XL413通过抑制DNA复制诱导细胞凋亡来抑制结肠癌细胞增殖  

XL413 inhibits colon cancer cell proliferation by inhibiting DNA replication and inducing cell apoptosis

作　　者：李倩[1] 胡蓉 袁平 Li Qian;Hu Rong;Yuan Ping(Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases,The Sixth Affiliated Hospital of Sun Yat-sen University,Guangdong Institute of Gastroenterology,Biomedical Innovation Center,The Sixth Affiliated Hospital of Sun Yat-sen University)

机构地区：[1]中山大学附属第六医院广东省结直肠盆底疾病研究重点实验室、广东省胃肠病学研究所,中山大学附属第六医院生物医学创新研究院,广州510655

出　　处：《重庆医科大学学报》2023年第7期753-759,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81773156)。

摘　　要：目的:探讨抑制细胞分裂周期蛋白7(cell division cycle 7,CDC7)的药物XL413对结直肠癌细胞的影响及其作用机制。方法:在多个结直肠癌细胞系中分别加入生理盐水和不同浓度的XL413处理72 h,用细胞活力检测试剂盒(cell counting Kit-8,CCK-8)检测细胞在450 nm处的吸光度值并计算细胞存活率和XL413的半抑制浓度(half maximal inhibitory concentration,IC50)值;Annexin V-FITC/PI双染法检测细胞凋亡;克隆形成实验检测细胞的再生能力;RNA-seq检测XL413对细胞转录组的影响;qRT-PCR法检测凋亡相关基因的mRNA表达量;Western blot检测DNA解旋微小染色体维持蛋白(minichromosome maintenance protein,MCM)复合体成员MCM2的磷酸化以及凋亡相关蛋白的表达水平。结果:与对照组相比,XL413处理组结直肠癌细胞的增殖、迁移、集落形成能力均下降(P=0.0015);细胞早期和晚期凋亡比例增高(P=0.0006);在基因转录表达水平上,XL413处理组的细胞凋亡标志B细胞淋巴瘤基因(B-cell lymphoma-2,BCL2)/BCL2相关X基因(BCL2 associated X,BAX)的比值下降(P=0.0087);在蛋白水平上,XL413抑制DNA解旋相关蛋白MCM2的磷酸化,从而抑制细胞DNA复制,同时促进凋亡相关蛋白的表达增高。结论:XL413可以通过降低DNA解旋相关蛋白MCM2的磷酸化阻碍DNA的复制,诱导结直肠癌细胞凋亡。该研究为XL413未来用于结直肠癌治疗提供了一定的理论依据。Objective:To investigate the effect of XL413,a drug inhibiting cell division cycle 7(CDC7),on colorectal cancer cells and its mechanism of action.Methods:Multiple colorectal cancer cell lines were treated with normal saline and XL413 at different concentrations for 72 hours.CCK-8 assay was used to measure the absorbance of cells at 450 nm and calculate cell viability and the halfmaximal inhibitory concentration(IC50)of XL413;Annexin V-FITC/PI double staining was used to measure cell apoptosis;colony formation assay was used to observe the regenerative ability of cells;RNA-seq was used to observe the effect of XL413 on cell transcriptome;qRT-PCR was used to measure the relative mRNA expression levels of apoptosis-related genes;Western blot was used to measure the phosphorylation level of minichromosome maintenance protein 2(MCM2),a member of the DNA unwinding minichromosome maintenance protein complex family,and the expression levels of apoptosis-related proteins.Results:Compared with the control group,the XL413 treatment group had significant reductions in the abilities of colorectal cancer cell proliferation,migration,and colony formation(P=0.0015)and a significant increase in the proportion of cells in the early and late stages of apoptosis(P=0.0006).As for the transcriptional expression level of genes,the XL413 treatment group had a significant reduction in the ratio of the cell apoptosis markers B-cell lymphoma-2(Bcl-2)to Bcl-2-associated X(P=0.0087),and at the protein level,XL413 inhibited the phosphorylation of the DNA unwinding-related protein MCM2 and thus DNA replication and promoted the increase in the expression of apoptosis-related proteins.Conclusion:XL413 can inhibit DNA replication by reducing the phosphorylation of the DNA unwinding-related protein MCM2 and induce the apoptosis of colorectal cancer cells.The study provides a theoretical basis for the application of XL413 in the treatment of colorectal cancer in the future.

关 键 词：XL413 结直肠癌细胞 半抑制浓度 DNA复制 凋亡 

分 类 号：R34[医药卫生—基础医学]