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A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells.

Suk FM, Lien GS, Huang WJ, Chen CN, Lu SY, Yang YC, Yan MD, Liang YC - Evid Based Complement Alternat Med (2013)

Bottom Line: First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners.Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells.These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 116, Taiwan.

ABSTRACT
Activating transcription factor-(ATF-) 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002) is a Taiwanese propolin G (PPG) derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). GS-002 also induced endoplasmic reticular (ER) stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), phosphorylated eukaryotic initiation factor 2 α (eIF2 α ), phosphorylated protein endoplasmic-reticular-resident kinase (PERK), and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK) signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

No MeSH data available.


Related in: MedlinePlus

The propolis derivative, GS-002, induced cell apoptosis in human hepatoma cells. Hep3B cells were treated with various concentrations of GS-002 for 24 h, and (a) the subG1 population was determined by a flow cytometric analysis; (b) apoptotic cells were determined by a flow cytometric analysis with annexin-V/PI staining; and (c) the DNA fraction was extracted and chromatographed by agarose gel electrophoresis. (d) Hep3B cells were treated with 20 μg/mL of GS-002 for the indicated time periods. Total cell lysates were used to detect the protein expression of full-length PPAR (FU-PPAR), cleaved form of PPAR (CF-PPAR), cleaved form of caspase-3 (CF-caspase-3), cleaved form of caspase-9 (CF-caspase-9), Bad, and α-tubulin by Western blotting.
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fig3: The propolis derivative, GS-002, induced cell apoptosis in human hepatoma cells. Hep3B cells were treated with various concentrations of GS-002 for 24 h, and (a) the subG1 population was determined by a flow cytometric analysis; (b) apoptotic cells were determined by a flow cytometric analysis with annexin-V/PI staining; and (c) the DNA fraction was extracted and chromatographed by agarose gel electrophoresis. (d) Hep3B cells were treated with 20 μg/mL of GS-002 for the indicated time periods. Total cell lysates were used to detect the protein expression of full-length PPAR (FU-PPAR), cleaved form of PPAR (CF-PPAR), cleaved form of caspase-3 (CF-caspase-3), cleaved form of caspase-9 (CF-caspase-9), Bad, and α-tubulin by Western blotting.

Mentions: Cell-cycle progression was analyzed by flow cytometry with PI staining. After treatment with GS-002 for 24 h, there was no significant cell-cycle change in the G1, S, or G2/M phases compared to control cells (Figure 3(a)). However, a marked increase in the subG1 apoptotic population was seen in cells treated with 20 μg/mL of GS-002. subG1 populations were 7.78% and 62.47% of control cells and cells treated with 20 μg/mL of GS-002, respectively. Cell death was also characterized using flow cytometry with PI and Annexin V-Alexa Fluor 488 staining of Hep3B cells. The lower right quadrant of the FACS histogram represents early apoptotic cells, which were stained with the green fluorescent Alexa488 dye, and the upper right quadrant of the FACS histogram represents late apoptotic cells, which were stained with both the red-green fluorescence PI and Alexa488 dyes. As shown in Figure 3(b), the late apoptotic cell population increased from 4.90% to 66.49% in cells treated with 20 μg/mL GS-002. We next questioned whether GS-002 induced apoptosis in Hep3B cells. After treatment of Hep3B cells with various concentrations of GS-002 for 24 h, genomic DNA from cells was subjected to agarose gel electrophoresis. DNA fragmentation ladders significantly increased as shown in Figure 3(c). We next determined the cleavage of PARP and activation of caspases in GS-002-treated cells. After treatment with GS-002 for 24 h, the cleavage of PARP and cleaved (i.e., activated) forms of caspases-3 and -9 and Bad were found in GS-002-treated cells in a dose-dependent manner (Figure 3(d)). These results suggest that GS-002 inhibited cell proliferation through activating an apoptotic pathway in human hepatoma cells.


