Study On Pharmacology And Mechanism Of Sponge Derived Compound BA Against Hepatocellular Carcinoma

ObjectiveCompound BA derived from marine sponge is an anti-tumor component with novel chemical structure, but its biological effect and anti-tumor mechanism are still unclear. This paper investigates anti-tumor effect and mechanism of compound BA derived from marine sponge on cell proliferation,cell migration and cell apoptosis in SMMC-7721cell line, and also explores its pharmacology and molecular mechanism in human Liver cancer xenograft.Methods1.In vitro study:①Effect of BA on cell proliferation:we observe the effect of BA on cell proliferation,including human hepatoma carcinoma cell line SMMC-7721, Huh-7, HepG2, and normal hepatocyte L02by MTT assay;②Effect of BA on cell migration:we determinate the function of BA on cell migration by transwell assay;③Effect of BA on cell apoptosis:we identify the function of BA on cell apoptosis by H&E staining assay, and Annexin V-FITC/PI flow cytometry;finally we discuss the expression of protein of Caspase3and Caspase9by Western Blot method;④Effect and mechanism of BA on inhibiting cell proliferation, migration and inducing apoptosis:we evaluate the role of BA on the expression of signal protein in PI3K/Akt pathway by Western Blot assay.2. In vivo study:①Establishment of human Liver cancer xenograft: we establish human Liver cancer xenograft by subcutaneously vaccination with0.1ml of1.0×108/ml SMMC-7721cell suspension in mice right axilla.②Drug treatment:we divide tumor xenografts into four groups when Liver tumors grow up to100～150mmJ, and then compound BA is administered by intraperitoneally injection at dose of Omg/kgBA,5mg/kgBA,10mg/kgBA and10mg/kg5-FU for5consecutive days.③Monitoring index:we measure the tumor size every two days, and calculate the inhibition rate of Liver tumor to evaluate the effect of BA on inhibiting tumor growth; record the mice weight and survival time to deliberate the effect of BA on quality of mice life; take blood by removalling eyeball and detect nude mice liver and kidney function; sacrifice mice and get the tumor and organ tissue, and have HE stain to deliberate tumor and organ pathologic change, and explore its anti-tumor mechanism in PI3K/Akt pathway by immunohistochemistry assay and Western Blot assay.Results1. In vitro effect of BA against hepatocellular carcinoma:①Effect of BA on inhibiting cell proliferation:BA can inhibit proliferation of series hepatoma carcinoma cell lines, including SMMC-7721, Huh-7, and HepG2cell lines. BA can inhibit proliferation of SMM-7721cell line in a dose and time dependent manner (p<0.05).②Effect of BA on inhibiting cell migration:The transference number of SMMC-7721cells treated by BA below control group (p<0.05).③Effect of BA on inducing cell apoptosis:BA can induce liver cell apoptosis, and the apoptosis rate is32.39%after24h treatment of BA at a concentration of16.00μmol/L;Effect of BA on apoptotic protein Caspase3and Caspase9:compared with blank group, BA can enhance the expression of Caspase3(p<0.05), and decrease the expression of Caspase9(p<0.05), they are both a concentration dependent manner.④The molecular antitumor mechanism of compound BA in vitro:Effect of BA on expression of protein in PI3K/Akt pathway: in SMMC-7721cell line after treatment of BA, BA can down-regulate the expression of protein PI3K and P-Akt (p<0.05), there is no significant effect on protein expression of Akt(p>0.05);Effect of BA combination LY294002or IGF-1on expression of protein in PI3K/Akt pathway:in SMMC-7721cell line, the expression of protein PI3K and P-Akt of BA combination LY294002group is lower BA group and LY294002group (p<0.05); BA combination IGF-1group can down-regulate the expression of protein PI3K and P-Akt (p<0.05) vs IGF-1group group (p<0.05)2. In vivo effect of BA against hepatocellular carcinoma mice model:①The pharmacology of BA against human liver xenograft: compound BA can significantly inhibit the growth of liver tumor at an effective dose of10mg/kg, the tumor inhibition rate of BA at a dose of5mg/kg is (58.24±27.51)%. However, BA can make no difference to the weight and organ function of mice.②Pathological change of liver tumor and organ tissue:HE result reveals that the area of necrosis in10mg/kg dose group increases, compared with the blank group(p<0.05); Liver, lungs, kidneys and spleen of treatment group were no obvious pathological changes vs control group.③The molecular antitumor mechanism of compound BA in vivo:Effect of BA on expression of protein in PI3K/Akt pathway:in liver tumor tissue after treatment of BA, BA can down-regulate the expression of protein PI3K and P-Akt (p<0.05). The possible antitumor mechanism of BA may be related to the PI3K/Akt pathway.Conclusion1. Compound BA exhibits anti-tumor activity in liver cancer cell line SMMC-7721.It can inhibite cell proliferation,migration and induce apoptosis in vitro.2. Compound BA exhibits specific pharmacology effect against liver cancer in vivo with an effective dose of5mg/kg.3. The mechanism of BA against liver cancer may be related to the blockage of PI3K/Akt signal pathway.