The Anti-tumor Effect Of Evodiamine On Human Gastric Cancer Cell Lines BGC-823 And SGC-7901 And Its Molecular Mechanisms

Objective:Evodiamine?EVO?is one of the main bioactive substances in Evodia rugosa.Modern pharmarcology research shows that evodiamine has a wide range of pharmacological activities in the immunomodulatory activities,anti-inflammatory and anti-tumor,and other aspects.This study was to explore the effect of EVO on the proliferation and apoptosis of gastric cancer cell lines BGC-823 and SGC-7901 cells and its possible molecular mechanism.Methods:The human gastric cancer cell lines BGC-823 and SGC-7901 were cultured in vitro,treated with different concentrations of EVO?5?M,7.5?M,10?M,12.5?M,15?M?for 24h,48h and 72h,respectively.CCK-8 assay was used to detect the effect of EVO on the proliferation of human gastric cancer cells BGC-823 and SGC-7901,and the IC₅₀value was calculated.The morphological changes of human gastric cancer cells treated with EVO for 24 hours were observed by inverted microscope,and the effect of EVO treatment for 24 hours on the clone formation ability of human gastric cancer cells was detected by clone formation assay.Flow cytometry?PI?was used to detect the effect of EVO treatment on the cell cycle of human gastric cancer cells.Flow cytometry?Annexin V-FITC+PI double staining?was used to detect the effect of EVO treatment on apoptosis of human gastric cancer cells.CFH-DA kits was used to detect the expression of ROS in human gastric cancer cells treated with EVO for 24h,and NAC?Antioxidant?was used to treat human gastric cancer cells lines BGC-823 and SGC-7901 for 24h.The proliferation activity and apoptosis rate of gastric cancer cells were detected,and the expression of cell cycle-related proteins and cell death-related proteins were detected by Western-blot.After further treatment of human gastric cancer cells BGC-823 and SGC-7901 with Z-VAD-fmk?Caspase broad-spectrum inhibitor?,Nec-1?RIP1 inhibitor?,AG14361 and?PARP-1 inhibitor?for 24h,the biological activity of related proteins was blocked.The proliferation activity and apoptosis rate of gastric cancer cells were detected.To observe whether it can reverse the cell death induced by EVO.Results:After treatment with EVO in vitro for 24h,48h and 72h,CCK-8 results showed that EVO could significantly inhibit the proliferation of human gastric cancer cells BGC-823 and SGC-7901 in a time-and dose-dependent manner compared with DMSO group.After 24 hours of EVO intervention,the number of suspended cells increased.The cell bodies were reduced,rounded and wrinkled,and no colonies were formed when they were separated from each other,and more cell fragments appeared.EVO could significantly decrease the colony number of human gastric cancer cells BGC-823 and SGC-7901?p<0.01?;FCM analysis showed that EVO mainly blocked cells in G2/M phase after treatment with 10?M EVO for 24h?p<0.001?and induced apoptosis of BGC-823 and SGC-7901 cells.And mainly late apoptosis?p<0.01?;EVO could induce a large number of ROS,in cells,but after NAC treatment for 24h,NAC almost completely blocked the inhibition of cell proliferation and cell death induced by EVO.It is suggested that EVO-induced cell death depends on the expression of ROS,and Western-blot results show that EVO can down-regulate the expression of cell cycle promoter protein Cdc25C,promote the expression of cell cycle suppressor protein p53,and up-regulate the protein expression of cyclin CyclinB1/Cdc2.At the same time,EVO could increase the cleavage of apoptosis-related proteins Caspase-3/9 and PARP-1 and induce apoptosis,activate Caspase-8 and c-FLIP,and increase the phosphorylation levels of RIP1 and RIP3,but had no significant effect on the ratio of Bcl-2/Bax.These results suggest that EVO can initiate exogenous apoptosis and programmed cell necrosis pathway and induce the death of gastric cancer cells in a time-dependent manner.However,treatment with inhibitors Z-VAD-fmk,Nec-1 and AG14361 for 24 h failed to reverse EVO-induced cell death,indicating the complexity and irreversibility of the type and mechanism of EVO-induced gastric cancer cell death.Conclusion:It is speculated that EVO can significantly inhibit the proliferation of gastric cancer cells and induce G2/M phase arrest and cell death in gastric cancer cells.The mechanism is that EVO induces exogenous cell apoptosis and programmed cell necrosis of gastric cancer cells through ROS-dependent pathway.