Effect Of Olaparib On The Colon Cancer Cell Lines With Low Expression Of ATM Gene

Objective Ataxia telangiectasia(ATM)protein kinase,as a tumor suppressor gene,whose mutation is closely related to the occurrence of the tumor,involved in the regulation of cell cycle,mismatch repair process of cell damage and cell repair and others.In recent years,the second-generation sequencing results of most tumors show that ATM genes are mutated in most human cancers,such as colonrectal cancer,lung cancer,prostate cancer and breast cancer and so on.Moreover,more and more studies have confirmed that PARP inhibitor Olaparib was sensitive to the solia tumor patients with low expression of ATM.Considering the key role of ATM gene in DNA damage repair process,this study is designed to investigate the expression of ATM m RNA and protein in colon cancer cell lines by real-time quantitative RT-PCR and Western blotting and screen out the cell line with low expression of ATM.After Olaparib targeted the corresponding colon cancer cell lines,we observe the effect of Olaparib on cell proliferation,cell invasion and migration and clonal formation.Methods 1.The m RNA and protein of ATM gene in human colon cancer HCT116,HCT8,HT29 and SW480 cell lines were detected by real-time reverse transcription and polymerase chain reaction(RT-PCR)and Western blot,respectively.By this way,we screened out the cell lines with low ATM expression;2.The colon cancer cell lines with low expression of ATM gene were treated with different concentrations of Olaparib(12.5?mol / L,25?mol / L,50?mol / L,100?mol / L and 200?mol / L).After 24 hours,48 hours and 72 hours,we detected the proliferate rate of cells by MTT.Then,the IC50 values of Olaparib was calculated by the software of SPSS 17.0;3.The colon cancer cell lines with low expression of ATM were randomly devided into two groups,including experimental group and blank control group which was the cultured naturally cell line without any treatment.According to the IC50 value of Olaparib,different concentration gradient of Olaparib(50?mol/L,100?mol/L,200?mol/L)after 48 h were set to treat the colon cell lines with low expression of ATM.Then,MTT and clonal formation were detected the proliferation of colon cancer cell lines.The cell invasion and migration ability were detected by Transwell chamber.Results 1.The results of RT-PCR and western blot showed that there all were m RNA and protein expression of ATM in all colon cancer cell lines.But the expression level of HCT116 cells was significantly lower than other cells,which performed a subsequent functional test;2.The IC50 values of Olaparib for HCT116 colon cancer cell lines after 48 h was 99.01±0.4?mol/L;3.MTT showed that the OD490 values of the cells in the experimental group including 50umol/L?100umol/L and 200umol/L after 48 h were 0.42±0.030,0.34±0.002,0.29±0.006,respectively,which were significantly lower than those in the blank control group(P<0.05);4.Plate cloning assay showed that the clonogenic ability of the experimental group was significantly lower than that of the control group.In other words,the cell growth of the experimental group was inhibited with statistically significant differences(P<0.05);5.Transwell experiments showed that the cell migration and invasion ability of the experimental group was significantly lower than that of the control group(P<0.05).Conclusion The expression of ATM in four colon cancer cells were different.RT-PCR and Western blot could be used to screen out the colon cancer cell lines with low expression of ATM;2.The IC50 value of low expression of colon cancer cell line was calculated by MTT,which provided some references for the subsequent functional experiment;3.Olaparib acted on colon cancer cells with low expression of ATM,thereby inhibiting cell growth and proliferation,promoting cell apoptosis,down-regulating cell invasion and migration ability,and reducing cell cloning ability;4.In conclusion,the sensitivity of Olaparib to colon cancer cells with low expression of ATM was obviously enhanced.