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

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=0.001 5）；细胞早期和晚期凋亡比例增高（P=0.000 6）；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=0.008 7）；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。

Abstract:

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 half-maximal 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=0.001 5） and a significant increase in the proportion of cells in the early and late stages of apoptosis（P=0.000 6）. 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=0.008 7），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.