinic acid and gymnasterkoreayne B, reduce oxidative damage induced by tert-butyl hydroperoxide or acetaminophen in HepG2 cells

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Eun Hye Jho

2013-10-01

Full Text Available We investigated the protective effects of Gymnaster koraiensisagainst oxidative stress-induced hepatic cell damage. We usedtwo different cytotoxicity models, i.e., the administration oftert-butyl hydroperoxide (t-BHP and acetaminophen, in HepG2cells to evaluate the protective effects of G. koraiensis. The ethylacetate (EA fraction of G. koraiensis and its major compound,3,5-di-O-caffeoylquinic acid (DCQA, exerted protective effectsin the t-BHP-induced liver cytotoxicity model. The EA fractionand DCQA ameliorated t-BHP-induced reductions in GSHlevels and exhibited free radical scavenging activity. The EAfraction and DCQA also significantly reduced t-BHP-inducedDNA damage in HepG2 cells. Furthermore, the hexane fractionof G. koraiensis and its major compound, gymnasterkoreayne B(GKB, exerted strong hepatoprotection in the acetaminopheninducedcytotoxicity model. CYP 3A4 enzyme activity wasstrongly inhibited by the extract, hexane fraction, and GKB. Thehexane fraction and GKB ameliorated acetaminophen-inducedreductions in GSH levels and protected against cell death. [BMBReports 2013; 46(10: 513-518

UHPLC-ESI-MS Analysis of Purified Flavonoids Fraction from Stem of Dendrobium denneaum Paxt. and Its Preliminary Study in Inducing Apoptosis of HepG2 Cells

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Chunhua Zhou

2018-01-01

Full Text Available Dendrobium denneaum paxt., which has been widely used for health prevention in traditional Chinese medicine (TCM, is one of the most popular tonic herbs in China. In order to analyze its flavonoids, characterization and antitumor activity of crude extract and flavonoids rich fractions from D. denneaum paxt. were investigated. Flavonoids extracted from D. denneaum paxt. were clearly enriched in fraction II after determining the total flavonoids content; there were 15 characteristic peaks which have been detected; ultra-high performance liquid chromatography-electrospray ionization/mass spectrometry (UHPLC-ESI-MS/MS was applied for structural elucidation of compounds. 13 characteristic peaks including flavonoid-O-glycosides and flavonoid-C-glycosides were determined or preliminarily characterized through comparing retention times and UV and MS spectra with standard compounds or documented literature. The antitumor activity of fraction II on human liver cancer cells HepG2 was investigated. MTT assay method was used to test the antiproliferation activity and to confirm the appropriate treatment concentration as well as inducing time. The morphological changes of the apoptosis cells after being induced by fraction II were observed by a Hoechst reagent and the apoptosis rate was tested by flow cytometry. The results showed that fraction II can inhibit HepG2 cells from proliferation in a dose-dependent and time-dependent manner. The apoptosis experiments indicated that fraction II can significantly induce apoptosis in HepG2 cells in a concentration over 50 μg/mL for 48 h and the most effective level was 150 μg/mL for 48 h.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

International Nuclear Information System (INIS)

Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

2009-01-01

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H 2 O 2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H 2 O 2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H 2 O 2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

Science.gov (United States)

Kim, Dae Jung; Kang, Yun Hwan; Kim, Kyoung Kon; Kim, Tae Woo; Park, Jae Bong

2017-01-01

BACKGROUND/OBJECTIVES Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment. PMID:28584574

Aglycemia keeps mitochondrial oxidative phosphorylation under hypoxic conditions in HepG2 cells

Czech Academy of Sciences Publication Activity Database

Plecitá-Hlavatá, Lydie; Ježek, Jan; Ježek, Petr

2015-01-01

Roč. 47, č. 6 (2015), s. 467-476 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GA13-02033S; GA MŠk(CZ) LH11055 Institutional support: RVO:67985823 Keywords : cancer mitochondria * non-canonical response to hypoxia * hypoxia-inducible factor * glutaminolysis * HepG2 cell s Subject RIV: ED - Physiology Impact factor: 2.080, year: 2015

Atorvastatin and fenofibrate increase apolipoprotein AV and decrease triglycerides by up-regulating peroxisome proliferator-activated receptor-α

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Huang, Xian-sheng; Zhao, Shui-ping; Bai, Lin; Hu, Min; Zhao, Wang; Zhang, Qian

2009-01-01

Background and purpose: Combining statin and fibrate in clinical practice provides a greater reduction of triglycerides than either drug given alone, but the mechanism for this effect is poorly understood. Apolipoprotein AV (apoAV) has been implicated in triglyceride metabolism. This study was designed to investigate the effect of the combination of statin and fibrate on apoAV and the underlying mechanism(s). Experimental approach: Hypertriglyceridaemia was induced in rats by giving them 10% fructose in drinking water for 2 weeks. They were then treated with atorvastatin, fenofibrate or the two agents combined for 4 weeks, and plasma triglyceride and apoAV measured. We also tested the effects of these two agents on triglycerides and apoAV in HepG2 cells in culture. Western blot and reverse transcription polymerase chain reaction was used to measure apoAV and peroxisome proliferator-activated receptor-α (PPARα) expression. Key results: The combination of atorvastatin and fenofibrate resulted in a greater decrease in plasma triglycerides and a greater increase in plasma and hepatic apoAV than either agent given alone. Hepatic expression of the PPARα was also more extensively up-regulated in rats treated with the combination. A similar, greater increase in apoAV and a greater decrease in triglycerides were observed following treatment of HepG2 cells pre-exposed to fructose), with the combination. Adding an inhibitor of PPARα (MK886) abolished the effects of atorvastatin on HepG2 cells. Conclusions and implications: A combination of atorvastatin and fenofibrate increased apoAV and decreased triglycerides through up-regulation of PPARα. PMID:19694729

Plasmatic concentration of organochlorine lindane acts as metabolic disruptors in HepG2 liver cell line by inducing mitochondrial disorder

Energy Technology Data Exchange (ETDEWEB)

Benarbia, Mohammed el Amine [LUNAM Université, Angers (France); Inserm 1063, Angers (France); Macherel, David [LUNAM Université, Angers (France); UMR 1345 IRHS, Angers (France); Faure, Sébastien; Jacques, Caroline; Andriantsitohaina, Ramaroson [LUNAM Université, Angers (France); Inserm 1063, Angers (France); Malthièry, Yves, E-mail: yves.malthiery@univ-angers.fr [LUNAM Université, Angers (France); Inserm 1063, Angers (France)

2013-10-15

Lindane (LD) is a persistent environmental pollutant that has been the subject of several toxicological studies. However, concentrations used in most of the reported studies were relatively higher than those found in the blood of the contaminated area residents and effects of low concentrations remain poorly investigated. Moreover, effects on cell metabolism and mitochondrial function of exposure to LD have received little attention. This study was designed to explore the effects of low concentrations of LD on cellular metabolism and mitochondrial function, using the hepatocarcinoma cell line HepG2. Cells were exposed to LD for 24, 48 and 72 h and different parameters linked with mitochondrial regulation and energy metabolism were analyzed. Despite having any impact on cellular viability, exposure to LD at plasmatic concentrations led to an increase of maximal respiratory capacity, complex I activity, intracellular ATP and NO release but decreased uncoupled respiration to ATP synthesis and medium lactate levels. In addition, LD exposure resulted in the upregulation of mitochondrial biogenesis genes. We suggest that, at plasmatic concentrations, LD acts as a metabolic disruptor through impaired mitochondrial function and regulation with an impact on cellular energetic metabolism. In addition, we propose that a cellular assay based on the analysis of mitochondria function, such as described here for LD, may be applicable for larger studies on the effects of low concentrations of xenobiotics, because of the exquisite sensitivity of this organelle. - Highlights: Our data clearly demonstrated in HepG2 cells that exposure at plasmatic low concentrations of LD were able to: • Impair mitochondrial function • Caused alteration on nucleo-mitochondrial cross-talk • Increase nitric oxide release and protein nitration • Impair cellular energetic metabolism and lipid accumulation.

Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells.

Science.gov (United States)

Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

2014-03-01

Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. Copyright © 2013. Published by

E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

International Nuclear Information System (INIS)

Hao, Hongying; Dong, Yanbin; Bowling, Maria T; Gomez-Gutierrez, Jorge G; Zhou, H Sam; McMasters, Kelly M

2007-01-01

PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells

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Kin Weng Kong

2016-01-01

Full Text Available Barringtonia racemosa is a tropical plant with medicinal values. In this study, the ability of the water extracts of the leaf (BLE and stem (BSE from the shoots to protect HepG2 cells against oxidative damage was studied. Five major polyphenolic compounds consisting of gallic acid, ellagic acid, protocatechuic acid, quercetin and kaempferol were identified using HPLC-DAD and ESI-MS. Cell viability assay revealed that BLE and BSE were non-cytotoxic (cell viabilities >80% at concentration less than 250 µg/ml and 500 µg/ml, respectively. BLE and BSE improved cellular antioxidant status measured by FRAP assay and protected HepG2 cells against H2O2-induced cytotoxicity. The extracts also inhibited lipid peroxidation in HepG2 cells as well as the production of reactive oxygen species. BLE and BSE could also suppress the activities of superoxide dismutase and catalase during oxidative stress. The shoots of B. racemosa can be an alternative bioactive ingredient in the prevention of oxidative damage.

Ananas comosus L. Leaf Phenols and p-Coumaric Acid Regulate Liver Fat Metabolism by Upregulating CPT-1 Expression

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Weidong Xie

2014-01-01

Full Text Available In this study, we aimed to investigate the effect and action mechanisms of pineapple leaf phenols (PLPs on liver fat metabolism in high-fat diet-fed mice. Results show that PLP significantly reduced abdominal fat and liver lipid accumulation in high-fat diet-fed mice. The effects of PLP were comparable with those of FB. Furthermore, at the protein level, PLP upregulated the expression of carnitine palmitoyltransferase 1 (CPT-1, whereas FB had no effects on CPT-1 compared with the HFD controls. Regarding mRNA expression, PLP mainly promoted the expression of CPT-1, PGC1a, UCP-1, and AMPK in the mitochondria, whereas FB mostly enhanced the expression of Ech1, Acox1, Acaa1, and Ehhadh in peroxisomes. PLP seemed to enhance fat metabolism in the mitochondria, whereas FB mainly exerted the effect in peroxisomes. In addition, p-coumaric acid (CA, one of the main components from PLP, significantly inhibited fat accumulation in oleic acid-induced HepG2 cells. CA also significantly upregulated CPT-1 mRNA and protein expressions in HepG2 cells. We, firstly, found that PLP enhanced liver fat metabolism by upregulating CPT-1 expression in the mitochondria and might be promising in treatment of fatty liver diseases as alternative natural products. CA may be one of the active components of PLP.

Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

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Azimahtol Hawariah LP

2007-04-01

Full Text Available Abstract Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml. The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity.

Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

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Song, Juquan; Zhang, Xiao-jun; Boehning, Darren; Brooks, Natasha C.; Herndon, David N.; Jeschke, Marc G.

2012-01-01

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-13C2-glycine and L-[ring-13C6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-13C2-glycine and L-[ring-13C6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (pisotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis. PMID:22298954

Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells

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An, Joo-Hee; Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; Kang, Eun-Jin; Choi, Kyung-Hee

2011-01-01

Highlights: → The actin binding protein Gelsolin (GSN) interacts with transcription factor p53. → GSN interacts with transactivation- and DNA binding domains of p53. → GSN represses transactivity of p53 via inhibition of nuclear translocation of p53. → GSN inhibits the p53-mediated apoptosis in hepatocarcinoma HepG2 cells. -- Abstract: As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

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Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth A S; Nallasamy, Palanisamy; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

2015-10-01

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity. © 2014 by the Society for Experimental Biology and Medicine.

Necrosis of HepG2 cancer cells induced by the vibration of magnetic particles

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Wang, Biran [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France); Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Bienvenu, Céline [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Mendez-Garza, Juan; Lançon, Pascal; Madeira, Alexandra [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France); Vierling, Pierre [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Di Giorgio, Christophe, E-mail: christophe.di-giorgio@unice.fr [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Bossis, Georges, E-mail: bossis@unice.fr [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France)

2013-10-15

Experiments of magnetolysis, i.e., destruction of cells induced with magnetic particles (MPs) submitted to the application of a magnetic field, were conducted on HepG2 cancer cells. We herein demonstrate the usefulness of combining anisotropic MPs with an alternative magnetic field in magnetolysis. Thus, the application of an alternative magnetic field of low frequency (a few Hertz) in the presence of anisotropic, submicronic particles allowed the destruction of cancer cells “in vitro”. We also show that a constant magnetic field is far less efficient than an oscillating one. Moreover, we demonstrate that, at equal particle volume, it is much more efficient to utilize spindle shaped particles rather than spherical ones. In order to get deeper insight into the mechanism of magnetolysis experiments, we performed a study by AFM, which strongly supports that the magnetic field induces the formation of clusters of particles becoming then large enough todamage cell membranes. - Highlights: • Magnetic force was applied on cancer cells through magnetic particles. • The penetration depth was predicted, both for spherical and ellipsoidal particles. • Alternative force was shown to damage the cells contrary to static force. • The effect of indentation of magnetic particles was compared to the one of AFM tips. • The damage was attributed to the formation of clusters of particles.

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

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Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

2017-08-01

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H 2 AX (γH 2 AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Rapamycin up-regulates triglycerides in hepatocytes by down-regulating Prox1.

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Kwon, Sora; Jeon, Ji-Sook; Kim, Su Bin; Hong, Young-Kwon; Ahn, Curie; Sung, Jung-Suk; Choi, Inho

2016-02-27

Although the prolonged use of rapamycin may cause unwanted side effects such as hyperlipidemia, the underlying mechanism remains unknown. Prox1 is a transcription factor responsible for the development of several tissues including lymphatics and liver. There is growing evidences that Prox1 participates in metabolism in addition to embryogenesis. However, whether Prox1 is directly related to lipid metabolism is currently unknown. HepG2 human hepatoma cells were treated with rapamycin and total lipids were analyzed by thin layer chromatography. The effect of rapamycin on the expression of Prox1 was determined by western blotting. To investigate the role of Prox1 in triglycerides regulation, siRNA and overexpression system were employed. Rapamycin was injected into mice for 2 weeks and total lipids and proteins in liver were measured by thin layer chromatography and western blot analysis, respectively. Rapamycin up-regulated the amount of triglyceride and down-regulated the expression of Prox1 in HepG2 cells by reducing protein half-life but did not affect its transcript. The loss-of-function of Prox1 was coincident with the increase of triglycerides in HepG2 cells treated with rapamycin. The up-regulation of triglycerides by rapamycin in HepG2 cells reverted to normal levels by the compensation of Prox1 using the overexpression system. Rapamycin also down-regulated Prox1 expression but increased triglycerides in mouse liver. This study suggests that rapamycin can increase the amount of triglycerides by down-regulating Prox1 expression in hepatocytes, which means that the mammalian target of rapamycin (mTOR) signaling is important for the regulation of triglycerides by maintaining Prox1 expression.

Apoptosis Induction by Polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and S-phase cell cycle arrest in HepG2 cell line.

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Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

2014-01-01

Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

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Mittal, Smriti P K; Khole, Swati; Jagadish, Nidhi; Ghosh, Debjani; Gadgil, Vijay; Sinkar, Vilas; Ghaskadbi, Saroj S

2016-11-01

Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3β by phosphorylation. Inactivated GSK-3β leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3β, together resulting in activation of HO-1. We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc. Copyright © 2016 Elsevier B.V. All rights reserved.

In vitro investigations of Cynara scolymus L. extract on cell physiology of HepG2 liver cells

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Gesine Löhr

2009-06-01

Full Text Available The objective of this study was the investigation of a potential influence of artichoke leaf extract (ALE on the cell physiology and gene expression of phase I/II enzymes of human liver cells HepG2 and investigation on potential cell protective effects against ethanol-induced cell toxicity against HepG2 cells. Cell biological assays under in vitro conditions using HepG2 liver cells and investigation of mitochondrial activity (MTT test, proliferation assay (BrdU incorporation ELISA, LDH as toxicity marker, gene expression analysis by RT-PCR and enzyme activity of glutationtransferase. Artichocke extract, containing 27% caffeoylquinic acids and 7% flavonoids induced mitochondrial activity, proliferation and total protein content under in vitro conditions in human liver cells HepG2. These effects could not be correlated to the well-known artichoke secondary compounds cynarin, caffeic acid, chlorogenic acid, luteolin and luteolin-7-O-glucoside. The flavones luteolin and luteolin-7-O-glucoside had inhibitory effects at 100 µg/mL level on HepG2 cells, with luteolin being a significant stronger inhibitor compared to the respective glucoside. Artichoke leaf extract had minor stimulating effect on gene expression of CYP1A2, while CYP3A4, GGT, GPX2, GSR and GST were slightly inhibited. GST inhibition under in vitro conditions was also shown by quantification of GST enzyme activity. Induction of gene expression of CYP1A2 was shown to be supraadditive after simultaneous application of ethanol plus artichoke extract. Artichoke leaf extract exhibited cell protective effects against ethanol-induced toxicity within cotreatment under in vitro conditions. Also H2O2 damage was significantly inhibited by simultaneous artichoke incubation. Pre- and posttreatments did not exert protective effects. DMSO-induced toxicity was significantly reduced by pre-, post- and cotreatment with artichoke extract and especially with luteolin-7-O-glucoside, indicating a direct

MicroRNA expression in the vildagliptin-treated two- and three-dimensional HepG2 cells.

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Yamashita, Yasunari; Asakura, Mitsutoshi; Mitsugi, Ryo; Fujii, Hideaki; Nagai, Kenichiro; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2016-06-01

Vildagliptin is an inhibitor of dipeptidyl peptidase-4 that is used for the treatment of type 2 diabetes mellitus. While vildagliptin can induce hepatic dysfunction in humans, the molecular mechanism has not been determined yet. Recent studies indicated that certain types of microRNA (miRNA) were linking to the development of drug-induced hepatotoxicity. In the present study, therefore, we identified hepatic miRNAs that were highly induced or reduced by the vildagliptin treatment in mice. MiR-222 and miR-877, toxicity-associated miRNAs, were induced 31- and 53-fold, respectively, by vildagliptin in the liver. While a number of miRNAs were significantly regulated by the orally treated vildagliptin in vivo, such regulation was not observed in the vildagliptin-treated HepG2 cells. In addition to the regular two-dimensional (2D) culture, we carried out the three-dimensional (3D) culturing of HepG2 cells. In the 3D-HepG2 cells, a significant reduction of miR-222 was observed compared to the expression level in 2D-HepG2 cells. A slight induction of miR-222 by vildagliptin was observed in the 3D-HepG2 cells, although miR-877 was not induced by vildagliptin even in the 3D-HepG2 cells. Further investigations are needed to overcome the discrepancy in the responsiveness of the miRNA expressions to vildagliptin between in vivo and in vitro. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Study of apoptotic mechanisms induced by all-trans retinoic acid and its 13-cis isomer on cellular lines of human hepato carcinoma Hep3B and HepG2

International Nuclear Information System (INIS)

Arce Vargas, Frederick

2006-01-01

Two cellular lines of liver cancer (Hep3B and HepG2) were incubated during different periods of time with some concentrations of two retinoic acid isomers (ATRA and 13-cis AR) and with 5-fu chemotherapeutic agents, cisplatin and paclitaxel. It was determined if these substances leaded cytotoxicity, apoptosis and if they modified the expression of different genes related to cellular death by apoptosis, in order to explain the hepatocellular carcinoma resistance to these drugs. HepG2 cells showed more resistance than Hep3B cells to 72 hours of treatment, as much ATRA as the 13-cis AR were toxic and produced apoptosis in two cellular lines. This type of cellular death seems to be mediated by a decrease in Bcl-xL concentration in Hep3B cells treated with both retinoids an increase in bax concentration in HepG2 cells treated with 13-cis AR. It were observed 3 and 8 proteolysis of procaspase in Hep3B cells, suggesting extrinsic via activation of the apoptosis, while cellular death in HepG2 cells seems to be independent of caspases. Cisplatin and paclitaxel leaded cytotoxicity to 48 hours of treatment, with significant differences between two cellular lines only in case of paclitaxel. Hep3B cells treated with cisplatin and HepG2 cells treated with paclytaxel suffered apoptosis. 5-FU produced toxicity only when it was used to high concentrations and the mechanism of cellular death induced by this agent seems to be primarily necrosis in Hep3B cells and apoptosis in HepG2. There was decrease in the Bcl-xL concentration in two cellular lines when it was treated with cisplatin and in HepG2 cells treated with 5-FU. Bax concentration there no was modified with no treatment. Activation of the 3 caspases seems to happen only in HepG2 cells with 5-FU and paclytaxel. These two agents, also, decreased the survivin concentration of HepG2 cells. Treatments of the three drugs produced an increase in the expression of this gen in Hep3B cells, which might explain partially the resistance

Phytol directly activates peroxisome proliferator-activated receptor α (PPARα) and regulates gene expression involved in lipid metabolism in PPARα-expressing HepG2 hepatocytes

International Nuclear Information System (INIS)

Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo

2005-01-01

The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPARα-specific activator. Phytol induced the increase in PPARα-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPARα. Moreover, the addition of phytol upregulated the expression of PPARα-target genes at both mRNA and protein levels in PPARα-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPARα ligand and that it stimulates the expression of PPARα-target genes in intact cells. Because PPARα activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism

Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPAR{sub β/δ} in HepG2 cells

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Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua [Comprehensive Laboratory, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Berggren-Söderlund, Maria; Nilsson-Ehle, Peter [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden); Zhang, Xiaoying, E-mail: zhangxy6689996@163.com [Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Xu, Ning, E-mail: ning.xu@med.lu.se [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden)

2014-02-28

Highlights: • Palmitic acid significantly inhibited APOM gene expression in HepG2 cells. • Palmitic acid could obviously increase PPARB/D mRNA levels in HepG2 cells. • PPAR{sub β/δ} antagonist, GSK3787, had no effect on APOM expression. • GSK3787 could reverse the palmitic acid-induced down-regulation of APOM expression. • Palmitic acid induced suppression of APOM expression is mediated via the PPAR{sub β/δ} pathway. - Abstract: It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPAR{sub β/δ}) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPAR{sub β/δ} pathway.

Cholesterol lowering effects of mono-lactose-appended β-cyclodextrin in Niemann–Pick type C disease-like HepG2 cells

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Keiichi Motoyama

2015-11-01

Full Text Available The Niemann–Pick type C disease (NPC is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD has been applied for the treatment of NPC. HP-β-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by actively targeted-β-CyD to hepatocytes is expected. In the present study, to deliver β-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended β-CyD (Lac-β-CyD and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-β-CyD (degree of substitution of lactose (DSL 1 significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-β-CyD was associated with NPC-like HepG2 cells higher than TRITC-β-CyD. In addition, TRITC-Lac-β-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-β-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-β-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

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Cao, Lei [Division of Experimental Vascular Research, Institute of Clinical Sciences in Lund, Lund University (Sweden); Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Ping, Na-Na; Cao, Yong-Xiao [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Li, Wei, E-mail: 13572512207@163.com [Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Cai, Yan [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi (China); Warfvinge, Karin; Edvinsson, Lars [Division of Experimental Vascular Research, Institute of Clinical Sciences in Lund, Lund University (Sweden)

2016-08-01

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ET{sub A} receptor mRNA and protein levels as well as contractile responses mediated by ET{sub A} receptors. The immunoreactivity of increased ET{sub A} receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ET{sub B} receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ET{sub A} receptor upregulation in rat cerebral arteries. - Highlights: • Cigarette smoke exposure induces ET{sub A} receptor upregulation in rat cerebral arteries. • U0126 can alleviate the receptor upregulation. • The mechanism relies on MEK/ERK1/2 pathway activation. • We may provide a new target for the

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

International Nuclear Information System (INIS)

Cao, Lei; Ping, Na-Na; Cao, Yong-Xiao; Li, Wei; Cai, Yan; Warfvinge, Karin; Edvinsson, Lars

2016-01-01

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ET A receptor mRNA and protein levels as well as contractile responses mediated by ET A receptors. The immunoreactivity of increased ET A receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ET B receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ET A receptor upregulation in rat cerebral arteries. - Highlights: • Cigarette smoke exposure induces ET A receptor upregulation in rat cerebral arteries. • U0126 can alleviate the receptor upregulation. • The mechanism relies on MEK/ERK1/2 pathway activation. • We may provide a new target for the treatment of SHS

HAb18G/CD147 is involved in TGF-β-induced epithelial-mesenchymal transition and hepatocellular carcinoma invasion.

Science.gov (United States)

Ru, Ning-Yu; Wu, Jiao; Chen, Zhi-Nan; Bian, Huijie

2015-01-01

Epithelial-mesenchymal transition (EMT) induced by the transforming growth factor beta (TGF-β) is involved in hepatocarcinogenesis and hepatocellular carcinoma (HCC) metastasis. HAb18G/CD147, a member of the immunoglobulin family, plays an important role in tumor invasion and metastasis. HAb18G/CD147 promotes EMT of hepatocytes through TGF-β signaling and is transcriptionally regulated by Slug. We investigated the role of HAb18G/CD147 in TGF-β-induced EMT in HCC invasion. Two human HCC cell lines, SMMC-7721 and HepG2, were used to determine the role of HAb18G/CD147 in EMT. Upregulation of HAb18G/CD147 induced by the high doses of TGF-β1 in SMMC-7721 (5 ng/mL) and HepG2 cells (10 ng/mL) (P CD147 upregulation was coupled with upregulation of Snail1 and Slug. CD147 knockout significantly decreased the expression of N-cadherin and vimentin, and colony formation ability of SMMC-7721 cells. TGF-β1 enhanced the migration capacity of SMMC-7721 cells, which was markedly attenuated by CD147 knockdown. Thus, HAb18G/CD147 is involved in TGF-β-induced EMT and HCC invasion. © 2014 International Federation for Cell Biology.

Cultivation of HepG2.2.15 on Cytodex-3

DEFF Research Database (Denmark)

Lupberger, Joachim; Mund, Andreas; Kock, Josef

2006-01-01

BACKGROUND/AIMS: Several novel systems are available to study human hepatitis B virus (HBV) replication in cell culture demanding for efficient cell culture based systems for HBV production. The aim was to enhance HBV production of the HBV stably producing cell line HepG2.2.15 by cultivation on s...

ATM phosphorylation in HepG2 cells following continuous low dose-rate irradiation

International Nuclear Information System (INIS)

Mei Quelin; Du Duanming; Chen Zaizhong; Liu Pengcheng; Yang Jianyong; Li Yanhao

2008-01-01

Objective: To investigate the change of ATM phosphorylation in HepG2 cells following a continuous low dose-rate irradiation. Methods: Cells were persistently exposed to low dose-rate (8.28 cGy/h) irradiation. Indirect immunofluorescence and Western blot were used to detect the expression of ATM phosphorylated proteins. Colony forming assay was used to observe the effect of a low dose-rate irradiation on HepG2 cell survival. Results: After 30 min of low dose-rate irradiation, the phosphorylation of ATM occurred. After 6 h persistent irradiation, the expression of ATM phosphorylated protein reached the peak value, then gradually decreased. After ATM phosphorylation was inhibited with Wortmannin, the surviving fraction of HepG2 cells was lower than that of the irradiation alone group at each time point (P<0.05). Conclusions: Continuous low dose-rate irradiation attenuated ATM phosphorylation, suggesting that continuous low dose-rate irradiation has a potential effect for increasing the radiosensitivity of HepG2 cells. (authors)

CdTe quantum dots with daunorubicin induce apoptosis of multidrug-resistant human hepatoma HepG2/ADM cells: in vitro and in vivo evaluation

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Shi Lixin

2011-01-01

Full Text Available Abstract Cadmium telluride quantum dots (Cdte QDs have received significant attention in biomedical research because of their potential in disease diagnosis and drug delivery. In this study, we have investigated the interaction mechanism and synergistic effect of 3-mercaptopropionic acid-capped Cdte QDs with the anti-cancer drug daunorubicin (DNR on the induction of apoptosis using drug-resistant human hepatoma HepG2/ADM cells. Electrochemical assay revealed that Cdte QDs readily facilitated the uptake of the DNR into HepG2/ADM cells. Apoptotic staining, DNA fragmentation, and flow cytometry analysis further demonstrated that compared with Cdte QDs or DNR treatment alone, the apoptosis rate increased after the treatment of Cdte QDs together with DNR in HepG2/ADM cells. We observed that Cdte QDs treatment could reduce the effect of P-glycoprotein while the treatment of Cdte QDs together with DNR can clearly activate apoptosis-related caspases protein expression in HepG2/ADM cells. Moreover, our in vivo study indicated that the treatment of Cdte QDs together with DNR effectively inhibited the human hepatoma HepG2/ADM nude mice tumor growth. The increased cell apoptosis rate was closely correlated with the enhanced inhibition of tumor growth in the studied animals. Thus, Cdte QDs combined with DNR may serve as a possible alternative for targeted therapeutic approaches for some cancer treatments.

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells.

Science.gov (United States)

Lamy, Evelyn; Kassie, Fekadu; Gminski, Richard; Schmeiser, Heinz H; Mersch-Sundermann, Volker

2004-01-15

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.

Anti-hepatocarcinoma effects of resveratrol nanoethosomes against human HepG2 cells

Science.gov (United States)

Meng, Xiang-Ping; Zhang, Zhen; Chen, Tong-sheng; Wang, Yi-fei; Wang, Zhi-ping

2017-02-01

Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Resveratrol (Res) has been widely investigated with its strong anti-tumor activity. However, its low oral bioavailability restricts its wide application. In this study, we prepared resveratrol nanoethosomes (ResN) via ethanol injection method. The in vitro anti-hepatocarcinoma effects of ResN relative to efficacy of bulk Res were evaluated on proliferation and apoptosis of human HepG2 cells. ResN were spherical vesicles and its particle diameter, zeta potential were (115.8 +/- 1.3) nm and (-12.8 +/- 1.9) mV, respectively. ResN exhibited significant inhibitory effects against human HepG2 cells by MTT assay, and the IC50 value was 49.2 μg/ml (105.4 μg/ml of Res bulk solution). By flow cytometry assay, there was an increase in G2/M phase cells treated with ResN. The results demonstrated ResN could effectively block the G2/M phase of HepG2 cells, which can also enhance the inhibitory effect of Res against HepG2 cells.

A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

International Nuclear Information System (INIS)

Oh, Seon-Hee; Lee, Byung-Hoon

2004-01-01

20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

Science.gov (United States)

Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation

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Fang Chen

2016-04-01

Full Text Available Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O, has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu and Cat D inhibitor (pepA inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05 in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment.

Science.gov (United States)

Chen, Miaojiao; Zhang, Jingjing; Hu, Fang; Liu, Shiping; Zhou, Zhiguang

2015-11-01

Accumulating evidence suggests an association between diabetes and cancer. Inflammation is a key event that underlies the pathological processes of the two diseases. Metformin displays anti-cancer effects, but the mechanism is not completely clear. This study investigated whether metformin regulated the microenvironment of macrophage polarization to affect the characteristics of HepG2 cells and the possible role of the Notch-signalling pathway. RAW264.7 macrophages were cultured alone or co-cultured with HepG2 cells and treated with metformin. We analysed classical (M1) and alternative (M2) gene expression in RAW264.7 cells using quantitative real-time polymerase chain reaction. Changes in mRNA and protein expressions of Notch signalling in both cell types were also detected using quantitative real-time polymerase chain reaction and Western-blotting analyses. The proliferation, apoptosis and migration of HepG2 cells were detected using Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Fitchburg, WI, USA), Annexin V-FITC/PI (7SeaPharmTech, Shanghai, China) and the cell scratch assay, respectively. Metformin induced single-cultured RAW264.7 macrophages with an M2 phenotype but attenuated the M2 macrophage differentiation and inhibited monocyte chemoattractant protein-1 (MCP-1) secretion in a co-culture system. The co-cultured group of metformin pretreatment activated Notch signalling in macrophages but repressed it inHepG2 cells. Co-culture also promoted the proliferation and migration of HepG2 cells. However, along with the enhanced apoptosis, the proliferation and the migration of HepG2 cells were remarkably inhibited in another co-culture system with metformin pretreatment. Metformin can skew RAW264.7 macrophages toward different phenotypes according to changes in the microenvironment, which may affect the inflammatory conditions mediated by macrophages, induce apoptosis and inhibit the proliferation and migration of HepG2

PDE7B is involved in nandrolone decanoate hydrolysis in liver cytosol and its transcription is up-regulated by androgens in HepG2

Directory of Open Access Journals (Sweden)

Emmanuel eStrahm

2014-05-01

Full Text Available Most androgenic drugs are available as esters for a prolonged depot action. However the enzymes involved in the hydrolysis of the esters have not been identified. There is one study indicating that PDE7B may be involved in the activation of testosterone enanthate. The aims are to identify the cellular compartments where the hydrolysis of testosterone enanthate and nandrolone decanoate occurs, and to investigate the involvement of PDE7B in the activation. We also determined if testosterone and nandrolone affect the expression of the PDE7B gene. The hydrolysis studies were performed in isolated human liver cytosolic and microsomal preparations with and without specific PDE7B inhibitor. The gene expression was studied in human hepatoma cells (HepG2 exposed to testosterone and nandrolone. We show that PDE7B serves as a catalyst of the hydrolysis of testosterone enanthate and nandrolone decanoate in liver cytosol. The gene expression of PDE7B was significantly induced 3- and 5- fold after 2 hours exposure to 1 µM testosterone enanthate and nandrolone decanoate, respectively. These results show that PDE7B is involved in the activation of esterified nandrolone and testosterone and that the gene expression of PDE7B is induced by supra-physiological concentrations of androgenic drugs.

Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells.

Science.gov (United States)

Zheng, Yingjuan; Zhao, Chao; Zhang, Naijian; Kang, Wenqin; Lu, Rongrong; Wu, Huadong; Geng, Yingxue; Zhao, Yaping; Xu, Xiaoyan

2018-04-01

The actions of thyroid hormone (TH) on lipid metabolism in the liver are associated with a number of genes involved in lipogenesis and lipid metabolism; however, the underlying mechanisms through which TH impacts on lipid metabolism remain to be elucidated. The present study aimed to investigate the effects of hyperthyroidism on the serum levels of the microRNA (miR) miR‑206 and the role of miR‑206 on TH‑regulated lipid metabolism in liver cells. Serum was obtained from 12 patients diagnosed with hyperthyroidism and 10 healthy control subjects. Human hepatoblastoma (HepG2) cells were used to study the effects of triiodothyronine (T3) and miR‑206 on lipid metabolism. Expression of miR‑206 in serum and cells was determined by reverse transcription‑quantitative polymerase chain reaction analysis. Lipid accumulation in HepG2 cells was assessed with Oil Red O staining. Suppression or overexpression of miR‑206 was performed via transfection with a miR‑206 mimic or miR‑206 inhibitor. Serum miR‑206 was significantly decreased in patients with hyperthyroidism compared with euthyroid controls. Treatment of HepG2 cells with T3 led to reduced total cholesterol (TC) and triglyceride (TG) content, accompanied by reduced miR‑206 expression. Inhibition of endogenous miR‑206 expression decreased intracellular TG and TC content in HepG2 cells. By contrast, overexpression of miR‑206 in HepG2 partially prevented the reduction in TG content induced by treatment with T3. In conclusion, serum miR‑206 expression is reduced in patients with hyperthyroidism. In addition, miR‑206 is involved in T3‑mediated regulation of lipid metabolism in HepG2 cells, indicating a role for miR‑206 in thyroid hormone‑induced disorders of lipid metabolism in the liver.

Statins Prevent Dextrose-Induced Endoplasmic Reticulum Stress and Oxidative Stress in Endothelial and HepG2 Cells.

Science.gov (United States)

Kojanian, Hagop; Szafran-Swietlik, Anna; Onstead-Haas, Luisa M; Haas, Michael J; Mooradian, Arshag D

Statins have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. However, antioxidant vitamins, unlike statins, are not as cardioprotective, and this paradox has been explained by failure of vitamin antioxidants to ameliorate endoplasmic reticulum (ER) stress. To determine whether statins prevent dextrose-induced ER stress in addition to their antioxidative effects, human umbilical vein endothelial cells and HepG2 hepatocytes were treated with 27.5 mM dextrose in the presence of simvastatin (lipophilic statin that is a prodrug) and pravastatin (water-soluble active drug), and oxidative stress, ER stress, and cell death were measured. Superoxide generation was measured using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride. ER stress was measured using the placental alkaline phosphatase assay and Western blot of glucose-regulated protein 75, c-jun-N-terminal kinase, phospho-JNK, eukaryotic initiating factor 2α and phospho-eIF2α, and X-box binding protein 1 mRNA splicing. Cell viability was measured by propidium iodide staining. Superoxide anion production, ER stress, and cell death induced by 27.5 mM dextrose were inhibited by therapeutic concentrations of simvastatin and pravastatin. The salutary effects of statins on endothelial cells in reducing both ER stress and oxidative stress observed with pravastatin and the prodrug simvastatin suggest that the effects may be independent of cholesterol-lowering activity.

Newly developed chitosan-silver hybrid nanoparticles: biosafety and apoptosis induction in HepG2 cells

International Nuclear Information System (INIS)

El-Sherbiny, Ibrahim M.; Salih, Ehab; Yassin, Abdelrahman M.; Hafez, Elsayed E.

2016-01-01

The present study reports the biosafety assessment, the exact molecular effects, and apoptosis induction of newly developed chitosan-silver hybrid nanoparticles (Cs–Ag NPs) in HepG2 cells. The investigated hybrid NPs were green synthesized using Cs/grape leaves aqueous extract (Cs/GLE) or Cs/GLE NPs as reducing and stabilizing agents. The successful formation of Cs/GLE NPs and Cs–Ag hybrid NPs has been confirmed by UV–Vis spectrophotometry, FTIR spectroscopy, XRD, and HRTEM. From the TEM analysis, the prepared Cs/GLE NPs are uniform and spherical with an average size of 150 nm, and the AgNPs (5–10 nm) were formed mainly on their surface. The UV–Vis spectra of Cs–Ag NPs showed a surface plasmon resonance (SPR) peak at about 450 nm confirming their formation. The synthesized Cs–Ag NPs were found to be crystalline as shown by XRD patterns with fcc phase oriented along the (111), (200), (220), and (311) planes. The cytotoxicity patterns, the antiproliferative activities, and the possible mechanisms of anticancer activity at molecular level of the newly developed Cs–Ag hybrid NPs were investigated. Cytotoxicity patterns of all the preparations demonstrated that the nontoxic treatment concentrations are ranged from 0.39 to 50 %, and many of the newly prepared Cs–Ag hybrid NPs showed high anticancer activities against HpG2 cells, and induced cellular apoptosis by downregulating BCL2 gene and upregulating P53.Graphical Abstract

Newly developed chitosan-silver hybrid nanoparticles: biosafety and apoptosis induction in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

El-Sherbiny, Ibrahim M., E-mail: ielsherbiny@Zewailcity.edu.eg; Salih, Ehab [Zewail City of Science and Technology, Center for Materials Science (Egypt); Yassin, Abdelrahman M. [Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, Biopharmaceutical Product Research Department (Egypt); Hafez, Elsayed E. [City of Scientific Research and Technology Applications, Plant Protection and Biomolecular Diagnosis Department (Egypt)

2016-07-15

The present study reports the biosafety assessment, the exact molecular effects, and apoptosis induction of newly developed chitosan-silver hybrid nanoparticles (Cs–Ag NPs) in HepG2 cells. The investigated hybrid NPs were green synthesized using Cs/grape leaves aqueous extract (Cs/GLE) or Cs/GLE NPs as reducing and stabilizing agents. The successful formation of Cs/GLE NPs and Cs–Ag hybrid NPs has been confirmed by UV–Vis spectrophotometry, FTIR spectroscopy, XRD, and HRTEM. From the TEM analysis, the prepared Cs/GLE NPs are uniform and spherical with an average size of 150 nm, and the AgNPs (5–10 nm) were formed mainly on their surface. The UV–Vis spectra of Cs–Ag NPs showed a surface plasmon resonance (SPR) peak at about 450 nm confirming their formation. The synthesized Cs–Ag NPs were found to be crystalline as shown by XRD patterns with fcc phase oriented along the (111), (200), (220), and (311) planes. The cytotoxicity patterns, the antiproliferative activities, and the possible mechanisms of anticancer activity at molecular level of the newly developed Cs–Ag hybrid NPs were investigated. Cytotoxicity patterns of all the preparations demonstrated that the nontoxic treatment concentrations are ranged from 0.39 to 50 %, and many of the newly prepared Cs–Ag hybrid NPs showed high anticancer activities against HpG2 cells, and induced cellular apoptosis by downregulating BCL2 gene and upregulating P53.Graphical Abstract.

PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

International Nuclear Information System (INIS)

Kan-o, Keiko; Matsumoto, Koichiro; Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi; Inoue, Hiromasa

2013-01-01

Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB

Ceramide-induced TCR up-regulation

DEFF Research Database (Denmark)

Menné, C; Lauritsen, Jens Peter Holst; Dietrich, J

2000-01-01

to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase......The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein...... kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected...

Protective Effect of Curcumin against Ionizing Radiation (IR)-induced Cytotoxicity and Genotoxicity in HepG2 Cells

Energy Technology Data Exchange (ETDEWEB)

Chung, Dong Min; Nasir Uddin, S. M.; Ryu, Tae Ho; Kang, Mi Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2013-10-15

Ionizing radiation (IR) has many practical applications such as medicine, foods, agricultures, industries, and research laboratories. However, the increasing use of radiation is associated with radiation accidents threatening human health. It is well known that exposure to IR gives rise to genomic alterations, mutagenesis, and cell death. IR is absorbed directly by DNA, leading to various DNA damages (single or double-strand breaks, base damage, and DNA-DNA or DNA-protein cross-linkages) in many living organisms. Therefore, the development of effective and nontoxic radioprotective agents is of considerable interest. Curcumin (C{sub 12}H{sub 20}O{sub 6}, structure is the major yellow component of Curcuma longa with biological activities (antioxidant, anti-proliferative and anti-inflammatory properties). It has been widely used as food and medicine for a long time. The aim of our present study is to investigate the protective effects of curcumin against IR-induced cytotoxicity and genotoxicity in cultured HepG2 cells.

Protective Effect of Curcumin against Ionizing Radiation (IR)-induced Cytotoxicity and Genotoxicity in HepG2 Cells

International Nuclear Information System (INIS)

Chung, Dong Min; Nasir Uddin, S. M.; Ryu, Tae Ho; Kang, Mi Young; Kim, Jin Kyu

2013-01-01

Ionizing radiation (IR) has many practical applications such as medicine, foods, agricultures, industries, and research laboratories. However, the increasing use of radiation is associated with radiation accidents threatening human health. It is well known that exposure to IR gives rise to genomic alterations, mutagenesis, and cell death. IR is absorbed directly by DNA, leading to various DNA damages (single or double-strand breaks, base damage, and DNA-DNA or DNA-protein cross-linkages) in many living organisms. Therefore, the development of effective and nontoxic radioprotective agents is of considerable interest. Curcumin (C 12 H 20 O 6 , structure is the major yellow component of Curcuma longa with biological activities (antioxidant, anti-proliferative and anti-inflammatory properties). It has been widely used as food and medicine for a long time. The aim of our present study is to investigate the protective effects of curcumin against IR-induced cytotoxicity and genotoxicity in cultured HepG2 cells

Influence of different chemical agents (H2O2, t-BHP and MMS) on the activity of antioxidant enzymes in human HepG2 and hamster V79 cells; relationship to cytotoxicity and genotoxicity.

Science.gov (United States)

Slamenova, D; Kozics, K; Melusova, M; Horvathova, E

2015-01-01

We investigated activities of antioxidant enzymes (AEs), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in human HepG2 and hamster V79 cells treated with a scale of concentrations of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP) and methyl methanesulfonate (MMS). Cytotoxicity and genotoxicity of these substances were evaluated simultaneously. We have found out that H2O2, t-BHP and MMS predictably induce significant concentration-dependent increase of DNA lesions in both cell lines. Cytotoxicity detected in V79 cells with help of PE test was in a good conformity with the level of DNA damage. MTT test has proved unsuitable, except for MMS-treated V79 cells. Compared with human cells HepG2, hamster cells V79 manifested approximately similar levels of SOD and CAT but ten times higher activity of GPx. Across all concentrations tested the most significant increase of activity of the enzyme CAT was found in H2O2- and t-BHP-treated HepG2 cells, of the enzyme SOD in t-BHP- and MMS-treated V79 cells, and of the enzyme GPx in H2O2-treated V79 cells. We suggest that stimulation of enzyme activity by the relevant chemical compounds may result from transcriptional or post-transcriptional regulation of the expression of the genes CAT, SOD and GPx. Several authors suggest that moderate levels of toxic reactants can induce increase of AEs activities, while very high levels of reactants can induce their decrease, as a consequence of damage of the molecular machinery required to induce AEs. Based on a great amount of experiments, which were done and described within this paper, we can say that the above mentioned principle does not apply in general. Only the reactions of t-BHP affected HepG2 cells were consistent with this idea.

Induction of apoptosis by pistachio (Pistacia vera L.) hull extract and its molecular mechanisms of action in human hepatoma cell line HepG2.

Science.gov (United States)

Fathalizadeh, J; Bagheri, V; Khorramdelazad, H; Kazemi Arababadi, M; Jafarzadeh, A; Mirzaei, M R; Shamsizadeh, A; Hajizadeh, M R

2015-11-30

Several important Pistacia species such as P. vera have been traditionally used for treating a wide range of diseases (for instance, liver-related disorders). There is a relative lack of research into pharmacological aspects of pistachio hull. Hence, this study was aimed at investigating whether pistachio rosy hull (PRH) extract exerts apoptotic impacts on HepG2 liver cancer cell line. In order to evaluate cell viability and apoptosis in response to treatment with the extract, MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining were performed, respectively. Moreover, molecular mechanism of apoptosis induced by the extract was determined using human apoptosis PCR array. Our findings showed that PRH extract treatment reduced cell viability (IC50 ~ 0.3 mg/ml) in a dose-dependent manner. Flow cytometric analysis revealed that the extract significantly induced apoptosis in HepG2 cells. In addition, quantitative PCR array results demonstrated the regulation of a considerable number of apoptosis-related genes belonging to the TNF, BCL2, IAP, TRAF, and caspase families. We observed altered expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. These results suggest that the aqueous extract of PRH possesses apoptotic activity through cytotoxic and apoptosis-inducing effects on HepG2 cells.

Galactomannan from Schizolobium amazonicum seed and its sulfated derivatives impair metabolism in HepG2 cells.

Science.gov (United States)

Cunha de Padua, Monique Meyenberg; Suter Correia Cadena, Silvia Maria; de Oliveira Petkowicz, Carmen Lucia; Martinez, Glaucia Regina; Rodrigues Noleto, Guilhermina

2017-08-01

This study evaluated the effects of native galactomannan from Schizolobium amazonicum seeds and its sulfated forms on certain metabolic parameters of HepG2 cells. Aqueous extraction from S. amazonicum seeds furnished galactomannan with 3.2:1 Man:Gal ratio (SAGM) and molar mass of 4.34×10 5 g/mol. The SAGM fraction was subjected to sulfation using chlorosulfonic acid to obtain SAGMS1 and SAGMS2 with DS of 0.4 and 0.6, respectively. Cytotoxicity of SAGM, SAGMS1, and SAGMS2 was evaluated in human hepatocellular carcinoma cells (HepG2). After 72h, SAGM decreased the viability of HepG2 cells by 50% at 250μg/mL, while SAGMS1 reduced it by 30% at the same concentration. SAGM, SAGMS1, and SAGMS2 promoted a reduction in oxygen consumption and an increase in lactate production in non-permeabilized HepG2 cells after 72h of treatment. These results suggest that SAGM, SAGMS1, and SAGMS2 could be recognized by HepG2 cells and might trigger alterations that impair its survival. These effects could be implicated in the modification of the oxidative phosphorylation process in HepG2 cells and activation of the glycolytic pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

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Li Y

2016-07-01

Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2 Cells

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Gabriel Yarmush

2016-01-01

Full Text Available Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2 by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro.

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Hurrell, Tracey; Ellero, Andrea Antonio; Masso, Zelie Flavienne; Cromarty, Allan Duncan

2018-02-21

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids. Copyright © 2018 Elsevier Ltd. All rights reserved.

Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo

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Baowei Li

2012-12-01

Full Text Available Salmonella typhimurium is probably most extensively studied tumor-targeting bacteria and SL7207 is one of its attenuated strains. SL7207 was first made for bacterial vaccine development and its therapeutic efficacy and safety for hepatocellular carcinoma has not been characterized. In this study, the inhibitory ability of SL7207-lux on human hepatoma HepG2 cells was tested in vitro and in vivo. A bacterial luminescent gene cluster (lux CDABE was transfected into SL7207 to better monitor the invasion of the bacteria. The results show that SL7207-lux can rapidly enter HepG2 cells and localize in the cytoplasm. This invasion represses cell proliferation and induces apoptosis. In vivo real-time invasion studies showed that the bacteria gradually accumulate in the tumor. This enrichment was confirmed by anatomic observation at 5 days after inoculation. About 40% of tumor growth was inhibited by SL7207-lux at 34 days post-treatment without significant loss of body weight. The area of necrosis of tumor tissue was clearly increased in the treated group. Bacterial quantification showed that the number of colony-forming units per gram of bacteria within tumor tissue was approximately 1000-fold higher than that of liver and spleen. These data suggest that attenuated S. typhimurium strain SL7207 has potential for the treatment of cancers.

Sagunja-Tang Improves Lipid Related Disease in a Postmenopausal Rat Model and HepG2 Cells

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Hiroe Go

2015-01-01

Full Text Available The present study was conducted to investigate the effect of Sagunja-tang on the lipid related disease in a rat model of menopausal hyperlipidemia and lipid accumulation in methyl-β-cyclodextrin-induced HepG2 cells. In in vivo study using menopausal hyperlipidemia rats, Sagunja-tang reduced retroperitoneal and perirenal fat, serum lipids, atherogenic index, cardiac risk factor, media thickness, and nonalcoholic steatohepatitis score, when compared to menopausal hyperlipidemia control rats. In HepG2 cells, Sagunja-tang significantly decreased the lipid accumulation, total cholesterol levels, and low-density/very-low-density lipoprotein levels. Moreover, Sagunja-tang reversed the methyl-β-cyclodextrin-induced decrease in the protein levels of critical molecule involved in cholesterol synthesis, sterol regulatory element binding protein-2, and low-density lipoprotein receptor and inhibited protein levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase as well as activity. Phosphorylation level of AMP-activated protein kinase was stimulated by Sagunja-tang. These results suggest that Sagunja-tang has effect on inhibiting hepatic lipid accumulation through regulation of cholesterol synthesis and AMPK activity in vitro. These observations support the idea that Sagunja-tang is bioavailable both in vivo and in vitro and could be developed as a preventive and therapeutic agent of hyperlipidemia in postmenopausal females.

Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/β-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells

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Perumal, NaveenKumar [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Perumal, MadanKumar [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 (United States); Kannan, Anbarasu [Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler, Texas (United States); Subramani, Kumar [Centre for Biotechnology, Anna University, Chennai 600025, Tamil Nadu (India); Halagowder, Devaraj [Department of Zoology, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Sivasithamparam, NiranjaliDevaraj, E-mail: profniranjali@gmail.com [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India)

2017-06-15

Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/β-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and β-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer. - Highlights: • Morin induced cytotoxicity in cultured HepG2 cells. • Morin activated hippo pathway via Mst1 activation in transfected HepG2 cells. • Morin suppressed Wnt/β-catenin signaling and induced G0/G1 cell cycle arrest. • Morin inhibited NF-κB signaling through Mst1 activation in transfected HepG2 cells. • Morin potentiates apoptosis through Mst1-JNK-caspase mediated mechanism in HepG2 cells.

Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/β-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells

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Perumal, NaveenKumar; Perumal, MadanKumar; Kannan, Anbarasu; Subramani, Kumar; Halagowder, Devaraj; Sivasithamparam, NiranjaliDevaraj

2017-01-01

Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/β-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and β-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer. - Highlights: • Morin induced cytotoxicity in cultured HepG2 cells. • Morin activated hippo pathway via Mst1 activation in transfected HepG2 cells. • Morin suppressed Wnt/β-catenin signaling and induced G0/G1 cell cycle arrest. • Morin inhibited NF-κB signaling through Mst1 activation in transfected HepG2 cells. • Morin potentiates apoptosis through Mst1-JNK-caspase mediated mechanism in HepG2 cells.

L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

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Tanaka, Yuma; Shimada, Masaya; Nagaoka, Satoshi

2014-02-14

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling. Copyright © 2014 Elsevier Inc. All rights reserved.

Quercitrin from Toona sinensis (Juss. M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation

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Van-Long Truong

2016-07-01

Full Text Available Quercitrin is found in many kinds of vegetables and fruits, and possesses various bioactive properties. The aim of the present study was to elucidate hepatoprotective mechanisms of quercitrin isolated from Toona sinensis (Juss. M.Roem. (syn. Cedrela sinensis Juss., using acetaminophen (APAP-treated HepG2 cell and animal models. In an in vitro study, quercitrin suppressed the production of reactive oxygen species and enhanced expression of nuclear factor E2-related factor 2 (Nrf2, activity of antioxidant response element (ARE-reporter gene, and protein levels of NADPH: quinone oxidoreductase 1 (NQO1, catalase (CAT, glutathione peroxidase (GPx, and superoxide dismutase 2 (SOD-2 in APAP-treated HepG2 cells. In an in vivo study, Balb/c mice were orally administered with 10 or 50 mg/kg of quercitrin for 7 days and followed by the injection with single dose of 300 mg/kg APAP. Quercitrin decreased APAP-caused elevation of alanine aminotransferase and aspartate aminotransferase levels, liver necrosis, the expression of pro-inflammatory factors including inducible nitric oxide synthase, cyclooxygenase 2 and inerleukin-1β, and phosphorylation of kinases including c-Jun N-terminal kinase and p38. Quercitrin restored protein levels of Nrf2, NQO1 and activities and expressions of CAT, GPx, SOD-2. The results suggested that quercitrin attenuates APAP-induced liver damage by the activation of defensive genes and the inhibition of pro-inflammatory genes via the suppressions of JNK and p38 signaling.

Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cynanchum atratum.

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Zhang, Jian; Ma, Lin; Wu, Zheng-Feng; Yu, Shu-Le; Wang, Lei; Ye, Wen-Cai; Zhang, Qing-Wen; Yin, Zhi-Qi

2017-06-01

Two new (1-2) and two known C 21 steroids (3-4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MTT assay showed that compounds 1-4 displayed obvious cytotoxic activities against HepG2 cells with IC 50 values ranging from 10.19μM to 76.12μM. Compounds 1-3 also exhibited cytotoxic effects in A549 cells with IC 50 values of 30.87-95.39μM. Compound 3 showed the antiproliferative activity via G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis. Western blotting analysis revealed that compound 3 could induce HepG2 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 expression, upregulating Bax protein expression, and activating caspase-9 and caspase-3. Copyright © 2017 Elsevier Inc. All rights reserved.

Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells.

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Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2016-10-19

Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin - another DPP-4 inhibitor - induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

Galangin suppresses HepG2 cell proliferation by activating the TGF-β receptor/Smad pathway

International Nuclear Information System (INIS)

Wang, Yajun; Wu, Jun; Lin, Biyun; Li, Xv; Zhang, Haitao; Ding, Hang; Chen, Xiaoyi; Lan, Liubo; Luo, Hui

2014-01-01

Galangin can suppress hepatocellular carcinoma (HCC) cell proliferation. In this study, we demonstrated that galangin induced autophagy by activating the transforming growth factor (TGF)-β receptor/Smad pathway and increased TGF-β receptor I (RI), TGF-βRII, Smad1, Smad2, Smad3 and Smad4 levels but decreased Smad6 and Smad7 levels. Autophagy induced by galangin appears to depend on the TGF-β receptor/Smad signalling pathway because the down-regulation of Smad4 by siRNA or inhibition of TGF-β receptor activation by LY2109761 blocked galangin-induced autophagy. The down-regulation of Beclin1, autophagy-related gene (ATG) 16L, ATG12 and ATG3 restored HepG2 cell proliferation and prevented galangin-induced apoptosis. Our findings indicate a novel mechanism for galangin-induced autophagy via activation of the TGF-β receptor/Smad pathway. The induction of autophagy thus reflects the anti-proliferation effect of galangin on HCC cells

Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

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Adriana M Zimnicka

Full Text Available Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX, a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2 also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC. In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

Linoleic acid-menthyl ester reduces the secretion of apolipoprotein B100 in HepG2 cells.

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Inoue, Nao; Yamano, Naomi; Sakata, Kotaro; Arao, Keisuke; Kobayashi, Takashi; Nagao, Toshihiro; Shimada, Yuji; Nagao, Koji; Yanagita, Teruyoshi

2009-01-01

The effect of linoleic acid-menthyl ester (LAME) on lipid metabolism were assessed in HepG2 cells. It is well known that high level of apolipoprotein (apo) B100 in the serum is risk for atherosclerosis. Although linoleic acid (LA) treatment and LA plus L-mentol treatment increased apo B100 secretion, LAME treatment significantly decreased apo B100 secretion in HepG2 cells compared with control medium. The hypolipidemic effect of LAME was attributable to the suppression of triglyceride synthesis in HepG2 cells. It is also known that the risk of coronary heart disease is negatively related to the concentration of serum apo A-1. In the present study, LAME treatment increased apo A-1 secretion as compared with LA treatment in HepG2 cells. These results suggest that mentyl-esterification of fatty acids may be beneficial in anti-atherogenic dietary therapy.

