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1.
Virol J ; 16(1): 80, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196105

RESUMO

BACKGROUND: Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. METHODS: Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. RESULTS: We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MßCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. CONCLUSIONS: Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.


Assuntos
Colesterol/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Vírus da Raiva/fisiologia , Internalização do Vírus , Linhagem Celular , Clorpromazina/farmacologia , Concentração de Íons de Hidrogênio , Proteínas do Nucleocapsídeo/genética , Interferência de RNA , Vírus da Raiva/efeitos dos fármacos
2.
Food Chem Toxicol ; 131: 110537, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150782

RESUMO

Programmed death ligand-1 (PD-L1) is an important immune checkpoint for cancer immunotherapy in clinic. In this study, we reported that platycodin D, a natural product isolated from an edible and medicinal plant Platycodon grandiflorus (Jacq.) A. DC., down-regulated the protein level of PD-L1 in lung cancer cells. Flow cytometry and immunofluorescence assay showed a weaker surface PD-L1 signal in NCI-H1975 cells after the incubation with platycodin D (10 µM) for 15 min compared to the control group. Jurkat T cells showed enhancive interleukin-2 secretion when co-cultured with platycodin D-treated NCI-H1975 cells, suggesting that platycodin D-induced PD-L1 reduction increases the activation of Jurkat T cells. An augmentation of PD-L1 protein was detected in the cell culture medium from platycodin D treatment group. Chlorpromazine (60 µM) almost abolished the platycodin D-mediated PD-L1 extracellular release and restored the membrane PD-L1. Finally, hemolysis assay exhibited that platycodin D-triggered PD-L1 extracellular release was independent of the hemolytic mechanism. Taken together, our study demonstrates that platycodin D reduces the protein level of PD-L1 in lung cancer cells via triggering its release into the cell culture medium, which sheds new light for the application of natural products in cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Humanos , Interleucina-2/metabolismo , Células Jurkat , Transporte Proteico/efeitos dos fármacos
3.
J Pharmacol Sci ; 140(2): 197-200, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31178327

RESUMO

Antipsychotics are often the first-line treatment for behavioral and psychological symptoms of dementia. However, the potential anticholinergic effects of antipsychotics could counteract the therapeutic effects of cholinesterase inhibitors used to treat dementia. We investigated the inhibitory effects of 26 antipsychotics on [N-Methyl-3H]scopolamine specific binding in mouse cerebral cortex. At 10-5 M, chlorpromazine, levomepromazine, prochlorperazine, timiperone, zotepine, pimozide, blonanserin, olanzapine, quetiapine, and clozapine inhibited [N-Methyl-3H]scopolamine binding by > 45%. Furthermore, the pKi values of chlorpromazine, levomepromazine, zotepine, olanzapine, and clozapine overlapped with their clinically achievable blood concentrations. Therefore, the anticholinergic properties of these antipsychotics could attenuate the effects of cholinesterase inhibitors.


Assuntos
Antipsicóticos/metabolismo , Antipsicóticos/farmacologia , Córtex Cerebral/metabolismo , Antagonistas Colinérgicos/metabolismo , Inibidores da Colinesterase/metabolismo , Receptores Muscarínicos/metabolismo , Escopolamina/metabolismo , Animais , Clorpromazina/farmacologia , Depressão Química , Interações de Medicamentos , Masculino , Metotrimeprazina/farmacologia , Camundongos Endogâmicos , Proclorperazina/farmacologia , Ligação Proteica
4.
Medicina (Kaunas) ; 55(5)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137751

