RESUMO
The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anoikis/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Tirosina Fosfatases/genética , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. We have developed two high-throughput methods for measuring heparanase activity and screening potential inhibitors. The first method involves coating fibroblast growth factor (FGF) on microtiter plates and capturing fluorescein isothiocyanate (FITC)-labeled heparin sulfate (HS), which is used as a substrate for heparanase digestion. Labeled HS fragments are released into the medium and quantitated by fluorescence intensity measurement. We have implemented this assay method into a Zeiss uHTS system and screened compound libraries for heparanase inhibitors. The second method involves labeling HS with biotin followed by FITC to generate a dual-labeled HS. The labeled material is bound to streptavidin-coated plates and used as a substrate for heparanase digestion. Both methods are sensitive and easily applicable to robotic systems. In addition, we have labeled both HS and biotin-HS with Eu-chelate, a fluorophore that exhibits long decay fluorescence. Assays using Eu-labeled HS and Eu-labeled biotin-HS have been developed and show higher sensitivity than those using FITC-labeled material. Furthermore, assays using Eu-chelate HS (or biotin-HS) should eliminate the interference of fluorescence compounds.
