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1.
Bull Environ Contam Toxicol ; 103(6): 796-801, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31676939

RESUMO

The aim of this study was to determine the cytotoxic and genotoxic effects of copper on the bivalve Perumytilus purpuratus. The individuals were exposed to three copper concentrations: 1, 30 and 45 µg L-1 for 24, 48 and 96 h. Lysosomal membrane stability in hemocytes was determined through the neutral red retention time (NRRT) and micronucleus (MN) frequency tests in hemocytes and gills. The results show that the NRRT decreased significantly at 30 µg L-1 after 48 h of exposure. The frequency of MN was significantly greater in gills after 24 h in all concentrations tested. Copper is cytotoxic from 30 µg L-1 and genotoxic from 1 µg L-1. The use of these biomarkers of effects in P. purpuratus is proposed as an early warning tool for monitoring in environmental assessment of coastal ecosystems impacted by mining activities.


Assuntos
Cobre/toxicidade , Monitoramento Ambiental/métodos , Brânquias/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mytilidae/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cobre/análise , Ecossistema , Biomarcadores Ambientais/efeitos dos fármacos , Brânquias/irrigação sanguínea , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Membranas Intracelulares/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Mytilidae/genética , Vermelho Neutro , Poluentes Químicos da Água/análise
2.
Int J Nanomedicine ; 14: 7003-7016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564862

RESUMO

Background: Yttria-stabilized zirconia (Y2O3/ZrO2) nanoparticles are one of the important nanoparticles extensively used in manufacturing of plastics, textiles, catalyst, etc. Still, the cytotoxic and apoptotic effects of yttria-stabilized zirconia nanoparticles have not been well identified on human skin keratinocyte (HaCaT) cells. Therefore, in this study, we have designed to examine the cytotoxic potential of yttria-stabilized zirconia nanoparticles in HaCaT cells. Methods: Prior to treatment, the yttria-stabilized zirconia nanoparticles were characterized by using different advanced instruments viz. dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM). Cell viability of HaCaT cells was measured by using MTS and NRU assays and viability of cells was reduced in a dose- and time-dependent manner. Results: Reduction in the viability of cells was correlated with the rise of reactive oxygen species generation, increased caspase-3, mitochondria membrane potential and evidence of DNA strand breakage. These were consistent with the possibility that mitochondria damage can play a significant role in the cytotoxic response. Moreover, the activity of oxidative enzymes such as lipid peroxide (LPO) was increased and glutathione was reduced in HaCaT cells exposed with yttria-stabilized zirconia nanoparticles. It is also important to indicate that HaCaT cells appear to be more susceptible to yttria-stabilized zirconia nanoparticles exposure after 24 hrs. Conclusion: This result provides a dose- and time-dependent apoptosis and genotoxicity of yttria-stabilized zirconia nanoparticles in HaCaT cells.


Assuntos
Apoptose , Dano ao DNA , Células Epiteliais/citologia , Nanopartículas Metálicas/química , Pele/citologia , Ítrio/química , Zircônio/química , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
3.
Aquat Toxicol ; 215: 105266, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401474

RESUMO

The persistent pollutants polybrominated diphenyl ethers (PBDEs) have been demonstrated to produce several negative effects on marine organisms. Although Mytilus galloprovincialis was extensively studied as model system, the effects of PBDEs on the innate immune system of mussels remains unclear. In this study, except for the control treatment, specimens of M. galloprovincialis were fed with microalgae treated with increasing concentrations of PBDEs (maximum level 100 ng L-1 of BDE-47 per day). BDE-47 treatment was maintained for 15 days and then the animals were fed with the same control diet, without contaminants, for 15 days. Samples of haemolymph (HL) were obtained at T0, T15 and T30 days of the experiment to evaluate different parameters related to immunity, such as neutral red retention time, and peroxidase, protease, antiprotease, lysozyme and bactericidal activities. BDE-47 exposure for 15 days affected both the stability of haemocytes and humoral parameters. In addition, the obtained results indicated that, at 30 days, after 15 days of culture without contaminant, the immune parameters were still affected, as some of them did not return to the basal levels, and others remained stimulated. Overall the results indicate that BDE-47 exposures at environmentally realistic levels may affect various aspects of immune function in M. galloprovincialis, acting as stressor that can compromise the general welfare.