Targeting and molecular imaging of HepG2 cells using surface-functionalized gold nanoparticles

International Nuclear Information System (INIS)

Rathinaraj, Pierson; Lee, Kyubae; Choi, Yuri; Park, Soo-Young; Kwon, Oh Hyeong; Kang, Inn-Kyu

2015-01-01

Mercaptosuccinic acid (M)-conjugated gold nanoparticles (GM) were prepared and characterized by transmission electron microscope and dynamic light scattering. M was used to improve the monodispersity and non-specific intracellular uptake of nanoparticles. Lactobionic acid (L) was subsequently conjugated to the GM to target preferentially HepG2 cells (liver cancer cells) that express asialoglycoprotein receptors (ASGPR) on their membrane surfaces and facilitate the transit of nanoparticles across the cell membrane. The mean size of lactobionic acid-conjugated gold nanoparticle (GL) was approximately 10 ± 0.2 nm. Finally, the Atto 680 dye (A6) was coupled to the nanoparticles to visualize their internalization into HepG2 cells. The interaction of surface-modified gold nanoparticles with HepG2 cells was studied after culturing cells in media containing the GM or L-conjugated GM (GL)

Targeting and molecular imaging of HepG2 cells using surface-functionalized gold nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Rathinaraj, Pierson [Auckland University of Technology, Institute of Biomedical Technologies (New Zealand); Lee, Kyubae; Choi, Yuri; Park, Soo-Young [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of); Kwon, Oh Hyeong [Kumoh National Institute of Technology, Department of Polymer Science and Engineering (Korea, Republic of); Kang, Inn-Kyu, E-mail: ikkang@knu.ac.kr [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of)

2015-07-15

Mercaptosuccinic acid (M)-conjugated gold nanoparticles (GM) were prepared and characterized by transmission electron microscope and dynamic light scattering. M was used to improve the monodispersity and non-specific intracellular uptake of nanoparticles. Lactobionic acid (L) was subsequently conjugated to the GM to target preferentially HepG2 cells (liver cancer cells) that express asialoglycoprotein receptors (ASGPR) on their membrane surfaces and facilitate the transit of nanoparticles across the cell membrane. The mean size of lactobionic acid-conjugated gold nanoparticle (GL) was approximately 10 ± 0.2 nm. Finally, the Atto 680 dye (A6) was coupled to the nanoparticles to visualize their internalization into HepG2 cells. The interaction of surface-modified gold nanoparticles with HepG2 cells was studied after culturing cells in media containing the GM or L-conjugated GM (GL)

SM22α-induced activation of p16INK4a/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of γ-radiation and doxorubicin in HepG2 cells

International Nuclear Information System (INIS)

Kim, Tae Rim; Lee, Hee Min; Lee, So Yong; Kim, Eun Jin; Kim, Kug Chan; Paik, Sang Gi; Cho, Eun Wie; Kim, In Gyu

2010-01-01

Research highlights: → SM22α overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of γ-radiation or doxorubicin promotes cellular senescence. → SM22α overexpression elevates p16 INK4a followed by pRB activation, but there are no effects on p53/p21 WAF1/Cip1 pathway. → SM22α-induced MT-1G activates p16 INK4a /pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22α) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22α overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22α overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of γ-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 μg/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21 WAF1/Cip1 induction or p16 INK4a /retinoblastoma protein (pRB) activation. SM22α overexpression in HepG2 cells elevated p16 INK4a followed by pRB activation, but did not activate the p53/p21 WAF1/Cip1 pathway. Moreover, MT-1G, which is induced by SM22α overexpression, was involved in the activation of the p16 INK4a /pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22α modulates cellular senescence caused by damaging agents via regulation of the p16 INK4a /pRB pathway in HepG2 cells and that these effects of SM22α are partially mediated by MT-1G.

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

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Hosseini, Farzaneh Sadat; Falahati-Pour, Soudeh Khanamani; Hajizadeh, Mohammad Reza; Khoshdel, Alireza; Mirzaei, Mohammad Reza; Ahmadirad, Hadis; Behroozi, Reza; Jafari, Nesa; Mahmoodi, Mehdi

2017-08-01

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC 50 value (inhibiting cell growth by 50%) was 149 μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.

Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

International Nuclear Information System (INIS)

Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H.

2006-01-01

The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis

Effect of Toxicants on Fatty Acid Metabolism in HepG2 Cells

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David Grünig

2018-04-01

Full Text Available Impairment of hepatic fatty acid metabolism can lead to liver steatosis and injury. Testing drugs for interference with hepatic fatty acid metabolism is therefore important. To find out whether HepG2 cells are suitable for this purpose, we investigated the effect of three established fatty acid metabolism inhibitors and of three test compounds on triglyceride accumulation, palmitate metabolism, the acylcarnitine pool and dicarboxylic acid accumulation in the cell supernatant and on ApoB-100 excretion in HepG2 cells. The three established inhibitors [etomoxir, methylenecyclopropylacetic acid (MCPA, and 4-bromocrotonic acid (4-BCA] depleted mitochondrial ATP at lower concentrations than cytotoxicity occurred, suggesting mitochondrial toxicity. They inhibited palmitate metabolism at similar or lower concentrations than ATP depletion, and 4-BCA was associated with cellular fat accumulation. They caused specific changes in the acylcarnitine pattern and etomoxir an increase of thapsic (C18 dicarboxylic acid in the cell supernatant, and did not interfere with ApoB-100 excretion (marker of VLDL export. The three test compounds (amiodarone, tamoxifen, and the cannabinoid WIN 55,212-2 depleted the cellular ATP content at lower concentrations than cytotoxicity occurred. They all caused cellular fat accumulation and inhibited palmitate metabolism at similar or higher concentrations than ATP depletion. They suppressed medium-chain acylcarnitines in the cell supernatant and amiodarone and tamoxifen impaired thapsic acid production. Tamoxifen and WIN 55,212-2 decreased cellular ApoB-100 excretion. In conclusion, the established inhibitors of fatty acid metabolism caused the expected effects in HepG2 cells. HepG cells proved to be useful for the detection of drug-associated toxicities on hepatocellular fatty acid metabolism.

20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells.

Science.gov (United States)

Ai, Hui-Han; Zhou, Zi-Long; Sun, Lu-Guo; Yang, Mei-Ting; Li, Wei; Yu, Chun-Lei; Song, Zhen-Bo; Huang, Yan-Xin; Wu, Yin; Liu, Lei; Yang, Xiao-Guang; Zhao, Yu-Qing; Bao, Yong-Li; Li, Yu-Xin

2017-11-01

The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH 3 -PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

Effects of the radiolysis products of sennoside A on HepG2 and PC-3 cell

International Nuclear Information System (INIS)

Kim, Dong Ho; Jo, Min Ho

2016-01-01

Radiolysis of sennoside A was carried out by gamma irradiation and the anti-cancer activities of the radiolysis product were evaluated. An aqueous solution of sennoside A was exposed to 0.5-3 kGy of gamma irradiation and the radiolysis products were analyzed by HPLC. A fraction of radiolysis product (RLF) of sennoside A was isolated and the RLF was presumed as a rhein-8-β-D-glucoside. The anticancer effect of the RLF was compared with the sennoside and rhein using a in vitro assay system of human prostate cancer cells (PC-3) and human hepatoma HepG2 cells. The cell viability of PC-3 and HepG2 cell was significantly decreased to 12.4±1.2% and 32.4±2.1%, respectively, by the treatment of 0.6 μM of RLF. The sennoside A (range from 0 to 25 μM) had no cytotoxic effect on PC-3 and HepG2 cells, while the rhein had the effect on HepG2 cells with a LD_5_0 at 80 μM

Effects of the radiolysis products of sennoside A on HepG2 and PC-3 cell

Energy Technology Data Exchange (ETDEWEB)

Kim, Dong Ho; Jo, Min Ho [Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2016-11-15

Radiolysis of sennoside A was carried out by gamma irradiation and the anti-cancer activities of the radiolysis product were evaluated. An aqueous solution of sennoside A was exposed to 0.5-3 kGy of gamma irradiation and the radiolysis products were analyzed by HPLC. A fraction of radiolysis product (RLF) of sennoside A was isolated and the RLF was presumed as a rhein-8-β-D-glucoside. The anticancer effect of the RLF was compared with the sennoside and rhein using a in vitro assay system of human prostate cancer cells (PC-3) and human hepatoma HepG2 cells. The cell viability of PC-3 and HepG2 cell was significantly decreased to 12.4±1.2% and 32.4±2.1%, respectively, by the treatment of 0.6 μM of RLF. The sennoside A (range from 0 to 25 μM) had no cytotoxic effect on PC-3 and HepG2 cells, while the rhein had the effect on HepG2 cells with a LD{sub 50} at 80 μM.

Airborne fine particulate matter induces an upregulation of endothelin receptors on rat bronchi

International Nuclear Information System (INIS)

Wang, Rong; Xiao, Xue; Cao, Lei; Shen, Zhen-xing; Lei, Ying; Cao, Yong-xiao

2016-01-01

Airborne fine particulate matter (PM2.5) is a risk factor for respiratory diseases. However, little is known about the effects of PM2.5 on bronchi. The present study investigated the effect of airborne PM2.5 on rat bronchi and the underlying mechanisms. Isolated rat bronchial segments were cultured for 24 h. Endothelin (ET) receptor-mediated contractile responses were recorded using a wire myograph. The mRNA and protein expression levels of ET receptors were studied using quantitative real-time PCR, Western blotting, and immunohistochemistry. The results demonstrated that ET A and ET B receptor agonists induced remarkable contractile responses on fresh and cultured bronchial segments. PM2.5 (1.0 or 3.0 μg/ml) significantly enhanced ET A and ET B receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction compared to the DMSO or fresh groups. PM2.5 increased the mRNA and protein expression levels of ET A and ET B receptors. U0126 (a MEK1/2 inhibitor) and SB203580 (a p38 inhibitor) significantly suppressed PM2.5-induced increases in ET B receptor-mediated contractile responses, mRNA and protein levels. SP600125 (a JNK inhibitor) and SB203580 significantly abrogated the PM2.5-induced enhancement of ET A receptor-mediated contraction and receptor expression. In conclusion, PM2.5 upregulates ET receptors in bronchi. ET B receptor upregulation is associated with MEK1/2 and p38 pathways, and the upregulation of ET A receptor is involved in JNK and p38 pathways. - Highlights: • Airborne PM2.5 induces bronchial hyperreactivity mediated with endothelin ET B and ET A receptors in rats. • PM2.5 increases mRNA and protein expressions of endothelin ET B and ET A receptors in bronchi. • The upregulation of ET B receptor is associated with MEK1/2 and p38 pathways. • The upregulation of ET A receptor is involved in JNK and p38 pathways. • The research provides novel understanding for PM2.5-associated respiratory diseases.

SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Kim, Tae Rim; Lee, Hee Min; Lee, So Yong; Kim, Eun Jin; Kim, Kug Chan [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Paik, Sang Gi [Department of Biology, School of Biosciences and Biotechnology, Chungnam National University, Daejeon (Korea, Republic of); Cho, Eun Wie, E-mail: ewcho@kribb.re.kr [Daejeon-KRIBB-FHCRC Cooperation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, In Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2010-09-10

Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.

Carboxymethyl chitin-glucan (CM-CG) protects human HepG2 and HeLa cells against oxidative DNA lesions and stimulates DNA repair of lesions induced by alkylating agents.

Science.gov (United States)

Slamenová, Darina; Kováciková, Ines; Horváthová, Eva; Wsólová, Ladislava; Navarová, Jana

2010-10-01

A large number of functional foods, including those that contain β-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H(2)O(2) and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H(2)O(2) but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay. Copyright © 2010 Elsevier Ltd. All rights reserved.

IGF-1 induces the epithelial-mesenchymal transition via Stat5 in hepatocellular carcinoma.

Science.gov (United States)

Zhao, Chuanzong; Wang, Qian; Wang, Ben; Sun, Qi; He, Zhaobin; Hong, Jianguo; Kuehn, Florian; Liu, Enyu; Zhang, Zongli

2017-12-19

It has been reported that the epithelial-mesenchymal transition (EMT) plays an important role in hepatocellular carcinoma (HCC). However, the relationship between the insulin-like growth factor-1 (IGF-1) and EMT of HCC was not fully elucidated. In the present work, we found that the expression of N-cadherin, Vimentin, Snail1, Snail2, and Twist1 was positively associated with IGF-1R expression, while E-cadherin expression was negatively associated with IGF-1 expression in human HCC samples. Furthermore, we observed that IGF-1 up-regulated the expression of N-cadherin, Vimentin, Snail1, Snail2 and Twist1, and down-regulated the expression of E-cadherin. In addition, Stat5 was induced in IGF-1-treated HepG2 and Hep3B cells, and Stat5 inhibition or siRNA significantly affected IGF-1-induced EMT in HepG2 and Hep3B cells. In conclusion, IGF-1 induces EMT of HCC via Stat5 signaling pathway. Thus, IGF-1/Stat5 can be recommended as a potential and novel therapeutic strategy for HCC patients.

Calcium channel alpha-2-delta-1 protein upregulation in dorsal spinal cord mediates spinal cord injury-induced neuropathic pain states.

Science.gov (United States)

Boroujerdi, Amin; Zeng, Jun; Sharp, Kelli; Kim, Donghyun; Steward, Oswald; Luo, Z David

2011-03-01

Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI-induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non-noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage-gated calcium channel alpha-2-delta-1 subunit (Ca(v)α2δ-1) proteins is effective in the management of SCI-induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Ca(v)α2δ-1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI-induced dysregulation of spinal Ca(v)α2δ-1 plays a contributory role in below-level allodynia development in a rat spinal T9 contusion injury model. We found that Ca(v)α2δ-1 expression levels were significantly increased in L4-6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI-induced Ca(v)α2δ-1 protein upregulation by intrathecal Ca(v)α2δ-1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI-induced Ca(v)α2δ-1 upregulation in spinal dorsal horn is a key component in mediating below-level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients. Spinal cord contusion injury caused increased calcium channel Ca(v)α2δ-1 subunit expression in dorsal spinal cord that contributes to neuropathic pain states. Copyright © 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Isolation of major phenolics from Launaea spinosa and their protective effect on HepG2 cells damaged with t-BHP.

Science.gov (United States)

Abdallah, Hossam; Farag, Mohamed; Osman, Samir; Kim, Da hye; Kang, Kyungsu; Pan, Cheol-Ho; Abdel-Sattar, Essam

2016-01-01

Some Launaea species (Asteraceae) are used traditionally to treat liver oxidative stress. The present study investigates the protective effects of isolated compounds from Launaea spinosa Sch. Bip. (Asteraceae) against oxidative stress on t-BHP-induced HepG2 cells. Major phenolic content from flowering aerial parts of L. spinosa was isolated and identified. The protective effects of isolated compounds (10 and 20 μM) against oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells were investigated through the measurement of aspartate aminotransferase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD) levels. A new phenolic compound identified as 2,3-diferulyl R,R-(+) methyl tartrate (6), in addition to five known metabolites, esculetin (1), esculetin-7-O-d-glucoside (cichoriin) (2), fertaric acid (3), acacetin-7-O-d-glucoside (4), and acacetin-7-O-d-glucuronic acid (5), were isolated. Oxidant-induced damage by 200 μM t-BHP in HepG2 cells was inhibited by compounds 1, 4, and 5 (10 and 20 μM), or quercetin (10 μM; positive control). The protective effects of compounds 1, 4, and 5 were associated with decreasing in AST, ALT, and SOD levels. Compound 4 (20 μM) decreased the AST level from 128.5 ± 13.9 to 7.9 ±1.8 U/mL. Meanwhile, compound 1 (20 μM) decreased ALT activity from 20.3 ± 7.0 to 7.6 ± 2.4 U/mL, while compound 5 decreased SOD levels from 41.6 ± 9.0 to 28.3 ± 3.4 mU/mg. The major phenolic compounds isolated from L. spinosa displayed a significant cytoprotective effect against oxidative stress, leading to maintenance of the normal redox status of the cell.

Study of apoptotic mechanisms induced by all-trans retinoic acid and its 13-cis isomer on cellular lines of human hepato carcinoma Hep3B and HepG2; Estudio de los mecanismos apoptoticos inducidos por el acido retinoico todo-trans y su isomero 13-cis en las lineas celulares de hepatocarcinoma humano Hep3B y HepG2

Energy Technology Data Exchange (ETDEWEB)

Arce Vargas, Frederick [Costa Rica

2006-07-01

Two cellular lines of liver cancer (Hep3B and HepG2) were incubated during different periods of time with some concentrations of two retinoic acid isomers (ATRA and 13-cis AR) and with 5-fu chemotherapeutic agents, cisplatin and paclitaxel. It was determined if these substances leaded cytotoxicity, apoptosis and if they modified the expression of different genes related to cellular death by apoptosis, in order to explain the hepatocellular carcinoma resistance to these drugs. HepG2 cells showed more resistance than Hep3B cells to 72 hours of treatment, as much ATRA as the 13-cis AR were toxic and produced apoptosis in two cellular lines. This type of cellular death seems to be mediated by a decrease in Bcl-xL concentration in Hep3B cells treated with both retinoids an increase in bax concentration in HepG2 cells treated with 13-cis AR. It were observed 3 and 8 proteolysis of procaspase in Hep3B cells, suggesting extrinsic via activation of the apoptosis, while cellular death in HepG2 cells seems to be independent of caspases. Cisplatin and paclitaxel leaded cytotoxicity to 48 hours of treatment, with significant differences between two cellular lines only in case of paclitaxel. Hep3B cells treated with cisplatin and HepG2 cells treated with paclytaxel suffered apoptosis. 5-FU produced toxicity only when it was used to high concentrations and the mechanism of cellular death induced by this agent seems to be primarily necrosis in Hep3B cells and apoptosis in HepG2. There was decrease in the Bcl-xL concentration in two cellular lines when it was treated with cisplatin and in HepG2 cells treated with 5-FU. Bax concentration there no was modified with no treatment. Activation of the 3 caspases seems to happen only in HepG2 cells with 5-FU and paclytaxel. These two agents, also, decreased the survivin concentration of HepG2 cells. Treatments of the three drugs produced an increase in the expression of this gen in Hep3B cells, which might explain partially the resistance

The Interactions between ZnO Nanoparticles (NPs and α-Linolenic Acid (LNA Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells

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Yiwei Zhou

2017-04-01

Full Text Available Background: Nanoparticles (NPs entering the biological environment could interact with biomolecules, but little is known about the interaction between unsaturated fatty acids (UFA and NPs. Methods: This study used α-linolenic acid (LNA complexed to bovine serum albumin (BSA for UFA and HepG2 cells for hepatocytes. The interactions between BSA or LNA and ZnO NPs were studied. Results: The presence of BSA or LNA affected the hydrodynamic size, zeta potential, UV-Vis, fluorescence, and synchronous fluorescence spectra of ZnO NPs, which indicated an interaction between BSA or LNA and NPs. Exposure to ZnO NPs with the presence of BSA significantly induced the damage to mitochondria and lysosomes in HepG2 cells, associated with an increase of intracellular Zn ions, but not intracellular superoxide. Paradoxically, the release of inflammatory cytokine interleukin-6 (IL-6 was decreased, which indicated the anti-inflammatory effects of ZnO NPs when BSA was present. The presence of LNA did not significantly affect all of these endpoints in HepG2 cells exposed to ZnO NPs and BSA. Conclusions: the results from the present study indicated that BSA-complexed LNA might modestly interact with ZnO NPs, but did not significantly affect ZnO NPs and BSA-induced biological effects in HepG2 cells.

Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

Science.gov (United States)

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effects of the peroxisome proliferator clofibric acid on superoxide dismutase expression in the human HepG2 hepatoma cell line.

Science.gov (United States)

Bécuwe, P; Bianchi, A; Keller, J M; Dauça, M

1999-09-15

We examined the effects of clofibric acid, a peroxisome proliferator, on the production of superoxide radicals, on the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and on the expression of superoxide dismutases (SODs) in the human HepG2 hepatoma cell line. To this end, HepG2 cells were treated for 1 or 5 days with 0.25, 0.50, or 0.75 mM clofibric acid. The production of superoxide radicals was only enhanced in HepG2 cells exposed for 5 days to the different clofibric acid concentrations. However, this overproduction of superoxide radicals was not accompanied by increased rates of lipid peroxidation, as the MDA and 4-HNE levels did not change significantly. Manganese (Mn) SOD activity was increased when HepG2 cells were treated for 1 day with 0.50 or 0.75 mM clofibric acid. For this duration of treatment, no change was observed in total SOD and copper/zinc (Cu/Zn) SOD activities. For a 5-day treatment, total SOD and MnSOD activities as well as the enzyme apoprotein and MnSOD mRNA levels increased whatever the clofibric acid concentration used. This transcriptional induction of the MnSOD gene was correlated with an activation of the activator protein-1 transcription factor for 1 and 5 days of treatment, but was independent of nuclear factor-kappa B and of peroxisome proliferator-activated receptor. On the other hand, the PP exerted very little effect if any on Cu,ZnSOD expression. In contrast to rodent data, PP treatment of human hepatoma cells induces MnSOD expression.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

Science.gov (United States)

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

Ku70 acetylation and modulation of c-Myc/ATF4/CHOP signaling axis by SIRT1 inhibition lead to sensitization of HepG2 cells to TRAIL through induction of DR5 and down-regulation of c-FLIP

DEFF Research Database (Denmark)

Kim, Mi-Ju; Hong, Kyung-Soo; Kim, Hak-Bong

2013-01-01

In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, a...

