Effect Of Cinobufacini Injection On Proliferation Invasiveness And Heterogeneous Adhesion Of Human Hepatoma HepG-2Cells Co-cultured With Human Lymphatic Endothelial Cells

Objective: Hepatocellular carcinoma is a world widely life-threatening disease,and metastasis is the leading cause of hepatocellular carcinoma related death. Aschemotherapy nonsensitive cancer, the partial response (PR) of hepatocellularcarcinoma to chemotherapy is less than20%up to now, also, the serious cytotoxicity ofchemotherapy on hepatocellular carcinoma limits its clinical use. Although clinicalobservation show that sorafenib is the first drug shown to extend survival inhepatocellular carcinoma, more appropriate use of sorafenib is recommended because ofserious adverse events or deaths in Japan. Clinical data show Cinobufacini injection, atraditional Chinese medication, can not only inhibit tumor growth and metastasis butalso improve the living quality of the patients with hepatocellular carcinoma. Howeverthe mechanism on it has not been identified. This program is performed to investigatethe effect of cinobufacini injection on malignant behaivior of hepatocellular carcinomaand the possible mechanism on it. Also, the influence of lymphatic endothelial cells onhepatoma cells is observed in the study.Methods: Co-culture system of human hepatoma HepG-2cells (HepG-2) andhuman lymphatic endothelial cells (HLEC) is established by means of transwell chamber,effects of cinobufacini injection with different concentration on HepG-2, HepG-2co-cultured with HLEC are observed respectively as following:(1)Cell proliferation chart is used to observe the proliferation capacity of HepG-2.(2)Invasiveness assay is used to observe the invasiveness capacity of HepG-2.(3)Matrigel assay is used to observe the heterogeneous adhesion capacity ofHepG-2.(4)Western-blot is used to analysis the expression of VEGF-C in HepG-2cells.(5)Western-blot is used to analysis the expression of MMP-2and MMP-9inHepG-2cells. Results:1. For HepG-2(1)Cinobufacini injection significantly inhibits HepG-2proliferation(P＜0.05) indose dependent ways.(2)Cinobufacini injection significantly inhibits HepG-2invasiveness (P＜0.05) indose dependent ways.(3)Cinobufacini injection significantly inhibits HepG-2heterogeneous adhesion (P＜0.05) in dose dependent ways.(4)Cinobufacini injection significantly descreases the expression of VEGF-C inHepG-2(P＜0.05) in dose dependent ways.(5)Cinobufacini injection significantly descreases the expression of MMP-2andMMP-9in HepG-2(P＜0.05) in dose dependent ways.2. For HepG-2cocultured with HLEC(1)Cinobufacini injection significantly inhibits proliferation of HepG-2coculturedwith HLEC (P＜0.05) in dose dependent ways.(2)Cinobufacini injection significantly inhibits invasiveness of HepG-2coculturedwith HLEC (P＜0.05) in dose dependent ways.(3)Cinobufacini injection significantly inhibits heterogeneous adhesion of HepG-2cocultured with HLEC (P＜0.05) in dose dependent ways.3.The capability of proliferation, invasiveness and heterogeneous adhesion ofHepG-2cocultured with HLEC are significantly stronger than those of HepG-2cells (P＜0.05).Conclusion:Cinobufacini injection can inhibit the capability of proliferation,invasiveness and heterogeneous adhesion of HepG-2, the down-regulation of VEGF-C,MMP-2and MMP-9might contribute the mechanism on it. HLEC cells can enhance thecapability of proliferation,invasiveness and heterogeneous adhesion of HepG-2cells.