Screening And Antitumor Effect Evaluation In Vitro Of Cripto-1 Inhibitors Targeting The 3'UTR

Malignant tumor has become a major public health issue facing our country and the world,and a serious threat to human health and life.With the development of science and technology,besides of the traditional surgical operation,radiotherapy and chemotherapy,cancer therapies also contain molecular targeting treatment,which has strong pertinence,smalldosage of administration,low toxic and side effects,high efficiency etc.and thus gradually becomes a research focus in the global cancer therapies area.Cripto-1 has low expressionin somatic cells of normal adults,but has high expressionin a variety of malignant tumors,so it playsa role of oncogene,and promotes tumorigenesis,transformation,migration,invasionandangiogenesis.Therefore,Cripto-1 can be used asscreening and therapeutic target for antitumor drugs.In the study,we firstly amplified a 1210 bp sequence of Cripto-1mRNA3'UTR from Hela cells mRNA,and inserted it into pGL3-control vector and constructed the pGL3-Cripto-1 mRNA 3'UTR luciferase reporter vector.Then we transfectedthe pGL3-Cripto-1 mRNA 3'UTR luciferase reporter vector into HEK-293 T cells,and screened over 300 kinds of traditional Chinese medicine compounds in our laboratory.The result showed that compound TI574 has a obvious inhibiting effect on the activity of Cripto-1 mRNA 3'UTR,and TI574 can inhibit the expression of Cripto-1 in both mRNA andprotein levels.Because Cripto-1 has promoting effect on tumorigenesis,so we tested antitumor activity of TI574.We first detected the effect of TI574 on cell vitality by MTT assay.The result showed that the inhibitingeffectofTI574 on cell viability in tumor cells are significantly higher than in normal cells.The IC10 and IC50 values of TI574 on normal cells GES-1 and L02 were significantly higher than those on three tumor cell lines including SGC-7901,HepG2 and Hela,which indicates that TI574 can perform anti-tumor effect under the doses of lower toxicity to normal cell,thussuggested that TI574 can be developed as a potential antitumor drug.Inducing tumor cell apoptosis and inhibiting tumor cell proliferation are the main approaches for compounds performits antitumor activity.Therefore,we analyzed the effect of compound TI574 on inducing tumor cell apoptosis.Annexin V-FITC/PI double parameter flow cytometry tests showed that TI574 can induce the apoptosis of Hela and HepG2 cells.Western blot showed that TI574 can activate caspase9 andcaspase3,downregulated Bcl-2,and upregulated Bax expression,these results suggest that TI574 can induce tumor cell apoptosis through the mitochondrial apoptosis pathway.Then we studied the effects of TI574 on cell proliferation.Flow cytometry showed that TI574 can induce S phase arrest in HepG2 and Hela cells.Western blot showed that TI574 can downregulate cell cycle regulating complex CyclinA/CDK2,upregulate p21 expression,therefore inhibit cell proliferation in Hela and HepG2 cells.Finally we tested the effects of TI574 on tumor cell migration and invasion.Wound healing test and transwell assay showed that TI574 can inhibit the migration and invasion of tumor cells.Western blot showed that TI574 can upregulate the expression of E-cadherin,and down regulate the expression of N-cadherin and Vimentin in tumor cells.The above results suggest that TI574 has antitumor activity in vitro.To further explore the correlation between the inhibitory effect of TI574 on the expression of Cripto-1 and the antitumor effect of TI574,we overexpressed Cripto-1in Hela cells and then treated with TI574.The results showed a decreased inhibitory effect of TI574 on cell proliferation,indicating that at least a part of the antitumor effect of TI574 was performed by inhibiting the expression of Cripto-1.In conclusion,we constructed an effective screening model for screeningof anti-tumor drugs targeting Cripto-1 mRNA 3'UTR,and obtained the compoundTI574,which can inhibit cell proliferation,migration and invasion in tumor cells,and induce tumor cellsapoptosis.This study provideda lead compound for antitumordrugs developmentand also provided a new approach for the treatment of tumors with high expression of Cripto-1.