Proteomic and ultrastructural analysis of Clara cell and type II alveolar epithelial cell-type lung cancer cells.

BACKGROUND: Currently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling. METHODS: Six human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed. RESULTS: NCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers. CONCLUSIONS: Cytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.

Identification of chaperones in a MPP+-induced and ATRA/TPA-differentiated SH-SY5Y cell PD model.

Parkinson's disease (PD) is characterized by the pathological accumulation of misfolded proteins. Molecular chaperones assist in the proper folding of proteins and removal of irreversibly misfolded proteins. This study aims to identify potential chaperones associated with protein misfolding and accumulation in PD. ATRA/TPA-differentiated SH-SY5Y cells were treated with 1 mM of MPP+ for 48 hours. Proteins were analyzed by 2D-DIGE followed by MALDI-ToF MS. The treatment of differentiated SH-SY5Y cells by MPP+ led to the unambiguous identification of 10 protein spots, which corresponds to six proteins. Among these six proteins, four were chaperone proteins including nucleophosmin (NPM1), chaperonin-containing TCP-1 subunit 2 (CCT2 or CCTß), heat shock 90 kDa protein 1 beta (HSP90AB1 or HSP90-ß), and tyrosin3/tryptopha5-monoxygenase activation protein, zeta polypeptide (14-3-3ζ, gene symbol: Ywhaz). To our knowledge, this is the first report that linked the upregulation of chaperones after MPP+ treatment with SH-SY5Y cells. However, the NPM1 protein was identified for the first time in the PD model. The upregulation of four chaperone proteins provided evidence that these chaperones have a complementary effect on protein misfolding in the pathogenesis of PD, and hold promise as a good therapeutic target for PD treatment.

Proteomic analysis of endothelial progenitor cells exposed to oxidative stress.

Endothelial progenitor cells (EPCs) repair vascular damage and participate in neovascularization. Accumulating evidence has demonstrated that EPCs have therapeutic potential in reactive oxygen species (ROS)-mediated vascular diseases. In this study, to investigate the effects of oxidative stress on EPCs, EPCs were treated with H2O2 at different final concentrations for 3 h. MTT assay, scratch-wound assay and Matrigel invasion assay revealed that cell proliferation, migration and tubule formation and function, respectively, were impaired under H2O2 stress in a concentration-dependent manner. To determine protein response to H2O2 stress, two-dimensional differential in-gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry were performed. The results revealed that triosephosphate isomerase and ADP-sugar pyrophosphatase were downregulated, while peroxiredoxin-2, thioredoxin-dependent peroxide reductase, mitochondrial (Prx­3), peroxiredoxin-6, EGF-containing fibulin-like extracellular matrix protein 1, vimentin and Rab GDP dissociation inhibitor α were upregulated in the H2O2-treated EPCs. To further confirm the results from mass spectrometry, the expression pattern of Prx-3 in response to H2O2 stress was examined by western blot analysis. The data presented in this study provide novel insight into the defensive mechanisms of EPCs and the pathways of oxidative damage in an oxidative environment.

α-lipoic acid protects dopaminergic neurons against MPP+-induced apoptosis by attenuating reactive oxygen species formation.

Reactive oxygen species (ROS) elicited by oxidative stress are widely recognized as a major initiator in the dege-neration of dopaminergic neurons distinctive of Parkinson's disease (PD). The interaction of ROS with mitochondria triggers sequential events in the mitochondrial cell death pathway, which is thought to be responsible for ROS-mediated neurodegeneration in PD. α-lipoic acid (LA) is a pleiotropic compound with potential pharmacotherapeutic value against a range of pathophysiological insults. Its protective actions against oxidative damage by scavenging ROS and reducing production of free radicals have been reported in various in vitro and in vivo systems. This study analyzed the ability of LA to protect PC12 neuronal cells from toxicity of 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which is known to kill dopaminergic neurons selectively and to cause severe parkinsonism-like symptoms in humans and primate animals. Our results demonstrate that the apoptosis of PC12 cells elicited by MPP+ could be significantly prevented by pretreatment with LA for 1 h. In addition, LA inhibits intercellular ROS levels and the mitochondrial transmembrane permeability, the key players in the pathogenesis of PD, thereby protecting dopaminergic neuronal cells against oxidative damage.