RESUMO

Background and Objectives: Lipid-based self-nanoemulsifying drug delivery systems (SNEDDS) have resurged the eminence of nanoemulsions by modest adjustments and offer many valuable opportunities in drug delivery. Chlorpromazine, an antipsychotic agent with poor aqueous solubility-with extensive first-pass metabolism-can be a suitable candidate for the development of SNEDDS. The current study was designed to develop triglyceride-based SNEDDS of chlorpromazine to achieve improved solubility, stability, and oral bioavailability. Materials and Methods: Fifteen SNEDDS formulations of each short, medium, and long chain, triglycerides were synthesized and characterized to achieve optimized formulation. The optimized formulation was characterized for several in vitro and in vivo parameters. Results: Particle size, zeta potential, and drug loading of the optimized SNEDDS (LCT14) were found to be 178 ± 16, -21.4, and 85.5%, respectively. Long chain triglyceride (LCT14) showed a 1.5-fold increased elimination half-life (p < 0.01), up to 6-fold increased oral bioavailability, and 1.7-fold decreased plasma clearance rate (p < 0.01) compared to a drug suspension. Conclusion: The findings suggest that SNEDDS based on long-chain triglycerides (LCT14) formulations seem to be a promising alternative for improving the oral bioavailability of chlorpromazine.


Assuntos
Disponibilidade Biológica , Clorpromazina/metabolismo , Emulsificantes/metabolismo , Administração Oral , Animais , Clorpromazina/farmacologia , Clorpromazina/uso terapêutico , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/normas , Emulsificantes/uso terapêutico , Ratos
5.
Dokl Biochem Biophys ; 484(1): 63-65, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012016

RESUMO

Using voltage-clamp technique, the involvement of sigma-1 receptors in the regulation of Na+ transport in frog skin by the immunomodulatory drug glutoxim was investigated. We have shown for the first time that preincubation of the frog skin with the sigma-1 receptor antagonists haloperidol and chlorpromazine attenuates the stimulatory effect of glutoxim on the Na+ transport. The results suggest the possible involvement of the sigma-1 receptors in the regulation of Na+ transport in frog skin epithelium by glutoxim.


Assuntos
Proteínas de Anfíbios/antagonistas & inibidores , Clorpromazina/farmacologia , Haloperidol/farmacologia , Oligopeptídeos/farmacologia , Receptores sigma/antagonistas & inibidores , Pele/metabolismo , Sódio/metabolismo , Proteínas de Anfíbios/metabolismo , Animais , Transporte de Íons/efeitos dos fármacos , Rana temporaria , Receptores sigma/metabolismo
6.
Nanoscale ; 11(13): 6377-6383, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30888365

RESUMO

Endogenous formaldehyde (FA) exists in many living cells and in inhomogeneous distribution in organelles. In particular, lysosomes play significant roles in FA generation and the biofunction of living cells. Herein, we developed a new ratiometric fluorescent nanoprobe, based on naphthalimide derivative (ND)-functionalized carbon dots (CDs), for monitoring endogenous FA in lysosomes. The fluorescence intensity (F535) of green-emitting ND at 535 nm serves as the response signal and the fluorescence intensity (F414) of blue-emitting CDs at 414 nm acts as the reference signal. The fluorescence intensity ratio (F535/F414) of the CD-ND probe is linearly correlated with FA concentration within the range of 1-40 µM in aqueous solution, and the detection limit (3σ/slope) is estimated to be 0.34 µM. As for practical application, this nanoprobe is utilized for the ratiometric fluorescence imaging of FA in live cells. Remarkably, this nanoprobe can specifically target and stain the lysosomes and detect exogenous and endogenous FA in HeLa cells. The new FA probe shows a superior lysosomal targeting ability with a Pearson's coefficient of 0.93, which is attributed to the macromolecular size and basic amine group functionalized surface of CD-ND.


Assuntos
Carbono/química , Corantes Fluorescentes/química , Formaldeído/análise , Lisossomos/metabolismo , Microscopia Confocal , Naftalimidas/química , Pontos Quânticos/química , Clorpromazina/farmacologia , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Limite de Detecção , Pontos Quânticos/metabolismo , Espectrometria de Fluorescência
7.
Microb Pathog ; 129: 250-256, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30742947

