Anti-inflammatory effect of transduced PEP-1-heme oxygenase-1 in Raw 264.7 cells and a mouse edema model

Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe²⁺), is up-regulated by several cellular stress and cell injuries, including inflammation, ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60 h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.     

Systemic analysis of heat shock response induced by heat shock and a proteasome inhibitor MG132

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF-1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins.     

Differentially expressed proteins in cerulein-stimulated pancreatic acinar cells: implication for acute pancreatitis

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.     

Proteomics analysis of kojic acid treated A375 human malignant melanoma cells

Although the toxicogenomics of kojic acid treated A375 human malignant melanoma cells has been elucidated, the proteomics of cellular response is still poorly understood. We performed proteomic analysis to investigate the anticancer effect of kojic acid on protein expression profile in A375 cells. A375 cells were treated with kojic acid at 8 microg/mL for 24, 48, and 72 h. With the use of 2-D PAGE and MALDI-Q-TOF MS and MS/MS analyses, proteomic profiles of A375 cells between control and kojic acid treatment were compared, and 30 differentially expressed proteins, containing 2 up-regulated proteins and 28 down-regulated proteins, were identified. Among these proteins, 17 isoforms of 5 identical proteins were observed and 11 chaperone proteins showed the high proportion of protein spots with 36.7% of total proteins. Bioinformatic tools were used to search for protein function and prediction of protein interaction. Sixteen differentially expressed proteins exhibited interaction network linked to the downstream regulations of p53 tumor suppressor and cell apoptosis, which may lead to suppress the melanogenesis and tumorigenesis of kojic acid treated A375 cells. In addition, GRP75, VIME and 2AAA were validated by Western blot analysis, whereas GRP75, 2AAA, HS90B, ENPL and KPYM were validated by RT-PCR. Therefore, these proteins play the important roles in cancer progression and may be potential biomarkers that are useful for diagnostic and therapeutic applications of malignant melanoma cancer.     

Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.     

Integrative proteomic and microRNA analysis of primary human coronary artery endothelial cells exposed to low-dose gamma radiation

High doses of ionising radiation significantly increase the risk of cardiovascular disease (CVD), the vascular endothelium representing one of the main targets. Whether radiation doses lower than 500 mGy induce cardiovascular damage is controversial. The aim of this study was to investigate radiation-induced expression changes on protein and microRNA (miRNA) level in primary human coronary artery endothelial cells after a single 200 mGy radiation dose (Co-60). Using a multiplex gel-based proteomics technology (2D-DIGE), we identified 28 deregulated proteins showing more than ±1.5-fold expression change in comparison with non-exposed cells. A great majority of the proteins showed up-regulation. Bioinformatics analysis indicated “cellular assembly and organisation, cellular function and maintenance and molecular transport” as the most significant radiation-responsive network. Caspase-3, a central regulator of this network, was confirmed to be up-regulated using immunoblotting. We also analysed radiation-induced alterations in the level of six miRNAs known to play a role either in CVD or in radiation response. The expression of miR-21 and miR-146b showed significant radiation-induced deregulation. Using miRNA target prediction, three proteins found differentially expressed in this study were identified as putative candidates for miR-21 regulation. A negative correlation was observed between miR-21 levels and the predicted target proteins, desmoglein 1, phosphoglucomutase and target of Myb protein. This study shows for the first time that a low-dose exposure has a significant impact on miRNA expression that is directly related to protein expression alterations. The data presented here may facilitate the discovery of low-dose biomarkers of radiation-induced cardiovascular damage.     

Profiling of differential protein expression in angiogenin-induced HUVECs using antibody-arrayed ProteoChip

ProteoChip has been developed as a novel protein microarray technology. So far it has been applied in new lead screening and molecular diagnostics and we expect its role to grow in the field of biology. Here, we investigated the application of ProteoChip for the study of differential protein expression profiles in angiogenin-induced human umbilical vein endothelial cells (HUVECs). Antibody microarrays constructed by immobilizing 60 distinct antibodies against signal-transducing proteins on ProteoChip base plates were used to analyze the expression pattern of cell-signaling proteins in HUVECs treated with angiogenin. The antibody microarray approach showed that angiogenin induced the up- and down-regulation of several cellular regulators related with cell proliferation. Changes in the expression of signaling proteins determined by antibody microarray were validated by Western blot analysis. In this experiment, ten up-regulated proteins and six down-regulated proteins were identified and confirmed by immunoblot analysis. Taken together, these data suggest that antibody microarrays using ProteoChip technology can be a powerful tool for high-throughput analysis of proteomes in biological samples.     

