The Inhibition Of Andrographolide On Gefitinib Resistantnon-Small Cell Lung Cancer

ObjectiveGefitinib(Gef)is the first targeted drug in the treatment of non-small cell lung cancer(NSCLC).It has the advantages of strong specificity,good tolerance and less adverse reactions.However,acquired drug resistance is the main reason for clinical treatment failure of gefitinib,and has become a bottleneck problem that restricts the further improvement of the treatment efficacy of advanced nonsmall cell lung cancer(NSCLC).Andrographolide(Andro)is the main effective ingredient of natural plant andrographolide,which has anti-tumor effect.The PC9/G cell line of non-small cell lung cancer resistant to gefitinib was used to preliminarily explore the inhibitory effect of Gef and Andro alone or in combination on the proliferation of PC9/G cells,and to screen the best way of combination of the two drugs.To explore the effect of Andro combined with Gef on the apoptosis of PC9/G cells,and clarify the role of apoptosis in Andro overcoming the Gef resistance of PC9/G cells.It is of great significance and value to study the methods to overcome the drug resistance of gefitinib for further improving the clinical treatment level of lung cancer.MethodsHuman non-small cell lung cancer cells(PC9,H1299),human normal lung tissue cells(Hpaepic)and human embryonic kidney epithelial cells(293T).The inhibitory effects of andrographolide,gefitinib alone and andrographolide combined with gefitinib on cell proliferation were detected by CCK8 experiment.The combination index(CI)was used to compare and evaluate the effect of the combination of the two drugs.The effects of andrographolide,gefitinib alone and andrographolide combined with gefitinib on apoptosis of non-small cell lung cancer cells were detected by flow cytometry.PC9/G cell lines were treated with andrographolide,gefitinib alone or andrographolide and gefitinib together.The proliferation of PC9/G cells was detected by CCK-8 assay,and the morphology of PC9/G cells was observed by Hoechst 33342 fluorescence staining;The apoptosis was analyzed by flow cytometry,the expression of apoptosis-related protein was detected by Western blot,the expression of apoptosis-related gene was detected by Q-PCR,and the change of mitochondrial membrane potential was detected by JC-1 fluorescence staining.Results(1)Compared with sensitive PC9 cell line,drug-resistant PC9/G cell line has changed in cell morphology and proliferation characteristics.The IC50 values of Andro and Gef for PC9/G are 271.2,respectively μ M and 80.3 μ M;Andro and Gef have synergistic effects(CI<1).(2)The results of flow cytometry showed that the apoptosis rate of PC9/G cells in Andro combined with Gef group was significantly higher than that in the control group(P<0.01).Compared with Gef alone group,the apoptosis rate of PC9/G cells in Andro combined with Gef group was significantly higher(P<0.01).Compared with Andro alone group,the apoptosis rate of PC9/G cells in Andro combined with Gef group was also significantly higher(P<0.05).(3)The Western Blot test results showed that compared with the control group,the expression of Bcl-2 in Andro combined Gef group was significantly lower,and the expression of Bax and cleaved caspase-3 was significantly higher(P<0.05).Compared with the Gefitinib alone group,the expression of Bcl-2 in Andro combined Gef group was significantly lower,and the expression of Bax and cleaved caspase-3 was significantly higher(P<0.05),In Andro combined with Gef group,the expression of Bcl-2 was significantly decreased,and the expression of Bax and cleaved-capase-3 was significantly increased(P<0.05).(4)The results of qRT-PCR showed that compared with the control group,the m RNA expression levels of Bcl-2,PCNA and Ki67 in Andro combined Gef group also showed a significant downward trend,and the expression levels of Bax,Caspase-3 and P53 were significantly higher(P<0.01).Compared with the gefitinib alone group,the m RNA expression levels of Bcl-2,PCNA and Ki67 in Andro combined Gef group were significantly lower,and the expression levels of Bax,Caspase-3 and P53 were significantly higher,The difference was significant(P<0.01).Compared with Andro monotherapy group,the m RNA expression level of Bcl-2,PCNA and Ki67 in Andro combined with Gef group decreased,and the expression of Bax,Caspase-3 and P53 increased,the difference was significant(P<0.05).(5)The results of Hochest33342 fluorescence staining showed that compared with the control group,the effect of Andro combined Gef group on inhibiting the proliferation of PC9/G was more significant(P<0.05).Compared with the gefitinib monotherapy group,the effect of Andro combined Gef group on inhibiting the proliferation of PC9/G was still significant(P<0.05).Compared with Andro monotherapy group,the effect of Andro combined Gef group on inhibiting the proliferation of PC9/G was more significant(P<0.05).(6)The results of flow cytometry showed that compared with the control group,Andro combined with Gef group had a significant effect on promoting apoptosis of PC9/G cells(P<0.05),and Andro combined with Gef group still had a significant effect on promoting apoptosis of PC9/G cells(P<0.05).Compared with Andro monotherapy group,Andro combined with Gef group promoted the apoptosis of PC9/G significantly(P<0.05)(7)The results of JC-1 fluorescence staining showed that andrographolide could significantly promote the apoptosis of PC9/G cells(P<0.05),while Andro combined with Gef group had more significant effect on inducing the apoptosis of PC9/G cells(P<0.05)Conclusions(1)Andro can significantly enhance the inhibitory effect of Gef on the proliferation of PC9/G cells,and Andro can significantly enhance the synergistic effect of Gef combined administration at the same time.(2)Andro and Gef jointly activate endogenous mitochondrial cell apoptosis.It is suggested that Andro may overcome the drug resistance of PC9/G cells mainly by inducing cell apoptosis and cooperating with Gef.