Institute of Integrated Medicine, Medicine College, Qingdao University, Qingdao, Shandong, 266071, China.
Department of Endocrinology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031, China.
Chem Biol Interact. 2022 Jan 5;351:109756. doi: 10.1016/j.cbi.2021.109756. Epub 2021 Nov 19.
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 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 levels ([Ca]i) were examined using Fluo-4 AM assay. Mitochondrial ROS ([ROS]m)levels were examined using Mitotracker Green/MitoSOX Red staining. Mitochondrial Ca ([Ca]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 chelation, confirming that EVO induces cell death through Ca overload. Pharmacological and genetic inhibition of TRPV1 could significantly attenuate Ca 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/Ca Pathway.
吴茱萸碱 (EVO) 是吴茱萸果实的主要活性成分之一，具有抗肿瘤作用（主要为细胞毒性作用），包括增殖抑制、细胞周期阻滞、细胞凋亡和转移抑制。我们的研究旨在解释 TRPV1/Ca 在 EVO 诱导的人胃癌细胞毒性中的作用机制。我们使用人胃癌细胞系 BGC-823 研究 EVO 诱导的细胞毒性。通过 CCK-8 测定法检测细胞活力。通过 Annexin V-FITC/PI 染色测定法检测细胞凋亡。通过 DCFH-DA 测定法检测细胞内 ROS ([ROS]i) 水平。通过 Mitotracker Green 染色检测线粒体形态。通过 JC-1 测定法检测线粒体膜电位 (Δψm)。通过 Fluo-4 AM 测定法检测细胞内 Ca 水平 ([Ca]i)。通过 Mitotracker Green/MitoSOX Red 染色检测线粒体 ROS ([ROS]m) 水平。通过 Mitotracker Green/Rhod-2 Red 染色检测线粒体 Ca ([Ca]m) 水平。通过 Western blot 检测蛋白水平。EVO 暴露会导致显著的 ROS 生成和细胞凋亡。EUK134 的预处理显著改善了 EVO 诱导的细胞凋亡。此外，EVO 暴露诱导 [ROS]i 生成和线粒体功能障碍，包括 [ROS]m 生成和 Δψm 耗散，而预先孵育鱼藤酮可显著减轻这些变化，表明 [ROS]m 是 EVO 诱导细胞内 ROS 生成的主要来源。重要的是，细胞内 Ca 螯合可显著改善 EVO 诱导的细胞毒性，证实 EVO 通过 Ca 过载诱导细胞死亡。TRPV1 的药理学和遗传学抑制可显著减轻 EVO 暴露引起的 Ca 内流、ROS 生成和细胞凋亡，而外源性 TRPV1 过表达可增强 EVO 诱导的细胞毒性。此外，线粒体钙单向转运体 (MCU) 的遗传抑制可减轻 EVO 诱导的细胞死亡和线粒体功能障碍。EVO 暴露诱导内质网 (ER) 应激，表现为暴露于 EVO 的细胞中 PERK/CHOP 的激活，而 EUK134 预处理可耗尽 PERK/CHOP 激活。我们的结果支持这样的概念，即 EVO 通过 TRPV1/Ca 途径诱导 ROS 依赖性细胞毒性。
