Analysis of in vitro anti-leukemia effect of 5-aza-2'-deoxycitydine / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycitydine (5-aza-2 dC) on the growth, differentiation and apoptosis of human acute myeloid leukemia(AML) cell line HL-60, and to explore the possible anti-leukemia mechanism of 5-aza-2 dC.@*METHODS@#HL-60 cells were treated by 5-aza-2 dC at various concentrations for different periods of time. The effect of 5-aza-2 dC on the growth of HL-60 cells were detected by MTT assay. The effect on the cell cycle and differentiation were detected by flow cytometry. The effect on the apoptosis were detected by Hochest33342 staining and flow cytometry. The expression of S100A8 and S100A9 was detected by reverse transcription-polymerase chain reaction (RT-PCR).@*RESULTS@#(1) 5-aza-2 dC inhibited the growth of HL-60 cells in a concentration- and time-dependent manner, and HL-60 cells were arrested at G2/M phases; (2) 5-aza-2 dC enhanced the expression of cell differentiation antigen CD11b at HL-60 cells, especially at the low drug concentration; (3) 5-aza-2 dC induced HL-60 cell apoptosis in a concentration- and time-dependent manner, especially at the high drug concentration; (4) 5-aza-2 dC increased the expression levels of S100A8 and S100A9 mRNA in HL-60 cells.@*CONCLUSION@#5-aza-2 dC can inhibit the growth of HL-60 cells accompanied with G2/M phase arrest, induce the differentiation and apoptosis of the cells, and increase the expression levels of S100A8 and S100A9 mRNA, which may be the anti-AML mechanism of 5-aza-2 dC.

Screening for differentially expressed proteins in nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis / 中南大学学报(医学版)

OBJECTIVE@#To search for the differentially expressed proteins of nasopharyngeal carcinoma (NPC),and provide scientific evidence for identifying molecular biomarkers for NPC.@*METHODS@#Laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of microdissected NPC and NNET, PDQuest software was applied to analyze 2-DE images,and the differential proteins between the 2 types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Western blot and immunohistochemistry of tissue microarray were used to detect the expression of the differential protein SCCA1 in NPC and NNET.@*RESULTS@#2-DE patterns of microdissected NPC and NNEC were established,and 36 differential proteins in the NPC and NNEC were identified,20 of which only expressed or up-regulated in NPC and 16 only expressed or up-regulated in NNET. The differentially expressed level of SCCA1 in the NPC and NNET was confirmed by Western blot and immunohistochemistry of tissue microarray.@*CONCLUSION@#Thirty-six differentially expressed proteins identified in this study may be associated with the carcinogenesis of NPC,and may be candidate molecular biomarkers for NPC.

Differential proteomic analysis of nasopharyngeal carcinoma cell lines 5-8F and 6-10B / 中南大学学报(医学版)

OBJECTIVE@#To compare the proteome difference of nasopharyngeal carcinoma (NPC) cell lines 5-8F and 6-10B, and to screen these proteins associated with NPC metastasis.@*METHODS@#Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins from NPC cell lines 5-8F and 6-10B with different metastatic potentials and same genetic background, respectively. PDQuest software was applied to analyze 2-DE images, and the differentially expressed protein spots between 5-8F and 6-10B were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The expression levels of partial identified proteins in the 2 cell lines were detected by Western blot.@*RESULTS@#2-DE maps of total proteins from 5-8F and 6-10B were established. A total of 65 differential protein spots in the 2 cell lines were found, and 15 non-redundant differential expression proteins were identified by MALDI-TOF-MS. Western blot showed that Annexin A1 and 14-3-3 protein sigma were differential expression proteins in 5-8F and 6-10B, which was consistent with the Results from the comparative proteomic analysis.@*CONCLUSION@#Fifteen non-redundant differential expression proteins are useful for studying the metastatic mechanism of NPC.

Mechanism of hepatocyte transformation by HCV NS3 using two-dimensional electrophoresis and mass spectrometry / 中南大学学报(医学版)

OBJECTIVE@#To investigate the proteome of hepatocyte transformation by hepatitis C virus (HCV) nonstructural protein 3 (NS3).@*METHODS@#Human hepatocyte line QSG7701 stably expressing HCV NS3 C-terminal deleted protein was constructed, which was named pRcHCNS3/QSG. Two-dimensional electrophoresis (2-DE) was used to separate the total protein of pRcHCNS3/QSG and pRcCMV transfected cells (pRcCMV/QSG) respectively. Differentially expressed protein spots were identified by mass spectrometry. Western blot confirmed the differentially expressed proteins.@*RESULTS@#2-DE profiles with high resolution and reproducibility were obtained. The average spots of pRcHCNS3/QSG and pRcCMV/QSG were (1183+/-77) and (1095+/-82) respectively, and (920+/-60) spots were matched. Twenty-one differentially expressed protein spots were chosen randomly and 15 were identified by mass spectrometry. Some proteins such as Ras, P38 and HD53 which were involved in signal transduction were increased in pRcHCNS3/QSG cells. Western blot also showed strong expression of phosphorylated P44/42 and P38 in pRcHCNS3/QSG cells. Other differentially expressed proteins were related to cell cycle regulation, immunoreaction, tumor invasion and metastasis, and liver metabolizability.@*CONCLUSION@#HCV NS3 might be involved in cell malignant transformation through affecting protein expression and signal transduction such as MAPK cascade. Further study on the signal transductions and their relationship would not only be helpful to explore the mechanism of HCV related HCC, but also provide a new idea for the molecular treatment of HCC.

Sorcin overexpression and multidrug resistance in human gastric cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To screen multidrug resistance (MDR) related proteins in human gastric cancer using proteomics technique.@*METHODS@#Two-dimensional electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant human gastric cancer cell line SGC7901/VCR and its counterpart SGC7901. PDQuest software was used to analyze 2-DE images, and the differential expression proteins between the 2 cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF. The differential expression level of sorcin, one of the identified proteins, was confirmed by western blot analysis. The effect of sorcin on the development of MDR of SGC7901/VCR was determined by antisense oligonucleotides (ASO) technique.@*RESULTS@#Sorcin as a high expression protein in SGC7901/VCR was identified and the suppression of sorcin expression by sorcin ASO could enhance the vincristine chemosensitivity in SGC7901/VCR.@*CONCLUSION@#Sorcin overexpression is related to MDR in human gastric cancer cell line SGC7901/VCR.

Mechanism of migration in CNE2 cells promoted by EBV-LMP1 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the mechanism of migration phenotype change induced by EBV-LMP1 in nasopharyngeal carcinoma (NPC) cell line CNE2.@*METHODS@#Retroviruses RV-LNSX, RV-LMP1, and RV-LMP1(TRADD) prepared previously were used to infect CNE2 cells. After selection with G418, the morphology, the ability of motion and migration in extracellular matrix, expression of LMP1 and E-Cadherin in transgenic cells were observed or detected. Meanwhile, pEcad-luc was respectively co-transfected with pLNSX, pLNSX-LMP1, and pLNSX-LMP1(TRADD), to examine the effect of LMP1 on the transcriptional activity of E-Cadherin promoter in 293 cells.@*RESULTS@#Compared with CNE2 and CNE2-LNSX cells, CNE2-LMP1 cells morphologically changed from typical epithelial appearance to long-spindle fibroblastic morphology with the concomitant loss of cell-to-cell contact, and relative migration of CNE2-LMP1 cells obviously increased (n=3, P< 0.05), while the expression of E-Cadherin was negative in CNE2-LMP1 cells. The transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin was suppressed by LMP1, and the level of suppression was correlated with the concentration of pLNSX-LMP1 (0.2,0.6 and 1.0 microg). LMP1(TRADD) didn't induce the changes of morphology and migration phenotype, nor suppress the transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin in CNE2 cells.@*CONCLUSION@#EBV-LMP1 promotes the migration and down-regulates the expression of E-Cadherin in CNE2 cells. The mechanism is that EBV-LMP1 suppresses the transcriptional activity of E-Cadherin promoter. TRADD of carboxyl terminus of LMP1 may be the main active domain to promote the migration in NPC cells.

