The Mechanism Of Jaspine B Derivative M-2 Ininducing Autophagy And Necroptosis In Gastric Cancer

Gastric cancer is the third leading cause of cancer death.Due to the lack of specific biological markers,the early detection rate is low,and the prognosis is poor.Jaspine B is a natural sphingolipid molecule found in Marine sponges Jaspis and Pachastrissa sp,which has significant biological activity.Therefore,the research on Jaspine B and its derivatives has become a hot target.In this project,we discussed the anti-tumor effect of Jaspine B derivative M-2 on gastric cancer cells and its molecular mechanism.The main conclusions are as follows:1.The anti-proliferation effect of M-2 on gastric cancer cells(MGC803,HGC27,SGC7901)and normal gastric epithelial cells(GES1)was detected by MTT.The results showed that M-2 inhibited the proliferation of gastric cancer cells(MGC803,HGC27,SGC7901)and had no toxicity to normal gastric epithelial cells(GES1).2.To determine the mechanism of M-2-induced gastric cancer cell death,we used flow cytometry to collect cells by Annexin v-FITC/PI double staining,and found that the dead cells were mainly concentrated in the regions of Annexin V-FITC+/PI+.Then,MTT method was used to detect the combination of the Caspases inhibitorZ-VAD-fmk and M-2,and it was found that Z-VAD-frm failed to reverse the mortality caused by M-2 compared with shikonin,the apoptosis-positive control.Finally,Hoechst staining was used to detect the changes in nuclear morphology after M-2 treatment,and it was found that compared with shikonin,the apoptosis-positive control,M-2 did not cause the changes in cell nucleus of apoptosis.These results indicated that M-2 caused non-apoptotic death of gastric cancer cells.3.According to the conclusion of the previous step,it was speculated that M-2 cause necroptosis of gastric cancer cells.Therefore,western blot was used to detect the changes in necroptosis protein expression after M-2 treatment of gastric cancer cells,and the results showed that necroptosis protein expression increased significantly.Moreover,MTT assay showed that RIP3-/-MGC cells were insensitive to M-2,while western blot assay showed no increase in RIP3 and p-MLKL expression after treatment of RIP3-/-MGC cells by M-2.These results showed that M-2 caused necroptosis of gastric cancer cells.4.Since vacuoles were observed in gastric cancer cells after treatment of M-2 under the microscope,it was speculated that M-2 might induce autophagy.In order to detect the occurrence of autophagy,co-immunoprecipitation was used to detect the dissociation of Beclinl and Bcl-xl after M-2 treatment of gastric cancer cells,and western blot revealed increased expression levels of LC3II and ATG family proteins.These results showed that M-2 induced autophagy in gastric cancer cells.5.MTT assay found that ML385 increased the inhibitory effect of M-2 on gastric cancer cells,indicating that M-2 might activate the Nrf2 pathway.Western blot showed increased p-Nrf2 and HO1 expression levels,and immunofluorescence and Nuclear and Cytoplasmic Protein Extraction tests showed that Nrf2 transferred from cytoplasm to nuclear localization,indicating that the Nrf2 pathway was activated.Western blot detection showed that ML385 further increased the necroptosis proteins RIP3 and p-MLKL caused by M-2,indicating that Nrf2 pathway inhibited necroptosis.These results suggest that M-2 activates Nrf2 pathway in gastric cancer cells.6.Existing literature showed that autophagy can activate Nrf2 by P62 competitively binding KEAP1.Therefore,it is speculated that M-2-induced autophagy can inhibit necroptosis by activating Nrf2 pathway.In order to link autophagy with Nrf2 pathway and necroptosis,early autophagy inhibitor 3-MA and late autophagy inhibitor BafAl were used in combination with M-2,respectively.MTT assay showed that 3-MA promoted the inhibitory effect of M-2 on gastric cancer cells,and BafAl reversed the inhibitory effect of M-2 on gastric cancer cells.Western blot detection showed that 3-MA inhibited the increase of p-Nrf2 induced by M-2 but promoted the increase of M-2 induced necroptosis proteins RIP3 and p-MLKL,while BafAl promoted the increase of M-2 induced RIP3 and p-MLKL.In addition,co-immunoprecipitation showed that 3-MA inhibited the increase of binding amount of M-2 induced P62 to KEAP1,while BafAl promoted the increase of binding amount of M-2 induced P62 to KEAP1.Finally,after transfection of siP62 with MGC803,siP62 further increased the death of gastric cancer cells induced by M-2.The above results showed that autophagy activated Nrf2 pathway through P62 to inhibit necroptosis.7.To detect the effect of M-2 on invasion and migration of tumor cells in vitro,we used Transwell and wound healing experiment.The Transwell showed that the number of cell invasioned in the M-2 treatment group was significantly lower than that in the control group,and the wound healing experiment showed that the area of wound healing in the M-2 treatment group was significantly lower than that in the control group.Moreover,western blot experiments showed that M-2 decreased the expression of Vimentin and increased the expression of E-Cadherin.These results indicated that M-2 significantly inhibited the invasion and migration of gastric cancer cells in vitro.8.Combined with all the above results,it can be concluded that M-2 induces autophagy and necroptosis in gastric cancer,and autophagy can activate Nrf2 pathway and inhibit necroptosis through P62.