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1.
Toxicol Lett ; 334: 94-101, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33010382

RESUMO

Silica dust mainly attacks alveolar macrophages (AMs). The apoptosis of AMs is correlated with the progress of silicosis. Our previous study showed that autophagic degradation was blocked in AMs from silicosis patients. However, the effects of nicotine on AM autophagy and apoptosis in silicosis are unknown. In this study, we collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups, according to the tuberous pathological changes observed by X-ray. The AMs from both groups were exposed to nicotine. We found increased levels of LC3, p62, and cleaved caspase-3, decreased levels of LAMP2, and damaged lysosomes after nicotine stimulation of the AMs from both groups. We also found that the autophagy inhibitor 3-methyladenine (3MA) inhibited nicotine-induced apoptosis in the AMs. Furthermore, 3MA reversed both the nicotine-induced decrease in Bcl-2 and the increase in Bax in both groups. These results suggest that nicotine may induce apoptosis by blocking AM autophagic degradation in human silicosis.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Nicotina/toxicidade , Silicose/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Humanos , Marcação In Situ das Extremidades Cortadas , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Dióxido de Silício/toxicidade , Silicose/metabolismo
2.
Int J Nanomedicine ; 15: 6779-6789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32982233

RESUMO

Purpose: Alzheimer's disease (AD) is a neurodegenerative disorder that manifests as abnormal behavior and a progressive decline in memory. Although the pathogenesis of AD is due to the excessive deposition of amyloid ß protein (Aß) outside the neurons in the brain, evidence suggests that tau proteins may be a better target for AD therapy. In neurodegenerative diseases, a decrease in autophagy results in the failure to eliminate abnormally deposited or misfolded proteins. Therefore, induction of autophagy may be an effective way to eliminate tau proteins in the treatment of AD. We investigated the effects of polyethylene glycol (PEG)-ceramide nanomicelles on autophagy and on tau proteins in N2a, a murine neuroblastoma metrocyte cell line. Methods: Ceramide is a sphingolipid bioactive molecule that induces autophagy. PEG-ceramide is a polymer that is composed of the hydrophobic chain of ceramide and the hydrophilic chain of PEG-2000. In this study, we prepared PEG-ceramide nanomicelles that were 10-20 nm in size and had nearly neutral zeta potential. Results: The results show that PEG-ceramide nanomicelles caused an increase in the LC3-II/LC3-I ratio, while p62 protein levels decreased. Confocal microscopy revealed a significant increase in the number of dots corresponding to autophagosomes and autolysosomes, which indicated autophagic activation. Moreover, PEG-ceramide nanomicelles induced tau degradation in N2a cells through autophagy. Conclusion: In summary, we have confirmed that PEG-ceramide nanomicelles enhanced autophagic flux and degraded overexpressed human tau proteins in N2a cells by regulating the autophagy pathway. Thus, PEG-ceramide nanomicelles show great promise as agents to induce autophagy and degrade tau proteins in the treatment of AD.


Assuntos
Autofagia/efeitos dos fármacos , Ceramidas/farmacologia , Nanoestruturas/química , Neurônios/efeitos dos fármacos , Proteínas tau/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Ceramidas/química , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos , Neuroblastoma/patologia , Neurônios/metabolismo , Polietilenoglicóis/química , Proteínas tau/genética
3.
Nat Commun ; 11(1): 4615, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934241

RESUMO

Integration of the unique advantages of the fields of drug discovery and drug delivery is invaluable for the advancement of drug development. Here we propose a self-delivering one-component new-chemical-entity nanomedicine (ONN) strategy to improve cancer therapy through incorporation of the self-assembly principle into drug design. A lysosomotropic detergent (MSDH) and an autophagy inhibitor (Lys05) are hybridised to develop bisaminoquinoline derivatives that can intrinsically form nanoassemblies. The selected BAQ12 and BAQ13 ONNs are highly effective in inducing lysosomal disruption, lysosomal dysfunction and autophagy blockade and exhibit 30-fold higher antiproliferative activity than hydroxychloroquine used in clinical trials. These single-drug nanoparticles demonstrate excellent pharmacokinetic and toxicological profiles and dramatic antitumour efficacy in vivo. In addition, they are able to encapsulate and deliver additional drugs to tumour sites and are thus promising agents for autophagy inhibition-based combination therapy. Given their transdisciplinary advantages, these BAQ ONNs have enormous potential to improve cancer therapy.


Assuntos
Aminoquinolinas/química , Antineoplásicos/química , Sistemas de Liberação de Medicamentos/métodos , Lisossomos/efeitos dos fármacos , Nanomedicina/métodos , Neoplasias/tratamento farmacológico , Aminoquinolinas/administração & dosagem , Aminoquinolinas/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Sistemas de Liberação de Medicamentos/instrumentação , Humanos , Nanomedicina/instrumentação , Nanopartículas/química , Neoplasias/fisiopatologia , Ratos , Ratos Sprague-Dawley
4.
Int J Nanomedicine ; 15: 6385-6399, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922007

RESUMO

Purpose: The mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions. Methods: In vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected. Results: In vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter's lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines. Conclusion: Our findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.


Assuntos
Portadores de Fármacos/química , Esomeprazol/farmacologia , Sistema Fagocitário Mononuclear/citologia , Nanopartículas/química , Células A549 , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Endocitose , Esomeprazol/farmacocinética , Esomeprazol/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Distribuição Tecidual/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
Ecotoxicol Environ Saf ; 204: 111069, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32758696

RESUMO

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/análogos & derivados , Mucosa Intestinal/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Oncorhynchus mykiss/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Saxitoxina/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Catalase/metabolismo , Dinoflagelados/metabolismo , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Frutos do Mar
6.
Int J Nanomedicine ; 15: 5803-5811, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32821102

RESUMO

Introduction: Photodynamic therapy (PDT), which induces tissue damage by exposing tissue to a specific wavelength of light in the presence of a photosensitizer and oxygen, is a promising alternative treatment that could be used as an adjunct to chemotherapy and surgery in oncology. Cell-penetrating peptides (CPPs) with high arginine content, such as protamine, have membrane translocation and lysosome localization activities. They have been used in an extensive range of drug delivery applications. Methods: We conjugated cell-penetrating peptides (CPPs) with methylene blue (MB) and then purification by FPLC. Synthesis structure was characterized by the absorbance spectrum, FPLC, Maldi-TOF, and then evaluated cell viability by cytotoxicity assay after photodynamic therapy (PDT) assay. An uptake imaging assay was used to determine the sites of MB and MB-Pro in subcellular compartments. Results: In vitro assays showed that MB-Pro has more efficient photodynamic activities than MB alone for the colon cancer cells, owing to lysosome rupture causing the rapid necrotic cell death. In this study, we coupled protamine with MB for high efficacy PDT. The conjugates localized in the lysosomes and enhanced the efficiency of PDT by inducing necrotic cell death, whereas PDT with non-coupled MB resulted in only apoptotic processes. Discussion: Our research aimed to enhance PDT by engineering the photosensitizers using CPPs coupled with methylene blue (MB). MB alone permeates through the cell membrane and distributes into the cytoplasm, whereas coupling of MB dye with CPPs localizes the MB through an endocytic mechanism to a specific organelle where the localized conjugates enhance the generation of reactive oxygen species (ROS) and induce cell damage.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Azul de Metileno/farmacologia , Fotoquimioterapia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Células HT29 , Humanos , Imageamento Tridimensional , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Azul de Metileno/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
7.
Cells ; 9(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854430

RESUMO

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.


Assuntos
Inibidores de Adenilil Ciclases/uso terapêutico , Adenilil Ciclases/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , AMP Cíclico/antagonistas & inibidores , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
8.
Ecotoxicol Environ Saf ; 205: 111062, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32846292

RESUMO

Carbon black (CB) and heavy metals are the main components of Particulate Matter (PM). Although the individual toxicities of CB and heavy metals have been extensively studied, the combined toxicity is much less understood. In this study, we choose the nano carbon black (CBNPs) and Pb2+ to simulate fine particles in the atmosphere and study the combined toxic effect on rat alveolar macrophages. The data showed that CBNPs could adsorb Pb2+ to form CBNPs-Pb2+ complex and displayed an altered physical properties by particle characterization. CBNPs-Pb2+ synergistically induced rat alveolar macrophages apoptosis and blocked autophagy flux compared with CBNPs and Pb2+ individually. Consistent with this, CBNPs-Pb2+ could impair the mitochondrial membrane potential (MMP), activate apoptotic signaling pathways, inhibit lysosomal function.