Assuntos
Exposição Ambiental , Éteres Difenil Halogenados/toxicidade , Mytilus/imunologia , Animais , Antibacterianos/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microalgas/fisiologia , Mytilus/efeitos dos fármacos , Mytilus/microbiologia , Peptídeo Hidrolases/metabolismo , Análise de Sobrevida , Poluentes Químicos da Água/toxicidade
4.
Biointerphases ; 14(2): 021002, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30884950

RESUMO

Recent developments in the field of fullerene C60 and its derivatives suggest its suitability in a wide range of applications ranging from photovoltaic instruments, development of solar based cells, cosmetics to enzyme inhibition treatment, and so on. These innovative applications raised possibilities of intentional or oblivious human-particle contact leading to possible deleterious effects on human health. The current study deals with the interaction of dextran functionalized fullerene C60 (Dex-C60) on Chinese Hamster Ovary cells. The results showed that the cell viability was not affected by Dex-C60 treatment even at higher concentrations. Treatment of Dex-C60 did not affect mitochondrial membrane potential and the integrity of lysosomal and cytoskeletal membrane. DNA ladder assay and nuclear staining showed that the DNA remains intact, and no fragmentation or nuclear condensation was visible. From flow cytometry analysis, the viable population of treated cells was seemed to be remaining similar to that of untreated cells. Hence, from the current result, it is concluded that Dex-C60 can be a potential candidate for various biomedical applications.


Assuntos
Células CHO/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Fulerenos/toxicidade , Polímeros/toxicidade , Animais , Cricetulus , DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos
5.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625978

RESUMO

The use of titanium suboxides, known as Magnéli phase TiOx, is expected to increase in the near future due to their desirable properties. In order to use Magnéli phase TiOx nanoparticles safely, it is necessary to know how nanoparticles interact with biological systems. In this study, the cytotoxicity of three different Magnéli TiOx nanoparticles was evaluated using human lung A549 cells and the results were compared with hazard data on two different TiO2 nanoparticles whose biological interactions have already been extensively studied. After A549 cells were exposed to nanoparticles, the metabolic activity was measured by the Resazurin assay, the amount of cellular proteins was measured by the Coomassie Blue assay, and lysosomal integrity was measured by the Neutral Red Uptake assay. In order to investigate possible modes of particle actions, intracellular Ca2+ level, reactive oxygen species (ROS) production, and photo-oxidative disruptions of lysosomal membranes were assessed. All experiments were performed in serum-containing and in serum-deprived cell culture mediums. In addition, the photocatalytic activity of Magnéli TiOx and TiO2 nanoparticles was measured. The results show that Magnéli TiOx nanoparticles increase intracellular Ca2+ but not ROS levels. In contrast, TiO2 nanoparticles increase ROS levels, resulting in a higher cytotoxicity. Although Magnéli TiOx nanoparticles showed a lower UV-A photocatalytic activity, the photo-stability of the lysosomal membranes was decreased by a greater extent, possibly due to particle accumulation inside lysosomes. We provide evidence that Magnéli TiOx nanoparticles have lower overall biological activity when compared with the two TiO2 formulations. However, some unique cellular interactions were detected and should be further studied in line with possible Magnéli TiOx application. We conclude that Magnéli phase nanoparticles could be considered as low toxic material same as other forms of titanium oxide particles.


Assuntos
Pulmão/patologia , Nanopartículas/toxicidade , Titânio/toxicidade , Células A549 , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Espaço Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Nanopartículas/ultraestrutura , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
6.
J Cell Biol ; 218(1): 83-96, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30442642

RESUMO

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.


