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1.
Signal Transduct Target Ther ; 5(1): 121, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641705
2.
Int J Antimicrob Agents ; 55(6): 106004, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32361028

RESUMO

SARS-coronavirus 2 is the causal agent of the COVID-19 outbreak. SARS-Cov-2 entry into a cell is dependent upon binding of the viral spike (S) protein to cellular receptor and on cleavage of the spike protein by the host cell proteases such as Cathepsin L and Cathepsin B. CTSL/B are crucial elements of lysosomal pathway and both enzymes are almost exclusively located in the lysosomes. CTSL disruption offers potential for CoVID-19 therapies. The mechanisms of disruption include: decreasing expression of CTSL, direct inhibition of CTSL activity and affecting the conditions of CTSL environment (increase pH in the lysosomes). We have conducted a high throughput drug screen gene expression analysis to identify compounds that would downregulate the expression of CTSL/CTSB. One of the top significant results shown to downregulate the expression of the CTSL gene is amantadine (10uM). Amantadine was approved by the US Food and Drug Administration in 1968 as a prophylactic agent for influenza and later for Parkinson's disease. It is available as a generic drug. Amantadine in addition to downregulating CTSL appears to further disrupt lysosomal pathway, hence, interfering with the capacity of the virus to replicate. It acts as a lysosomotropic agent altering the CTSL functional environment.  We hypothesize that amantadine could decrease the viral load in SARS-CoV-2 positive patients and as such it may serve as a potent therapeutic decreasing the replication and infectivity of the virus likely leading to better clinical outcomes. Clinical studies will be needed to examine the therapeutic utility of amantadine in COVID-19 infection.


Assuntos
Amantadina/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Catepsina L/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Catepsina B/metabolismo , Catepsina L/genética , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Pandemias
3.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140423, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32247787

RESUMO

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.


Assuntos
Catepsinas/metabolismo , Cisteína/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral/fisiologia , Aloenxertos , Animais , Neoplasias da Mama/metabolismo , Carcinogênese , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L , Catepsinas/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Intestinais/metabolismo , Camundongos , Neoplasias/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/genética
4.
Chem Commun (Camb) ; 56(14): 2119-2122, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-31970364

RESUMO

We develop a simple fluorescence method for the sensitive detection of cathepsin B activity based on the integration of a peptide-DNA conjugate with multiple cyclic signal amplification. This method can detect cathepsin B activity with an extremely low detection limit of 8.1 × 10-12 g mL-1 and a large dynamic range of 4 orders of magnitude from 1 × 10-11 to 1 × 10-7 g mL-1, and it can even measure cathepsin B activity at the single-cell level. This method can be further used for the screening of cathepsin B inhibitors.


Assuntos
Catepsina B/análise , DNA/química , Técnicas de Amplificação de Ácido Nucleico , Peptídeos/química , Catepsina B/metabolismo , DNA/genética , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Imagem Óptica , Peptídeos/genética , Peptídeos/metabolismo
5.
Nat Commun ; 11(1): 229, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932607

RESUMO

Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal lumen, lysosomal hydrolases promote tissue remodeling when delivered to the extracellular space and cell death when released to the cytosol. Here, we show that spatially and temporally controlled lysosomal leakage contributes to the accurate chromosome segregation in normal mammalian cell division. One or more chromatin-proximal lysosomes leak in the majority of prometaphases, after which active cathepsin B (CTSB) localizes to the metaphase chromatin and cleaves a small subset of histone H3. Stabilization of lysosomal membranes or inhibition of CTSB activity during mitotic entry results in a significant increase in telomere-related chromosome segregation defects, whereas cells and tissues lacking CTSB and cells expressing CTSB-resistant histone H3 accumulate micronuclei and other nuclear defects. These data suggest that lysosomal leakage and chromatin-associated CTSB contribute to proper chromosome segregation and maintenance of genomic integrity.


Assuntos
Catepsina B/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Lisossomos/metabolismo , Mitose , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/patologia , Segregação de Cromossomos/genética , Feminino , Inativação Gênica , Histonas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , Metáfase , Camundongos , Mitose/genética , Permeabilidade , Telômero/metabolismo
6.
Nat Commun ; 11(1): 517, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980631

RESUMO

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Neurogênese , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina/metabolismo , Sequência de Aminoácidos , Catepsina B/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Proteínas de Membrana/química , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Nucleotidiltransferases/química , Organoides/metabolismo
7.
Nature ; 578(7795): 419-424, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996848

RESUMO

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.


