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(-)-Epigallocatechingallate induces apoptosis in B lymphoma cells via caspase-dependent pathway and Bcl-2 family protein modulation.

Wang J, Xie Y, Feng Y, Zhang L, Huang X, Shen X, Luo X - Int. J. Oncol. (2015)

Bottom Line: (-)-Epigallocatechingallate (EGCG) as a representative polyphenol has attracted increasing attention due to its diversified effects, especially its potential as an agent for the prevention or treatment of certain cancers.In agreement, EGCG upregulated the mRNA expression of Fas and Bax while downregulating Bcl-2.Protein expression levels of Bax, activated caspase-3, -7, -8, and -9, and PARP were increased, while Bcl-2 protein levels were reduced by EGCG treatment.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Affiliated Tumour Hospital of Guangxi Medical University, Nanning 530021, P.R. China.

ABSTRACT
(-)-Epigallocatechingallate (EGCG) as a representative polyphenol has attracted increasing attention due to its diversified effects, especially its potential as an agent for the prevention or treatment of certain cancers. However, the molecular mechanisms of EGCG-induced apoptosis in B lymphoma cells are unclear. The aim of this study was to investigate the effect of EGCG on proliferation and apoptosis in the B lymphoma cell lines Jeko-1 and Raji, and determine the underlying mechanisms. Cell proliferation and cytotoxicity were determined by the cell counting kit (CCK-8) assay; apoptosis was assessed by flow cytometry using the Annexin V-PE/7AAD double staining; Fas, Bcl-2 and Bax mRNA expression levels were determined by real-time PCR; caspase activity was measured by the caspase activity assay kit; the expression levels of apoptosis-associated proteins were determined by western blot analysis. We demonstrated that EGCG induced growth inhibition and apoptosis in a dose- and time-dependent manner. In agreement, EGCG upregulated the mRNA expression of Fas and Bax while downregulating Bcl-2. Protein expression levels of Bax, activated caspase-3, -7, -8, and -9, and PARP were increased, while Bcl-2 protein levels were reduced by EGCG treatment. Taken together, EGCG induces B lymphoma cell apoptosis by triggering caspase-dependent intrinsic (mitochondrial) and extrinsic (death receptor) pathways. These findings suggest that EGCG may be a potential agent for the treatment of B lymphoma.

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EGCG upregulates Bcl-2 and Bax expression, and caspase-9 activation. (A) Jeko-1 or Raji cells were treated with EGCG at different concentrations (0, 20, 40 and 60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis, with appropriate antibodies. β-actin was used as a loading control. (B) Relative band intensities of Bcl-2, Bax and activated caspase-9. (C) The cells were pretreated with the general caspase inhibitor Z-VAD-FMK (10 μM) for 1 h and incubated with EGCG (60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis to determine the inhibition of EGCG-induced caspase-9 activation. *p<0.05, compared with the control group (0 μg/ml); #p<0.05, compared with the previous group. (D) Inhibition of caspase-9 activation shown by relative band intensity detected by western blot analysis using appropriate antibodies. β-actin was used as a loading control. The values represent mean ± SD from three independent experiments. *p<0.05, compared with the EGCG-treatment group.
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f7-ijo-46-04-1507: EGCG upregulates Bcl-2 and Bax expression, and caspase-9 activation. (A) Jeko-1 or Raji cells were treated with EGCG at different concentrations (0, 20, 40 and 60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis, with appropriate antibodies. β-actin was used as a loading control. (B) Relative band intensities of Bcl-2, Bax and activated caspase-9. (C) The cells were pretreated with the general caspase inhibitor Z-VAD-FMK (10 μM) for 1 h and incubated with EGCG (60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis to determine the inhibition of EGCG-induced caspase-9 activation. *p<0.05, compared with the control group (0 μg/ml); #p<0.05, compared with the previous group. (D) Inhibition of caspase-9 activation shown by relative band intensity detected by western blot analysis using appropriate antibodies. β-actin was used as a loading control. The values represent mean ± SD from three independent experiments. *p<0.05, compared with the EGCG-treatment group.

Mentions: In order to further confirm the involvement of the mitochondrial pathway, the most important and common apoptosis pathway, in EGCG-induced apoptosis in Jeko-1 and Raji cells, caspase-9 activity and expression levels of Bcl-2 family proteins Bcl-2 and Bax were assessed. Treatment of both cell lines with EGCG resulted in increased activity of caspase-9 (Fig. 7A and B). The expression of the anti-apoptotic protein Bcl-2 was downregulated significantly, whereas that of the pro-apoptotic protein Bax was upregulated. Importantly, these changes were also EGCG dose-dependent. When cells were pretreated with the general caspase inhibitor Z-VAD-FMK, caspase-9 activity was decreased compared with the EGCG-treated group as indicated in Fig. 7C and D. These results suggested that EGCG triggers the mitochondrial pathway, regulating Bcl-2 family proteins to induce apoptosis in Jeko-1 and Raji cells.


