RESUMEN

Deubiquitinating protease USP7 is a promising therapeutic target for cancer treatment, and interest in developing USP7 inhibitors has greatly increased. In the present study, we reported a series of natural pentacyclic triterpenes with USP7 inhibitory activity in vitro. Among them, both the ursane triterpenes and oleanane triterpenes were more active than the lupine triterpenes, whereas ursolic acid was the most potent with IC50 of 7.0±1.5 µmol/L. Molecular docking studies showed that ursolic acid might occupy the ubiquitin binding pocket of USP7, with the 17-carboxyl group and 3-hydroxyl group playing a vital role in the USP7-ursolic acid interaction. Using the cellular thermal shift assay, we demonstrated that ursolic acid interacted with USP7 in RPMI8226 human myeloma cells. Ursolic acid dose-dependently inhibited the proliferation of the myeloma cells with IC50 of 6.56 µmol/L, accompanied by reductions in USP7 substrates such as MDM2, UHRF1 and DNMT1. Overexpression of USP7 partially, but significantly attenuated ursolic acid-induced cell death as well as downregulation of MDM2, UHRF1 and DNMT1. In conclusion, we demonstrate for the first time that pentacyclic triterpenes represent a novel scaffold for developing USP7 inhibitors and that USP7 inhibition contributes to the anti-cancer effect of ursolic acid.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Triterpenos Pentacíclicos/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad , Triterpenos/antagonistas & inhibidores , Triterpenos/farmacología , Ubiquitina-Proteína Ligasas , Peptidasa Específica de Ubiquitina 7/biosíntesis , Ácido Ursólico

RESUMEN

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, ß-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

Asunto(s)

Inhibidores Enzimáticos/administración & dosificación , Ácido Oleanólico/análogos & derivados , Neoplasias Ováricas/tratamiento farmacológico , Peptidasa Específica de Ubiquitina 7/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/química , Ácido Oleanólico/farmacología , Neoplasias Ováricas/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Although 1,25­dihydroxyvitamin D3 (VD3) is regarded as a promising inducing agent for leukemia cell differentiation, it is not as effective an agent as all­trans­retinoic acid, and its usefulness is also limited by the adverse effects of hypercalcemia. The aim of the present study was to determine whether combining VD3 with adenanthin, a peroxiresoxin I (Prx I)­targeting natural compound, improves the efficacy of VD3. Cell viability was assessed using a trypan blue exclusion assay and flow cytometry was used to evaluate the expression of cell surface markers, CD11b/CD14, and the level of reactive oxygen species (ROS). Wright's staining was used to examine morphological changes and RNA­interference was used to knockdown Prx I and p65 gene expression. Protein expression was determined by western blot analysis. The results demonstrated that adenanthin markedly enhanced VD3­induced cell differentiation of leukemia NB4 cells, as evidenced by the increased percentage of CD11b­ and CD14­positive cells, the mature morphology of the monocytes and the increased phagocytic ability. Consistent with these results, knockdown of Prx I, but not nuclear factor­κB (p65), enhanced VD3­induced cell differentiation. The combinatorial effects of adenanthin and VD3 were shown to be associated with the ROS­CCAAT­enhancer­binding protein (C/EBP)ß axis, since N­acetylcysteine, a ROS scavenger, was able to abrogate the differentiation­enhancing effects of adenanthin, and the knockdown of C/EBPß also inhibited the combinatorial effects of adenanthin and VD3. In addition, co­treatment with adenanthin and VD3 was able to induce differentiation in other non­acute promyelocytic leukemia cells and primary leukemia cells. In conclusion, the results of the present study revealed a novel role for Prx I in VD3­induced cell differentiation, and suggested that targeting Prx I may represent a novel strategy to enhance VD3­induced leukemia cell differentiation.

Asunto(s)

Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , Peroxirredoxinas/antagonistas & inhibidores , Adulto , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Diterpenos de Tipo Kaurano/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Promielocítica Aguda/metabolismo , Monocitos/efectos de los fármacos , Monocitos/patología , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirement. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II-V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPß axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition.

Asunto(s)

Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/patología , Peroxirredoxinas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Animales , Western Blotting , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Ratones , Ratones Transgénicos , Resonancia por Plasmón de Superficie , Células Tumorales Cultivadas

RESUMEN

The normal epithelial cell-specific-1 (NES1) gene, also named as KLK10, is recognised as a novel putative tumour suppressor in breast cancer, but few studies have focused on the function of KLK10 in human prostate cancer. Our study confirms that the expression of KLK10 in prostate cancer tissue and cell lines (PC3, DU145, and LNCaP clone FGC) is low. Given that the androgen-independent growth characteristic of the PC3 cell line is more similar to clinical castration-resistant prostate cancer, we studied the role of KLK10 in PC3. In vitro and in vivo assays showed that over-expressing KLK10 in PC3 could decelerate tumour proliferation, which was accompanied with an increase in apoptosis and suppression of glucose metabolism. The related proteins, such as Bcl-2 and HK-2, were down-regulated subsequently. Furthermore, by up-regulating Bcl-2 or HK-2 respectively in the PC3-KLK10 cell line, we observed a subsequent increase of cell proliferation and a synchronous up-regulation of HK-2 and Bcl-2. Besides, KLK10 expression was also increased by Bcl-2 and HK-2, which suggests that there is a negative feedback loop between KLK10 and Bcl-2/HK-2. Thus, our results demonstrated that KLK10 may function as a tumour suppressor by repressing proliferation, enhancing apoptosis and decreasing glucose metabolism in PC3 cells.

Asunto(s)

Apoptosis/genética , Glucosa/metabolismo , Calicreínas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Caspasa 3/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fluorodesoxiglucosa F18 , Regulación Neoplásica de la Expresión Génica , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Calicreínas/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Microtomografía por Rayos X

RESUMEN

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

Asunto(s)

Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Ácido Oleanólico/análogos & derivados , Neoplasias Ováricas/patología , Antineoplásicos/química , División Celular/efectos de los fármacos , Línea Celular Tumoral , Ditiotreitol/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes erbB-2 , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estructura Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Ácido Oleanólico/antagonistas & inhibidores , Ácido Oleanólico/química , Ácido Oleanólico/farmacología , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor ErbB-2/biosíntesis , Relación Estructura-Actividad , Transfección , Regulación hacia Arriba/efectos de los fármacos