The Inhibition Effect Of Epigallocatechin-3-gallate(EGCG) On Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the worldand the third highest cause of cancer-related death in humans. Surgical intervention hasbeen the most effective therapeutic modality for improving survival of patients.However, the surgical resection showed low5-year survival due to the high rate ofmetastasis. Epigallocatechin-3-gallat (EGCG) has been studied for inhibiting themetastatic activities on some cancer cells. In this in vitro study, we mainly focused ondetermination of the inhibitory effect of EGCG on LM6cells HCC, a hepatocellularcarcinoma with high metastasis. The aim of this study is to identify the effect andplausible mechanism of EGCG on human HCC from cellular changes to molecularcharacterizations including proteomics study. Our results may lead to further study onutilizing EGCG for clinical application.Three major objectives were proposed in this study. First，EGCG was examined todetermine the inhibitory effect on human hepatocellular cancer cells (HCCLM6) andnon-cancerous liver cells (HL-7702) though inducing apoptosis, and gene expressionwas measured to elucidate the molecular mechanism. Growth inhibition rate wasexamined by MTT, Flow cytometric (FCM) analysis were performed to assess apoptoticactivity, cell cycle, and mitochondrial membrane potential (MMP). The geneexpressions of Bcl-2, NF-kB, Bax, P53, caspase-9, caspase-3and cytochrome-c weremeasured by Western-blotting. Results showed that EGCG (10-100g/ml) effectivelyinhibited the proliferation of HCCLM6cells (p<0.01). A much higher minimum doseof EGCG (80μg/ml) was required to significantly inhibit the growth of the HL7702livercells (P<0.01). EGCG at10μg/ml and30μg/ml induced apoptosis, decreasedmitochondrial membrane potential, and caused a G0/G1-phase cell cycle arrest onHCCLM6cells, but no effect on non-cancerous liver cells (HL-7702). The effect ofEGCG on HCCLM6cells was associated with decreased expressions of Bcl-2and NF-kB significantly. Expressions of Bax, P53, caspase-9and caspase-3were increased,and cytochrome c were increased. Those results implied that EGCG selectively inducedapoptosis in cancerous cells with minimal effect on non-cancerous cells. EGCG inducedHCCLM6cells apoptosis predominantly through the mitochondria-mediated apoptosispathway. This study showed EGCG as an anticancer agent in the treatment of HCC.Secondly, EGCG was examined for the metastisis inhibition effect on humanhepatocellular cancer cells (HCCLM6). Migration and invasion assay for the metastaticbehavior of HCCLM6cells were conducted. The anti-metastatic mechanism of EGCGwas investigated using RT-PCR and gelatin zymography analysis. The results showedthat EGCG induced apoptosis and inhibited the metastasis of HCCLM6cells. Theanti-metastatic mechanism of EGCG therefore might be associated with the inhibitionof MMP-2and MMP-9activities and transcriptional level. The present study suggestedEGCG as an anticancer agent in the treatment of HCC.Finally, in order to to further investigate the inhibition mechanism of EGCG onHCCLM6cells and to study the effect of EGCG on HCCLM6cell proteomics,proteomics analysis was applied to compare the changes protein expressions before andafter EGCG treatment. There were35proteins were identified repetitively as stronglyassociated with EGCG, including11increased proteins and24decreased proteins. Fourmost significantly changed proteins were further analyzed by MALDI-TOF/MS.FUBP1, HSPB1, CH60and NPM proteins were identified by peptide data base analysis.The four proteins were reported as metastisis-and apoptosis-associated proteins,therefore our study provided evidence that EGCG exhibited anti-cancer activitiesthrough promoting apoptosis and inhibited metastisis.