Study On The Anti-breast Cancer Metastasis And Anti-angiogenesis Effect, Target Identification, And Molecular Mechanism Of ION-31a

The newest global cancer report published by world health organization in 2020,declared that both of the morbidity and mortality in China ranked the first among the world.Among them,the morbidity(24.5%)and mortality(15.5%)of breast cancer top the list in femeal patients,which become the number one killer of women’s health.In our previous study,we found an active compound ION-31a with potential anti-metastasis activity through the derivative synthesis of ionone alkaloid.In present research,the therapeutic target,anti-metastasis activity and underlying molecular mechanism of ION-31a in breast cancer was investigated in vitro and in vivo.First,the direct therapeutic target of ION-31a was investigated.The active probe synthesized by ION-31a was used to fish the target.Extracted protein was separated by two dimensional electrophoresis.Then high abundance,biological function matched protein which obtained from proteomics analysis that carried out by high resolution mass spectrum,was further evaluated through molecular docking simulation with ION-31a.Then the interaction and strength between ION-31a with candidate protein was identified by surface plasmon resonance(SPR)analysis.The result showed that ION-31a interacted with HSP90αspecifically,and K_Dvalue was16.9μM,which corresponding to the pharmacology experiments in vitro.Therefore,HSP90αwas one of the major targets of ION-31a.Secondly,anti-metastasis activity and the target-based mechanism of ION-31a was investigated in vitro.The results showed that ION-31a inhibited the migration,invasion and adhesion of MDA-MB-231 and 4T1 cells in dose dependent manner.By using the western blot and ELISA,the regulation of ION-31a on the expression and phosphorylation of major client protein of HSP90 and the protein implicated in the signaling pathway was investigated.The results identified that ION-31a downregulated the expression level of client protein HIF-1αin dose dependent manner,then downregulated the expression level of target gene VEGF.Decreased phosphorylation of receptor VEGFR2 elicit the downregulation of phosphorylation level of signaling molecules in downstream signaling pathway,such as AKT,m TOR,PKCζ,FAK and ERK 1/2,which inhibited themigration,invasion and adhesion of breast cancer cells.In addition,the treatment of DMOG which act as the activator of HIF-1α,significantly suppress the anti-migration capacity of ION-31a.The result demonstrated that ION-31a inhibited breast cancer metastasis through HIF-1α/VEGFR2/AKT pathway.Thirdly,anti-angiogenesis and the target-based molecular mechanism of ION-31a was investigated in vitro.The results showed that ION-31a inhibited the VEGF induced migration,invasion,adhesion and tube formation of HUVECs in dose dependent manner.The results of western blot showed that ION-31a downregulated the phosphorylation level of VEGFR2 and the signaling molecules in downstream signaling pathway,such as AKT,p38MAPK,FAK and ERK 1/2,which play the angiogenesis role in HUVECs.At last,anti-metastasis,anti-angiogenesis and the underling mechanism of ION-31a was investigated in vivo.For evaluating the effect of ION-31a on the tumor growth and lung metastasis,pulmonary metastatic orthotopic transplantation model of4T1 cells was established by Balb/c mice.The tumor volume,tumor weight and living animal imaging results indicated that ION-31a inhibited the growth of orthotopic tumor and lung metastasis in dose dependent manner.The treatment of ION-31a(100 mg/kg)showed significant superior efficacy to the treatment of positive control sorafenib(50 m/kg).H&E staining showed that lung tissue of control group was infiltrated with lots of tumor cells addicted to double dye,the alveolar structure reduced as well.The treatment of ION-31a(50 mg/kg)and sorafenib(50mg/kg)reduced the infiltration of tumor cells in lung tissues and keep the intergrity of alveolar structure.The lung tissue of mice treated with ION-31a(100 mg/kg)showed few infiltration of tumor cells and clear alveolar structure.The results of immunohistochemistry indicated that the tumor tissue showed apparent positive staining of endothelial cells under the treatment of ION-31a(25 mg/kg),which were obviously reduced by the treatment of ION-31a(50 mg/kg)and sorafenib(50 mg/kg).The tumor tissue showed few positive staining of CD31 in ION-31a(100 mg/kg)treatment group.The above results demonstrated that ION-31a could inhibited the lung metastais and angiogenesis in orthotopic tumor of breast cancer in dose dependent manner.The functional protein and signaling molecules related to the mechanism investigation in vitro were examined by western blot and ELISA in orthotopic tumor model.The results indicated that ION-31a downregulated the expression level of HIF-1αand VEGF,the phosphorylation level of VEGFR2 and phosphorylation level of signaling molecules in downstream signaling pathway including AKT,m TOR,ERK 1/2 and integrinβ1.In conclusion,on the basis of the target research and mechanism research in vitro and in vivo,the mian mechanism of ionone alkaloid derivative ION-31a was interacting with N-terminal domain of HSP90αdirectly and downregulated the expression level of client protein HIF-1α,and downregulated the phosphorylation level of the downstream signaling molecules AKT,m TOR,PKCζ,FAK,p38MAPK and ERK 1/2 through HIF-1α/VEGF/VEGFR2 signaling pathway,which functioned anti-metastasis and anti-angiogenesis effect in breast cancer.