RESUMO
Calcitriol, vitamin D3 (VD3), and structurally related VD3 analogues are inhibitors of Hh signaling in multiple contexts and are promising anti-cancer agents in Hh-dependent forms of cancer; however, the cellular mechanisms through which these compounds regulate Hh signal transmission are not clearly defined. Previous studies in this area have implicated both Smoothened, a key mediator of Hh signaling, and the vitamin D receptor (VDR) as potential mediators of Hh inhibition for this class of seco-steroids. We have performed a series of in vitro studies to more fully probe the cellular mechanisms that govern seco-steroid-mediated inhibition of Hh signaling. Our results support a role for both the Hh and VDR pathways in this process, as well as the possibility that other, as yet unidentified proteins, are also central to seco-steroid-mediated inhibition of Hh signaling.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores de Calcitriol/metabolismo , Secoesteroides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , CamundongosRESUMO
Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.
Assuntos
DNA Polimerase Dirigida por DNA , Sistemas de Liberação de Medicamentos , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotidiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/química , Nucleotidiltransferases/genética , Proteínas/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Itraconazole (ITZ) is an FDA-approved member of the triazole class of antifungal agents. Two recent drug repurposing screens identified ITZ as a promising anticancer chemotherapeutic that inhibits both the angiogenesis and hedgehog (Hh) signaling pathways. We have synthesized and evaluated first- and second-generation ITZ analogues for their anti-Hh and antiangiogenic activities to probe more fully the structural requirements for these anticancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anticancer properties of the ITZ scaffold.
Assuntos
Antifúngicos/uso terapêutico , Antineoplásicos/uso terapêutico , Itraconazol/uso terapêutico , Animais , Linhagem Celular , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos C3H , RNA Mensageiro/genética , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
A structure-activity relationship study for a series of vitamin D3-based (VD3) analogues that incorporate aromatic A-ring mimics with varying functionality has provided key insight into scaffold features that result in potent, selective Hedgehog (Hh) pathway inhibition. Three analogue subclasses containing (1) a single substitution at the ortho or para position of the aromatic A-ring, (2) a heteroaryl or biaryl moiety, or (3) multiple substituents on the aromatic A-ring were prepared and evaluated. Aromatic A-ring mimics incorporating either single or multiple hydrophilic moieties on a six-membered ring inhibited the Hh pathway in both Hh-dependent mouse embryonic fibroblasts and cultured cancer cells (IC50 values 0.74-10 µM). Preliminary studies were conducted to probe the cellular mechanisms through which VD3 and 5, the most active analogue, inhibit Hh signaling. These studies suggested that the anti-Hh activity of VD3 is primarily attributed to the vitamin D receptor, whereas 5 affects Hh inhibition through a separate mechanism.
Assuntos
Colecalciferol/análogos & derivados , Proteínas Hedgehog/antagonistas & inibidores , Animais , Células Cultivadas , Colecalciferol/química , Avaliação Pré-Clínica de Medicamentos , Proteínas Hedgehog/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Relação Estrutura-AtividadeRESUMO
Hes1 is a key transcriptional regulator primarily controlled by the Notch signaling pathway, and recent studies have demonstrated both an oncogenic and tumor suppressor role for Hes1, depending on the cell type. Small molecules that activate and inhibit Hes1 activity hold promise as future anticancer chemotherapeutics. We have utilized a cell-based dual luciferase assay to identify modulators of Hes1 expression in a medium-throughput format. A modest screen was performed in HCT-116 colon cancer cell lines, and two small molecules were identified and characterized as Hes1 regulators. Compound 3 induced Hes1 expression and exhibited anticancer effects in pulmonary carcinoid tumor cells, a cell type in which the upregulated Notch/Hes1 signaling plays a tumor suppressive role. Treatment of HCT-116 cells with compound 12 resulted in Hes1 downregulation and antitumor effects.
Assuntos
Aminoacridinas/química , Compostos de Anilina/química , Antineoplásicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Cumarínicos/química , Proteínas de Homeodomínio/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Aminoacridinas/farmacologia , Aminoacridinas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Tumor Carcinoide/tratamento farmacológico , Tumor Carcinoide/metabolismo , Tumor Carcinoide/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Células HCT116 , Proteínas de Homeodomínio/metabolismo , Humanos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Fatores de Transcrição HES-1 , Regulação para Cima/efeitos dos fármacosRESUMO
Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.