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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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Z-VAD-FMK reverses the EGCG-induced activation of caspase-3, -7 and PARP. (A) Jeko-1 or Raji cells were pretreated with Z-VAD-FMK (10 μM) for 1 h and incubated with 60 μg/ml EGCG for 24 h. Equal amounts of total protein were examined by western blot analysis using the indicated antibodies. β-actin was used as a loading control. (B) Relative intensity of activated caspase-3, -7 and PARP. The values represent mean ± SD from three times independent experiments. *p<0.05, compared with the EGCG-treatment group.
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f4-ijo-46-04-1507: Z-VAD-FMK reverses the EGCG-induced activation of caspase-3, -7 and PARP. (A) Jeko-1 or Raji cells were pretreated with Z-VAD-FMK (10 μM) for 1 h and incubated with 60 μg/ml EGCG for 24 h. Equal amounts of total protein were examined by western blot analysis using the indicated antibodies. β-actin was used as a loading control. (B) Relative intensity of activated caspase-3, -7 and PARP. The values represent mean ± SD from three times independent experiments. *p<0.05, compared with the EGCG-treatment group.

Mentions: Previous studies have proposed that potential novel agents should mainly inhibit growth and induce apoptosis in different lymphoma cells. Activation of caspases is a central process of the 2 major apoptosis pathways, and activated caspases provide a link between cell signaling and apoptotic execution (24). It has been reported that green tea polyphenols induce cell death by caspase-3 activation (33). We investigated the molecular mechanisms of EGCG induced Jeko-1 and Raji cells growth inhibition, and found a dose-dependent increase of caspase-3 and -7 activation (Fig. 3). PARP is associated with DNA damage in apoptosis and is the major downstream substrate of caspase-3 (34). PARP activation increased overtly with EGCG concentration as shown in Fig. 3. Furthermore, the activation of caspase-3, -7 and PARP was inhibited by the general caspase inhibitor Z-VAD-FMK (Fig. 4). These results indicated that EGCG-induced apoptosis is caspase-dependent in Jeko-1 and Raji cells. This finding was consistent with a previous study demonstrating that EGCG suppresses VEGF-R phosphorylation and induces apoptosis by increasing the activity of caspase-3 and PARP in chronic lymphocytic leukemia B cells (35).


(-)-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)

Z-VAD-FMK reverses the EGCG-induced activation of caspase-3, -7 and PARP. (A) Jeko-1 or Raji cells were pretreated with Z-VAD-FMK (10 μM) for 1 h and incubated with 60 μg/ml EGCG for 24 h. Equal amounts of total protein were examined by western blot analysis using the indicated antibodies. β-actin was used as a loading control. (B) Relative intensity of activated caspase-3, -7 and PARP. The values represent mean ± SD from three times 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

f4-ijo-46-04-1507: Z-VAD-FMK reverses the EGCG-induced activation of caspase-3, -7 and PARP. (A) Jeko-1 or Raji cells were pretreated with Z-VAD-FMK (10 μM) for 1 h and incubated with 60 μg/ml EGCG for 24 h. Equal amounts of total protein were examined by western blot analysis using the indicated antibodies. β-actin was used as a loading control. (B) Relative intensity of activated caspase-3, -7 and PARP. The values represent mean ± SD from three times independent experiments. *p<0.05, compared with the EGCG-treatment group.
Mentions: Previous studies have proposed that potential novel agents should mainly inhibit growth and induce apoptosis in different lymphoma cells. Activation of caspases is a central process of the 2 major apoptosis pathways, and activated caspases provide a link between cell signaling and apoptotic execution (24). It has been reported that green tea polyphenols induce cell death by caspase-3 activation (33). We investigated the molecular mechanisms of EGCG induced Jeko-1 and Raji cells growth inhibition, and found a dose-dependent increase of caspase-3 and -7 activation (Fig. 3). PARP is associated with DNA damage in apoptosis and is the major downstream substrate of caspase-3 (34). PARP activation increased overtly with EGCG concentration as shown in Fig. 3. Furthermore, the activation of caspase-3, -7 and PARP was inhibited by the general caspase inhibitor Z-VAD-FMK (Fig. 4). These results indicated that EGCG-induced apoptosis is caspase-dependent in Jeko-1 and Raji cells. This finding was consistent with a previous study demonstrating that EGCG suppresses VEGF-R phosphorylation and induces apoptosis by increasing the activity of caspase-3 and PARP in chronic lymphocytic leukemia B cells (35).

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