The Mechanism Of 5,7-Two Methoxy Flavone Against Liver Cancer

Part 1:Activity and mechanism of 5,7-DMF against hepatocellular carcinoma cell line HepG2Objective:To investigate the possible mechanism of the 5,7-DMF against the activity of hepatocellular carcinoma cell line HepG2.Methods:The hepatoma cell line HepG2 was inoculated on the 6 pore plate,the density of cell was 2 x 105 per hole,divided into medium treatment group(medium 1%DMSO),5,7-DMF treatment groups(10uM group,25uM group and 50uM group),each group was divided into four subgroups:0 hour,24 hour,48 hour and 72 hour.The hepatoma cell line HepG2 was inoculated at the medium with a corresponding concentration 5,7-DMF for culture,the morphology of HepG2 cells was observed under light microscope,and the cell activity of HepG2 treatment with different concentrations of 5,7-DMF at the corresponding time was measured by MTT colorimetric assay;the hepatoma cell line HepG2 was cultured with 5,7-DMF of 0,10,25 and 50 Um respectively for 24h,and the distribution of cell cycle was measured by flow cytometry;the HepG2 was cultured with concentration of 25 uM 5,7-DMF,the levels of cells ROS was detected by using the DCFH-DA method respectively in 0,6,12,24,48 and 72h,and intracellular ??m level was measured by using the DiOC6 method;the HepG2 was cultured at difference concentration of 5,7-DMF(10 uM,25 uM and 50 uM)for 48h,the apoptosis was detected by using the tunnel method and to observed the colony formation under light microscope.Results:After treatment with 5,7-DMF,the number of HepG2 cells decreased and the cell morphology changed obviously;With the extension of 5,7-DMF processing time and increase in concentration,the activity of HepG2 decreased more obviously,and IC50 was 25 uM.After treatment with 5,7-DMF,the sub G1 pahase cell populations increased with increasing 5,7-DMF concentration;the intracellular level of ROS increased gradually with the extension of time,while the intracellular level of ??m decreased gradually with the extension of time;the level of apoptosis increased with the increase concentration of 5,7-DMF,and the level of cell aggregation decreased with the increase concentration of 5,7-DMF.Conclusion:5,7-DMF can reduce the vitality of HepG2,and the IC50 was 25uM.5,7-DMF reduce the vitality of HepG2 maybe through arrest the cell cycle,increased intracellular ROS levels and decreased the level of ??m,reduce the formation of cell colony,resulting in increased cell apoptosis.Part 2:Effect of 5,7-DMF on the tumorigenesis of hepatoma cell line HepG2 in nude miceObjective:To investigate the anti-tumor mechanism of 5,7-dimethoxyflavone in nude mice.Methods:Thirty-six BALB/c nude mice were inoculated with HepG2 cells on the first day after the operation.The rats were divided into 6 groups according to the method of administration and dosage.(1)NS:NS10il/(2)5,7-DMF(10iM)group(D1):intraperitoneal injection of 5,7-DMF at 10il/(4)5,7-DMF(50iM)group(D3):intraperitoneal injection of 5,7-DMF,10il/mouse;(6)5,7-DMF(50iM)+ 5-FU group(D3 + FU):intraperitoneal injection of 5-FU 20mg/kg + 5,7-DMF 10il/only.The above groups were once every 3 days for 5 consecutive times before inoculation,2,5,8,11,14,and 17 days after inoculation.The tumor growth of BALB/c nude mice in each group and the general condition of mice,Electronic balance measurement of the weight of mice,the average weight of mice were plotted curve.The growth of HepG2 cells was observed on the 5th,8th,11th,14th,17th day after subcutaneous inoculation of HepG2 cells.The length and width of the tumor were measured by vernier calipers.The curves of tumor volume were plotted.Average plus or minus standard deviation expressed(-?�s){Tumor volume calculation formula:V =(A � B2)/2,A is the long diameter,B is the short diameter}.Twenty-seven days after the inoculation of the tumor cells,all the BALB/c nude mice were sacrificed by the neck method.The subcutaneous tumors in the nude mice were completely peeled off and photographed.The sections were stained with HE and observed under a microscope.Results:In weight,,the weight of NS,D1,D2 and D3 groups after inoculation were no different,D3+FU and FU groups were no different.The tumor volume of subcutaneous tumor in nude mice in each group increased at the end of the experiment compared with the fifth day after inoculation,which was the most obvious in NS group,followed by D1 group,last by D3+FU group.Conclusion:This study found that 5,7-DMF in nude mice have anti-liver cancer cell growth and concentration-dependent,the higher the concentration,the more obvious anti-tumor function,5,7-DMF combined with 5-FU have joint resistance Function of tumor growth.