Effect Of NF-?B Signaling Pathway On Inhibition Of Human Esophageal Carcinoma Cells By Procyanidins

Objective:We aimed to systematically analyze the effect of procyanidins on the nuclear factor kappa B?NF-?B?pathway using data available from recent studies.Methods:Two of our researchers searched electronic databases such as Pub Med,China National Knowledge Infrastructure,and Wanfang Data using the search string “the NF kappa B OR NF-?B OR nuclear factor kappa B AND procyanidins.” They then independently performed meta-analysis on the results,using NF-?B DNA binding activity and the expression levels of inhibitory kappa B,phosphorylated inhibitory kappa B,and nuclear factor kappa B protein as outcome indicators.Results:Compared with results from the activated NF-?B model group,procyanidins effectively elevated the expression of inhibitory kappa B?standardized mean difference,SMD = 6.20;P = 0.03?,inhibited phosphorylation of inhibitory kappa B,and down-regulated phosphorylated inhibitory kappa B?SMD =-3.73,P = 0.03?.NF-?B monomer p65 levels decreased?SMD =-5.21,P = 0.02?.Procyanidins also reduced NF-?B DNA binding activity?SMD =-5.42,P < 0.0001?and weakened the expression of NF-?B protein in cells?SMD =-3.31,P < 0.00001?.Conclusion:we confirmed by a Meta-Analysis that procyanidins effectively inhibit activation of NF-?B,an effect that could be influenced by dose and duration of exposure.Objective:To explore the mechanism of Grape Seed Proanthocyanidins Extract?GSPE?against human esophageal squamous carcinoma cells and the role of NF-?B signal pathway in it,so as to provide reference for the anti-cancer mechanism of procyanidins.Methods:This study was performed on human esophageal squamous cell carcinoma cells?ECA 109?cells according to GSPE?0,25,50,and 80 ?g/m L?and BAY 11-7082?0 and 10 ?mol/L?.Dose and intervention time?12,24,48h?were divided into 24 groups.The survival rate of the cells was measured by MTT assay,and the apoptosis rate of the cells was measured by flow cytometry;the expression levels of IL-6,COX-2,CRP,PGE₂,Bax,and Bcl-2 in the ECA109 cell homogenate were determined by ELISA.q RT-PCR assayed the m RNA levels of I?B-?,IKK?,IKK?,NF-?Bp65,NF-?Bp50,and caspase-3 in ECA109 cells;Western-Blot assays IKK?/?,I?B-?,and p-I?B-in ECA109 cells.?,NF-?B p65,NF-?B p50 protein expression levels.One-Way-ANOVA was used to compare the multiple groups that met the conditions.Results:1.With the increase of GSPE intervention dose and the prolongation of time,the survival rate of ECA109 cells decreased from 99.17 + 0.74,98.19 + 0.81,97.38 + 0.59 to 20.71 + 0.94,12.32 +1.07,9.14 + 2.19,P < 0.05.2.The apoptosis rate increased from 0% to 87.3%?P < 0.05?with the increase of GSPE dose,and increased from 43.42% to 82.8%?P < 0.05?in BAY 11-7082 groups with different concentrations of GSPE.3.When the concentration of GSPE was 25?g/m L,compared with the control group,the migration distance of ECA109 cells was not statistically significant,but with the increase of GSPE concentration,the migration distance of ECA cells was 50?g/m L and 80?g/m L.Compared with the control group significantly shorter.Compared with the control group,the cells in the GSPE group that passed through the Transwell chamber were significantly reduced.With the increase of the GSPE concentration,the number of ECA109 cells passing through the Transwell chamber was reduced.While BAY11-7082 with the final concentration of GSPE of 25,50,80 ?g/m L and 10?mol/m L was added to transwell lower chamber culture cells for 24 hours,it was observed that compared with the control group,each concentration of GSPE+BAY11-7082 inhibited capacity of cell metastasis.4.Compared with the control group,the expression levels of inflammatory factors?IL-6,COX-2,PGE₂,and CRP?decreased with different concentrations of GSPE at 12 h?P < 0.05?,and the anti-apoptotic protein Bcl-2 expression level from 19.55±1.80 Decreased to 12.19±1.55?P=0.021?,the expression of apoptosis protein Bax increased from6.95±1.17 to 11.08±1.81?P=0.016?;and the expression of inflammatory factors and anti-apoptotic proteins increased as the intervention time prolonged.The decrease in the level is more pronounced and the expression of apoptotic proteins rises.Compared with GSPE group,after BAY 11-7082 was applied,the expression of inflammatory factors and anti-apoptosis protein was significantly decreased?P<0.05?,while the apoptotic factor was increased?P<0.05?.5.The results of q RT-PCR showed that the expression of IKK?,IKK?,NF-?Bp65,NF-?Bp50m RNA was higher in ECA109 cells without any intervention,but the m RNA expression of caspase-3 was lower,but 24 hours after the intervention with GSPE.The levels of IKK-?,IKK?,NF-?B p65,and NF-?B p50 m RNA I?B-? m RNA were decreased?P<0.05?.The greater the intervention dose,the more inhibited the m RNA expression of these factors by GSPE.Compared with the GSPE group,the levels of I?B-?,NF-?B p65,and NF-?Bp50 m RNA were lower than those in the GSPE group after BAY 11-7082 was applied?P<0.05?,and IKK m RNA levels were not significance statistically changed.6.Western-Blot results showed that compared with the blank control group,GSPE could reduce the ratios of IKK?/?,I?B-?,P-I?B-?,NF-?Bp65,NF-?Bp50 and ?-actin?P<0.05?.),increase the ratio of caspase-3 to ?-actin?P<0.05?and the larger the dose,the more obvious the effect.Compared with GSPE group,the ratios of I?B-?,P-I?B-?,NF-?Bp65,NF-?Bp50 and ?-actin were reduced in BAY 11-7082?P<0.05?,caspase-3 and ?-actin.The ratio increased?P<0.05?.Conclusion:GSPE inhibits the growth of cancer cells by inducing apoptosis in human esophageal squamous carcinoma cell ECA109.The NF-?B signaling pathway plays an important role in inhibiting the growth of ECA109 cells by GSPE.GSPE reduces the expression of inflammatory cytokines IL-6,COX-2,PGE₂ and CRP in cells by inhibiting NF-?B pathway,promotes apoptosis protein Bax,inhibits anti-apoptotic protein Bcl-2 and then activates caspase-3.Eventually apoptosis of esophageal cancer cell ECA109 was induced,and proliferation,migration,and invasion were inhibited.