Berberine Inhibits The Migration Of EGFR-TKIs Resistant Cell Lines In Non-small Cell Lung Cancer By Down-regulating CCL2

Background and objective:Epidermal growth factor receptor-tyrosine kinase inhibitors（EGFR-TKIs）are a key milestone in targeted therapy for non-small cell lung cancer,which significantly improves patient’s quality of life.However,drug resistance is a serious obstacle for the clinical application of EGFR-TKIs.Accumulating studies demonstrated that overexpression of chemokine CCL2（CC motif chemokine ligand 2）is related to drug resistance to various anti-tumor drugs.Our previous GEO database analysis showed that the expression of CCL2 is significantly increased in gefitinib resistant cells,strongly indicates that CCL2 plays an important role in development of EGFR-TKIs resistance.Berberine（BBR）is an isoquinoline quaternary ammonium alkaloid isolated from a variety of medicinal plants,which exhibits anti-inflammatory,antioxidant,anti-tumor effects.In addition,studies reported that berberine inhibits the expression of CCL2.We propose that berberine may affect the development of EGFR-TKIs resistance by inhibiting the expression of CCL2.The aim of this study is to clarify the role of CCL2 in the development of EGFR-TKIs resistance,and to investigate the role and mechanism of berberine in EGFR-TKIs-resistant cells.Methods:1.Firstly,we constructed the stable cell lines which is resistant to Gefitinib and Osimertinib by using the concentration gradient method in non-small cell lung cancer HCC827 cell lines and named them as HCC827-GR and HCC827-OR.Then,we identified the IC50 value of HCC827-GR and HCC827-OR cell lines to gefitinib and Osimertinib,as well as the drug resistance index by using MTT assay to verify whether the resistant stable cell line is successfully constructed.2.MTT,clone formation and Transwell assays were implied to observe the biological behavior changes,such as proliferation and migration ability of HCC827-GR and HCC827-OR cell lines.Western blot and immunofluorescence assay was used to observe the changes of PI3K/AKT,MAPK and EMT signaling pathways in HCC827-GR and HCC827-OR cell lines to study the mechanism of the development of EGFR-TKIs resistance.3.To analyze the changes of gene expression of gefitinib-resistant cells in the GEO database GSE122005 data set using bioinformatics technology.Then the expression level of CCL2 in EGFR-TKIs-resistant cells were confirmed by q q RT-PCR and Western blot.We constructed the CCL2 overexpressed stable cell lines by using o CCL2 overexpression plasmids.Furthermore,the IC50 and the drug resistance index of CCL2-overexpressed cell lines against Gefitinib and Osimertinib was calculated by using MTT assay.Then,we investigated the proliferation,migration ability,PI3K/AKT,MAPK and EMT signaling pathways of CCL2 to clarify the role of CCL2 in EGFR-TKIs resistance.4.The HCC827-GR and HCC827-OR cell lines and CCL2-overexpressing stable cell lines were treated with berberine,and then the effects of berberine on the migration ability of drug-resistant cell lines were observed by wound healing and Transwell assay.Then,we explored the expression of CCL2 and the changes of EMT signaling pathways using q RT-PCR,Western blot and immunofluorescence assay.Results:1.The IC50 of HCC827 cells to Gefitinib was 5.415x10^-7μM,and the IC50 of HCC827-GR was 11.89μM,and the resistance index（RI）=22018518;The IC50 of HCC827 cells to Osimertinib was 3.835x10^-5μM,and the IC50 of HCC827-OR was10.41μM,and the resistance index（RI）=271447,which highly suggest that we successfully constructed HCC827-GR and HCC827-OR cell lines.2.Compared to HCC827 cells,the proliferation rate of HCC827-GR and HCC827-OR cell lines were significantly slowly.Notably,we found that the migration ability of HCC827-GR and HCC827-OR was significantly increased in compared to the HCC827 cells.In addition,we observed that the expression level of Vimentin in HCC827-GR and HCC827-OR was significantly increased,while the level of E-cadherin was significantly decreased.3.The bioinformatic analysis results demonstrated that gefitinib-resistant cells has 1362 differential genes in compared to sensitive cells,including 636 down-regulated genes and 726 up-regulated genes（︱log FC︱>1,P<0.05）,in which CCL2 ranked second in the up-regulated genes.The results of KEGG analysis showed that the up-regulated genes are closely related to the chemokine signaling pathway.Remarkably,we found the expression of CCL2 was significantly increased in HCC827-GR and HCC827-OR cell lines compared with HCC827,P<0.01.4.Western blot results showed that the CCL2 overexpression plasmid tag protein DDK was significantly expressed,which indicated that HCC827-CCL2overexpressed cells were successfully constructed.Additionally,we found that the IC50 of HCC827-Empty cells to Gefitinib and Osimertinib were 0.02975μM and0.002625μM,respectively,and the IC50 of HCC827-CCL2 overexpressed cells to gefitinib and Osimertinib were 5.209μM and 933.7μM,and the resistance indices were 175.09 and 355695,respectively.These findings strongly implied that the sensitivity of CCL2 overexpressed cells to EGFR-TKIs is significantly decreased（P<0.001）.Furthermore,we detected that the proliferation and migration ability of the CCL2 overexpressed cell line was significantly increased（P<0.001）.Th Immunofluorescence results showed that the expression level of E-Cadherin was decreased,and the expression level of Vimentin was increased in CCL2-overexpressed cells in compared with HCC827-Empty cells.5.We found that berberine inhibited the proliferation of HCC827-GR and HCC827-OR cell lines in a time-concentration-dependent manner.However,low concentration（12.5μM）of berberine did not affect the proliferation ability of cells at24 h.Hence,we used 12.5μM berberine to investigate the effects of berberine on migration ability of EGFR-TKIs resistant cells.Interestingly,we observed that berberine could not only inhibit the migration of HCC827-GR and HCC827-OR cell lines（P<0.01）,but also significantly inhibit the migration of CCL2-overexpressed cells（P<0.01）.In addition,berberine could significantly reduce the expression level of CCL2 in HCC827-GR and HCC827-OR cell lines（P<0.01）.Both Western blot and immunofluorescence results showed that berberine could up-regulate the expression of E-cadherin and down-regulate the expression of Vimentin in HCC827-GR and HCC827-OR cell lines and CCL2 overexpressed cells.Conclusion:1.HCC827 cells,which resistant to gefitinib and Osimertinib,exhibits slowly proliferation ability and enhanced migration ability.2.CCL2 is overexpressed in Gefitinib and Osimertinib-resistant cells.Overexpression of CCL2 reduces the drug sensitivity of HCC827 cells to gefitinib and Osimertinib,and results in enhanced cell proliferation and migration and epithelial mesenchymal transformation.3.Increased expression of CCL2 may be one of the mechanisms of HCC82 cells leading to resistance of EGFR-TKIs.4.Berberine may inhibit the migration of lung cancer EGFR-TKIs acquired resistance cells through inhibiting the CCL2-EMT signaling pathway.