Study On The Mechanism Of A New Compound 1,2,3-triazole-Jaspine B Hybrids Inhibited The MGC803 Cells Proliferation

Recently,more and more interesting focus on screen the active compounds from natural resources.Jaspine B was isolated from the marine sponge Pachastrissa sp.and Jaspis sp.,has significant antitumor activity,can bind to 1,2,3-Triazoles group and synthesis a new compounds 1,2,3-triazole-Jaspine B hybrids using click chemistry method,which was named LHM in following experiment.We found it can inhibit the proliferation of gastric cancer MGC803.Therefore,the aim of this study is to investigate the LHM inhibiting the proliferation of gastric cancer cell line MGC803,and try to clarify its antitumor molecular mechanismFirstly,,we use the fluorescence microscopy and Hoechst33342-PI to observe the changes in cell morphology after treatment with different concentration of LHM for 24 h and 48 h.Some proliferation associated protein's expression were detected by Western blotting The autophagy and necroptosis complexs were tested by immunoprecipitation after treatment with LHM.The results showed that the cell proliferation inhibition ratio was increased after treatment with LHM for 24 h in a dose-dependent manner.In terms with cell morphology,we can observe the cell vacuolization phenomenon upon the surface of cell MGC803,cell membrance had ruptured at low concentration;and yet in the presence of high concentration of compound,the cell status was condensed into a mission and suspended in the medium.Western blotting results suggested that apoptosis related proteins such as caspase family proteins including caspase-3,8,9 and Bcl-2 family proteins contain Bid,Bax,Bcl-2,Bcl-xl and so on,the level of these proteins expression had not been significant changes with low concentrations LHM treatment;nevertheless,in the presence of high concentration of LHM,these proteins expression were stimulated.In order to understand its antitumor mechanism,we further examed some autophagy and necroptosis related proteins expression,the autophagy related proteins contain LC3 and Beclin1 were activated in a dose and time dependent manner,,the necrosis related proteins including RIP1,RIP3,MLKL and p-MLKL,their expression were increased.at the same time,the complexes including Beclin1-BCL-2\Bcl-XL,FADD-Cas8-RIP1-RIP3 and RIP1-RIP3-MLKL,obvious changement were observed.these suggested that autophagy and necroptosis may have taken part in the process.Using their relative inhibitors,results showed that a large extent autophagy and necroptosis should be involved this processing.Meantime,we also measured the generation of reactive oxygen species by flow cytometry after treatment with LHM,the ROS level of MG C803 cell was increased,it exhibited that ROS exactly play a key role in the process.The MAPK signaling pathway was involved through activing ERK and JNK pathway,and JNK may induce the autophagy.In conclusion,the proliferation of gastric cancer cell line MGC803 were inhibited by LHM,autophagy and necroptosis might participate in process of cell death;reactive oxygen species could be involved in autophagy and necroptosis;JNK might induce autophagy.