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1.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443472

RESUMO

Feruloylacetone (FER) is a natural degradant of curcumin after heating, which structurally reserves some functional groups of curcumin. It is not as widely discussed as its original counterpart has been previously; and in this study, its anticancer efficacy is investigated. This study focuses on the suppressive effect of FER on colon cancer, as the efficacious effect of curcumin on this typical cancer type has been well evidenced. In addition, demethoxy-feruloylacetone (DFER) was applied to compare the effect that might be brought on by the structural differences of the methoxy group. It was revealed that both FER and DFER inhibited the proliferation of HCT116 cells, possibly via suppression of the phosphorylated mTOR/STAT3 pathway. Notably, FER could significantly repress both the STAT3 phosphorylation and protein levels. Furthermore, both samples showed capability of arresting HCT116 cells at the G2/M phase via the activation of p53/p21 and the upregulation of cyclin-B. In addition, ROS elevation and changes in mitochondrial membrane potential were revealed, as indicated by p-atm elevation. The apoptotic rate rose to 36.9 and 32.2% after being treated by FER and DFER, respectively. In summary, both compounds exhibited an anticancer effect, and FER showed a greater proapoptotic effect, possibly due to the presence of the methoxy group on the aromatic ring.


Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Curcumina/farmacologia , Mitocôndrias/efeitos dos fármacos , Estirenos/farmacologia , Antineoplásicos/química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Curcumina/química , Curcumina/metabolismo , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/agonistas , Fase G2/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Fenol/química , Fenol/farmacologia , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Estirenos/química , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/agonistas
2.
Cell Death Dis ; 12(7): 691, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244479

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there still remains a lack of effective diagnostic and therapeutic targets for this disease. Increasing evidence demonstrates that RNA modifications play an important role in the progression of HCC, but the role of the N7-methylguanosine (m7G) methylation modification in HCC has not been properly evaluated. Thus, the goal of the present study was to investigate the function and mechanism of the m7G methyltransferase WD repeat domain 4 (WDR4) in HCC as well as its clinical relevance and potential value. We first verified the high expression of WDR4 in HCC and observed that upregulated WDR4 expression increased the m7G methylation level in HCC. WDR4 promoted HCC cell proliferation by inducing the G2/M cell cycle transition and inhibiting apoptosis in addition to enhancing metastasis and sorafenib resistance through epithelial-mesenchymal transition (EMT). Furthermore, we observed that c-MYC (MYC) can activate WDR4 transcription and that WDR4 promotes CCNB1 mRNA stability and translation to enhance HCC progression. Mechanistically, we determined that WDR4 enhances CCNB1 translation by promoting the binding of EIF2A to CCNB1 mRNA. Furthermore, CCNB1 was observed to promote PI3K and AKT phosphorylation in HCC and reduce P53 protein expression by promoting P53 ubiquitination. In summary, we elucidated the MYC/WDR4/CCNB1 signalling pathway and its impact on PI3K/AKT and P53. Furthermore, the result showed that the m7G methyltransferase WDR4 is a tumour promoter in the development and progression of HCC and may act as a candidate therapeutic target in HCC treatment.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Ligação ao GTP/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sorafenibe/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Ciclina B1/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nature ; 596(7870): 138-142, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290405

RESUMO

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.


Assuntos
Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separase/química , Separase/metabolismo , Motivos de Aminoácidos , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/ultraestrutura , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinases relacionadas a CDC2 e CDC28/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Microscopia Crioeletrônica , Ciclina B1/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Securina/ultraestrutura , Separase/antagonistas & inibidores , Separase/ultraestrutura , Especificidade por Substrato
4.
Methods Mol Biol ; 2329: 143-164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085221

RESUMO

A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteína Quinase CDC2/metabolismo , Sistema Livre de Células/metabolismo , Ciclina B1/metabolismo , Anáfase , Ciclina B1/genética , Células HEK293 , Humanos , Mitose , Mutação , Fosforilação , Proteólise , Ubiquitinação
5.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071893

