Research On Inhibition Of Tumor Metastasis And Angiogenesis As Well As Mechanism Of Two Peptides From Abalone

Abalone?hallotis discus hannai?is a precious seafood.It has been reported that biological active substances derived from abalone have anti-oxidation,anti-inflammatory,anti-bacterial,anti-thrombosis and anti-cancer potential and so on.However,there were few studies that have been conducted to assess whether it has anti-cancer potential.In this study,we evaluated the anti-tumor?anti-metastasis and anti-angiogenesis?effect of two peptides including BAB?EMDEAQDPSEW?and AATP?KVEPQDPSEW?from abalone boiled by-products and intestine on human fibrosarcoma HT1080 cells and human umbilical vein cells?HUVEC?,respectively.In addition,the interaction of the two peptides and pro-angiogenesis factor?including HIF-1?and VEGFR-2?were analyzed by molecular docking.This research mainly focuses on two parts.The anti-metastatic effect of BAB and AATP:1.MTT assay was utilized to assess the cytotoxicity of BAB and AATP on HT1080cells and HUVEC.The result showed that all tested concentrations?10,20,50 and 100?M?of AATP have no cytotoxicity on HT1080 cells and HUVEC,which revealed that AATP is non-toxic to tumor cells as well as nontumor cells.Therefore,10-100?M of AATP could be used for further experiments.2.The indicated concentrations?20,50 and 100?M?of BAB and AATP of the ability of cell colony were determined by colony formation assay.And the colony formation assay indicated that the number of colonies of HT1080 cells were sharply reduced by BAB and AATP treatment compared with untreated group,suggesting BAB and AATP exerted inhibitory effect on tumor cells.3.The wound healing assay and cancer cell spheroid assay were used to estimate the impact of AATP on cancer cells migration and invasion.The results showed that the migration and invasion of HT1080 cells was were obviously down-regulated by BAB and AATP treatment in a dose-and time-dependent manner.4.In order to determine the anti-metastatic ability of BAB and AATP,we investigated the transcriptional levels of MMPs including MMP-1,-2,-3,-9,-13,activity and protein expression of MMP-2 and-9 in HT1080 cells by Real-Time quantitative reverse transcription-PCR?qPCR?,zymography analysis and western blotting analysis.All results showed that PMA stimulation significantly upregulated MMPs RNA expression,MMP-2/-9 activity and expression,whereas BAB treatment efficiently decreased the levels of MMP-1,-3,-9,-13,the activity and expression of MMPd-9 under PMA stimulation,but the level of MMP-2 activity and expression has no significant change.And AATP treatment efficiently decreased the levels of MMP-1,-2,-3,-9,-13,the activity and expression of MMP-2 and-9 under PMA stimulation.5.MAPK and NF-?B signal pathways are related to the expressions of numerous genes that modulate tumor promotion,angiogenesis,metastasis and MMPs expressions.To determine the effect of AATP on MAPK and NF-?B signal pathway in HT1080 cells,the western blotting analysis,p65 translocation and NF-?B activation assay?EMSA?were conducted.The results of western blotting assay indicated that BAB and AATP treatment markedly suppressed PMA-induced phosphorylation activation of ERK and JNK in dose dependent compared with PMA-induced group.Moreover,in the p65translocation and EMSA analysis,BAB and AATP dramatically suppressed p65 nuclear translocation and binding with DNA.The anti-angiogenic effect of BAB and AATP:1.Under hypoxia environment induced by CoCl₂,the level of VEGF of cancer cells secreted into the culture medium was measured by ELISA.The ELISA results showed that BAB and AATP dose-dependently inhibit VEGF release of cancer cells.2.VEGF is downstream target of HIF-1?.Therefore,we detected expressions of HIF-1?and AKT/mTOR/p70S6K signal pathway,which is related of angiogenesis.The results showed that BAB and AATP treatment effectively inhibit expression of HIF-1?via blocking AKT/mTOR/p70S6K signaling in a concentration-dependent manner.3.Tube formation analysis was employed to investigate the anti-angiogenic effect of BAB and AATP on HUVEC.The results showed that BAB?50 and 100 M?and AATP?50 and 100 M?attenuated tube formation of HUVEC.4.We investigated the potential of BAB and AATP against HIF-1?and VEGFR-2binding affinity using molecular docking approach.BAB and AATP can combine the active site of HIF-1?and VEGFR-2,which contribute to suppression of activity of HIF-1?and VEGFR-2.