Protection of betulin against cadmium-induced apoptosis in hepatoma cells

International Nuclear Information System (INIS)

Oh, Seon-Hee; Choi, Jeong-Eun; Lim, Sung-Chul

2006-01-01

The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G /G 1 arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation. Taken together, it was found that Cd can induce apoptosis via the Fas-dependent and -independent apoptosis pathways. However, the observed protective effects of BT were clearly more sensitive to Fas-expressing HepG2 cells than to Fas-deficient Hep3B cells

Effect of Genistein and 17-β Estradiol on the Viability and Apoptosis of Human Hepatocellular Carcinoma HepG2 cell line

Directory of Open Access Journals (Sweden)

Masumeh Sanaei

2017-01-01

Full Text Available Background: One of the most lethal cancers is hepatocellular carcinoma (HCC. Genistein (GE is a choice compound for treatment of certain types of cancer. Phytoestrogens are plant derivatives that bear a structural similarity to 17-β estradiol (E2 and act in a similar manner. They are a group of lipophillic plant compounds with tumorigenic and antitumorigenic effects. E2 has stimulatory and inhibitory effects on cancer cell lines. This study was designed to investigate the antiproliferative and apoptotic effects of GE and E2 on the HCC HepG2 cell line. Materials and Methods: HepG2 cells were cultured and treated with various concentrations of GE and E2 and then 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromideand flow cytometry assay were performed to determine cell viability and apoptosis. Results: GE and E2 induced apoptosis and inhibited cell growth significantly. Reduction of cell viability by 50% required 20 μM E2 for E2-treatment groups and 20 μMGE for GE-treatment groups. The percentage of the GE-treated apoptotic cells was reduced by about 35%, 42%, and 47% (P < 0.001 and that of E2-treated groups 34%, 39%, and 42% (P < 0.001 after 24, 48, and 72 h, respectively. Conclusions: Our experimental work clearly demonstrated that GE and E2 exhibited significant antiproliferative and apoptotic effects on human HCC HepG2 cells.

Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes.

Science.gov (United States)

Yang, Jin-ting; Tang, Li-hui; Liu, Yun-qing; Wang, Yin; Wang, Lie-ju; Zhang, Feng-jiang; Yan, Min

2015-05-01

The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 μg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 μg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 μg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 μg/ml) (P0.05). In conclusion, pretreatment with cisplatin (50 μg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

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Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2014-01-01

The Pharmacological potential, such as antioxidant, anti-inflammatory, and antibacterial activities of Portulaca oleracea (PO) and Petroselinum sativum (PS) extracts are well known. However, the preventive properties against hepatocellular carcinoma cells have not been explored so far. Therefore, the present investigation was designed to study the anticancer activity of seed extracts of PO and PS on the human hepatocellular carcinoma cells (HepG2). The HepG2 cells were exposed with 5-500 μg/ml of PO and PS for 24 h. After the exposure, cell viability by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay, and cellular morphology by phase contrast inverted microscope were studied. The results showed that PO and PS extracts significantly reduced the cell viability of HepG2 in a concentration dependent manner. The cell viability was recorded to be 67%, 31%, 21%, and 17% at 50, 100, 250, and 500 μg/ml of PO, respectively by MTT assay and 91%, 62%, 27%, and 18% at 50, 100, 250, and 500 μg/ml of PO, respectively by NRU assay. PS exposed HepG2 cells with 100 μg/ml and higher concentrations were also found to be cytotoxic. The decrease in the cell viability at 100, 250, and 500 μg/ml of PS was recorded as 70%, 33%, and 15% by MTT assay and 63%, 29%, and 17%, respectively by NRU assay. Results also showed that PO and PS exposed cells reduced the normal morphology and adhesion capacity of HepG2 cells. HepG2 cells exposed with 50 μg/ml and higher concentrations of PO and PS lost their typical morphology, become smaller in size, and appeared in rounded bodies. Our results demonstrated preliminary screening of anticancer activity of Portulaca oleracea and Petroselinum sativum extracts against HepG2 cells, which can be further used for the development of a potential therapeutic anticancer agent.

Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

DEFF Research Database (Denmark)

Stervbo, Ulrik; Vang, Ole; Bonnesen, Christine

2006-01-01

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell...... proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 μM in both HL-60 and HepG2 cells. When...... the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 μM for HL-60 cells and 60 μM for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore...

(3'R)-hydroxytabernaelegantine C: A bisindole alkaloid with potent apoptosis inducing activity in colon (HCT116, SW620) and liver (HepG2) cancer cells.

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Paterna, Angela; Gomes, Sofia E; Borralho, Pedro M; Mulhovo, Silva; Rodrigues, Cecília M P; Ferreira, Maria-José U

2016-12-24

Tabernaemontana elegans Stapf. (Apocynaceae) is a medicinal plant traditionally used in African countries to treat cancer. To discover new apoptosis inducing lead compounds from T. elegans and provide scientific validation of the ethnopharmacological use of this plant. Through fractionation, (3'R)-hydroxytaberanelegantine C (1), a vobasinyl-iboga bisindole alkaloid, was isolated from a cytotoxic alkaloid fraction of the methanol extract of T. elegans roots. Its structure was identified by spectroscopic methods, mainly 1D and 2D NMR experiments. Compound 1 was evaluated for its ability to induce apoptosis in HCT116 and SW620 colon and HepG2 liver carcinoma cells. The cell viability of compound 1 was evaluated by the MTS and lactate dehydrogenase (LDH) assays. Induction of apoptosis was analyzed through Guava ViaCount assay, by flow cytometry, caspase-3/7 activity assays and evaluation of nuclear morphology by Hoechst staining. To determine the molecular pathways elicited by 1 exposure, immunoblot analysis was also performed. (3'R)-hydroxytaberanelegantine C (1) displayed strong apoptosis induction activity as compared to 5-fluorouracil (5-FU), the most used anticancer agent in colorectal cancer treatment. In the MTS assay, compound 1 exhibited IC 50 values similar or lower than 5-FU in the three cell lines tested. The IC 50 value of 1 was also calculated in CCD18co normal human colon fibroblasts. The lactate dehydrogenase assay showed increased LDH release by compound 1, and the Guava ViaCount assay revealed that 1 significantly increased the incidence of apoptosis to a further extent than 5-FU. Moreover, the induction of apoptosis was corroborated by evaluation of nuclear morphology by Hoechst staining and caspase-3/7 activity assays of 1 treated cells. As expected, in immunoblot analysis, compound 1 treatment led to poly(ADP-ribose) polymerase cleavage. This was accompanied by decreased anti-apoptotic proteins Bcl-2 and XIAP steady state levels in all three cancer

Upregulated Expression of a Unique Gene by Hepatitis B x Antigen Promotes Hepatocellular Growth and Tumorigenesis

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Zhaorui Lian

2003-05-01

Full Text Available Hepatitis B x antigen (HBxAg is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HBxAg that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HBxAg-positive and HBxAg-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11, was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HBxAg-positive compared to HBxAg-negative HepG2 cells. Costaining was observed in infected liver (P<.01. URG11 stimulated cell growth in culture (P<.01, anchorage-independent growth in soft agar (P<.001, and accelerated tumor formation (P<.01, and yielded larger tumors (P<.02 in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HBxAg that may promote the development of hepatocellular carcinoma.

Exogenous regucalcin suppresses the growth of human liver cancer HepG2 cells in vitro.

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Yamaguchi, Masayoshi; Murata, Tomiyasu

2018-04-05

Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

Protective Effects of Maillard Reaction Products of Whey Protein Concentrate against Oxidative Stress through an Nrf2-Dependent Pathway in HepG2 Cells.

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Pyo, Min Cheol; Yang, Sung-Yong; Chun, Su-Hyun; Oh, Nam Su; Lee, Kwang-Won

2016-09-01

Whey protein concentrate (WPC), which contains α-lactalbumin and β-lactoglobulin, is utilized widely in the food industry. The Maillard reaction is a complex reaction that produces Maillard reaction products (MRPs), which are associated with the formation of antioxidant compounds. In this study, the hepatoprotection activity of MRPs of WPC against oxidative stress through the nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant pathway in HepG2 cells was examined. Glucose-whey protein concentrate conjugate (Glc-WPC) was obtained from Maillard reaction between WPC and glucose. The fluorescence intensity of Glc-WPC increased after 7 d compared to native WPC, and resulted in loss of 48% of the free amino groups of WPC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of Glc-WPC showed the presence of a high-molecular-weight portion. Treatment of HepG2 cells with Glc-WPC increased cell viability in the presence of oxidative stress, inhibited the generation of intracellular reactive oxygen species by tert-butyl hydroperoxide (t-BHP), and increased the glutathione level. Nrf2 translocation and Nrf2, reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-quinone oxidoreductase 1 (NOQ1), heme oxygenase-1 (HO-1), glutamate-L-cysteine ligase (GCL)M and GCLC mRNA levels were increased by Glc-WPC. Also, Glc-WPC increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). The results of this study demonstrate that Glc-WPC activates the Nrf2-dependent pathway through the phosphorylation of ERK1/2 and JNK in HepG2 cells, and induces production of antioxidant enzymes and phase II enzymes.

Upregulation of LYAR induces neuroblastoma cell proliferation and survival.

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Sun, Yuting; Atmadibrata, Bernard; Yu, Denise; Wong, Matthew; Liu, Bing; Ho, Nicholas; Ling, Dora; Tee, Andrew E; Wang, Jenny; Mungrue, Imran N; Liu, Pei Y; Liu, Tao

2017-09-01

The N-Myc oncoprotein induces neuroblastoma by regulating gene transcription and consequently causing cell proliferation. Paradoxically, N-Myc is well known to induce apoptosis by upregulating pro-apoptosis genes, and it is not clear how N-Myc overexpressing neuroblastoma cells escape N-Myc-mediated apoptosis. The nuclear zinc finger protein LYAR has recently been shown to modulate gene expression by forming a protein complex with the protein arginine methyltransferase PRMT5. Here we showed that N-Myc upregulated LYAR gene expression by binding to its gene promoter. Genome-wide differential gene expression studies revealed that knocking down LYAR considerably upregulated the expression of oxidative stress genes including CHAC1, which depletes intracellular glutathione and induces oxidative stress. Although knocking down LYAR expression with siRNAs induced oxidative stress, neuroblastoma cell growth inhibition and apoptosis, co-treatment with the glutathione supplement N-acetyl-l-cysteine or co-transfection with CHAC1 siRNAs blocked the effect of LYAR siRNAs. Importantly, high levels of LYAR gene expression in human neuroblastoma tissues predicted poor event-free and overall survival in neuroblastoma patients, independent of the best current markers for poor prognosis. Taken together, our data suggest that LYAR induces proliferation and promotes survival of neuroblastoma cells by repressing the expression of oxidative stress genes such as CHAC1 and suppressing oxidative stress, and identify LYAR as a novel co-factor in N-Myc oncogenesis.

Protective effect of polysaccharide from maca (Lepidium meyenii) on Hep-G2 cells and alcoholic liver oxidative injury in mice.

Science.gov (United States)

Zhang, Lijun; Zhao, Qingsheng; Wang, Liwei; Zhao, Mingxia; Zhao, Bing

2017-06-01

To study the characterization and hepatoprotective activity of polysaccharide from maca (Lepidium meyenii), the main polysaccharide from maca (MP-1) was obtained by DEAE-52 cellulose column. The average molecular weight of MP-1 was 1067.3kDa and the polysaccharide purity was 91.63%. In order to assess the antioxidant activities of MP-1, four kinds of methods were used, including scavenging hydroxyl radical, DPPH, superoxide anion radical, and FRAP, and the results indicated high antioxidant activities. Furthermore, hepatoprotective activity of MP-1 was studied both in vitro and vivo. In vitro, the alcohol induced Hep-G2 cells model was established to evaluate the protective effect of MP-1, which demonstrated MP-1 can alleviate alcohol damage in Hep-G2 cells. In vivo, the Institute　of　Cancer　Researcch (ICR) mice were used to evaluate hepatoprotecive effects of MP-1 on alcoholic liver disease (ALD). Supplement with MP-1 supressed the triglyceride level both in serum and in hepatic tissue. In addition, MP-1 ameliorated serous transaminases increase induced by alcohol, including aspartate transaminase, alanine aminotransferase, and γ-glutamyl transpeptidase. Moreover, MP-1 also dramatically increased the superoxide dismutase, glutathione peroxidase, and glutathione s-transferase levels in alcoholic mice. Meantime, histopathologic results MP-1 lighten inflammation induced by alcohol. These results indicate that MP-1 possesses hepatoprotective activity against hepatic injury induced by alcohol. Copyright © 2017 Elsevier B.V. All rights reserved.

ANTIPROLIFERATIVE AND APOPTOTIC EFFECTS OF THE ESSENTIAL OIL OF ORIGANUM ONITES AND CARVACROL ON HEP-G2 CELLS

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Özlem TOMSUK

2011-08-01

Full Text Available The essential oil Origanum onites L. and its phenolic constituent carvacrol were examined for their cytotoxic and apoptotic effects in a human hepatocellular carcinoma cells Hep-G2. WST-1 and neutral red uptake assays were performed to determine the inhibitory effects of the oil and carvacrol on the growth of the cells. Possible induction of apoptosis by Origanum oil and carvacrol was further investigated by acridine orange/ethidium bromide (AO/EB staining. Results showed that the Ori- ganum oil and carvacrol was significantly cytotoxic and induced apoptosis in Hep-G2 cells. IC₅₀ value of essential oil and carvacrol was found about 0,009% (v/v and 500 μM, respectively. After incuba- tion of the cells with Origanum oil and carvacrol, characteristics of apoptotic morphology such as chromatin condensation, shrinkage of the cells and cytoplasmic blebbing was observed. In conclusion, both essential oil and its major constituent carvacrol significantly exhibited cytotoxic and apoptotic activities in hepatocellular carcinoma cells, indicating its potential for use as an anticancer agent.

Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

Science.gov (United States)

Yokobori, Kosuke; Kobayashi, Kaoru; Azuma, Ikuko; Akita, Hidetaka; Chiba, Kan

2017-10-01

Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Copper excess in liver HepG2 cells interferes with apoptosis and lipid metabolic signaling at the protein level.

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Liu, Yu; Yang, Huarong; Song, Zhi; Gu, Shaojuan

2014-12-01

Copper is an essential trace element that serves as an important catalytic cofactor for cuproenzymes, carrying out major biological functions in growth and development. Although Wilson's disease (WD) is unquestionably caused by mutations in the ATP7B gene and subsequent copper overload, the precise role of copper in inducing pathological changes remains poorly understood. Our study aimed to explore, in HepG2 cells exposed to copper, the cell viability and apoptotic cells was tested by MTT and Hoechst 33342 stainning respectively, and the signaling pathways involved in oxidative stress response, apoptosis and lipid metabolism were determined by real time RT-PCR and Western blot analysis. The results demonstrate dose- and time-dependent cell viability and apoptosis in HepG2 cells following treatment with 10 μM, 200 μM and 500 μM of copper sulfate for 8 and 24 h. Copper overload significantly induced the expression of HSPA1A (heat shock 70 kDa protein 1A), an oxidative stress-responsive signal gene, and BAG3 (BCL2 associated athanogene3), an anti-apoptotic gene, while expression of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), a lipid biosynthesis and lipid metabolism gene, was inhibited. These findings provide new insights into possible mechanisms accounting for the development of liver apoptosis and steatosis in the early stages of Wilson's disease.

Osmopriming-induced salt tolerance during seed germination of alfalfa most likely mediates through H2O2 signaling and upregulation of heme oxygenase.

Science.gov (United States)

Amooaghaie, Rayhaneh; Tabatabaie, Fatemeh

2017-07-01

The present study showed that osmopriming or pretreatment with low H 2 O 2 doses (2 mM) for 6 h alleviated salt-reduced seed germination. The NADPH oxidase activity was the main source, and superoxide dismutase (SOD) activity might be a secondary source of H 2 O 2 generation during osmopriming or H 2 O 2 pretreatment. Hematin pretreatment similar to osmopriming improved salt-reduced seed germination that was coincident with the enhancement of heme oxygenase (HO) activity. The semi-quantitative RT-PCR confirmed that osmopriming or H 2 O 2 pretreatment was able to upregulate heme oxygenase HO-1 transcription, while the application of N,N-dimethyl thiourea (DMTU as trap of endogenous H 2 O 2 ) and diphenyleneiodonium (DPI as inhibitor of NADPHox) not only blocked the upregulation of HO but also reversed the osmopriming-induced salt attenuation. The addition of CO-saturated aqueous rescued the inhibitory effect of DMTU and DPI on seed germination and α-amylase activity during osmopriming or H 2 O 2 pretreatment, but H 2 O 2 could not reverse the inhibitory effect of ZnPPIX (as HO inhibitor) or Hb (as CO scavenger) that indicates that the CO acts downstream of H 2 O 2 in priming-driven salt acclimation. The antioxidant enzymes and proline synthesis were upregulated in roots of seedlings grown from primed seeds, and these responses were reversed by adding DMTU, ZnPPIX, and Hb during osmopriming. These findings for the first time suggest that H 2 O 2 signaling and upregulation of heme oxygenase play a crucial role in priming-driven salt tolerance.

The role of the vascular endothelial growth factor/vascular endothelial growth factor receptors axis mediated angiogenesis in curcumin-loaded nanostructured lipid carriers induced human HepG2 cells apoptosis

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Fengling Wang

2015-01-01

Full Text Available Background: Curcumin (diferuloylmethane, the active constituent of turmeric extract has potent anti-cancer properties have been demonstrated in hepatocellular carcinoma (HCC. However, its underlying molecular mechanism of therapeutic effects remains unclear. Vascular endothelial growth factor (VEGF and its receptors (VEGFRs have crucial roles in tumor angiogenesis. Purpose: The goal of this study was to investigate the role of the VEGF/VEGFRs mediated angiogenesis during the proliferation and apoptosis of human HepG2 hepatoma cell line and the effect of curcumin-loaded nanostructured lipid carriers (Cur-NLC. Materials and Methods: The proliferation of HepG2 cells was determined by methyl thiazolyl tetrazolium after exposure to Cur-NLC and native curcumin. Apoptosis was quantified by flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodide staining. Cellular internalization of Cur-NLC was observed by fluorescent microscope. The level of VEGF was detected by enzyme-linked immunosorbent assay kits. The expression of VEGFRs was quantified by Western blotting. Results: Cur-NLC was more effective in inhibiting the proliferation and enhancing the apoptosis of HepG2 cells than native curcumin. Fluorescent microscope analysis showed that HepG2 cells internalized Cur-NLC more effectively than native curcumin. Furthermore, Cur-NLC down-regulated the level of VEGF and the expression of VEGFR-2, but had a slight effect on VEGFR-1. Conclusion: These results clearly demonstrated that Cur-NLC was more effective in anti-cancer activity than the free form of curcumin. These studies demonstrate for the 1 st time that Cur-NLC exerts an antitumor effect on HepG2 cells by modulating VEGF/VEGFRs signaling pathway.

Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

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Sakineh Kazemi Noureini

2015-02-01

Full Text Available Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT gene (p < 0.01 and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002. However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02 and p21 (p < 0.01 genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations.

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

International Nuclear Information System (INIS)

Ellsworth, J.L.; Brown, C.; Cooper, A.D.

1988-01-01

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column

Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

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Yoon TH

2012-03-01

Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

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Zhang, Guojun [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Xu, Mingkai, E-mail: mkxu@iae.ac.cn [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Zhang, Huiwen [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Song, Yubo [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Wang, Jian; Zhang, Chenggang [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China)

2016-12-15

Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis

HepG2 cells biospecific extraction and HPLC-ESI-MS analysis for screening potential antiatherosclerotic active components in Bupeuri radix.

Science.gov (United States)

Liu, Shuqiang; Tan, Zhibin; Li, Pingting; Gao, Xiaoling; Zeng, Yuaner; Wang, Shuling

2016-03-20

HepG2 cells biospecific extraction method and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis was proposed for screening of potential antiatherosclerotic active components in Bupeuri radix, a well-known Traditional Chinese Medicine (TCM). The hypothesis suggested that when cells are incubated together with the extracts of TCM, the potential bioactive components in the TCM should selectively combine with the receptor or channel of HepG2 cells, then the eluate which contained biospecific component binding to HepG2 cells was identified using HPLC-ESI-MS analysis. The potential bioactive components of Bupeuri radix were investigated using the proposed approach. Five compounds in the saikosaponins of Bupeuri radix were detected as these components selectively combined with HepG2 cells, among these compounds, two potentially bioactive compounds namely saikosaponin b1 and saikosaponin b2 (SSb2) were identified by comparing with the chromatography of the standard sample and analysis of the structural clearance characterization of MS. Then SSb2 was used to assess the uptake of DiI-high density lipoprotein (HDL) in HepG2 cells for antiatherosclerotic activity. The results have showed that SSb2, with indicated concentrations (5, 15, 25, and 40 μM) could remarkably uptake dioctadecylindocarbocyanine labeled- (DiI) -HDL in HepG2 cells (Vs control group, *PESI-MS analysis is a rapid, convenient, and reliable method for screening potential bioactive components in TCM and SSb2 may be a valuable novel drug agent for the treatment of atherosclerosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of miR-155 in fluorooctane sulfonate-induced oxidative hepatic damage via the Nrf2-dependent pathway

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Wan, Chong; Han, Rui; Liu, Limin [Laboratory of Environment and Health, College of Life Sciences, University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049 (China); Zhang, Fang, E-mail: zhangfang@ucas.ac.cn [Laboratory of Environment and Health, College of Life Sciences, University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049 (China); Li, Fang [Laboratory of Environment and Health, College of Life Sciences, University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049 (China); Xiang, Mingdeng [State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012 (China); Ding, Wenjun, E-mail: dingwj@ucas.ac.cn [Laboratory of Environment and Health, College of Life Sciences, University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049 (China)

2016-03-15

Studies demonstrated that perfluorooctane sulfonate (PFOS) tends to accumulate in the liver and is capable to cause hepatomegaly. In the present study, we investigated the roles of miR-155 in PFOS-induced hepatotoxicity in SD rats and HepG2 cells. Male SD rats were orally administrated with PFOS at 1 or 10 mg/kg/day for 28 days while HepG2 cells were treated with 0–50 μM of PFOS for 24 h or 50 μM of PFOS for 1, 3, 6, 12 or 24 h, respectively. We found that PFOS significantly increased the liver weight and serum alanine transaminase (ALT) and aspartate amino transferase (AST) levels in rats. Morphologically, PFOS caused actin filament remodeling and endothelial permeability changes in the liver. Moreover, PFOS triggered reactive oxygen species (ROS) generation and induced apoptosis in both in vivo and in vitro assays. Immunoblotting data showed that NF-E2-related factor-2 (Nrf2) expression and activation and its target genes were all suppressed by PFOS in the liver and HepG2 cells. However, PFOS significantly increased miR-155 expression. Further studies showed that pretreatment of HepG2 cells with catalase significantly decreased miR-155 expression and substantially increased Nrf2 expression and activation, resulting in reduction of PFOS-induced cytotoxicity and oxidative stress. Taken together, these results indicated that miR-155 plays an important role in the PFOS-induced hepatotoxicity by disrupting Nrf2/ARE signaling pathway. - Highlights: • PFOS is capable to cause hepatotoxicity. • PFOS triggers ROS generation and induces apoptosis both in vivo and in vitro assays. • PFOS-induced ROS inhibits Nrf2 expression and its transactivation function. • PFOS promotes miR155 expression in liver and HepG2 cells. • miR-155 is involved in PFOS-induced hepatotoxicity by disrupting Nrf2/ARE pathway.

Combination Treatment of Deep Sea Water and Fucoidan Attenuates High Glucose-Induced Insulin-Resistance in HepG2 Hepatocytes

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He, Shan; Peng, Wei-Bing; Zhou, Hong-Lei

2018-01-01

Insulin resistance (IR) plays a central role in the development of several metabolic diseases, which leads to increased morbidity and mortality rates, in addition to soaring health-care costs. Deep sea water (DSW) and fucoidans (FPS) have drawn much attention in recent years because of their potential medical and pharmaceutical applications. This study investigated the effects and mechanisms of combination treatment of DSW and FPS in improving IR in HepG2 hepatocytes induced by a high glucose concentration. The results elucidated that co-treatment with DSW and FPS could synergistically repress hepatic glucose production and increase the glycogen level in IR-HepG2 cells. In addition, they stimulated the phosphorylation levels of the components of the insulin signaling pathway, including tyrosine phosphorylation of IRS-1, and serine phosphorylation of Akt and GSK-3β. Furthermore, they increased the phosphorylation of AMPK and ACC, which in turn decreased the intracellular triglyceride level. Taken together, these results suggested that co-treatment with DSW and FPS had a greater improving effect than DSW or FPS alone on IR. They might attenuate IR by targeting Akt/GSK-3β and AMPK pathways. These results may have some implications in the treatment of metabolic diseases. PMID:29393871

Galactosylated poly(ε-caprolactone) membrane promoted liver-specific functions of HepG2 cells in vitro

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Zhang, Yan, E-mail: zhang_yan@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Zhang, Yi [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Chen, Min; Zhou, Yan [The State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 (China); Lang, Meidong, E-mail: mdlang@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China)

2014-08-01

The lack of pendant functional groups on the PCL backbone has been a great challenge for surface bioactivation of poly(ε-caprolactone) (PCL). In the present study, covalently galactosylated PCL (GPCL) was developed through coupling between the amino-functionalized PCL (NPCL) and the lactobionic acid (LA) and its potential application in maintenance of physiological functions of HepG2 cells was further evaluated. The structure and properties of GPCL were explored by {sup 1}H NMR, FT-IR, GPC and DSC. Moreover, the incorporation of galactose ligands onto GPCL membranes not only promoted higher wettability, but also radically changed surface morphology in comparison with PCL and NPCL according to the contact angle measurement and atomic force microscopy. When HepG2 cells were seeded onto these membranes, the cells on GPCL membranes showed more pronounced cell adhesion and tended to form aggregates during the initial adhesion stage and then progressively grew into multi-layer structures compared to those without galactose ligands by the observation with fluorescence microscope and scanning electron microscopy. Furthermore, live–dead assay and functional tests demonstrated that HepG2 cells on GPCL membranes had superior viability and maintained better liver-specific functions. Collectively, GPCL has great potential for hepatic tissue engineering scaffolds. - Graphical abstract: The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction. The galactosylated functionalized PCL scaffold is a potential candidate for liver tissue engineering. - Highlights: • The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. • The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction. �

Surface Grafted Glycopolymer Brushes to Enhance Selective Adhesion of HepG2 Cells

DEFF Research Database (Denmark)

Chernyy, Sergey; Jensen, Bettina Elisabeth Brøgger; Shimizu, Kyoko

2013-01-01

on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic...... studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP...... process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside...

Ginseng (Panax quinquefolius and Licorice (Glycyrrhiza uralensis Root Extract Combinations Increase Hepatocarcinoma Cell (Hep-G2 Viability

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David G. Popovich

2011-01-01

Full Text Available The combined cytoactive effects of American ginseng (Panax quinquefolius and licorice (Glycyrrhiza uralensis root extracts were investigated in a hepatocarcinoma cell line (Hep-G2. An isobolographic analysis was utilized to express the possibility of synergistic, additive or antagonistic interaction between the two extracts. Both ginseng and licorice roots are widely utilized in traditional Chinese medicine preparations to treat a variety of ailments. However, the effect of the herbs in combination is currently unknown in cultured Hep-G2 cells. Ginseng (GE and licorice (LE extracts were both able to reduce cell viability. The LC50 values, after 72 h, were found to be 0.64 ± 0.02 mg/mL (GE and 0.53 ± 0.02 mg/mL (LE. An isobologram was plotted, which included five theoretical LC50s calculated, based on the fixed fraction method of combination ginseng to licorice extracts to establish a line of additivity. All combinations of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5 produced an effect on Hep-G2 cell viability but they were all found to be antagonistic. The LC50 of fractions 1/3, 1/2, 2/3 were 23%, 21% and 18% above the theoretical LC50. Lactate dehydrogenase release indicated that as the proportion of GE to LE increased beyond 50%, the influence on membrane permeability increased. Cell-cycle analysis showed a slight but significant arrest at the G1 phase of cell cycle for LE. Both GE and LE reduced Hep-G2 viability independently; however, the combinations of both extracts were found to have an antagonistic effect on cell viability and increased cultured Hep-G2 survival.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

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Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

Hyperglycemia and anthocyanin inhibit quercetin metabolism in HepG2 cells

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A high glucose (Glu) milieu promotes generation of reactive oxygen species, which may not only cause cellular damage, but also modulate phase II enzymes that are responsible for the metabolism of flavonoids. Thus, we examined the effect of a high Glu milieu on quercetin (Q) metabolism in HepG2 cells...

Role of vitamin C transporters and biliverdin reductase in the dual pro-oxidant and anti-oxidant effect of biliary compounds on the placental-fetal unit in cholestasis during pregnancy

International Nuclear Information System (INIS)

Perez, Maria J.; Castano, Beatriz; Jimenez, Silvia; Serrano, Maria A.; Gonzalez-Buitrago, Jose M.; Marin, Jose J.G.

2008-01-01

Maternal cholestasis causes oxidative damage to the placental-fetal unit that may challenge the outcome of pregnancy. This has been associated with the accumulation of biliary compounds able to induce oxidative stress. However, other cholephilic compounds such as ursodeoxycholic acid (UDCA) and bilirubin have direct anti-oxidant properties. In the present study we investigated whether these compounds exert a protective effect on cholestasis-induced oxidative stress in placenta as compared to maternal and fetal livers, and whether this is due in part to the activation of anti-oxidant mechanisms involving vitamin C uptake and biliverdin/bilirubin recycling. In human placenta (JAr) and liver (HepG2) cells, deoxycholic acid (DCA) similar rates of free radical generation. In JAr (not HepG2), the mitochondrial membrane potential and cell viability were impaired by low DCA concentrations; this was partly prevented by bilirubin and UDCA. In HepG2, taurocholic acid (TCA) and UDCA up-regulated biliverdin-IXα reductase (BVRα) and the vitamin C transporter SVCT2 (not SVCT1), whereas bilirubin up-regulated both SVCT1 and SVCT2. In JAr, TCA and UDCA up-regulated BVRα, SVCT1 and SVCT2, whereas bilirubin up-regulated only SVCT2. A differential response to these compounds of nuclear receptor expression (SXR, CAR, FXR and SHP) was found in both cell types. When cholestasis was induced in pregnant rats, BVRα, SVCT1 and SVCT2 expression in maternal and fetal livers was stimulated, and this was further enhanced by UDCA treatment. In placenta, only BVRα was up-regulated. In conclusion, bilirubin accumulation and UDCA administration may directly and indirectly protect the placental-fetal unit from maternal cholestasis-induced oxidative stress

Oleuropein potentiates anti-tumor activity of cisplatin against HepG2 through affecting proNGF/NGF balance.

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Sherif, Iman O; Al-Gayyar, Mohammed M H

2018-04-01

Oleuropein is considered as a new chemotherapeutic agent in human hepatocellular carcinoma (HCC) while, its exact underlying molecular mechanism still not yet explored. In addition, cisplatin is a standard anticancer drug against solid tumors with toxic side effects. Therefore, we conducted this study to assess antitumor activity of oleuropein either alone or in combination with cisplatin against HepG2, human HCC cell lines, via targeting pro-NGF/NGF signaling pathway. HepG2 cells were treated with cisplatin (20, 50, 100 μM) and oleuropein (100, 200, 300 and 400 μM) as well as some of the cells were treated with 50 μM cisplatin and different concentrations of oleuropein. Gene expressions of nerve growth factor (NGF), matrix metalloproteinase-7 (MMP-7) and caspase-3 were evaluated by real time-PCR. In addition, protein levels of NGF and pro-form of NGF (pro-NGF) were measured by ELISA while, nitric oxide (NO) content was determined colorimetrically. Cisplatin treatment showed a significant elevation of NO content and pro-NGF protein level with a marked reduction of NGF protein level in addition to the upregulation of caspase-3 along with downregulation of MMP-7 gene expressions in a dose-dependent manner. However, the combination of 50 μM cisplatin and 200 μM oleuropein showed the most potent effect on the molecular level when compared with oleuropein or cisplatin alone. Our results showed for the first time that the anti-tumor activity of oleuropein against HCC could be attributed to influencing the pro-NGF/NGF balance via affecting MMP-7 activity without affecting the gene expression of NGF. Concurrent treatment with both oleuropein and cisplatin could lead to more effective chemotherapeutic combination against HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells

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Liang G

2018-01-01

Full Text Available Gaofeng Liang,1,2,* Shu Kan,2,* Yanliang Zhu,3 Shuying Feng,1 Wenpo Feng,1 Shegan Gao1,4 1Medical College, Henan University of Science and Technology, Luoyang, China; 2Department of Biomedical Engineering, University of California Berkeley, California, CA, USA; 3State Key laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 4Henan Key Laboratory of Cancer Epigenetics, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China *These authors contributed equally to this work Introduction: Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells. Methods: In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation. Results: The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1–Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle. Conclusion: Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of mi

Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

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Hesham M. Korashy

2012-01-01

Full Text Available Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2 and human breast (MCF7 cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways.

Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells

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U.S. Environmental Protection Agency — Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. This dataset is associated with the following publication:...

Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells

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U.S. Environmental Protection Agency — The data set is a matrix of cellular biochemical (metabolites) in HepG2 cells treated with various metal oxide nanomaterials composed of CeO2, SiO2 and CuO. This...

Radiosensitization by inhibiting survivin in human hepatoma HepG2 cells to high-LET radiation

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Wu Qingfeng; Li Ping; Gong Li; Hao Jifang; Dai Zhongying; Matsumoto, Yoshitaka; Furusawa, Yoshiya

2011-01-01

In this study, whether survivin plays a direct role in mediating high-linear energy transfer (LET) radiation resistance in human hepatoma cells was investigated. Small interfering RNA (siRNA) targeting survivin mRNA was designed and transfected into human hepatoma HepG2 cells. Real-time polymerase chain reaction (PCR) and western blotting analyses revealed that survivin expression in HepG2 cells decreased at both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA. Caspase-3 activity was determined with a microplate reader assay as well. Following exposure to high-LET carbon ions, a reduced clonogenic survival effect, increased apoptotic rates and caspase-3 activity were observed in the cells treated with the siRNA compared to those untreated with the siRNA. The cells with transfection of the survivin-specific siRNA also increased the level of G 2 /M arrest. These results suggest that survivin definitely plays a role in mediating the resistance of HepG2 cells to high-LET radiation and depressing survivin expression might be useful to improve the therapeutic efficacy of heavy ions for radioresistant solid tumors. (author)

Aryl hydrocarbon receptor activation and CYP1A induction by cooked food-derived carcinogenic heterocyclic amines in human HepG2 cell lines.

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Sekimoto, Masashi; Sumi, Haruna; Hosaka, Takuomi; Umemura, Takashi; Nishikawa, Akiyoshi; Degawa, Masakuni

2016-11-01

The ability of nine cooked food-derived heterocyclic aromatic amines (HCAs), such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methylpyrido[12-a:3',2'-d]imidazole (Glu-P-1), 2-amino-pyrido[12-a:3',2'-d]imidazole hydrochloride (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP), to activate human aryl hydrocarbon receptor (hAhR) was examined using a HepG2-A10 cell line, which has previously established from human hepatocarcinoma-derived HepG2 cells for use in hAhR-based luciferase reporter gene assays. Trp-P-1, Trp-P-2, AαC, MeAαC, IQ and MeIQx showed a definite ability to induce not only luciferase (hAhR activation) in HepG2-A10 cells but also cytochrome P450 (CYP)1A1/1A2 mRNAs in HepG2 cells, while such the ability of Glu-P-1, Glu-P-2, and PhIP was very low. In addition, all the HCAs examined, especially MeAαC and MeIQx, had a definite capacity for inhibiting the activity of ethoxyresorfin O-deethylase (CYP1As, especially CYP1A1). The present findings demonstrate that all the HCAs examined have the ability to activate hAhR and its target genes, and further confirm that these HCAs become good substrates for human CYP1A subfamily enzyme(s). Copyright © 2016 Elsevier Ltd. All rights reserved.

Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence.

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Chen, Zhenzhen; Li, Qingling; Sun, Qianqian; Chen, Hao; Wang, Xu; Li, Na; Yin, Miao; Xie, Yanxia; Li, Hongmin; Tang, Bo

2012-06-05

Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

The ERK1/2 Inhibitor U0126 Attenuates Diabetes-Induced Upregulation of MMP-9 and Biomarkers of Inflammation in the Retina

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Ghulam Mohammad

2013-01-01

Full Text Available This study was conducted to determine the expression of matrix metalloproteinase-9 (MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1 in a time-dependent manner and the effect of extracellular-signal-regulated kinases-1/2 (ERK1/2 inhibition on the expressions of MMP-9, TIMP-1, and inflammatory biomarkers in the retinas of diabetic rats. The expression of MMP-9 was quantified by zymography, and the mRNA level of MMP-9 and TIMP-1 was quantified by RT-PCR. The expression of inducible nitric oxide synthase (iNOS, interleukin-6 (IL-6, and tumor necrosis factor-alpha (TNF-α was examined by Western blot analysis. MMP-9 expression was significantly higher in diabetic rat retinas compared to controls at all time points.TIMP-1 expression was nonsignificantly upregulated at 1week of diabetes and was significantly downregulated at 4 and 12 weeks of diabetes. Intravitreal administration of the ERK1/2 inhibitor U0126 prior to induction of diabetes decreased ERK1/2 activation, attenuated diabetes-induced upregulation of MMP-9, iNOS, IL-6, and TNF-α and upregulated TIMP-1 expression. In MMP-9 knockout mice, diabetes had no effect on retinal iNOS expression and its level remained unchanged. These data provide evidence that ERK1/2 signaling pathway is involved in MMP-9, iNOS, IL-6, and TNF-α induction in diabetic retinas and suggest that ERK1/2 can be a novel therapeutic target in diabetic retinopathy.

Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

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Minta Maria

2014-12-01

Full Text Available The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES and ethinyloestradiol (EE2 and two androgens: testosterone propionate (TP and trenbolone (TREN on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide reduction assay, lysosomal activity (neutral red uptake assay, total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3 and 3.7-5.2 μg/mL (HepG2; EE2 2.1-14.3 μg/mL (Balb/c 3T3 and 1.8-7.8 μg/mL (HepG2; TP-14.9-17.5 μg/mL (Balb/c 3T3, and 63.9- 100 μg/mL (HepG2; and TREN 11.3-31.4 μg/mL (Balb/c 3T3 and 12.5-59.4 μg/mL (HepG2. The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

9-cis-retinoic acid increases apolipoprotein AI secretion and mRNA expression in HepG2 cells.

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Haghpassand, M; Moberly, J B

1995-10-01

HepG2 cells were studied as a model for regulation of hepatic apolipoprotein AI (apo AI) secretion and gene expression by 9-cis-retinoic acid. HepG2 cells cultured on plastic dishes were exposed to 9-cis-retinoic acid (9-cis-RA) for 48 h with a complete media change at 24 h. Apo AI mass in cultured media was determined by ELISA, by quantitative immunoblotting and by steady-state 35S-methionine labeling. Messenger RNA levels were determined by RNase protection using probes for apo AI and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). 9-cis-RA increased secretion of apo AI by 52% at doses of 10 and 1 microM (6.3 +/- 0.6 vs. 4.2 +/- 0.3; P G3PDH mRNA was slightly decreased (14%, P < 0.05). Thus, 9-cis-RA stimulates apo AI expression in HepG2 cells, suggesting a role for retinoids in activating endogenous apo AI gene expression.

