RESUMEN
Hepatocellular carcinoma (HCC) is the fifth most common neoplasm in the world. Chronic inflammation of liver and associated wound healing processes collectively contribute to the development of cirrhosis which further progresses to dysplastic nodule and then to HCC. Etiological mediators and ongoing manipulations at cellular level in HCC are well established; however, key protein interactions and genetic alterations involved in stepwise hepatocarcinogenic pathways are seldom explored. This study aims to unravel novel targets of HCC and repurpose the FDA-approved drugs against the same. Genetic data pertinent to different stages of HCC were retrieved from GSE6764 dataset and analyzed via GEO2R. Subsequently, protein-protein interaction network analysis of differentially expressed genes was performed to identify the hub genes with significant interaction. Hub genes displaying higher interactions were considered as potential HCC targets and were validated thorough UALCAN and GEPIA databases. These targets were screened against FDA-approved drugs through molecular docking and dynamics simulation studies to capture the drugs with potential activity against HCC. Finally, cytotoxicity of the shortlisted drug was confirmed in vitro by MTT assay. CDC20 was identified as potential druggable target. Docking, binding energy calculations, and dynamic studies revealed significant interaction exhibited by Labetalol with CDC20. Further, in MTT assay, Labetalol demonstrated an IC50 of 200.29 µg/ml in inhibiting the cell growth of HepG2 cell line. In conclusion, this study discloses a series of key genetic underpinnings of HCC and recommends the pertinence of labetalol as a potential repurposable drug against HCC.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Biología Computacional/métodos , Reposicionamiento de Medicamentos , Neoplasias Hepáticas/tratamiento farmacológico , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/fisiología , Humanos , Labetalol/farmacología , Cirrosis Hepática/etiología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Simulación del Acoplamiento Molecular , Mapas de Interacción de ProteínasRESUMEN
OBJECT: Find potential therapeutic targets of triple-negative breast cancer (TNBC) patients by bioinformatics. Screen ideal natural ligand that can bind with the potential target and inhibit it by using molecular biology. METHODS: Bioinformatics and molecular biology were combined to analyze potential therapeutic targets. Differential expression analysis identified the differentially expressed genes (DEGs) between TNBC tissues and non-TNBC tissues. The functional enrichment analyses of DEGs shown the important gene ontology (GO) terms and pathways of TNBC. Protein-protein interaction (PPI) network construction screened 20 hub genes, while Kaplan website was used to analyze the relationship between the survival curve and expression of hub genes. Then Discovery Studio 4.5 screened ideal natural inhibitors of the potential therapeutic target by LibDock, ADME, toxicity prediction, CDOCKER and molecular dynamic simulation. RESULTS: 1,212 and 353 DEGs were respectively found between TNBC tissues and non-TNBC tissues, including 88 up-regulated and 141 down-regulated DEGs in both databases. 20 hub genes were screened, and the higher expression of CDC20 was associated with a poor prognosis. Therefore, we chose CDC20 as the potential therapeutic target. 7,416 natural ligands were conducted to bind firmly with CDC20, and among these ligands, ZINC000004098930 was regarded as the potential ideal ligand, owing to its non-hepatotoxicity, more solubility level and less carcinogenicity than the reference drug, apcin. The ZINC000004098930-CDC20 could exist stably in natural environment. CONCLUSION: 20 genes were regarded as hub genes of TNBC and most of them were relevant to the survival curve of breast cancer patients, especially CDC20. ZINC000004098930 was chosen as the ideal natural ligand that can targeted and inhibited CDC20, which may give great contribution to TNBC targeted treatment.
Asunto(s)
Proteínas Cdc20/antagonistas & inhibidores , Biología Computacional/métodos , Mapas de Interacción de Proteínas/genética , Transcriptoma/genética , Neoplasias de la Mama Triple Negativas , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ligandos , Simulación de Dinámica Molecular , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
Apcin is one of the few compounds that have been previously reported as a Cdc20 specific inhibitor, although Cdc20 is a very promising drug target. We reported here the design, synthesis, and biological evaluations of 2,2,2-trichloro-1-aryl carbamate derivatives as Cdc20 inhibitors. Among these derivatives, compound 9f was much more efficient than the positive compound apcin in inhibiting cancer cell growth, but it had approximately the same binding affinity with apcin in SPR assays. It is possible that another mechanism of action might exist. Further evidence demonstrated that compound 9f also inhibited tubulin polymerization, disorganized the microtubule network, and blocked the cell cycle at the M phase with changes in the expression of cyclins. Thus, it induced apoptosis through the activation of caspase-3 and PARP. In addition, compound 9f inhibited cell migration and invasion in a concentration-dependent manner. These results provide guidance for developing the current series as potential new anticancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Carbamatos/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Diaminas/farmacología , Moduladores de Tubulina/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Carbamatos/síntesis química , Carbamatos/metabolismo , Proteínas Cdc20/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diaminas/síntesis química , Diaminas/metabolismo , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Células Hep G2 , Humanos , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/metabolismoRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase that initiates anaphase and mitotic exit. APC/C is activated by Cdc20 and inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin as a small molecule ligand of Cdc20 that inhibits APC/CCdc20 and prolongs mitosis. Here we find that apcin paradoxically shortens mitosis when SAC activity is high. These opposing effects of apcin arise from targeting of a common binding site in Cdc20 required for both substrate ubiquitination and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Carbamatos/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Diaminas/farmacología , Mitosis/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Células HCT116 , Células HeLa , Humanos , Nocodazol/farmacología , Proteínas Nucleares/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Imagen de Lapso de Tiempo , UbiquitinaciónRESUMEN
Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G2/M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G2/M transition and thereby controls mitosis and cell division.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Ciclo Celular , Ciclina B1/química , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Mitosis , Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/genética , Células HEK293 , Células HeLa , Humanos , Huso Acromático , Especificidad por SustratoRESUMEN
Osteosarcoma (OS) is one of the aggressive malignancies for young adults. Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in OS, suggesting that inhibition of Cdc20 could be a novel strategy for the treatment of OS. Since Cdc20 inhibitors have side effects, it is important to discover the new CDC20 inhibitors with non-toxic nature. In the present study, we determine whether natural agent diosgenin is an inhibitor of Cdc20 in OS cells. We performed MTT, FACS, Wound healing assay, Transwell, Western blotting, transfection assays in our study. We found diosgenin inhibited cell growth and induced apoptosis. Moreover, diosgenin exposure led to inhibition of cell migration and invasion. Notably, diosgenin inhibited the expression of Cdc20 in OS cells. Overexpression of Cdc20 abrogated the inhibition of cell growth and invasion induced by diosgenin. Our data reveal that inhibition of Cdc20 by diosgenin could be helpful for the treatment of patients with OS.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Proteínas Cdc20/antagonistas & inhibidores , Diosgenina/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Invasividad Neoplásica , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patologíaRESUMEN
Small interfering RNA (siRNA) delivered to silence overexpressed genes associated with malignancies is a promising targeted therapy to decrease the uncontrolled growth of malignant cells. To create potent delivery agents for siRNA, here we formulated additive polyplexes of siRNA using linoleic acid-substituted polyethylenimine and additive polymers (hyaluronic acid, poly(acrylic acid), dextran sulfate, and methyl cellulose) and characterized their physicochemical properties and effectiveness. Incorporating polyanionic polymer along with anionic siRNA in polyplexes was found to decrease the ζ-potential of polyplexes but enhance the cellular delivery of siRNA. The CDC20 and survivin siRNAs delivered by additive polyplexes showed promising efficacy in breast cancer MDA-MB-231, SUM149PT, MDA-MB-436, and MCF7 cells. However, the side effects of the siRNA delivery were observed in nonmalignant cells, and a careful formulation of siRNA/polymer polyplexes was needed to minimize side effects on normal cells. Because the efficacy of siRNA delivery by additive polyplexes was independent of breast cancer phenotypes used in this study, these polyplexes could be further developed to treat a wide range of breast cancers.
Asunto(s)
Neoplasias de la Mama/terapia , Proteínas Cdc20/antagonistas & inhibidores , Ácido Hialurónico/química , Polietileneimina/química , Polímeros/química , ARN Interferente Pequeño/genética , Survivin/antagonistas & inhibidores , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Cdc20/genética , Proliferación Celular , Femenino , Silenciador del Gen , Humanos , Polielectrolitos , ARN Interferente Pequeño/química , Survivin/genética , Células Tumorales CultivadasRESUMEN
Osteosarcoma (OS) is the most common primary malignant bone tumor worldwide, primarily affecting children and adolescents. The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that is activated by its coactivator Cdc20 during the metaphaseanaphase transition. Apcin is a novel cellpermeable molecule that blocks the interaction between APC/C and Cdc20. Cdc20 overexpression has been reported in various malignancies and plays an oncogenic role in tumorigenesis and tumor progression. In the present study, the antitumor properties of apcin, an inhibitor of Cdc20, was investigated in OS cell lines. In addition, the possible molecular target by which apcin mediates cell death was explored. Apcin was demonstrated to inhibit OS cell growth and induce significant apoptosis. The invasion and mobile abilities of OS cells were also markedly suppressed by apcin treatment. Furthermore, Bim and p21 were upregulated in OS cells following apcin treatment. The results of the present study indicated that apcin may have therapeutic potential as a treatment for OS and that Cdc20 may be a promising molecular target for chemotherapy.
Asunto(s)
Carbamatos/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Diaminas/farmacología , Osteosarcoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Progresión de la Enfermedad , Humanos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1), known as IDO, catabolizes tryptophan through kynurenine pathway, whose activity is correlated with impaired clinical outcome of colorectal cancer. Here we showed that 1-L-MT, a canonical IDO inhibitor, suppressed proliferation of human colorectal cancer cells through inducing mitotic death. Our results showed that inhibition of IDO decreased the transcription of CDC20, which resulted in G2/M cycle arrest of HCT-116 and HT-29. Furthermore, 1-L-MT induced mitochondria injuries and caused apoptotic cancer cells. Importantly, 1-L-MT protected mice from azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon carcinogenesis, with reduced mortality, tumor number and size. What is more, IDO1-/- mice exhibited fewer tumor burdens and reduced proliferation in the neoplastic epithelium, while, 1-L-MT did not exhibit any further protective effects on IDO-/- mice, confirming the critical role of IDO and the protective effect of 1-L-MT-mediated IDO inhibition in CRC. Furthermore, 1-L-MT also alleviated CRC in Rag1-/- mice, demonstrating the modulatory effects of IDO independent of its role in modulating adaptive immunity. Taken together, our findings validated that the anti-proliferation effect of 1-L-MT in vitro and the prevention of CRC in vivo were through IDO-induced cell cycle disaster of colon cancer cells. Our results identified 1-L-MT as a promising candidate for the chemoprevention of CRC.
Asunto(s)
Proteínas Cdc20/antagonistas & inhibidores , Colitis/complicaciones , Neoplasias del Colon/prevención & control , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Mitosis , Triptófano/farmacología , Animales , Apoptosis , Azoximetano/toxicidad , Carcinógenos/toxicidad , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Ciclo Celular , Proliferación Celular , Colitis/inducido químicamente , Neoplasias del Colon/enzimología , Neoplasias del Colon/etiología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Proteínas de Homeodominio/fisiología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Triptófano/química , Triptófano/uso terapéutico , Células Tumorales CultivadasRESUMEN
