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.
Highlights
Kansenone-induced apoptosis is likely to be mediated through the death receptor and mitochondrial pathways, as evidenced by up-regulation of Bax, apoptosis-inducing factor (AIF), the adaptor molecule apoptotic protease activating factor 1 (Apaf-1), and cytochrome c, caspase-3, caspase-9, caspase-8 activity, Fas receptor (FasR), Fas ligand (FasL), NF-κB, and tumor necrosis factor (TNF) receptor-1 (TNFR1) mRNA
Kansenone is a compound isolated from a traditional Chinese medicine plant kansui
The expression of cytochrome c was up-regulated along with the increase of caspase-9 and caspase-3 (Figure 10a,b). These results suggested that kansenone increased the expression of Bax, AIF, Apfa-1, cytochrome c, caspase-3, caspase-9, and decreased Bcl-2 expression in IEC-6 cells in time- and dose-dependent manners, respectively, indicating kansenone induced cell apoptosis via the mitochondrial-mediated pathway
Summary
Fangfang Cheng 1, Yanjing Yang 2, Li Zhang 1,*, Yudan Cao 1, Weifeng Yao 1, Yuping Tang 1,*. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources
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