A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells.

Suk FM, Lien GS, Huang WJ, Chen CN, Lu SY, Yang YC, Yan MD, Liang YC - Evid Based Complement Alternat Med (2013)

The propolis derivative, GS-002, induced cell apoptosis in human hepatoma cells. Hep3B cells were treated with various concentrations of GS-002 for 24 h, and (a) the subG1 population was determined by a flow cytometric analysis; (b) apoptotic cells were determined by a flow cytometric analysis with annexin-V/PI staining; and (c) the DNA fraction was extracted and chromatographed by agarose gel electrophoresis. (d) Hep3B cells were treated with 20 μg/mL of GS-002 for the indicated time periods. Total cell lysates were used to detect the protein expression of full-length PPAR (FU-PPAR), cleaved form of PPAR (CF-PPAR), cleaved form of caspase-3 (CF-caspase-3), cleaved form of caspase-9 (CF-caspase-9), Bad, and α-tubulin by Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814109&req=5

fig3: The propolis derivative, GS-002, induced cell apoptosis in human hepatoma cells. Hep3B cells were treated with various concentrations of GS-002 for 24 h, and (a) the subG1 population was determined by a flow cytometric analysis; (b) apoptotic cells were determined by a flow cytometric analysis with annexin-V/PI staining; and (c) the DNA fraction was extracted and chromatographed by agarose gel electrophoresis. (d) Hep3B cells were treated with 20 μg/mL of GS-002 for the indicated time periods. Total cell lysates were used to detect the protein expression of full-length PPAR (FU-PPAR), cleaved form of PPAR (CF-PPAR), cleaved form of caspase-3 (CF-caspase-3), cleaved form of caspase-9 (CF-caspase-9), Bad, and α-tubulin by Western blotting.
Mentions: Cell-cycle progression was analyzed by flow cytometry with PI staining. After treatment with GS-002 for 24 h, there was no significant cell-cycle change in the G1, S, or G2/M phases compared to control cells (Figure 3(a)). However, a marked increase in the subG1 apoptotic population was seen in cells treated with 20 μg/mL of GS-002. subG1 populations were 7.78% and 62.47% of control cells and cells treated with 20 μg/mL of GS-002, respectively. Cell death was also characterized using flow cytometry with PI and Annexin V-Alexa Fluor 488 staining of Hep3B cells. The lower right quadrant of the FACS histogram represents early apoptotic cells, which were stained with the green fluorescent Alexa488 dye, and the upper right quadrant of the FACS histogram represents late apoptotic cells, which were stained with both the red-green fluorescence PI and Alexa488 dyes. As shown in Figure 3(b), the late apoptotic cell population increased from 4.90% to 66.49% in cells treated with 20 μg/mL GS-002. We next questioned whether GS-002 induced apoptosis in Hep3B cells. After treatment of Hep3B cells with various concentrations of GS-002 for 24 h, genomic DNA from cells was subjected to agarose gel electrophoresis. DNA fragmentation ladders significantly increased as shown in Figure 3(c). We next determined the cleavage of PARP and activation of caspases in GS-002-treated cells. After treatment with GS-002 for 24 h, the cleavage of PARP and cleaved (i.e., activated) forms of caspases-3 and -9 and Bad were found in GS-002-treated cells in a dose-dependent manner (Figure 3(d)). These results suggest that GS-002 inhibited cell proliferation through activating an apoptotic pathway in human hepatoma cells.

Bottom Line: First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners.Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells.These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 116, Taiwan.

ABSTRACT
Activating transcription factor-(ATF-) 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002) is a Taiwanese propolin G (PPG) derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). GS-002 also induced endoplasmic reticular (ER) stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), phosphorylated eukaryotic initiation factor 2 α (eIF2 α ), phosphorylated protein endoplasmic-reticular-resident kinase (PERK), and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK) signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

No MeSH data available.


Related in: MedlinePlus