Assuntos
Lisossomos/metabolismo , Poliaminas/metabolismo , ATPases Translocadoras de Prótons/deficiência , ATPases Translocadoras de Prótons/genética , Animais , Biocatálise , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Catepsina B/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Endocitose , Humanos , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Fenótipo , Poliaminas/toxicidade , ATPases Translocadoras de Prótons/metabolismo , Espermidina/metabolismo , Espermina/metabolismo
8.
Chemosphere ; 242: 124959, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31669990

RESUMO

Long-term exposure to arsenic can cause liver injury and fibrosis. The activation of hepatic stellate cells (HSCs) plays an essential role in the process of liver fibrosis. We found that NaAsO2 caused liver damage and fibrosis in vivo, accompanied by excessive collagen deposition and HSCs activation. In addition, NaAsO2 upregulated autophagy flux, elevated the level of cytoplasmic cathepsin B (CTSB), and activated the NOD-like receptors containing pyrin domain 3 (NLRP3) inflammasome in a subtle way. Consistent with these findings in vivo, we demonstrated that NaAsO2-induced activation of HSCs depended on CTSB-mediated NLRP3 inflammasome activation in HSC-t6 cells and rats primary HSCs. Moreover, inhibition of autophagy decreased the cytoplasmic CTSB and alleviated the activation of the NLRP3 inflammasome, thereby attenuating the NaAsO2-induced HSCs activation. In summary, these results indicated that NaAsO2 induced HSCs activation via autophagic-CTSB-NLRP3 inflammasome pathway. These findings may provide a novel insight into the potential mechanism of NaAsO2-induced liver fibrosis.


Assuntos
Arsênico/toxicidade , Autofagia , Catepsina B/metabolismo , Células Estreladas do Fígado/metabolismo , Inflamassomos/fisiologia , Cirrose Hepática/induzido quimicamente , Animais , Arsênico/metabolismo , Inflamassomos/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos
9.
Chem Pharm Bull (Tokyo) ; 68(3): 212-215, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189762

RESUMO

Anti-cancer chemotherapy with good efficacy and fewer side effects is highly desirable. A drug delivery system comprising a cancer-targeting module and a cytotoxic agent connected with a cleavable linker is promising for reducing side effects. The development of a cleavable linker satisfying the requirements of both stability and cleavability, however, is difficult, especially when a carbonate moiety is used for conjugating the linker to a hydroxy group in a drug of interest. We herein report a new stable linker comprising carbamate and ester spacers, which can be introduced on a hydroxy group of a drug. This linker is more stable in aqueous neutral buffer than a corresponding carbonate-type linker, and releases a payload anti-cancer drug, SN-38, through a two-step sequence upon cathepsin B treatment. This linker may have potential use in other drug delivery systems to lower side effects by selectively transporting cytotoxic drugs to tumor cells.


Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Oxigênio/química , Antineoplásicos/análise , Antineoplásicos/metabolismo , Carbamatos/química , Catepsina B/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Liberação Controlada de Fármacos , Ésteres/química , Humanos , Irinotecano/análise , Irinotecano/química , Irinotecano/metabolismo
10.
Toxicol Lett ; 321: 32-43, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31862506

RESUMO

Cadmium (Cd) is an important environmental pollutant. Previous studies have shown that Cd can induce liver cell injury; however, the toxicity mechanisms of Cd have not been fully elucidated. This study aimed to further confirm the hepatotoxic effects of Cd in mouse liver cells by various methods both in vivo and in vitro. In addition, it found that Cd induced autophagy but also caused autophagy blockade, and autophagy blockade intensified Cd-induced injury in liver cells. Subsequently, the study investigated the effects of Cd on lysosomes and found that Cd induced lysosomal acidification, promoted the expression of lysosomal-associated membrane protein 2 and lysosomal hydrolase cathepsin B both in vivo and in vitro, and enhanced the lysosomal degradation capacity. It indicated that Cd triggered lysosomal activation. However, the fusion of autophagosomes with lysosomes was inhibited by Cd both in vivo and in vitro. Next, the expression of Rab7, a key protein that regulates autophagosome-lysosome fusion, was examined. Cd was found to inhibit Rab7 expression both in vivo and in vitro. In conclusion, the results indicated that Cd obstructed the autophagic flux by inhibiting the fusion of autophagosomes with lysosomes, thus exacerbating the Cd-induced hepatotoxicity. Moreover, the molecular mechanism of Cd-induced inhibition of autophagosome-lysosome fusion is probably related to the Cd-induced downregulation of Rab7.


