Effects Of Curcumin On The Expression Of VEGF And HIF-1 In Hepatocellular Carcinoma BEL-7402 Cells And Its Mechanism

ObjectivesCurcumin is a phenol pigment which is abstracted from the curcuma rhizome of curcuma genus in ginger family. It is the main component of curcuma and a natural phenol antioxidant, and also an anti-mutant and a anti-carcinogenic promoting agent. The anti-tumor mechanisms of curcumin are miscellaneous,its inhibition on the formation of new vessels is an important mechanism for the anti-tumor effect of curcumin. It is proved that curcumin can down-regulate the expression of VEGF gene in EAT(ehrlich ascites tumor) cells,and decrease the level of VEGF in cells, it is the mechanism of curcumin inhibiting the tumor angiogenesis.Accordingly, the main aims of our study are as follows: (1)To observe the effects of curcumin on the expression of VEGF in human hepatocellular carcinoma BEL-7402 cells; (2) To observe the effects of curcumin on the expression of HIF-1αin human hepatocellular carcinoma BEL-7402 cells;(3)To observe the proteasome and p53 in the effect of curcumin on the expression of HIF-1αprotein by adopting a proteasome inhibitor MG132 and p53-null human hepatocellular carcinoma Hep3B cell line;(4) To observe the effect of curcumin on the expression of VEGF, HIF-1αin PI3K and MAPK signal transduction pathways by adopting PI3K inhibitor LY294002,MEK inhibitor U0126 and mTOR/FRAP inhibitor rapamycin.and furtherly,to reveal the mechanism of curcumin inhibiting tumor angiogenesis.Methods1. The effects of curcumin on the expression of VEGF and HIF-1αin human hepatocellular carcinoma BEL-7402 cells (1)Human hepatocellular carcinoma BEL-7402 cells were treated with different concentrations under hypoxic conditions for 6 hours.(2)Human hepatocellular carcinoma BEL-7402 cells were treated with 10-μmol/L of curcumin under hypoxic conditions for 0-8 hours,The cell proliferation and cell viability were assayed by WST-8 and the trypan blue dye exclusion methods. The expressions of VEGF and HIF-1αin human hepatocellular carcinoma BEL-7402 cells were determined by RT-PCR and Western Blot, Statistical analysis was performed by student t test.2. Involvement of proteasome and p53 in the inhibition effects of HIF-1αprotein levels in human hepatocellular carcinoma BEL-7402 cells induced by curcumin(1)Human hepatocellular carcinoma BEL-7402 cells were treated with 0,10μmol/L of curcumin, 10μmol/L of MG-132, 10μmol/L of curcumin plus 10μmol/L of MG-132 under hypoxic condition for 6 hours.(2)Human hepatocellular carcinoma BEL-7402 and Hep3B cells were treated with different concentrations under hypoxic condition for 6 hours. The cell proliferation and cell viability was assayed by WST-8 and the trypan blue dye exclusion methods. The expressions of HIF-1αprotein in human hepatocellular carcinoma BEL-7402 cells were determined by Western Blot,Statistical analysis was performed by student t test.3. The effects of PI3K and MAPK signal transduction pathways on the expression of VEGF and HIF-1αin human hepatocellular carcinoma BEL-7402 cells(1)Adding 0, 10μmol/L of curcumin into the 6-well plates with a cover slip which have seeded in human hepatocellular carcinoma BEL-7402 cells,were cultured under hypoxic conditions for 6 hours, the protein expression of VEGF and HIF-1αwas detected by immunochemistry.(2)Human hepatocellular carcinoma BEL-7402 cells were respectively pretreated with 20μmol/L,50μmol/L of LY294002,10μmol/L,20μmol/L of U0126,5ng/ml,10ng/ml of rapamycin for 30 minutes, followed by treatment with 10μmol/L of curcumin,and alone treated with 0, 10μmol/L of curcumin as control group ,the cells were cultured under hypoxic conditions for 6 hours.the expression of VEGF,mRNA and HIF-1αwere detected by RT-PCR and Western Blot.(3)Human hepatocellular carcinoma BEL-7402 cells were treated with different concentrations of curcumin and 25μmol/L of LY294002 under hypoxic conditions for 6 hours the phosphorylation of AKT and total AKT were detected by Western Blot, Statistical analysis were performed by student t test and Pearson related analysis.Results1. The effects of curcumin on the expression of VEGF and HIF-1αin human hepatocellular carcinoma BEL-7402 cells(1)The cell proliferation rate and cell viability were no significant difference between control group and different concentrations of curcumin groups under hypoxic conditions for 6 hours, P>0.05;Curcumin can gradually down-regulate the expression of HIF-1αprotein,VEGF protein and VEGF mRNA with the rise of concentrations. Significant difference were compared with the control group, P<0.01;The expression of HIF-1αmRNA were no significant difference between control and different concentrations of curcumin, P>0.05.