Mechanism Of Apatinib Inducing Breast Cancer Cells Apoptosis By Inhibiting Erk1/2 Signaling Pathway And Activating Autophagy

Objective Exploring the functions of Apatinib in inhibiting breast cancer cell proliferation,inducing apoptosis,blocking cells cycle,as well as activating autophagy.Establishing the taget of Apatinib relationship between VEGFR-2 and Erk1/2 signaling pathway,which can study the mechanism of Apatinib inducing breast cancer cells apoptosis by regulating Erk1/2 signaling pathway and activating autophagy.Methods In this study,we cultured the breast cancer cell lines MDA-MB-231 and MCF-7 in vitro,the cell proliferation rate was detected by the CCK-8;The anti-invasion effects of apatinib in MDA-MB-231 and MCF-7 breast cancer cell lines were tested by wound-healing at 24、48 and 72 hours;cell apoptosis rate and cell cycle were determined by flow cytometry;mRFP-eGFP-LC3 plasmid transfected breast cancer cell lines to detect autophagy.More over,we constructed and analyzed the target of apatinib through the String database that the protein interaction network between VEGFR-2 and MAPK;some protein related about apoptosis,such as PARP1,Cleaved-PAPR1,Cleaved-Caspase3,Bcl-2,Bax,autophagy,LC3、P62 and Beclin1 included,as well as signaling pathway p-Erk1/ 2-Erk1/2were analysed by Western Blot.Results 1.The proliferation rate of breast cancer cells of the different concentration groups,apatinib signficantly inhibited MDA-MB-231 and MCF-7 cell proliferation in a dose and time-dependent minner(all P<0.001).2.There was significant difference in which the wound-healing ratios of MDA-MB-231 cells in the experimental group at 24 h,48h,and 72 h were(3.1±0.4)%,(8.8±1.7)%,(33.8±2.5)%;meanwhile,the scratch healing ratios of MCF-7 cells in the experimental group at 24 h and 48 h were(15.04±3.07)% and(24.62±5.17)%,respectively,both lower than the blank group(all P<0.001).3.Compared with the blank group,flow cytometry detection of the apoptosis rate of MDA-MB-231 and MCF-7 cells increased with the concertration of apatinib growing(all P<0.001);western blot analysis of apoptosis-related proteins showed that the concentration of apatinib increased,the apoptosis rate of MDA-MB-231 cells and MCF-7 cells increased(all P<0.001),however,the relative expression of apoptotic protein,such as Cleaved-PARP1 and Cleaved-Caspase3 decreased(all P<0.05).4.Compared with the blank group,there was significantly difference that apatinib blocked MDA-MB-231 cells and MCF-7 cells in G0/G1 phase detected by flow cytometry assay(all P<0.05).5.The results of dual-fluorescence mRFP-eGFP-LC3 system detection of autophagy showed: observed by laser confocal microscope,apatinib activated the autophagy of breast cancer cells MDA-MB-231 and MCF-7;western blot detected autophagy-related protein results showed: MDA-MB-231 cells,MCF-7 cells,compared with the blank group,the concentration of apatinib increased,the related expression of autophagy protein such as LC3II/LC3I、P62 protein increased in a concentration-dependent manner(all P<0.001),the relative expression of Beclin1 protein decreased in a concentration-dependent manner,too(all P<0.001).6.After treating breast cancer cells with the autophagy inhibitor 3-Methyladenine(3MA10m M)for 8 hours,then changed the complete medium or Apatinib(20μM)to treat with breast cancer cells for 24 hours.Flow cytometry detected the rate of apoptosis: compared with the blank group,cell apoptosis rate increased in Apatinib group and 3MA+apatinib group at MDA-MB-231 cells and MCF-7 cells(all P<0.001),and the apoptosis rate of 3MA+apatinib group was higher than which of Apatinib’s group(P<0.05).Western blot detection of programmed cell death and autophagy-related proteins showed that: compared with the blank group,MDA-MB-231 cells,the expression of Cleaved-Caspase3 、 LC3II/LC3 I protein in Apatinib group and 3MA+apatinib group increased(all P<0.01);MCF-7 cells,the relative expression of LC3II/LC3 I protein increased(all P< 0.05)、Cleaved-PARP1,Bcl-2/Bax protein decreased in 3MA group、Apatinib group and 3MA+Apatinib group(all P<0.01),7.The autophagy inhibitor Bafilomycin A1(BafA1 4n M)treated with breast cancer cells for 8 hours and changed the complete medium or Apatinib(20μM)to treat breast cancer for24 hours.Compared with the blank group,MDA-MB-231,MCF-7 cells,apoptosis rate increased in BafA1 group、Apatinib group and BafA1+apatinib group(all P<0.001);In MDA-MB-231 cells,compared with the blank group,relative transcript level LC3II/LC3 I protein increased 、PARP1 protein decreased in BafA1,Apatinib,and BafA1+apatinib groups(all P<0.05),the relative expression level of Cleaved-Caspase3 protein increased in the Apatinib group and the BafA1+Apatinib group(all P<0.01).In MCF-7 cells,compared with NC group,the relation of expression LC3II/LC3 I protein decreased in BafA1 group and BafA1+Apatinb group,and the relative expression of LC3II/LC3 I protein increased in Apatinib group(all P<0.001),the relative expression of P62 protein decreased in BafA1,Apatinib,and BafA1+Apatinib groups(all P < 0.01),the relative expression of Cleaved-PARP1 protein decreased in BafA1 group,and the relative expression of Cleaved-PARP1 protein increased in Apatinib group cells(all P < 0.001),the relative expression of Cleaved-Caspase3 protein increased in BafA1 and Apatinib groups,and the relative expression of Cleaved-Caspase3 decreased in BafA1+Apatinib group cells(all P<0.05).8.Bioinformatics analysis was analyzed the molecular interaction network between VEGFR-2 protein and MAPK signaling pathway protein.Compared with the blank group,the relative expression of p-Erk1/2 protein decreased in the experimental group(P<0.01),at the same time,the expression of p-Erk1/2 protein decreased in the Apatinib group,3MA+Apatinbi group,and BafA1+Apatinbi group in MDA-MB-231 cells(all P<0.05);the expression of p-Erk1/2 decreased in PD98059 group 、 Apatinib group and PD98059+Apatinib(all P<0.001),Erk1/2 and Cleaved-Caspase3 protein decreased in the Apatinib group and PD98059+Apatinib group(all P<0.01),the relative expression of LC3II/LC3 I protein increased in the Apatinib group(P<0.007),PARP1 decreased in the Apatinib group and PD98059+Apatinib group(P<0.01 和 P<0.001).Compared with the blank group,the expression of p Erk1/2 and total Erk1/2 proteins decreased in the MCF-7 cell experimental group(P<0.001 and P<0.05);the relative expression of p Erk1/2 protein decreased in Apatinib group、3MA+Apatinib、BafA1+Apatinib group(all P<0.05),the relative expression of p Erk1/2 and Cleaved-Caspase3 proteins decreased in the Apatinib group(all P<0.001),and the relative expression of LC3II/LC3 I protein increased in the PD98059+Apatinib group(all P<0.05).Conclusion Apatinib inhibits the proliferation of breast cancer cells MDA-MB-231 and MCF-7,and the rate of proliferation is negatively correlated with drug concentration and in a time-dependent manner;Apatinib induces breast cancer cell apoptosis and the rate of apoptosis is positively correlated with drug concentration,and block the G0/G1 phase of MDA-MB-231 and MCF-7;Apatinib activates the autophagy of MDA-MB-231 and MCF-7cells.3MA and BafA1,as autophagy inhibitors,cooperate with apatinib induce apoptosis of breast cancer cells MDA-MB-231 and MCF-7;Apatinib inhibits p-Erk1/2-MAPK signaling pathway and activates breast cancer cell autophagy in order to promote apoptosis by specifically binding to VEGFR-2.