| In recent years,the morbidity and mortality of malignant tumors remains high,which threatens human's life and health seriously.Although there are many ways to treat malignant tumors,such as surgical treatment,radiotherapy and chemotherapy,however,all of these mathods have some deficiencies.Whereas,targeted drug therapy,due to its strong specificity,small toxicity,low dose and so on,gradually becomes the main method to treat malignant tumor.Cripto-1 is a typical oncogene,which plays an important role in tumor cell migration,invasion,and tumor angiogenesis.Meanwhile,it was not or low expressed in normal human tissues and cells,whereas it was highly expressed in malignant tumors cells,thus Cripto-1 can be used as a target for screening of anti-tumor drugs.1.Screening and identification of the inhibitors of Cripto-1 expression targeting promoter and mRNA 3'UTRIn this study,the Cripto-1 promoter fragment was amplified from the cell genome,and linking it with the pGL3-basic vector.The luciferase activity detection result shown that the fragment we amplified has promoter activity,so it can be used to perform the follow-up compounds screening experiment.Secondly,more than 400 compounds were screened through the Cripto-1 promoter and Cripto-1 mRNA 3 'UTR luciferase reporter screening model,the results showed that compounds T150 and T173 significantly inhibited the activity of Cripto-1 promoter and Cripto-1 mRNA 3 'UTR respectively and also inhibited the expression of Cripto-1 protein.2.The in vitro antitumor activities of the inhibitors of Cripto-1 expressionSince the Cripto-1 can promote the occurrence of tumors,we detected the antitumor activity of T150 and T173.The effects of T150 and T173 on the cell riability were examined by MTT assay,and the results showed that both two compounds can inhibited tumor cell viabilities.Furthermore the compound T150 can perform it's antitumor activity in a concentration which has low toxicity to normal cells.3.The cell biological mechanisms involving in the antitumor activities of the inhibitors of Cripto-1 expressionInhibition of tumor cell proliferation and induction of tumor cell apoptosis are the main pathways of compounds perform it's anti-tumor activities.Therefore,we further study the proliferation inhibitory effect of T150 and T173 on the tumor cells.Flow cytometry results showed that T150 and T173 can induce Hela cell cycle arest at S phase.And Western blot results indicated that both T150 and T173 can down-regulate the expression of CyclinA/CDK2,up-regulate the expression of p21,and further influenced the normal cell cycle progression and inhibited cell proliferation.Then,we detected the effects of T150 and T173 on tumour cell apoptosis,DAPI staining result shown that there were many apoptotic bodies in the cells treated with two compounds.The proportion of apoptotic cells treated by T150 and T173 increased significantly by flow cytometry.Western blot results showed that T150 and T173 activated caspase-3 and caspase-9 protein,the expression of Pro apoptotic protein Bax increased,and the expression of apoptosis inhibitor protein Bcl-2 decreased,suggesting that these two compounds could induce cell apoptosis via mitochondrial pathway in Hela cells.Finally,the effects of T150 and T173 on tumor cell migration were also examined.The results showed that T150 and T173 could significantly inhibit the migration of tumor cells.It was found that the expression of epithelial cell marker E-cadherin increased,and the expression of stromal cell markers N-cadherin and vimentin decreased in the protein detection related to migration.These results suggest that T150 and T173 possess antitumor activity in vitro.To sum up,this study provided an effective anti-tumor drug screening model targeting Cripto-1 and found that the compounds T150 and T173 can influence tumor cell morphology,inhibit tumor cell proliferation and migration,induce tumor cell apoptosis.This research provided a new way for the research and treatment of Cripto-1 high expression tumor. |
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