Assuntos
Citoesqueleto/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Membranas Intracelulares/ultraestrutura , Microscopia/métodos , Microtúbulos/ultraestrutura , Animais , Células COS , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Imagem Molecular/métodos , Nocodazol/farmacologia , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Imagem com Lapso de Tempo/estatística & dados numéricos , Moduladores de Tubulina/farmacologia
7.
Nat Commun ; 9(1): 5358, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560896

RESUMO

Surface receptor and transporter protein down-regulation is assumed to be exclusively mediated by the canonical multivesicular body (MVB) pathway and ESCRTs (Endosomal Sorting Complexes Required for Transport). However, few surface proteins are known to require ESCRTs for down-regulation, and reports of ESCRT-independent degradation are emerging, suggesting that alternative pathways exist. Here, using Saccharomyces cerevisiae as a model, we show that the hexose transporter Hxt3 does not require ESCRTs for down-regulation conferring resistance to 2-deoxyglucose. This is consistent with GFP-tagged Hxt3 bypassing ESCRT-mediated entry into intralumenal vesicles at endosomes. Instead, Hxt3-GFP accumulates on vacuolar lysosome membranes and is sorted into an area that, upon fusion, is internalized as an intralumenal fragment (ILF) and degraded. Moreover, heat stress or cycloheximide trigger degradation of Hxt3-GFP and other surface transporter proteins (Itr1, Aqr1) by this ESCRT-independent process. How this ILF pathway compares to the MVB pathway and potentially contributes to physiology is discussed.


Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Desoxiglucose/farmacologia , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
8.
PLoS One ; 13(9): e0204532, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30240452

RESUMO

PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdIns(3,5)P2, is crucial for mammalian cell proliferation and viability. Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized. We demonstrate here for the first time that apilimod powerfully inhibited in vitro synthesis of PtdIns5P along with that of PtdIns(3,5)P2. HPLC-based resolution of intracellular phosphoinositides (PIs) revealed that apilimod triggered a marked reduction of both lipids in the context of intact cells. Notably, there was also a profound rise in PtdIns3P resulting from arrested PtdIns3P consumption for PtdIns(3,5)P2 synthesis. As typical for PIKfyve inhibition and the concomitant PtdIns(3,5)P2 reduction, apilimod induced the appearance of dilated endomembrane structures in the form of large translucent cytoplasmic vacuoles. Remarkably, bafilomycin A1 (BafA1) fully reversed the aberrant cell phenotype back to normal and completely precluded the appearance of cytoplasmic vacuoles when added prior to apilimod. Inspection of the PI profiles ruled out restoration of the reduced PtdIns(3,5)P2 pool as a molecular mechanism underlying BafA1 rescue. Rather, we found that BafA1 markedly attenuated the PtdIns3P elevation under PIKfyve inhibition. This was accompanied by profoundly decreased endosomal recruitment of fusogenic EEA1. Together, our data demonstrate that apilimod inhibits not only PtdIns(3,5)P2 but also PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by BafA1 through a mechanism associated, in part, with reduction in both PtdIns3P levels and EEA1 membrane recruitment.


Assuntos
Antineoplásicos/farmacologia , Endossomos/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Triazinas/farmacologia , Animais , Células COS , Citoplasma/efeitos dos fármacos , Citoplasma/patologia , Citoplasma/fisiologia , Endossomos/patologia , Endossomos/fisiologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Membranas Intracelulares/patologia , Membranas Intracelulares/fisiologia , Macrolídeos/farmacologia
9.
Exp Cell Res ; 371(1): 139-150, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30098331

RESUMO

Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.


Assuntos
Benzoquinonas/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Lactamas Macrocíclicas/farmacologia , Proteína Quinase C/genética , Receptor ErbB-2/genética , Acetato de Tetradecanoilforbol/farmacologia , Linhagem Celular Tumoral , Clatrina/genética , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Microscopia Imunoeletrônica , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Transdução de Sinais , Ubiquitinação/efeitos dos fármacos
10.
Mol Biol Cell ; 29(19): 2303-2316, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30024290

RESUMO

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.


