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Suppression of E-cadherin mediates gallotannin induced apoptosis in Hep G2 hepatocelluar carcinoma cells.

Han HJ, Kwon HY, Sohn EJ, Ko H, Kim B, Jung K, Lew JH, Kim SH - Int. J. Biol. Sci. (2014)

Bottom Line: Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin.Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells.Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

View Article: PubMed Central - PubMed

Affiliation: 2. Graduate School of East-West Medical Science, Kyung Hee University, Yongin 449-701, Republic of Korea.

ABSTRACT
Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

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Gallotannin increased sub-G1 population and regulated apoptotic genes in Hep G2 and Chang cells. (A) Effect of gallotannin on sub G1 population by FACS analysis with propidium iodide (PI) staining. Hep G2 and Chang cells were treated with various concentrations of gallotannin (0, 4, 8 μM) for 48 h and stained with PI using Flow cytometry. (B) Bar graphs for sub-G1 population in Hep G2 and Chang cells. ***, p < 0.001, vs untreated control. (C) Effect of gallotannin on apoptotic proteins in Hep G2 and Chang cells. Cell lysates from gallatonnin treated Hep G2 and Chang cells were subjected for western blotting for PARP, pro-caspase 9 and 3, Bcl2 and β-Actin. (D) Apoptotic cells were detected by TUNEL assay. Cells in the absence or presence of gallotannin were stained with TUNEL-FITC (Green) and PI (Red). Data represent means ± S.D.
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Figure 2: Gallotannin increased sub-G1 population and regulated apoptotic genes in Hep G2 and Chang cells. (A) Effect of gallotannin on sub G1 population by FACS analysis with propidium iodide (PI) staining. Hep G2 and Chang cells were treated with various concentrations of gallotannin (0, 4, 8 μM) for 48 h and stained with PI using Flow cytometry. (B) Bar graphs for sub-G1 population in Hep G2 and Chang cells. ***, p < 0.001, vs untreated control. (C) Effect of gallotannin on apoptotic proteins in Hep G2 and Chang cells. Cell lysates from gallatonnin treated Hep G2 and Chang cells were subjected for western blotting for PARP, pro-caspase 9 and 3, Bcl2 and β-Actin. (D) Apoptotic cells were detected by TUNEL assay. Cells in the absence or presence of gallotannin were stained with TUNEL-FITC (Green) and PI (Red). Data represent means ± S.D.

Mentions: In order to confirm whether the cytotoxicity of gallotannin in Hep G2 or Chang cells was due to apoptosis induction, cell cycle analysis using flow cytometry with PI staining was performed. As shown in Figure 2 A and B, gallotannin significantly increased the accumulation of sub-G1 apoptotic portion in a dose-dependent manner in Hep G2 and Chang cells compared to untreated control. Consistently, Western blotting revealed that gallotannin cleaved PARP and attenuated the expression of pro-caspase 9/3, Bcl2 and integrin β1 in a dose dependent manner in Hep G2 or Chang cells (Figure 2C). Also, we confirmed that gallotannin induced apoptosis in Hep G2 or Chang cells by TUNEL assay (Figure 2D).


Suppression of E-cadherin mediates gallotannin induced apoptosis in Hep G2 hepatocelluar carcinoma cells.

Han HJ, Kwon HY, Sohn EJ, Ko H, Kim B, Jung K, Lew JH, Kim SH - Int. J. Biol. Sci. (2014)

Gallotannin increased sub-G1 population and regulated apoptotic genes in Hep G2 and Chang cells. (A) Effect of gallotannin on sub G1 population by FACS analysis with propidium iodide (PI) staining. Hep G2 and Chang cells were treated with various concentrations of gallotannin (0, 4, 8 μM) for 48 h and stained with PI using Flow cytometry. (B) Bar graphs for sub-G1 population in Hep G2 and Chang cells. ***, p < 0.001, vs untreated control. (C) Effect of gallotannin on apoptotic proteins in Hep G2 and Chang cells. Cell lysates from gallatonnin treated Hep G2 and Chang cells were subjected for western blotting for PARP, pro-caspase 9 and 3, Bcl2 and β-Actin. (D) Apoptotic cells were detected by TUNEL assay. Cells in the absence or presence of gallotannin were stained with TUNEL-FITC (Green) and PI (Red). Data represent means ± S.D.
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Related In: Results  -  Collection

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Figure 2: Gallotannin increased sub-G1 population and regulated apoptotic genes in Hep G2 and Chang cells. (A) Effect of gallotannin on sub G1 population by FACS analysis with propidium iodide (PI) staining. Hep G2 and Chang cells were treated with various concentrations of gallotannin (0, 4, 8 μM) for 48 h and stained with PI using Flow cytometry. (B) Bar graphs for sub-G1 population in Hep G2 and Chang cells. ***, p < 0.001, vs untreated control. (C) Effect of gallotannin on apoptotic proteins in Hep G2 and Chang cells. Cell lysates from gallatonnin treated Hep G2 and Chang cells were subjected for western blotting for PARP, pro-caspase 9 and 3, Bcl2 and β-Actin. (D) Apoptotic cells were detected by TUNEL assay. Cells in the absence or presence of gallotannin were stained with TUNEL-FITC (Green) and PI (Red). Data represent means ± S.D.
Mentions: In order to confirm whether the cytotoxicity of gallotannin in Hep G2 or Chang cells was due to apoptosis induction, cell cycle analysis using flow cytometry with PI staining was performed. As shown in Figure 2 A and B, gallotannin significantly increased the accumulation of sub-G1 apoptotic portion in a dose-dependent manner in Hep G2 and Chang cells compared to untreated control. Consistently, Western blotting revealed that gallotannin cleaved PARP and attenuated the expression of pro-caspase 9/3, Bcl2 and integrin β1 in a dose dependent manner in Hep G2 or Chang cells (Figure 2C). Also, we confirmed that gallotannin induced apoptosis in Hep G2 or Chang cells by TUNEL assay (Figure 2D).

Bottom Line: Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin.Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells.Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

View Article: PubMed Central - PubMed

Affiliation: 2. Graduate School of East-West Medical Science, Kyung Hee University, Yongin 449-701, Republic of Korea.

ABSTRACT
Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

Show MeSH
Related in: MedlinePlus