Inhibitory Effect Of Capillaris On Human Hepatocellular Carcinoma Cells And Transplanted Liver Cancer In Mice

Objective To explore the inhibition effects of Aplysin against Hepatic carcinoma and possibly related mechanism of Aplysin on the proliferation of human hepatioma Hep G2 cel s and on tumor growth in H22 mice.Methods1.Hep G2 cells were cultured in DMEM/HIGH GLUCOSE medium with 10% FBS and incubate at 37℃ with 5% CO2.Then the cells were treated with Aplysin at different concentrations(20mg/L、40mg/L、60mg/L、80mg/L、100mg L and 120mg/L).CCK-8 assay were used to examine the effect of Aplysin on Hep G2 cells proliferartion after intervening 24 h and 48 h. Fluorescence microcopy was used to observe the apoptosis of Hep G2 cells via Annexin V-FITC/PI staining. 2. Kun Ming mice were randomly divided into four groups: the model group, the Aplysin group(25,50,100mg?kg-1?d-1)and each group had ten mice.H22 Cells were inoculated subcutaneously into left anteromedial of KM mice. Except for the model group,all the mice in the other 3 groups were treated with ig Aplysin of different dosage on the second day and executed after 15 days. The model group were treated with ig equal volume soybean oil. The mice were weighted and taken blood sample from eyes at the 24 h after the last treatment. We weighed the tumor which was stripped completely and calculated the tumor inhibition rate. Simultaneously the expression of MMP9, VEGF and PCNA in tumor tissue was determined by immunohistochemistry. And the level of IL-6 and TNF-α in serum was measured by ELISA.Results1.Evaluation of inhibition effect of Aplysin against human hepatoma Hep G2 cel s in vitro Cell growth and proliferation of Hep G2 cells were inhibited significantly after intervention of Aplysin.CCK-8 assay indicated a dose- as well as time-dependent inhibition in Aplysin- intervention Hep G2 cells(P<0.05),and the IC25 and IC50 of 24 h were 65.36mg/L and 103.77mg/L. After treated with 65mg/L and 105mg/L Aplysin for 24 h, fluorescence microscopy showed that the extent of apoptosis increased with an increasing Aplysin concentration(P<0.05). 2.Evaluation of inhibition effect of Aplysin on H22 xenografts of liver cancer in vivo Aplysin could decrease the tumor weight significantly in a dose-dependent manner, and the tumor inhibition rate was 28.31%, 33.84%, 42.96% respectively. The expression of MMP-9 in Aplysin groups, especially in 50 mg?kg-1?d-1 and 100mg?kg-1?d-1 groups, were significant lower than model group(P<0.05). Compared with model group, the expression of VEGF and PCNA were obviously inhibited with a dose-effect relationship in all Aplysin groups(P<0.05). In Mid and high-dose groups, the concentration of IL-6 and TNF-α in serum were higher than model group(P<0.05).Conclusion1. Based on the results of CCK-8 assay and Annexin V-FITC/PI staining experiment, it is initially proved that Aplysin could suppress the proliferation and induce the apoptosis of human hepatoma Hep G2 cells. 2. Aplysin could inhibit tumor growth by suppressing extracellular matrix degradation and angiogenesis, and improving the immune capacity in mice. 3. The results show that Aplysin is expected to become a novel anticarcinogen for its effective inhibition on Hepatoma cells. But whether Aplysin would effectively cure liver cancer still need more and further experiments to confirm.