Assuntos
IMP Desidrogenase/química , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Glucose/deficiência , Guanosina/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Cinética , Síndrome de Lesch-Nyhan/patologia , Camundongos , Ácido Micofenólico/farmacologia , Transporte Proteico , Ribonucleotídeos/farmacologia
11.
Cell Microbiol ; 20(11): e12889, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29993167

RESUMO

Miltefosine is an important drug for the treatment of leishmaniasis; however, its mechanism of action is still poorly understood. In these studies, we tested the hypothesis that like in cancer cells, miltefosine's efficacy in leishmaniasis is due to its inhibition of Akt activation in host cells. We show using pharmacologic agents that block Akt activation by different mechanisms and also using an inducible knockdown approach that miltefosine loses its efficacy when its access to Akt1 is limited. Interestingly, limitation of Akt activation results in clearance of established Leishmania infections. We then show, using fluorophore-tagged probes that bind to phosphoinositides, that Leishmania parasitophorous vacuole membranes (LPVMs) display the relevant phosphoinositides to which Akt can be recruited and activated continuously. Taken together, we propose that the acquisition of PI(4) P and the display of PI (3,4)P2 on LPVMs initiate the machinery that supports continuous Akt activation and sensitivity to miltefosine.


Assuntos
Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Fosfatidilinositóis/metabolismo , Fosforilcolina/análogos & derivados , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antiprotozoários/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Leishmaniose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Fosforilcolina/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Vacúolos/efeitos dos fármacos
12.
Cell Death Dis ; 9(7): 780, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006504

RESUMO

The cellular recycling pathway of autophagy plays a fundamental role in adaptive responses to nutrient deprivation and other forms of stress under physiological and pathological conditions. However, autophagy can also be a double-edge sword during certain bacterial infections (such as urinary tract infections) and in cancer, where it can be hijacked by the pathogens and cancer cells, respectively, to promote their own survival. Thus, autophagy modulation can potentially have multiple effects in multiple contexts and this property can be leveraged to improve outcomes. In this report, we identify that a broad-spectrum antibiotic, 2-((3-(3, 6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl) amino)-2-(hydroxymethyl) propane-1, 3-diol (DCAP) modulates autophagy. We employed combined biochemical, fluorescence microscopy and correlative light electron microscopy approaches to demonstrate that DCAP treatment blocks autophagy at the late stages by preventing autophagolysosome maturation and interrupting the autophagic flux. We further show that, DCAP significantly reduces UPEC infection in urinary tract epithelial cells via inhibition of autophagy. Finally, we reveal that DCAP enhances the anticancer activity of the histone acetyltransferase (HDAC) inhibitor, vorinostat, which has been reported to increase susceptibility to bacterial infections as a common adverse effect. Collectively, our data support the concept that DCAP represents a valuable chemical scaffold for the development of an innovative class of bactericidal autophagy inhibitors for treatment of urinary tract infections and/or for adjuvant therapy in cancer treatment.


Assuntos
Aminofenóis/farmacologia , Antibacterianos/uso terapêutico , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/fisiologia , Vorinostat/farmacologia , Antibacterianos/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular Tumoral , Infecções por Escherichia coli/microbiologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos
13.
Oncogene ; 37(38): 5205-5220, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29849119

RESUMO

mTOR is an important regulator of cell growth and forms two complexes, mTORC1/2. In cancer, mTOR signaling is highly activated, and the regulation of this signaling, as an anti-cancer strategy, has been emphasized. However, PP242 (inhibitor of mTORC1 and mTORC2) alone did not induce human renal carcinoma cell death. In this study, we found that PP242 alone did not alter cell viability, but combined curcumin and PP242 treatment induced cell death. Combined PP242 and curcumin treatment induced Bax activation and decreased expression of Mcl-1 and Bcl-2. Furthermore, co-treatment with PP242 and curcumin-induced the downregulation of the Rictor (an mTORC2 complex protein) and Akt protein levels, and ectopic overexpression of Rictor or Akt inhibited PP242 plus curcumin induced cell death. Downregulation of Rictor increased cytosolic Ca2+ release from endoplasmic reticulum, which led to lysosomal damage in PP242 plus curcumin-treated cells. Furthermore, damaged lysosomes induced autophagy. Autophagy inhibitors markedly inhibited cell death. Finally, combined curcumin and PP242 treatment reduced tumor growth and induced cell death in xenograft models. Altogether, our results reveal that combined PP242 and curcumin treatment could induce autophagy-mediated cell death by reducing the expression of Rictor and Akt in renal carcinoma cells.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Curcumina/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Camundongos , Permeabilidade/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/farmacologia , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo
14.
Bull Exp Biol Med ; 165(1): 36-39, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29796812