The F-box protein FBXO31 is a tumor suppressor that is encoded in 16q24.3, for which there is loss of heterozygosity in various solid tumors. FBXO31 serves as the substrate-recognition component of the SKP/Cullin/F-box protein class of E3 ubiquitin ligases and has been shown to direct degradation of pivotal cell-cycle regulatory proteins including cyclin D1 and the p53 antagonist MDM2. FBXO31 levels are normally low but increase substantially following genotoxic stress through a mechanism that remains to be determined. Here we show that the low levels of FBXO31 are maintained through proteasomal degradation by anaphase-promoting complex/cyclosome (APC/C). We find that the APC/C coactivators CDH1 and CDC20 bind to a destruction-box (D-box) motif present in FBXO31 to promote its polyubiquitination and degradation in a cell-cycle-regulated manner, which requires phosphorylation of FBXO31 on serine-33 by the prosurvival kinase AKT. Following genotoxic stress, phosphorylation of FBXO31 on serine-278 by another kinase, the DNA damage kinase ATM, results in disruption of its interaction with CDH1 and CDC20, thereby preventing FBXO31 degradation. Collectively, our results reveal how alterations in FBXO31 phosphorylation, mediated by AKT and ATM, underlie physiological regulation of FBXO31 levels in unstressed and genotoxically stressed cells.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclosoma-Complejo Promotor de la Anafase/genética , Antígenos CD , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Puntos de Control del Ciclo Celular , Daño del ADN , Proteínas F-Box/química , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Modelos Biológicos , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/química , UbiquitinaciónRESUMEN
Glioma remains one of the most aggressive and lethal cancers in central nervous system. Temozolomide (TMZ) is the most commonly used chemotherapeutic agent in gliomas. However, therapeutic benefits of TMZ could be very limited and all patients would finally suffer from tumor progression as the tumors develop resistance to TMZ. In this study, we aim to investigate the underlying mechanism of chemoresistance in glioma cell line and to identify whether there is still a close link between epithelial-mesenchymal transition (EMT) and TMZ resistance in gliomas. The real-time RT-PCR and Western blotting were used to measure the expression of EMT markers in TMZ-resistant cells. The migration and invasion assays were conducted to detect the cell motility activity in TMZ-resistant cells. The transfection was used to down-regulate the Cdc20 expression. The student t-test was applied for data analysis. We established stable TMZ-resistant glioma cells and designated as TR. Our results revealed that TR cells exhibited a significantly increased resistance to TMZ compared with their parental cells. Moreover, TMZ-resistant cells had acquired EMT-like changes. For the mechanism study, we measured a significant increased expression of CDC20 and decreased expression of Bim in TR cells. Moreover, upon suppression of CDC20 by shRNA transfection, TR cells underwent a reverse of EMT features. Importantly, knockdown of CDC20 enhanced the drug sensitivity of TR cells to TMZ. Our results suggested that inactivation of CDC20 could contribute to the future therapy that possibly overcomes drug resistance in human cancers.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Proteínas Cdc20/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Temozolomida/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/metabolismo , Glioma/patología , Humanos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Recent studies revealed that mutations in SPOP (Speckle-type POZ protein) occur in up to 15% of patients with prostate cancer. However, the physiological role of SPOP in regulating prostate tumorigenesis remains elusive. Here, we identified the Cdc20 oncoprotein as a novel ubiquitin substrate of SPOP. As such, pharmacological inhibition of Cullin-based E3 ligases by MLN4924 could stabilize endogenous Cdc20 in cells. Furthermore, we found that Cullin 3, and, to a less extent, Cullin 1, specifically interacted with Cdc20. Depletion of Cullin 3, but not Cullin 1, could upregulate the abudance of Cdc20 largely via prolonging Cdc20 half-life. Moreover, SPOP, the adaptor protein of Cullin 3 family E3 ligase, specifically interacted with Cdc20, and promoted the poly-ubiquitination and subsequent degradation of Cdc20 in a degron-dependent manner. Importantly, prostate cancer-derived SPOP mutants failed to interact with Cdc20 to promote its degradation. As a result, SPOP-deficient prostate cancer cells with elevated Cdc20 expression became resistant to a pharmacological Cdc20 inhibitor. Therefore, our results revealed a novel role of SPOP in tumorigenesis in part by promoting the degradation of the Cdc20 oncoprotein.
