CHK1 inhibition overcomes gemcitabine resistance in non-small cell lung cancer cell A549.

Abstract

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.