穿心莲内酯衍生物抗H2O2诱导胰岛RIN-mβ细胞凋亡的蛋白组学研究

采用蛋白组学方法筛选穿心莲内酯衍生物(AL-1)抗过氧化氢诱导胰岛RIN-mβ细胞凋亡的差异蛋白质分子并探讨其作用分子机制.结果显示:AL-1浓度依赖性地提高H₂O₂处理的胰岛RIN-mβ细胞的存活率.经蛋白组学研究分析,成功地鉴定了18个与凋亡、应激等相关的蛋白,包括Prohibitin、Shmt2、RhoGDP-dissociationinhibitor-1、Galectin-1、Cyt b5、Hsps等;与对照组(H₂O₂)相比,处理组(AL-1+H₂O₂)中,有9个表达上调的蛋白和9个表达下调的蛋白.AL-1通过调控与细胞凋亡、应激等相关的蛋白发挥其抗H₂O₂诱导的凋亡作用.     

TAB182对电离辐射诱发细胞周期G2/M阻滞的调节作用

TAB182是一个端锚聚合酶1（tankyrase 1）结合蛋白,它在体外能够被tankyrase 1发生二磷酸腺苷核糖基化(PAR)修饰,其生物学功能目前尚不明确.本研究发现,TAB182蛋白水平受电离辐射诱导表达,HeLa细胞经过4 Gy照射处理时,TAB182在2 h表达含量最高; 经过不同剂量照射处理,2 h后2 Gy、4 Gy照射剂量组HeLa细胞中TAB182的表达有明显增加. 通过shRNA沉默HeLa细胞中TAB182基因表达,导致其对4 Gy及以下剂量 辐射的敏感性增加,但对8 Gy大剂量照射的敏感性没有明显变化. 与对照组相比,4 Gy照射诱发TAB182基因沉默细胞的G2/M期阻滞时间显著延长.抑制TAB182表达导致细胞中DNA损伤反应蛋白DNA PKcs、ATM、Chk2的表达水平显著降低. 实验结果提示,TAB182蛋白参与放射DNA损伤信号反应和调控细胞周期G₂/M进程.     

Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death

Up-regulation of several stress proteins such as heat-shock proteins and glucose-regulated proteins participate in tolerance against environmental stress. Previously, we found that protein-disulfide isomerase (PDI) is specifically up-regulated in response to hypoxia/brain ischemia in astrocytes. In addition, the overexpression of this gene into neurons protects against apoptotic cell death induced by hypoxia/brain ischemia. To address the detailed function of PDI, we screened for proteins that interact with PDI using the yeast two-hybrid system. We report here that PDI interacts with ubiquilin, which has a ubiquitin-like domain and a ubiquitin-associated domain. Interestingly, ubiquilin is also up-regulated in response to hypoxia in glial cells with a time course similar to that of PDI induction. In hypoxia-treated glial cells, the endogenous ubiquilin and PDI were almost completely co-localized, suggesting that ubiquilin is an endoplasmic reticulum-associated protein. Overexpression of this gene in neuronal cells resulted in significant inhibition of the DNA fragmentation triggered by hypoxia, but not that induced by nitric oxide or staurosporine. Moreover, ubiquilin has the ability to attenuate CHOP induction by hypoxia. These observations suggested that ubiquilin together with PDI have critical functions as regulatory proteins for CHOP-mediated cell death, and therefore up-regulation of these proteins may result in acquisition of tolerance against ischemic stress in glial cells.     

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.     

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.     

Protein profile in HBx transfected cells: A comparative iTRAQ-coupled 2D LC-MS/MS analysis

The x protein of HBV (HBx) has been involved in the development of hepatocellular carcinoma (HCC), with a possible link to individual genotypes. Nevertheless, the underlying mechanism remains obscure. In this study, we aim to identify the HBx-induced protein profile in HepG2 cells by LC-MS/MS proteomics analysis. Our results indicated that proteins were differentially expressed in HepG2 cells transfected by HBx of various genotypes. Proteins associated with cytoskeleton were found to be either up-regulated (MACF1, HMGB1, Annexin A2) or down-regulated (Lamin A/C). These may in turn result in the decrease of focal adhesion and increase of cell migration in response to HBx. Levels of other cellular proteins with reported impact on the function of extracellular matrix (ECM) proteins and cell migration, including Ca²⁺-binding proteins (S100A11, S100A6, and S100A4) and proteasome protein (PSMA3), were affected by HBx. The differential protein profile identified in this study was also supported by our functional assay which indicated that cell migration was enhanced by HBx. Our preliminary study provided a new platform to establish a comprehensive cellular protein profile by LC-MS/MS proteomics analysis. Further downstream functional assays, including our reported cell migration assay, should provide new insights in the association between HCC and HBx.     

Proteomic identification of heat shock protein 70 as a candidate target for enhancing apoptosis induced by farnesyl transferase inhibitor

Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 microM manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 microM quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.     

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.