Contribution of endothelial progenitor cells to neovascularization (Review).

Endothelial progenitor cells (EPCs) are a cell population mobilized from bone marrow into the peripheral circulation and recruited into sites of vessel injury to participate in blood vessel formation in both physiological and pathological conditions. Due to the lack of unique surface markers and different isolation methods, EPCs represent heterogeneous cell populations including cells of myeloid or endothelial origin. Evidence suggests that EPCs play a critical role in postnatal blood vessel formation and vascular homeostasis and provide a promising therapy for vascular disease. However, the mechanisms by which EPCs participate in new vessel formation are still incompletely understood. We review the process of EPCs in neovascularization including EPC mobilization, migration, adhesion and effect on new vessel formation, in an attempt to better understand the underlying mechanisms and to provide potential effective management for the treatment of patients with vascular disease.

Beneficial effects of statins on endothelial progenitor cells.

In recent years, endothelial progenitor cells (EPCs) have been demonstrated to play an important role during tissue vascularization and endothelium homeostasis in adults. In addition, EPCs have been implicated in the pathophysiology of cardiovascular and cerebrovascular disease, such that a decreased number of EPCs may not only be a risk indicator but also a potential therapeutic target. Of the many agents that have been examined to increase EPCs and enhance their function, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are one of the most intriguing. Accumulated evidence has demonstrated that statins promote EPC mobilization, proliferation, migration, adhesion, differentiation and reduce senescence and apoptosis independent of their serum lipid-lowering effect. This review summarizes the understanding of current mechanisms explaining the myriad of beneficial effects of statins on EPCs and discusses future challenges for studies involving statins and subpopulations of EPCs. However, the pharmacologic mechanisms of action of statins on EPCs remain at the cellular level, whereas the putative molecular mechanisms await further studies.

Proteomic analysis of oxidative modification in endothelial colony-forming cells treated by hydrogen peroxide.

Endothelial progenitor cells (EPCs) which circulate in the peripheral blood and reside in blood vessels are proven to promote the repair of damaged endothelium and improve the function of endothelial cells after vascular injury. Recently, EPCs have been extensively studied as risk biomarkers and a potential therapeutic tool for cardiovascular disease. It is known that oxidative stress is one of the most important pathogenetic factors impairing endothelial function. During the repair process after endothelial injury, EPCs are exposed to oxidative stress. In this study, we treated endothelial colony-forming cells (ECFCs) with hydrogen peroxide (H2O2) as an oxidative stress model and observed the changes in cytology and morphology of ECFCs. In addition, we investigated the alterations in oxidative levels of proteins associated with H2O2-induced morphological and cytological changes in ECFCs by proteomic analysis of oxidative modification. The results showed that H2O2 treatment led to a decreased proliferation, increased apoptosis and impaired tube-forming ability of ECFCs in a dose-dependent manner. Five proteins with upregulated oxidative levels were identified successfully. The upregulated oxidative levels of these five proteins may be responsible for the dysfunction of ECFCs under oxidative stress. Our results may provide some novel insights into the molecular mechanisms of oxidative stress action on ECFCs.

HDAC6 α-tubulin deacetylase: a potential therapeutic target in neurodegenerative diseases.

