3-O-(2′E, 4′Z-decadienoyl)-20-O-acetylingenol induces apoptosis in intestinal epithelial cells of rats via mitochondrial pathway. (4th November 2015) - Gao, Jing
Abstract: Ethnopharmacological relevance: Euphorbia kansui is a traditional Chinese medicine widely used for the treatment of edema, ascite and asthma in China for centuries. However, its serious gastrointestinal toxicity restricted its safe clinical application. 3- O -(2′ E, 4′ Z -decadienoyl)-20- O -acetylingenol (3EZ, 20Ac-ingenol), a diterpenoid compound derived from kansui, has obvious gastrointestinal cytotoxicity in cells. Until now, its gastrointestinal cytotoxic mechanism is mostly unknown. This study focused on elucidating the cytotoxic mechanism of 3EZ, 20Ac-ingenol in intestinal epithelial cells of rats (IEC-6 cells) to guide safer application of this herb in clinic. Materials and methods: 3EZ, 20Ac-ingenol was isolated from the EtOAc extract of kansui. Cell morphology was detected by inverted phase contrast microscope and transmission electron microscope (TEM). Cell apoptosis was examined by Annexin V-FITC/PI dual-staining or Hoechst staining. ROS generation was detected with DCFH-DA staining by laser scanning confocal microscope. MMP change was examined with JC-1 staining by high content screening (HCS). Further, the release of cytochrome c, the expressions of Bax, Bcl-2, AIF and Apaf-1 were analyzed by western blot and the activities of caspase-3, 8, 9 were determined by ELISA. Additionally, cell cycle analysis was performed to detect the effects of 3EZ, 20Ac-ingenol on cell cycle in IEC-6 cells by flow cytometry. Results: The study showed that 3EZ, 20Ac-ingenol significantly reduced IEC-6 cells viability in a dose-dependent manner and the IC50 value was 5.74 μg/mL. Consistently, 3EZ, 20Ac-ingenol could elevate reactive oxygen species (ROS), disrupt mitochondrial membrane potential (MMP), induce the release of cytochrome c from mitochondria to cytosol, enhance the expressions of Bax, AIF and Apaf-1, suppress the expression of Bcl-2, then activate caspase-3, 8, 9 cascade, and subsequently result in apoptosis. Additionally, 3EZ, 20Ac-ingenol also could cause G2/M phase arrest in IEC-6 cells. Conclusions: The results indicated that 3EZ, 20Ac-ingenol induced the cytotoxicity of IEC-6 cells depends on induction of cell apoptosis via mitochondrial pathway and cell cycle arrest. Graphical abstract: