Effects Of EGCG On Regulating NF-κB Induced By TNF-α And Inducing Apoptosis

Objective: To survey the effects of epigallocatechin-3-gallate (EGCG) on inducing apoptosis of human gastric carcinoma cells SGC-7901 and subsequently approach whether the molecular mechanism relates to regulating NF-κB.Methods: Hoechst staining method was used to display the apoptosis morphological change of SGC-7901 cell induced by EGCG. After being treated by 10ng/ml TNF-α, a inductor of regulating NF-κB, Western Blot was used to detect the disposition of NF-κB. The same method was used to detect the effect of EGCG, too. Apoptosis of SGC-7901 cell induced by EGCG was determined using flow cytometry to analyse apoptosis rate. DNA agarose gel electrophoresis was used to detect the fragments of SGC-7901 cell's DNA .Results: Hoechst staining method showed that EGCG could significantly induce apoptosis of partial SGC-7901.After being exposed to EGCG, the cell nucleus changed to shiver and pallor. After being treated by 10ng/ml TNF-αfor different time(30,60,90,120min), Western Blot showed that the NF-κB in the cell nucleus increased obviously and displayed a platform after being treated 60 minutes. Being treated by 60μg/ml EGCG for different time (0,12,24,36,48h) before the 10ng/ml TNF-αfor 60min , Western Blot showed that the NF-κB in the cell nucleus decreased obviously and displayed a platform after being treated for 24h. Being treated by EGCG at various concentrations of 40,60,80,100μmol/l for 24 h before the 10ng/ml TNF-αfor 60min , Western Blot showed that the NF-κB in the cell nucleus decreased with a dose-dependent manner. Analysis by flow cytometry showed that the percentages of apoptosis cells were (3.73±0.15)% exposed to 10ng/ml TNF-αfor 60min.The data was (16.63±0.15)% exposed to 60μg/ml EGCG for 24h before the 10ng/ml TNF-αfor 60min .It was (21.47±0.12) % exposed to 100μmol/L Pyrrolidine dithiocarbamate (PDTC ,a suppressor of NF-κB) for 30 minutes before the 10ng/ml TNF-αfor 60min. In DNA agarose gel electrophoresis ,"Ladder"bands occurred after being treated by EGCG (60μg/ml , for 24h) or PDTC (100μmol/L ,for 30min) before 10ng/ml TNF-αfor 60min.Conclusion: EGCG could induce apoptosis of human gastric cell SGC-7901. EGCG could inhibit the entering of NF-κB to the cell nucleus induced by TNF-α.The effects of inducing apoptosis coulde be related to the effects of inhibition the entering of NF-κB.