Effect Of Cinobufacini Injection On Lymphangiogenesis Of Human Lymphatic Endothelial Cells Co-cultured With Human Hepatoma HepG-2Cells

Objective: Lymphatic metastasis is regarded as the earlier stage of tumormetastasis, and no effective medication is available currently. Recent researches revealthat lymphangiogenesis plays essential roles in tumor lymphatic metastasis andVEGF-C/VEGFR-3passway regulates lymphangiogenesis specifically. Clinical datashows that cinobufacini injection, a Chinese medication, inhibits lymphatic metastasisin patients with variety of tumors. However, little is known on the mechanism. Thisprogram is performed to investigate the effect of cinobufacini injection on tumorlymphangiogenesis and the possible mechanism on it.Methods: Co-culture system of human lymphatic endothelial cells (HLEC) andhuman hepatoma HepG-2cells (HepG-2) is established by transwell chamber, effects ofcinobufacini injection with different concentration on HLEC, HLEC co-cultured withHepG-2are observed respectively as following:(1)Cell growth curve is used to observethe effect of HepG-2and cinobufacini injection on the proliferation ofHLEC.(2)Migration assay is used to observe the effect of HepG-2and cinobufaciniinjection on the migration of HLEC.(3)Matrigel assay is used to observe the effect ofHepG-2and cinobufacini injection on the tube-like structure formation ofHLEC.(4)Western-blot is used to analysis the expression of VEGFR-3in HLEC underthe action of cinobufacini injection.(5)Western-blot is used to analysis the expression ofHGF in HLEC under the action of cinobufacini injection.Results:1. Effect of Cinobufacini Injection on HLEC.(1) Cinobufacini injectionsignificantly inhibits HLEC proliferation(P＜0.05),in dose dependent ways.(2)Cinobufacini injection significantly inhibits HLEC migration (P＜0.05),in dosedependent ways.(3) Cinobufacini injection significantly inhibits HLEC tube-likestructure formation (P＜0.05),in dose dependent ways.(4) Cinobufacini injection significantly descreases the expression of VEGFR-3in HLEC(P＜0.05), in dosedependent ways.(5) Cinobufacini injection significantly descreases the expression ofHGF in HLEC(P＜0.05), in dose dependent ways.2. Effect of Cinobufacini Injection onHLEC cultured with HepG-2cells(1)Cinobufacini injection significantly inhibitsproliferation of HLEC cultured with HepG-2cells (P＜0.05),in dose dependentways.(2)Cinobufacini injection significantly inhibits migration of HLEC cultured withHepG-2cells (P＜0.05),in dose dependent ways.(3)Cinobufacini injection significantlyinhibits tube-like structure formation of HLEC cultured with HepG-2cells (P＜0.05),indose dependent ways.3.HepG-2cells can promote the proliferation, migration andtube-like structure formation of HLEC(P＜0.05).Conclusion：By comparing above results,we know that cinobufacini injection caninhibit lymphangiogenesis of HLEC, the down-regulation of VEGFR-3and HGF mightcontribute the mechanism on it. HepG-2cells can enhance the lymphangiogenesis ofHLEC.