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Tang C.,Key Laboratory of Cancer Proteomics | Tang C.,Central South University | Xiao Y.,Key Laboratory of Cancer Proteomics | Ruan L.,Key Laboratory of Cancer Proteomics | And 6 more authors.
Journal of Central South University (Medical Sciences) | Year: 2010

Objective To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Methods Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia ( AML ) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6. 5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. Results F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment. Conclusion Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.

Li X.,Central South University | Hu R.,Key Laboratory of Cancer Proteomics | Qu J.,Key Laboratory of Cancer Proteomics | He Q.,Key Laboratory of Cancer Proteomics | And 5 more authors.
Journal of Central South University (Medical Sciences) | Year: 2012

Objective: To identify proteins associated with nasopharyngeal carcinoma (NPC) metastasis, and provide scientific basis for the prevention and cure of NPC. Methods: A two-dimensional gel electrophoresis and mass spectrometry were performed to screen for differential proteins between highly metastatic 5-8F and non-metastatic 6-1OB NPC cell lines. Western blot was used to confirm the differential proteins. We used siRNA to inhibit the expression of differential protein nm23-Hl to determine the association of nm23-H1 with NPC in vitro invasive ability. Immunohistochemistry and statistics were used to evaluate the correlation of nm23-Hl expression with clinicopathological features and clinical outcomes in paraffin-embedded archival tissues including 93 cases of primary NPC and 20 cases of cervical lymphonode metastatic NPC (LMNPC). Results: A total of 15 differential proteins in the 2 cell lines were identified by a proteomic approach, and 3 differential proteins were selectively confirmed. Downregulation of nm23-H1 by siRNA significantly increased the in vitro invasive ability of 6-10B. Significant nm23-H1 downregulation was observed in LMNPC compared with primary NPC. nm23-H1 downregulation in primary NPC was positively correlated with lymphonode and distant metastasis, advanced clinical stage and recurrence. Survival curves showed that patients with nm23-H1 downregulation in primary NPC had a poor prognosis. Multivariate analysis confirmed that nm23-H1 expression level in primary NPC was an independent prognostic indicator. Conclusion: nm23-H1 behaves as a metastasis suppressor in NPC, and nm23-H1 downregulation is a biomarker for poor NPC prognosis.

Min L.,Central South University | He S.,University of South China | Chen Q.,Central South University | Peng F.,Key Laboratory of Cancer Proteomics | And 2 more authors.
Toxicology Mechanisms and Methods | Year: 2011

This work aimed to investigate the cellular response of human airway epithelial cells (A549) to oxidative stress induced by benzo(a)pyrene [B(a)P]. Levels of intracellular reactive oxygen species (ROS) and lipid peroxidation were investigated in A549 cells treated with varying concentrations of B(a)P. A comparative proteomic analysis of total proteins was performed in cells treated with 1 μM B(a)P [B(a)P-1] and untreated cells. The expression of Mn superoxide dismutase (Mn SOD), one of the identified down-regulated proteins in B(a)P-1 cells, was then analyzed by Western blotting. The total antioxidant activity, total superoxide dismutase activity, catalase (CAT) activity, and glutathione reductase (GR) activity were all analyzed after B(a)P treatment. Our results demonstrated that 1 μM B(a)P could induce ROS generation and lead to lipid peroxidation in A549 cells, and 23 differentially expressed proteins were identified. The expression levels of Mn SOD and the total SOD were induced at 0.1 μM and suppressed at 1 μM and 10 μM. Up-regulation of CAT and GR activity resulted in an increase in total antioxidant activity in A549 after exposure to B(a)P. These findings provide a basis for understanding the mechanisms of mitochondrial dysfunction and perturbation of antioxidant status induced by B(a)P on airway epithelial cells. © 2011 Informa Healthcare USA, Inc.

Wei R.,Central South University | Xie Y.,Zhuhai People S Hospital of Guangdong | Yang D.,Central South University | He L.,Central South University | And 2 more authors.
Journal of Central South University (Medical Sciences) | Year: 2010

Objective To establish 2-dimensional electrophoresis (2-DE) graph of A549 and A549/DDP cell lines, to identify the differentially expressed proteins, and to screen multidrug resistance (MDR) related proteins in human lung adenocarcinoma. Methods The total proteins of A549 and A549/DDP cells were obtained , and were extracted and separated by 2-DE. PDQuest software was applied to analyze the 2-DE images, and the differential proteins of the 2 types of cells were identified by matrix - assisted laser desorption/ionization time of flight mass spectrometry ( MALDITOF-MS) . Western blot was used to determine the expression levels of the 4 proteins. Results We established 2-DE maps of total proteins from A549 and A549/DDP. A total of 40 differential protein spots in the 2 cell lines were found , and 23 differential expression proteins were identified by MALDI-TOF-MS. Western blot showed that heat shock protein beta-1 ,annexin A4 , cofilin 1, vimentin were differential expression proteins in A549 and A549/DDP,which was consistent with the results of the comparative proteomic analysis. Conclusion The 23 differential expression proteins in human lung adenocarcinoma are useful for studying the MDR mechanism of lung adenocarcinoma.

Ye L.,Central South University | Zhang G.-Y.,Central South University | Liu T.,Central South University | Chen X.-M.,Central South University | And 4 more authors.
Progress in Biochemistry and Biophysics | Year: 2010

This research aimed to construct a new combination gene vector: pcDNA3.1 (-)VEGF-siRNA/ yCDglyTK, study its expression quality and lethal effet in human gastric cancer cell line SGC7901. First, RNA interference (RNAi) targeting vascular endothelial growth factor (VEGF) was applied to construct interfering plasmid pGenesil-VEGF-siRNA. Then, the siRNA expression cassette (including U6 promotor ) was amplified by PCR and subcloned into pcDNA3.1 (-)CV-yCDglyTK to build a new combination gene plasmid: pcDNA3.1(-) VEGF-siRNA/yCDglyTK. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. All of the three plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expressions of yCDglyTK and VEGF were detected by RT-PCR and Western-blot. MTT assays were applied to determine the cytotoxic effect of plasmids in the presence of 5-FC. Restriction enzyme digestion and gene sequencing confirmed the combination gene vector pcDNA3.1 (-)VEGF-siRNA/yCDglyTK was constructed successfully. RT-PCR, Western-blot showed expression of yCDglyTK and inhibition of VEGF in SGC7901 cells transfected with the combined gene plasmid, which were the most sensitive to 5-FC in the MTT assays. The combination gene vector pcDNA3.1(-)VEGF-siRNA/yCDglyTK was constructed successfully. It was tentatively confirmed that RNAi targeting VEGF could synergize with suicide gene therapy.

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