rBTI联合奥沙利铂促进结肠癌细胞凋亡

rBTI Combined with Oxaliplatin Promotes Apoptosis in Colon Cancer Cells

该文探讨了重组荞麦胰蛋白酶抑制剂(rBTI)联合奥沙利铂对结肠癌细胞HCT116和DLD-1凋亡的影响。首先通过细胞增殖实验筛选rBTI和奥沙利铂对结肠癌细胞的有效作用浓度,选取奥沙利铂存活率大于80%的浓度作为联合用药的浓度;然后采用MTT、形态学观察、克隆形成、流式细胞术、Western blot以及qRT-PCR检测了细胞增殖、形态变化、克隆形成、凋亡情况以及Bcl-2、Bax、Cleaved Caspase-3的蛋白质及m RNA的表达情况;进一步通过活性氧检测、分子对接以及Western blot分析了联合用药后MAPK信号通路的变化情况。结果显示,rBTI和奥沙利铂均可以抑制结肠癌细胞增殖且具有剂量依赖性;药物联合作用于结肠癌细胞后,以协同作用的方式显著抑制结肠癌细胞的增殖;与单独用药组相比,联合用药组细胞数量明显减少,收缩变圆,体积减小;细胞克隆形成减少,细胞凋亡数量增加;Cleaved Caspase-3、Bax的蛋白和m RNA表达量上升,Bcl-2表达量下降;活性氧含量上升;rBTI和奥沙利铂均可以与MAPK信号通路关键蛋白紧密结合,并能改变关键蛋白的表达。总之,rBTI联合奥沙利铂可以促进结肠癌HCT116和DLD-1细胞凋亡。

In this paper,the effects of rBTI(recombinant buckwheat trypsin inhibitor)combined with Ox-aliplatin on apoptosis of colon cancer cells HCT116 and DLD-1 were investigated.Firstly,the effective concentra-tion of rBTI and Oxaliplatin on colon cancer cells were screened by cell proliferation assay,and the concentration with a survival rate of more than 80%was selected as the concentration of the combined drug.MTT,morphological observation,clonogenesis,flow cytometry,Western blot and qRT-PCR were used to detect cell proliferation,mor-phology,clonogenesis,apoptosis,Bcl-2,Bax,Cleaved Caspase-3 protein and mRNA expression.Furthermore,the changes of MAPK signaling pathway were analyzed by reactive oxygen species detection,molecular docking and Western blot.The results showed that both rBTI and Oxaliplatin could inhibit the proliferation of colon cancer cells in a dose-dependent manner.The proliferation of colon cancer cells was significantly inhibited by the synergistic action of the drugs.Compared with the single treatment group,the number of cells in the combined treatment group decreased,the contraction became round and the volume decreased.The cell clonal formation decreased and the number of apoptosis increased.Cleaved Caspase-3 and Bax protein and mRNA expressions increased,and Bcl-2 expression decreased.The expression of reactive oxygen species increased;both rBTI and Oxaliplatin could bind closely to the key proteins of MAPK signaling pathway,and could change the expression of key proteins.In con-clusion,rBTI combined with Oxaliplatin could promote apoptosis of colon cancer HCT116 and DLD-1 cells.