Experiment Study Of Curcumin Inhibit Expression Via IGF-1R Pathway In Hypoxia SMMC-7721 Human Hepatocellular Carcinoma Cells

Part 1Objective To establish a hypoxic model of cultured human SMMC-7721 cells in vitro by the cobalt choride(Co Cl2), And study the effect of hypoxic on the proliferatio and expressionaof protein and m RNA of IGF-1R、HIF-1a、VEGF Protein and m RNA in SMMC-7721 cells.Methods Human SMMC-7721 cells was used in this study.Using the cobalt choride(Co Cl2) to simulate anoxie circumstance SMMC-7721.Cells were incubited at different concentration and time.The proliferation of SMMC-7721 cells was determined by CCK-8 assay. Examined expression of VEGF protein using Western blot or ELISA. Expressiona of Protein and m RNA of IGF-1R 、HIF-1a、VEGF was detected by Western blot and RT-PCR respectively.Result CCK-8 showed Co Cl2 inhibited the cells proliferation of SMMC-7721 cells in a dose-and time-dependent manner. cell apopotic rate increased with the extension of hypoxia incubation time and enhanced with the increase of Co Cl2 concentration elasa : Protein expression of VEGF enhanced with the increase of Co Cl2 concentration when the cell were treated with Co Cl2 for 12 h. and they reached their peak at 200mmol/L, the protein expression of VEGF also increased with the extension of hypoxia incubation time. P<0.05. Western-blotting and RTPCR measuring protein and m RNA as follows:compared with the control group HIF-1a ptotein and expressiona of Protein and m RNA of IGF-1R VEGF increased with the extension of hypoxia incubation time and concentrate on Expressiona of protein and m RNA of VEGF had significant correlations with Co Cl2 concentration and hypoxia incubation time.Conclusion The effect of hypoxia could increase expressiona of protein and m RNA of IGF-1R、VEGF also increase expressiona levels of m RNA of HIF-1a.Expressiona of protein and m RNA of VEGF had significant correlations with C-oCl2 concentration and hypoxia incubation time.Part 2Objective To explore whether curcumin mediated the inhibition of VEGF exp-ression via IGF-1 pathway in hypoxia-induced SMMC-7721 cells.Methods CCK-8 assay wsa used conduct The effect of curcumin on cell survival in hypoxia induced SMMC-7721 cells.Western-blotting and ELASA were used to measured the HIF-la、IGF-1 R、VEGF、P-Akt and P-Erkl/2 expressions.cells were divided into 6 groups. A group as control.other cells were incubated with curcumin cells were either left untreated or treated with MAPK inhibitor PD98059 、PI3K inhibitor LY294002 alone or in combination.The proli-feration of SMMC-7721 cells was determined by CCK-8 assay. Examined expression of VEGF protein using Western blot or ELISA. Expressiona of Protein and m RNA of IGF-1R、HIF-1a、VEGF was detected by Western blot and RT-PCR respectively.Result When the curcumin concentration was 10 μmol /L,It inhibited the cells proliferation of SMMC-7721 cells treated with curcumin for 12 h.elasa: compare with A group B vegf protein is higher, compare with group B VEGF protein in group C、D、E、F decreased.Western blot: compare with B the level of VEGF protein in group C、D、E is lower,group F is the lowest.RT-PCR compare with group B m RNA level of IGF-1R and VEGF in group C、D、E、F decreased.Conclusion Curcumin suppressed VEGF expression mainly by blocking the activation of IGF/PI3 K signaling pathway in hypoxia induced SMMC-7721 cells.