(-)-Epigallocatechingallate induces apoptosis in B lymphoma cells via caspase-dependent pathway and Bcl-2 family protein modulation.

Wang J, Xie Y, Feng Y, Zhang L, Huang X, Shen X, Luo X - Int. J. Oncol. (2015)

EGCG upregulates Bcl-2 and Bax expression, and caspase-9 activation. (A) Jeko-1 or Raji cells were treated with EGCG at different concentrations (0, 20, 40 and 60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis, with appropriate antibodies. β-actin was used as a loading control. (B) Relative band intensities of Bcl-2, Bax and activated caspase-9. (C) The cells were pretreated with the general caspase inhibitor Z-VAD-FMK (10 μM) for 1 h and incubated with EGCG (60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis to determine the inhibition of EGCG-induced caspase-9 activation. *p<0.05, compared with the control group (0 μg/ml); #p<0.05, compared with the previous group. (D) Inhibition of caspase-9 activation shown by relative band intensity detected by western blot analysis using appropriate antibodies. β-actin was used as a loading control. The values represent mean ± SD from three independent experiments. *p<0.05, compared with the EGCG-treatment group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356505&req=5

f7-ijo-46-04-1507: EGCG upregulates Bcl-2 and Bax expression, and caspase-9 activation. (A) Jeko-1 or Raji cells were treated with EGCG at different concentrations (0, 20, 40 and 60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis, with appropriate antibodies. β-actin was used as a loading control. (B) Relative band intensities of Bcl-2, Bax and activated caspase-9. (C) The cells were pretreated with the general caspase inhibitor Z-VAD-FMK (10 μM) for 1 h and incubated with EGCG (60 μg/ml) for 24 h. Equal amounts of total protein were examined by western blot analysis to determine the inhibition of EGCG-induced caspase-9 activation. *p<0.05, compared with the control group (0 μg/ml); #p<0.05, compared with the previous group. (D) Inhibition of caspase-9 activation shown by relative band intensity detected by western blot analysis using appropriate antibodies. β-actin was used as a loading control. The values represent mean ± SD from three independent experiments. *p<0.05, compared with the EGCG-treatment group.
Mentions: In order to further confirm the involvement of the mitochondrial pathway, the most important and common apoptosis pathway, in EGCG-induced apoptosis in Jeko-1 and Raji cells, caspase-9 activity and expression levels of Bcl-2 family proteins Bcl-2 and Bax were assessed. Treatment of both cell lines with EGCG resulted in increased activity of caspase-9 (Fig. 7A and B). The expression of the anti-apoptotic protein Bcl-2 was downregulated significantly, whereas that of the pro-apoptotic protein Bax was upregulated. Importantly, these changes were also EGCG dose-dependent. When cells were pretreated with the general caspase inhibitor Z-VAD-FMK, caspase-9 activity was decreased compared with the EGCG-treated group as indicated in Fig. 7C and D. These results suggested that EGCG triggers the mitochondrial pathway, regulating Bcl-2 family proteins to induce apoptosis in Jeko-1 and Raji cells.

Bottom Line: (-)-Epigallocatechingallate (EGCG) as a representative polyphenol has attracted increasing attention due to its diversified effects, especially its potential as an agent for the prevention or treatment of certain cancers.In agreement, EGCG upregulated the mRNA expression of Fas and Bax while downregulating Bcl-2.Protein expression levels of Bax, activated caspase-3, -7, -8, and -9, and PARP were increased, while Bcl-2 protein levels were reduced by EGCG treatment.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Affiliated Tumour Hospital of Guangxi Medical University, Nanning 530021, P.R. China.

ABSTRACT
(-)-Epigallocatechingallate (EGCG) as a representative polyphenol has attracted increasing attention due to its diversified effects, especially its potential as an agent for the prevention or treatment of certain cancers. However, the molecular mechanisms of EGCG-induced apoptosis in B lymphoma cells are unclear. The aim of this study was to investigate the effect of EGCG on proliferation and apoptosis in the B lymphoma cell lines Jeko-1 and Raji, and determine the underlying mechanisms. Cell proliferation and cytotoxicity were determined by the cell counting kit (CCK-8) assay; apoptosis was assessed by flow cytometry using the Annexin V-PE/7AAD double staining; Fas, Bcl-2 and Bax mRNA expression levels were determined by real-time PCR; caspase activity was measured by the caspase activity assay kit; the expression levels of apoptosis-associated proteins were determined by western blot analysis. We demonstrated that EGCG induced growth inhibition and apoptosis in a dose- and time-dependent manner. In agreement, EGCG upregulated the mRNA expression of Fas and Bax while downregulating Bcl-2. Protein expression levels of Bax, activated caspase-3, -7, -8, and -9, and PARP were increased, while Bcl-2 protein levels were reduced by EGCG treatment. Taken together, EGCG induces B lymphoma cell apoptosis by triggering caspase-dependent intrinsic (mitochondrial) and extrinsic (death receptor) pathways. These findings suggest that EGCG may be a potential agent for the treatment of B lymphoma.

Show MeSH
Related in: MedlinePlus