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Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G.

Kim WH, Song HO, Choi HJ, Bang HI, Choi DY, Park H - Int J Mol Sci (2012)

Bottom Line: Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax).EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid).Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, 344-2, Shinyong-dong, Iksan, Chonbuk 570-749, South Korea; E-Mails: woong621@gmail.com (W.-H.K.); sea5328@daum.net (H.-O.S.); rerived@empal.com (H.-J.C.).

ABSTRACT
Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

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Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 105 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. (A) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; (B) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; (C) Cytochrome c. These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
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f3-ijms-13-11912: Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 105 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. (A) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; (B) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; (C) Cytochrome c. These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.

Mentions: To investigate the molecular mechanism by which EG induced apoptosis in HL-60 cells, we examined the expression of several apoptosis-associated proteins by western blotting. We found that EG treatment of HL-60 cells decreased the expression of Bcl-2 at 75 μM EG, and increased Bax and truncated Bid (tBid) expression at 24 h (Figure 3A). Also, caspase-9 expression increased after treatment of 75 μM EG for 24 h (Figure 3B). Caspase-8 expression did not increase until 6 h after treatment of 50 μM or 75 μM EG and increased at 12 h after treatment of 50 μM or 75 μM EG (Figure 3B). Caspase-3 expression increased at 12 h and 24 h after treatment of 50 μM or 75 μM EG (Figure 3B). AIF, Endo G and cytochrome c expression increased at 24 h after treatment of 75 μM EG (Figure 3B,C). These results suggest that EG induced apoptotic death in HL-60 cells via activation of the caspase enzyme cascade and through mitochondrial pathways.


Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G.

Kim WH, Song HO, Choi HJ, Bang HI, Choi DY, Park H - Int J Mol Sci (2012)

Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 105 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. (A) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; (B) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; (C) Cytochrome c. These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472783&req=5

f3-ijms-13-11912: Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 105 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. (A) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; (B) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; (C) Cytochrome c. These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
Mentions: To investigate the molecular mechanism by which EG induced apoptosis in HL-60 cells, we examined the expression of several apoptosis-associated proteins by western blotting. We found that EG treatment of HL-60 cells decreased the expression of Bcl-2 at 75 μM EG, and increased Bax and truncated Bid (tBid) expression at 24 h (Figure 3A). Also, caspase-9 expression increased after treatment of 75 μM EG for 24 h (Figure 3B). Caspase-8 expression did not increase until 6 h after treatment of 50 μM or 75 μM EG and increased at 12 h after treatment of 50 μM or 75 μM EG (Figure 3B). Caspase-3 expression increased at 12 h and 24 h after treatment of 50 μM or 75 μM EG (Figure 3B). AIF, Endo G and cytochrome c expression increased at 24 h after treatment of 75 μM EG (Figure 3B,C). These results suggest that EG induced apoptotic death in HL-60 cells via activation of the caspase enzyme cascade and through mitochondrial pathways.

Bottom Line: Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax).EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid).Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, 344-2, Shinyong-dong, Iksan, Chonbuk 570-749, South Korea; E-Mails: woong621@gmail.com (W.-H.K.); sea5328@daum.net (H.-O.S.); rerived@empal.com (H.-J.C.).

ABSTRACT
Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

Show MeSH
Related in: MedlinePlus