[Show abstract][Hide abstract] ABSTRACT: Objective:
To develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic analysis.
A subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry.
Four extracted fractions clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, integrin-β1, histone H1 and cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by mass spectrometry as cytoskeleton and/or its associated proteins.
The subcellular proteome sequential fractionation method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2013; 33(5):698-702.
[Show abstract][Hide abstract] ABSTRACT: A novel electrochemical biosensor was developed for the analysis of BRAF V600E mutation in colorectal cancer cell samples based on a dual amplification strategy of amplification-refractory mutation system (ARMS) PCR and multiple enzyme labels. The labeled amplicons were conjugated on Fe(3)O(4)/Au nanoparticles using Au-S linkages. Alkaline phosphatases were then loaded onto the nanoparticles through biotin-streptavidin interactions. The resultant composite nanoparticles were characterized by transmission electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. In the presence of 2-phospho-l-ascorbic acid, the mutant alleles were quantified on a screen-printed carbon electrode (SPCE) from the anodic current of the enzymatic product, ascorbic acid. BRAF V600E mutant alleles concentrations as low as 0.8% were successfully determined in an excess of wild-type background. In a cell-line dilution model, the proposed method was more sensitive than were DNA sequencing and agarose gel electrophoresis. This work demonstrates a new strategy for sensitivly detecting BRAF V600E variations. It can pave the way for analyzing other rare mutations in complex cancer samples because of its high sensitivity, simplicity, low cost, and easy validation of assay procedures.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effect of simulated microgravity on the proliferation of human monocytic cells THP-1 and the expression of tissue factor (TF) mRNA.
THP-1 cells were cultured under a simulated microgravity environment using the rotating cell culture system (RCCS). The changes in the cell proliferation after microgravity culture were assessed by cell counting and cell cycle analysis with flow cytometry. RT-PCR was used to detect the changes in the expression of TF mRNA in THP-1 cells.
Culture under simulated microgravity resulted in a significant decrease in the cell number of THP-1 cells in comparison with that of the control cells (P<0.01). After a 24-h culture under microgravity, the G0-Gl phase cells increased from the control level of (46.57∓1.64)% to (67.64∓2.71)% (P<0.05). The cells in both groups showed a low level of TF mRNA expression in the absence of LPS stimulation. A 4-h stimulation with LPS caused up-regulated expression of TF mRNA in both cells, but the microgravity group showed a significantly smaller increase in the expression (2.301∓0.179) than the control group (9.210∓1.328) (P<0.05).
Microgravity can inhibit the proliferation of THP-1 cells and suppress the cellular expression of TF mRNA.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 06/2011; 31(6):1020-2.
[Show abstract][Hide abstract] ABSTRACT: Deciphering the molecular basis of esophageal cancer metastasis requires adequate experimental models. Epithelial-mesenchymal transition (EMT) is the hallmark of tumor metastasis. As a promoter of the malignant progression of esophageal cancer, epidermal growth factor (EGF) has been shown to induce EMT in several cell lines. In this study we examined the effects of EGF on esophageal carcinoma EC109 cells. We found that EGF at high concentration induced the cells to undergo morphological change, exhibit higher invasive and metastatic potential, as well as change in the expression of lineage markers. This EMT model might facilitate mechanistic studies of esophageal cancer metastasis.
Cancer letters 10/2010; 296(1):88-95. DOI:10.1016/j.canlet.2010.03.020 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPCs) contribute to tumor angiogenesis and growth. We aimed to determine whether inhibitors of differentiation 1 (Id1) were expressed in circulating EPCs of patients with ovarian cancer, whether Id1 could mediate EPCs mobilization and recruitment, and, if so, what underlying signaling pathway it used.
Circulating EPCs cultures were from 25 patients with ovarian cancer and 20 healthy control subjects. Id1 and integrin α4 expression were analyzed by real-time reverse transcription-polymerase chain reaction and western blot. EPCs proliferation, migration, and adhesion were detected by MTT, transwell chamber, and EPCs-matrigel adhesion assays. Double-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. Positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA).
Id1 and integrin α4 expression were increased in EPCs freshly isolated from ovarian cancer patients compared to those obtained from healthy subjects. siRNA-mediated Id1 downregulation substantially reduced EPCs function and integrin α4 expression. Importantly, Inhibition of PI3K/Akt inhibited Id1 and integrin α4 expression, resulting in the decreasing biological function of EPCs.
Id1 induced EPCs mobilization and recruitment is mediated chiefly by the PI3K/Akt signaling pathway and is associated with activation of integrin α4.
BMC Cancer 08/2010; 10(1):459. DOI:10.1186/1471-2407-10-459 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate the clinical significance of genomic amplification of the human telomerase RNA gene (TERC) for cervical cancer screening and explore whether it can serve as a biomarker to improve the specificity of human papillomavirus (HPV) DNA testing for cervical cancer screening.
One hundred twenty women, including 20 cases of normal (control), 14 cases of cervical intraepithelial neoplasia grade I (CIN I), 35 cases of CIN II, 36 cases of CIN III, and 15 cases of squamous cervical cancer diagnosed by histopathologic evaluation, were subjected to cytopathologic examination, TERC detection by fluorescence in situ hybridization (FISH), and HPV DNA testing by Hybrid Capture II.