RESUMO

The direct effect of 5 mM L-NAME and 0.1 mM sodium nitroprusside on activity of lysosomal cysteine proteinases and permeability of lysosomal membrane was studied in vitro after 1, 2, and 4 h of incubation. Isolated from the liver of intact female rats lysosome suspensions were used. Both substances reduced total activity of cathepsin H and did not affect cathepsin B at all time intervals. L-NAME increased cathepsin L activity at all incubation times, while sodium nitroprusside increased activity of this enzyme after 2-h incubation and reduced it incubation after 4-h incubation. L-NAME demonstrated a membrane-destabilizing effect in in vitro experiments, while sodium nitroprusside on the contrary stabilized lysosomal membranes.


Assuntos
Cisteína Proteases/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Membranas Intracelulares/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Ratos , Ratos Wistar
15.
Carbohydr Polym ; 192: 95-103, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29691039

RESUMO

In this study, the fungicidal effects and detailed action of chitosan against Ceratocystis fimbriata were evaluated. The results demonstrated that chitosan exhibited strong antifungal activity that restricted the mycelium extension and changed the hyphal morphology. Fluorescein diacetate (FDA) and propidium iodide (PI) double-staining directly visualized decreased cell viability in response to chitosan treatment. Investigation of the PI influx showed that chitosan induced irreversible cell membrane damage. The efflux of potassium ions from the cytosol into the extracellular matrix demonstrated that chitosan induced the leakage of intracellular components. Massive intracellular bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] accumulation indicated the dissipation of membrane potential. Furthermore, chitosan clearly decreased the activity of H+/K+ ATPase. Fluorescence microscopy revealed that the fluorescence distribution and intensity of fluorescein isothiocyanate (FITC) changed as the incubation time increased. These results indicate that chitosan exerts a fungicidal effect via its ability to disturb fungal membranes.


Assuntos
Antifúngicos/farmacologia , Ascomicetos/citologia , Ascomicetos/efeitos dos fármacos , Quitosana/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Ascomicetos/crescimento & desenvolvimento , Citoplasma/efeitos dos fármacos , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Potássio/metabolismo
16.
J Med Chem ; 61(9): 3889-3907, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29648811

RESUMO

Antimicrobial peptides are an important weapon against invading pathogens and are potential candidates as novel antibacterial agents, but their antifungal activities are not fully developed. In this study, a set of imperfectly amphipathic peptides was developed based on the imperfectly amphipathic palindromic structure R n(XRXXXRX)R n ( n = 1, 2; X represents L, I, F, or W), and the engineered peptides exhibited high antimicrobial activities against all fungi and bacteria tested (including fluconazole-resistant Candida albicans), with geometric mean (GM) MICs ranging from 2.2 to 6.62 µM. Of such peptides, 13 (I6) (RRIRIIIRIRR-NH2) that was Ile rich in its hydrophobic face had the highest antifungal activity (GMfungi = 1.64 µM) while showing low toxicity and high salt and serum tolerance. It also had dramatic LPS-neutralizing propensity and a potent membrane-disruptive mechanism against microbial cells. In summary, these findings were useful for short AMPs design to combat the growing threat of drug-resistant fungal and bacterial infections.


Assuntos
Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Sequências Repetidas Invertidas , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Candida albicans/citologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Camundongos , Modelos Moleculares , Peptídeos/toxicidade , Conformação Proteica , Células RAW 264.7
17.
Sci Adv ; 4(1): eaap8258, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29387794

RESUMO

Positive-strand RNA viruses replicate their genomes in membrane-bounded cytoplasmic complexes. We show that endoplasmic reticulum (ER)-linked genomic RNA replication by brome mosaic virus (BMV), a well-studied member of the alphavirus superfamily, depends on the ER luminal thiol oxidase ERO1. We further show that BMV RNA replication protein 1a, a key protein for the formation and function of vesicular BMV RNA replication compartments on ER membranes, permeabilizes these membranes to release oxidizing potential from the ER lumen. Conserved amphipathic sequences in 1a are sufficient to permeabilize liposomes, and mutations in these sequences simultaneously block membrane permeabilization, formation of a disulfide-linked, oxidized 1a multimer, 1a's RNA capping function, and productive genome replication. These results reveal new transmembrane complexities in positive-strand RNA virus replication, show that-as previously reported for certain picornaviruses and flaviviruses-some alphavirus superfamily members encode viroporins, identify roles for such viroporins in genome replication, and provide a potential new foundation for broad-spectrum antivirals.


