Expression Of Telomerase Activity In Esophageal Carcinoma And Inhibition On Telomerase Activity By EGCG

Objectives:Esophageal carcinoma(EC) is one of the most commonmalignant digestive tumors in the world. The incidence of EC is high in China,which lines the second in digestive gastrointestinal cancers, and it is a greatthreaten to human health. Therefore, it is significance to investigate thepathogenesis of EC, search new therapy target and effective curative medicine.In recent years, it has been convincingly documented that telomere andtelomerase have intimate relationships with the occurrence and developmentof malignant tumor.Consequently, this study principally investigated the expression andsignificance of hTERT protein in people from high incidence area ofesophageal cancer, researched the expression and clinical significance oftelomerase activity in esophageal carcinoma,dysplasia and normal esophagealtissue from esophageal squamous cell carcinoma patients. Aslo, Eca109cellswere cultured in vitro and intervented with EGCG, the effect of EGCG wasstudyed by analysis of the cell apoptosis and the expression of telomreaseactivity. It is intended to reveal the relationship between telomerase activityand esophageal carcinoma, and provide experimental basis for chemotherapyof esophageal carcinoma.Methods:1The expression of hTETR were examined by s-p immunohistochemicalstain (IHC) in36cases of normal mucosa and50cases of dysplasia ofesophageal squamous epithelium and36cases of esophageal squamous cellcarcinoma.2Telomerase activity of30esophageal carcinoma tissues includingesophageal carcinoma tissues paired adjacent carcinoma tissues and normalmucosa were analyzed by TRAP-PCR argentation.3Esophageal carcinoma Eca109cells were cultured and intervened by EGCG with various concentrations(0,25,50,100,200,400mg/L) anddifferent times(24h,48h,72h), respectively. Cell growth inhibition wasdetected by MTT, and the concentration of half inhibition was calculated.4Eca109cells were treated with different concentrations of EGCG(0,25,50,100mg/L) for24,48,72hours, apoptosis and cycle of cells wereanalyzed by flow cytometry (FMC).0mg/L EGCG as the control group. Theexperiment was repeated by3times.5Eca109cells were treated with different concentrations of EGCG (0,25,50,100mg/L) for24,48,72hours, and telomerase activity of cells wereanalyzed by TRAP-PCR argentation.0mg/L EGCG as the control group. Theexperiment was repeated by3times.Results:1The expression of hTERT was examined by s-p immunohistochemicalstain (IHC) in122cases of esophageal mucosa,4cases were weakly positivein36normal mucosa,16cases were weakly positive and6cases werestrongly positive in50dysplasia of esophageal squamous epithelium,4caseswere weakly positive and28were strongly positive in36ESCC. The resultsshowed that the expression of hTERT in esophageal carcinoma (88.89%) wassignificantly higher than that in dysplasia of esophageal squamous (44.00%)and normal mucosa (11.11%)(P<0.05).2The telomerase activity of30clinical specimens were examined byTRAP-argentation. The results showed that the positive percentage in theesophageal carcinoma group (86.67%) was significantly higher than that inadjacent carcinoma group (23.33%) and normal mucosa (0.00%)(P<0.05).3Eca109cells were treated with0mg/L,25mg/L,50mg/L,100mg/L,200mg/L and400mg/L EGCG for12h,24h,48h respectively, thegrowth of cells was inhibited significantly in a time-and dose-dependentfashion. The IC50of Eca109cells of24h,48h and72h were162.9829mg/L,138.8385mg/L and109.6057mg/L respectively(P<0.01).4Apoptosis were analyzed by flow cytometry. Eca109cells were treatedwith EGCG (0,25,50and100mg/L) for24h,48h and72h. The apoptosis rate (AP):0.43±0.10%,6.01±0.67%,11.80±0.25%,16.54±0.84%(24h),0.59±0.12%,8.12±1.13%,13.99±0.90%,24.35±4.50%(48h),0.88±0.19%,10.82±0.25%,20.84±1.71%,40.01±3.44%(72h). Apoptosis rate in treatmentgroup were significantly higher than control group(P<0.01). The alterationoccured in a dose and time dependent manner.5Telomerase activity of cells were analyzed by TRAP-argentation. Aftersilver staining, the positive outcome showed up straps with a spacer of6bp.With the same concentration (25mg/L), telomerase activity of cells treated for24h expressed positive,48h group expressed weakly positive,72h groupexpressed negative. Along with the increasing of the concentration of EGCG,the telomerase activity decreased. With the same action time (24h), telomeraseactivity of cells treated with25mg/L EGCG expressed positive,50mg/L groupexpressed weakly positive,100mg/L group expressed negative. Along with theincreasing of action time, the telomerase activity decreased.Conclusions:1The positive rate of hTRET was significantly higher in esophagealcarcinoma tissues than that in squamous epithelium dysplasia tissues andnormal esophageal mucosa, it indicated that the expression of telomerase isrelated to the occurrence and development of malignant tumor.2Telomerase activity in esophageal carcinoma was significantly higherthan that in adjacent carcinoma tissues and normal mucosa. It is significanceto assay the telomerase activity of esophageal carcinoma.3EGCG can induce the apoptosis of Eca109cells by reducing theactivity of telomerase,this may be one of the important mechanisms of teapolyphenol against tumor.