The Role And Mechanism Of Apurinic/Aprimidinic Endonuclease 1 In Resistance Of NSCLC To EGFR-TKI

Background: Non-small cell lung cancer(NSCLC)is one of the leading malignant tumors in the world.Combination chemotherapy with platinum based regimens has been the only treatment option for patients with advanced NSCLC until a few years ago,but the emergence of resistance is the main reason for limiting the improvement in outcomes.Deletion mutations of the exon 19(especially E746-A750 censored)and substitutemutation of exon 21(amino acid substitution at codon 858 substitution of arginine for leucine(L858R))is the two most common EGFR mutations activating epidermal growth factor receptor of two kinds of mutations,conferring epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)therapeutic sensitivity.Compared with EGFR wild type(wild-type,WT)patients with NSCLC,patients with NSCLC with above mutations have a higher response rate to the treatment of EGFR-TKI(response rates RR)(up to 70%),have a longer the median survival period(up to 24-30 months).Compared with wild-type EGFR,these mutations lead to an increased affinity of ATP-binding pocket of EGFR for EGFR-TKI,thus making NSCLC patients more sensitive to EGFR-TKI treatment.Dual function gene APE1 plays an important role in tumor resistance,and its DNA damage repair activity is affected by redox state.Apurinic apyrimidinic endonuclease(Apurinic aprimidinic endonuclease 1,APE1)is also known as redox factor(Redox,factor-1,Ref-1),which has two independent domains: redox domain at the C end and DNA repair function domain at the N terminal.One hand,APE1 is a core repair enzyme in the pathway of DNA base excision repair(base excision,repair,BER).APE1 cleave DNA single strand at the 5 'end of the main chain of DNA in apurinic apyrimidinic(AP)sites in single strand breaks open form(single strand break,SSB),and treat the 3' ends in order to facilitate the incorporation of Pol beta and the correct nucleotide of DNA.AP endonuclease activity is essential for successful repair of BER,whereas APE1 provides more than 95% AP endonuclease activity in cells.It is more critical that the APE1 number at the AP site of DNA as the repair substrate reflects the damage degree of DNA directiy,so APE1 is a key protein sensing DNA base damage and its repair capacity correspond to ability of DNA base damage directly.On the other hand,APE1 also has redox activity,which is responsible to DNA-binding activity of the key transcription factors,including AP-1,NF-kappa B,Egr-1,p53,HIF-1,as well as transcription factors associated with mitochondrial function such as NRF1 and TFAM.In the previous study,we found that expression levels of APE1 in lung cancer,colorectal cancer,myeloma,hepatocellular carcinoma,osteosarcoma tumor cells can be induced to rise by ionizing radiation,and knockdown of APE1 can also significantly improve sensitivity of the lung cancer cell to treatment.At present,a number of research evidence support that APE1 is an important determinant of intrinsic sensitivity of tumor cell to antitumumor therapy,and so it is also a potential molecular target for cancer therapy.Based on the correlation between DNA damage and sensitivity to antitumor therapy,at present most of scholars are of the view that the DNA repair activity of APE1 may be a key function involved in regulation of sensitivity to antitumor therapy.Our previous study found that the combination of AT-101 as inhibitor of dual function of APE1 with EGFR-TKI significantly sensitizes NSCLC cells with acquired resistance to EGFR-TKIs,which imply that APE1 may promote generation of resistance of NSCLC to EGFR-TKIs.Objective: This paper mainly discusses role and mechanism of the apurinic / apyrimidinic endonuclease 1 / redox factor-1(APE1/Ref-1)in the acquired resistance of NSCLC to EGFR-TKIs by in vivo and in vitro experiments.Methods: To EGFR-TKI acquired drug resistance cells(HCC-827 IR and PC-9ER)and primary drug resistance cell(NHI-H1975),AT-101,APE1,E3330 or MK2206 combined with EGFR-TKI drugs(Icotinib and Erlotinib)to treat;Investigate the expression level of APE1 by cell transfection experiments regulating intracellular APE1 by siRNA-APE1 and siRNA-CON plasmid;Investigate inhibition level of cell proliferation by CCK-8 assay and colony formation assay;Cell apoptosis was detected by flow cytometry;Investigate the subcellular distribution of APE1 by confocal immunofluorescence;Investigate expression levels BCL-2,Bax,APE1,p-Akt and Akt genesby by Western blot.Results: At first,after treatment of acquired drug resistance cells(HCC-827 IR and PC-9ER)and primary drug resistance cell(NHI-H1975)with EGFR-TKI(Icotinib and Erlotinib)combined with AT-101,we found that combination of EGFR-TKI with AT-101,compared with EGFR-TKI alone,sensitivity of HCC-827 IR,PC-9ER and NHI-H1975 to the EGFR-TKI drugs increased obviously by clone formation assay and CCK-8 assay;By Western blot experiment,we found combination of EGFR-TKI with AT-101 significantly inhibited EGFR-TKI induced P-Akt levels;After transfection using siRNA-CON and siRNA-APE1 plasmids,EGFR-TKI drug(Icotinib and Erlotinib)treatment of acquired drug resistance cells(HCC-827 IR and PC-9ER)and primary drug resistance cell(NHI-H1975),found that after the inhibition of the expression of APE1,the sensitivity of PC-9ER,HCC-827 IR cells and NHI-H1975 cells to EGFR-TKI drug increased obviously,and induced P-Akt levels by EGFR-TKI was significantly decreased obviously.Secondly,by combining E3330 or APE1 inhibitor III with EGFR-TKI(Icotinib and Erlotinib)to treat acquired drug resistance cells(HCC-827 IR and PC-9ER)and primary drug resistance cells(NHI-H1975),we found that EGFR-TKI combined with E3330,compared with EGFR-TKI alone and EGFR-TKI combined with APE1 inhibitor III,sensitivity of HCC-827 IR,PC-9ER and NHI-H1975 to the EGFR-TKI drugs increased significantly by CCK-8 proliferation assay;EGFR-TKI combined with E3330 inhibited EGFR-TKI induced p-Akt level is most obviously;we combined MK2206 and EGFR-TKI(Icotinib and Erlotinib)drugs to treated above cells,the result showed that this joint significantly enhanced the sensitivity of HCC-827 IR,PC-9ER and NHI-H1975 cells to EGFR-TKI drugs.Finally,after establishment of xenograft tumor of HCC-827 IR cells in nude mice animal model,combined treatment of Icotinib and AT-101 to xenograft tumor results in decreased the growth rate,combination with both significantly inhibited growth of tumor,decreased tumor weight in nude mice;and immunohistochemical experiments showed that combination with both significantly inhibited the level of p-Akt in xenograft tumor tissue of nude mice.Conclusion: Inhibiting to APE1 or redox activity of APE1 significantly inhibited p-Akt levels induced by EGFR-TKI and sensitized resistant cells of NSCLC to EGFR-TKI;APE1,especially its redox activity,played an important role in promoting resistance of NSCLC to EGFR-TKI.