Application And Research Of Drug Relocation Strategy In Screening Castration Resistant Prostate Cancer Drugs

Research background and purpose The incidence of prostate cancer in China is increasing rapidly,and the proportion of patients with advanced disease is higher and the prognosis is worse.For patients with advanced prostate cancer,the current treatment is mainly dependent on endocrine therapy,including drug castration treatment,anti-androgen therapy,a new generation of androgen biosynthesis inhibitor treatment,can effectively control the progress of patients with disease.But the vast majority of patients will still appear castration resistant,and accompanied by metastasis.Therefore,the use of existing castrated prostate cancer molecular biology research basis,continue to develop new and effective treatment of drugs,is still the urgent task of this field.However,new drug research and development is still faced with long research and development cycle,human,capital cost is high,the probability of success is low.In the new drug research and development under the constraints of a variety of unfavorable factors,as well as precision medical treatment of individual needs,drug relocation(Drug Reposition)strategy,also known as the old drug new,that is,the existing mining of unknown drugs New use,because of its high input-output efficiency,has now become the current anti-cancer drug research and development of one of the important strategies.The public database CMap(CMap)is a drug research platform based on drug gene expression profile,which is one of the important tools in drug relocation research.The purpose of this study is to apply drug relocation strategy,based on gene expression profile,screening treatment of castrated resistance prostate cancer drugs,and to study its mechanism.Experimental method Data from the NCBI website on the GEO free shared database Download the data set GSE32269 covering the gene expression profiles of the hormone-sensitive prostate cancer in situ lesion tissue and the ovariectomized prostate cancer bone metastases.Using the R language software,the differential genes of the two groups were analyzed by linear regression and empirical Bayesian method(limma).Parallel GO analysis and KEGG pathway analysis.The differential gene was then transformed into a probe number,introduced into the CMap database platform,and the gene expression profile was compared and scored.We selected the prostate cancer cell line PC3 alignment results to p ≤ 0.05,enrichment ≤ 0.8,mean ≤-0.6 for drug screening criteria,screening a total of nine candidate drugs.Combined with CCK8 cell proliferation assay,and drug information(Drugbank,Super Target),to determine the candidate drug guanabenz.In vitro experiments,mainly through CCK8 cell proliferation test,cell cloning experiments,flow cytometry detection of apoptosis,cell scratches experiments,transwell chamber shuttle test(with or without matrigel covered pore membrane),western blot experiment,RT-q PCR experiment,Immunohistochemistry,immunofluorescence and other clear guanabenz on the castration of prostate cancer inhibition of cell proliferation,clonal formation,promote cell apoptosis,inhibition of cell migration,invasion and other malignant biological phenotype.In vitro experiments,we constructed PC3 cells that stabilized luciferase and m Cherry fluorescent protein.The mice were injected with subcutaneous xenografts based on PC3 cells and tibial plateau.The growth of subcutaneous xenografts was monitored by vernier caliper.The tumor load of the lung and tibia was measured.Experimental results(1)Part one Analysis of gene expression profile data set GSE32269,found that the two types of tissue gene expression was significantly different in the castrated prostate cancer bone metastases,93 of them were significantly up-regulated expression,149 genes were significantly down-regulated.GO analysis showed that the differential gene was mainly enriched in extracellular domain,structural molecular activity,cell adhesion and so on.KEGG pathway analysis differs mainly in enrichment of extracellular matrix interactions,focal adhesion,glutathione metabolism,amino sugar and nucleotide glucose metabolism.Gene corresponding probe number into the CMap database,the gene expression spectrum comparison,score.We selected the prostate cancer cell line PC3 alignment results to p ≤ 0.05,enrichment ≤ 0.8,mean ≤-0.6 for drug screening criteria,screening a total of nine candidate drugs.(2)Part two CCK8 and clone formation experiments showed that guanabenz was significantly inhibited by ovariectomized prostate cancer cell proliferation,clonal formation,and in a concentration-dependent manner.Annexin V-FITC / PI marker double-labeled flow detection showed that guanabenz was significantly promoted by apoptosis and cycle arrest in ovariectomized prostate cancer cells.Cell scratch test and transwell chamber shuttle test showed that guanabenz was able to inhibit the migration and invasion of prostate cancer cells.RT-q PCR,Western blot and immunofluorescence assay showed that guanabenz was significantly inhibited by epithelial stromal changes in prostate cancer cells.Based on PC3 cell subcutaneous xenografts,tibial plateau injection of bone metastases in nude mice model experiments showed that guanabenz was significantly inhibited by inhibition of tumor growth and metastatic tumor load growth.(3)Part three RT-q PCR and Western blot showed that GADD34 m RNA and protein expression in ovariectomized prostate cancer were significantly higher than that in androgen-dependent prostate cancer cells and normal prostate epithelial cells.The expression of GADD34 in ovariectomized prostate cancer cells was higher than that in androgen-dependent prostate cancer and normal prostate cancer epithelial cells.And guanabenz can inhibit the dephosphorylation of p-ei F2α.When knocked down GADD34,the proliferation of castrated prostate cancer cells was inhibited,but at the same time,in vitro,in vivo experiments have found knocking down GADD34,the tumor cells on the sensitivity of guanabenz is also significantly reduced.Conclusion(1)ovariectomized prostate cancer has a great change in gene expression of hormone-sensitive prostate cancer,and it is feasible and effective to carry out drug repositioning based on this differential gene expression profile.(B)guanabenz in vivo,in vitro inhibition of prostate cancer cell proliferation,metastasis,and promote its apoptosis.(C)guanabenz mainly by combining GADD34(growth arrest and DNA damage inducible gene 34)to play the role of anti-cancer.