The Effect Of EGCG On Human Blymphoma Cells And The Research Of Mechanisms

Backgroud:The aim of this study was to investigate the effect of (—)-Epigallocatechin gallate (EGCG) on proliferation and apoptosis in the two kinds of B lymphoma cell lines Jeko-1 and Raji, and further determine the molecular mechanisms.Methods:The Jeko-1 and Raji cells were divided as control group, EGCG treated group and inhibitor with EGCG treated group to experiment. The cell counting kit (CCK-8) assay measured cell proliferation and cytotoxicity; the flow cytometry measured apoptosis rates using the Annexin V-PE/7AAD double staining; real-time PCR determined mRNA expression of Fas, Bcl-2 and Bax; caspase-8 activity was measured by the colorimetry; the protein expression levels of apoptosis-associated were measured by Western blot.Results:1. EGCG significantly inhibited the Jeko-1 and Raji cells proliferation, and the effect of EGCG-induced apoptosis in a dose-and time-dependent manner. The IC50 of EGCG-treated 24h with Jeko-1 and Raji cells respectively was 68.65 and 62.16μg/ml.2. The apoptosis rates of the control group and EGCG-treated group were 5.43%±1.34%,27.64%±3.13%,56.63%±7.85% and 77.9%±0.7% in Jeko-1 cells; while were 9.56%±2.87%,23.04%± 11.02%,61.43%±8.61% and 72.78% ±2.44% in Raji cells; the apoptosis rates of EGCG-treated groups were significantly higher than control group (p<0.05).3. The results of RT-PCR showed that in Jeko-1 cells the expressions of Fas mRNA were 1.88±0.06 times,2.38±0.04 times and 5.17±0.36 times that of control group; Bax mRNA were 1.53±0.25 times,1.75±0.08 times and 2.25± 0.28 times that of control group; Bcl-2 mRNA were 0.73±0.02 times,0.68± 0.03 times and 0.54±0.02 times that of control group; while in Raji cells, the expressions of Fas mRNA were 1.09±0.02 times,1.16±0.01 times and 1.24± 0.03 times that of control group; Bax mRNA were 1.19±0.06 times,1.32±0.04 times and 1.46±0.08 times that of control group; Bcl-2 mRNA were 0.8±0.08 times,0.57±0.11 times and 0.35±0.1 times that of control group. Fas and Bax mRNA were upregulated by EGCG, while Bcl-2 was downregulated, and the difference was statistically significant compared with control group (p<0.05).4. The results of caspase-8 activity showed that in Jeko-1 cells the activations of caspase-8 were 1.25±0.2 times,2.14±0.15 times and 2.72±0.22 times that of control group; while in Raji cells were 0.98±0.04 times,1.16± 0.04 times and 1.28±0.07 times that of control group; in other words, the activations of caspase-8 in EGCG-treated groups were significantly higher than control group (p<0.05).5. The results of western blot suggested that in Jeko-1 cells the relative expression levels ofcaspase-3 were at least 7.32 times than control group (0.19 ±0.03), caspase-7 were at least 2.47 times then control group (0.15±0.07), PARP were at least 1.33 times than control group (0.12±0.01), caspase-9 were at least 1.61 times than control group (0.18±0.04), Bax were at least twice than control group (0.5±0.03), Bcl-2 were decreasd at least 11.19% than control group (1.34±0.34); while in Raji cells the relative expression levels of caspase-3 were at least 1.29 times than control group (0.07±0.03), caspase-7 were at least 1.53 times than control group (0.77±0.04), PARP were at least 13 times than control group (0.10±0.04), caspase-9 were 6.25 times than control group (0.16±0.02), Bax were at least 2.76 times than control group (0.58± 0.13), Bcl-2 were decreased at least 47.91% than control group (0.48±0.03). EGCG activated caspase-3,-7,-9 and PARP and increased Bax, while Bcl-2 protein levels were reduced. All the difference of EGCG-treated groups were statistically significant compared with control group (p<0.05). The caspase inhibitor Z-VAD-FMK could inhibite caspase activation induced by EGCG All the difference of inhibitor with EGCG group were statistically significant compared with EGCG-treated group (p<0.05).Conclusion:EGCG could induce the B cell lymphoma cells apoptosis and inhibite cells proliferation, through triggering caspase-dependent intrinsic (mitochondrial) and extrinsic (death receptor) pathways. The results suggested that EGCG may act as a potential agent for the treatment of B cell lymphoma.