Jianjun Sui

Protein expression profiles in osteoblasts in response to differentially shaped hydroxyapatite nanoparticles.

The use of synthetic hydroxyapatite as bone substitute calls for the knowledge of the influence on adjacent cells. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of human osteoblast cell line incubated separately with various nano sized hydroxyapatite powders with different shapes and chemical compositions using iTRAQ-coupled 2D LC-MS/MS approach. In the present study, we investigated several intracellular signaling molecules involved in calcium regulation to analyze how osteoblast cells respond to dissimilar HA nanoparticles. It was found there was a significant decrease in cell population after adding the HA nanoparticles to the osteoblasts. Our results combining proteomics analysis and RT-PCR validation on targeted genes involved in calcium regulation confirmed the differences in the cellular response to dissimilar HA nanoparticles.

Cytotoxicity Evaluation of Oxidized Single-Walled Carbon Nanotubes and Graphene Oxide on Human Hepatoma HepG2 cells: An iTRAQ-Coupled 2D LC-MS/MS Proteome Analysis

Because of their attractive chemical and physical properties, graphitic nanomaterials and their derivatives have gained tremendous interest for applications in electronics, materials, and biomedical areas. However, few detailed studies have been performed to evaluate the potential cytotoxicity of these nanomaterials on living systems at the molecular level. In the present study, our group exploited the isobaric tagged relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) approach with the purpose of characterizing the cellular functions in response to these nanomaterials at the proteome level. Specifically, the human hepatoma HepG2 cells were selected as the in vitro model to study the potential cytotoxicity of oxidized single-walled carbon nanotubes (SWCNTs) and graphene oxide (GO) on the vital organ of liver. Overall, 30 differentially expressed proteins involved in metabolic pathway, redox regulation, cytoskeleton formation, and cell growth were identified. Based on the protein profile, we found oxidized SWCNTs induced oxidative stress and interfered with intracellular metabolic routes, protein synthesis, and cytoskeletal systems. Further functional assays confirmed that oxidized SWCNTs triggered elevated level of reactive oxygen species (ROS), perturbed the cell cycle, and resulted in a significant increase in the proportion of apoptotic cells. However, only moderate variation of protein levels for the cells treated with GO was observed and functional assays further confirmed that GO was less cytotoxic in comparison to oxidized SWCNTs. These finding suggested that GO was more biocompatible and could be a promising candidate for bio-related applications.

Comparative Proteomics Analysis of Vascular Smooth Muscle Cells Incubated with S- and R-Enantiomers of Atenolol Using iTRAQ-coupled Two-dimensional LC-MS/MS

Atenolol is a β1-selective drug, which exerts greater blocking activity on β1-adrenoreceptors than on β2-adrenoreceptors, with the S-enantiomer being more active than R-enantiomer. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using an iTRAQ-coupled two-dimensional LC-MS/MS approach. Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b5 reductase, and α-N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with the S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b5 reductase in the intracellular anabolic activity was validated by NAD+/NADH assay with a higher ratio of NAD+/NADH correlating with a higher proportion of NAD+. The down-regulation of the calcium-binding proteins was possibly involved in the lower intracellular Ca2+ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca2+ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modeling. Our preliminary results thus provide molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV‐induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2‐D LC‐MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell‐based HBV replication was established in both RPHs and HepG2 cells. HBV replication‐induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl‐tRNA synthetase, were identified by 2‐D LC‐MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis‐associated proteins identified in our study may eventually lead to novel anti‐angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.

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.

Protein profile in neuroblastoma cells incubated with S-and R-enantiomers of ibuprofen by iTRAQ-coupled 2-D LC-MS/MS analysis : Possible action of induced proteins on Alzheimer's disease

Ibuprofen is a member of the proprionic acid group of nonsteroidal anti‐inflammatory drugs (NSAID), with the S‐enantiomer being more active than the R‐enantiomer. It has been shown to display protective effects against neuroinflammation, which is linked to the pathogenesis of several neurodegenerative disorders, including Alzheimers disease (AD). While its prophylactic effect on AD has been suggested, a comprehensive understanding of its mechanism of action remains unclear. Using iTRAQ‐coupled 2‐D LC‐MS/MS analysis, we report here the first study of protein profiles of neuroblastoma cells incubated separately with the two enantiomers of ibuprofen. Three types of cellular proteins, including metabolic enzymes, signaling molecules and cytoskeletal proteins, displayed changes. The changes in the level of a number of enzymes involved in fatty acid synthesis and antioxidant activity in cells incubated with the S‐enantiomer were further supported by the real‐time PCR analysis as well as the reduced level of reactive oxygen species in cells incubated with the S‐enantiomer of ibuprofen. Our findings, therefore, provide the possible mechanism of ibuprofen‐induced proteins on AD, and the beneficial effects of ibuprofen in reducing the development of AD.

Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials : an iTRAQ-coupled 2-D LC-MS/MS analysis

Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ‐coupled 2‐D LC‐MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.

Comparative proteomic analysis of extracellular proteins reveals secretion of T-kininogen from vascular smooth muscle cells in response to incubation with s-enantiomer of propranolol.

Propranolol, a nonselective beta blocker, exerts blocking activity both on beta1 adrenoceptors and beta2 ones, with the S-enantiomer being more active than the R-enantiomer. The aim of the study was to investigate the secreted proteins with differential protein expression levels in culture medium of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of propranolol using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional LC-MS/MS approach. Our results indicated that secretion of T-kininogen by S-enantiomer of propranolol incubated cells was greatly enhanced as compared with that of R-enantiomer incubated cells or control cells. It can be inferred that the S-enantiomer of propranolol will induce more Ile-Ser-bradykinin (BK) (T-kinin), the vasoactive peptides. This therefore provides molecular evidence and possible link of T-kininogen with treatment of cardiovascular disease associated with propranolol treatment.

Cytotoxicity of single-walled carbon nanotubes on human hepatoma HepG2 cells: An iTRAQ-coupled 2D LC–MS/MS proteome analysis

Single-walled carbon nanotubes (SWCNTs) and its derivatives are promising candidates for applications in electronics, energy, materials and biomedical areas. However, with the growing potential biomedical applications and the rising societal concerns on nanosafety, mechanistic understanding of the interactions between nanomaterials and living systems has become imperative. In the present study, our group applied the iTRAQ-coupled 2D LC-MS/MS approach to analyze the protein profile change of mammalian cells in response to SWCNTs. Specifically, the human hepatoma HepG2 cells were chosen as the in vitro model to study the potential cytotoxicity of SWCNTs on the vital organ of liver. Overall 51 differentially expressed proteins that involved in metabolic pathway, redox regulation, signaling pathway, cytoskeleton formation and cell growth were identified. We found SWCNTs triggered the up-regulation of metabolic enzymes, heat shock proteins and proteins involved in redox regulation, which indicated SWCNTs could induce oxidative stress, perturb protein synthesis and interfere cellular metabolism. Our data also suggested that SWCNTs might induce the activation of apoptosis signal-regulating kinase 1, and finally lead to stress-induced apoptosis. The comparative protein profile obtained here provided molecular evidence on the cellular functions in response to SWCNTs, which should very useful to elucidate the cytotoxicity caused by those nanomaterials.

Investigation of Multiwall Carbon Nanotube Modified Hydroxyapatite on Human Osteoblast Cell Line Using iTRAQ Proteomics Technology

Hydroxyapatite (HA) is a bioactive ceramic material with a chemical composition similar to natural bone, and carbon nano tubes (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. This study reported an attempt to use a commercially multiwalled CNT strengthen brittle hydroxyapatite bioceramics. Using iTRAQ-coupled 2D LCMS/ MS analysis, we report the first study of protein profile in osteoblasts from human osteoblastic cell line incubated separately on HA with and without strengthening multiwall CNT surfaces. Sixty proteins were identified and quantified simultaneously at the initial culturing stage of 3 days. The results were validated by Western blotting for selected proteins: Fetuin-A, Elongation factor II and Peroxiredoxin VI. Fetuin-A showed up-regulation, and Peroxiredoxin VI gave down-regulation in the osteoblasts cultured on HA based ceramic surfaces. Similar regulation was expressed by the protein of Elongation factor II on the phase pure HA surfaces as compared to the control group cultured on the polystyrene substrate. Relatively high EF 2 expression was detected on the phase the surfaces of CNT strengthen HA samples.