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Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

Lan YH, Chiang JH, Huang WW, Lu CC, Chung JG, Wu TS, Jhan JH, Lin KL, Pai SJ, Chiu YJ, Tsuzuki M, Yang JS - Evid Based Complement Alternat Med (2012)

Bottom Line: Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis.ESE promoted the mitochondria-dependent and death-receptor-associated protein levels.Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, China Medical University, Taichung 404, Taiwan.

ABSTRACT
Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

ESE altered the protein abundance-associated with mitochondria- and death-receptor-dependent apoptotic signaling in HCT 116 cells. Cells were treated with ESE (50 μg/mL) for 0, 6, 12, 24 h, and total protein, cytosolic, and mitochondrial lysates were prepared and subjected to Western blotting analysis. The membranes were incubated with (a) anti-Bcl-2, anti-Bax, anti-Bid and anti-PUMA antibodies; (b) anti-Fas, anti-FasL, anti-DR4 and anti-DR5 antibodies; (c) anticytochrome c antibody. The blot was also probed with anti-Actin and anti-Complex V antibodies to confirm equal loading of samples. Each band was quantified using ImageJ software.
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fig4: ESE altered the protein abundance-associated with mitochondria- and death-receptor-dependent apoptotic signaling in HCT 116 cells. Cells were treated with ESE (50 μg/mL) for 0, 6, 12, 24 h, and total protein, cytosolic, and mitochondrial lysates were prepared and subjected to Western blotting analysis. The membranes were incubated with (a) anti-Bcl-2, anti-Bax, anti-Bid and anti-PUMA antibodies; (b) anti-Fas, anti-FasL, anti-DR4 and anti-DR5 antibodies; (c) anticytochrome c antibody. The blot was also probed with anti-Actin and anti-Complex V antibodies to confirm equal loading of samples. Each band was quantified using ImageJ software.

Mentions: To assess the alterations in apoptosis-related protein levels in ESE-treated HCT 116 cells, we administered ESE at the concentration of 50 μg/mL for 0, 6, 12, and 24 h in HCT 116 cells and then evaluated the protein levels by Western blot analysis. Figure 4(a) shows that ESE promoted a decrease of Bcl-2 level (an antiapoptotic protein) and the increases of proapoptotic protein levels of Bax and PUMA in HCT 116 cells. Also, treatment of ESE showed that the level of Bid was downregulated in HCT 116 cells (Figure 4(a)). As shown in Figure 4(b), ESE enhanced the death receptor pathway-associated protein levels (Fas, DR4 and DR5) in HCT 116 cells. Alternatively, the level of cytochrome c from cytosolic fraction is upregulated and from mitochondrial fraction is downregulated in ESE-treated HCT 116 cells (Figure 4(c)). Collectively, these results suggest that both intrinsic (mitochondria) and extrinsic (death-receptor-) dependent pathways contributed to ESE-provoked apoptotic death in HCT 116 cells.


Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

Lan YH, Chiang JH, Huang WW, Lu CC, Chung JG, Wu TS, Jhan JH, Lin KL, Pai SJ, Chiu YJ, Tsuzuki M, Yang JS - Evid Based Complement Alternat Med (2012)

ESE altered the protein abundance-associated with mitochondria- and death-receptor-dependent apoptotic signaling in HCT 116 cells. Cells were treated with ESE (50 μg/mL) for 0, 6, 12, 24 h, and total protein, cytosolic, and mitochondrial lysates were prepared and subjected to Western blotting analysis. The membranes were incubated with (a) anti-Bcl-2, anti-Bax, anti-Bid and anti-PUMA antibodies; (b) anti-Fas, anti-FasL, anti-DR4 and anti-DR5 antibodies; (c) anticytochrome c antibody. The blot was also probed with anti-Actin and anti-Complex V antibodies to confirm equal loading of samples. Each band was quantified using ImageJ software.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303801&req=5

fig4: ESE altered the protein abundance-associated with mitochondria- and death-receptor-dependent apoptotic signaling in HCT 116 cells. Cells were treated with ESE (50 μg/mL) for 0, 6, 12, 24 h, and total protein, cytosolic, and mitochondrial lysates were prepared and subjected to Western blotting analysis. The membranes were incubated with (a) anti-Bcl-2, anti-Bax, anti-Bid and anti-PUMA antibodies; (b) anti-Fas, anti-FasL, anti-DR4 and anti-DR5 antibodies; (c) anticytochrome c antibody. The blot was also probed with anti-Actin and anti-Complex V antibodies to confirm equal loading of samples. Each band was quantified using ImageJ software.
Mentions: To assess the alterations in apoptosis-related protein levels in ESE-treated HCT 116 cells, we administered ESE at the concentration of 50 μg/mL for 0, 6, 12, and 24 h in HCT 116 cells and then evaluated the protein levels by Western blot analysis. Figure 4(a) shows that ESE promoted a decrease of Bcl-2 level (an antiapoptotic protein) and the increases of proapoptotic protein levels of Bax and PUMA in HCT 116 cells. Also, treatment of ESE showed that the level of Bid was downregulated in HCT 116 cells (Figure 4(a)). As shown in Figure 4(b), ESE enhanced the death receptor pathway-associated protein levels (Fas, DR4 and DR5) in HCT 116 cells. Alternatively, the level of cytochrome c from cytosolic fraction is upregulated and from mitochondrial fraction is downregulated in ESE-treated HCT 116 cells (Figure 4(c)). Collectively, these results suggest that both intrinsic (mitochondria) and extrinsic (death-receptor-) dependent pathways contributed to ESE-provoked apoptotic death in HCT 116 cells.

Bottom Line: Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis.ESE promoted the mitochondria-dependent and death-receptor-associated protein levels.Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, China Medical University, Taichung 404, Taiwan.

ABSTRACT
Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus