The Novel RIP3 Agonist CY6 Activates RIP3-P62 To Induce Necroptosis Through Defective Autophagy In Gastric Cancer

Jaspine B was isolated from the marine sponge Pachastrissa sp.and Jaspis sp.,has significant anticancer activity,can bind to 1,2,3-triazoles group and synthesis the new compound 1,2,3-triazole-jaspine B hybrids,which was named CY6 in following experiment.The present study aimed to determine whether RIP3 was activated by CY6 and CY6 effectively inhibits the proliferation of gastric cancer cells and to investigate the mechanism underlying its anticancer functions.Among them,the MTT assay was used to detect the gastric cancer cytotoxicity;western blotting was used to detect the change of protein expression by CY6 treatment;the complexes of RIP3-P62 and Beclin-1-Bcl-2/Bcl-xl were detected by co-immunoprecipitation assay;LC3-immunofluorescence was used to observe autophagic flux.In addition,we introduced RIP3^-/-MGC803 cell line through lentiviral packaging.The main results were showed as follows:1.The proliferation of gastric cancer cells?MGC803,HGC27 and SGC7901?were effectively inhibited in a dose-dependent manner after treatment with CY6.MGC803 cells showed the most sensitivity to CY6 treatment,and CY6 did not have toxicity in GES1.These results suggested that CY6 has selective cytotoxicity against gastric cancer cells versus normal human gastric epithelial cells in vitro.2.Hoechst?BF?/PI double staining was used to detect the morphological changes of MGC803 cells after treatment of CY6.In the absence of CY6,MGC803cells exhibited round dark green nuclei stained with BF,whereas MGC803 cells treated with CY6 had dark green and red nuclei of normal size,indicating uptake of both dyes by necrotic cells.The expression of RIP1 and RIP3 were increased by CY6-treatedingrastriccancercells.Moreover,endogenousRIP3co-immunoprecipitated with endogenous RIP1 in MGC803 cells when subjected to CY6 for 24 h.Taken together,these data suggest that CY6 induced necroptosis.3.Significant cell death was observed at 24 h with different concentrations of CY6?0-8?M?treatment in MGC803 and SGC7901 cells by microscopic observation of cell morphology.The expression of autophagy key proteins the Beclin-1,LC3 and ATGs were increased in a dose-dependent manner by CY6-treated in MGC803 and SGC7901 cells.The anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xl can inhibit autophagy via binding to Beclin-1,and the complex consisting of Bcl-2/Beclin-1 or Bcl-xl/Beclin-1 was reduced in CY6-treated cells.These data suggested that CY6induced autophagy in gastric cancer cells.4.The autolysosome protein P62 participates in the process of autophagy degradation.Interestingly,we found P62 was increased by CY6 comparing with control both in MGC803 and SGC7901 cells,indicating that autophagy was blocked before autolysosome degradation.CY6-induced LC3 puncta formation was examined in MGC803 cells,which is another hallmark of autophagy.On treatment of MGC803cells with 4?M CY6 for different times,a granular pattern due to strong accumulation of LC3 what was observed in the autophagy and developed a large area with times,suggesting that CY6 triggered autophagic flux and stimulated autophagy.Altogether,these results provide strong evidence suggesting that CY6 induced defective autophagy in gastric cancer cells.5.A clear mobility shift of RIP3 was detected by CY6,indicating that RIP3 was phosphorylated by CY6.Studies have shown that P62 can be combined with RIP protein.Indeed,we found that RIP3 co-immunoprecipitated with P62 under CY6-treated conditions.6.We introduced RIP3 knockdown?KD?of MGC803 cells here.CY6 was treated in wild type?WT?and RIP3 KD MGC803 cells respectively.It was found that CY6-induced cell death and the change of autophagy protein expression were completely blocked by RIP3 knockdown.The results showed that RIP3 was the target of CY6.7.MGC803 xenografts were constructed in nude mice.It was found that tumor growth was inhibited by CY6.In addition,the upregulation of RIP3,P62 and Beclin-1 expression,the dissociation of the Beclin-1-Bcl-2/Bcl-xl complex and the formation of the P62-RIP3 complex were detected after CY6 treatment in vivo.The results showed that CY6 inhibited tumor proliferation and induced autophagy and necroptosis in vivo.Indeed,compound CY6 was used as RIP3 agonist in gastric cancer cells.Necroptosis was induced by CY6 through defective autophagy and RIP3-P62interaction.