NS-398协同顺铂对人肺腺癌细胞生长影响的机制研究

Study of mechanism in growth inhibition of human lung adenocarcinoma with combined use of NS-398 and cisplatin

目的明确COX-2抑制剂NS-398对顺铂肺腺癌细胞增殖抑制作用的影响,探讨NS-398与顺铂的联合抑制肺腺癌细胞增殖的作用机制。方法采用MTT比色法检测NS-398和顺铂联合作用对肺腺癌细胞增殖的影响,荧光染色法(吖啶橙和溴化乙啶)和流式细胞法观察肺腺癌细胞凋亡凋亡率和细胞周期的改变,二维聚丙烯酰胺凝胶电泳分离NS-398和顺铂联合作用肺腺癌细胞的蛋白质,质谱分析法(MOL-DI-TOF-MS)和数据库查询鉴定其中部分差异蛋白质。结果NS-398对顺铂IC50降低的百分率在43.34%～69.61%之间,两药联合作用为轻度协同作用～协同作用;NS-398与顺铂联合组作用24和48 h诱导肺腺癌细胞的凋亡率增高。NS-398与顺铂联合组表达量下调的差异蛋白点为22个,表达量上调蛋白点为2个,只在NS-398与顺铂联合组出现的蛋白点25个,NS-398与顺铂联合组消失的蛋白点17个。选择其中17个表达量下调蛋白点、2个表达量上调蛋白点进行质谱鉴定,所鉴定的差异蛋白质中部分为细胞骨架蛋白,部分为与细胞增殖和凋亡相关的蛋白,部分为细胞代谢途径相关酶。结论NS-398可以增强顺铂对肺腺癌细胞增殖的抑制作用,两种药物具有协同作用,肺腺癌细胞凋亡作用的增强可能参与了NS-398与顺铂协同抑制肺腺癌细胞增殖的作用机制。热休克蛋白90和磷酸丙糖异构酶可能参与NS-398对顺铂肺腺癌细胞增殖抑制作用的增强。

[Objective] To study the effect of a selective COX-2 inhibitor NS-398 combined with chemotherapy agent cisplatin on the proliferation of human lung adenocareinoma cell lines, ＂ and the mechanism of the combined use of the two agents. [Methods] the effect of synergistic use of NS-398 and cisplatin on the proliferation of human lung adenocareinoma cell lines was discussed by MTI＂ growth assay, the apoptosis assay and the cell cycle distribu- tion were analyzed by fluorescence staining of Acridine orange （AO） and Ethidium bromide （EB） and by the flow cy- tometry. The differential expressed proteins in the human lung adenocareinoma cells with the combined use of NS- 398 and eisplatin were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis （2-DE）, then 19 proteins among them were identified by peptide mass fingerprint （PMF） based on matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching. [Results] NS-398 in combination with cisplatin resulted in a reduction of eisplatin＇ IC50 by 43.34%-69.61%, the effects of NS-398 combined with cisplatin ranged from slight synergism to synergism. The apoptotie ratios at 24 h and 48 h were higher than that of the control group. In the group with the combined use of NS-398 and cisplatin there were 22 down-regnlated proteins, and 2 up-regulated proteins compared with the control group, and the changes didn＇t occurred in the cisplatin group or NS-398 group as well. There were 25 proteins only appearing in the combined group, whereas 17 proteins disappeared in the combined group. 19 proteins among the proteins were selected on the basis of the intensity and the significant difference in abundance of changes and identified by means of MALDITOF-MS. Some proteins identified were cytokreratins, some proteins were associated with proliferation and apoptosis, others were involved in the metabolism of the cells. [Conclusion] Combination studies showed synergistic interactions for NS-398 with eisplatin. NS-398 would enhance the chemosensitivity of lung adenocareinoma cells to cisplatin. The enhanced apoptosis may be involved in the synergistic effect of NS-398 and cisplatin in the growth inhibition of tung adenocareinoma cells. The down-regnlation of HSP 90 and Triosephosphate-isomerase were related to the enhanced growth inhibition of the lung-cancer ceils by the combined use of the two agents.