Mechanism Of Aloe-emodin’s Effect On The Proliferation Of Endometrial Cancer Cells

Objective:Screening the effective components of aloe vera which have the activity of inhibiting endometrial cancer,using network pharmacology screening the composition effect on endometrial cancer may target,through the experiment in vitro,explore the aloe ingredient,possible mechanisms of inhibition of endometrial carcinoma,active ingredients for the study of aloe has provided the new mentality inhibitory effect of endometrial carcinoma.Research Methods:(1)Four species of Aloe vera(Aloe-Emodin,Aloin,Aloesin,Esculetin)were found out by literature search.In vitro experiments were conducted to observe the same concentration gradient(0,5,10,20,40,80μmol/L)were applied to three endometrial cancer cell lines(Ishikawa,AN3 Ca,HEC-1-B cells),respectively,and the most effective inhibition of endometrial cancer cell activity was screened.(2)Based on multiple TCM and disease databases,network pharmacology was used to screen the potential targets of the active ingredient acting on endometrial cancer,and the biological functions of the potential targets were analyzed to build the relationship network among the targets,and GO and KEGG enrichment analysis was conducted according to the core targets.(3)The core targets obtained through network pharmacology were verified by qPCR,and related signaling pathways were explored by Western Blot according to the core targets.Results:(1)CCK-8 showed that Aloe-Emodin had the strongest inhibitory activity against three endometrial cancer cells(Ishikawa,An3 Ca,and HEC-1-B),compared with Aloin,Aloesin,and Escupelin.(2)Based on network pharmacology,it was found that there were 7 core targets of AE on endometrial cancer:TP53,Casp3,MYC,CDKN1A,PTGS2,CCNB1 and CDK1.The molecular functions of the core target are the regulation of cyclin-dependent serine/threonine kinase activity and the binding of ubiquitin-like protein ligases.Biological processes include the regulation of cyclin-dependent serine/threonine kinase activity,DNA damage response,and cell cycle arrest caused by the signal transduction of p53 mediators.The cellular components include cyclin-dependent protein kinase holoenzyme complex,mitochondrial matrix,and transferase complex to transfer phosphorus-containing groups.The core targets are mainly related to the p53 signaling pathway and cell cycle.(3)It was assumed that the 7 core targets screened by network pharmacology were all effective targets of AE for inhibiting endometrial cancer cells,which were verified by qPCR assay.The results showed that 6 of the 7 core targets(TP53,MYC,CDKN1A,PTGS2,CCNB1,CDK1)mRNA expression of AE inhibited endometrial cancer cells met the expectation.The expression of MYC was significantly decreased with the increase of dosing concentration,while the expression of CDKN1A was significantly increased with the increase of dosing concentration.Contrary to expectations,Casp3 expression was not the target of AE inhibition of AN3 Ca cells,although the changes were obvious.The predicted targets were mainly related to cell cycle,suggesting that AE might inhibit endometrial cancer by blocking cell cycle and thereby inhibiting cell proliferation,which was consistent with the phenotypic results obtained by CCK-8 method in the first part.(4)Explore the possible pathway related to MYC target in the process of inhibition of endometrial cancer cell proliferation by AE by Western Blot.The protein expression changes of c-myc,β-catenin,GSK-3β,p-GSK-3β(Ser9),Akt and p-Akt in endometrial cancer cells after AE treatment were analyzed.The results showed that the expression of c-myc,β-catenin and p-GSK-3β(Ser9)decreased with the increase of AE concentration(5μmol/L,P<0.5;10,20μmol/L,p<0.01),was concentration dependent;the expression of p-Akt increased significantly with the increase of AE concentration(10μmol/L,p<0.01;20 mu mol/L,p<0.001),not as expected.(5)After adding AE into AN3 CA cells and adding GSK-3β inhibitors CHIR99021 and AR-A01441872h,OD value was detected by CCK-8 method.The results showed that the cell activity recovered obviously after adding the inhibitor.These results suggest that AE may inhibit the proliferation of endometrial cancer cells through GSK-3β/β-catenin/c-myc pathway.Conclusion:(1)AE is an active component of aloe vera with strong inhibitory effect on endometrial cancer.(2)Seven core targets of AE on endometrial cancer were obtained based on network pharmacology:TP53,Casp3,MYC,CDKN1A,PTGS2,CCNB1 and CDK1.(3)qPCR assay confirmed that 6 of the 7 core targets of AE inhibiting endometrial cancer cells(TP53,MYC,CDKN1A,PTGS2,CCNB1,CDK1)mRNA expression met the expectation.AE may play an inhibitory role in endometrial cancer through these targets.(4)AE may inhibit the proliferation of endometrial cancer cells through GSK-3β/β-catenin pathway.