The Relationship Between The Effect Of Human Hepatocellular Carcinoma Cell Induced By EGCG And The Pathway Of PI3K-AKT

Objective Discuss the growth, proliferation and apoptosis of humanHCC cell lines, which are cultured in vitro and induced byEpigallocatechin-3-gallate (EGCG), and explore the correlative signalingmolecules’ variation of mRNA and protein in the signaling pathway ofPI3K-AKT.Methods The cell growth inhibition ability of EGCG was firstdetermined by MTT assay. The apoptosis induced by EGCG was investigatedby AO/EB staining. The cell cycle and apoptosis distribution were analyzed byflow cytometer. Detect the variance of signaling molecules EGFR, PI3K, AKTand NF-κB on mRNA level by real time PCR. The expression of AKT and itsphosphorylation at Ser473were detected by western blotting after theSMMC7721cells were treated with EGCG and combined with inhibitor(LY294002) or activator (IGF-1) of PI3K.Results The optimal EGCG concentration treated on HepG2,SMMC7721and Sk-hep1cells were74.7,59.6,61.3μg/ml and the most suitabletime were48hour. The adherent cells of experimental group were significantlyless than the control group, but apoptotic cells increased.[HepG2:(16.33±3.51 vs3.67±1.15, P=0.004, P＜0.01)，SMMC7721:(15.33±3.06vs3.33±2.08,P=0.005, P＜0.01)，Sk-hep1:(18.33±2.31vs2.33±2.08, P=0.001, P＜0.01)].The S phrase of the cell cycle in experimental group is significantly higher thancontrol group (49.7%±1.2%vs22.1%±1.5%, P＜0.05). The apoptosis rate ofexperimental group (25.1%±4.2%) is significantly higher than control group(9.0%±2.7%)(P＜0.05). Real time-PCR: The mRNA expression level ofEGFR, PI3K, AKT and NF-κB were76.3%±4.0%,68.3%±4.0%,71.0%±5.6%and57.3%±7.1%in experimental group, which were significantly lessthan the control group (100.0%±0.0%, P＜0.05). Western Blot: The expressionof AKT protein were1.23±0.06(control),0.53±0.11(LY294002),2.14±0.14(IGF-1),0.75±0.12(EGCG),1.37±0.09(EGCG+LY294002),1.56±0.12(EGCG+IGF-1). The expression of its phosphorylation at Ser473were0.70±0.11(control),0.33±0.09(LY294002),1.12±0.16(IGF-1),0.25±0.18(EGCG),0.71±0.07(EGCG+LY294002),0.75±0.08(EGCG+IGF-1). Thegroup of LY294002and EGCG were significantly less than control group thatwas significantly less than IGF-1group, but the group of EGCG+LY294002andEGCG+IGF-1were significantly higher than EGCG group (P＜0.05).Conclusion1. EGCG inhibits cell growth, induces apoptosis and causesS phase arrest in SMMC7721cell line;2. The possible mechanism of inhibitinggrowth, inducing apoptosis and causing S phase arrest may be suppressing thePI3K-AKT pathway in hepatocellular carcinoma.