RESUMO

Aqueous chlorpromazine solutions exposed to 266 nm generated as fourth harmonic of Nd:YAG pulsed laser along time intervals from 1 min to 240 min were investigated for their antimicrobial activity against planktonic and adherent Gram-positive bacterial strains. Qualitative and quantitative assays based on microbiological methods and flow cytometry assays were performed to establish the minimum inhibitory and minimum biofilm eradication concentrations and to reveal some of the possible mechanisms of antimicrobial activity. Optimal irradiation conditions and combinations of photoproducts for achieving the best antimicrobial and antibiofilm effects are suggested. It was confirmed that chlorpromazine solutions irradiated for 15 min and 30 min have the best antimicrobial and antibiofilm activity against Staphylococcus aureus ATCC 6538, methicillin susceptible Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Enterococcus faecium 17-VAR, Enterococcus faecalis 2921, and Bacillus subtilis 6633. Flow cytometry revealed that two of the possible mechanisms of the antimicrobial activity of irradiated chlorpromazine are the inhibition of efflux pumps activity and induction of cellular membrane lesions.


Assuntos
Biofilmes/efeitos dos fármacos , Clorpromazina/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Terapia com Luz de Baixa Intensidade/métodos , Citometria de Fluxo , Testes de Sensibilidade Microbiana
8.
Chem Biol Interact ; 302: 28-35, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30703373

RESUMO

The major obstacle in current cancer therapy is the existence of cancer stem cells (CSCs), which are responsible for therapeutic resistance and contribute to metastasis and recurrence. Identification of reliable biomarkers for diagnostic and therapeutic targets is necessary for drug development and cancer treatment. In this study, we identified that the antipsychotic chlorpromazine (CPZ) exhibited potent anti-breast cancer and anti-CSC capabilities. Treatment with CPZ suppressed stemness properties including mammosphere formation, aldehyde dehydrogenase (ALDH) activity, and stemness-related gene expressions in breast cancer cells and CSCs. Moreover, CPZ increased the susceptibility of breast cancer MCF7 cells and drug-resistant MCF7/ADR cells when combined with chemotherapies. Mechanistically, we identified that CPZ suppressed yes-associated protein (YAP) through modulating Hippo signaling and promoting proteasomal degradation of YAP. Elevated expression of YAP was confirmed to be crucial for stemness-related gene expressions, and was associated with invasiveness and stem-like signatures in breast cancer patients. Moreover, overexpression of YAP conferred poor outcomes particularly of basal-like breast cancer patients. Our data showed that YAP is a promising therapeutic target for breast CSCs, and CPZ has the potential to be a repurposed drug for breast cancer treatment.


Assuntos
Antipsicóticos/farmacologia , Clorpromazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Neurosci Res ; 146: 54-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30296459

RESUMO

Hyperactivity and impulsivity are common symptoms in several psychiatric disorders. Although dysfunction of Na+, K+-ATPase has been reported to be associated with the psychiatric disorders, it is not clear whether inhibition of Na+, K+-ATPase causes behavioral effects, including hyperactivity and impulsivity, in mice. Here, we evaluated the effect of intracerebroventricular (icv) injection of ouabain, an inhibitor of Na+, K+-ATPase, on hyperactivity and impulsivity in mice. At seven days after icv injection, ouabain-injected mice displayed the increase in the distance traveled in the open field arena in the open field test and the increase in the number of head-dipping behavior in the cliff avoidance test. Chlorpromazine or haloperidol, typical antipsychotics, reduced the hyperactivity and impulsivity in ouabain-injected mice. On the other hand, neither lithium carbonate nor valproate, established mood-stabilizing drugs, improved hyperactivity and impulsivity in our mouse model. Furthermore, ouabain-injected mice exhibited the increase in the number of c-fos-positive cells in the nucleus accumbens and the prefrontal cortex but not in the ventral tegmental area, which was reduced by haloperidol. These results suggest that the dysfunction of Na+, K+-ATPase causes hyperactivity and impulsivity via hyperactivation of dopamine D2 receptor-mediated signaling pathway, causing disturbed neuronal circuits in mice.