(2)The cell proliferation rate and cell viability were no significant difference between control group and 10μmol/L of curcumin under hypoxic conditions for 0-8 hours groups, P>0.05.Curcumin can gradually up-regulate the expression of HIF-1αprotein,VEGF protein and VEGF mRNA with the extension of time. Significant difference were compared with the control group, P<0.01;The expression of HIF-1αmRNA was no significant difference between control group and 10-μmol/L of curcumin under hypoxic condition for 0-8 hours groups, P>0.05.2. Involvement of proteasome and p53 in the inhibition effects of HIF-1 protein levels in human hepatocellular carcinoma BEL-7402 cells induced by curcumin(1) The expression of HIF-1αprotein in human hepatoceular carcinoma BEL-7402 cells treated with 10μmol/L of curcumin group under hypoxic condition for 6 hours was lower than MG132 plus curcumin group,the expression of HIF-1αprotein in cells treated with 10μmol/ of MG-132 group was lower than MG132 plus curcumin group, the expression of HIF-1αprotein in cells treated with 10μmol/ of curcumin group was lower than 0μmol/L of curcumin group, the expression of HIF-1αprotein in cells treated with 10μmol/ of MG-132 group was higher than 0μmol/L of curcumin group,they were significant difference, P <0.01.(2)Curcumin decreased HIF-1αprotein levels in BEL-7402 cells in a concentration-dependent manner, the effect of curcumin on expression of HIF-1αprotein was significant difference between control and different concentrations in BEL-7402 cells,P <0.01, While the effect of curcumin on expression of HIF-1αprotein were no significant difference between control and different concentrations in Hep3B cells, P >0.05.3.The effects of PI3K and MAPK signal transduction pathways on the expression of VEGF and HIF-1αin human hepatocellular carcinoma BEL-7402 cells(1)The expression of HIF-1αand VEGF protein in human hepatocellular carcinoma BEL-7402 cells treated with 10μmol/L of curcumin group under hypoxic condition for 6 hours was obviously lower than control group, the expression of HIF-1αprotein was positively correlated with the expression of VEGF protein, r =0.965, P <0.01.(2) The expression of VEGF protein,mRNA and HIF-1αprotein were lower in 10μmol/L of curcumin plus 25μmol/L of LY294002 group,10μmol/L of curcumin plus 50μmol/L of LY294002 group,10μmol/L of curcumin plus 5ng/ml rapamycin group and 10μmol/L of curcumin plus 10ng/ml rapamycin group than in 10μmol/L of curcumin group, they were significant difference, P <0.01; The expression of VEGF protein,mRNA and HIF-1αprotein were no significant difference between 10μmol/L of curcumin plus U0126 10μmol/L group,10μmol/L of curcumin plus U0126 20μmol/L group and 10μmol/L of curcumin group,P > 0.05.(3) The expression of phosphorylation of AKT protein in human hepatocellular carcinoma BEL-7402 cells treated with different concentrations of curcumin and 25μmol/L of LY294002 under hypoxic condition for 6 hours gradually decrease, 25μmol/L of LY294002 can block the expression of the phosphorylated AKT protein ,but it cann't influence on the expression of the total AKT protein,and it cann't block the expression of the total AKT protein.Conclusions(1)Curcumin can inhibit the expression of VEGF mRNA and protein in human hepatocellular carcinoma BEL-7402 cells.(2)Inhibitory effect of curcumin on expression of HIF-1αin human hepatocellular carcinoma BEL-7402 cells were through post-transcriptional mechanisms,not through cell death or cell proliferation.(3)Curcumin inhibit the expression of HIF-1αprotein via the proteasome pathway and p53 in human hepatocellular carcinoma BEL-7402 cells.(4)Inhibitory effect of curcumin on expression of VEGF in human hepatocellular carcinoma BEL-7402 cells was mostly through inhibiting the expression of HIF-1αprotein. Curcumin inhibit the expression of VEGF and HIF-1αprotein in human hepatocellular carcinoma BEL-7402 cells by PI3K/AKT/FRAP signal transduction pathway,not by MAPK signal transduction pathway.