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A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro.

Cheng F, Yang Y, Zhang L, Cao Y, Yao W, Tang Y, Ding A - Int J Mol Sci (2015)

Bottom Line: RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis.Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis.In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China. cff19870524@163.com.

ABSTRACT
Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

No MeSH data available.


Related in: MedlinePlus

Confocal laser scanning microscopy (CLSM) images of IEC-6 cells incubated with Hoechst 33342/Annexin V-FITC/PI under a magnification of 100. (a) and the fluorescence intensity analysis after IEC cells incubated with Hoechst 33342/Annexin V-FITC/PI (b). Compared with corresponding control group, *p < 0.05, **p < 0.01.
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ijms-16-18956-f006: Confocal laser scanning microscopy (CLSM) images of IEC-6 cells incubated with Hoechst 33342/Annexin V-FITC/PI under a magnification of 100. (a) and the fluorescence intensity analysis after IEC cells incubated with Hoechst 33342/Annexin V-FITC/PI (b). Compared with corresponding control group, *p < 0.05, **p < 0.01.

Mentions: Programmed cell death, short for apoptosis, is related to many physiological growth control mechanisms that regulate cell proliferation and tissue homeostasis. It is a major form of cell death [30,31]. Flow cytometry was used to quantitatively analyze the apoptotic effect of kansenone in vitro via Annexin V-FITC/PI dual staining assay (Figure 5a). Phosphatidylserine (PS) moieties flip out from the inside of the cell membrane when apoptosis is triggered leading to specific binding of Annexin V- FITC to PS of cells in the early stage of apoptosis [32]. PI stains the necrotic cells and late apoptotic cells. As shown in Figure 5b, both the percentages of early apoptotic cells and late apoptotic cells increased with increases in kansenone concentration (4, 8 and 16 μg·mL−1) for IEC-6 cells. The percentages of total apoptotic cells treated with kansenone obviously changed from 23.48% to 40.43% when the concentration increased from 4 to 16 μg·mL−1 after 48 h treatment, whereas the proportion of apoptotic cells was only 10.37% in the control. The percentage of total apoptotic cells demonstrated that kansenone effectively promotes cell apoptosis in a concentration-dependent manner. This conclusion was further confirmed by Hoechst 33342/Annexin V-FITC/PI triple staining analysis under fluorescence microscope. In this assay, another dye Hoechst 33342 was introduced to characterize the cells nucleic. Hoechst 33342 could easily enter into apoptotic cells, but not into cells in good condition. As seen in Figure 6a, the number of cells with blue fluorescence from Hoechst 33342 gradually decreased from low to high concentrations of kansenone, indicating that cell proliferation was remarkably suppressed after kansenone treatment, consistent with the results in Figure 2. As illustrated in Figure 6b, the fluorescence intensity of Hoechst 33342, FITC and PI was significantly enhanced when IEC-6 cells were treated with kansenone at high concentrations of about 16 μg·mL−1, indicating that high concentration kansenone induced significant cell apoptosis, as expected in the Annexin V-FITC/PI dual staining assay.


A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro.

Cheng F, Yang Y, Zhang L, Cao Y, Yao W, Tang Y, Ding A - Int J Mol Sci (2015)

Confocal laser scanning microscopy (CLSM) images of IEC-6 cells incubated with Hoechst 33342/Annexin V-FITC/PI under a magnification of 100. (a) and the fluorescence intensity analysis after IEC cells incubated with Hoechst 33342/Annexin V-FITC/PI (b). Compared with corresponding control group, *p < 0.05, **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581281&req=5

ijms-16-18956-f006: Confocal laser scanning microscopy (CLSM) images of IEC-6 cells incubated with Hoechst 33342/Annexin V-FITC/PI under a magnification of 100. (a) and the fluorescence intensity analysis after IEC cells incubated with Hoechst 33342/Annexin V-FITC/PI (b). Compared with corresponding control group, *p < 0.05, **p < 0.01.
Mentions: Programmed cell death, short for apoptosis, is related to many physiological growth control mechanisms that regulate cell proliferation and tissue homeostasis. It is a major form of cell death [30,31]. Flow cytometry was used to quantitatively analyze the apoptotic effect of kansenone in vitro via Annexin V-FITC/PI dual staining assay (Figure 5a). Phosphatidylserine (PS) moieties flip out from the inside of the cell membrane when apoptosis is triggered leading to specific binding of Annexin V- FITC to PS of cells in the early stage of apoptosis [32]. PI stains the necrotic cells and late apoptotic cells. As shown in Figure 5b, both the percentages of early apoptotic cells and late apoptotic cells increased with increases in kansenone concentration (4, 8 and 16 μg·mL−1) for IEC-6 cells. The percentages of total apoptotic cells treated with kansenone obviously changed from 23.48% to 40.43% when the concentration increased from 4 to 16 μg·mL−1 after 48 h treatment, whereas the proportion of apoptotic cells was only 10.37% in the control. The percentage of total apoptotic cells demonstrated that kansenone effectively promotes cell apoptosis in a concentration-dependent manner. This conclusion was further confirmed by Hoechst 33342/Annexin V-FITC/PI triple staining analysis under fluorescence microscope. In this assay, another dye Hoechst 33342 was introduced to characterize the cells nucleic. Hoechst 33342 could easily enter into apoptotic cells, but not into cells in good condition. As seen in Figure 6a, the number of cells with blue fluorescence from Hoechst 33342 gradually decreased from low to high concentrations of kansenone, indicating that cell proliferation was remarkably suppressed after kansenone treatment, consistent with the results in Figure 2. As illustrated in Figure 6b, the fluorescence intensity of Hoechst 33342, FITC and PI was significantly enhanced when IEC-6 cells were treated with kansenone at high concentrations of about 16 μg·mL−1, indicating that high concentration kansenone induced significant cell apoptosis, as expected in the Annexin V-FITC/PI dual staining assay.

Bottom Line: RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis.Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis.In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China. cff19870524@163.com.

ABSTRACT
Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

No MeSH data available.


Related in: MedlinePlus