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1.
Gene ; 760: 144989, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717307

RESUMO

Kinesin 14 family member KIFC1 is a mitotic kinesin which contains a C-terminal motor domain and plays a vital role for clustering the amplified centrosomes. Overexpression of KIFC1 in prostate cancer (PCa) cells showed resistance to docetaxel (DTX). The present study revealed that small KIFC1 inhibitor AZ82 suppresed the transcription and translation of KIFC1 significantly in PCa cells. AZ82 inhibited the KIFC1 expression both in the cytoplasm and nucleus of PCa cells. Inhibition of KIFC1 by AZ82 caused multipolar mitosis in PCa cells via de-clustering the amplified centrosomes and decreased the rate of cancer cell growth and proliferation. Moreover, depletion of KIFC1 reduced cells entering the cell cycle and caused PCa cells death through apoptosis by increasing the expression of Bax and Cytochrome C. Thereby, KIFC1 silencing and inhibition decreased the PCa cells survival by inducing multipolar mitosis as well as apoptosis, suggesting inhibition of KIFC1 using AZ82 might be a strategy to treat PCa by controlling the cancer cell proliferation.


Assuntos
Alanina/análogos & derivados , Centrossomo/efeitos dos fármacos , Cinesina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Piridinas/farmacologia , Alanina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/metabolismo , Dineínas/metabolismo , Humanos , Cinesina/genética , Cinesina/metabolismo , Masculino , Mitose/efeitos dos fármacos , Miosinas/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
2.
Cell Physiol Biochem ; 54(2): 303-320, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32259417

RESUMO

BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/toxicidade , Tetraploidia , Antimitóticos/farmacologia , Antimitóticos/toxicidade , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Sinergismo Farmacológico , Humanos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/farmacologia , RNA Interferente Pequeno , Moduladores de Tubulina/farmacologia , Vincristina/farmacologia
3.
J Med Chem ; 63(9): 4685-4700, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32290657

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Carbamatos/farmacologia , Proteínas Cdc20/antagonistas & inibidores , Diaminas/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Carbamatos/síntese química , Carbamatos/metabolismo , Proteínas Cdc20/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diaminas/síntese química , Diaminas/metabolismo , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo
4.
Mutat Res ; 852: 503169, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265043

RESUMO

The phycotoxins, okadaic acid (OA) and dinophysistoxins 1 and 2 (DTX-1 and -2), are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP) in humans. Data on the in vivo acute toxicity of the OA-group toxins show some differences and the European Food Safety Authority (EFSA) has determined toxicity equivalent factors (TEFs) of one for the reference toxin, OA, as well as for DTX-1 and 0.6 for DTX-2. However, recent in vitro studies indicated that DTX-1 seems to be more toxic than OA. As OA was described as apoptotic and aneugenic compound, we analyzed the DNA damage responses induced by the 3 toxins through γH2AX and pH3 biomarkers on proliferative HepaRG cells using High Content Analysis. We quantitatively examined the responses for γH2AX and pH3 by benchmark dose analyzing (BMD) using PROAST software. We found that the three toxins increased both γH2AX- and pH3-positive cells populations in a concentration-dependent manner. The 3 toxins induced mitotic arrest, characteristic of aneugenic compounds, as well as DNA strand-breaks concomitantly to cytotoxicity. BMD analysis showed that DTX-1 is the most potent inducer of DNA damage, followed by OA and DTX-2. The quantitative genotoxic data provided in this study are additional findings for reconsidering the estimated TEFs of this group of phycotoxins.


