Abstract

Evodiamine (EVO), a key active ingredient of the fruit of Evodiae fructus , is provided with antitumor effects (mainly cytotoxic effect) including proliferation inhibition, cell cycle arrest, apoptosis , and metastasis inhibition . Our study aims to explain the underlying role of TRPV1/Ca 2+ in EVO-induced cytotoxicity in human gastric cancer cells. Human gastric cancer line BGC-823 was used to study EVO-induced cytotoxicity. Cell viability was examined using CCK-8 assay. Apoptosis was examined using Annexin V-FITC/PI staining assay. Intracellular ROS ([ROS]i) levels were examined using DCFH-DA assay. Mitochondrial morphology was examined using Mitotracker Green staining. Mitochondrial membrane potential (Δψm) were examined using JC-1 assay. Intracellular Ca 2+ levels ([Ca 2+ ]i) were examined using Fluo-4 AM assay. Mitochondrial ROS ([ROS]m)levels were examined using Mitotracker Green/MitoSOX Red staining. Mitochondrial Ca 2+ ([Ca 2+ ]m)levels were examined using Mitotracker Green/Rhod-2 Red staining. The protein levels was detected by Western blot . EVO exposure causes significant ROS generation and apoptotic cell death . Pretreatment of EUK134 significantly ameliorated EVO-induced apoptotic cell death. Furthermore, EVO exposure induced [ROS]i generation and mitochondrial dysfunction, including [ROS]m generation and Δψm dissipation, which can be significantly attenuated by pre-incubation of rotenone indicating that [ROS]m is the main source of EVO-induced intracellular ROS generation. Importantly, EVO-induced cytotoxicity was significantly ameliorated by intracellular Ca 2+ chelation, confirming that EVO induces cell death through Ca 2+ overload. Pharmacological and genetic inhibition of TRPV1 could significantly attenuate Ca 2+ influx, ROS generation and apoptotic cell death induced by EVO exposure, while exogenous TRPV1 overexpression could aug