Establishment of protein expression profile of human normal colonic epithelia / 中南大学学报(医学版)

OBJECTIVE@#To establish a protein expression profile of human normal colonic epithelia.@*METHODS@#Two-dimensional gel electrophoresis (2-DE) was applied to separate the total proteins of 20 human normal colonic epithelial tissues. The expression proteins in the human normal colonic epithelia were identified by both matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization tandem mass spectrometry (ESI-Q-TOF), and the biological function and subcellular locations of the identified proteins were analyzed by bioinformatics.@*RESULTS@#A 2-DE reference map of human normal colonic epithelium was established. On the 2-DE map, 1020+/-50 protein spots were detected, 204 protein spots representing 162 non-redundant proteins were identified, and 37 proteins had posttranslational modification. The identified proteins were categorized into several protein groups according to their functions or subcellular locations, whose data were available at our website (http://www.xyproteomics.org).@*CONCLUSION@#A protein expression profile of human normal colonic epithelia is established for the first time, which provides useful information for investigating the physiological functions and pathologic process of colonic epithelia.

Comparative proteome analysis of human lung squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>This study was designed to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous cell carcinoma tissue and paired tumor-adjacent normal bronchial epithelial tissue, and to identify differential expression of tumor-associated proteins by using proteome analysis.</p><p><b>METHODS</b>Comparative proteome analysis of human lung squamous carcinoma and paired normal bronchial mucosa adjacent to tumors from 20 cases were carried out. Total proteins of the carcinoma tissue and normal bronchial mucosa were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULTS</b>(1) Seventy-six differentially expressed proteins were screened by analyzing the electrophoretic maps of the 20 carcinoma and control mucosa tissues. (2) Sixty-eight differential proteins were identified by peptide mass fingerprinting (PMF). Some proteins were products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. (3) The expression of three proteins mdm2, c-Jun and EGFR, correlated with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results showed that the expression of mdm2, c-Jun and EGFR were up-regulated in lung squamous carcinomas, whereas down-regulated in control normal mucosa. It was consistent with our proteome analysis results. Those results suggested that those proteins may play roles in the carcinogenesis of lung squamous carcinoma.</p><p><b>CONCLUSION</b>sixty-eight differentially expressed proteins were successfully characterized by comparative proteome analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of lung squamous carcinoma, as well as to improve patients' prognosis and provide a new clue for carcinogenesis research of lung squamous cell carcinoma.</p>

Identification of aging related proteins in human normal colonic epithelium / 中南大学学报(医学版)

OBJECTIVE@#To explore the molecular mechanisms of colonic epithelial aging related proteins and aged colonic epithelial susceptibility to tumor.@*METHODS@#The proteins of normal human colonic epithelial tissue from young and old people were separated by 2-dimensional gel electrophoresis (2DGE), respectively. Then gels were stained by silver, scanned by imagescanner and analyzed with PDQuest software. The differentially expressed protein spots of colonic epithelium between the old and the young groups were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching.@*RESULTS@#Well-resolved and reproducible 2DGE maps of normal human colonic epithelium from the young and the old were acquired. Nineteen more than 2 fold differentially expressed protein spots were identified representing 17 different proteins by MALDI-TOF-MS. The functions of these proteins involve in metabolism, energy generation, transportation, antioxidation, translation and protein folding.@*CONCLUSION@#Seventeen aging related proteins of human colonic epithelium identified indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium. These data provided useful clues for elucidating the mechanisms of colonic epithelial aging and aged colonic epithelial susceptibility to cancer.