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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-affected cytotoxicity and apoptosis is mediated through alterations of p53 downstream signals in p53 siRNA-transfected HCT 116 cells. (a) The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 12 h, and total protein was prepared and subjected to Western blotting analysis. The membranes were incubated with anti-p53, anti-Fas, anti-PUMA, anticaspase-8 and, anti-caspase-3 antibodies. The blot was also probed with anti-Actin antibody to confirm equal loading of samples. Each band was quantified using ImageJ software. The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 24 h, cell viability was determined by MTT assay (b) and apoptotic cells were assessed by TUNEL assayand flow cytometric analysis (c). The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to control sample.
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fig6: ESE-affected cytotoxicity and apoptosis is mediated through alterations of p53 downstream signals in p53 siRNA-transfected HCT 116 cells. (a) The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 12 h, and total protein was prepared and subjected to Western blotting analysis. The membranes were incubated with anti-p53, anti-Fas, anti-PUMA, anticaspase-8 and, anti-caspase-3 antibodies. The blot was also probed with anti-Actin antibody to confirm equal loading of samples. Each band was quantified using ImageJ software. The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 24 h, cell viability was determined by MTT assay (b) and apoptotic cells were assessed by TUNEL assayand flow cytometric analysis (c). The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to control sample.

Mentions: Therefore, our results showed that the induction of p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 due to ESE treatment was correlated with the decrease in p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 protein levels by the transfection with p53 siRNA in HCT 116 cells (Figure 6(a)). We found that ESE-reduced viability in HCT 116 cells was nearly enhanced after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(b). We also found that ESE induced apoptosis in HCT 116 cells was nearly prevented after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(c). Taken together, these results suggest that p53 activation is an important factor in ESE-induced apoptosis of HCT 116 cells, which is mediated through ROS productions (oxidative stress) and ATM/p53/Fas-dependent signaling pathways.


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-affected cytotoxicity and apoptosis is mediated through alterations of p53 downstream signals in p53 siRNA-transfected HCT 116 cells. (a) The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 12 h, and total protein was prepared and subjected to Western blotting analysis. The membranes were incubated with anti-p53, anti-Fas, anti-PUMA, anticaspase-8 and, anti-caspase-3 antibodies. The blot was also probed with anti-Actin antibody to confirm equal loading of samples. Each band was quantified using ImageJ software. The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 24 h, cell viability was determined by MTT assay (b) and apoptotic cells were assessed by TUNEL assayand flow cytometric analysis (c). The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to control sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: ESE-affected cytotoxicity and apoptosis is mediated through alterations of p53 downstream signals in p53 siRNA-transfected HCT 116 cells. (a) The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 12 h, and total protein was prepared and subjected to Western blotting analysis. The membranes were incubated with anti-p53, anti-Fas, anti-PUMA, anticaspase-8 and, anti-caspase-3 antibodies. The blot was also probed with anti-Actin antibody to confirm equal loading of samples. Each band was quantified using ImageJ software. The p53 siRNA or control siRNA-transfected HCT 116 cells were treated with ESE (50 μg/mL) for 24 h, cell viability was determined by MTT assay (b) and apoptotic cells were assessed by TUNEL assayand flow cytometric analysis (c). The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to control sample.
Mentions: Therefore, our results showed that the induction of p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 due to ESE treatment was correlated with the decrease in p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 protein levels by the transfection with p53 siRNA in HCT 116 cells (Figure 6(a)). We found that ESE-reduced viability in HCT 116 cells was nearly enhanced after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(b). We also found that ESE induced apoptosis in HCT 116 cells was nearly prevented after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(c). Taken together, these results suggest that p53 activation is an important factor in ESE-induced apoptosis of HCT 116 cells, which is mediated through ROS productions (oxidative stress) and ATM/p53/Fas-dependent signaling pathways.

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