LXR agonist increases apoE secretion from HepG2 spheroid, together with an increased production of VLDL and apoE-rich large HDL

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Koike Kazuhiko

2011-08-01

Full Text Available Abstract Background The physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR. Methods and Results We investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well. Conclusions LXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism.

GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells.

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ZhenZhen Hu

Full Text Available BACKGROUND: Epidermal growth factor (EGF signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Analysis of changes in energy and redox states in HepG2 hepatoma and C6 glioma cells upon exposure to cadmium

International Nuclear Information System (INIS)

Yang, M.S.; Yu, L.C.; Gupta, R.C.

2004-01-01

The energy and redox states of the HepG2 hepatoma and the C6 glioma cells were studied by quantifying the levels of ATP, ADP, AMP, GSH, and GSSG. These values were used to calculate the energy charge potential (ECP = [ATP + 0.5ADP]/TAN), total adenosine nucleotides (TAN = ATP + ADP + AMP), total glutathione (TG = [GSH + GSSG]/TAN), and the redox state (GSH/GSSG ratio). For comparison between cell types, the level of each energy metabolite (ATP, ADP, and AMP) was normalized against TAN of the respective cell. The results showed that ATP:ADP:AMP ratio was 0.76:0.11:0.13 for the HepG2 cells and 0.80:0.11:0.09 for the C6 glioma cells. ECP was 0.81 ± 0.01 and 0.85 ± 0.01 for the HepG2 and the C6 glioma cells, respectively. GSH/GSSG ratio was 2.66 ± 0.16 and 3.63 ± 0.48 for HepG2 and C6 glioma cells, respectively. TG was 3.2 ± 0.54 for the HepG2 cells and 2.43 ± 0.18 for the C6 glioma cells, indicating that the level of total glutathione is more than two to three times higher than the total energy metabolites in these cell lines. Following a 3-h incubation in medium containing different concentrations of Cd, there was a dose-dependent decrease in cell viability. The 3-h LC 50 for the HepG2 cells was 0.5 mM and that for the C6 glioma cells was 0.4 mM. Cellular TAN decreased with a decrease in cell viability. Upon careful analysis of the energy state, there was a significant increase in relative amount of ATP and decrease in ADP and AMP in both cells as Cd concentration increased from 0 to 0.1, 0.2, and 0.6 mM. However, ECP in both cell lines increased, which indicated that the level of high energy phosphate was adequate. There was also a significant increase in TG and a significant decrease in GSH/GSSG in the C6 glioma cells when cells were exposed to as low as 0.1 mM Cd, which suggested that the cellular redox state was compromised. The HepG2 cells, on the other hand, showed no significant change in both TG and GSH/GSSG level until Cd concentration reached 0.6 m

Trigeminal nerve injury-induced thrombospondin-4 up-regulation contributes to orofacial neuropathic pain states in a rat model.

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Li, K-W; Kim, D-S; Zaucke, F; Luo, Z D

2014-04-01

Injury to the trigeminal nerve often results in the development of chronic pain states including tactile allodynia, or hypersensitivity to light touch, in orofacial area, but its underlying mechanisms are poorly understood. Peripheral nerve injury has been shown to cause up-regulation of thrombospondin-4 (TSP4) in dorsal spinal cord that correlates with neuropathic pain development. In this study, we examined whether injury-induced TSP4 is critical in mediating orofacial pain development in a rat model of chronic constriction injury to the infraorbital nerve. Orofacial sensitivity to mechanical stimulation was examined in a unilateral infraorbital nerve ligation rat model. The levels of TSP4 in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 spinal cord (Vc/C2) from injured rats were examined at time points correlating with the initiation and peak orofacial hypersensitivity. TSP4 antisense and mismatch oligodeoxynucleotides were intrathecally injected into injured rats to see if antisense oligodeoxynucleotide treatment could reverse injury-induced TSP4 up-regulation and orofacial behavioural hypersensitivity. Our data indicated that trigeminal nerve injury induced TSP4 up-regulation in Vc/C2 at a time point correlated with orofacial tactile allodynia. In addition, intrathecal treatment with TSP4 antisense, but not mismatch, oligodeoxynucleotides blocked both injury-induced TSP4 up-regulation in Vc/C2 and behavioural hypersensitivity. Our data support that infraorbital nerve injury leads to TSP4 up-regulation in trigeminal spinal complex that contributes to orofacial neuropathic pain states. Blocking this pathway may provide an alternative approach in management of orofacial neuropathic pain states. © 2013 European Pain Federation - EFIC®

Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

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Liu, Maoxi; Du, Kunli; Fu, Zhongxue; Zhang, Shouru; Wu, Xingye

2015-01-01

Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

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Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

2017-03-01

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

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Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A.

2013-01-01

Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive

Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

Energy Technology Data Exchange (ETDEWEB)

Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Simon, Felipe; Riedel, Claudia [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile); Ferrick, David [Seahorse Bioscience, Billerica, MA (United States); Elorza, Alvaro A., E-mail: aelorza@unab.cl [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile)

2013-08-02

Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

β2-Adrenergic Receptor-Mediated HIF-1α Upregulation Mediates Blood Brain Barrier Damage in Acute Cerebral Ischemia

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Yanyun Sun

2017-08-01

Full Text Available Disruption of the blood brain barrier (BBB within the thrombolytic time window is an antecedent event to intracerebral hemorrhage in ischemic stroke. Our recent studies showed that 2-h cerebral ischemia induced BBB damage in non-infarcted area and secreted matrix metalloproteinase-2 (MMP-2 accounted for this disruption. However, the factors that affect MMP-2 secretion and regulate BBB damage remains unknown. Since hypoxia-inducible factor-1 alpha (HIF-1α was discovered as a mater regulator in hypoxia, we sought to investigate the roles of HIF-1α in BBB damage as well as the factors regulating HIF-1α expression in the ischemic brain. in vivo rat middle cerebral artery occlusion (MCAO and in vitro oxygen glucose deprivation (OGD models were used to mimic ischemia. Pretreatment with HIF-1α inhibitor YC-1 significantly inhibited 2-h MCAO-induced BBB damage, which was accompanied by suppressed occludin degradation and vascular endothelial growth factor (VEGF mRNA upregulation. Interestingly, β2-adrenergic receptor (β2-AR antagonist ICI 118551 attenuated ischemia-induced BBB damage by regulating HIF-1α expression. Double immunostaining showed that HIF-1α was upregulated in ischemic neurons but not in astrocytes andendothelial cells. Of note, HIF-1α inhibition with inhibitor YC-1 or siRNA significantly prevented OGD-induced VEGF upregulation as well as the secretion of VEGF and MMP-2 in neurons. More importantly, blocking β2-AR with ICI 118551 suppressedHIF-1α upregulation in ischemic neurons and attenuated occludin degradation induced by the conditioned media of OGD-treatedneurons. Taken together, blockade of β2-AR-mediated HIF-1α upregulation mediates BBB damage during acute cerebral ischemia. These findings provide new mechanistic understanding of early BBB damage in ischemic stroke and may help reduce thrombolysis-related hemorrhagic complications.

Metabolomic effects in HepG2 cells exposed to CeO2, SiO2 and CuO nanomaterials.

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To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for three days to 5 different CeO2 (either 30 or 100 ug/ml), 3 SiO2 based (30 ug/ml) or 1 CuO (3 ug/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metab...

A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in O. tsutsugamushi-Infected Human Macrophages.

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Ching-Ying Wu

Full Text Available Orientia (O. tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE inhibitors on TNF-α production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB, but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70 expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response.

APPLICATION OF CDNA MICROARRAY TECHNOLOGY TO IN VITRO TOXICOLOGY AND THE SELECTION OF GENES FOR A REAL TIME RT-PCR-BASED SCREEN FOR OXIDATIVE STRESS IN HEP-G2 CELLS

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Large-scale analysis of gene expression using cDNA microarrays promises therapid detection of the mode of toxicity for drugs and other chemicals. cDNAmicroarrays were used to examine chemically-induced alterations of geneexpression in HepG2 cells exposed to oxidative ...

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

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Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

PPARγ activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

International Nuclear Information System (INIS)

Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Orlov, Sergey V.; Perevozchikov, Andrej P.

2010-01-01

Research highlights: → PPARγ activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. → Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1-LXRβ complex. → Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex. → Activation of PPARγ leads to increasing of the level of LXRβ associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRβ binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1/LXRβ complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRβ and MEK1/2 in regulation of ABCA1 mRNA and protein expression.

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

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Kim, Ji Eun; Yoo, Hyun Ju; Gu, Ja Yoon; Kim, Hyun Kyung

2016-01-01

The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.

The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1

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Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

2016-01-01

Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation. PMID:27624558

Saponarin activates AMPK in a calcium-dependent manner and suppresses gluconeogenesis and increases glucose uptake via phosphorylation of CRTC2 and HDAC5.

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Seo, Woo-Duck; Lee, Ji Hae; Jia, Yaoyao; Wu, Chunyan; Lee, Sung-Joon

2015-11-15

This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake. Copyright © 2015 Elsevier Ltd. All rights reserved.

Shifts in dietary carbohydrate-lipid exposure regulate expression of the non-alcoholic fatty liver disease-associated gene PNPLA3/adiponutrin in mouse liver and HepG2 human liver cells.

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Hao, Lei; Ito, Kyoko; Huang, Kuan-Hsun; Sae-tan, Sudathip; Lambert, Joshua D; Ross, A Catharine

2014-10-01

Patatin-like phospholipase domain containing 3 (PNPLA3, adiponutrin) has been identified as a modifier of lipid metabolism. To better understand the physiological role of PNPLA3/adiponutrin, we have investigated its regulation in intact mice and human hepatocytes under various nutritional/metabolic conditions. PNPLA3 gene expression was determined by real-time PCR in liver of C57BL/6 mice after dietary treatments and in HepG2 cells exposed to various nutritional/metabolic stimuli. Intracellular lipid content was determined in HepG2 cells after siRNA-mediated knockdown of PNPLA3. In vivo, mice fed a high-carbohydrate (HC) liquid diet had elevated hepatic lipid content, and PNPLA3 mRNA and protein expression, compared to chow-fed mice. Elevated expression was completely abrogated by addition of unsaturated lipid emulsion to the HC diet. By contrast, in mice with high-fat diet-induced steatosis, Pnpla3 expression did not differ compared to low-fat fed mice. In HepG2 cells, Pnpla3 expression was reversibly suppressed by glucose depletion and increased by glucose refeeding, but unchanged by addition of insulin and glucagon. Several unsaturated fatty acids each significantly decreased Pnpla3 mRNA, similar to lipid emulsion in vivo. However, Pnpla3 knockdown in HepG2 cells did not alter total lipid content in high glucose- or oleic acid-treated cells. Our results provide evidence that PNPLA3 expression is an early signal/signature of carbohydrate-induced lipogenesis, but its expression is not associated with steatosis per se. Under lipogenic conditions due to high-carbohydrate feeding, certain unsaturated fatty acids can effectively suppress both lipogenesis and PNPLA3 expression, both in vivo and in a hepatocyte cell line. Copyright © 2014 Elsevier Inc. All rights reserved.

SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells.

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Gorgani-Firuzjaee, Sattar; Khatami, Shohreh; Adeli, Khosrow; Meshkani, Reza

2015-09-04

Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia. Copyright © 2015 Elsevier Inc. All rights reserved.

Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines.

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Al-Sheddi, Ebtesam Saad; Farshori, Nida Nayyar; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2015-01-01

Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, anti- bacterial, and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against human liver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, the present study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549 cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After the exposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed a concentration-dependent significant reduction in the percentage cell viability and an alteration in the cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and 54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2 cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56% by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrations were found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT and NRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, and appeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resulted in significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-κB in H1299 human lung cancer cells

International Nuclear Information System (INIS)

Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

2009-01-01

Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (Gαi1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of Gαi1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of Gαi1QL. Gαi1 induced the transcription of Bcl-2 by activation of NF-κB, which resulted from an increase in NF-κB p50 protein. We conclude that Gαi1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-κB activation.

Germacrane sesquiterpenes isolated from the rhizome of Curcuma xanthorrhiza Roxb. inhibit UVB-induced upregulation of MMP-1, -2, and -3 expression in human keratinocytes.

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Park, Ji-Hae; Mohamed, Mohamed Antar Aziz; Jung, Ye-Jin; Shrestha, Sabina; Lee, Tae Hoon; Lee, Chang-Ho; Han, Daeseok; Kim, Jiyoung; Baek, Nam-In

2015-10-01

Four sesquiterpenes were isolated from the rhizome of Curcuma xanthorrhiza Roxb.: furanodiene (1), germacrone (2), furanodienone (3), and 13-hydroxygermacrone (4). Importantly, this was the first time compounds 1 and 4 were isolated from this plant. The chemical structures of these compounds were determined using 1D- and 2D-nuclear magnetic resonance, infrared spectroscopy, and electron ionization mass spectrometry analyses. Among the isolated compounds, compounds 2 and 4 inhibited UVB-induced upregulation of the mRNA and protein expression levels of MMP-1, MMP-2, and MMP-3 in human keratinocytes (HaCaT). Moreover, this upregulation occurred in a dose-dependent manner over the range of 1-10 μM for each compound.

[Inhibitory effect of Biejiajian pills on HepG2 cell xenograft growth and expression of β-catenin and Tbx3 in nude mice].

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Wen, Bin; Sun, Hai-Tao; He, Song-Qi; LA, Lei; An, Hai-Yan; Pang, Jie

2016-02-01

To explore the molecular mechanism by which Biejiajian pills inhibit hepatocellular carcinoma in a nude mouse model bearing HepG2 cell xenograft. The inhibitory effect of Biejiajian pills on the growth of HepG2 cell xenograft in nude mice was observed. Immunohistochemical method was used to examine proliferating cell nuclear antigen (PCNA) expression in HepG2 cell xenograft, and TUNEL method was employed to detect the cell apoptosis; the expression levels of β-catenin and Tbx3 were measured by Western blotting. Biejiajian pills significantly suppressed the growth of HepG2 cell xenograft in nude mice. The tumor-bearing mice treated with a high and a moderate dose of Biejiajian pills showed significantly increased apoptosis rate of the tumor cells [(22.9±1.220)% and (14.7±0.50)%, respectively] compared with the control group [(5.5±0.90)%, Ppills significantly decreased the expressions of PNCA, β-catenin, and Tbx3 in the cell xenograft (Ppills can inhibit the growth of HepG2 cell xenograft in nude mice and promote tumor cell apoptosis possibly by inhibiting PNCA expression and the Wnt/β-catenin signaling pathway.

Gas6 induces cancer cell migration and epithelial–mesenchymal transition through upregulation of MAPK and Slug

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Lee, Yunhee [Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of); Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Lee, Mira [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, Semi, E-mail: semikim@kribb.re.kr [Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of); Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of)

2013-04-26

Highlights: •We investigated the molecular mechanisms underlying Gas6-mediated cancer cell migration. •Gas6 treatment and subsequent Axl activation induce cell migration and EMT via upregulation of Slug. •Slug expression mediated by Gas6 is mainly through c-Jun and ATF-2 in an ERK1/2 and JNK-dependent manner. •The Gas6/Axl-Slug axis may be exploited as a target for anti-cancer metastasis therapy. -- Abstract: Binding of Gas6 to Axl (Gas6/Axl axis) alters cellular functions, including migration, invasion, proliferation, and survival. However, the molecular mechanisms underlying Gas6-mediated cell migration remain poorly understood. In this study, we found that Gas6 induced the activation of JNK and ERK1/2 signaling in cancer cells expressing Axl, resulting in the phosphorylation of activator protein-1 (AP-1) transcription factors c-Jun and ATF-2, and induction of Slug. Depletion of c-Jun or ATF-2 by siRNA attenuated the Gas6-induced expression of Slug. Slug expression was required for cell migration and E-cadherin reduction/vimentin induction induced by Gas6. These results suggest that Gas6 induced cell migration via Slug upregulation in JNK- and ERK1/2-dependent mechanisms. These data provide an important insight into the molecular mechanisms mediating Gas6-induced cell migration.

Data on HepG2 cells changes following exposure to cadmium sulphide quantum dots (CdS QDs

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Laura Paesano

2017-04-01

Full Text Available The data included in this paper are associated with the research article entitled "Markers for toxicity to HepG2 exposed to cadmium sulphide quantum dots; damage to mitochondria" (Paesano et al. [1]. The article concerns the cytotoxic and genotoxic effects of CdS QDs in HepG2 cells and the mechanisms involved. In this dataset, changes in expression levels of candidate genes are reported, together with details concerning synthesis and properties of CdS QDs, additional information obtained through literature survey, measures of the mitochondrial membrane potential and the glutathione redox state.

Glycyrrhizin, silymarin, and ursodeoxycholic acid regulate a common hepatoprotective pathway in HepG2 cells.

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Hsiang, Chien-Yun; Lin, Li-Jen; Kao, Shung-Te; Lo, Hsin-Yi; Chou, Shun-Ting; Ho, Tin-Yun

2015-07-15

Glycyrrhizin, silymarin, and ursodeoxycholic acid are widely used hepatoprotectants for the treatment of liver disorders, such as hepatitis C virus infection, primary biliary cirrhosis, and hepatocellular carcinoma. The gene expression profiles of HepG2 cells responsive to glycyrrhizin, silymarin, and ursodeoxycholic acid were analyzed in this study. HepG2 cells were treated with 25 µM hepatoprotectants for 24 h. Gene expression profiles of hepatoprotectants-treated cells were analyzed by oligonucleotide microarray in triplicates. Nuclear factor-κB (NF-κB) activities were assessed by luciferase assay. Among a total of 30,968 genes, 252 genes were commonly regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid. These compounds affected the expression of genes relevant various biological pathways, such as neurotransmission, and glucose and lipid metabolism. Genes involved in hepatocarcinogenesis, apoptosis, and anti-oxidative pathways were differentially regulated by all compounds. Moreover, interaction networks showed that NF-κB might play a central role in the regulation of gene expression. Further analysis revealed that these hepatoprotectants inhibited NF-κB activities in a dose-dependent manner. Our data suggested that glycyrrhizin, silymarin, and ursodeoxycholic acid regulated the expression of genes relevant to apoptosis and oxidative stress in HepG2 cells. Moreover, the regulation by these hepatoprotectants might be relevant to the suppression of NF-κB activities. Copyright © 2015 Elsevier GmbH. All rights reserved.

Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells

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To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for 3 days to five different CeO2 (either 30 or 100 μg/ml), 3 SiO2 based (30 μg/ml) or 1 CuO (3 μg/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metabol...

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

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Park Raekil

2006-01-01

Full Text Available Abstract Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi and that interleukin 1 alpha (IL-1 alpha up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM. Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

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Zhang, Xinyuan; Li, Qingling; Chen, Zhenzhen; Li, Hongmin; Xu, Kehua; Zhang, Lisheng; Tang, Bo

2011-03-21

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells. This journal is © The Royal Society of Chemistry 2011

Oroxylin A regulates glucose metabolism in response to hypoxic stress with the involvement of Hypoxia-inducible factor-1 in human hepatoma HepG2 cells.

