The Effect Of Tablysin-15 Against Metastatic Breast Cancer And Bone Destruction Diseases Via Blocking Integrin α_vβ₃

BackgroundIn 2020,the number of women breast cancer cases in worldwide has surpassed that of lung cancer,becoming the world’s number one cancer incidence,with an estimated 2.2614 million new cases,with a mortality rate of 6.9%.In China,breast cancer is also the most diagnosed malignant tumor in women,and the number of new breast cancer cases among women is about 416,400.The number of new cases of breast cancer ranked fourth,less than lung cancer,colorectal cancer and gastric cancer.Inflammatory bone destruction disease is a type of bone loss disease caused by microbial products or inflammatory factors,including rheumatoid arthritis,periodontitis and so on.Approximately 200 million people worldwide suffer from diseases caused by excessive bone loss.Therefore,it’s important for the study of drugs for the treatment of breast cancer and bone destruction.ObjectiveTablysin-15,an RGD-containing disintegrin from the salivary gland of the horsefly Tabanus yao,which is a high-affinity antagonist of integrin αvβ3.This article aims to use molecular biology and pharmacological research methods to investigate the in vivo and in vitro effects and underling mechanism of Tablysin-15 on metastatic breast cancer cell lines MDA-MB-435s,MDA-MB-231 and osteoclasts with high expression of integrin αvβ3.In order to provide a theoretical basis for the application of Tablysin-15 to breast cancer and bone destruction diseases.Methods1.MDA-MB-435s,MDA-MB-231 and MCF-7 cells were used as cell models,cell adhesion experiment,antibody competitive binding experiment,and molecular docking were conducted to verify whether Tablysin-15 binds to integrin αvβ3;MTT,CTL Luminescence method was used to detect the effect of Tablysin-15 on the proliferation of MDA-MB-435s,MDA-MB-231 and MCF-7 cells;DAPI staining,DNA Ladder,Western Blot and PI staining were used to detect the effect of Tablysin-15 on cell apoptosis and cell cycle.Western Blot assay was used to detect the effect of Tablysin-15 on related signal pathways.Cell migration assay,transwell invasion assay,qRT-PCR and gelatin zymography assay were used to evaluate the effect of Tablysin-15 on cell motility.In vivo tumor xenograft mouse model was used to detect the anti-tumor activity of Tablysin-15.2.The RANKL-induced osteoclast formation as a model,TRAP staining,F-actin staining and bone resorption pit assay were used to evaluate the effect of Tablysin-15 on osteoclastogenesis and bone resorption.Western Blot,qRT-PCR,and antibody competitive binding assay were used to determin the effect of Tablysin-15 on the signal pathways related to osteoclastogenesis.The LPS-induced mouse bone destruction model was constructed to evaluate the protective effect of Tablysin-15 on the bone destruction.Results1.Tablysin-15 inhibits the proliferation,migration and invasion of MDA-MB-435s and MDA-MB-231 cells but not MCF-7 cells by binding to integrinαvβ3.The inhibition of cell proliferation by Tablysin-15 can be attributed to cell cycle arrest in G0/G1 phase,rather than apoptosis or necrosis.In addition,Tablysin-15 down-regulates the mRNA expression of MMP-2/9,VEGF and COX-2,and up-regulates the mRNA expression of TIMP-1/2.Moreover,while increasing the expression of the CDK inhibitor p21wafl/C1,Tablysin-15 inhibited the expression of CDK2,CDK6,Cyclin D1 and Cyclin E.Tablysin-15 also inhibited the phosphorylation of FAK,Akt,GSK-3β and ERK,and the nuclear translocation of NF-κB p65.Finally,Tablysin-15 exhibited effective anti-breast cancer activity in vivo.2.Tablysin-15 concentration-dependently inhibited RANKL-induced osteoclast formation,F-actin ring formation and bone resorption.Tablysin-15 binds to αvβ33 integrin to inhibit the activation of FAK-related signaling pathways and MAPK,NF-κB and Akt-NFATc1.In addition,Tablysin-15 inhibits the expression of Cofilin and DC-STAMP,which are responsible for cytoskeleton reorganization and cell fusion,while reducing the expression of genes related to osteoclast formation such as MMP-9,TRAP,CTSK and c-Src.However,Tably sin-15 increases the phosphorylation of Cofilin.Finally,Tablysin-15 significantly inhibited LPS-induced bone loss in vivo.ConclusionsTablysin-15 can inhibit the proliferation and migration of metastatic breast cancer in vitro.In vivo,Tablysin-15 can inhibit the growth of tumors;Tablysin-15 can inhibit the formation of osteoclasts and bone resorption capacity in vitro.In vivo,Tablysin-15 can relieve acute inflammatory bone destruction.Therefore,Tablysin-15 will be an excellent candidate drug molecule for the treatment of breast cancer and bone destruction diseases.