Assuntos
Inibidores Enzimáticos/toxicidade , Histonas/genética , Mutagênicos/toxicidade , Ácido Okadáico/toxicidade , Piranos/toxicidade , Benchmarking , Biomarcadores/metabolismo , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histonas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitose/efeitos dos fármacos , Testes de Mutagenicidade , Fosforilação/efeitos dos fármacos , Software
5.
Mutat Res ; 850-851: 503147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247562

RESUMO

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Fritillaria/química , Extratos Vegetais/farmacologia , Aurora Quinase B/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Instabilidade Cromossômica/genética , Colo/efeitos dos fármacos , Colo/patologia , Citocinese/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a Y-Box/genética
6.
Nat Chem Biol ; 16(5): 546-555, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152539

RESUMO

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.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Carbamatos/farmacologia , Proteínas Cdc20/antagonistas & inibidores , Diaminas/farmacologia , Mitose/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Células HCT116 , Células HeLa , Humanos , Nocodazol/farmacologia , Proteínas Nucleares/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Telomerase/genética , Telomerase/metabolismo , Imagem com Lapso de Tempo , Ubiquitinação
7.
Mutat Res ; 819-820: 111694, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32120135

RESUMO

Precise execution of the cell division cycle is vital for all organisms. The Cyclin dependent kinases (CDKs) are the main cell cycle drivers, however, their activities must be precisely fine-tuned to ensure orderly cell cycle progression. A major regulatory axis is guarded by WEE1 kinase, which directly phosphorylates and inhibits CDK1 and CDK2. The role of WEE1 in the G2/M cell-cycle phase has been thoroughly investigated, and it is a focal point of multiple clinical trials targeting a variety of cancers in combination with DNA-damaging chemotherapeutic agents. However, the emerging role of WEE1 in S phase has so far largely been neglected. Here, we review how WEE1 regulates cell-cycle progression highlighting the importance of this kinase for proper S phase. We discuss how its function is modulated throughout different cell-cycle stages and provide an overview of how WEE1 levels are regulated. Furthermore, we outline recent clinical trials targeting WEE1 and elaborate on the mechanisms behind the anticancer efficacy of WEE1 inhibition. Finally, we consider novel biomarkers that may benefit WEE1-inhibition approaches in the clinic.


Assuntos
Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/genética , Replicação do DNA , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Proteínas Tirosina Quinases/genética , Antineoplásicos/uso terapêutico , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Ensaios Clínicos como Assunto , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Humanos , Mitose/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de Sinais
8.
PLoS One ; 15(3): e0230633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32208440

RESUMO

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.


Assuntos
Degranulação Celular/efeitos dos fármacos , Lectinas/farmacologia , Proteínas de Plantas/farmacologia , Animais , Artocarpus/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Interleucina-6/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Mitose/efeitos dos fármacos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ratos
9.
Cancer Res ; 80(8): 1693-1706, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32054769

RESUMO

A significant therapeutic challenge for patients with cancer is resistance to chemotherapies such as taxanes. Overexpression of LIN9, a transcriptional regulator of cell-cycle progression, occurs in 65% of patients with triple-negative breast cancer (TNBC), a disease commonly treated with these drugs. Here, we report that LIN9 is further elevated with acquisition of taxane resistance. Inhibiting LIN9 genetically or by suppressing its expression with a global BET inhibitor restored taxane sensitivity by inducing mitotic progression errors and apoptosis. While sustained LIN9 is necessary to maintain taxane resistance, there are no inhibitors that directly repress its function. Hence, we sought to discover a druggable downstream transcriptional target of LIN9. Using a computational approach, we identified NIMA-related kinase 2 (NEK2), a regulator of centrosome separation that is also elevated in taxane-resistant cells. High expression of NEK2 was predictive of low survival rates in patients who had residual disease following treatment with taxanes plus an anthracycline, suggesting a role for this kinase in modulating taxane sensitivity. Like LIN9, genetic or pharmacologic blockade of NEK2 activity in the presence of paclitaxel synergistically induced mitotic abnormalities in nearly 100% of cells and completely restored sensitivity to paclitaxel, in vitro. In addition, suppressing NEK2 activity with two distinct small molecules potentiated taxane response in multiple in vivo models of TNBC, including a patient-derived xenograft, without inducing toxicity. These data demonstrate that the LIN9/NEK2 pathway is a therapeutically targetable mediator of taxane resistance that can be leveraged to improve response to this core chemotherapy. SIGNIFICANCE: Resistance to chemotherapy is a major hurdle for treating patients with cancer. Combining NEK2 inhibitors with taxanes may be a viable approach for improving patient outcomes by enhancing mitotic defects induced by taxanes alone.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Quinases Relacionadas a NIMA/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Paclitaxel/farmacologia , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Senescência Celular , Centrossomo/enzimologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Xenoenxertos , Humanos , Mitose/genética , Quinases Relacionadas a NIMA/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Paclitaxel/administração & dosagem , Taxa de Sobrevida , Taxoides/administração & dosagem , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
10.
Sci Rep ; 10(1): 355, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31942016

