Study On The Mechanism Of Jaspine B Derivative ZE-2 Inhibiting The Proliferation Of Gastric Cancer Cells

Jaspine B isolated from marine sponges is a natural compound and has the positive antitumor activity against a variety of cancer cells.Due to the specificity of the structure of Jaspine B,a set of Jaspine B derivatives have been synthesized and found to have obvious activity against some cancer cells,which can limit the proliferation of them.However,the anticancer mechanism has not been studied in depth,and there are few studies on the internal signaling pathways of gastric cancer cells.In this research,the Jaspine B derivative named ZE-2 has a significant growth inhibition on gastric cancer cells.The article mainly explores how this compound inhibits the proliferation of gastric cancer cells and the signal transduction process of the compound to induce gastric cancer cells death.The reseach findings are as follows:1.MTT assay detected that ZE-2 inhibited the growth of gastric cancer cells?MGC803,HGC27 and SGC7901?,in which MGC803 was the most obviously inhibited and ZE-2 had no obvious toxicity to the normal human gastric epithelial cells GES-1.Annexin V-FITC/PI staining flow cytometry results showed that ZE-2induced apoptosis in gastric cancer cells.The increase of the pro-apoptosis protein Actived-caspase3 and CF-PAPR which was detected by Western blotting also indicated that ZE-2 induced the occurrence of apoptosis.2.The decrease of the mitochondrial membrane potential of gastric cancer cells?MGC803 and HGC27?was detected by JC-1 fluorescent probe.Western blotting assay showed that the expression of the pro-apoptotic protein Bax was increased,and the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL was down-regulated,indicating that ZE-2 induced apoptosis through the internal mitochondrial pathway.3.The morphological changes of MGC803 treated by ZE-2 for 24 h were observed by inverted microscope,and the result is that the surface of the cells was vacuolated,rich in small vesicles.Western blotting analysis detected that the expression of autophagy related proteins LC3-II,Beclin-1,Atg3 and Atg12 were increased,indicating that ZE-2 caused the occurrence of autophagy.4.Western blotting assay showed that the phosphorylation of JNK and ERK proteins of the MAPK pathway was up-regulated,and the JNK inhibitor SP600125and the ERK inhibitor SCH772984 reversed the expression of LC3-II protein induced by ZE-2.Immunoprecipitation experiments results showed that ZE-2 can dissociate the Beclin-1-Bcl-2/Bcl-xL protein complex and SP600125 and SCH772984 can inhibit the separation of Beclin-1-Bcl-2/Bcl-xL complex.These results indicate that ZE-2 induced autophagy through the JNK/ERK-Bcl-2/Beclin-1/Bcl-xL cascade reaction.5.Western blotting results showed that the expression of P62 protein increased with the prolongation of ZE-2 treatment time.Due to the specificity of the P62structure,P62 interacts with various binding partners and acts as a signaling center for various cellular signal transduction cascades.Immunoprecipitation detected that when MGC803 was incubated ZE-2?4?M?with for 6 h,P62 bound to Keap1,and HO-1and NQO1,Nrf2 the downstream proteins,were makedly increased.The nuclear separation experiments showed that the expression of Nrf2 in the nucleus gradually increased with time and reached the highest at 6 h,but its expression in the cytoplasm decreased in a gradual manner.The staining immunofluorescence of Nrf2 experiment showed that Nrf2 was concentrated in the nucleus.The above results indicated that ZE-2 activated the Nrf2 protection mechanism during the gastric cancer cell death6.Explore the relationship between the P62-Nrf2 pathway and apoptosis,Immunoprecipitation showed that SP600125 and SCH772984 can reduce the binding of P62 to Keap1 and down-regulate the expression of P62 and Nrf2 downstream proteins HO-1 and NQO1.Annexin V-FITC/PI staining flow cytometry showed that SP600125andSCH772984increasedtheapoptosisinducedbyZE-2.Immunoprecipitation showed that the early inhibitor of autophagy LY294002 reduced the expression of P62 and HO-1.MTT showed that LY294002 reduced the cell survival rate induced by ZE-2 and Beclin-1^-/-cell was easier to die under the treatment of ZE-2.MTT assay results showed that P62 siRNA could further enhance ZE-2 induced cell death.Immunoprecipitation showed that the autophagy late inhibitor BafA1 could increase the binding degree of P62 to Keap1 protein.The results of MTT assay showed that BafA1 could limit the cell death induced by ZE-2.The above data indicated that JNK/ERK induced the production of autophagy,increasing the expression of P62 which activated the Nrf2 signaling pathway,which limited the occurrence of apoptosis to some extent.In summary,ZE-2 induces apoptotic death of gastric cancer cells through the internal mitochondrial pathway,and induces autophagy in gastric cancer cells through JNK/ERK pathway,which increases the expression of P62 protein and further activates Nrf2 signaling pathway to resist ZE-2 induced apoptosis.However,with the prolongation of time or the increase of ZE-2 concentration,the degree of apoptosis increase and cell death occurre in gastric cancer cells,which restricte cell survival.