Evodiamine induces ROS-Dependent cytotoxicity in human gastric cancer cells via TRPV1/Ca2+ pathway

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/Ca2+ 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 (Delta psi m) were examined using JC-1 assay. Intracellular Ca2+ levels ([Ca2+]i) were examined using Fluo-4 AM assay. Mitochondrial ROS ([ROS]m)levels were examined using Mitotracker Green/MitoSOX Red staining. Mitochondrial Ca2+ ([Ca2+]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 Delta psi m 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 Ca2+ chelation, confirming that EVO induces cell death through Ca2+ overload. Pharmacological and genetic inhibition of TRPV1 could significantly attenuate Ca2+ influx, ROS generation and apoptotic cell death induced by EVO exposure, while exogenous TRPV1 overexpression could augment the EVO-induced cytotoxicity. Moreover, genetic inhibition of mitochondrial calcium uniporter (MCU) attenuated EVO-induced cell death and mitochondrial dysfunction. EVO exposure induced endoplasmic reticulum (ER) stress demonstrated by the activation of PERK/CHOP in cells exposed to EVO, and PERK/CHOP activation was depleted by EUK134 pre-treatment. Our results support the concept that EVO induces ROS-dependent cytotoxicity via TRPV1/Ca2+ Pathway.

Automated summary

Evodiamine (EVO) from Evodiae fructus has antitumor effects on gastric cancer cells, mainly through cytotoxicity, including inhibition of proliferation, cell cycle arrest, apoptosis, and metastasis. This study aimed to investigate the role of TRPV1/Ca2+ in EVO-induced cytotoxicity in human gastric cancer cells. The results showed that EVO exposure led to ROS generation and apoptotic cell death, which could be ameliorated by pretreatment with EUK134. EVO exposure also induced ROS generation and mitochondrial dysfunction, mainly through mitochondrial ROS generation, which could be attenuated by rotenone pretreatment. Moreover, EVO-induced cytotoxicity could be significantly ameliorated by chelation of intracellular Ca2+, confirming that EVO induces cell death through Ca2+ overload. Inhibition of TRPV1 attenuated Ca2+ influx, ROS generation, and apoptotic cell death induced by EVO exposure, while overexpression of TRPV1 augmented EVO-induced cytotoxicity. Furthermore, inhibition of mitochondrial calcium uniporter (MCU) attenuated EVO-induced cell death and mitochondrial dysfunction. EVO exposure also induced endoplasmic reticulum stress, which was depleted by EUK134 pretreatment.