RESUMO
Prostate cancer (PC) is the second most prevalent cancer among men in Western societies, and those who develop metastatic castration-resistant PC (CRPC) invariably succumb to the disease. The need for effective treatments for CRPC is a pressing concern, especially due to limited durable responses with currently employed therapies. Here, we demonstrate the successful application of a high-throughput gene-expression profiling assay directly targeting genes of the androgen receptor pathway to screen a natural products library leading to the identification of 17ß-hydroxywithanolides 1-5, of which physachenolide D (5) exhibited potent and selective in vitro activity against two PC cell lines, LNCaP and PC-3. Epoxidation of 5 afforded physachenolide C (6) with higher potency and stability. Structure-activity relationships for withanolides as potential anti-PC agents are presented together with in vivo efficacy studies on compound 6, suggesting that 17ß-hydroxywithanolides are promising candidates for further development as CRPC therapeutics.
Assuntos
Androgênios/metabolismo , Antineoplásicos/química , Produtos Biológicos/química , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Vitanolídeos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Xenoenxertos , Ensaios de Triagem em Larga Escala , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Camundongos SCID , Transplante de Neoplasias , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Vitanolídeos/síntese química , Vitanolídeos/farmacologiaRESUMO
High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes.