Anti-Tumor Activity Evaluation And Anti Gastric Cancer Cells Mechanism Exploration Of 1,2,4-Triazine Derivatives

ObjectiveIn this study,two series of 1,2,4-triazine derivatives were screened for antitumor activity in vitro.Their effects on the proliferation,apoptosis and the mechanism were investigated.This study could provide the basis for design and synthesis of1,2,4-triazine derivatives with high anti-cancer activity and low toxicity.Methods1.Detection of anti-tumor activityThe MTT assay method was used to screen the anti-tumor activity of the two series of 1,2,4-triazine derivative compounds in vitro,and the anti-tumor effects of the two series of compounds were evaluated.2.The research of anti-tumor mechanism（1）The effects of the compound on the anti-proliferation of cells were detected by methods such as clone formation,growth curve and flow cytometry assays.（2）Flow cytometry was used to detect the changes of tumor cell apoptosis rate and mitochondrial membrance potential with the treatment of the compound and to investigate the effect of compound on the pro-apoptosis effect of tumor cells.（3）Western Blot was used to detect the expression changes of apoptosis-related proteins or proteins related to NEDDylation or MAPK after the tumor cells were treated with compounds,so as to explore the signaling pathways of the compound applied to tumor cells.Results1.In the serie Ⅰ of synthetic compounds,compound V-11 showed the best activity against gastric cell MGC-803,IC₅₀ of which is 8.22μmol/L.2.Results of clone formation,growth curve and flow cytometry assays showed that compound V-11 inhibited the proliferation of MGC-803 cells and blocked the cells in G₂/M phase.Results of Western Blot showed that compound V-11 inhibited the NEDDylation of E1,E2,E3 enzymes.As the result,the levels of NEDDylation substrate protein p21 and ATF4 were increased,and the level of downstream proteins CHOP,DR5 and Noxa were increased as well.With the flow cytometry detection,the apoptosis rate increased as the concentration rised.Results from Western blot showed that with the increase of compound concentration,the expression of endogenous apoptosis-related protein Bax protein was up-regulated.The expression of Bcl-2protein and XIAP protein was down-regulated,the level of cleaved-caspase3 and cleaved-caspase9 were increased,and the apoptosis substrate protein PARP was cleaved.2.In the 1,2,4-triazine derivative serie Ⅱ,the results of MTT assay showed that the inhibitory effect of compound V-11 on gastric cancer cell MGC-803 was higher than the other four tumor cells,and its IC₅₀ was 0.35μmol/L.Results of clone formation,growth curve and flow cytometry assays showed that compound Ⅱ-13 inhibited the proliferation of gastric cancer cells MGC-803 cells and SGC-7901 cells,cells were arrested at G₂/M phase.Western blot results showed that compound Ⅱ-13 did not inhibit the NEDDylation process of MGC-803 cells.The results of flow cytometry showed that as the concentration of compound Ⅱ-13 increased,the level of reactive oxygen species（ROS）increased,the mitochondrial membrane potential decreased and the rate of apoptosis increased.With the co-incubation of antioxidant NAC,these results induced by compound Ⅱ-13 can be reversed by NAC.Western Blot results show that with the increase of the concentration of compound Ⅱ-13,the p-c-raf,p-MEK1/2,p-ERK1/2,p-P90RSK and c-Myc proteins in the ERK signaling pathway were decreased;the levels of endogenous apoptosis pathway proteins cleaved-Caspase3/9 and cleaved-PARP increased;DR5 and cleaved-Caspase8 in the extrinsic apoptosis pathway were up-regulated.Similarly,with co-incubation of antioxidant NAC,changes in these proteins caused by compound Ⅱ-13 were reversed.Western Blot and the si RNA technology showed that after the compound Ⅱ-13 acted on gastric cancer cells,the expression level of p-ERK1/2 decreased,which in turn increased the expression of DR5 protein and promoted the occurrence of apoptosis.Conclusions1.Compounds V-11 and Ⅱ-13 are two representative compounds of1,2,4-triazine derivatives.The anti-tumor activity in vitro of 1,2,4-triazine derivatives were significantly improved by chalcone active fragments.The anti-tumor activity of compound Ⅱ-13 is better than that of V-11.2.The anti-tumor mechanism of compounds V-11 and Ⅱ-13 were different:Compound V-11 inhibits the degradation of substrate protein p21 and ATF4 protein by inhibiting the NEDDylation process of gastric cancer cell MGC-803,thereby induced intrinsic apoptosis of MGC-803 cells;compound Ⅱ-13 did not significantly inhibit the NEDDylation process of gastric cancer cells,but via inducing the generation of ROS,it inhibited the p-ERK pathway in the MAPK pathway.Compound Ⅱ-13 down-regulated p-ERK1/2 then up-regulated the DR5 protein in the extrinsic apoptosis induced apoptosis of gastric cancer cells MGC-803 and SGC-7901.