RESUMEN
Increased survival of cancer cells mediated by high levels of ionizing radiation (IR) reduces the effectiveness of radiation therapy for non-small cell lung cancer (NSCLC). In the present study, danshensu which is a selected component of traditional oriental medicine (TOM) compound was found to reduce the radioresistance of NSCLC by inhibiting the nuclear factor-κB (NF-κB) pathway. Of the various TOM compounds reported to inhibit the IR activation of NF-κB, danshensu was chosen as a final candidate based on the results of structural comparisons with human metabolites and monoamine oxidase B (MAOB) was identified as the putative target enzyme. Danshensu decreased the activation of NF-κB by inhibiting MAOB activity in A549 and NCI-H1299 NSCLC cells. Moreover, it suppressed IR-induced epithelial-to-mesenchymal transition, expressions of NF-κB-regulated prosurvival and proinflammatory genes, and in vivo radioresistance of mouse xenograft models. Taken together, this study shows that danshensu significantly reduces MAOB activity and attenuates NF-κB signaling to elicit the radiosensitization of NSCLC.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Lactatos/farmacología , Neoplasias Pulmonares/terapia , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/genética , Tolerancia a Radiación/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Medicamentos Herbarios Chinos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de la radiación , Rayos gamma/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Medicina Tradicional de Asia Oriental , Ratones , Ratones Desnudos , Monoaminooxidasa/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism.
Asunto(s)
Calorimetría/métodos , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/enzimología , Solanum tuberosum/enzimología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Unión Competitiva , Western Blotting , Glucosa-1-Fosfato Adenililtransferasa/química , Glucofosfatos/química , Glucofosfatos/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , Proteínas de Plantas/química , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
The hedgehog (Hh) signalling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis and progression of a variety of aggressive human cancers including skin cancer. Despite its importance, little is known about its role in photoageing, a type of UV-induced skin lesions. In this study, we investigated the involvement of Hh signalling in the photoageing process as well as the use of an Hh-regulating alkaloid compound as a novel therapeutic drug to regulate photoageing in keratinocytes. We found that UVB induced Hh signalling by the expression of Hh ligands and Hh-mediated transcription factors, respectively. Moreover, UVB-induced Hh activation relied on mitogen-activated protein kinase (p38, ERK and JNK) activity and inflammatory responses (upregulation of COX-2, IL-1ß, IL-6 and TNF-α), resulting in premature senescence and photoageing in vitro and in vivo. Notably, a selected Hh inhibitor, evodiamine, mediated photoageing blockade in a mouse skin model. Taken together, our findings demonstrated that Hh signalling is associated with UVB-induced photoageing, while pharmacological inhibition of Hh signalling significantly reduced experimental photoageing, indicating its potential for use as a therapeutic target for this disease.
Asunto(s)
Anilidas/uso terapéutico , Flavonoides/uso terapéutico , Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/uso terapéutico , Quinazolinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Envejecimiento de la Piel/efectos de los fármacos , Rayos Ultravioleta/efectos adversos , Envejecimiento/genética , Envejecimiento/metabolismo , Anilidas/farmacología , Animales , Línea Celular , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Citocinas/biosíntesis , Citocinas/genética , Evaluación Preclínica de Medicamentos , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Proteínas Hedgehog/biosíntesis , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiología , Humanos , Inflamación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Piridinas/farmacología , Quinazolinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Envejecimiento de la Piel/genética , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Potato (Solanum tuberosum) multicystatin (PMC) is a unique cystatin composed of eight repeating units, each capable of inhibiting cysteine proteases. PMC is a composite of several cystatins linked by trypsin-sensitive (serine protease) domains and undergoes transitions between soluble and crystalline forms. However, the significance and the regulatory mechanism or mechanisms governing these transitions are not clearly established. Here, we report the 2.2-Å crystal structure of the trypsin-resistant PMC core consisting of the fifth, sixth, and seventh domains. The observed interdomain interaction explains PMC's resistance to trypsin and pH-dependent solubility/aggregation. Under acidic pH, weakening of the interdomain interactions exposes individual domains, resulting in not only depolymerization of the crystalline form but also exposure of cystatin domains for inhibition of cysteine proteases. This in turn allows serine protease-mediated fragmentation of PMC, producing â¼ 10-kD domains with intact inhibitory capacity and faster diffusion, thus enhancing PMC's inhibitory ability toward cysteine proteases. The crystal structure, light-scattering experiments, isothermal titration calorimetry, and site-directed mutagenesis confirmed the critical role of pH and N-terminal residues in these dynamic transitions between monomer/polymer of PMC. Our data support a notion that the pH-dependent structural regulation of PMC has defense-related implications in tuber physiology via its ability to regulate protein catabolism.
Asunto(s)
Cistatinas/química , Proteínas de Plantas/química , Solanum tuberosum/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Cistatinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO(4)(-)-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, alpha-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.
Asunto(s)
Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Proteínas de Plantas/química , Solanum tuberosum/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Cistatinas/genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Solanum tuberosum/citología , Solanum tuberosum/metabolismoRESUMEN
(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.