Ke Zhi-Yin, Fu Tian, Wang Xue-Chun, Ma Xuan, Yin Hai-Han, Wang Wen-Xuan, Liu Yong-Jun, Liang Ai-Ling
Department of Biochemistry and Molecular Biology & Department of Clinical Biochemistry, Guangdong Provincial Key Laboratory of Medical Immunology and Molecular Diagnosis, Guangdong Medical University, Dongguan, China.
Department of Clinical Laboratory, Zhanjiang Central Hospital, Zhanjiang, China.
Mol Cell Oncol. 2025 Apr 9;12(1):2488537. doi: 10.1080/23723556.2025.2488537. eCollection 2025.
The purpose of the study is mainly to investigate anti proliferation of non-small cell lung cancer A549 cells and its mechanism by inhibition of CHK1 expression combined with gemcitabine. The mRNA and protein levels of genes were analyzed by RT-qPCR and Western blot, respectively. Cell viability was detected by CCK-8 assay and clone formation assay. The detection of the cell cycle was used by Annexin V/7-amino-actinomycin D apoptosis detection kit. Analysis of DNA damage was done by immunofluorescence and alkaline comet assay. The results showed that inhibition of CHK1 and gemcitabine combination significantly reduced the proliferation ability of the two cell lines. We also revealed the degradation of full-length PARP and reduced Bcl-2/Bax ratio on increased apoptosis. Inhibition of CHK1 expression leads to DNA damage, induces phosphorylation of γ-H2AX, and affects the repair of homologous recombination ability through Rad51. Mechanistically, gemcitabine increased phosphorylation-ATR and phosphorylation-CHK1, indicating activation of the DNA repair system and ATR-CHK1-CDC25A pathway. Inhibition of CHK1 resulted in increased synthesis of CDK2/Cyclin A2 and CDK2/Cyclin E1 complexes, and more cells entered the subsequent cell cycle, leading to S phase arrest and mitotic catastrophe. We identified inhibition of CHK1 as a potential treatment for NSCLC and confirmed that inhibition of this kinase could overcome acquired gemcitabine resistance.
本研究的目的主要是通过抑制CHK1表达联合吉西他滨来研究其对非小细胞肺癌A549细胞的抗增殖作用及其机制。分别通过RT-qPCR和蛋白质免疫印迹法分析基因的mRNA和蛋白质水平。通过CCK-8法和克隆形成试验检测细胞活力。使用膜联蛋白V/7-氨基放线菌素D凋亡检测试剂盒检测细胞周期。通过免疫荧光和碱性彗星试验进行DNA损伤分析。结果表明，抑制CHK1与吉西他滨联合使用可显著降低两种细胞系的增殖能力。我们还发现全长PARP降解以及凋亡增加时Bcl-2/Bax比值降低。抑制CHK1表达导致DNA损伤，诱导γ-H2AX磷酸化，并通过Rad51影响同源重组能力的修复。机制上，吉西他滨增加了磷酸化-ATR和磷酸化-CHK1，表明DNA修复系统和ATR-CHK1-CDC25A通路被激活。抑制CHK1导致CDK2/Cyclin A2和CDK2/Cyclin E1复合物的合成增加，更多细胞进入后续细胞周期，导致S期阻滞和有丝分裂灾难。我们确定抑制CHK1是一种潜在的非小细胞肺癌治疗方法，并证实抑制这种激酶可以克服获得性吉西他滨耐药性。