Assuntos
Antivirais/farmacologia , Organelas/virologia , Vírus de RNA/fisiologia , Replicação Viral , Bromovirus/efeitos dos fármacos , Bromovirus/fisiologia , Dissulfetos/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/virologia , Organelas/efeitos dos fármacos , Permeabilidade , Vírus de RNA/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
Artigo em Inglês | MEDLINE | ID: mdl-29408432

RESUMO

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L-1 for IONPs and 1 mg L-1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future.


Assuntos
Poluentes Ambientais/toxicidade , Compostos Férricos/toxicidade , Hemócitos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Zeolitas/toxicidade , Administração Oral , Animais , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Monitoramento Ambiental , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/química , Compostos Férricos/administração & dosagem , Compostos Férricos/química , /ultraestrutura , Hemócitos/metabolismo , Hemócitos/ultraestrutura , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Carbonilação Proteica/efeitos dos fármacos , Propriedades de Superfície , Fatores de Tempo , Zeolitas/administração & dosagem , Zeolitas/química
19.
Artigo em Inglês | MEDLINE | ID: mdl-29311064

RESUMO

Plasmodium falciparum infections leading to malaria have severe clinical manifestations and high mortality rates. Chloroquine (CQ), a former mainstay of malaria chemotherapy, has been rendered ineffective due to the emergence of widespread resistance. Recent studies, however, have unveiled a novel mode of action in which low-micromolar levels of CQ permeabilized the parasite's digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization, and DNA degradation. These phenotypes implicate the DV as an alternative target of CQ and suggest that DV disruption is an attractive target for exploitation by DV-disruptive antimalarials. In the current study, high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box (2015) was performed to select compounds which disrupt the DV membrane, as measured by the leakage of intravacuolar Ca2+ using the calcium probe Fluo-4 AM. The hits were further characterized by hemozoin biocrystallization inhibition assays and dose-response half-maximal (50%) inhibitory concentration (IC50) assays across resistant and sensitive strains. Three hits, MMV676380, MMV085071, and MMV687812, were shown to demonstrate a lack of CQ cross-resistance in parasite strains and field isolates. Through systematic analyses, MMV085071 emerged as the top hit due to its rapid parasiticidal effect, low-nanomolar IC50, and good efficacy in triggering DV disruption, mitochondrial degradation, and DNA fragmentation in P. falciparum These programmed cell death (PCD)-like phenotypes following permeabilization of the DV suggests that these compounds kill the parasite by a PCD-like mechanism. From the drug development perspective, MMV085071, which was identified to be a potent DV disruptor, offers a promising starting point for subsequent hit-to-lead generation and optimization through structure-activity relationships.


Assuntos
Antimaláricos/farmacologia , Cálcio/metabolismo , Ensaios de Triagem em Larga Escala , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Vacúolos/efeitos dos fármacos , Compostos de Anilina/química , Antimaláricos/química , Cloroquina/química , Cloroquina/farmacologia , Cristalização , Bases de Dados de Produtos Farmacêuticos , Resistência a Medicamentos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Hemeproteínas/química , Hemeproteínas/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Permeabilidade , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Vacúolos/metabolismo , Vacúolos/parasitologia , Xantenos/química
20.
Nucleic Acids Res ; 46(4): 1601-1613, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29361039

RESUMO

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure-activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.


Assuntos
Oligonucleotídeos/metabolismo , Pirazinas/farmacologia , Piridinas/farmacologia , Células HeLa , Humanos , Membranas Intracelulares/efeitos dos fármacos , Oligonucleotídeos/análise , Pirazinas/química , Piridinas/química , Relação Estrutura-Atividade
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