TERC amplification was significantly associated with cytopathologic diagnosis (P < 0.001) and histopathologic evaluation (P < 0.001). The positive rate of TERC gain was significantly higher in patients with CIN III or squamous cervical cancer than in patients with CIN I or those in the control group (P < 0.001). The specificity and positive predictive value of FISH for detecting CIN II or more severe cervical lesions (> or = CIN II) were obviously higher than those of HPV DNA testing (97.1% vs 52.9%, 98.7% vs 83.8%).
TERC amplification analyzed by FISH may serve as an adjunctive test to HPV DNA testing for improving the specificity and positive predictive value of cervical cancer screening.
International Journal of Gynecological Cancer 08/2010; 20(6):912-7. DOI:10.1111/IGC.0b013e3181e5c424 · 1.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating bone marrow-derived endothelial progenitor cells (EPCs) have been reported to participate in tumor angiogenesis and growth; however, the role of circulating EPCs in tumor progression is controversial. The role of circulating EPCs in ovarian cancer progression and angiogenesis has not yet been investigated.
The number of circulating EPCs in the peripheral blood in 25 healthy volunteers and 42 patients with ovarian cancer was determined by flow cytometry. EPCs were defined by co-expression of CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). In addition, we determined CD34 and VEGFR2 mRNA levels by real-time reverse transcription-polymerase chain reaction. Plasma levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were determined by enzyme-linked immunosorbent assay.
Circulating levels of EPCs were significantly increased in ovarian cancer patients, correlating with tumor stage and residual tumor size. Higher levels of EPCs were detected in patients with stage III and IV ovarian cancer than in patients with stage I and II disease. After excision of the tumor, EPCs levels rapidly declined. Residual tumor size greater than 2 cm was associated with significantly higher levels of EPCs. In addition, high circulating EPCs correlated with poor overall survival. Pretreatment CD34 mRNA levels were not significantly increased in ovarian cancer patients compared with healthy controls; however, VEGFR2 expression was increased, and plasma levels of VEGF and MMP-9 were also elevated.
Our results demonstrate the clinical relevance of circulating EPCs in ovarian cancer. EPCs may be a potential biomarker to monitor ovarian cancer progression and angiogenesis and treatment response.
Journal of Experimental & Clinical Cancer Research 03/2010; 29(1):27. DOI:10.1186/1756-9966-29-27 · 4.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian epithelial serous cystadenocarcinoma (OESC) is the most lethal of all gynecologic malignancies. In this report, we performed comparative proteomic study of normal ovarian epithelial and OESC tissue in order to establish OESC related protein atlas, and to identify tumor-associated proteins which may be important target proteins for clinical diagnosis and therapy and/or closely correlated to OESC pathogenesis. Six proteins were found significantly differentially expressed between normal ovarian epithelial and OESC tissues. The marked expression differences of selected proteins were further confirmed by Western blotting and immunohistochemistry. The correlation of prx-II expression with clinico-pathological features was also investigated.
Cancer letters 03/2009; 275(1):109-16. DOI:10.1016/j.canlet.2008.10.019 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perinatal hypoxia and ischemia (HI) are a significant cause of mortality and morbidity. To understand the molecular mechanisms for HI-induced brain damage, here we used a proteomic approach to analyze the alteration and modification of proteins in neonatal mouse brain 24 h after HI treatment. Significant changes of collapsin response mediator proteins (CRMPs) were observed in HI brain. CRMPs are a family of cytosolic proteins involved in axonal guidance and neuronal outgrowth. We found that CRMP2, CRMP4 and CRMP5 proteins were altered post-translationally after HI treatment. Mass spectrometric and Western blot analyses detected hypophosphorylated CRMP proteins after HI. Further analysis of CRMP kinases indicated inactivation of cyclin dependent kinase 5 (CDK5), a priming kinase of CRMPs and a neuronal specific kinase that plays pivotal roles in neuronal development and survival. The reduction of CDK5 activity was associated with underexpression of its activator p35. Taken together, our findings reveal HI-induced dephosphorylation of CRMPs in neonatal brain and suggest a novel mechanism for this modification. Hypophosphorylated CRMPs might be implicated in the pathogenesis of HI-related neurological disorders.
Journal of Proteome Research 07/2008; 7(6):2507-15. DOI:10.1021/pr800108k · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Two dimensional polyacrylamide gel electrophoresis (2DE) has been a core technology of current proteomics for its high resolution and ability to detect proteins with post-translational modifications. This technology has been widely applied in numerous biomedical research projects including biomarker discovery and drug development. The application of narrow-range immobilized pH gradient strips (IPGs) and advanced detection methodologies have increased the resolution of 2DE technology. However, 2DE still suffers from its limitation in unambiguous detection of proteins of lowabundance. To improve this, sample preparation is the first and key step for successful 2DE analysis. This article summarizes current progresses in sample handling and prefractionation aiming to enrich proteins of interest according to their special physical and chemical properties so that the application of 2DE can be greatly extended.
Current Proteomics 11/2005; 2(4):259-268. DOI:10.2174/157016405775201757 · 0.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular and cellular mechanisms underlying tumor metastasis are still elusive. The identification of protein molecules with their expressions correlated to the metastatic process would help to understand the metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions and clinical management of cancer. Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. Proteomic technology has been widely used in biomarker discovery and pathogenetic studies including tumor metastasis. This article provides a brief review of the application of proteomics in identifying molecular factors in tumor metastasis process. The combination of proteomics with other experimental approaches in biochemistry, cell biology, molecular genetics and chemistry, together with the development of new technologies and improvements in existing methodologies will continue to extend its application in studying cancer metastasis.