RESUMO

Insulin-like growth factor 1 receptor (IGF1R), a receptor-type tyrosine kinase, transduces signals related to cell proliferation, survival, and differentiation. We recently reported that OSI-906, an IGF1R inhibitor, in combination with the Aurora B inhibitor ZM447439 suppresses cell proliferation. However, the mechanism underlying this suppressive effect is yet to be elucidated. In this study, we examined the effects of combination treatment with OSI-906 and ZM447439 on cell division, so as to understand how cell proliferation was suppressed. Morphological analysis showed that the combination treatment generated enlarged cells with aberrant nuclei, whereas neither OSI-906 nor ZM447439 treatment alone caused this morphological change. Flow cytometry analysis indicated that over-replicated cells were generated by the combination treatment, but not by the lone treatment with either inhibitors. Time-lapse imaging showed mitotic slippage following a severe delay in chromosome alignment and cytokinesis failure with furrow regression. Furthermore, in S-trityl-l-cysteine-treated cells, cyclin B1 was precociously degraded. These results suggest that the combination treatment caused severe defect in the chromosome alignment and spindle assembly checkpoint, which resulted in the generation of over-replicated cells. The generation of over-replicated cells with massive aneuploidy may be the cause of reduction of cell viability and cell death. This study provides new possibilities of cancer chemotherapy.


Assuntos
Aurora Quinase B/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/metabolismo , Imidazóis/farmacologia , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Benzamidas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteólise , Quinazolinas/farmacologia , Receptor IGF Tipo 1/metabolismo
6.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33819340

RESUMO

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.


Assuntos
Proteínas Cdc20/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Mitose/fisiologia , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Fuso Acromático/metabolismo
7.
Biochim Biophys Acta Mol Cell Res ; 1868(7): 119044, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33865884

RESUMO

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.


Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Aneuploidia , Animais , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Ciclina B1/metabolismo , Feminino , Meiose/fisiologia , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Oócitos/metabolismo , Corpos Polares/metabolismo , Fuso Acromático/metabolismo
8.
Cancer Med ; 10(8): 2732-2739, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710778

RESUMO

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor, and vasohibin-2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere-forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.


Assuntos
Proteínas Angiogênicas/genética , Carboxipeptidases/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sistemas CRISPR-Cas , Carboxipeptidases/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ciclina B1/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Tubulina (Proteína)/metabolismo , Tirosina/genética , Tirosina/metabolismo
9.
Theranostics ; 11(10): 4809-4824, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754029

RESUMO

Purpose: Advanced prostate cancer (PCa) has limited treatment regimens and shows low response to chemotherapy and immunotherapy, leading to poor prognosis. Histone modification is a vital mechanism of gene expression and a promising therapy target. In this study, we characterized WD repeat domain 5 (WDR5), a regulator of histone modification, and explored its potential therapeutic value in PCa. Experimental Design: We characterized specific regulators of histone modification, based on TCGA data. The expression and clinical features of WDR5 were analyzed in two dependent cohorts. The functional role of WDR5 was further investigated with siRNA and OICR-9429, a small molecular antagonist of WDR5, in vitro and in vivo. The mechanism of WDR5 was explored by RNA-sequencing and chromatin immunoprecipitation (ChIP). Results: WDR5 was overexpressed in PCa and associated with advanced clinicopathological features, and predicted poor prognosis. Both inhibition of WDR5 by siRNA and OICR-9429 could reduce proliferation, and increase apoptosis and chemosensitivity to cisplatin in vitro and in vivo. Interestingly, targeting WDR5 by siRNA and OICR-9429 could block IFN-γ-induced PD-L1 expression in PCa cells. Mechanistically, we clarified that some cell cycle, anti-apoptosis, DNA repair and immune related genes, including AURKA, CCNB1, E2F1, PLK1, BIRC5, XRCC2 and PD-L1, were directly regulated by WDR5 and OICR-9429 in H3K4me3 and c-Myc dependent manner. Conclusions: These data revealed that targeting WDR5 suppressed proliferation, enhanced apoptosis, chemosensitivity to cisplatin and immunotherapy in PCa. Therefore, our findings provide insight into OICR-9429 is a multi-potency and promising therapy drug, which improves the antitumor effect of cisplatin or immunotherapy in PCa.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/genética , Proliferação de Células/genética , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias da Próstata/genética , Idoso , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Compostos de Bifenilo/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Di-Hidropiridinas/farmacologia , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Survivina/genética , Survivina/metabolismo , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/genética
10.
Phytomedicine ; 85: 153537, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33744595

RESUMO

BACKGROUND: Valtrate is a novel epoxy iridoid ester isolated from Chinese herbal medicine Valeriana jatamansi Jones with anti-proliferative activity against various human cancer cell lines. However, its efficacy and molecular mechanisms against pancreatic cancer (PC) cells are largely unclear. PURPOSE: To investigate the anti-cancer effects of valtrate on PC cell lines and its underlying mechanisms. METHODS: MTT assay was first performed to detect the effect of valtrate on cell viability in human PC cell lines and normal pancreatic epithelial cells HPDE. Cell apoptosis and cycle phase assay were detected by flow cytometry. The relative mRNA expressions of Bax, Bcl-2, c-Myc, and CyclinB1 were tested by quantitative PCR (qPCR) assay. The expression of relative proteins was detected by Western blotting (WB). A PANC-1luc cells xenograft mouse model in nu/nu female mice was used to elucidate the effect of valtrate on tumor growth in vivo. RESULTS: Valtrate significantly inhibited the growth of PC cells without affecting the growth of normal pancreatic epithelial cells HPDE, induced significant apoptosis and cell cycle arrest in G2/M phase. Moreover, valtrate inhibited the tumor growth of PC cell PANC-1 in xenograft mice by 61%. Further mechanism study demonstrated that valtrate could increase the expression level of Bax, suppress Bcl-2 as well as c-Myc and Cyclin B1, inhibit the transcriptional activity of Stat3, while valtrate decreased the expression level of Stat3 and phosphated-Stat3 (Tyr705) and induced the high molecular aggregation of Stat3. Molecular docking analysis predicted that valtrate might interact with Cys712 of Stat3 protein. Valtrate could also induce a transient depleted intracellular glutathione (GSH) level and increased reactive oxygen species (ROS). NAC (N-acetylcysteine), a reducer reversed valtrate-induced the depletion of Stat3, p-Stat3, c-Myc, and Cyclin B1. CONCLUSION: Valtrate exerts anti-cancer activity against PC cells by directly targeting Stat3 through a covalent linkage to inhibit Stat3 activity, which causes apoptosis and cell cycle arrest.


Assuntos
Iridoides/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Valeriana/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Mol Carcinog ; 60(4): 265-278, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33634905

RESUMO

Ubiquitin-specific protease 39 (USP39) is frequently overexpressed in a variety of cancers, and involved in the regulation of various biological processes, such as cell proliferation, cell cycle progression, apoptosis and pre-messenger RNA splicing. Nevertheless, the biological roles and mechanisms of USP39 in colon cancer remain largely unknown. In this study, we analyzed whether USP39 can be a molecular target for the treatment of colon cancer. Whilst overexpression of USP39 was detected in human colon cancer tissues and cell lines, USP39 knockdown was observed to inhibit the growth and subcutaneous tumor formation of colon cancer cells. Further analysis showed that USP39 knockdown can stabilize p21 by prolonging the half-life of p21 and by upregulating the promoter activity of p21. The RS domain and USP domain of USP39 were found to play an essential role. Additionally, our findings revealed that USP39 plays a regulatory role in the proliferation of colon cancer cells by the p53/p21/CDC2/cyclin B1 axis in a p21-dependent manner. Taken together, this study provided the theoretical basis that may facilitate the development of USP39 as a novel potential target of colon cancer therapy.


Assuntos
Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Camundongos , Regiões Promotoras Genéticas , Domínios Proteicos , Estabilidade Proteica , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/química , Regulação para Cima
12.
Sci Rep ; 11(1): 2157, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495511

RESUMO

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.


Assuntos
Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo , RecQ Helicases/deficiência , Autofagia , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Fibroblastos/enzimologia , Fibroblastos/patologia , Fase G1 , Humanos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Mitose , Espécies Reativas de Oxigênio/metabolismo , RecQ Helicases/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima
13.
Biochimie ; 182: 108-119, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33421501

