Inhibitory Effect Of ACM On Angiogenesis And Its Possible Mechanism In Hepatocellular Carcinoma

ObjectiveThis study aims to investigate the anti-angiogenesis effect of ACM on SMMC-7721 cells with high metastatic potential in vitro,and in vivo and to explore its probable mechanismMethods(1)MTT assay was used to investigate the inhibitory effect of ACM on the proliferation of 5 hepatocellular carcinoma cell lines,Human Umbilical Vein Endothelial Cells(HUVECs)and human hepatocytes L-02(2)Clone formation assays were used to measure the inhibition effect of ACM on formation of HUVECs and SMMC-7721 cells clone(3)Wound healing,transwell migration and invasion assays were used to measure the effects of ACM on migration and invasion of HUVECs and SMMC-7721 cells(4)An assay of endothelial cells tubes in three dimensional Matrigel cultures of HUVECs was used to assess the anti angiogenic potential of ACM(5)Rat aortic rings were used to investigate the inhibitory effect of ACM on capillary formation of aortic rings(6)Immunofluorescence assay was used to detect the effect of ACM on the VEGFR2 phosphorylation in HUVECs and SMMC-7721 cells(7)The zebrafish model was used to investigate the effect of ACM on zebrafish intravascular angiogenesis(8)The SMMC-7721 xenografted nude mice model was used to investigate the antiangiogenic effect of ACM in vivo(9)The effect of ACM on the expression of angiogenesis-related proteins in HUVECs and SMMC-7721 cells,as well as SMMC-7721 cell xenografts was detected by Western blottingResults(1)The results of MTT assay showed that ACM could inhibit the proliferation of Bel-7402,HepG2,Huh7,Hep3B,SMMC-7721,HUVECs and L-02 cells;And ACM inhibited HUVECs and SMMC-7721 cells proliferation in a time-and concentration-dependent manner.(2)Clone formation assay showed that ACM could inhibit the clone formation of HUVECs and SMMC-7721 cells in a concentration-dependent manner.(3)Wound healing,Transwell migration/invasion experiments showed that ACM could significantly inhibit the migration and invasion of HUVECs and SMMC-7721 cells in a dose-dependent manner.(4)Rat aortic rings experiment showed that ACM could inhibit the formation of aortic capillaries(5)The results of tube formation assay showed that ACM could significantly inhibit tube formation of HUVECs in a concentration-dependent manner.(6)Immunofluorescence results showed that ACM could reduce VEGFR2 phosphorylation in HUVECs and SMMC-7721 cells in a concentration-dependent manner(7)The zebrafish experimental results showed that ACM could inhibit zebrafish intravascular angiogenesis in a concentration-dependent manner(8)The results of SMMC-7721 xenografts in nude mice showed that ACM could effectively inhibit the growth of xenografts and has no significant effect on organ indexes.The result of immunohistochemistry showed that ACM could down-regulate the expression of CD31.The results of HE staining showed that ACM had no toxic effect on the liver and kidney of nude mice and could promote tumor necrosis(9)Western blotting results showed that ACM could reduce VEGFR2 and STAT3 phosphorylation in HUVECs and SMMC-7721 cells,and reduce PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways phosphorylation ACM could down-regulate the expression of VEGFR2,CD31 and reduce STAT3,Akt,ERK phosphorylation in the tumor tissues of SMMC-7721 xenograftsConclusion:In this paper,we could make a conclusion that ACM can inhibit the angiogenesis of hepatocellular carcinoma,and its possible mechanism is mediated through the VEGF/VEGFR2 signaling pathway both in vitro and in vivo.