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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 increased ROS production and contributed to p53-correlated ATM/Fas apoptotic signaling in HCT 116 cells. (a) Treatment with ESE (50 μg/mL) for the indicated times (0, 2, and 4 h) was subjected to ROS productions by flow cytometry as described in Section 2. (b) Pretreatment with NAC (10 mM, a scavenger of ROS), or caffeine (1 mM, an inhibitor of ATM) in ESE-treated HCT 116 cells restored the cell viability by MTT assay. The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to ESE treatment. (c) ESE elevated the protein levels of ATM, phosphorylated ATM (Ser1981), p53, phosphorylation, and p53 (Ser15) by Western blotting. (d) Effects of ESE on Fas mRNA level in HCT 116 cells, and the total RNA was extracted from each treatment of ESE (50 μg/mL) on HCT 116 cells for 0, 6, and 12 h. RNA samples were reverse transcribed into cDNA and quantified with real-time PCR as described in Section 2. The ratios of Fas mRNA/GAPDH are presented. 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 (0 h) sample.
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fig5: ESE increased ROS production and contributed to p53-correlated ATM/Fas apoptotic signaling in HCT 116 cells. (a) Treatment with ESE (50 μg/mL) for the indicated times (0, 2, and 4 h) was subjected to ROS productions by flow cytometry as described in Section 2. (b) Pretreatment with NAC (10 mM, a scavenger of ROS), or caffeine (1 mM, an inhibitor of ATM) in ESE-treated HCT 116 cells restored the cell viability by MTT assay. The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to ESE treatment. (c) ESE elevated the protein levels of ATM, phosphorylated ATM (Ser1981), p53, phosphorylation, and p53 (Ser15) by Western blotting. (d) Effects of ESE on Fas mRNA level in HCT 116 cells, and the total RNA was extracted from each treatment of ESE (50 μg/mL) on HCT 116 cells for 0, 6, and 12 h. RNA samples were reverse transcribed into cDNA and quantified with real-time PCR as described in Section 2. The ratios of Fas mRNA/GAPDH are presented. 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 (0 h) sample.

Mentions: Results shown in Figure 5(a) revealed that ESE promoted the level of intracellular ROS production (2 h treatment: 81.26 ± 2.39%; 4 h treatment: 90.34 ± 4.18%) in HCT 116 cells by flow cytometry and a specific fluorescent probe, H2DCFDA for determining ROS level. Cells pretreated with N-acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) significantly reduced ESE-induced growth inhibition effect (Figure 5(b)). Previous studies have stated that p53 gene and its phosphorylation at the Ser15 interacted Fas/CD95 activation when cell apoptosis occur [7, 8, 30]. To elucidate the crucial roles of ATM, p53 and Fas in HCT 116 cells after treatment with ESE, the protein levels of ATM, p-ATMSer1981, p53, and p-p53Ser15 expression were investigated by Western blot analysis. Our results showed that ESE increased the protein levels of ATM, p-ATMSer1981, p53 and p-p53Ser15 in HCT 116 cells as can be seen in Figure 5(b). Our further study investigated if p53 affects the Fas expression in ESE-treated HCT 116 cells. We hypothesized that ESE induces apoptosis through the increase of Fas/CD95 by p53-dependent transcriptional activation. real-time PCR analysis was performed to determine whether the induction of Fas/CD95 protein level by ESE was due to increased the level of mRNA. As shown in Figure 5(d), the 6 and 12 h treatment of HCT 116 cells with ESE (50 μg/mL) led to an increase in mRNA levels of Fas/CD95. Our results indicate that ESE increased the protein level of Fas/CD95 through the p53-dependent regulation of transcription levels.


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 increased ROS production and contributed to p53-correlated ATM/Fas apoptotic signaling in HCT 116 cells. (a) Treatment with ESE (50 μg/mL) for the indicated times (0, 2, and 4 h) was subjected to ROS productions by flow cytometry as described in Section 2. (b) Pretreatment with NAC (10 mM, a scavenger of ROS), or caffeine (1 mM, an inhibitor of ATM) in ESE-treated HCT 116 cells restored the cell viability by MTT assay. The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to ESE treatment. (c) ESE elevated the protein levels of ATM, phosphorylated ATM (Ser1981), p53, phosphorylation, and p53 (Ser15) by Western blotting. (d) Effects of ESE on Fas mRNA level in HCT 116 cells, and the total RNA was extracted from each treatment of ESE (50 μg/mL) on HCT 116 cells for 0, 6, and 12 h. RNA samples were reverse transcribed into cDNA and quantified with real-time PCR as described in Section 2. The ratios of Fas mRNA/GAPDH are presented. 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 (0 h) sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: ESE increased ROS production and contributed to p53-correlated ATM/Fas apoptotic signaling in HCT 116 cells. (a) Treatment with ESE (50 μg/mL) for the indicated times (0, 2, and 4 h) was subjected to ROS productions by flow cytometry as described in Section 2. (b) Pretreatment with NAC (10 mM, a scavenger of ROS), or caffeine (1 mM, an inhibitor of ATM) in ESE-treated HCT 116 cells restored the cell viability by MTT assay. The values presented are the mean ± S.D. (n = 3) from three independent experiments. ***P < 0.001 shows a significant different when compared to ESE treatment. (c) ESE elevated the protein levels of ATM, phosphorylated ATM (Ser1981), p53, phosphorylation, and p53 (Ser15) by Western blotting. (d) Effects of ESE on Fas mRNA level in HCT 116 cells, and the total RNA was extracted from each treatment of ESE (50 μg/mL) on HCT 116 cells for 0, 6, and 12 h. RNA samples were reverse transcribed into cDNA and quantified with real-time PCR as described in Section 2. The ratios of Fas mRNA/GAPDH are presented. 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 (0 h) sample.
Mentions: Results shown in Figure 5(a) revealed that ESE promoted the level of intracellular ROS production (2 h treatment: 81.26 ± 2.39%; 4 h treatment: 90.34 ± 4.18%) in HCT 116 cells by flow cytometry and a specific fluorescent probe, H2DCFDA for determining ROS level. Cells pretreated with N-acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) significantly reduced ESE-induced growth inhibition effect (Figure 5(b)). Previous studies have stated that p53 gene and its phosphorylation at the Ser15 interacted Fas/CD95 activation when cell apoptosis occur [7, 8, 30]. To elucidate the crucial roles of ATM, p53 and Fas in HCT 116 cells after treatment with ESE, the protein levels of ATM, p-ATMSer1981, p53, and p-p53Ser15 expression were investigated by Western blot analysis. Our results showed that ESE increased the protein levels of ATM, p-ATMSer1981, p53 and p-p53Ser15 in HCT 116 cells as can be seen in Figure 5(b). Our further study investigated if p53 affects the Fas expression in ESE-treated HCT 116 cells. We hypothesized that ESE induces apoptosis through the increase of Fas/CD95 by p53-dependent transcriptional activation. real-time PCR analysis was performed to determine whether the induction of Fas/CD95 protein level by ESE was due to increased the level of mRNA. As shown in Figure 5(d), the 6 and 12 h treatment of HCT 116 cells with ESE (50 μg/mL) led to an increase in mRNA levels of Fas/CD95. Our results indicate that ESE increased the protein level of Fas/CD95 through the p53-dependent regulation of transcription levels.

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