Anti-breast Cancer Efficacy Of CXCR4 Antagonist Polypeptide E5 Combined With Chemotherapeutic Drugs And Screening Of Axl Antagonist Polypeptides

Objective:It has been widely recognized that tumor microenvironment plays a critical role in tumor development and progression,and now has become the new target of drug resistance.In particular,stroma derived factor 1?(SDF-la,or termed as CXCL12),and its cognate receptor,the chemokine receptor 4(CXCR4)and the receptor tyrosine kinase Axl and its ligand Gas6 are two very key receptor-ligand pairs in the cross-talking between tumor cells and their microenvironment.Blocking the interaction between CXCR4 and CXCL12 can inhibit the tumor progression,angiogenesis and metastasis of breast cancer.Furthermore,Axl antagonist is able to reverse drug resistance.The purpose of this research is extension of tumor treatment indications of E5 a CXCR4 antagonist developed by our laboratory;apply E5 on breast cancer,investigating whether E5 could sensitize breast cancer cells to chemotherapeutics and revealing the underlying mechanisms.Meanwhile,screen new peptides with high binding capacity to Axl,determin the interaction of peptides to different tumor cells in vitro and explor its relationship with chemoresistance.Methods:The expression of Axl in human acute myeloid leukemia cells(HL60,U937,THP-1,NB-4),human chronic myeloid leukemia cells K562 and human breast cancer cell MCF7 which were exposed to chemotherapy drug and variety drug resistance strains was tested by flow cytometry to evaluated the relationship between Axl and drug resistance.The affinity of a series of peptide to K562 and MCF7/R was examined through flow cytometry to screen peptides with high binding capacity to Axl.Establish U937 resistant strains(U937R)by gradually increasing the concentration of Cytarabine to repeated stimulation of U937 cells.The affinity of E5 to murine breast cancer cell line(4T1),human umbilical vein endothelial cell line(HUVEC)and murine stromal cell line(MS-5)was detected by flow cytometry to evaluated specificity of E5.CCK-8 assay was conducted to examine the cytotoxic effect of E5 to 4T1,HUVEC and MS-5 cells.Utilize Annexin V-FITC/PI double staining and westernblot to evaluate the cell-killing effect of E5 to 4T1cell.Transwell assay were performed to examine the effect of E5 on CXCL12 and MS-5-induced 4T1 and HUVEC cells migration.CCK-8 assay was used to examine the effect of E5 on MS-5-induced 4T1 cell adhesion.The cell-killing effect of E5 combined with multiple drugs(Paclitaxel,Elemene,Cisplatin)to 4T1 cell in the co-culture system(Co-incubated 4T1 cell with MS-5 cell or MS-5 conditional medium or CXCL12)was determined by CCK-8 assay.The combination effect of E5 with paclitaxel and cyclophosphamide was evaluated on tumor-bearing mice in breast cancer model,through monitoring the tumor sizes of the mice.Westernblot was used to detecte the expression of CXCR4 protein,CXCR4 associated downstream signaling protein p-Akt,p-Erk and the endothelial cell marker CD31 protein of the tumor tissue to evaluate the mechanism of E5 facilitating the antitumor effect of chemotherapeutic drug.Pharmacokinetic study of E5 was conducted in the female Balb/c mice after subcutaneous injection of FITC-labeled E5.The storage stability of E5 in vitro was detected by high performance liquid chromatography.Results:(1)E5 has specific affinity to breast cancer cell(4T1)and vascular endothelium cell(HUVEC)which express CXCR4;E5 has cytotoxicity to breast cancer cells in a concentration dependent manner;When E5 concentration was under 25 ?M,there was no effect on the viability of 4T1 cells;as concentration was higher than 25?M,E5 can induce 4T1 cells apoptosis.E5 can effectively inhibit the migration of 4T1 or HUVEC cells mediated by CXCL12 or MS-5 conditional medium and the adhesion of 4T1 cells to stromal cells.E5 can enhance the sensitivity of 4T1 cells to chemotherapeutics(Paclitaxel,Elemene,Cisplatin)when co-incubated with MS-5 cell or MS-5 conditional medium or CXCL12.E5 can reduce the CXCR4,pAkt,pErk and CD31 protein expression level and inhibit tumor angiogenesis to improve the anti-tumor effect of paclitaxel and cyclophosphamide.E5 was mainly cleared through hepatic metabolic pathway;the concentration of E5 peaked in the blood after 2 h of administration;the half-life of E5 in mice was around 10 h.E5 can be preserved 6 days at 37 ?,and 5 months at 4 ?.(2)Chemotherapy drugs temporary stimulation can induce the expression of Axl in U937 and K562 cell surface;Axl ligand Gas6 can increase the expression of Axl in K562 cell surface.Meanwhile,screen out U937 ARA resistant strain,U937R.Compared with normal cells,the change of the expression of Axl in HL60,U937 and K562 drug-resistant strains(HL60/A,U937R,K562/R)was different;there is litter change in HL60/A,a slight increase in U937R but a significant reduce in K562/R.The Axl expression in MCF7/R,a solid tumors resistant strains,was significantly higher than MCF7.In addition,screen out a peptide with high affinity with Axl from K562 and MCF7/R cells high expressing Axl and lay a good foundation for the follow-up studies.Conclusion:E5 can inhibit MS-5 cell-mediated cell migration,adhesion and resistance to chemotherapy,improving the effect of chemotherapeutic drugs for the treatment of breast cancer modle through blocking the CXCR4/CXCL12 axis.E5 can inhibit tumor angiogenesis by blocking the recruitment of vascular endothelial progenitor cell.E5 has a better body stability with a half-life about 10 h.E5 was easily degradable at 37 ?,but can be stored for 5 months at 4 ?.Peptides A2-7 has a high affinity with Axl in the surface of K562 and MCF7/R cells,and it may be a potentially Axl antagonistic peptide.