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目的探讨应用γ分泌酶抑制剂(Gamma-secretase inhibitor,GSI)阻断Notch1信号通路后对人结肠癌耐药细胞株SW480/L-OHP化疗敏感性的影响。方法应用人结肠癌细胞系SW480,以反复诱导、逐渐递增奥沙利铂(L-OHP)浓度的方法,体外构建耐药细胞株SW480/L-OHP。采用CCK-8法检测细胞株对L-OHP的耐药指数,RT-PCR法检测survivin、notch1、hes1、cyclinD1基因mRNA的转录,Western blot检测Notch1和Hes1蛋白的表达。将耐药细胞分成对照组、L-OHP组(3.0μmol/L)、GSI组(10μmol/L)和GSI+L-OHP组,分别处理后,检测各组细胞的抑制率、凋亡率及survivin、notch1、hes1、cyclinD1基因mRNA的转录水平和Notch1、Nicd、Hes1蛋白的表达水平。结果与SW480亲本细胞相比,SW480/L-OHP细胞中survivin、notch1、hes1和cyclinD1基因mRNA转录水平及Notch1和Hes1蛋白表达水平均上调。GSI组耐药细胞的增殖受到抑制,GSI+L-OHP组抑制作用更明显;GSI组耐药细胞survivin、hes1、cyclinD1基因mRNA转录水平下调,Nicd和Hes1蛋白表达水平下调,GSI+L-OHP组下调更明显。结论 GSI通过阻断Notch1胞内段Nicd的释放,影响下游Hes1基因和蛋白的表达,从而抑制SW480/L-OHP细胞增殖,提高其化疗敏感性;且与L-OHP联合使用具有协同作用。
Objective To investigate the effect of gamma-secretase inhibitor (GSI) on chemosensitivity of human colon cancer cell line SW480 / L-OHP after blocking Notch1 signaling pathway. Methods The human colorectal cancer cell line SW480 was used to establish the drug-resistant cell line SW480 / L-OHP in vitro by gradually increasing the concentration of oxaliplatin (L-OHP). The drug resistance index of L-OHP was detected by CCK-8 assay. The transcription of survivin, notch1, hes1 and cyclinD1 mRNA was detected by RT-PCR and the expression of Notch1 and Hes1 protein by Western blot. The drug-resistant cells were divided into control group, L-OHP group (3.0μmol / L), GSI group (10μmol / L) and GSI + L-OHP group. survivin, notch1, hes1, cyclinD1 mRNA and Notch1, Nicd, Hes1 protein levels. Results Compared with SW480 parental cells, the mRNA transcription level of survivin, notch1, hes1 and cyclinD1 and the expression of Notch1 and Hes1 in SW480 / L-OHP cells were up-regulated. The GSI + L-OHP inhibited the proliferation of GSI + L-OHP group. The mRNA transcription levels of survivin, hes1 and cyclinD1 in GSI group were down-regulated while the expressions of Nicd and Hes1 were down-regulated Group down more pronounced. Conclusions GSI can inhibit the proliferation of SW480 / L-OHP cells and increase the chemosensitivity by blocking the release of Nicd in the intracellular domain of Notch1 and affecting the expression of downstream Hes1 gene and protein. Synergistic effect of GSI with L-OHP was also observed.