RESUMO
Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of â¼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Exossomos/metabolismo , Neoplasias da Próstata/química , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Masculino , Neoplasias da Próstata/classificação , Neoplasias da Próstata/metabolismo , Técnica de Seleção de Aptâmeros , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
BACKGROUND: Currently available 5α-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel 5α-reductase, 5α-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP. METHODS: A new protein with potential 5α-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry. RESULTS: The relative mRNA expression for the three 5α-reductase enzymes in clinical samples of CR-CaP was 5α-reductase-3 â« 5α-reductase-1> 5α-reductase-2. Recombinant 5α-reductase-3 protein incubations converted testosterone, 4-androstene-3,17-dione (androstenedione) and 4-pregnene-3,20-dione (progesterone) to dihydrotestosterone, 5α-androstan-3,17-dione, and 5α-pregnan-3,20-dione, respectively. 5α-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of 5α-reductase-1 and 2). CONCLUSION: Human prostate tissues contain a third 5α-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low.