RESUMO

Cell proliferation during seed germination is determinant for an appropriate seedling establishment. The present work aimed to evaluate the participation of two maize B-type Cyclins during germination and under the stimulus of two simple sugars: sucrose and glucose. We found out that the corresponding genes, ZmCycB1;2 and ZmCycB2;1, increased their expression at 24 h of germination, but only ZmCycB1;2 responded negatively to sugar type at the highest sugar concentration tested (120 mM). Also, CycB1;2 showed differential protein levels along germination in response to sugar, or its absence. Both CycBs interacted with CDKA;1 and CDKB1;1 by pull down assays. By an immunoprecipitation approach, it was found that each CycB associated with two CDKB isoforms (34 and 36 kDa). A higher proportion of CycB1;2-CDKB-36kDa was coincident to an increased kinase activity in the presence of sugar and particularly in glucose treatment at 36 h of imbibition. CycB1;2-CDKB activity increased in parallel to germination advance and this was dependent on sugar: glucose > sucrose > No sugar treatment. At RAM, CycB1;2 was more abundant in nuclei on Glucose at late germination; DNA-CycB1;2 colocalization was parallel to CycB1;2 inside the nucleus. Overall, results point out CycB1;2 as a player on promoting proliferation during germination by binding a specific CDKB isoform partner and changing its cellular localization to nuclei, co-localizing with DNA, being glucose a triggering signal.


Assuntos
Ciclina B1/metabolismo , Ciclina B2/metabolismo , Germinação/fisiologia , Glucose/metabolismo , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Zea mays/metabolismo , Ciclina B1/genética , Ciclina B2/genética , Glucose/genética , Proteínas de Plantas/genética , Zea mays/genética
14.
Mol Cell Endocrinol ; 525: 111140, 2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33359304

RESUMO

Previous studies have shown that CCNB1 affects the invasiveness of pituitary adenomas, and it is of great significance to find the upstream mechanism of regulating CCNB1.In this study, we explored a significantly overexpressed circRNA in invasive pituitary adenomas. Based on bioinformatics analysis and mechanism experiments, we determined that circNFIX (has-circ_0005660) affects cell invasion, migration and proliferation in pituitary adenomas by sponging miR-34a-5p through CCNB1. In pituitary adenoma tissues, the expression of circNFIX and CCNB1 was upregulated, while miR-34a-5p expression was downregulated. The silencing of circNFIX or overexpression of miR-34a-5p inhibited cell invasion, migration and proliferation. Inhibition of miR-34a-5p expression reversed the inhibitory effect of circNFIX silencing on the progression of pituitary adenoma. In conclusion, CircNFIX affects cell invasion, migration, and proliferation in pituitary adenomas by sponging miR-34a-5p through CCNB1. Therefore, circNFIX is expected to serve as a potential target for the treatment of pituitary adenomas.


Assuntos
Adenoma/genética , Ciclina B1/metabolismo , Progressão da Doença , MicroRNAs/metabolismo , Neoplasias Hipofisárias/genética , RNA Circular/metabolismo , Adenoma/diagnóstico por imagem , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ciclina B1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Invasividade Neoplásica , Neoplasias Hipofisárias/diagnóstico por imagem , RNA Circular/genética
15.
J Cell Sci ; 133(23)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33148609

RESUMO

Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate times to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein pumilio 1 (Pum1) regulate the translation of target mRNAs and progression of oocyte maturation. Pum1 was shown to bind to Mad2 (also known as Mad2l1) and cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and cyclin B1 RNA granules in mouse oocytes. These Pum1 aggregates were dissolved prior to the translational activation of target mRNAs, possibly through phosphorylation. Stabilization of Pum1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.


Assuntos
Biossíntese de Proteínas , Peixe-Zebra , Animais , Ciclina B/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Meiose/genética , Camundongos , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética
16.
Sci Rep ; 10(1): 18788, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139756

RESUMO

Di-isononyl phthalate (DiNP), a common plasticizer used in polyvinyl chloride products, exhibits endocrine-disrupting capabilities. It is also toxic to the brain, reproductive system, liver, and kidney. However, little is known about how DiNP impacts the gastrointestinal tract (GIT). It is crucial to understand how DiNP exposure affects the GIT because humans are primarily exposed to DiNP through the GIT. Thus, this study tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. To test this hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 to 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. DiNP exposure significantly increased histological damage in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels, increased Tnf expression, decreased a cell cycle regulator (Ccnb1), and increased apoptotic factors (Aifm1 and Bcl2l10) in the colon compared to control. Colon-extracted lipids revealed that DiNP exposure significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in adult female mice.


