title: Retinoic acid inhibits endometrial cancer cell growth via multiple genomic mechanisms.
abstract: Previous studies have indicated that retinoic acid (RA) may be therapeutic for endometrial cancer. However, the downstream target genes and pathways triggered by ligand-activated RA receptor α (RARα) in endometrial cancer cells are largely unknown. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and immunoblotting assays were used to assess the roles of RA and the RA agonist (AM580) in the growth of endometrial cancer cells. Illumina-based microarray expression profiling of endometrial Ishikawa cells incubated with and without AM580 for 1, 3, and 6 h was performed. We found that both RA and AM580 markedly inhibited endometrial cancer cell proliferation, while knockdown of RARα could block AM580 inhibition. Knockdown of RARα significantly increased proliferating cell nuclear antigen and BCL2 protein levels. Incubation of Ishikawa cells with or without AM580 followed by microarray expression profiling showed that 12 768 genes out of 47 296 gene probes were differentially expressed with significant P values. We found that 90 genes were the most regulated genes with the most significant P value (P<0.0001) using F-test. We selected four highly regulated genes with diverse functions, namely G0S2, TNFAIP2, SMAD3, and NRIP1. Real-time PCR verified that AM580 highly regulated these genes, whereas chromatin immunoprecipitation-PCR assay demonstrated that ligand-activated RARα interacted with the promoter of these genes in intact endometrial cancer cells. AM580 also significantly altered 18 pathways including those related to cell growth, differentiation, and apoptosis. In conclusion, AM580 treatment of Ishikawa cells causes the differential expression of a number of RARα target genes and activation of signaling pathways. These pathways could, therefore, mediate the carcinogenesis of human endometrial cancer.
authors.affiliation: Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, 303 East Superior Street, Suite 4-131, Chicago, Illinois 60611-3095, USA. y-cheng@northwestern.edu
authors.forename: You-Hong
authors.initials: YH
authors.lastname: Cheng
authors.forename: Hiroki
authors.initials: H
authors.lastname: Utsunomiya
authors.forename: Mary Ellen
authors.initials: ME
authors.lastname: Pavone
authors.forename: Ping
authors.initials: P
authors.lastname: Yin
authors.forename: Serdar E
authors.initials: SE
authors.lastname: Bulun
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001565:Benzoates / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016889:Endometrial Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D004717:Endometrium / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D005260:Female; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D016678:Genome / Q000187:drug effects; D006801:Humans; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D015398:Signal Transduction / Q000187:drug effects; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D001565:Benzoates; C000607071:RARA protein, human; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D013764:Tetrahydronaphthalenes; C068073:Am 580; D014212:Tretinoin