Assuntos
Antagonistas dos Receptores de Dopamina D2/farmacologia , Ouabaína/farmacologia , Receptores de Dopamina D2/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Clorpromazina/farmacologia , Modelos Animais de Doenças , Genes fos/efeitos dos fármacos , Genes fos/fisiologia , Haloperidol/farmacologia , Comportamento Impulsivo/efeitos dos fármacos , Comportamento Impulsivo/fisiologia , Infusões Intraventriculares , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Toxicol Appl Pharmacol ; 363: 131-141, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529166

RESUMO

Physico-chemical characteristics of nanoparticles have been shown to alter the uptake and toxicity of nanoparticles. This study investigated the uptake of six gold nanoparticles (AuNPs) into the human bronchial epithelial cell line BEAS-2B. The AuNPs studied included colloidal citrate-stabilised AuNPs of 14 nm in diameter; and 14 nm AuNPs conjugated to functional groups via polyethylene glycol (PEG), namely hydroxyl-PEG (POH), carboxyl-PEG (PCOOH), biotin-PEG (PBtn), nitrilotriacetic acid-PEG (PNTA), and azide-PEG (PAZ). Uptake was visualised by dark field microscopy using the CytoViva Hyperspectral Imaging system and after a 2 hour incubation at 37 °C, uptake was observed in cells treated with the citrate-stabilised and PCOOH AuNPs. However, no uptake was observed for the POH, PBtn, PNTA, or PAZ AuNPs, even after 24 h of incubation. An investigation into the energy dependence of uptake of the citrate-stabilised and PCOOH AuNPs showed that uptake was an active process. Cells pre-treated with either chlorpromazine or genistein as endocytosis inhibitors for clathrin- and caveolae-mediated pathways respectively, prior to addition of AuNPs, suggested a caveolae-dependent mechanism of endocytosis. These results further support recent findings on the mechanism of intracellular uptake and localisation and the subsequent toxicity of nanoparticles.


Assuntos
Cavéolas/metabolismo , Endocitose/efeitos dos fármacos , Células Epiteliais/metabolismo , Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Brônquios/citologia , Linhagem Celular , Clorpromazina/farmacologia , Ácido Cítrico/química , Coloides , Células Epiteliais/citologia , Genisteína/farmacologia , Ouro/química , Humanos , Nanopartículas Metálicas/química , Polietilenoglicóis/química
11.
J Biol Regul Homeost Agents ; 32(6): 1443-1450, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30574748

RESUMO

This study aimed to assess the molecular mechanism of the histone deacetylase inhibitor (HDACI) valproate acid (VPA) alone or in combination with the antipsychotic drug chlorpromazine in the epigenetic regulation of schizophrenia. A total of 60 perinatal CD-SD rats were divided in a control group (16 animals) and a schizophrenia model group (44 animals). For the schizophrenia model group the rats received phencyclidine (PCP) 10 mg/kg/day by intradermal injection on days 7, 9, and 11 after birth. The model was confirmed by the Morris water test in 40 rats. The control and model rats were divided into 7 groups. The Real Time PCR assay was used to detect the mRNA expression changes of GABA system gene [GABBR1 (GABA B receptor 1)], GAD1 (glutamic acid decarboxylase1), GAD2 (glutamic acid decarboxylase2), Lipase metabolic key enzyme LPL (lipoprotein lipase) gene, glutamate neurotransmitter gene GRIA2 (AMPA subtype glutamate receptors 2), inward rectifier potassium channel members KCNJ4 (potassium voltage-gated channel subfamily J member 4) and neuropeptide signal gene TAC1 (tachykinin precursor 1,TAC1) in four brain regions: the prefrontal cortex (PC), the amygdala (AM), the caudate-putamen (CPU) and the hippocampus (HIP). The platform arrival time of PMV and PMVC groups was significantly reduced compared to the PM group, the reduction being more significant in the PMV group. In the four brain regions of the epigenetic animal model of schizophrenia, the expression of GABBR1, GAD1, and GAD2 genes increased significantly. Following administration of HDACI VPA, the mRNA expression of this gene in the four brain regions decreased or approached normal levels. GABBR1 GAD1 and GAD2 are likely to be the target genes affected by the HDACI VPA.


