Effects And Mechanisms Of Neddylation Inhibitor MLN4924 On The Proliferation Of Endometrial Cells

Purpose:Endometrial cancer is a serious threat to human health.It’s urgent to find a new therapeutic target of anti-tumor molecular therapy with wide spectrum,high efficiency and low toxicity.Neddylation pathway is a novel protein post-translational modification Studies have shown that neddylation pathway is overactivated in a variety of human primary tumor tissues.It can promote the development of tumor by activating CRLs(Cullin Ring ligases)to cause the degradation of CRL tumor suppressor protein substrates.Neddylation inhibitor MLN4924 has significant antitumor effect in both vitro and vivo.However,the effect and mechanism of MLN4924 on the proliferation of endometrial cancer cells are rarely reported.The aim of this study is to reveal the mechanism of MLN4924 in killing endometrial cells.Methods:Immunoblotting(IB)was used to determine the effect of MLN4924 on the neddylation pathway in endometrial cells.ATPlite、CCK8 and classical clone formation analysis were used to evaluate the effect of MLN4924 on the proliferation of endometrial cells.Effects of MLN4924 on cell cycle,DNA damage response,cell apoptosis and degradation of CRL substrates were analyzed by immunoblotting(IB)and flow cytometry.Finally,the cell phenotype rescue experiment was performed by siRNAResults:1.TCGA database shows that mRNA levels of NEDD8,UBE2M and UBE2F in the tumor tissue are higher than that in the tumor adjacent tissue.Immunoblotting analysis(IB)showed that the levels of global protein neddylation and cullins(Cullin 1,2,3,4a,4b,5,7,9)were inhibited under MLN4924 treatment in endometrial cancer cell lines(Ishikawa and HEC-1-A).2.CCK8 and ATPlite data showed that the cell viability of MLN4924 treatment group was lower than that of the control group in a dose dependent manner.The results of clone formation showed that the number of cell clones in MLN4924 treatment group was significantly lower than that in the control group.3.PI staining data indicated that the cell cycle of MLN4924 treated group was blocked in G2/M phase.WB results showed that the expression levels of p21,p27 and Weel in MLN4924 treated group were higher than that in the control group.Besides,the expression levels of ORC1,CDT1 and p-H2AX were obviously higher than that in the control group.4.According to the analysis of apoptosis by annexin V-FITC/PI staining,the proportion of apoptosis in MLN4924 treatment group was obviously higher than that in the control group.The higher the concentration of MLN4924 is,the higher the proportion of apoptosis is.WB results showed that the expression levels of C-PARP and C-casp-3 in MLN4924 treated cells were significantly increased,and the expression level of Noxa in MLN4924 treated cells was significantly up-regulated.After down-regulated Noxa by siRNA,the expression level of C-PARP,C-casp-3 and p-H2AX in MLN4924 treated cells were rescued.Conclusions:1.Neddylation pathway was over-activated in clinical samples of endometrial cancer.MLN4924 can significantly block neddylation pathway in endometrial cancer cells.2.MLN4924 significantly inhibited the proliferation of endometrial cancer cells.3.MLN4924 induced G2 phase cell arrest,DNA damage response and apoptosis of endometrial cells,thus ultimately killing tumor cells.4.MLN4924 activated the apoptotic pathway partly through pro-apoptotic protein Noxa in endometrial cells.Knockdown of Noxa can significantly rescue the apoptotic phenotype of endometrial cells.