Inhibitory Effect And Mechanism Of The Active Ingredient Of Betulinic Acid On Hepatocarcinoma Cells

Liver cancer is a common disease caused by many factors, which has extremely high fatality in face of mankind nowadays. As people of their own health and the increasing attention due to the rising number of deaths year by year, to find an effective way of treatment and prevention of hepatocarcinoma is imminent. However, surgery, radiation therapy and chemical therapy have their own limitations, individual gene therapy is still an experimental treatment, and biological treatment is more as an auxiliary therapy. Targeted therapy is a new breakthrough for the treatment of tumor i n recent years. M ore and more scholars begi n to I ook to the cancer stem cells (CSC). To find anti-cancer medicine from Chinese medicine has caused the industry consensus at home and abroad. The paper will selective study the anti-CSC machanism of effective constitutent of FuZhengHuaYuJieDu (FZHYJD) formula from the angle of Wnt pathway.The first part:To compare the anti-cancer effect in vivo of FZHYJD including astragalus and ginsengEstablishing ICR tumor-burdened mouse model by cultivating H22 ascites cells, setting up the model group, positive control group and two groups of different formula of FZHYJD. After inoculation of H22 ascites cells, we filled the stomachs of model mice with distilled water every day, injected control mice with cyclophosphamide, and drenched mice of two experimental group daily with different formula of FZHYJD respectively. In the meantime, the volume of tumors was measured with caliper. Ten days later, after the mice were executed, the tumor, spleen and thymus were stripped. Then we calculated tumor inhibition rate, thethymus index and spleen index of mice. The experiment results showed that two different formula had no significant influence on anti-cancer rate of H22 hepatoma-bearing mice, but all three results showed that the tumor inhibition rate of group consisting of astragalus was bigger than that of formula concluding ginseng, the inhibitory rate reached 42.70%; two kinds of prescription had no influence on the thymus index and spleen index. The results suggested that FZHYJD formula consisting astragalus had good effect.The second part:To contrast the anti-cancer effective constituent from FZHYJD prescription in vitroIn order to contrast the differences between chemical components of FZHYJD prescription, we apply method of CCK8, and human hepatocarcinoma cell line HepG2, Huh7, and human being carcinomaA549 cell to research the anti-tumor effect in vitro of specnuezhenide, curcumin, ursolic acid, oleanolic acid, SI, S2, S3, betulinic acid, hawthorn acid, and corosolic add. The results revealed that specnuezhenide had no inhibiting effect on HepG2 cells and even accelerated cell proliferation; curcumin, ursolic acid, oleanolic caid, S1, S2, S3, betulinic acid, hawthorn acid, and corosolic acid had certain effects on proliferative activity of HepG2, Huh7 and A549 cell lines. Among those constituents IC50 of hawthorn add were 37.30μmol·L-1、70.26μmol·L-1 and 136.64μmol·L-1 respectively, IC50 of betulinic acid were 11.29μmol·L-1、34.67μmol·L-1 and 75.29μmol·L-1,and IC50 of corosolic acid were 11.29μmol·L-1、34.67μmol·L-1 and 75.29μmol·L-1. The above suggested that betulinic acid, hawthorn acid, corosolic acid of FZHYJD had better anti-tumor effects.The third part:To detect the action of ursolic acid from FZHYJD prescription on Wnt pathway of liver cancer cells.CCK8 assay, flow cytometry assay were used to detect the proliferative activity, cell cycle and apoptosis of HepG2 cells. The expression status of protein of β-catenin, GSK-3β, pGSK-3p, CyclinD1, C-Myc were detected with method of western blot assay. The above results indicated that after treated with ursolic acid of 40μmol·L-1, cells in phases of G1/G0(P<0.01) and G2/M(P<0.05) decreased significantly, and cells in S phase(P<0.01) were markly increased. Cell apoptosis rate after exposing to ursolic acid of 10,20,40μmol·L-1 elevated remarkably and it also showed evidently dose dependence. The expression of β-catenin(P<0.01), pGSK-3β(P<0.01), CyclinD1(P<0.05), C-Myc(P<0.01) were decreased significantly. Those demonstrated that ursolic acid performed anti-cancer effect through inhibiting Wnt signaling path.The forth part:To test the influence of ursolic acid from FZHYJD prescription on LCSCs.Enriching liver cancer stem cells successfully through serum-free culture method, proving self-renewal capacity of LCSCs by coony formation assay, and detecting stem cell markers of LCSCs through flow cytometer. It turned out that LCSCs had certain ability of self-renewal. CCK8 assay was used to test the action of ursolic acid on LCSCs’ proliferative activity; flow cytometry assay was applyed to inspect cell cycle and apoptosis of LCSCs after dealed with ursolic acid. The above experiment indicated that ursolic acid had anti-proliferative effect on LCSCs, and it actually worked best in 48 hours. And the cells in G1/G0(P<0.05) and G2/M(P<0.05) phase decreased obviously, the apoptosis rate of LCSCs became bigger with the increase of ursolic acid concentration, which presented a certain dose-effect relationship.The topic research showed, ursolic acid, that is from Chinese sage of FZHYJD prescription performed a certain proliferative inhibition effects on hepatocellular carcinoma HepG2 cell line and its cancer stem cells, which was related with the suppression of Wnt signaling path.