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1.
Int J Mol Sci ; 23(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35887096

RESUMO

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.


Assuntos
Aurora Quinase A/metabolismo , Desacetilase 6 de Histona/metabolismo , MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Animais , Aurora Quinase A/genética , Cílios/genética , Células Epiteliais , Camundongos , MicroRNAs/genética , Doença Pulmonar Obstrutiva Crônica/genética , Tubulina (Proteína)/genética
2.
Invest New Drugs ; 37(5): 1044-1051, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30680583

RESUMO

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1ß1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1ß1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Sarcoma Experimental/tratamento farmacológico , Venenos de Víboras/farmacologia , Animais , Apoptose , Proliferação de Células , Embrião de Galinha , Membrana Corioalantoide , Integrina alfa1beta1/antagonistas & inibidores , Camundongos , Neovascularização Patológica/patologia , Sarcoma Experimental/irrigação sanguínea , Sarcoma Experimental/patologia , Células Tumorais Cultivadas
3.
Cancers (Basel) ; 12(9)2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854244

RESUMO

Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression-downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.

4.
J Vis Exp ; (153)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31789312

RESUMO

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microtúbulos/fisiologia , Fuso Acromático/fisiologia , Células HeLa , Humanos , Proteínas Luminescentes/análise , Microscopia Confocal/métodos , Microtúbulos/química , Fuso Acromático/química , Proteína Vermelha Fluorescente
5.
Cell Cycle ; 15(6): 799-811, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27029528

RESUMO

Progression of cell cycle is associated with changes in K(+) channel expression and activity. In this study, we report that Kv10.1, a K(+) channel that increases cell proliferation and tumor growth, is regulated at the transcriptional level by the pRb/E2F1 pathway. De-repression of E2F1 by HPV-E7 oncoprotein leads to increased expression of Kv10.1. In proliferating cells, E2F1 transcription factor binds directly to the Kv10.1 promoter during (or close to) G2/M, resulting in transient expression of the channel. Importantly, this happens not only in cancer cells but also in non-transformed cells. Lack of Kv10.1 in both cancer and non-transformed cells resulted in prolonged G2/M phase, as indicated by phosphorylation of Cdk1 (Y15) and sustained pRb hyperphosphorylation. Our results strongly suggest that Kv10.1 expression is coupled to cell cycle progression and facilitates G2/M progression in both healthy and tumor cells.


Assuntos
Fator de Transcrição E2F1/genética , Canais de Potássio Éter-A-Go-Go/genética , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteína do Retinoblastoma/genética , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Fator de Transcrição E2F1/metabolismo , Embrião de Mamíferos , Canais de Potássio Éter-A-Go-Go/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Transporte de Íons , Camundongos , Camundongos Knockout , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Potássio/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Transcrição Gênica
6.
Neurochem Int ; 62(8): 1048-54, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23579020

RESUMO

UNLABELLED: The aggregates of amyloid beta peptides (Aßs) are regarded as one of the main pathological hallmarks of Alzheimer's disease (AD). An imbalance between the rates of synthesis and clearance of Aßs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аßs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aß40 and Aß42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aß40 and Aß42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aß40 (Km=37.5 µM, kcat/Km=1.7×10(3)M(-1)sec(-1)) and Aß42 (Km=138.4 µM, kcat/Km=1.90×10(2)M(-1)sec(-1)). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20mM phosphate buffer, pH 7.4, 37°C by 50-80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30-40%. CONCLUSION: for the first time it was shown that the Aß40 and Aß42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Dipeptidil Peptidase 4/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Ligação Proteica , Proteólise
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