RESUMO

Breast cancer patients are commonly treated with taxane (e.g. docetaxel) chemotherapy, despite poor outcomes and eventual disease relapse. We previously identified the Bcl-2-associated death promoter (BAD) as a prognostic indicator of good outcome in taxane-treated breast cancer patients. We also demonstrated that BAD expression in human breast carcinoma cells generated larger tumors in mouse xenograft models. These paradoxical results suggest that BAD-expressing tumors are differentially sensitive to taxane treatment. We validated this here and show that docetaxel therapy preferentially reduced growth of BAD-expressing xenograft tumors. We next explored the cellular mechanism whereby BAD sensitizes cells to docetaxel. Taxanes are microtubule inhibiting agents that cause cell cycle arrest in mitosis whereupon the cells either die in mitosis or aberrantly exit (mitotic slippage) and survive as polyploid cells. In response to docetaxel, BAD-expressing cells had lengthened mitotic arrest with a higher proportion of cells undergoing death in mitosis with decreased mitotic slippage. Death in mitosis was non-apoptotic and not dependent on Bcl-XL interaction or caspase activation. Instead, cell death was necroptotic, and dependent on ROS. These results suggest that BAD is prognostic for favourable outcome in response to taxane chemotherapy by enhancing necroptotic cell death and inhibiting the production of potentially chemoresistant polyploid cells.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Docetaxel/uso terapêutico , Genes bcl-2 , Proteína de Morte Celular Associada a bcl/genética , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Mitose/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Fosforilação Oxidativa , Prognóstico , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Lett ; 473: 130-138, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904486

RESUMO

All-trans retinoic acid (ATRA) is known to be a potent inhibitor of FLT3-ITD acute myeloid leukemia (AML) cells, although the exact mechanism remains unclear. In this work, we report that ATRA causes fatal mitotic catastrophe in FLT3-ITD AML cells by degrading Chk1 kinase, and therefore preventing DNA damage repair. In order to explore a further enhancement in the inhibitory effect of ATRA on FLT3-ITD AML cells, we investigated the suitability of a combination of ATRA and DNA damage drug SN38. In vitro experiments showed that this combinatorial approach effectively inhibited the proliferation of FLT3-ITD cells and induced cell apoptosis in AML. In vivo experiments confirmed that the combination could substantially improve the anti-tumor effect of SN38. Taken together, our results indicate that ATRA down-regulates Chk1 in FLT3-ITD AML cells, and the combination of ATRA and SN38 significantly improves the anti-tumor effect of either ATRA or SN38 when used alone.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Tretinoína/farmacologia , Adulto , Idoso , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Pré-Escolar , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Irinotecano/farmacologia , Irinotecano/uso terapêutico , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , Sequências de Repetição em Tandem/genética , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/uso terapêutico , Tretinoína/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genética
12.
J Biol Chem ; 295(8): 2359-2374, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896573

RESUMO

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.