Histone deacetylases (HDACs), or lysine deacetylases (KDAC), are epigenetic regulators that catalyze the removal of acetyl moieties from the tails of lysine residues of histones and other proteins. To date, eighteen HDAC family members (HDAC1-11 and SIRT1-7) have been identified and grouped into four classes according to their homology to yeast histone deacetylases. HDACs play an important role in regulating gene transcription as well as a variety of cellular functions. Recent studies have found that HDAC6 (α-tubulin deacetylase) has the novel ability to capture α-tubulin as a substrate and regulate the physiological level of its acetylated form. In addition, a growing body of evidence suggests that α-tubulin deacetylase plays a critical role in the cellular response to the accumulation of misfolded and aggregated proteins, which are a prominent pathological feature common to many age-related neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. Therefore, the role of α-tubulin deacetylase and its potential as a therapeutic target for neurodegenerative diseases are areas of rapidly expanding investigation. Here we review the research of the role played by HDAC6 in the regulation of tubulin modification and aggresome formation. We also summarize the specific inhibitors of HDAC6 and address reports that implicate HDAC6 in various neurodegenerative disorders.

Proteomics analysis of methylglyoxal-induced neurotoxic effects in SH-SY5Y cells.

Reactive carbonyl compounds contribute to aging, Alzheimer's disease (AD) and other neurodegenerative diseases. Among these compounds, methylglyoxal (MG) can yield advanced glycation end products (AGEs), which are crucial in AD pathogenesis. However, the molecular and biochemical mechanisms of MG neurotoxicity are not completely understood. In the present study, SH-SY5Y cells were treated with MG to induce cell death. 2-D Fluorescence Difference Gel Electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry were employed to determine the changes in protein levels in these cells compared with vehicle-treated cells. Proteomics analysis revealed that 49 proteins were differentially expressed in MG-treated SH-SY5Y cells, of which 16 were upregulated and 33 were downregulated. Among them, eight proteins were identified unambiguously. The significant changes in protein levels of actin, immunoglobulin lambda light chain and protein phosphatase 2 were noteworthy given their functional roles in AD pathogenesis. Taken together, our results suggest that multiple pathways are potentially involved in MG-induced neuron death.

Proteomics analysis of MPP+-induced apoptosis in SH-SY5Y cells.

Accumulating evidence suggests that oxidative stress plays a pivotal role in dopaminergic neurodegeneration. However, the kinds of proteins involved in the response to oxidative stress remain unclear. In the present study, SH-SY5Y cells were treated with neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) to induce apoptotic neuronal injury. 2D-DIGE followed by MALDI-TOF-MS was used to determine the changing protein levels. Proteomics analysis revealed that 22 proteins were differentially altered in MPP(+)-treated SH-SY5Y cells, of which 7 were up-regulated proteins and 15 were down-regulated proteins, respectively. Three protein spots were unambiguously identified as sorcin, annexin V, and ribosomal protein P0. The three proteins showed a significant increase in level, suggesting a role in MPP(+)-induced apoptosis. The functional roles of these three proteins collectively indicate that multiple mechanisms are pertinent in the underlying pathogenesis of Parkinson's disease (PD), such as apoptosis, calcium homeostasis, and DNA insults.

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI.

BACKGROUND: Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. RESULTS: To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. CONCLUSIONS: It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

OBJECTIVE: To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. DATA SOURCES: The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). STUDY SELECTION: After searching the literature, 60 articles were selected to address this review. RESULTS: The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. CONCLUSIONS: Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis.

Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled. Proteomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 microM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and calcium-binding protein 1 were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.

A genetic study of the NOS3 gene for ischemic stroke in a Chinese population.

We recruited 560 unrelated patients with ischemic stroke and 153 unrelated controls to undertake a genetic analysis for association between the NOS3 gene and ischemic stroke. All the subjects were Chinese of Han descent. Because the NOS3 gene spans about 21 kb of DNA and contains 26 exons, we selected a single nucleotide polymorphism (SNP) rs3918181, an A to G base change located in intron 14 of the gene, as a DNA marker. PCR-based restriction fragment length polymorphism analysis was applied to genotype rs3918181 (RsaI site). The chi-square (chi(2)) goodness-of-fit test showed that the genotypic distributions of the marker were not deviated from Hardy-Weinberg equilibrium in both the patient group (chi(2) = 0.166, p = 0.684) and the control group (chi(2) = 0.421, p = 0.517). The cocaphase analysis showed allelic association of rs3918181 with ischemic stroke in males (chi(2) = 4.04, p = 0.044, OR = 1.43, 95% CI 1.01 approximately 2.02) and frequency of allele A was significantly higher in male patients than male control subjects. The chi(2) test revealed genotypic association between rs3918181 and ischemic stroke in males (chi(2) = 4.26, df = 1, p = 0.039, OR = 1.61, 95% CI 1.02 approximately 2.53) but not in females. The present work suggests that rs3918181 is associated with ischemic stroke in male patients. This finding gives further evidence in support of the eNOS association with ischemic stroke in the Chinese population.

Proteomic changes of PC12 cells treated with proteasomal inhibitor PSI.

Growing evidences suggest that the ubiquitin-proteasome system dysfunction may play an important role in the pathogenesis of Parkinson's disease (PD). In order to investigate the protein changes caused by ubiquitin-proteasome dysfunction in PD, we used a proteomic approach to determine the different protein levels in PC12 cells following proteasomal inhibitor PSI treatment. Twenty-four hour treatment of PC12 cells with PSI induced cell apoptosis and the appearance of cytoplasmic Lewy body-like eosinophilic inclusions, thus recapitulating two primary features of PD. Six protein spots whose contents were changed in response to PSI administration were unambiguously identified as: 94 kDa glucose-regulated protein (GRP94), heat shock 70 kDa protein 5 (GRP78), heat shock 27 kDa protein 1 (Hsp27), aldehyde reductase 1 (aldose reductase), p47 protein and beta-galactoside-binding lectin (galectin-1). They are mainly related with endoplasmic reticulum stress, cellular metabolism and defensive response against toxicity with the last two whose function is unknown in this model. Out of these proteins, some were described for the first time in relation to proteasomal inhibition and PD. These results may provide a valuable clue to the further exploration of the pathogenetic mechanism of PD.

Proteomic analysis of kidney in fluoride-treated rat.

The recent development of proteomic techniques has enabled investigators to directly examine the population of proteins present in biological systems. We first report here the proteomic changes of renal protein induced by fluoride. To investigate molecular mechanisms of renal injury induced by fluoride, proteins were isolated from rat kidney and profiled by two-dimensional gel electrophoresis (2DE). With the analysis of Image-Master 2D Elite software, 141 up-regulated and eight down-regulated protein spots in 2DE gels of fluoride-treated group were gained by comparison to the control group, 13 of which were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The identified proteins are mainly related with cell proliferation, metabolism and oxidative stress, and provide a valuable clue to explore the mechanism of renal fluorosis. This study also shows that the proteomic techniques were powerful in fluoride toxical field.

Clinical and experimental pathology of Moyamoya disease.

OBJECTIVE: To investigate the etiology, pathology, and mechanism of pathogenesis of Moyamoya disease. METHODS: A total of 15 human autopsies were analyzed. In addition, in order to create an animal model of the disease, 21 Japanese rabbits were divided randomly into two groups and subjected to injections of horse serum either intravenously or locally in the area of the sympathetic ganglia. Pathological and immunohistochemical characteristics were observed. RESULTS: The pathological features of the autopsies and the animal models both involved intima hyperplasia and stenosis or even occlusion of the lumen in the terminal ends of the internal carotid artery and the anterior and middle cerebral arteries. Disconnections or even breakages of the inner layer of the lumen were also observed, without an obvious inflammatory response. Hyperplasic smooth muscle cells of the medial membrane had extended inward through broken portions of the internal elastic lamina, with intima cell hyperplasia resulting in lumen stenosis. The hyperplastic vascular walls were positive for IgG and IgM. CONCLUSIONS: The etiology of Moyamoya disease may involve allergic angiitis. A possible mechanism is that proximal portions of the circle of Willis first develop chronic stenosis or occlusion, leading to compensatory small vessel proliferation, which perforates into the cerebral parenchyma.