Assuntos
Chumbo/toxicidade , Macrófagos Alveolares/efeitos dos fármacos , Nanopartículas/toxicidade , Fuligem/toxicidade , Poluentes Atmosféricos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia , Lisossomos/efeitos dos fármacos , Metais Pesados/farmacologia , Material Particulado/farmacologia , Ratos , Transdução de Sinais
9.
Biomed Pharmacother ; 130: 110582, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763818

RESUMO

Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Lisossomos/efeitos dos fármacos , Pandemias , Pneumonia Viral/tratamento farmacológico , Anti-Inflamatórios/farmacocinética , Antivirais/efeitos adversos , Antivirais/farmacocinética , Arritmias Cardíacas/induzido quimicamente , Azitromicina/farmacocinética , Azitromicina/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cloroquina/farmacocinética , Cloroquina/uso terapêutico , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapêutico , Membranas Intracelulares/fisiologia , Lisossomos/química , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Fosfolipídeos/metabolismo , Tensoativos/farmacocinética , Internalização do Vírus
10.
Cell Death Dis ; 11(8): 656, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814759

RESUMO

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Morte Celular/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Hidroxicloroquina/farmacologia , Mesilato de Imatinib/farmacologia , Lisossomos/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , RNA Viral/efeitos dos fármacos , Células Vero , Carga Viral/efeitos dos fármacos
11.
Cell Signal ; 73: 109706, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32629149

RESUMO

Chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) have been thrust into our everyday vernacular because some believe, based on very limited basic and clinical data, that they might be helpful in preventing and/or lessening the severity of the pandemic coronavirus disease 2019 (COVID-19). However, lacking is a temperance in enthusiasm for their possible use as well as sufficient perspective on their effects and side-effects. CQ and HCQ have well-known properties of being diprotic weak bases that preferentially accumulate in acidic organelles (endolysosomes and Golgi apparatus) and neutralize luminal pH of acidic organelles. These primary actions of CQ and HCQ are responsible for their anti-malarial effects; malaria parasites rely on acidic digestive vacuoles for survival. Similarly, de-acidification of endolysosomes and Golgi by CQ and HCQ may block severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) integration into host cells because SARS-CoV-2 may require an acidic environment for its entry and for its ability to bud and infect bystander cells. Further, de-acidification of endolysosomes and Golgi may underly the immunosuppressive effects of these two drugs. However, modern cell biology studies have shown clearly that de-acidification results in profound changes in the structure, function and cellular positioning of endolysosomes and Golgi, in signaling between these organelles and other subcellular organelles, and in fundamental cellular functions. Thus, studying the possible therapeutic effects of CQ and HCQ against COVID-19 must occur concurrent with studies of the extent to which these drugs affect organellar and cell biology. When comprehensively examined, a better understanding of the Janus sword actions of these and other drugs might yield better decisions and better outcomes.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Endossomos/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidroxicloroquina/uso terapêutico , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Malária/tratamento farmacológico , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico
12.
Arterioscler Thromb Vasc Biol ; 40(9): 2054-2069, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640907

RESUMO

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.


Assuntos
Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Catepsinas/biossíntese , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catepsinas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Exocitose , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout para ApoE , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , Via Secretória , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
13.
Cells ; 9(6)2020 06 13.
Artigo em Inglês | MEDLINE | ID: covidwho-603067

RESUMO

There is no vaccine or specific antiviral treatment for COVID-19, which is causing a global pandemic. One current focus is drug repurposing research, but those drugs have limited therapeutic efficacies and known adverse effects. The pathology of COVID-19 is essentially unknown. Without this understanding, it is challenging to discover a successful treatment to be approved for clinical use. This paper addresses several key biological processes of reactive oxygen, halogen and nitrogen species (ROS, RHS and RNS) that play crucial physiological roles in organisms from plants to humans. These include why superoxide dismutases, the enzymes to catalyze the formation of H2O2, are required for protecting ROS-induced injury in cell metabolism, why the amount of ROS/RNS produced by ionizing radiation at clinically relevant doses is ~1000 fold lower than the endogenous ROS/RNS level routinely produced in the cell and why a low level of endogenous RHS plays a crucial role in phagocytosis for immune defense. Herein we propose a plausible amplification mechanism in immune defense: ozone-depleting-like halogen cyclic reactions enhancing RHS effects are responsible for all the mentioned physiological functions, which are activated by H2O2 and deactivated by NO signaling molecule. Our results show that the reaction cycles can be repeated thousands of times and amplify the RHS pathogen-killing (defense) effects by 100,000 fold in phagocytosis, resembling the cyclic ozone-depleting reactions in the stratosphere. It is unraveled that H2O2 is a required protective signaling molecule (angel) in the defense system for human health and its dysfunction can cause many diseases or conditions such as autoimmune disorders, aging and cancer. We also identify a class of potent drugs for effective treatment of invading pathogens such as HIV and SARS-CoV-2 (COVID-19), cancer and other diseases, and provide a molecular mechanism of action of the drugs or candidates.


