Effect Of Erlotinib On Radiosensitivity Of Human Non-small Cell Lung Cancer Cells And Its Mechanism

Objective:Epidermal growth factor receptor plays an important role in radiotherapy resistance of non-small cell lung cancer.This study was to investigate the effect of epidermal growth factor receptor tyrosine kinase inhibitor on radiosensitivity of human non-small cell lung cancer cells and its possible mechanism.Methods:Three human non-small cell lung cancer cells A549,H1299,PC9 were cultured in vitro.CCK-8 was used to detect the antiproliferative activity of erlotinib on three cells,IC50 and IC20 were calculated.The IC20 of each cell was used as the drug action concentration for subsequent experiments.According to whether erlotinib was combined with erlotinib before X-ray irradiation,the three cells were divided into blank control group?NC?,erlotinib group?E?,irradiation group?R?and radiotherapy+erlotinib group?E+R?4 groups.Clonogenic radiation survival assays were performed to detect the effects of irradiation with or without erlotinib on three cells,and the radiosensitivity parameters were calculated,the cell survival curve was ploted.Flow cytometry was used to identified the apoptosis and cell cycle distribution of each group.The expression levels of EGFR/PI3K/AKT pathway,Rab25 gene,apoptosis-related protein mRNA,miR125a-5p and MALAT1 were detected by qRT-PCR.Western blot was used to measure the expression of EGFR/PI3K/AKT pathway,Rab25 and apoptosis-related proteins in each group.To explore the possible mechanism of erlotinib radiosensitization to non-small cell lung cancer cells.Results:1.The CCK-8 results showed that erlotinib had proliferation inhibition effects on all three cells.The IC50 of A549 cells was?18.167?3.683??mol/L,IC20 was?0.860?0.196??mol/L,the IC50 of H1299 cells was?27.297?4.220??mol/L,IC20 was?3.324?0.253??mol/L,The IC50 of PC9 cells was?0.137?0.024??mol/L,and IC20was?0.011?0.002??mol/L.PC9 cells are more sensitive to erlotinib than the other two cells.2.The results of Clonogenic radiation survival assays demonstrated that the combination of erlotinib before X-ray irradiation could reduce the number of clones of A549 and H1299 cells and reduce the colony formation rate.At the same time,the D_q,D₀ and SF₂ of the two cells combined with the erlotinib group were smaller than the irradiation group,and the SER values were 1.248 and 1.244.The difference was statistically significan.However,PC9 cells showed no similar results to A549 and H1299 cells.There was no significant difference in D_q,D₀ and SF₂ between the radiotherapy+erlotinib group and irradiation group.The SER value was 1.053.These data suggested that erlotinib could increase the killing effect of radiation on A549cells and H1299 cells,but didn't increase the effect on PC9 cells.3.Flow cytometry results revealed that erlotinib increased the apoptosis rate of A549and H1299 cells induced by radiation,but could not increase the apoptosis rate of PC9cells induced by radiation.The cell cycle results showed that erlotinib blocked the A549,H1299,and PC9 cells in the G₀/G₁ phase,which reduced the S phase ratio.When combined with radiation,A549,H1299 cells have a higher proportion of G₀/G₁phase,less proportion of S phase,and increase the proportion of G₂/M phase.However,compared with the simple irradiation group,erlotinib combined with radiation for PC9 cells did not significantly change the G₀/G₁ phase ratio,S phase ratio,and G₂/M phase ratio of PC9 cells.4.The results of qRT-PCR suggested that compared with the R group,the expression of AKT mRNA in the E+R group of A549 cells decreased,the expression of PARP gene mRNA in the E+R group of H1299 cells increased,and The expression of EGFR gene mRNA was decreased in PC9 cells E and E+R groups.There was no significant difference in the expression of miR125a-5p and MALAT1 between the different group of three cells.5.Western blot results indicated that erlotinib could inhibit the expression of pEGFR and pAKT in three cells,but the single irradiation did not cause significant changes in pEGFR and pAKT.Compared with the erlotinib group,the expression of pEGFR and pAKT in A549 and H1299 cells was further inhibited after irradiation,and the expression of pEGFR and pAKT in PC9 cells was not further decreased.Compared with the erlotinib group and the irradiation group,erlotinib combined irradiation increased the expression of Active Caspase 3 and Cleaved PARP in A549 and H1299cells,the expression of EGFR was inhibited and Caspase 3 was increased in A549cells.but the expression of Active Caspase 3 and Cleaved PARP in PC9 cells were not increased.Due to the low expression of Rab25 in A549 cells and H1299 cells,the expression of these two cellular proteins were not detected by Western blot.For PC9cells,erlotinib did not cause significant changes in Rab25 protein expression,and irradiation inhibited Rab25 protein expression,but erlotinib combined with radiation did not further inhibit Rab25 protein expression.Conclusion:Erlotinib increased radiosensitivity of EGFR wild-type NSCLC cells A549 and H1299,but did not increase PC9 radiosensitivity of EGFR mutant NSCLC cells.The possible mechanism is that erlotinib combined with radiation can further inhibit EGFR/PI3K/AKT pathway in EGFR wild-type NSCLC cells A549 and H1299cells,change cell cycle progression and increase apoptotic cells.