Asunto(s)
Proteínas Cdc20/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/enzimología , Proteínas Represoras/metabolismo , Ubiquitinación , Antineoplásicos/farmacología , Carbamatos/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Diaminas/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Masculino , Terapia Molecular Dirigida , Proteínas Nucleares/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Pirimidinas/farmacología , Proteínas Represoras/genética , Factores de Tiempo , TransfecciónRESUMEN
The role of cell division cycle 20 (CDC20) was investigated in chemoresistance to decetaxel and the underlying mechanisms in metastatic castration-resistant prostate cancer (mCRPC). MTT assays were performed to determine effects of siRNA-mediated CDC20 knockdown on cell proliferation and anticancer activity of docetaxel. Western blot analyses were conducted to detect changes of Akt and Wnt signaling. Furthermore, in vivo growth of PCa was examined in nude mice treated with siCDC20 or docetaxel alone or in combination. CDC20 was overexpressed in mCRPC cells. Knockdown of CDC20 suppressed cell proliferation and enhanced anticancer effect of docetaxel with IC50 reducing from 0.358 to 0.188 µg/ml in PC3 cells and 0.307 to 0.162 µg/ml in DU145 cells (P<0.01). While no change of Akt signaling was observed, inhibition of Wnt/ß-catenin signaling was detected upon CDC20 silencing. Xenograft tumor growth was significantly reduced in nude mice by CDC20 inhibition. The additional treatment of siCDC20 achieved better anticancer effects than that of docetaxel alone. Silencing of CDC20 may be a new strategy to improve chemosensitization to docetaxel in mCRPC.
Asunto(s)
Proteínas Cdc20/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/prevención & control , Taxoides/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor , Western Blotting , Proliferación Celular , Docetaxel , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/secundario , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rottlerin, isolated from a medicinal plant Mallotus phillippinensis, has been demonstrated to inhibit cellular growth and induce cytoxicity in glioblastoma cell lines through inhibition of calmodulin-dependent protein kinase III. Emerging evidence suggests that rottlerin exerts its antitumor activity as a protein kinase C inhibitor. Although further studies revealed that rottlerin regulated multiple signaling pathways to suppress tumor cell growth, the exact molecular insight on rottlerin-mediated tumor inhibition is not fully elucidated. In the current study, we determine the function of rottlerin on glioma cell growth, apoptosis, cell cycle, migration and invasion. We found that rottlerin inhibited cell growth, migration, invasion, but induced apoptosis and cell cycle arrest. Mechanistically, the expression of Cdc20 oncoprotein was measured by the RT-PCR and Western blot analysis in glioma cells treated with rottlerin. We observed that rottlerin significantly inhibited the expression of Cdc20 in glioma cells, implying that Cdc20 could be a novel target of rottlerin. In line with this, over-expression of Cdc20 decreased rottlerin-induced cell growth inhibition and apoptosis, whereas down-regulation of Cdc20 by its shRNA promotes rottlerin-induced anti-tumor activity. Our findings indicted that rottlerin could exert its tumor suppressive function by inhibiting Cdc20 pathway which is constitutively active in glioma cells. Therefore, down-regulation of Cdc20 by rottlerin could be a promising therapeutic strategy for the treatment of glioma.
Asunto(s)
Acetofenonas/farmacología , Benzopiranos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/fisiología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Glioma/patología , Humanos , Invasividad Neoplásica , ARN Interferente Pequeño/genéticaRESUMEN
Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdh1/genética , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/genética , Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Antineoplásicos/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas Cdh1/antagonistas & inhibidores , Proteínas Cdh1/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Perfilación de la Expresión Génica , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Interferencia de ARNRESUMEN
In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Mitosis , Fosfoproteínas/metabolismo , Secuencias de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Antígenos CD , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Apoenzimas/metabolismo , Sitios de Unión , Cadherinas/química , Cadherinas/metabolismo , Cadherinas/ultraestructura , Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestructura , Microscopía por Crioelectrón , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Activación Enzimática , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/ultraestructura , Fosforilación , Unión Proteica , Conformación Proteica , Tosilarginina Metil Éster/farmacologíaRESUMEN
Over 90% of patients with hepatocellular carcinoma (HCC) are diagnosed at an advanced stage. This study investigated the antitumor efficacy of the inhibition of cell division cycle protein 20 (CDC20) and heparanase (HPSE) expression in Hepa1-6 mouse hepatoma cells. Cell viability was measured by the MTT assay. Cell cycle was analyzed by cytometry. The invasion assay was performed using the Transwell chamber. The orthotopic liver tumor model was established by inoculating the livers of immunocompetent Kunming mice with Hepa1-6 cells. The MTT assay showed that 50 and 100 nM CDC20 siRNA-1 and HPSE siRNA-2 significantly reduced Hepa1-6 cell viability with the combination of CDC20 and HPSE siRNA being the most effective. Silencing of CDC20 or both CDC20 and HPSE expression significantly induced G2/M phase cell cycle arrest in Hepa1-6 HCC cells. Silencing HPSE expression significantly inhibited the invasion ability of Hepa1-6 cells with the combination of CDC20 and HPSE silencing being more effective than HPSE alone. Silencing CDC20 and HPSE expression significantly inhibited HCC tumor growth in the orthotopic liver tumor model, but the combination was most effective. Silencing CDC20 and HPSE expression activated cell apoptosis and autophagy. In conclusion, targeting inhibition of both CDC20 and HPSE expression is an ideal strategy for HCC therapy.