Assuntos
Espectrometria de Massas , Mitose , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Histonas/metabolismo , Humanos , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia
13.
Cell Biol Int ; 44(3): 744-754, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31769558

RESUMO

Recent epidemiologic studies pointed out a significant correlation between dietary monosodium glutamate (MSG) and increased body mass index. Corroborating evidences came from animal studies depicting a clear association between dietary MSG intake and increased abdominal fat, dyslipidemia, adipocyte hypertrophy, and total body weight gain. Taken together with the inferred absence of conspicuous hypothalamic neuropathies the hallmark of disease etiopathogenesis in MSG-obese animals, these animal studies with dietary MSG strongly argue for the presence of an alternative non-neuronal route for MSG to mediate its adipose tissue-specific phenotype and body weight gain. On the basis of this hypothesis, we investigated the direct effect of physiologically relevant low (100 µM), moderate (250 µM), and high dosages (2.5 and 25 mM) of MSG on distinct phases of adipocyte differentiation. MSG-dependent changes in cell proliferation and lipid accumulation were analyzed by cell proliferation assays, flow cytometry, and biochemical methods, respectively. Physiologically relevant high dosages MSG demonstrated a significant potential in reducing MCE and thereof adipogenic capacity of preadipocytes in a dose-dependent manner by restricting the availability of critical mitogenic proteins, CCAAT/enhancer-binding protein ß (CEBPß), and the mitotic cyclin B. Our findings warrant further investigations to unravel the effect of long-term dietary MSG intake on capacity of preadipocytes in different fat depots to undergo mitotic clonal expansion and hyperplasia in rodent models and human subjects, respectively.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Glutamato de Sódio/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Camundongos , Obesidade/metabolismo
14.
Cell Mol Life Sci ; 77(6): 1031-1047, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31562563

RESUMO

AURKA is a serine/threonine kinase overexpressed in several cancers. Originally identified as a protein with multifaceted roles during mitosis, improvements in quantitative microscopy uncovered several non-mitotic roles as well. In physiological conditions, AURKA regulates cilia disassembly, neurite extension, cell motility, DNA replication and senescence programs. In cancer-like contexts, AURKA actively promotes DNA repair, it acts as a transcription factor, promotes cell migration and invasion, and it localises at mitochondria to regulate mitochondrial dynamics and ATP production. Here we review the non-mitotic roles of AURKA, and its partners outside of cell division. In addition, we give an insight into how structural data and quantitative fluorescence microscopy allowed to understand AURKA activation and its interaction with new substrates, highlighting future developments in fluorescence microscopy needed to better understand AURKA functions in vivo. Last, we will recapitulate the most significant AURKA inhibitors currently in clinical trials, and we will explore how the non-mitotic roles of the kinase may provide new insights to ameliorate current pharmacological strategies against AURKA overexpression.


Assuntos
Aurora Quinase A/metabolismo , Neoplasias/metabolismo , Animais , Aurora Quinase A/análise , Aurora Quinase A/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Especificidade por Substrato
15.
Sci China Life Sci ; 63(3): 419-428, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31420851

RESUMO

LIN9 functions to regulate cell mitotic process. Dysregulation of LIN9 expression is associated with development of human cancers. In this study we assessed the association of LIN9 expression with paclitaxel resistance and clarified the underlying mechanisms for the first time. LIN9 expression in breast cancer tissues was retrieved from publicly available online databases and statistically analyzed. Human TNBC cell lines MDA-MB-231 and MDA-MB-468 and their corresponding paclitaxel-resistant sublines 231PTX and 468PTX were used to assess the expression of LIN9 by qRT-PCR and Western blot, cell growth by cell counting, cell viability by MTS assay, and cell apoptosis by flow cytometry. The data showed that high LIN9 expression in breast cancer patients receiving chemotherapy was related to poor overall survival (OS). LIN9 expression was upregulated in paclitaxel-resistant TNBC cells compared to their parental cells. Knockdown of LIN9 or treatment of paclitaxel-resistant TNBC cells with a bromo- and extra-terminal domain inhibitor (BETi) JQ1 which also decreased LIN9 expression enhanced the sensitivity of paclitaxel-resistant TNBC cells to paclitaxel. Mechanistically, decreased LIN9 in resistant cell lines reduced tumor cell viability, promoted multinucleated cells formation and induced tumor cell apoptosis, potentially by directly regulating microtubule-binding protein CCSAP. In conclusion, high LIN9 expression contributed to poor clinical outcomes and paclitaxel resistance in TNBC and BETi, targeting LIN9 expression, could be a reversible drug for PTX-resistant TNBC patients.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Mitose/efeitos dos fármacos , Paclitaxel/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Oncogene ; 39(2): 454-468, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31492900

