A Novel Indole Microtubule Protein Polymerization Inhibitor Study On Antitumor Activity In Vitro

Microtubules are widely present in the cells of eukaryotes and have a very close relationship with the occurrence and development of tumor cells.It is also considered to be an attractive target for chemotherapy drugs to treat cancer.Based on the preliminary work,our research group designed and synthesized a new class of steroid derivatives,and selected the activity-optimized compound 2803 to study its mechanism of action on gastric cancer cells MGC803.The main research of this paper is as follows:1.The antiproliferative activity of compound 2803 against human esophageal cancer cells（EC109）,human prostate cancer cells（PC3）,and human gastric cancer cells（MGC803）was examined by MTT assay.The results showed that 2803 had the most significant effect on MGC803 with an IC₅₀ of 1.59μmol/L.2.To verify the inhibitory effect of compounds on tubulin,we tested them by immunofluorescence,immunoblotting and molecular docking techniques.The results of immunofluorescence showed that with the increase of 2803concentration,the ability to depolymerize microtubules gradually increased,and the spindle could not form at 2μmol/L.The EBI results showed that after a certain concentration,it could competitively bind colchicum after 2803.Base site;docking of 1SA0 molecule by MOE,the results showed that 2803 can occupy the binding site of colchicine and form a good interaction with tubulin.3.Further explore 2803 of the methods of death that act on cells,and the results of flow cytometry showed that the apoptosis rate of compound 2803induced MGC803 at concentrations of 2μmol/L had reached 65.2%.There are significant nuclear changes after DAPI staining,such as breakage and condensation.In the comet experiment,the tail length of the comet appeared significantly longer.These results all indicate the presence of apoptosis after 2803acting cells.In the Western Blot test,anti-apoptosis protein Bcl-2 and Bcl-xl appeared downwards,while the apoptosis protein Bax increased,two executors Caspase-3/7 and its substrate protein PARP were also significantly cut.In addition,both death receptor DR5 and activated Caspase-8 showed an increase.These results show that 2803 induced apoptosis of MGC803 by external source and endogenous combined pathway.4.Due to the accumulation of DR5,it is suspected that the effect of 2803 on MGC803 may be related to NEDDylation.In the NEDD8 binding assay,Ubc12-NEDD8,NAE1-NEDD8,and Cullins-NEDD8 were all inhibited by 2803.Then we examined the expression of cyclin p21 regulated by NEDDylation,and the results showed that it accumulated to a large extent.5.The accumulation of p21 can induce cell cycle block.First,from the flow results,2803 of the exact dose-dependent block cell cycle,the percentage of the G2/M period increased from 20%to 60%.The results of clonal formation showed that the cell colony in the experimental group decreased obviously in turn.However,the results of the Western Blot showed that the CDK1,CyclinB1 and CyclinB2 affecting the G2 period did not decrease,while the protein p-Histone H3 associated with the M period was significantly increased,suggesting that 2803inhibited cell proliferation and blocked the cycle during the M period.6.MAPK is an important signaling pathway involved in cell proliferation.It has three main pathways,and the results show that p-c-Jun is not regulated by2803,and both ERK and p38 pathways are inhibited.Among them,the ERK pathway was most inhibited.The upstream protein of the ERK pathway was found to be c-Raf,MEK1/2,and ERK1/2 were inhibited and the initial Ras was not affected.In further testing,it was found that 2803 can directly interact with Ras.This indicates that 2803 interacts with Ras to affect the phosphorylation of Raf by Ras,thereby inhibiting the activation of downstream MEK1/2 and ERK1/2,resulting in inhibition of cell proliferation.To sum up,indole derivative 2803 can be used as a potential microtubule protein inhibitor for targeted anti-tumor research.In addition,it inhibits the NEDDylation process of MGC803 and inhibits the ERK pathway of MAPK through interaction with Ras.The final compound 2803 plays an anti-tumor role by inducing apoptosis of gastric cancer cell MGC803 and inhibiting its proliferation.