RESUMO
Nucleophosmin (NPM), a multifunctional nucleolar phosphoprotein is dysregulated in human malignancies leading to anti-apoptosis and inhibition of differentiation. We evaluated the precise three-dimensional structure of NPM based on the highly conserved structure of Xenopus NO38 and its requirement to form dimers and pentamers via its N-terminal domain (residues, 1-107). We hypothesized that a small molecular inhibitor (SMI) that could disrupt the formation of dimers would inhibit aberrant NPM function(s) in cancer cells. Molecular modeling, pharmacophore design, in silico screening and interactive docking identified NSC348884 as a putative NPM SMI that disrupts a defined hydrophobic pocket required for oligomerization. NSC348884 inhibited cell proliferation at an IC(50) of 1.7-4.0 muM in distinct cancer cell lines and disrupted NPM oligomer formation by native polyacrylamide gel electrophoresis assay. Treatment of several different cancer cell types with NSC348884 upregulated p53 (increased Ser15 phosphorylation) and induced apoptosis in a dose-dependent manner that correlated with apoptotic markers: H2AX phosphorylation, poly(ADP-ribose) polymerase cleavage and Annexin V labeling. Further, NSC348884 synergized doxorubicin cytotoxicity on cancer cell viability. The data together show that NSC348884 is an SMI of NPM oligomer formation, upregulates p53, induces apoptosis and synergizes with chemotherapy. Hence, an SMI to NPM may be a useful approach to anticancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Citotoxinas/química , Dimerização , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes p53/efeitos dos fármacos , Humanos , Indóis/administração & dosagem , Indóis/química , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/farmacologia , Nucleofosmina , Homologia de Sequência do Ácido Nucleico , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
