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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 cell cycle and induction of apoptosis in HL-60 cells. Cells were incubated with 50 μM or 75 μM EG for 24 h or 48 h. (A) Cells were examined for DNA content by staining with Propidium iodide (PI) and analysis by flow cytometry. M1: Sub G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase; (B) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy; (C) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by ethidium bromide staining. M: DNA molecular weight marker, Con: control, Adria: adriamycin. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
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f2-ijms-13-11912: Effect of EG on the cell cycle and induction of apoptosis in HL-60 cells. Cells were incubated with 50 μM or 75 μM EG for 24 h or 48 h. (A) Cells were examined for DNA content by staining with Propidium iodide (PI) and analysis by flow cytometry. M1: Sub G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase; (B) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy; (C) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by ethidium bromide staining. M: DNA molecular weight marker, Con: control, Adria: adriamycin. These results presented are representative of data obtained from three independent experiments carried out in triplicate.

Mentions: To determine whether the cytotoxicity of EG for HL-60 cells was a result of apoptosis, we analyzed the DNA content and cell cycle distribution of EG-treated cells by flow cytometric analysis of PI-stained cells. EG treatment increased the proportion of cells in subG1 phase, indicative of a reduction in DNA content, in a concentration- and time-dependent manner. Treatment of cells for 24 h or 48 h with 50 μM or 75 μM EG increased the percentage of cells in the subG1 phase from a baseline of 2.9% to 26.5% or 52.6%, respectively (Figure 2A). Similarly, HL-60 cells treated with 50 μM, 75 μM, or 100 μM EG for 48 h showed a dose-dependent increase in apoptosis, as measured by the DAPI assay (Figure 2B). Finally, DNA fragmentation in the EG-treated cells was confirmed by agarose gel electrophoresis, which showed the presence of DNA laddering, a marker of apoptosis, in EG-treated HL-60 cells (Figure 2C).


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 cell cycle and induction of apoptosis in HL-60 cells. Cells were incubated with 50 μM or 75 μM EG for 24 h or 48 h. (A) Cells were examined for DNA content by staining with Propidium iodide (PI) and analysis by flow cytometry. M1: Sub G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase; (B) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy; (C) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by ethidium bromide staining. M: DNA molecular weight marker, Con: control, Adria: adriamycin. 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

f2-ijms-13-11912: Effect of EG on the cell cycle and induction of apoptosis in HL-60 cells. Cells were incubated with 50 μM or 75 μM EG for 24 h or 48 h. (A) Cells were examined for DNA content by staining with Propidium iodide (PI) and analysis by flow cytometry. M1: Sub G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase; (B) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy; (C) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by ethidium bromide staining. M: DNA molecular weight marker, Con: control, Adria: adriamycin. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
Mentions: To determine whether the cytotoxicity of EG for HL-60 cells was a result of apoptosis, we analyzed the DNA content and cell cycle distribution of EG-treated cells by flow cytometric analysis of PI-stained cells. EG treatment increased the proportion of cells in subG1 phase, indicative of a reduction in DNA content, in a concentration- and time-dependent manner. Treatment of cells for 24 h or 48 h with 50 μM or 75 μM EG increased the percentage of cells in the subG1 phase from a baseline of 2.9% to 26.5% or 52.6%, respectively (Figure 2A). Similarly, HL-60 cells treated with 50 μM, 75 μM, or 100 μM EG for 48 h showed a dose-dependent increase in apoptosis, as measured by the DAPI assay (Figure 2B). Finally, DNA fragmentation in the EG-treated cells was confirmed by agarose gel electrophoresis, which showed the presence of DNA laddering, a marker of apoptosis, in EG-treated HL-60 cells (Figure 2C).

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