Assuntos
Antivirais/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Compostos Heterocíclicos/uso terapêutico , Hidrocarbonetos Halogenados/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Animais , Antivirais/uso terapêutico , Infecções por Coronavirus/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Lisossomos/efeitos dos fármacos , Pandemias , Fagocitose , Pneumonia Viral/metabolismo , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais
14.
Cells ; 9(6)2020 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545714

RESUMO

There is no vaccine or specific antiviral treatment for COVID-19, which is causing a global pandemic. One current focus is drug repurposing research, but those drugs have limited therapeutic efficacies and known adverse effects. The pathology of COVID-19 is essentially unknown. Without this understanding, it is challenging to discover a successful treatment to be approved for clinical use. This paper addresses several key biological processes of reactive oxygen, halogen and nitrogen species (ROS, RHS and RNS) that play crucial physiological roles in organisms from plants to humans. These include why superoxide dismutases, the enzymes to catalyze the formation of H2O2, are required for protecting ROS-induced injury in cell metabolism, why the amount of ROS/RNS produced by ionizing radiation at clinically relevant doses is ~1000 fold lower than the endogenous ROS/RNS level routinely produced in the cell and why a low level of endogenous RHS plays a crucial role in phagocytosis for immune defense. Herein we propose a plausible amplification mechanism in immune defense: ozone-depleting-like halogen cyclic reactions enhancing RHS effects are responsible for all the mentioned physiological functions, which are activated by H2O2 and deactivated by NO signaling molecule. Our results show that the reaction cycles can be repeated thousands of times and amplify the RHS pathogen-killing (defense) effects by 100,000 fold in phagocytosis, resembling the cyclic ozone-depleting reactions in the stratosphere. It is unraveled that H2O2 is a required protective signaling molecule (angel) in the defense system for human health and its dysfunction can cause many diseases or conditions such as autoimmune disorders, aging and cancer. We also identify a class of potent drugs for effective treatment of invading pathogens such as HIV and SARS-CoV-2 (COVID-19), cancer and other diseases, and provide a molecular mechanism of action of the drugs or candidates.


Assuntos
Antivirais/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Compostos Heterocíclicos/uso terapêutico , Hidrocarbonetos Halogenados/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Animais , Antivirais/uso terapêutico , Infecções por Coronavirus/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Lisossomos/efeitos dos fármacos , Pandemias , Fagocitose , Pneumonia Viral/metabolismo , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais
15.
Cell Prolif ; 53(7): e12813, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32515860

RESUMO

OBJECTIVES: Accumulating studies have investigated the PM2.5-induced pulmonary toxicity, while gaps still remain in understanding its toxic mechanism. Due to its high specific surface area and adsorption capacity similar to nanoparticles, PM2.5 acts as a significant carrier of metals in air and then leads to altered toxic effects. In this study, we aimed to use CBs and Ni as model materials to investigate the autophagy changes and pulmonary toxic effects at 30 days following intratracheal instillation of CBs-Ni mixture. MATERIALS AND METHODS: Groups of mice were instilled with 100 µL normal saline (NS), 20 µg CBs, and 4 µg Ni or CBs-Ni mixture, respectively. At 7 and 30 days post-instillation, all the mice were weighed and then sacrificed. The evaluation system was composed of the following: (a) autophagy and lysosomal function assessment, (b) trace element biodistribution observation in lungs, (c) pulmonary lavage biomedical analysis, (d) lung histopathological evaluation, (e) coefficient analysis of major organs and (f) CBs-Ni interaction and cell proliferation assessment. RESULTS: We found that after CBs-Ni co-exposure, no obvious autophagy and lysosomal dysfunction or pulmonary toxicity was detected, along with complete clearance of Ni from lung tissues as well as recovery of biochemical indexes to normal range. CONCLUSIONS: We conclude that the damaged autophagy and lysosomal function, as well as physiological function, was repaired at 30 days after exposure of CBs-Ni. Our findings provide a new idea for scientific assessment of the impact of fine particles on environment and human health, and useful information for the comprehensive treatment of air pollution.


Assuntos
Autofagia/efeitos dos fármacos , Carbono/efeitos adversos , Pneumopatias/induzido quimicamente , Pulmão/efeitos dos fármacos , Metais/efeitos adversos , Animais , Linhagem Celular , Pulmão/metabolismo , Pneumopatias/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Células RAW 264.7 , Distribuição Tecidual
16.
Int J Antimicrob Agents ; 56(2): 106044, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32522674

