达沙替尼联合奥沙利铂协同抑制胃癌细胞增殖和迁移

Dasatinib combined with oxaliplatin to inhibit proliferation and migration of gastric cancer cells

目的:评价Src酪氨酸激酶抑制剂达沙替尼联合奥沙利铂抑制胃癌细胞体外增殖、迁移等恶性生物学行为的效应。方法:实时定量-聚合酶链反应(RT-PCR)及蛋白质印迹法(Western blot)检测Src及其活性形式p-Src在胃癌细胞株中的基础表达;蛋白质印迹技术观察不同剂量奥沙利铂作用于胃癌细胞或作用不同时间后,细胞内Src及p-Src的变化规律;细胞计数试剂盒(CCK-8)方法评价胃癌细胞暴露于不同浓度奥沙利铂和达沙替尼后的生长抑制率,分别计算2种药物的半数抑制浓度(IC50),并使用Calcusyn 2.0软件计算联合作用指数(CI);细胞集落形成实验、流式细胞术和划痕实验观察上述药物对胃癌细胞体外增殖、细胞周期、凋亡和迁移能力的影响。结果:胃癌细胞株SGC-7901、BGC-823、MKN-28的p-Src/Src基础表达比值较高;奥沙利铂可明显上调胃癌细胞SGC-7901、BGC-823、MKN-28的p-Src,并呈时间依赖性(r2=0.96、0.85、0.94);达沙替尼和奥沙利铂在胃癌细胞中的CI均<1;与单药组比较,达沙替尼联合奥沙利铂组细胞集落形成数最少(P=0.015);划痕实验结果显示,联合用药组划痕愈合能力最弱(P=0.000);达沙替尼单药对细胞周期影响不明显,但两药联合组对比奥沙利铂组,G1期细胞数目增多(P=0.002),S期和G2期细胞数目减少(P=0.02)。结论:奥沙利铂可上调胃癌细胞p-Src表达,并呈时间依赖性,Src酪氨酸激酶抑制剂达沙替尼联合奥沙利铂可协同抑制胃癌细胞体外增殖和迁移。

Objective To study the effects of Src tyrosine kinase inhibitor dasatinib combined with oxaliplatin on the malignant biological behavior of gastric cancer cells such as proliferation and migration in vitro. Methods RT-PCR was used to detect the baseline expression of Src and its active form p-Src in gastric cancer cells. The effect of different doses of oxaliplatin of different time duration on changes of Src and its active form p-Src was detected by Western blot. After exposed to different doses of oxaliplatin and dasatinib, cell counting kit 8 （CCK-8）was used to detect cell growth inhibition rate, and calculate the half-inhibitory concentration （IC50）. Then Calcusyn 2.0 software was used to calculate the combined action index （CI）. The effects on cell proliferation, cell cycle, apoptosis and migration of gastric cancer cells were determined by cell colony formation, cell cycle, apoptosis, and scratch tests. Results The basal p-Src/Src ratios of SGC- 7901, BGC-823, MKN-28 cells were high. Oxaliplatin induced the up-regulation of expression of p-Src in SGC-7901, BGC-823, MKN-28 cells in a time dependent manner, the squared correlation coefficient （r2） between p-SRC/SRC and time were 0.96,0.85,0.94,respectively. The CI value of dasatinib and oxaliplatin was less than 1, suggesting a synergisticeffect of dasatinib with oxaliplatin on growth of gastric： cancer cells. Dasatinib combined with oxaliplatin had tile minimum number of colonies （P=0.015）. Scratch test showed that wound healing in combined dn, g group was the weakest （P=0.000）. I）asatinib had no obvious effect on cell cycle, but the two drugs combined as compared with oxaliplatin alone, the number of cells in G1 phase increased （P=0.02） and the number of cells in S phase and G, phase decreased. Conclusions Oxaliplatio could up-regulate the expression of p-Src in gastric cancer cells in a time dependent manner, dasatinib combined with nxaliplatin could synergically inhibit the proliferation and migration of gastric cancer cells in vitro.