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1.
Biomed Res Int ; 2019: 6125068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531359

RESUMO

Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Processamento Alternativo/efeitos dos fármacos , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Precursores de RNA/metabolismo
2.
PLoS One ; 14(2): e0212210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779812

RESUMO

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.


Assuntos
Fase G1/efeitos dos fármacos , Glucocorticoides/farmacologia , Células Secretoras de Insulina/metabolismo , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fase S/efeitos dos fármacos , Adulto , Idoso , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Feminino , Humanos , Células Secretoras de Insulina/citologia , Masculino , Pessoa de Meia-Idade , Ratos
3.
Theriogenology ; 123: 22-29, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30273737

RESUMO

Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca2+ and Zn2+ levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different time intervals. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with PKCa + CDK1i for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different times to a Zn2+ chelator (TPEN) following Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximise oocyte activation and embryo development. Next, we observed that treatment with PKCa + CDK1i for 4 h after TPEN for 15 min decreased embryo development compared to TPEN alone. Lastly, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs 49.2%) and blastocyst from cleaved embryos (65.5% vs 46.2%) compared to CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol compared with CT-245 and the non-activated (N-A) group. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a fast and effective protocol for activation of porcine oocytes.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Suínos/fisiologia , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Etilaminas/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Quinase C-alfa , Piridinas/farmacologia
4.
Cell Chem Biol ; 26(1): 121-130.e5, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30472117

RESUMO

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Entropia , Inibidores de Proteínas Quinases/farmacologia , Proteína Quinase CDC2/isolamento & purificação , Proteína Quinase CDC2/metabolismo , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Ressonância de Plasmônio de Superfície
5.
Elife ; 72018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30422115

RESUMO

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.


Assuntos
Antineoplásicos/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Ovarianas/patologia , Fenilenodiaminas/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Pirimidinas/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos SCID , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Fenilenodiaminas/administração & dosagem , Pirimidinas/administração & dosagem , Resultado do Tratamento
6.
Cell Death Dis ; 9(11): 1100, 2018 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-30368521

RESUMO

Spinal Muscular Atrophy (SMA) is caused by genetic mutations in the SMN1 gene, resulting in drastically reduced levels of Survival of Motor Neuron (SMN) protein. Although SMN is ubiquitously expressed, spinal motor neurons are one of the most affected cell types. Previous studies have identified pathways uniquely activated in SMA motor neurons, including a hyperactivated ER stress pathway, neuronal hyperexcitability, and defective spliceosomes. To investigate why motor neurons are more affected than other neural types, we developed a spinal organoid model of SMA. We demonstrate overt motor neuron degeneration in SMA spinal organoids, and this degeneration can be prevented using a small molecule inhibitor of CDK4/6, indicating that spinal organoids are an ideal platform for therapeutic discovery.


Assuntos
Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Neurônios Motores/efeitos dos fármacos , Organoides/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Colágeno/química , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Combinação de Medicamentos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Laminina/química , Modelos Biológicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteoglicanas/química , Transdução de Sinais , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
7.
Biosci Rep ; 38(6)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30341238

RESUMO

It was previously reported that the expression of CD274 was down-regulated in psoriatic epidermis, leading to immune disorders of psoriasis. However, the regulatory mechanisms of CD274 were rarely elucidated. We aimed to explore the regulatory mechanisms of CD274. Skin samples were collected from 18 patients with psoriasis vulgaris and 9 healthy participants for RNA sequencing. Candidate genes were chosen based on degree and k-core difference of genes in the co-expression network. The relations between candidate genes and CD274 were validated by flow cytometry and real-time PCR in primary human epidermal keratinocytes. The therapeutic effect of indirubin was assessed in an imiquimod-treated mouse model. Interferon-γ (IFN-γ), cyclin-dependent kinase (CDK) 1, Toll-like receptor 3 (TLR3), TLR4 and interleukin (IL)-17A were considered as candidate genes. In primary human epidermal keratinocytes, the level of CD274 was obviously increased under the stimulation of IFN-γ and CDK1 inhibitor (indirubin), independent of TLR4, TLR3 or IL-17A. Indirubin alleviated the severity of psoriatic mice in a CD274-dependent manner. Co-expression network analysis served as an effective method for the exploration of molecular mechanisms. We demonstrated for the first time that CD274 was the regulator of indirubin-mediated effect on mouse psoriasis-like skin lesion based on co-expression network analysis, contributing to the alleviation of mouse psoriasis-like skin lesion.