Asunto(s)
Proteínas Cdc20/antagonistas & inhibidores , Proteínas Cdc20/metabolismo , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Interferencia de ARN/fisiología , Animales , Proteínas Cdc20/genética , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , TransfecciónRESUMEN
The genetic integrity of each organism depends on the faithful segregation of its genome during mitosis. To meet this challenge, a cellular surveillance mechanism, termed the spindle assembly checkpoint (SAC), evolved that monitors the correct attachment of chromosomes and blocks progression through mitosis if corrections are needed. While the central role of the SAC for genome integrity is well established, its functional dissection has been hampered by the limited availability of appropriate small molecule inhibitors. Using a fluorescence polarization-based screen, we identify Mad2 inhibitor-1 (M2I-1), the first small molecule inhibitor targeting the binding of Mad2 to Cdc20, an essential protein-protein interaction (PPI) within the SAC. Based on computational and biochemical analyses, we propose that M2I-1 disturbs conformational dynamics of Mad2 critical for complex formation with Cdc20. Cellular studies revealed that M2I-1 weakens the SAC response, indicating that the compound might be active in cells. Thus, our study identifies the SAC specific complex formation between Mad2 and Cdc20 as a protein-protein interaction that can be targeted by small molecules.
Asunto(s)
Proteínas Cdc20/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas Mad2/antagonistas & inhibidores , Proteínas Mad2/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Cdc20/antagonistas & inhibidores , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Simulación del Acoplamiento Molecular , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismoRESUMEN
The Anaphase Promoting Complex (APC, also called APC/C) regulates cell cycle progression by forming two closely related, but functionally distinct E3 ubiquitin ligase sub-complexes, APC(Cdc20) and APC(Cdh1), respectively. Emerging evidence has begun to reveal that Cdc20 and Cdh1 have opposing functions in tumorigenesis. Specifically, Cdh1 functions largely as a tumor suppressor, whereas Cdc20 exhibits an oncogenic function, suggesting that Cdc20 could be a promising therapeutic target for combating human cancer. However, the exact underlying molecular mechanisms accounting for their differences in tumorigenesis remain largely unknown. Therefore, in this review, we summarize the downstream substrates of Cdc20 and the critical functions of Cdc20 in cell cycle progression, apoptosis, ciliary disassembly and brain development. Moreover, we briefly describe the upstream regulators of Cdc20 and the oncogenic role of Cdc20 in a variety of human malignancies. Furthermore, we summarize multiple pharmacological Cdc20 inhibitors including TAME and Apcin, and their potential clinical benefits. Taken together, development of specific Cdc20 inhibitors could be a novel strategy for the treatment of human cancers with elevated Cdc20 expression.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Cdc20/metabolismo , Neoplasias/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis , Encéfalo/citología , Encéfalo/fisiología , Proteínas Cdc20/antagonistas & inhibidores , Ciclo Celular , Diferenciación Celular , Cilios/fisiología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Plasticidad Neuronal , Neuronas/citología , Neuronas/fisiología , UbiquitinaciónRESUMEN
The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.