3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol induces apoptosis in intestinal epithelial cells of rats via mitochondrial pathway

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.

Keywords: 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol); Apoptosis; Caspase activation; Cell cycle arrest; Cytochrome c release; Mitochondria; Reactive oxygen species (ROS).