Assuntos
Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Animais , Autofagossomos/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Catepsina B/metabolismo , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Concentração de Íons de Hidrogênio , Fígado/metabolismo , Fígado/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Proteólise , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo
11.
Oxid Med Cell Longev ; 2019: 5637075, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885803

RESUMO

Age-related macular degeneration (AMD) is characterized by retinal pigment epithelial (RPE) cell dysfunction beginning at early stages of the disease. The lack of an appropriate in vitro model is a major limitation in understanding the mechanisms leading to the occurrence of AMD. This study compared human-induced pluripotent stem cell- (hiPSC-) RPE cells derived from atrophic AMD patients (77 y/o ± 7) to hiPSC-RPE cells derived from healthy elderly individuals with no drusen or pigmentary alteration (62.5 y/o ± 17.5). Control and AMD hiPSC-RPE cell lines were characterized by immunofluorescence, flow cytometry, and electronic microscopy. The toxicity level of iron after Fe-NTA treatment was evaluated by an MTT test and by the detection of dichloro-dihydro-fluorescein diacetate. Twelve hiPSC-RPE cell lines (6 AMD and 6 controls) were used for the experiment. Under basal conditions, all hiPSC-RPE cells expressed a phenotypic profile of senescent cells with rounded mitochondria at passage 2. However, the treatment with Fe-NTA induced higher reactive oxygen species production and cell death in hiPSC-RPE AMD cells than in hiPSC-RPE Control cells. Interestingly, functional analysis showed differences in lysosomal activity between the two populations. Indeed, Cathepsin B activity was higher in hiPSC-RPE AMD cells compared to hiPSC-RPE Control cells in basal condition and link to a pH more acidic in this cell population. Moreover, oxidative stress exposure leads to an increase of Cathepsin D immature form levels in both populations, but in a higher proportion in hiPSC-RPE AMD cells. These findings could demonstrate that hiPSC-RPE AMD cells have a typical disease phenotype compared to hiPSC-RPE Control cells.


Assuntos
Catepsina B/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Lisossomos/metabolismo , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Atrofia , Morte Celular , Células Cultivadas , Senescência Celular , Compostos Férricos , Humanos , Concentração de Íons de Hidrogênio , Ácido Nitrilotriacético/análogos & derivados , Estresse Oxidativo , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia
12.
Plast Reconstr Surg ; 144(6): 1338-1349, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31764649

RESUMO

BACKGROUND: The authors have previously shown that an embryonic stem cell-like population within keloid-associated lymphoid tissues in keloid lesions expresses components of the renin-angiotensin system that may be dysregulated. The authors hypothesized that cathepsins B, D, and G are present within the embryonic stem cell-like population in keloid lesions and contribute to bypass loops of the renin-angiotensin system. METHODS: 3,3'-Diaminobenzidine immunohistochemical staining for cathepsins B, D, and G was performed on formalin-fixed paraffin-embedded sections in keloid tissue samples of 11 patients. Immunofluorescence immunohistochemical staining was performed on three of these keloid tissue samples, by co-staining with CD34, tryptase, and OCT4. Western blotting, reverse transcription quantitative polymerase chain reaction, and enzyme activity assays were performed on five keloid tissue samples and four keloid-derived primary cell lines to investigate protein and mRNA expression, and functional activity, respectively. RESULTS: 3,3'-Diaminobenzidine immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 15 keloid tissue samples. Immunofluorescence immunohistochemical staining showed localization of cathepsins B and D to the endothelium of microvessels within the keloid-associated lymphoid tissues and localization of cathepsin G to the tryptase-positive perivascular cells. Western blotting confirmed semiquantitative levels of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines. Reverse transcription quantitative polymerase chain reaction showed quantitative transcriptional activation of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines and cathepsin G in keloid tissue samples. Enzyme activity assays demonstrated functional activity of cathepsins B and D. CONCLUSION: Cathepsins B, D, and G are expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues of keloid lesions and may act to bypass the renin-angiotensin system, suggesting a potential therapeutic target using renin-angiotensin system modulators and cathepsin inhibitors.


