Screening And Antitumor Effect Evaluation In Vitro Of TSP50 Inhibitors Targeting The 3’UTR

In recent years,the incidence and mortality rates of gynecologic malignancies have been on a significant rise,and have gradually become an important factor threatening women’s life and health.Among many gynecologic malignancies,epithelial ovarian cancer(EOC)is the most common subtype of ovarian cancer,and its mortality rate is the highest among gynecologic malignancies.The high recurrence rate of EOC and resistance to chemotherapy have become major barriers to prolonging the survival of patients with advanced ovarian cancer.Therefore,there is an urgent need to develop new anti-cancer drugs to improve the prognosis of EOC patients.Testes-specific protease 50(TSP50)is a proto-oncogene that is highly expressed only in the testis but is aberrantly expressed in breast,gastric,cervical,colorectal and laryngeal cancers.It has been shown that down-regulation of TSP50 expression can inhibit cell proliferation and induce apoptosis,which is a potential target for tumor therapy.1.Screening and identification of compounds targeting the 3’UTR of TSP50 mRNAIn this study,pGL3-TSP50 3’UTR plasmid(luciferase reporter vector)was transfected into HEK-293 T cells to construct a screening model targeting TSP50 mRNA 3’UTR,and small molecule compounds that inhibit TSP50 expression were screened from our laboratory’s listed drug library by detecting luciferase activity.The results showed that compound A453 could inhibit luciferase activity,and the inhibitory effect of A453 on TSP50 protein expression was further confirmed by Western blot.2.Antitumor effect and selection of action concentration of A453To further determine the effective action concentration of A453 on epithelial ovarian cancer cell lines,its effect on the cell viability of normal ovarian epithelial cells(HOSEpi C)and the corresponding tumor cells(A2780)was firstly examined.Then,the drug concentration of 5 μg/m L was determined for the subsequent study based on the IC10 value of A453 on HOSEpi C and the IC50 value on A2780.To confirm whether the inhibitory effect of A453 on A2780 cells was related to its inhibition of TSP50 expression,we treated TSP50 overexpressed A2780 cells with A453.It was found that TSP50 overexpression could rescue the inhibitory effect of A453 on A2780 cell viability.The anti-tumor effect of A453 was revealed to be at least partially mediated by the inhibition of TSP50 expression.3.In vitro anti-tumor mechanism study of A453To investigate the in vitro anti-tumor mechanism of A453,we investigated the effects of A453 on apoptosis,proliferation,migration and expression of angiogenesisrelated factors in A2780 cells.First,the apoptosis of A2780 cells was detected by TUNEL staining after treatment with A453,and the results showed that A453 obviously promoted the apoptosis of A2780 cells.Meanwhile,the results of Annexin V/PI double staining method by flow cytometry also confirmed the above conclusion.Further Western blot analysis results showed that A453 activated Caspase-9,Caspase-8 and Caspase-3 and activated endogenous and exogenous apoptotic pathways,thus promoting apoptosis in A2780 cells.After that,the effect of A453 on the DNA synthesis ability of A2780 cells was examined by Brd U doping method.The results showed that A453 had a significant inhibitory effect on the proliferation of A2780 cells.It was also found that A453 could induce G1 phase block and inhibit cell proliferation in A2780 cells by down-regulating the expression of Cyclin D1,Cyclin E,CDK2 and CDK4 and up-regulating the expression of p21.Finally,effect of A453 on the migration of A2780 cells was examined by cell scratch assay and Transwell assay.The results showed that A453 could significantly inhibit the migration of A2780 cells.Afterwards,we found that E-cadherin expression was up-regulated and Vimentin and N-cadherin expression levels were decreased after treating A2780 cells with A453 by Western blot assay.Meanwhile,the expressions of VEGF and HIF-1α were significantly down-regulated,suggesting that they may inhibit neovascularization.In summary,this study utilized a screening model targeting TSP50 mRNA 3’UTR to screen small molecule compound A453 with in vitro antitumor activity,which promoted apoptosis and significantly inhibited the proliferation and migration of A2780 cells.This study provides a theoretical basis for the clinical treatment of epithelial ovarian cancer.