RESUMO

The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Taxoides/farmacologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Ligação Proteica
17.
Int J Cancer ; 146(4): 1086-1098, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31286496

RESUMO

Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Ciclina E/genética , Cistadenocarcinoma Seroso/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Ciclina E/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Amplificação de Genes , Humanos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Gradação de Tumores , Proteínas Oncogênicas/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia
18.
Exp Cell Res ; 386(2): 111720, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31738907

RESUMO

CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCFSKP2 and APCCDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APCCDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Proteínas Cdh1/genética , Proteínas Cdh1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Genes Reporter , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fase S/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Imagem com Lapso de Tempo
19.
Ann N Y Acad Sci ; 1466(1): 73-82, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31814150

RESUMO

The asymmetric inheritance of NUMB during mitosis determines future daughter cell fates in multiple model organisms. NUMB asymmetric inheritance has also been postulated for hematopoietic stem cell (HSC) divisions but remained controversial until recently. To reconcile conflicting reports, we revisited the evidence for asymmetric inheritance of NUMB during HSC divisions. We demonstrate that previously used strategies to identify dividing cells in fixed samples suffer from multiple systematic errors. Nonmitotic cells in close proximity are frequently mistaken as dividing cells, while mitotic cells are not detected. Furthermore, microtubule depolymerization by either nocodazole or low temperatures prevents the reliable detection of mitosis and introduces mitotic artifacts. Without artificial microtubule depolymerization and by the use of reliable mitotic markers, we find NUMB differences in daughter cells to be reduced and restricted to cells with low NUMB expression and thus low signal over background. This bias fits the expected random distribution of simulated noise data, suggesting that the putative asymmetric inheritance of NUMB in HSCs could be merely technical noise. We conclude that functionally relevant asymmetric inheritance of NUMB and other factors in mitotic HSCs and other cells cannot be conclusively demonstrated using snapshot data and requires alternative approaches, such as continuous quantitative single-cell analysis.


Assuntos
Divisão Celular Assimétrica/fisiologia , Diferenciação Celular , Divisão Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Divisão Celular Assimétrica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Padrões de Herança/efeitos dos fármacos , Padrões de Herança/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mitose/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Nocodazol/farmacologia , Polimerização/efeitos dos fármacos , Distribuição Tecidual , Moduladores de Tubulina/farmacologia
20.
Bull Environ Contam Toxicol ; 104(2): 245-252, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31858153

RESUMO

Textile effluent treatment methods use biological and chemical treatments to reduce the toxicity and to comply with standard effluent discharge limits. However, trace amounts of pollutants can affect the biological organisms in the receiving environment. The present study used Allium cepa bio assay to assess the cytotoxic and genotoxic effects of treated textile effluents discharged to the natural environment. The results of the bioassay indicated that treated textile effluents can induce alterations in the mitotic index. Also nuclear buds, bi nuclei, condensed nuclei, were recorded in the bioassay and the severity of them decreased towards downstream of the effluent discharge point. Therefore, it can be concluded that even the discharged effluents comply with the standard limits, there is a possibility of causing cytotoxic and genotoxic effects in the organisms living in the natural environment.


Assuntos
Resíduos Industriais/análise , Indústria Têxtil , Poluentes Químicos da Água/toxicidade , Núcleo Celular/efeitos dos fármacos , Dano ao DNA , Monitoramento Ambiental , Mitose/efeitos dos fármacos , Cebolas/citologia , Cebolas/efeitos dos fármacos , Cebolas/genética , Testes de Toxicidade Aguda , Eliminação de Resíduos Líquidos , Poluentes Químicos da Água/análise
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