ABSTRACT
Multidrug resistance (MDR) is one of the main causes leading to the failure in cancer treatment. Differential proteins between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its cisdiamminedichloroplatinum (CDDP)-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC. EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation. The stable isotope labeling with amino acids in cell culture (SILAC) was used to label EC9706 and EC9706/CDDP with heavy and light medium, separately. Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS) and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP. Compared to parental EC9706, EC9706/CDDP manifested phenotypes of slow proliferation, cell pleomorphology, atypia and increased resistant-index 3.23. Seventy-four differential proteins identified in the present study belongs to various families with multiple functions, such as cytoskeleton (20%), energy metabolism (11%), transcription regulation and DNA repair (11%), redox homeostasis (9.5%), protein biosynthesis and mRNA processing (12%), ribosome constituent (8.1%), molecular chaperone (8.1%), immunity/inflammation (5.4%), intracellular transport (5.4%) and nucleosome assembly (2.7%), which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways. The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.
ABSTRACT
Objective:To identify differentially expressed proteins related with malignant transformation of esophageal squamous cell carcinoma (ESCC) using proteomic analysis. Methods:Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization timE-of flight mass spectrometry (MALDI-TOF-MS) in combination with protein database searching were used to determine and identify differentially expressed proteins in esophageal cancer cell lines (EC1, EC18, and EC109) and immortal cell line (NECA-E6E7-hTERT). Western blotting and immunocytochemistry were used to verify the differential expression of annexin 2 in esophageal cancer cell lines and immortal cell line (NECA-E6E7-hTERT). Real-time fluorogentic quantitative PCR(RFQ-PCR) was performed to analyze the expression level of annexin A2 mRNA.Results: A total of 15 differentially expressed proteins were identified with more than 5 folds difference. Among them three proteins were down-regulated and 12 proteins were up-regulated. Western blotting and immunocytochemical analysis verified the down-regulation of annexin A2 protein in ESCC cell lines. However, differential expression pattern of annexin A2 mRNA was not consistant with its protein expression in ESCC cell lines and immortal cell line (NECA-E6E7-hTERT). Conclusion:The findings provide important clues for identifying the candidate biomarkers for high-risk population screening and early diagnosis of ESCC. Post-translative regulation/modification contributes to the down-regulation of annexin A2 protein.
ABSTRACT
Aim To establish a resistant human osteosarcoma cell line(Saos-2/ADM1and Saos-2/ADM4)from the Saos-2 cell line and study its resistant mechanisms.Methods Saos-2 cells were pulse exposed in gradually increased dose of ADM culturemedium.The sensitivity of the resistance drug from the Saos-2、Saos-2/ADM1 and Saos-2/ADM4 cell lines to ADM、DDP、EPI、MTX、THP and PTX was measured by MTT assay.The morphology and ultramicro structure of the cell lines were observed by optical microscopy and transmission electronic microscopy.The expression level of MDR1 mRNA、MRP mRNA and their proteins P-gp、MRP was examined by reverse transcription polymerase chain reaction and immunohistochemistry.Results Resistance cell lines were established after 167 days.The resistance index to methotrexate of the Saos-2/ADM1and Saos-2/ADM4 cell lines to ADM was 49.8 and 74.6 times than that of Saos-2 respectively.The two cell lines had resistance to MTX、IFO、EPI、THP and PTX(P0.05).Disordered structure of the Saos-2/ADM1and Saos-2/ADM4 cells was observed through microscopy.The cells appeared in coenocytic.The decrease of cell villus and the increase of nucleoli were observed through transmission electronic microscopy.The proliferation ability of Saos-2/ADM1and Saos-2/ADM4 cells decreased significantly.MDR1 mRNA、MRP mRNA、P-gp and MRP showed positive staining in resistance cell lines.Conclusion The genes of MDR1 mRNA、MRP mRNA and their corresponding proteins participated in the formation of multidrug resistance in resistant adriamycin cell line.These newly-described resistant osteosarcoma cell lines were useful models for further characterization of drug resistance in osteosarcoma and for the development of treatment protocol.