卵巢上皮性癌细胞系铂类药物耐药相关蛋白的比较蛋白质组分析

Proteomic analysis of human ovarian cancer cell lines and their platinum-resistant clones

目的对卵巢上皮性癌(卵巢癌)细胞系进行铂类药物耐药相关蛋白的比较蛋白质组分析。方法应用双向凝胶电泳和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)寻找对铂类药物敏感(SKOV3和A2780)及耐药(SKOV3/CDDP、SKOV3/CBP、A2780/CDDP 和 A2780/CBP)卵巢癌细胞系的差异表达蛋白质。RT-PCR、蛋白印迹法分别验证差异表达蛋白质在各细胞系中 mRNA和蛋白的表达水平。结果共发现5种蛋白质(膜联蛋白 A3、破解蛋白、辅酶Ⅱ依赖的异柠檬酸脱氢酶1、谷胱甘肽转移酶 omega 1和丝切蛋白1)在3种及3种以上耐药细胞中均有差异表达,膜联蛋白A3的表达差异最显著。膜联蛋白 A3基因的 mRNA 和蛋白表达水平,SKOV3/CDDP、SKOV3/CBP、A2780/CDDP 和 A2780/CBP 细胞较 SKOV3和 A2780细胞明显升高。结论应用蛋白质组学技术对SKOV3和 A2780细胞系及其4种耐药细胞系进行比较蛋白质组分析,共识别鉴定出5种蛋白质,膜联蛋白 A3、破解蛋白、辅酶Ⅱ依赖的异柠檬酸脱氢酶1、谷胱甘肽转移酶 omega 1和丝切蛋白1可能参与卵巢癌铂类药物耐药机制的形成。

Objective To perform comparative proteomic analysis of human ovarian cancer cell lines for detecting platinum-resistance associated proteins. Methods The total proteins of two sensitive （SKOV3 and A2780） and four resistant （SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP） human ovarian cancer cell lines were separated by two-dimensional gel electrophoresis （2-DE）. The differentially expressed proteins were analyzed using image analysis software, stained with Coomassie Brilliant Blue, then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. The mRNA and protein levels of the differentially expressed protein which was most significant in all of the four resistant cell lines were validated by RT-PCR and western blotting, respectively. Results Five proteins were found to be significant in four cell lines. Annexin A3 and destrin were up-regulated and nicotinamide-adenine dinucleotide phosphate （NADP）- dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was not changed. However, cofilin 1 represented a different trend. In the two resistant sublines of SKOV3, cofilin 1 had a down-regulation, but it had an up-regulation in the cell lines induced from SKOV3. The expression of annexin A3 was up-regulated by 3 - 20 fold and the results of RT- PCR and western blotting showed complete consistency with that by 2-DE. Conclusions Proteomic techniques are useful to the identification of the resistance-associated proteins in ovarian cancer platinumresistant cell lines and five candidates have been found. The five differential proteins might become hopeful candidate biomarkers for resistance.