Assuntos
Catepsinas/metabolismo , Células-Tronco Embrionárias/química , Queloide/metabolismo , Western Blotting , Catepsina A/metabolismo , Catepsina B/metabolismo , Catepsina G/metabolismo , Linhagem Celular/citologia , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Cell Physiol Biochem ; 53(3): 550-572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31529928

RESUMO

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Macrófagos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células da Medula Óssea/citologia , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Células Cultivadas , Colesterol/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
14.
World Neurosurg ; 130: e117-e126, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31371266

RESUMO

BACKGROUND: This study was aimed at evaluating the gene expression levels of 4 genes in the intracranial aneurysm wall and comparing them with extracranial arteries. The analysis was done using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Also, a correlation of the differential genetic expression was done with various patient clinical and radiologic factors. METHODS: The quantitative assessment of ribonucleic acid levels was done with RT-PCR and was validated with IHC. The genes studied were collagen 1A2 (COL1A2), tissue inhibitor of metalloproteinase 4 (TIMP4), cathepsin B (CTSB), and alpha-1 antitrypsin (α-1 AT). The analysis was done on 24 aneurysm sacs and superficial temporal/occipital artery samples from patients undergoing surgical clipping. RESULTS: The mean fold change of COL1A2 in the aneurysm sample was 8.89, that of TIMP4 was 10.16, that of CTSB was 1.02, and that of α-1 AT was 1.46 when compared with normal control vessel on PCR. On semiquantitative IHC, COL1A2 was 94.44%, α-1 AT was 77.8% overexpressed, CTSB was positive in 50%, and the expression of TIMP4 was 94.4% underexpressed in aneurysmal walls. There was no statistically significant correlation between patient profile and gene expression. CONCLUSIONS: On RT-PCR and IHC analysis, COL1A2 and α-1 AT were overexpressed, CTSB was marginally overexpressed, and TIMP4 had equivocal expression in the aneurysmal sac when compared with the normal extracranial vessel. This is the first study of its kind in the Indian population with the largest sample size on live human patients.


Assuntos
Artérias/metabolismo , Catepsina B/metabolismo , Colágeno Tipo I/metabolismo , Expressão Gênica , Aneurisma Intracraniano/genética , Aneurisma Intracraniano/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , alfa 1-Antitripsina/metabolismo , Adulto , Idoso , Artéria Carótida Externa/metabolismo , Catepsina B/genética , Colágeno Tipo I/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Pescoço/irrigação sanguínea , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Couro Cabeludo/irrigação sanguínea , Inibidores Teciduais de Metaloproteinases/genética , Adulto Jovem , alfa 1-Antitripsina/genética
15.
Inorg Chem ; 58(18): 12334-12347, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31464130

RESUMO

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Catepsina B/antagonistas & inibidores , Invasividade Neoplásica/prevenção & controle , Nitroquinolinas/farmacologia , Rutênio/farmacologia , Antineoplásicos/química , Neoplasias da Mama/patologia , Catepsina B/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Modelos Moleculares , Invasividade Neoplásica/patologia , Nitroquinolinas/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Rutênio/química
16.
Chem Soc Rev ; 48(16): 4361-4374, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31294429

RESUMO

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Ácidos/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Catepsina B/metabolismo , Dissulfetos/química , Estabilidade de Medicamentos , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo
17.
Int J Nanomedicine ; 14: 4309-4317, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354262

