Study On The Stability Of EG And Its Mechanism Of Inhibiting Tumor Cell Proliferatio

Objective:To investigate the stability of emodin-8-O-β-D-glucopyranoside in various physicochemical conditions and biological matrix environment.To research the antitumor effect of EG on human hepatoma HepG2 cells,human neuroblastoma SH-SY5Y cells and human colorectal cancer HCT116 cells in vitro,and declare its antitumor mechanism.To research the antitumor effect of EG on mouse hepatoma H22 cells and human colorectal cancer HCT116 cells in vivo.Methods:The stability study of EG was performed under different physicochemical conditions(light,temperature,pH,medium,both high pressure and high temperature),and with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),liver microsomes and intestinal flora,and HPLC analytical method was used to detect the stability of EG.The morphology of HepG2,SH-SY5Y and HCT116 were observed by inverted microscope and Giemsa staining.The effect of EG on the cell viability of tumor cells was detected by MTT assay.Flow cytometry was used to detect the effect of EG on cell cycle,cell apoptosis and cell proliferation of tumor cells.Cell senescence of tumor cells was analysed byβ-galactosidase staining.Clone formation test was used to detect the effect of EG on the clone formation of tumor cells.The mRNA and protein expression of cell cycle related genes was detected by qRT-PCR and Western blotting in tumor cells.The in vivo effect of EG on mouse hepatoma H22 cells was tested by using tumor bearing mouse model,and the effect of EG on the viscera index of mouse was studied.The in vivo effect of EG on human colorectal cancer HCT116 cells was tested by using xenograft mouse model,and the effect of EG on the viscera index of mouse was studied.Results:1.EG was stable in the physicochemical conditions(light,temperature,pH,medium,high pressure and temperature conditions),with SIF and liver microsomes,respectively under experiment condition,but was unstable in SGF and intestinal flora.EG was degraded by 60%in SGF after 2 h incubation,and there was a metabolite peak at the retention time of emodin.EG was degraded by 80%in intestinal flora after 4 h incubation.EG was degraded in SGF while not degraded in SGF without pepsin,which suggests that the degradation of EG is due to the action of pepsin.2.EG significantly inhibited the cell viability of HepG2,SH-SY5Y and HCT116 in doseand time-dependent manner.The IC50 of EG on HCT116 cells in 24,48 and 72 h were 396.17,325.92 and 287.15 μg/mL respectively.The IC50 of EG on HepG2 cells in 48 and 72 h were 331.27 and 224.36 μg/mL respectively.The IC50 of EG on SH-SY5Y cells in 72 h was about 413.32 μg/mL.EG caused G1 phase cells’ratio accumulated on SH-SY5Y cells while EG had no apoptosis and aging effect on three cells.EG inhibited colony forming of HepG2,SH-SY5Y and HCT116 cells significantly.EG inhibited the expression of phosphorylated pRbS780 and pRbS807/811 proteins in SH-SY5Y cells;EG inhibited the expression of Rb protein and its phosphorylated pRbS780 proteins in HCT116 cells;EG up-regulated the expression of Pdcd4 protein and down regulated the expression of mutP53 protein in SH-SY5Y,HCT116 and HepG2 cells.3.EG inhibited tumor growth of H22 in vivo and the tumor weight inhibition rate of 5 mg/kg EG was 58.83%,which was the same as that of 5-Fu positive drug(53.64%).EG inhibited tumor growth of HCT116 in vivo and the tumor volume inhibition rate of 2,4,8 mg/kg EG were 48.49%,43.24%and 43.34%respectively,which was the same as that of 5-Fu positive drug(46.60%).EG had no significant effect on viscera index of mouse.Conclusion:1.EG was stable in the physicochemical conditions(light,temperature,pH,medium,high pressure and temperature conditions),with SIF and liver microsomes,respectively.EG had not been degraded in 1640 medium or 1640 medium containing 10%serum,indicating that the in vitro antitumor activity of EG is caused by EG itself.But EG was unstable in SGF and intestinal flora,indicating that oral administration of EG should be avoided in vivo.2.EG had inhibitory effects on HepG2,SH-SY5Y and HCT116 cells,and the mechanism was related to the inhibition of the expression of cycle related protein Rb and its phosphorylated protein pRbS780 and pRbS807/811,as well as up-regulating Pdcd4 protein and down regulating mutP53 protein.3.EG could inhibit H22 tumor growth in H22 tumor bearing mouse,and had no significant effect on spleen index and thymus index.EG inhibited the growth of human colorectal cancer HCT116 in xenografts mouse,and had no significant effect on organ index.