Assuntos
Antígeno B7-H1/genética , Proteína Quinase CDC2/genética , Psoríase/tratamento farmacológico , Anormalidades da Pele/tratamento farmacológico , Adolescente , Adulto , Idoso , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Epiderme/patologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Interferon gama/genética , Interleucina-17/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Cultura Primária de Células , Psoríase/genética , Psoríase/patologia , Análise de Sequência de RNA/métodos , Anormalidades da Pele/genética , Anormalidades da Pele/patologia , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética , Adulto Jovem
8.
Theranostics ; 8(14): 3737-3750, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30083256

RESUMO

Rationale: Hepatocellular carcinoma (HCC) is an aggressive malignant solid tumor wherein CDK1/PDK1/ß-Catenin is activated, suggesting that inhibition of this pathway may have therapeutic potential. Methods: CDK1 overexpression and clinicopathological parameters were analyzed. HCC patient-derived xenograft (PDX) tumor models were treated with RO3306 (4 mg/kg) or sorafenib (30 mg/kg), alone or in combination. The relevant signaling of CDK1/PDK1/ß-Catenin was measured by western blot. Silencing of CDK1 with shRNA and corresponding inhibitors was performed for mechanism and functional studies. Results: We found that CDK1 was frequently augmented in up to 46% (18/39) of HCC tissues, which was significantly associated with poor overall survival (p=0.008). CDK1 inhibitor RO3306 in combination with sorafenib treatment significantly decreased tumor growth in PDX tumor models. Furthermore, the combinatorial treatment could overcome sorafenib resistance in the HCC case #10 PDX model. Western blot results demonstrated the combined administration resulted in synergistic down-regulation of CDK1, PDK1 and ß-Catenin as well as concurrent decreases of pluripotency proteins Oct4, Sox2 and Nanog. Decreased CDK1/PDK1/ß-Catenin was associated with suppression of epithelial mesenchymal transition (EMT). In addition, a low dose of RO3306 and sorafenib combination could inhibit 97H CSC growth via decreasing the S phase and promoting cells to enter into a Sub-G1 phase. Mechanistic and functional studies silencing CDK1 with shRNA and RO3306 combined with sorafenib abolished oncogenic function via downregulating CDK1, with downstream PDK1 and ß-Catenin inactivation. Conclusion: Anti-CDK1 treatment can boost sorafenib antitumor responses in PDX tumor models, providing a rational combined treatment to increase sorafenib efficacy in the clinic.


Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Quinolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/administração & dosagem , Tiazóis/administração & dosagem , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Xenoenxertos , Humanos , Camundongos SCID , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sobrevida , Resultado do Tratamento , beta Catenina
9.
J Biol Chem ; 293(40): 15524-15537, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30135207

RESUMO

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and in vitro kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteína Quinase CDC2/genética , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Proteína Oncogênica pp60(v-src)/genética , Paclitaxel/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteína Oncogênica pp60(v-src)/antagonistas & inibidores , Proteína Oncogênica pp60(v-src)/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Poliploidia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo
10.
Cell Cycle ; 17(12): 1513-1523, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30045664

RESUMO

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner. Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacos
11.
Placenta ; 66: 57-64, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29884303

RESUMO

AIMS: The human placental syncytiotrophoblast (STB) cells play essential roles in embryo implantation and nutrient exchange between the mother and the fetus. STBs are polyploid which are formed by fusion of diploid cytotrophoblast (CTB) cells. Abnormality in STBs formation can result in pregnancy-related disorders. While a number of genes have been associated with CTB fusion the initial events that trigger cell fusion are not well understood. Primary objective of this study was to enhance our understanding about the molecular mechanism of placental cell fusion. METHODS: FACS and microscopic analysis was used to optimize Forskolin-induced fusion of BeWo cells (surrogate of CTBs) and subsequently, changes in the expression of different cell cycle regulator genes were analyzed through Western blotting and qPCR. Immunohistochemistry was performed on the first trimester placental tissue sections to validate the results in the context of placental tissue. Effect of Cyclin Dependent Kinase 1 (CDK1) inhibitor, RO3306, on BeWo cell fusion was studied by microscopy and FACS, and by monitoring the expression of human Chorionic Gonadotropin (hCG) by Western blotting and qPCR. RESULTS: The data showed that the placental cell fusion was associated with down regulation of CDK1 and its associated cyclin B, and significant decrease in DNA replication. Moreover, inhibition of CDK1 by an exogenous inhibitor induced placental cell fusion and expression of hCG. CONCLUSION: Here, we report that the placental cell fusion can be induced by inhibiting CDK1. This study has a high therapeutic significance to manage pregnancy related abnormalities.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Fusão Celular , Linhagem Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Replicação do DNA , Regulação para Baixo , Feminino , Humanos , Camundongos , Placenta/citologia , Placenta/metabolismo , Gravidez , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Especificidade da Espécie , Trofoblastos/efeitos dos fármacos
12.
Nucleic Acids Res ; 46(13): 6544-6560, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29901724