RESUMO

Background: The intraoperative visualization of tumor cells is a powerful modality for surgical treatment of solid tumors. Since the completeness of tumor excision is closely correlated with the survival of patients, probes that can assist in distinguishing tumor cells are highly demanded. Purpose: In the present study, a fluorescent probe JF1 was synthesized for imaging of tumor cells by conjugating a substrate of cathepsin B (quenching moiety) to Oregon Green derivative JF2 using a self-immolative linker. Methods: JF1 was then loaded into the folate-PEG modified CaCO3 nanoparticles. The folate receptor-targeted, pH-dependent, and cathepsin B activable CaCO3 nanoprobe was test in vitro and in vivo for tumor imaging. Results: CaCO3 nanoprobe demonstrated good stability and fast lighting ability in tumors under low pH conditions. It also showed lower fluorescence background than the single cathepsin B dependent fluorescent probe. The pH-dependent and cathepsin B controlled "turn-on" property enables precise and fast indication of tumor in vitro and in vivo. Conclusion: This strategy of controlled drug delivery enables in vivo imaging of tumor nodules with a high signal-to-noise ratio, which has great potential in surgical tumor treatment.


Assuntos
Carbonato de Cálcio/química , Catepsina B/metabolismo , Diagnóstico por Imagem , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Animais , Corantes Fluorescentes/química , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Camundongos , Nanopartículas/ultraestrutura , Neoplasias/patologia , Especificidade de Órgãos , Polietilenoglicóis/química
18.
Nat Commun ; 10(1): 3051, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296864

RESUMO

Treatment of liver metastasis experiences slow progress owing to the severe side effects. In this study, we demonstrate a strategy capable of eliminating metastatic cancer cells in a selective manner. Nucleus-targeting W18O49 nanoparticles (WONPs) are conjugated to mitochondria-selective mesoporous silica nanoparticles (MSNs) containing photosensitizer (Ce6) through a Cathepsin B-cleavable peptide. In hepatocytes, upon the laser irradiation, the generated singlet oxygen species are consumed by WONPs, in turn leading to the loss of their photothermally heating capacity, thereby sparing hepatocyte from thermal damage induced by the laser illumination. By contrast, in cancer cells, the cleaved peptide linker allows WONPs and MSNs to respectively target nucleus and mitochondria, where the therapeutic powers could be unleashed, both photodynamically and photothermally. This ensures the energy production of cancer cells can be abolished. We further assess the underlying molecular mechanism at both gene and protein levels to better understand the therapeutic outcome.


Assuntos
Portadores de Fármacos/metabolismo , Neoplasias Hepáticas/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Catepsina B/metabolismo , Núcleo Celular/metabolismo , Portadores de Fármacos/química , Feminino , Células HCT116 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Injeções Intravenosas , Lasers , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Fosforilação Oxidativa , Óxidos/química , Fotoquimioterapia/instrumentação , Espécies Reativas de Oxigênio , Dióxido de Silício/química , Resultado do Tratamento , Tungstênio/química , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biochimie ; 166: 270-285, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31302164

RESUMO

Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes. Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with ß-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5 cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway. We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.


Assuntos
Catepsina B/metabolismo , Catepsina L/metabolismo , Cílios/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células Epiteliais da Tireoide/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Linhagem Celular , Transporte Proteico/efeitos dos fármacos , Células Epiteliais da Tireoide/citologia
20.
Food Chem ; 297: 125012, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253295

RESUMO

To obtain better understanding of the formation mechanisms of bitterness and adhesiveness, protease activities, proteolysis index and protein degradation were investigated among raw, normal and defective hams. Normal and defective hams both showed a decrease in cathepsin B and B + L activities compared with raw ham, while higher residual activities were observed in defective ham. Approximate 1.2-fold values of proteolysis index were observed in defective ham than in normal ham, indicating that cathepsin B and B + L activities were key contributors in degrading muscle proteins of dry-cured ham. 322 proteins were identified by label-free proteomics, and 49 down-regulated proteins were found in the comparison between normal and defective hams. Creatine kinase, myosin, α-actinin and troponin-T showed the most intense response to bitterness and adhesiveness of dry-cured ham, confirmed by partial least squares regression analysis. Myosin could be a suitable biomarker to monitor bitterness and adhesiveness of dry-cured ham.


Assuntos
Produtos da Carne/análise , Proteômica/métodos , Paladar/fisiologia , Adesividade , Animais , Catepsina B/metabolismo , Catepsina L/metabolismo , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Regulação para Baixo , Análise dos Mínimos Quadrados , Proteínas/análise , Proteólise , Suínos , Espectrometria de Massas em Tandem , Troponina T/análise
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