Expression Of Twist And Resistance-associated Proteins In Breast Cancer And Differential Proteomic Analysis Of The Taxol Resistant MCF-7 Cell Lines

Objestive:To detect the expression levels of the transcription factor Twist and resistance-associated proteins(MRP,P-gp,LRP. TOPOⅡ)in human breast cancer tissues and taxol resistant MCF-7cell lines(MCF-7/Taxol)by immunohistochemistry;The differential protein expression between MCF-7 cell line and MCF-7/Taxol cell lines was analyzed by 2-DE(two-dimensional electrophoresis)to provide experimental evidence to further reveal the mechanism of taxol resistance in breast cancer and explore new molecular targets for drug-resistant tumors.Methods:1.Immunohistochemical methods were used to analyze expression levels of Twist and MRP. P-gp,LRP,TOPOⅡin the breast cancerous and pericancerous tissues comparatively. 2.Twist and MRP,P-gp,LRP,TOPOⅡexpression levels were detected in human breast cancer cell line and its taxol-resistant variant,MCF-7/Taxol by immunohistochemical.3.Total proteins of MCF-7 and MCF-7/Taxol were extracted and 2-DE based proteomics methods were used to analyze the differential expression proteins. In this study,the first dimension was carried out by IPG pH gradient isoelectric focusing system and the second dimension was separated by vertical SDS-PAGE followed by Silver staining.The 2-DE patterns were matched and analyzed by ImageMaster 2D platinum software to find differentially expressed proteins spots.Results:1.Expressions of Twist and resistance-associated proteins were significantly different in cancerous and pericancerous tissues.Expressions of Twist and MRP. TOPOⅡ. P-gp in breast cancerous tissues were higher than that of pericancerous tissues(P<0.05),while LRP was lower (P<0.05) in pericancerous tissues.2.The positive particles of Twist,MRP,TOPOⅡ,P-gp increased and deepened in MCF-7/Taxol compared with the parental MCF-7cells;however the situation was reversed for LRP. 3.High resolution and reproducibility of 2-DE technique for separation of MCF-7 proteins was established.The 2-DE patterns were similar between the MCF-7 and taxol-resistant MCF-7/Taxol cell lines;There were 269 spots on MCF-7 pattern and 170 spots on MCF-7/Taxol pattern.128 pairs were matched between the two patterns and the match rate was 75.3%.13 protein spots were differentially expressed between the two cell lines.Conclusion:1.The high expressions of Twist and MRP,TOPOⅡ,P-gp were highly consistent in human breast cancer tissues, suggesting that Twist relates to the drug resistance in human breast cancer.2. Expressions of Twist,MRP,TOPⅡ,P-gp protein gradually increased in the process of taxol-resistance induction, indicating that Twist play an important role in acquired taxol resistance of breast cancer and this role may be correlate with the high expression of resistance-associated proteins.3.Preliminarily 2-DE analysis indicated that there are 13 differential expression proteins between MCF-7 and MCF-7/Taxol cell lines,four of them might beβ-gonadotropin, fructose 1,6-bisphosphatase, Annexin A2 and Ras-related nuclear protein,which involved in cancer metastasis,energy metabolism and cell proliferation.The result provides evidence for further study of the mechanism of drug resistance in breast cancer.