RESUMO

Cyclin-dependent kinase 1 (Cdk1) is indispensable for embryonic stem cell (ESC) maintenance and embryo development. Even though some reports have described a connection between Cdk1 and Oct4, there is no evidence that Cdk1 activity is directly linked to the ESC pluripotency transcription program. We recently reported that Aurkb/PP1-mediated Oct4 resetting is important to cell cycle maintenance and pluripotency in mouse ESCs (mESCs). In this study, we show that Cdk1 is an upstream regulator of the Oct4 phosphorylation state during cell cycle progression, and it coordinates the chromatin associated state of Oct4 for pluripotency-related gene expression within the cell cycle. Upon entry into mitosis, Aurkb in the chromosome passenger complex becomes fully activated and PP1 activity is inhibited downstream of Cdk1 activation, leading to sustaining Oct4(S229) phosphorylation and dissociation of Oct4 from chromatin during the mitotic phase. Cdk1 inhibition at the mitotic phase abnormally results in Oct4 dephosphorylation, chromosome decondensation and chromatin association of Oct4, even in replicated chromosome. Our study results suggest a molecular mechanism by which Cdk1 directly links the cell cycle to the pluripotency transcription program in mESCs.


Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição Genética , Animais , Aurora Quinase B/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Divisão Celular/genética , Células Cultivadas , Fase G2/genética , Humanos , Camundongos , Fosforilação , Proteína Fosfatase 1/metabolismo
13.
Tumour Biol ; 40(4): 1010428318770957, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29663854

RESUMO

OBJECTIVES: Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells. METHODS: Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis. RESULTS: Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation. CONCLUSION: These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.


Assuntos
Proteína Quinase CDC2/genética , Neoplasias Colorretais/radioterapia , Proteínas Proto-Oncogênicas B-raf/genética , Tolerância a Radiação/genética , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/biossíntese , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Células HCT116 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Células HT29 , Humanos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiação Ionizante , Radiossensibilizantes/farmacologia
14.
Bioorg Chem ; 78: 149-157, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29567429

RESUMO

A series of novel 1,3,4-trisubstituted pyrazole derivatives were synthesized and evaluated for their cytotoxic activity against three different cancer cell lines namely HCT116, UO-31 and HepG2. Compounds 3b, 3d, 7b and 9 showed excellent anticancer activity against all the tested cancer cell lines and had better cytotoxic activities than the reference drug, Sorafenib. Therefore, these compounds were chosen to be further evaluated in a panel of HCC cell lines. Among them, 3b and 7b were the most active compounds against HCC cells used here. Further studies on the mechanism demonstrated that 3b and 7b induced apoptosis in addition to induction of cell cycle arrest at G2/M phase in HepG2 and Huh7 cells. Consistent with these results, caspase-3 assay was done and the results revealed that the pro-apoptotic activity of the target compounds could be due to the stimulation of caspases-3. In addition, CDK1 inhibition assay was done and it was found that compounds 3b and 7b inhibited CDK1 activities with IC50 values of 2.38 and 1.52 µM, respectively. Finally, pyrazole derivatives 3b and 7b showed potent bioactivities, indicating that these compounds could be potent anticancer drugs in the future.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Desenho de Drogas , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspase 3/biossíntese , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
15.
Mol Cancer Res ; 16(3): 378-389, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29233910

RESUMO

The BRAFV600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAFV600E colorectal cancer cells. BRAFV600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAFV600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy.Implications: CDK1 is a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16(3); 378-89. ©2017 AACR.


Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Azetidinas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Proteína Quinase CDC2/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HT29 , Humanos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Mutação , Piperidinas/administração & dosagem , Compostos de Piridínio/administração & dosagem , Quinolinas , Tiazóis , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Bioorg Med Chem Lett ; 27(23): 5332-5336, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29074254

RESUMO

The discovery of a class of diheteroaromatic amines based on LY2835219 as cyclin-dependent kinase (CDK1/4/6) inhibitors was described. The series was found to have much more improved CDK1 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. The synthesis and structure-activity relationship studies of these compounds were reported. One promising compound was selected to evaluate as a novel lead compound after in vitro and in vivo profiling.


Assuntos
Aminas/farmacologia , Antineoplásicos/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Aminas/administração & dosagem , Aminas/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
17.
Sci Rep ; 7(1): 14077, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29074977

RESUMO

Control of protein turnover is critical for meiotic progression. Using RiboTag immunoprecipitation, RNA binding protein immunoprecipitation, and luciferase reporter assay, we investigated how rates of mRNA translation, protein synthesis and degradation contribute to the steady state level of Cyclin B1 and B2 in mouse oocytes. Ribosome loading onto Ccnb1 and Mos mRNAs increases during cell cycle reentry, well after germinal vesicle breakdown (GVBD). This is followed by the translation of reporters containing 3' untranslated region of Mos or Ccnb1 and the accumulation of Mos and Cyclin B1 proteins. Conversely, ribosome loading onto Ccnb2 mRNA and Cyclin B2 protein level undergo minimal changes during meiotic reentry. Degradation rates of Cyclin B1 or B2 protein at the GV stage are comparable. The translational activation of Mos and Ccnb1, but not Ccnb2, mRNAs is dependent on the RNA binding protein CPEB1. Inhibition of Cdk1 activity, but not Aurora A kinase activity, prevents the translation of Mos or Ccnb1 reporters, suggesting that MPF is required for their translation in mouse oocytes. Conversely, Ccnb2 translation is insensitive to Cdk1 inhibition. Thus, the poised state that allows rapid meiotic reentry in mouse GV oocytes may be determined by the differential translational control of two Cyclins.


Assuntos
Ciclina B1/metabolismo , Ciclina B2/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Regiões 3' não Traduzidas , Animais , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Promotor de Maturação/metabolismo , Meiose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oócitos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Proteólise , Proteínas Proto-Oncogênicas c-mos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
18.
Int J Oncol ; 51(6): 1661-1673, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29075787

RESUMO

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.


Assuntos
Anticarcinógenos/farmacologia , Aspirina/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Neoplasias Colorretais/prevenção & controle , Hidroxibenzoatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ácido Salicílico/farmacologia , Acetilação , Proteína Quinase CDC2/metabolismo , Neoplasias Colorretais/enzimologia , Ciclina B1/metabolismo , Células HCT116 , Humanos , Simulação de Acoplamento Molecular , Ácido Salicílico/metabolismo
19.
Comput Biol Chem ; 70: 175-185, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28950208

RESUMO

Pelargonidin is an anthocyanidin isolated from plant resources. It shows strong cytotoxicity toward various cancer cell lines, even though the carcinogenesis-modulating pathway of pelargonidin is not yet known. One of our previous reports showed that pelargonidin arrests the cell cycle and induces apoptosis in HT29 cells. Flowcytometry and immunoblot analysis confirmed that pelargonidin specifically inhibits the activation of CDK1 and blocks the G2-M transition of the cell cycle. In addition, DNA fragmentation was observed along with induction of cytochrome c release-mediated apoptosis. Hence, the aim of the present study was to investigate the molecular mechanism of pelargonidin's action on cell cycle regulators CDK1, CDK4, and CDK6 as well as the substrate-binding domain of DNMT1 and DNMT3A, which regulate the epigenetic signals related to DNA methylation. The results of docking analysis, binding free energy calculation, and molecular dynamics simulation correlated with the experimental results, and pelargonidin showed a specific interaction with CDK1. In this context, pelargonidin may also inhibit the recognition of DNA and catalytic binding by DNMT1 and DNMT3A. The HOMO-LUMO analysis mapped the functional groups of pelargonidin. Prediction of pharmacological descriptors suggested that pelargonidin can serve as a multitarget inhibitor for cancer treatment.


Assuntos
Antocianinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Modelos Moleculares , Antocianinas/química , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Humanos , Teoria Quântica , Termodinâmica
20.
Cell Rep ; 20(9): 2227-2237, 2017 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-28854370

RESUMO

The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.


Assuntos
Testes Genéticos , Genoma , Haploidia , Amidas/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diploide , Camundongos , Mitose/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pâncreas/citologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
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