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Dai, Qinsheng; Yin, Qian; Wei, Libin; Zhou, Yuxin; Qiao, Chen; Guo, Yongjian; Wang, Xiaotang; Ma, Shiping; Lu, Na

2016-08-01

Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. In this study, we investigated the influence and signaling ways of oroxylin A affecting cancer cell energy metabolism under hypoxia. The data showed that oroxylin A remarkably reduced the generation of lactate and glucose uptake under hypoxia in HepG2 cells. Moreover, oroxylin A inhibited HIF-1α expression and its stability. The downstream targets (PDK1, LDHA, and HK II), as well as their mRNA levels were also suppressed by oroxylin A under hypoxia. The silencing or the overexpression of HIF-1α assays suggested that HIF-1α is required for metabolic effect of oroxylin A in HepG2 cells during hypoxia. Furthermore, oroxylin A could reduce the expression of complex III in mitochondrial respiratory chain, and then decrease the accumulation of ROS at moderate concentrations (0-50 µM) under hypoxia, which was benefit for its inhibition on glycolytic activity by decreasing ROS-mediated HIF-1 expression. Besides, oroxylin A didn't cause the loss of MMP under hypoxia and had no obvious effects on the expression of OXPHOS complexes, suggesting that oroxylin A did not affect mitochondrial mass at the moderate stress of oroxylin A. The suppressive effect of oroxylin A on glycolysis led to a significantly repress of ATP generation, for ATP generation mostly depends on glycolysis in HepG2 cells. This study revealed a new aspect of glucose metabolism regulation of oroxylin A under hypoxia, which may contribute to its new anticancer mechanism. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor γ, controls hepatitis B virus replication

International Nuclear Information System (INIS)

Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

2011-01-01

In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin, liver X receptor α (LXRα), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPARγ and C/EBPα. Conversely, HBV replication was upregulated by adiponectin and PPARγ agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

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Du Kejun; Chai Yubo; Hou Lichao; Chang Wenhui; Chen Suming; Luo Wenjing; Cai Tongjian; Zhang Xiaonan; Chen Nanchun; Chen Yaoming; Chen Jingyuan

2008-01-01

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2

Interferon-β-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

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Takada, Eiko; Shimo, Kuniaki; Hata, Kikumi; Abiake, Maira; Mukai, Yasuo; Moriyama, Masami; Heasley, Lynn; Mizuguchi, Junichiro

2005-01-01

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-β induced apoptosis and the loss of mitochondrial membrane potential (ΔΨm) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-β-induced loss of ΔΨm, suggesting that the interaction of IFN-β-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-β induced a sustained activation of c-Jun NH 2 -terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-β-induced apoptosis and loss of ΔΨm were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-β-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-β but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-β-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

International Nuclear Information System (INIS)

Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

2014-01-01

Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

Energy Technology Data Exchange (ETDEWEB)

Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

2014-02-15

Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

PPARγ agonists upregulate sphingosine 1-phosphate (S1P) receptor 1 expression, which in turn reduces S1P-induced [Ca(2+)]i increases in renal mesangial cells.

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Koch, Alexander; Völzke, Anja; Puff, Bianca; Blankenbach, Kira; Meyer Zu Heringdorf, Dagmar; Huwiler, Andrea; Pfeilschifter, Josef

2013-11-01

We previously identified peroxisome proliferator-activated receptor gamma (PPARγ) agonists (thiazolidinediones, TZDs) as modulators of the sphingolipid metabolism in renal mesangial cells. TZDs upregulated sphingosine kinase 1 (SK-1) and increased the formation of intracellular sphingosine 1-phosphate (S1P), which in turn reduced the expression of pro-fibrotic connective tissue growth factor. Since S1P also acts as extracellular ligand at specific S1P receptors (S1PR, S1P1-5), we investigated here the effect of TZDs on S1PR expression in mesangial cells and evaluated the functional consequences by measuring S1P-induced increases in intracellular free Ca(2+) concentration ([Ca(2+)]i). Treatment with two different TZDs, troglitazone and rosiglitazone, enhanced S1P1 mRNA and protein expression in rat mesangial cells, whereas S1P2-5 expression levels were not altered. Upregulation of S1P1 mRNA upon TZD treatment was also detected in human mesangial cells and mouse glomeruli. PPARγ antagonism and promoter studies revealed that the TZD-dependent S1P1 mRNA induction involved a functional PPAR response element in the S1P1 promoter. Pharmacological approaches disclosed that S1P-induced [Ca(2+)]i increases in rat mesangial cells were predominantly mediated by S1P2 and S1P3. Interestingly, the transcriptional upregulation of S1P1 by TZDs resulted in a reduction of S1P-induced [Ca(2+)]i increases, which was reversed by the S1P1/3 antagonist VPC-23019, the protein kinase C (PKC) inhibitor PKC-412, and by S1P1 siRNA. These data suggest that PPARγ-dependent upregulation of S1P1 leads to an inhibition of S1P-induced Ca(2+) signaling in a PKC-dependent manner. Overall, these results reveal that TZDs not only modulate intracellular S1P levels but also regulate S1PR signaling by increasing S1P1 expression in mesangial cells. © 2013.

Trovafloxacin-induced replication stress sensitizes HepG2 cells to tumor necrosis factor-alpha-induced cytotoxicity mediated by extracellular signal-regulated kinase and ataxia telangiectasia and Rad3-related

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Beggs, Kevin M.; Maiuri, Ashley R.; Fullerton, Aaron M.; Poulsen, Kyle L.; Breier, Anna B.; Ganey, Patricia E.; Roth, Robert A.

2015-01-01

Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress

Pim kinases are upregulated during Epstein-Barr virus infection and enhance EBNA2 activity

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Rainio, Eeva-Marja; Ahlfors, Helena; Carter, Kara L.; Ruuska, Marja; Matikainen, Sampsa; Kieff, Elliott; Koskinen, Paeivi J.

2005-01-01

Latent Epstein-Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt's lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth

Monascus-fermented red mold dioscorea protects mice against alcohol-induced liver injury, whereas its metabolites ankaflavin and monascin regulate ethanol-induced peroxisome proliferator-activated receptor-γ and sterol regulatory element-binding transcription factor-1 expression in HepG2 cells.

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Cheng, Chih-Fu; Pan, Tzu-Ming

2018-03-01

Alcoholic hepatitis is a necroinflammatory process that is associated with fibrosis and leads to cirrhosis in 40% of cases. The hepatoprotective effects of red mold dioscorea (RMD) from Monascus purpureus NTU 568 were evaluated in vivo using a mouse model of chronic alcohol-induced liver disease (ALD). ALD mice were orally administered vehicle (ALD group) or vehicle plus 307.5, 615.0 or 1537.5 mg kg -1 (1 ×, 2 × and 5 ×) RMD for 5 weeks. RMD lowered serum leptin, hepatic total cholesterol, free fatty acid and hepatic triglyceride levels and increased serum adiponectin, hepatic alcohol dehydrogenase and antioxidant enzyme levels. Furthermore, ankaflavin (AK) and monascin (MS), metabolites of RMD fermented with M. purpureus 568, induced peroxisome proliferator-activated receptor-γ expression and the concomitant suppression of ethanol-induced elevation of sterol regulatory element-binding transcription factor-1 and TG in HepG2 cells. These results indicate the hepatoprotective effect of Monascus-fermented RMD. Moreover, AK and MS were identified as the active constituents of RMD for the first time and were shown to protect against ethanol-induced liver damage. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Biochemical Effects of six Ti02 and four Ce02 Nanomaterials in HepG2 cells

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Abstract The potential mammalian hepatotoxicity of nanomaterials were explored in dose-response and structure-activity studies with human hepatic HepG2 cells exposed to between 10 and 1000 ug/ml of six different TiO2 and four CeO2 nanomaterials for 3 days. Var...

Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

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Galloway, D C; Blake, D G; Shepherd, A G; McLellan, L I

1997-11-15

We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Science.gov (United States)

Ruiz-Aracama, Ainhoa; Peijnenburg, Ad; Kleinjans, Jos; Jennen, Danyel; van Delft, Joost; Hellfrisch, Caroline; Lommen, Arjen

2011-05-20

In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

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Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

2010-04-01

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

The effects of herbal composition Gambigyeongsinhwan (4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty rats and HepG2 cells.

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Yoon, Seolah; Kim, Jeongjun; Lee, Hyunghee; Lee, Haerim; Lim, Jonghoon; Yang, Heejeong; Shin, Soon Shik; Yoon, Michung

2017-01-04

Hepatic steatosis has risen rapidly in parallel with a dramatic increase in obesity. The aim of this study was to determine whether the herbal composition Gambigyeongsinhwan (4) (GGH(4)), composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata, regulates hepatic steatosis and inflammation. The effects of GGH(4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty (OLETF) rats and HepG2 cells were examined using Oil red O, hematoxylin and eosin, and toluidine blue staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. Administration of GGH(4) to OLETF rats improved hepatic steatosis and lowered serum levels of alanine transaminase, total cholesterol, triglycerides, and free fatty acids. GGH(4) increased mRNA levels of fatty acid oxidation enzymes (ACOX, HD, CPT-1, and MCAD) and decreased mRNA levels of lipogenesis genes (FAS, ACC1, C/EBPα, and SREBP-1c) in the liver of OLETF rats. In addition, infiltration of inflammatory cells and expression of inflammatory cytokines (CD68, TNFα, and MCP-1) in liver tissue were reduced by GGH(4). Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, but GGH(4) inhibited lipid accumulation by regulating the expression of hepatic fatty acid oxidation and lipogenic genes. GGH(4) also increased PPARα reporter gene expression. These effects of GGH(4) were similar to those of the PPARα activator fenofibrate, whereas the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on lipid accumulation in HepG2 cells. These results suggest that GGH(4) inhibits obesity-induced hepatic steatosis and that this process may be mediated by regulation of the expression of PPARα target genes and lipogenic genes. GGH(4) also suppressed obesity

Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

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Katherine R Cronin

Full Text Available BACKGROUND: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. METHODOLOGY/PRINCIPAL FINDINGS: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001 at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. CONCLUSIONS/SIGNIFICANCE: Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

HNF-4α regulated miR-122 contributes to development of gluconeogenesis and lipid metabolism disorders in Type 2 diabetic mice and in palmitate-treated HepG2 cells.

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Wei, Shengnan; Zhang, Ming; Yu, Yang; Xue, Huan; Lan, Xiaoxin; Liu, Shuping; Hatch, Grant; Chen, Li

2016-11-15

Hepatocyte Nuclear Factor-4α (HNF-4α) is a key nuclear receptor protein required for liver development. miR-122 is a predominant microRNA expressed in liver and is involved in the regulation of cholesterol and fatty acid metabolism. HNF-4α is know to regulate expression of miR-122 in liver. We examined how HNF-4α regulated gluconeogenesis and lipid metabolism through miR-122 in vivo and in vitro. Expression of miR-122, HNF-4α, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), sterol response elementary binding protein-1 (SREBP-1), fatty acid synthase-1 (FAS-1), carnitine palmitoyltransferase-1 (CPT-1) and acetyl Coenzyme A carboxylase alpha (ACCα) were determined in livers of Type 2 diabetic mice and in insulin resistant palmitate-treated HepG2 cells. CPT-1 and phosphorylated ACCα expression were significantly decreased in livers of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. In contrast, expression of miR-122, HNF-4α, PEPCK, G6Pase, SREBP-1, FAS-1 and ACCα were significantly elevated in liver of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. Expression of HNF-4α increased whereas siRNA knockdown of HNF-4α decreased miR-122 levels in HepG2 cells compared to controls. In addition, expression of HNF-4α in HepG2 cells increased PEPCK, G6Pase, SREBP-1, FAS-1, ACCα mRNA and protein expression and decreased CPT-1 and p-ACCα mRNA and protein expression compared to controls. Addition of miR-122 inhibitors attenuated the HNF-4α mediated effect on expression of these gluconeogenic and lipid metabolism proteins. The results indicate that HNF-4α regulated miR-122 contributes to development of the gluconeogenic and lipid metabolism alterations observed in Type 2 diabetic mice and in palmitate-treated HepG2 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells.

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Li, Kai; Ding, Shijia; Chen, Ke; Qin, Dongdong; Qu, Jialin; Wang, Sen; Sheng, Yanrui; Zou, Chengcheng; Chen, Limin; Tang, Hua

2013-01-01

The hepatitis B virus X (HBx) protein has long been recognized as an important transcriptional transactivator of several genes. Human aldo-keto reductase family 1, member C1 (AKR1C1), a member of the family of AKR1CS, is significantly increased in HBx-expressed cells. This study aimed to investigate the possible mechanism of HBx in regulating AKR1C1 expression in HepG2.2.15 cells and the role of AKR1C1 for HBV-induced HCC. RT-PCR was performed to detect AKR1C1 expression on mRNA level in HepG2 and HepG2.2.15 cell. The promoter activity of AKR1C1 was assayed by transient transfection and Dual-luciferase reporter assay system. The AKR1C1 promoter sequence was screened using the TFSEARCH database and the ALIBABA 2.0 software. The potential transcription factors binding sites were identified using 5' functional deletion analysis and site-directed mutagenesis. In this study, we found that HBx promoted AKR1C1 expression in HepG2.2.15 cells. Knockdown of HBx inhibited AKR1C1 activation. The role of HBx expression in regulating the promoter activity of human AKR1C1 gene was analyzed. The 5'functional deletion analysis identified that the region between -128 and -88 was the minimal promoter region of HBx to activate AKR1C1 gene expression. Site-directed mutagenesis studies suggested that nuclear factor-Y (NF-Y) plays an important role in this HBx-induced AKR1C1 activation. In HepG2.2.1.5 cell, HBx can promote AKR1C1 promoter activity and thus activates the basal transcription of AKR1C1 gene. This process is mediated by the transcription factor NF-Y. This study explored the mechanism for the regulation of HBV on AKR1C1 expression and has provided a new understanding of HBV-induced HCC.

The 3-D Culture and In Vivo Growth of the Human Hepatocellular Carcinoma Cell Line HepG2 in a Self-Assembling Peptide Nanofiber Scaffold

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Wu, M.; Yang, Z.; Liu, Y.; Liu, B.; Zhao, X.

2010-01-01

We report the use of the RADA16-I scaffold to mimic the ECM microenvironment and support tumor cell adherence and survival. Cellular morphology, proliferation, adhesion ability, and in vivo tumor formation were studied in the human hepatocellular carcinoma cell line HepG2 in the 3-D RADA16-I scaffold. No significant differences in HepG2 cell proliferation, adhesion, and albumin secretion were observed in the peptide scaffold compared to collagen I. Furthermore, the HepG2 cells pre cultured in the peptide scaffold showed a higher proliferation rate and formed significantly bigger tumors when compared to cells grown on a traditional 2D monolayer, suggesting that the 3-D RADA16-I scaffold can mimic the tumor microenvironment and promote a malignant phenotype in HepG2 cells. Our results indicate that the RADA16-I scaffold can serve as an ideal model for tumorigenesis, growth, local invasion, and metastasis.

Crude Flavonoid Extract of Medicinal Herb Zingibar officinale Inhibits Proliferation and Induces Apoptosis in Hepatocellular Carcinoma Cells.

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Elkady, Ayman I; Abu-Zinadah, Osama A; Hussein, Rania Abd El Hamid

2017-07-05

There is an urgent need to improve the clinical management of hepatocellular carcinoma (HCC), one of the most common causes of global cancer-related deaths. Zingibar officinale is a medicinal herb used throughout history for both culinary and medicinal purposes. It has antioxidant, anticarcinogenic, and free radical scavenging properties. Previously, we proved that the crude flavonoid extract of Z. officinale (CFEZO) inhibited growth and induced apoptosis in several cancer cell lines. However, the effect of the CFEZO on an HCC cell line has not yet been evaluated. In this study, we explored the anticancer activity of CFEZO against an HCC cell line, HepG2. CFEZO significantly inhibited proliferation and induced apoptosis in HepG2 cells. Typical apoptotic morphological and biochemical changes, including cell shrinkage and detachment, nuclear condensation and fragmentation, DNA degradation, and comet tail formation, were observed after treatments with CFEZO. The apoptogenic activity of CFEZO involved induction of ROS, depletion of GSH, disruption of the mitochondrial membrane potential, activation of caspase 3/9, and an increase in the Bax/Bcl-2 ratio. CFEZO treatments induced upregulation of p53 and p21 expression and downregulation of cyclin D1 and cyclin-dependent kinase-4 expression, which were accompanied by G2/M phase arrest. These findings suggest that CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of HCC.

Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells.

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Pezdirc, Marko; Žegura, Bojana; Filipič, Metka

2013-09-01

Heterocyclic aromatic amines (HAAs) are potential human carcinogens formed in well-done meats and fish. The most abundant are 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ). HAAs exert genotoxic activity after metabolic transformation by CYP1A enzymes, that is well characterized, however the genomic and intervening responses are not well explored. We have examined cellular and genomic responses of human hepatoma HepG2 cells after 24h exposure to HAAs. Comet assay revealed increase in formation of DNA strand breaks by PhIP, MeIQx and IQ but not 4,8-DiMeIQx, whereas increased formation of micronuclei was not observed. The four HAAs up-regulated expression of genes encoding metabolic enzymes CYP1A1, CYP1A2 and UGT1A1 and expression of TP53 and its downstream regulated genes CDKN1A, GADD45α and BAX. Consistent with the up-regulation of CDKN1A and GADD45α the cell-cycle analysis showed arrest in S-phase by PhIP and IQ, and in G1-phase by 4,8-DiMeIQx and MeIQx. The results indicate that upon exposure to HAAs the cells respond with the cell-cycle arrest, which enables cells to repair the damage or eliminate them by apoptosis. However, elevated expression of BCL2 and down-regulation of BAX may indicate that HAAs could suppress apoptosis meaning higher probability of damaged cells to survive and mutate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impairment of Akt activity by CYP2E1 mediated oxidative stress is involved in chronic ethanol-induced fatty liver

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Tao Zeng

2018-04-01

Full Text Available Protein kinase B (PKB/Akt plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD. Previous studies suggest that cytochrome P4502E1 (CYP2E1 plays causal roles in the pathogenesis of alcoholic fatty liver (AFL. We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K and phosphatase and tensin homologue deleted on chromosome ten (PTEN, and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1 protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA and 4-hydroxynonenal (4-HNE-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ, an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2 cells compared with the negative control HepG2 (NC-HepG2 cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1 significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver. Keywords

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2012-02-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2011-05-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells

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To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of Ce02 (0.3, 3 and 30 µg/mL). The two Ce02 nanopartices had dry primary particle sizes of 8 nanometers {(M) made b...

Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

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Shukla, Shatrunajay [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi ‐110062 (India); Sharma, Ankita [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Pandey, Vivek Kumar [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Academy of Scientific and Innovative Research (India); Raisuddin, Sheikh [Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi ‐110062 (India); Kakkar, Poonam, E-mail: kakkarp59@gmail.com [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Academy of Scientific and Innovative Research (India)

2016-01-15

-mediated apoptosis. - Highlights: • Berberine inhibits sirtuins at transcriptional as well as at translational level. • Sirtuins inhibition leads to acetylation of non-histone proteins, FoxO1/3a and p53. • Acetylated FoxO and p53 transcriptionally upregulate BH3-only proteins Bim and PUMA respectively. • BH3-only proteins trigger mitochondrial dysfunction culminating into apoptosis. • SRT-1720 (SIRT-1 activator) partially restores Bax/Bcl-2 ratio and reduces berberine induced cytotoxicity in HepG2 cells.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

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Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist

Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

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Pelorosso, Facundo Germán; Halperin, Ana Verónica; Palma, Alejandro Martín; Nowak, Wanda; Errasti, Andrea Emilse; Rothlin, Rodolfo Pedro

2007-02-01

Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

Erbb2 up-regulation of ADAM12 expression accelerates skin cancer progression.

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Rao, Velidi H; Vogel, Kristen; Yanagida, Jodi K; Marwaha, Nitin; Kandel, Amrit; Trempus, Carol; Repertinger, Susan K; Hansen, Laura A

2015-10-01

Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness. © 2014 Wiley Periodicals, Inc.

Cellular trafficking of thymosin beta-4 in HEPG2 cells following serum starvation.

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Giuseppina Pichiri

Full Text Available Thymosin beta-4 (Tβ4 is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could

ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.