Hedyotis Diffusa Willd Inhibits Lymphangiogenesis In Colorectal Cancer Via Regulating VEGF-C/PI3K/AKT Pathway

Objective:Through the observation of Hedyotis Diffusa Willd(HDW)on the expression of VEGF-C and metastasis of colorectal cancer(CRC),and the PI3K/AKT signaling pathway mediated by VEGF-C based on the study of the effect of HDW on endothelial cells lymphangiogenesis lymphatic.To investigate the mechanism of HDW of lymphangiogenesis in HLEC,which was based on VEGF-C via regulating PI3K/AKT pathway.So as to provide new insight into the molecular mechanism of the anti-metastatic activity of HDW in CRC lymphangiogenesis and to provide theoretical basis for the clinical application of HDW.Methods:(1)HDW was extracted with ethanol.Human metastatic CRC cell line HCT116 and HCT-8 were treated with different concentration of EEHDW for a certain period of time,cell ability by MTT assay and Cloning formation was examined to estimate cell survival,Wound Healing was used to evaluate cell migration,and Western Blot was performed to detect the secretion level or the expression level of VEGF-C in the culture medium or in the cytoplasm.(2)Human lymphatic endothelial cells(HLECs)were received different concentration of EEHDW treatment for a certain time-point,Cloning formation was used to evaluate cell survival,AnnexinV/PI Staining and Cell DNA Content detected by Flow Cytometry(FCM)were used to estimate cell apoptosis and proliferation,Transwell assay was performed to evaluate cell migration,Tube Formation assay was used to evaluate lymphatic vessel formation ability.(3)Exogenous-VEGF-C-stimulated HLECs were treated with different concentration of EEHDW,to study the effect of EEHDW on VEGF-C-mediated lymphangiogenesis,by MTT assay to estimate cell viability,Cloning formation to estimate cell survival,FCM to observe cell cycle and cell apoptosis,Transwell assay to evaluate cell migration,Tube Formation assay to evaluate lymphatic vessel formation ability and Western Blot to determine the expression of p-PI3K?PI3K?p-AKT?AKT?VEGFR-3?MMP-2?MMP-9 in HLECs.Results:(1)MTT assay indicated that EEHDW could remarkablely decrease the amount of alive cells of HCT116 and HCT-8 in dose-dependent manners.Cloning formation showed that EEHDW inhibited the cell survival,Wound healing and Transwell assay suggested that EEHDW inhibited the migration of the CRC cell lines.Western Blot showed that EEHDW could reduce the secretion level or the expression level of the lymphangiogenesis facilitated factor-VEGF-C in the culture medium or in the cytoplasm.(2)MTT assay and Cloning formation showed that different concentration of EEHDW could reduce cell survival of HLECs,Cell DNA Content showed that EEHDW did not have significant effect on cell cycle,Annexin V/PI staining indicated that EEHDW did not have significant effect on cell apoptosis.Transwell assay suggested that EEHDW markedly inhibit cell migration of HLECs in a dose-dependent manner.Tube Formation assay showed that EEHDW remarkablely suppressed the lymphatic vessel formation ability.(3)MTT,Cloning,Transwell and Tube Formation assays showed that the exogenous-VEGF-C-stimulated HLECs got increased cell viability,enhanced migration and lymphatic vessel formation abilities,while EEHDW treatment could significantly ameliorate the situations.Western Blot indicated that the expression of p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2,MMP-9 were up-regulated after exogenous VEGF-C stimulated,and down-regulated by EEHDW treatment.However,the exogenous-VEGF-C-stimulated HLECs did not have significant effect on cell cycle and cell apoptosis.Conclusions:EEHDW can inhibit the metastasis and the expression of VEGF-C on colorectal cancer cells,inhibit the growth and migration of colorectal cancer cells and the growth,migration and lumen formation of lymphatic endothelial cells.EEHDW can inhibit the activation of VEGF-C mediated PI3K/AKT pathway,which is one of the most important mechanism to inhibit lymphangiogenesis in colorectal cancer.