Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.     

Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line HepG2

Hepatitis B virus (HBV) infection is a worldwide health problem and may develop to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. To investigate the global proteome responses of liver‐derived cells to HBV infection and IFNα treatment, 2‐DE and MS‐based analysis were performed to compare the proteome changes between HBV stably transfected cell line HepG2.2.15 and its parental cell line HepG2, as well as HepG2.2.15 before and after IFNα treatment (5000 IU/mL for 72 h). Compared to HepG2, 12 of 18 down‐regulated and 27 of 32 up‐regulated proteins were identified in HepG2.2.15. After IFNα treatment, 6 of 7 down‐regulated and 11 of 14 up‐regulated proteins were identified. Differentially expressed proteins caused by HBV infection were involved with cytoskeletal matrix, heat shock stress, kinases/signal transduction, protease/proteasome components, etc. Prohibitin showed a dose‐dependent up‐regulation during IFNα treatment and might play a potent role in anti‐HBV activities of IFNα by enhancing the crossbinding p53 expression to achieve the apoptosis of HBV infected liver cells. Down‐regulation of interferon‐stimulated gene 15 (ISG15) in HepG2.2.15 and recovery by IFNα suggested its relationship with IFNα's anti‐HBV effect.     

Regulation of Survivin and Bcl‐2 in HepG2 Cell Apoptosis Induced by Quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in a variety of human cancer cells. However, the regulation of survivin and Bcl‐2 on the quercetin‐induced cell‐growth inhibition and apoptosis in cancer cells remains unclear. In the present study, we report that quercetin can inhibit proliferation and induce apoptosis in HepG2 cells in dose‐ and time‐dependent manner. Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) staining showed that HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin. Cell‐cycle analysis reveals a significant increase of the proportion of cells in G₀/G₁ phase. We also demonstrate that the levels of survivin and Bcl‐2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin by immunocytochemistry analysis. Relative activity of caspase‐3 and caspase‐9 increased significantly. These data clearly indicate that quercetin‐induced apoptosis is associated with caspase activation, and the levels of survivin and Bcl‐2. Our results indicate that the expression of survivin may be associated with Bcl‐2 expression, and the inhibition expression of survivin, in conjunction with Bcl‐2, might cause more pronounced apoptotic effects. Together, concurrent down‐regulated survivin and Bcl‐2 play an important role in HepG2 cell apoptosis induced by quercetin.     

A comparative proteomic analysis of HepG2 cells incubated by S(−) and R(+) enantiomers of anti‐coagulating drug warfarin

Warfarin is a commonly prescribed oral anti‐coagulant with narrow therapeutic index. It interferes with vitamin K cycle to achieve anti‐coagulating effects. Warfarin has two enantiomers, S(?) and R(+) and undergoes stereoselective metabolism, with the S(?) enantiomer being more effective. We reported that the intracellular protein profile in HepG2 cells incubated with S(?) and R(+) warfarin, using iTRAQ‐coupled 2‐D LC‐MS/MS. In samples incubated with S(?) and R(+) warfarin alone, the multi‐task protein Protein SET showed significant elevation in cells incubated with S(?) warfarin but not in those incubated with R(+) warfarin. In cells incubated with individual enantiomers of warfarin in the presence of vitamin K, protein disulfide isomerase A3 which is known as a glucose‐regulated protein, in cells incubated with S(?) warfarin was found to be down‐regulated compared to those incubated with R(+) warfarin. In addition, Protein DJ‐1 and 14‐3‐3 Proteinσ were down‐regulated in cells incubated with either S(?) or R(+) warfarin regardless of the presence of vitamin K. Our results indicated that Protein DJ‐1 may act as an enzyme for expression of essential enzymes in vitamin K cycle. Taken together, our findings provided molecular evidence on a comprehensive protein profile on warfarin–cell interaction, which may shed new lights on future improvement of warfarin therapy.     

Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

Honokiol inhibits HepG2 migration via down‐regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis

Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti‐tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture‐based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase‐activating‐like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53‐fold down‐regulated under 10 μg/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti‐tumor activity.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type‐1 (HSV‐1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV‐1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV‐1 replication, 2‐DE and LC‐MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock‐ and HSV‐1‐infected HEp‐2 cells revealed a total of 103 protein spot changes. Of these, 63 were up‐regulated and 40 down‐regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV‐1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV‐1 infection.     

裂蹄木层孔菌子实体水提物诱导HepG2细胞凋亡的初步研究

研究裂蹄木层孔菌子实体水提物(WEPL)对人类克隆肝癌细胞系HepG2生长的作用。用裂蹄木层孔菌子实体水提物处理HepG2细胞后,噻唑蓝法(MTT法)可见浓度和时间依赖性抑制细胞增殖;电镜下观察凋亡小体的出现,流式细胞仪技术显示Annexin-Ⅴ染色呈阳性,都证明了HepG2细胞发生了凋亡。RT-PCR和Western Blot分析证实WEPL刺激Bax表达量上调、Bcl-2表达量下调进而诱导了细胞凋亡。结果表明WEPL诱发的克隆人类肝癌细胞系HepG2的细胞凋亡可能是通过上调Bax、下调Bcl-2活性来实现的。     

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells

Human HepG2 cells were exposed to six TiO₂ nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 μg/mL) for 3 days. Some of these canonical pathways changed by nano‐TiO₂ in vitro treatments have been already reported in the literature, such as NRF2‐mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer‐related signaling pathways. More importantly, this genomic analysis of nano‐TiO₂ treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2‐mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.     

The effect of adrenocorticotropic hormone on alpha‐2‐macroglobulin in osteoblasts derived from human mesenchymal stem cells

Nowadays, alpha‐2‐macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic‐related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10^?8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real‐time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma‐carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up‐regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up‐regulates A2M, which promotes osteoblastic differentiation probably through TGF‐β induction.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

Down‐regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L^?1, 50 μL), the combination with metformin (60 mmol L^?1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.     

Comparative two‐dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two‐dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P‐value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC‐MS/MS. Alpha‐amylase, CREB‐binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down‐regulated in ASD. Increased expression of proto‐oncogene Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin‐inducible protein precursor, Mucin‐16, Ca binding protein migration inhibitory factor‐related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.     

The algal metabolite yessotoxin affects heterogeneous nuclear ribonucleoproteins in HepG2 cells

The dinoflagellate metabolite yessotoxin (YTX) is produced by several species of algae and accumulates in marine food chains, leading to concerns about possible affects on aquaculture industries and human health. In mice used for toxicity testing, YTX is lethal by the intraperitoneal route, but is considerably less toxic when orally administered. The mode of action of YTX and its potential effect on humans is unclear and we therefore conducted the first proteomic analysis of the effects of this compound. We used 2‐DE to examine protein changes in HepG2 cell cultures exposed to 1.4 μM YTX for 3, 12.5, 18 and 24 h. After selecting proteins that changed more than three‐fold after YTX exposure, 55 spots were deemed significantly affected by the toxin (p<0.05). Major groups of affected proteins include members from the heterogeneous nuclear ribonucleoprotein (hnRNP), lamin, cathepsin and heat shock protein families that often are associated with apoptosis. We therefore confirmed apoptosis using Annexin‐V‐FLUOS staining of phosphatidylserine exposed at the surface of apoptotic cells. Ingenuity pathways analysis also indicated effects on pathways involved in protein processing, cell cycling and cell death.