RESUMO

While the coronavirus disease 2019 (COVID-19) pandemic advances, the scientific community continues to struggle in the search for treatments. Several improvements have been made, including discovery of the clinical efficacy of chloroquine (CQ) in patients with COVID-19, but effective treatment protocols remain elusive. In the search for novel treatment options, many scientists have used the in-silico approach to identify compounds that could interfere with the key molecules involved in entrance, replication or dissemination of severe acute respiratory syndrome coronavirus-2. However, most of the identified molecules are not available as pharmacological agents at present, and assessment of their safety and efficacy could take many months. This review took a different approach based on the proposed pharmacodynamic model of CQ in COVID-19. The main mechanism of action responsible for the favourable outcome of patients with COVID-19 treated with CQ seems to be related to a pH-modulation-mediated effect on endolysosomal trafficking, a characteristic of chemical compounds often called 'lysosomotropic agents' because of the physico-chemical properties that enable them to diffuse passively through the endosomal membrane and undergo protonation-based trapping in the lumen of the acidic vesicles. This review discusses lysosomotropic and lysosome targeting drugs that are already in clinical use and are characterized by good safety profiles, low cost and wide availability. Some of these drugs -particularly azithromycin and other macrolides, indomethacin and some other non-steroidal anti-inflammatory drugs, proton pump inhibitors and fluoxetine - could provide additional therapeutic benefits in addition to the potential antiviral effect that is still to be confirmed by well-controlled clinical trials. As some of these drugs have probably been used empirically in the treatment of COVID-19, it is hoped that colleagues worldwide will publish patient data to enable evaluation of the potential efficacy of these agents in the clinical context, and rapid implementation in therapeutic protocols if they are shown to have a beneficial effect on clinical outcome.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Lisossomos/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Antivirais/farmacologia , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Humanos , Pandemias , Pneumonia Viral/virologia , Replicação Viral/efeitos dos fármacos
17.
Toxicol Appl Pharmacol ; 401: 115090, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32512069

RESUMO

Copper dyshomeostasis is involved in the pathogenesis of Alzheimer's disease (AD). Microglia play a major role in the proteolytic clearance of oligomeric ß-amyloid (Aßo). Here, we investigated whether Cu(II) affects microglial Aßo clearance and whether this effect involves autophagy-lysosomal pathway. Microtubule associated protein 1 light chain 3 (LC3)-II and p62 protein levels and autophagic flux in Cu(II)-treated microglia were detected. Aßo clearance was detected by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. In vivo, Cu(II) and Aßo were injected into mouse hippocampus to evaluate Aß clearance. The results showed that Cu(II) inhibited phagocytic uptake and intracellular degradation of Aßo in microglial cultures. Additionally, Cu(II) elevated LC3-II and p62 protein levels and impaired autophagic flux. It also inhibited transcription factor EB (TFEB) expression and lysosomal biogenesis. Moreover, Cu(II) activated mammalian target of rapamycin kinase (mTOR), an upstream signaling of TFEB. The mTOR inhibitor PP242 ameliorated Cu(II)-impaired TFEB expression, lysosomal biogenesis, autophagic flux, and Aßo clearance in microglia. In vivo, Cu(II) inhibited microglial Aßo clearance in mouse hippocampus, an effect accompanied with activation of mTOR and impairment of TFEB expression and lysosomal biogenesis. Collectively, our results suggest that Cu(II) reduces microglial Aßo clearance through disrupting lysosomal biogenesis and autophagic flux. This effect could involve modulation of mTOR-TFEB axis and was prevented by pharmacological antagonism of mTOR. This study reveals a novel mechanism for Cu(II) involvement in AD. Our results implicate that rescue of Cu(II)-impaired autophagy-mediated lysosomal degradation may provide a new strategy to benefit multiple neurodegenerative disorders.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cobre/toxicidade , Lisossomos/metabolismo , Microglia/metabolismo , Fragmentos de Peptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
18.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591533

RESUMO

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Assuntos
Autofagia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fenótipo , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/patologia , Fatores de Tempo
19.
Exp Parasitol ; 216: 107939, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32535115

RESUMO

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.


Assuntos
Glucosilceramidase/antagonistas & inibidores , Leishmania/fisiologia , Macrófagos/parasitologia , Fagocitose , Animais , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Doença de Gaucher/complicações , Doença de Gaucher/fisiopatologia , Glucosilceramidase/efeitos dos fármacos , Glucosilceramidase/genética , Inositol/análogos & derivados , Inositol/farmacologia , Leishmania infantum/fisiologia , Leishmania major/fisiologia , Leishmania mexicana/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos
20.
Nat Struct Mol Biol ; 27(7): 605-614, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541897

RESUMO

Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.


Assuntos
Descoberta de Drogas/métodos , Fatores Imunológicos/farmacologia , Proteínas/metabolismo , Humanos , Fatores Imunológicos/química , Ácidos Indolacéticos/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Ubiquitinação/efeitos dos fármacos
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