Experimental Study On EGFR-TKI Resistant Non-Small Cell Lung Cancer With Antiangiogenic Drugs

Purpose and background:In recent years,more and more clinical studies have shown that the combination of antiangiogenic drugs can significantly prolong the progression-free period of disease in patients with first-line resistance to epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI).Tumor angiogenesis is closely related to tumor growth,invasion of peripheral tissues and organs and distant metastasis.However,anti-angiogenic drugs can effectively inhibit tumor angiogenesis,destroy tumor microenvironment by blocking the nutritional supply of tumor cells,and thus inhibit the proliferation and invasion of tumor cells.Relevant in vitro experiments indicated that vascular endothelial growth factor receptor(VEGFR)is not only expressed in vascular endothelial cells,but also in lung adenocarcinoma cells,and tumor cells can secrete vascular endothelial growth factor(VEGF)spontaneously,which indicates that anti-VEGF has a direct inhibitory effect on tumor cells.EGFR and VEGFR share a common downstream signaling pathway,and the inhibition of EGFR can also inhibit the secretion of VEGF.The combination of the two can inhibit both EGFR and VEGFR signaling pathways,so as to enhance the anti-tumor effect of the drug.Therefore,after the first generation of EGFR-TKI appears drug resistance,it may become a new therapeutic approach to continue the treatment with the combination of the original molecular targeted drugs and anti-angiogenic drugs.Therefore,the addition of antiangiogenic drugs may become a new treatment method for patients with drug resistance after first-generation EGFR-TKI.The purpose of this study was to study in vitro the effect of molecular targeting combined with anti-angiogenic drugs on the proliferation of EGFR-TKI resistant NSCLC,the formation of vascular mimicry and the changes of related protein molecules,and to explore the anti-tumor effect and mechanism of the combination of the above drugs in EGFR-TKI resistant cell lines.Methods:(1)PC-9GR and HCC827GR cell lines were induced by gefitinib at long-term low dose from PC-9 and HCC827 cell lines of lung adenocarcinoma cells with EGFR19 exon-deletion as the parent strains.PC-9GR carried T790M mutation,while HCC827GR had no T790M mutation,H-1975 carried EGFR 21 exon L858R mutation and T790M mutation.(2)Selected PC-9 GR,H-1975,HCC827GR as experimental object,set up the negative control group(no drugs),positive control groups(gefitinib monotherapy group,AZD 9291 monotherapy group,anlotinib monotherapy group,bevacizumab monotherapy group)and experimental groups(anlotinib combined with gefitinib group,AZD 9291 combined with anlotinib group,bevacizumab combined with gefitinib group,bevacizumab combined with AZD 9291 group);(3)the IC50 value of antiangiogenesis in combination with EGFR-TKI and both alone in each cell line was detected by CCK-8 method;(4)The combination index(CI)of different cell lines were calculated by CompuSyn software to judge the effect of the combined drug regimen;(5)Vascular mimicry was used to observe the influence of each cell line on the formation of angiogenic mimicry under the antiangiogenesis in combination with EGFR-TKI and both alone.(6)Western blot was used to detect the molecular changes of anti angiogenic signal transduction pathway in each cell line,such as p-FGFR,Akt,p-Akt,Erk and p-Erk,and to explore the anti-tumor mechanism of anti angiogenic drugs on EGFR-TKI resistant non-small cell lung cancer cells.Results:(1)Among the three gefitinib-resistant lung adenocarcinoma cell lines,anlotinib and bevacizumab inhibite EGFR mutation-positive lung cancer cells.The combination of anlotinib and gefitinib can reverse the resistance of drug-resistant lung cancer cells to gefitinib,and the combination of anlotinib and bevacizumab with AZD 9291 can enhance the antitumor activity of the drug against drug-resistant lung cancer cells.(2)The inhibition of PC-9GR and H-1975 by high concentration of anlotinib and bevacizumab combined with gefitinib is superior to that of the low-concentration of anlotinib and bevacizumab combination scheme,with statistically significant difference(p<0.05).In HCC827GR cell line,there is no significant difference in the inhibitory effect of gefitinib combined with bevacizumab at different concentrations.The inhibition of PC-9GR,H-1975 and HCC827GR by combining high concentration of anlotinib and bevacizumab with AZD 9291 is better than combining low concentration of anlotinib and bevacizumab with AZD 9291.(3)Anlotinib and bevacizumab combined with gefitinib and AZD 9291 has synergistic effect on the inhibition of tumor cell proliferation,and there is a dose dependence between the synergistic effect and the dose of the drugs.(4)In vitro experiment,anlotinib and bevacizumab combine with gefitinib or AZD 9291 can significantly inhibite the formation of vascular mimicry in PC-9GR and HCC827GR cell lines.But for H-1975,anlotinib and bevacizumab combine with geitinib or AZD 9291 has no inhibitory effect on vascular mimicry.(5)The expression of p-Akt in PC-9GR,H-1975 and HCC827GR cell lines decrease when anlotinib and bevacizumab combine with gefitinib and AZD 9291.The expression of p-Erk in PC-9GR and H-1975 cell lines decrease when anlotinib combines with gefitinib and AZD 9291.In HCC827GR cell line,there is no effect on the expression of p-Erk by combination of anlotinib and bevacizumab with gefitinib and AZD 9291.Conclusion:The combination of anti-angiogenic drugs and molecular-targeted drugs can reduce the drug resistance of first-generation EGFR-TKI resistant tumor cells and improve the anti-tumor activity of third-generation EGFR-TKI.Anti-angiogenesis combined with gefitinib and AZD 9291 can inhibit the proliferation of EGFR mutation-positive tumor cells.The two have a synergistic effect,which is related to the concentration of anti-angiogenesis drugs and molecular-targeted drugs.It is suggested that anlotinib and bevacizumab has a better inhibitory effect on angiopoiesis in the cells with exon 19 deletion.The combination of anlotinib and gefitinib or AZD 9291 can inhibit the proliferation of T790M positive tumor cells by inhibiting Akt and Erk signaling pathways.In tumors without T790M mutation,the combination of anlotinib and bevacizumab with gefitinib and AZD 9291 mainly inhibit the proliferation of tumor cells by inhibiting the Akt signaling pathway.