The Role And Mechanism Of TRPV1/Ca²⁺ In The Apoptosis In Human Gastric Adenocarcinoma Cells Induced By Evodiamine

Objective:To explore the mechanism of TRPV1,Ca²⁺,ROS and MCU in evodiamine（EVO）-induced apoptosis in human gastric adenocarcinoma cells.This study elucidated the molecular mechanism of EVO cytotoxicity from the perspective of TRPV1 mediated apoptosis,provided a new basis for revealing EVO’s cytotoxicity to tumor cells,provided toxicological data support for scientific and rational use of EVO,and provided a basis for finding an appropriate dose window.It also provides a new choice and molecular biological evidence for the clinical treatment of human gastric adenocarcinoma.Methods:From 2019 to 2021,33 cases of human gastric adenocarcinoma（moderately differentiated and poorly differentiated）were randomly selected,and immunohistochemical staining was performed on their cancer tissues and corresponding adjacent tissues to determine the expression of target protein TRPV1.CCK-8 method was used to detect the relative cell viability of human normal gastric epithelial cells GES-1 and human gastric adenocarcinoma cell BGC-823 after treatment with different concentrations of EVO,and to detect the pretreatment with inhibitors Capsazepine,BAPTA-AM,EUK-134,Rotenone,Ru360,2-APB and KN93,the relative viability of BGC-823 cells after TRPV1 and MCU knockdown were detected by EVO.The ROS and Ca²⁺levels in DCFH-DA and FLou-4 AM cells were detected by flow cytometry.The ROS and Ca²⁺levels in mitochondria after Mito Tracker+Mito Sox and Mito Tracker+Rhod2 staining were observed by inverted fluorescence microscope.The expression of TRPV1 on bgc-823 cells was observed by laser tool.The morphological changes of mitochondria after treated with 10μmol/L EVO for 24h were observed.Lentivirus transfected TRPV1 and MCU proteins on human gastric adenocarcinoma cell BGC-823.Western-blot was used to detect the expression of related target proteins.Results:EVO had cytotoxicity to Human gastric adenocarcinoma cell BGC-823 and human gastric epithelial immortal line GES-1 with IC50 of 10.57μmmol/L and 13.78μmmol/L,respectively.Euk-134 can inhibit EVO-induced apoptosis（P<0.001）,inhibit EVO-induced intracellular ROS level（P<0.001）,EVO induces ROS-dependent cell apoptosis;After EVO was applied to BGC-823 cells,mitochondrial function was impaired,mitochondrial morphology was changed,fragments increased（P<0.01）,aspect ratio increased（P<0.05）,and mitochondrial membrane potential decreased（P<0.001）.Rotenone could inhibit EVO-induced decreased cell viability（P<0.001）.Increased intracellular ROS levels（P<0.001）,Mito-ROS levels（P<0.001）,and EVO-induced mitochondrial membrane potential decline（P<0.001）.Mitochondrial membrane permeability pore inhibitor Cs A could inhibit EVO-induced cell viability decline（P<0.05）.The high ROS level from mitochondria is one of the causes of EVO-induced cytotoxicity.Increased intracellular Ca²⁺is critical for EVO-induced ROS production and cytotoxicity.BAPTA-am inhibits EVO-induced apoptosis（P<0.01）,evo-induced increase in intracellular Ca²⁺levels（P<0.001）,and EVO-induced decrease in mitochondrial membrane potential（P<0.01）.KN93 could not inhibit EVO-induced cell viability decline（P>0.05）.Immunohistochemical results showed that TRPV1 was expressed in the cancer tissues and adjacent tissues of 66 patients,as well as in the cell membranes and organelles of BGC-823.Capsazepine inhibited EVO-induced cytotoxicity（P<0.001）and evo-induced intracellular Ca²⁺increase（P<0.001）.After TRPV1 knockdown,EVO-induced BGC-823 cytotoxicity decreased（P<0.001）and ROS decreased（P<0.001）.After TRPV1overexpression,EVO-induced BGC-823 cytotoxicity increased（P<0.001）and ROS increased（P<0.001）.TRPV1 controlled EVo-induced ROS production and cytotoxicity.After MCU knockdown,The EVO-induced cytotoxicity of BGC-823 decreased（P<0.01）,ROS decreased（P<0.001）,EVO-induced mitochondrial membrane potential decreased（P<0.001）,MCU is responsible for EVO-induced ROS production and cytotoxicity;2-APB inhibited EVO-induced cell viability decline（P<0.001）,and er stress was one of the causes of EVO-induced cell death.Wester-blot results showed that:EVO can induce significantly increased expression levels of cl-PARP（P<0.001）,Cl-Caspase3（P<0.001）,P-DRP1（P<0.001）,P-CAMKII（P<0.001）,CHOP（P<0.001）.The protein PERK expression level was significantly decreased（P<0.001）.EUK-134 and Rotenone inhibited the increase of EVO-induced apoptosis-related proteins Cl-PARP1（P<0.001,P<0.01）and Cl-Caspase3（P<0.001,P<0.001）.Rotenone inhibited evo-induced elevation of P-Drp1 protein expression（P<0.001）,while BAPTA-AM inhibited EVO-induced elevation of P-CAMKII（P<0.001）.EUK-134 inhibited EVO-induced decrease in protein PERK（P<0.001）and increase in CHOP（P<0.001）.Conclusions:EVO induces apoptosis of oxidative stress-dependent human gastric adenocarcinoma cells through TRPV1/Ca²⁺pathway.