Assuntos
Colo/imunologia , Colo/metabolismo , Disruptores Endócrinos/efeitos adversos , Ácidos Ftálicos/efeitos adversos , Plastificantes/efeitos adversos , Animais , Apoptose/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular/genética , Colo/efeitos dos fármacos , Colo/patologia , Ciclina B1/metabolismo , Disruptores Endócrinos/toxicidade , Estradiol/metabolismo , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Ácidos Ftálicos/administração & dosagem , Ácidos Ftálicos/toxicidade , Plastificantes/administração & dosagem , Plastificantes/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
Mol Med Rep ; 22(6): 4899-4908, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33174033

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Ubiquitin­specific protease 12 (USP12) is specifically upregulated in the tumor tissues of patients with HCC compared with the corresponding adjacent normal tissues. However, the relationship between USP12 and the growth of HCCs is not fully understood. In the present study, USP12 was knocked down in HCC cell lines to investigate its effects on proliferation and apoptosis. The results showed that USP12­knockdown could inhibit the proliferation and promote apoptosis in HCC cell lines. Flow cytometry analysis also showed that USP12 could induce cell cycle arrest at the G2/M stage. In vivo experiments showed that USP12­knockdown could suppress tumor growth in mice, and immuno­blotting revealed that USP12 could induce G2/M arrest through the cyclin dependent kinase 1/cyclinB1 axis, and trigger apoptosis via the p38/mitogen­activated protein kinase pathway. These data strongly indicate that USP12 is a potential target for the treatment of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Apoptose/genética , Proteína Quinase CDC2/metabolismo , Carcinoma Hepatocelular/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina B1/metabolismo , Feminino , Citometria de Fluxo/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Cells ; 9(9)2020 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-32961751

RESUMO

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.


Assuntos
Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina B1/genética , Ciclina B1/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Zinostatina/farmacologia
19.
Mol Med Rep ; 22(2): 1527-1535, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32627020

RESUMO

Tubeimoside-1 (TBMS1), a traditional Chinese herb extracted from Bolbostemma paniculatum (Maxim.), induces apoptosis in a number of human cancer cell lines. TBMS1 has been reported to induce apoptosis in human glioma cells, however the mechanism remains to be elucidated. The present study explored TBMS1­induced PI3K/Akt­related pathways in human glioma cells. The human glioma U251 and the human astrocyte (HA) cell lines were treated with various concentrations of TBMS1. MTT assays were conducted to analyze cell viability. Cell cycle distribution and the rate of apoptosis were assessed using flow cytometry. BrdU incorporation and Hoechst 33342 staining were performed to analyze the cell cycle and apoptosis, respectively. Western blotting was performed to investigate protein expression levels. The results demonstrated that TBMS1 reduced cell viability in human glioma cells U251 by suppressing Akt phosphorylation. Subsequently, TBMS1 inhibited DNA synthesis and induced G2/M phase arrest by targeting the PI3K/Akt/p21 and the cyclin­dependent kinase 1/cyclin B1 signaling cascades. In addition, TBMS1 triggered apoptosis via the PI3K/Akt­mediated Bcl­2 signaling pathway. These results demonstrated that TBMS1 prevented the progression of gliomas via the PI3K/Akt­dependent pathway, which provided a theoretical basis for in vivo studies to use TBMS1 as potential therapy for the prevention of cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Glioma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Astrócitos , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glioma/tratamento farmacológico , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
20.
Cancer Med ; 9(17): 6322-6329, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32692487

RESUMO

Targeting mitotic kinases is an emerging anticancer approach with promising preclinical outcomes. Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl), is an important mitotic kinase that regulates mitotic progression of normal or transformed cells by blocking the activity of tumor suppressor protein phosphatase 2A (PP2A). MASTL upregulation has now been detected in multiple cancer types and associated with aggressive clinicopathological features. Apart, an aberrant MASTL activity has been implicated in oncogenic transformation through the development of chromosomal instability and alteration of key oncogenic signaling pathways. In this regard, recent publications have revealed potential role of MASTL in the regulation of AKT/mTOR and Wnt/ß-catenin signaling pathways, which may be independent of its regulation of PP2A-B55 (PP2A holoenzyme containing a B55-family regulatory subunit). Taken together, MASTL kinase has emerged as a novel target for cancer therapeutics, and hence development of small molecule inhibitors of MASTL may significantly improve the clinical outcomes of cancer patients. In this article, we review the role of MASTL in cancer progression and the current gaps in this knowledge. We also discuss potential efficacy of MASTL expression for cancer diagnosis and therapy.


Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Quinase CDC2/metabolismo , Transformação Celular Neoplásica , Instabilidade Cromossômica , Ciclina B1/metabolismo , Dano ao DNA , Reparo do DNA , Progressão da Doença , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Via de Sinalização Wnt
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