Screening Of Antitumor Drugs Targeting 3'UTR Of TSP50 MRNA And Analysis Of Its Antitumor Effect

Malignant tumors are seriously threatening human's life.Tumor treatment methods includes surgical treatment,radiotherapy,chemotherapy,immunotherapy,and molecularly targeted therapies.Among them,molecular targeted therapy has attracted more and more attention because of its advantages of strong targeting,small dose,and high efficiency.However,most targets exist in both tumor cells and normal cells,the drugs targeting these targets are harmful to human body,which prompts us to develop more novel drugs specific targeting tumor.Testes-specific protease 50 is a proto-oncogene.It is specifically expressed highly in the testis and lower in other normal tissues,such as brain,stomach,kidney,liver,etc.Studies have shown that TSP50 is highly expressed in variety of malignancies,including breast cancer,and associated with a poor prognosis of malignancy.Down-regulation of TSP50 expression can inhibit the tumor formation and tumor cell proliferation,migration and invasion.Therefore,the drugs targeting TSP50 may have less toxicity to normal tissue.Although TSP50 targeting drug screening has achieved preliminary results,but we still need to continue it.In this study,we choose TSP50 mRNA 3'UTR as a target to screened over 800 small molecule compounds,and further clarified the anti-tumor activity of the selected inhibitors of TSP50 expression.1.Screening and Identification of the Inhibitors of TSP50 Expression Targeting TSP50 mRNA 3'UTRWe transfected cells with pGL3-TSP50 3'UTR and treated the cells with 810 small molecule compounds individually,then we found that compounds A132 and A246 both significantly inhibited the luciferase activity of the cells,and can inhibit the expression of TSP50 protein in breast cancer.2.Antitumor Effects of Targeted CompoundsBefore investigating the antitumor effects of compounds A132 and A246,we first determined the optimal concentration of compounds based on its IC10 value to normal cells and IC50 value to tumor cells.We detected the effects of A132 and A246 on cell viabilities by MTT method,and found that both of compounds can inhibit the viabilities of the tumor cells such as MDA-MB-231,HepG2 and SGC-7901 in 5 ?g/mL concentration which have low toxicities to normal cells MCF-10 A,L02 and GES-1.Therefore we finally choose 5 ?g/mL for subsequent studies.To further explore whether the inhibitory effects of A132 and A246 on tumor cell viability related to its inhibitory effect on TSP50 expression,we overexpressed the TSP50 in MDA-MB-231 cells,and analyzed the cell viability,the results shown that the inhibitory effect of A132 and A246 on cell viabilities were significantly rescued by TSP50 overexpression.These results indicated that the in vitro antitumor effects of A132 and A246 at least partially via inhibiting the expression of TSP50.3.Antitumor Mechanisms of Targeted CompoundsThen we detected the antitumor activity of A132 and A246.We first investigate the effects of A132 and A246 on cell apoptosis in MDA-MB-231 cells.After DAPI staining,obvious apoptotic bodies were observed in cells after treatment with compounds for 48 hours.And Annexin V-FITC/PI double labeling flow cytometry results showed that A132 and A246 could significantly induce the apoptosis of MDA-MB-231 cells.The western blot results showed that A132 and A246 could activated Caspase3,down-regulated the expression of Bcl-2 and up-regulated the expression of Bax.These results suggest that A132 and A246 may induce apoptosis of MDA-MB-231 cells through mitochondrial pathway.Then we examined the effect of compound A132 and A246 on the proliferation of MDA-MB-231 cells.BrdU incorporation assay showed that both A132 and A246 could significantly inhibit the DNA synthesis of MDA-MB-231 cells,and the inhibitory effect was dose-dependent.We further detected the effects of compounds on cell cycle by using flow cytometry.and the results showed that both A132 and A246 could induce G2/M arrest.Western blot analysis showed that A132 and A246 could down-regulated the expression of CyclinB-CDK1 complex and cycle arrest protein p21.Therefore,compounds A132 and A246 can inhibit cell proliferation via inducing cell cycle arrest.Finally,we examined the effect of A132 and A246 on tumor cell migration by wound healing test and the results showed that A132 and A246 could significantly inhibit the migration of MDA-MB-231 cells in a time-dependent manner.In summary,in this study,we identified two antitumor compounds A132 and A246 by using a screening model targeting TSP50 mRNA 3'UTR.Both of A132 and A246 could induce tumor cell apoptosis,and inhibit their proliferation and migration.Our study layed a foundation for the development of antitumor drugs targeting TSP50.