Assuntos
Clorpromazina/farmacologia , Epigênese Genética , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Ácido Valproico/farmacologia , Animais , Feminino , Glutamato Descarboxilase/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/metabolismo
12.
Int J Nanomedicine ; 13: 7549-7563, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532538

RESUMO

Purpose: The aim of this study was to show enhanced anticancer activity of paclitaxel (Ptx) incorporated into solid lipid nanoparticles (SLNs) and reveal reversal of multidrug resistance (MDR) by SLNs mediated by increased uptake through different entry mechanisms from that in drug-sensitive cells. Methods: Anticancer activity of Ptx incorporated in SLNs (Ptx-SLNs) was measured in the drug-sensitive human breast cancer cell line MCF7 and its MDR variant MCF7/ADR. Cellular uptake of cargo molecules in SLNs was compared using Ptx-SLNs and rhodamine 123-loaded SLNs (Rho-SLNs) in both cell lines. In addition, endocytic uptake was evaluated using genistein (Gen) and chlorpromazine (Cpz) as inhibitors of clathrin- and caveola-mediated endocytosis, respectively. Results: Ptx-SLNs showed remarkably enhanced anticancer activity in MCF7/ADR compared to Ptx delivered in dimethyl sulfoxide (DMSO) and Cremophor EL-based vehicles. SLNs significantly increased intracellular uptake of Ptx and Rho in MCF7/ADR. Western blotting demonstrated that clathrin was expressed in both cell lines, while caveolin 1 was expressed only in MCF7/ADR. In MCF7/ADR, uptake of Ptx-SLNs and Rho-SLNs was reduced by Gen, while there was no change by Cpz, suggesting the involvement of caveola-mediated endocytosis. Size reduction of Rho-SLNs through high-pressure homogenization (Rho-SLNs) appeared to cause a shift of the endocytosis mechanism from a clathrin-independent pathway to a clathrin-dependent one. In contrast to MCF7/ADR, the uptake of SLNs into MCF7 was not changed by Gen or Cpz, suggesting involvement of clathrin- and caveola-independent mechanism for the entry of SLNs. Conclusion: MDR was reversed by incorporating drug into SLNs, and the reversal was mediated by increased uptake of SLNs evading efflux pumps in MDR cells. The enhanced uptake could also be due to the use of different endocytosis pathways by SLNs in MDR cells from drug-sensitive cancer cells.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Endocitose , Lipídeos/química , Nanopartículas/química , Paclitaxel/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Células Madin Darby de Rim Canino , Paclitaxel/farmacologia , Tamanho da Partícula , Verapamil/farmacologia
13.
Mol Pharm ; 15(12): 5585-5590, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30351959

RESUMO

Nanosized extracellular vesicles (EVs) possess the natural machinery needed to enter selectively and transmit complex molecular messages efficiently into targeted cells. The intracellular fate of the vesicular cargos depends on the route of internalization. Therefore, understanding the mechanism of attachment and subsequent intake of these vesicles (before and after exerting any modification) is imperative. Here the extent of communication, the uptake kinetics, and the pathways of endothelial EVs into endothelial cells in the presence of specific pharmacological inhibitors were assessed by imaging flow cytometry. The results showed that the uptake of endothelial EVs into endothelial cells was largely an energy-dependent process using predominantly a receptor-mediated, clathrin-dependent pathway.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Exossomos/metabolismo , Nanopartículas/metabolismo , Animais , Linhagem Celular , Clorpromazina/farmacologia , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Exossomos/ultraestrutura , Citometria de Fluxo/métodos , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Nistatina/farmacologia
14.
J Phys Chem B ; 122(49): 11571-11578, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30247032

RESUMO

We present a simple approach to calculate the kinetic properties of lipid membrane crossing processes from biased molecular dynamics simulations. We demonstrate that by using biased simulations, one can obtain highly accurate kinetic information with significantly reduced computational time with respect to unbiased simulations. We describe how to conveniently calculate the transition rates to enter, cross, and exit the membrane in terms of the mean first passage times. To obtain free energy barriers and relaxation times from biased simulations only, we constructed Markov models using the dynamic histogram analysis method (DHAM). The permeability coefficients that are calculated from the relaxation times are found to correlate highly with experimentally evaluated values. We show that more generally, certain calculated kinetic properties linked to the crossing of the membrane layer (e.g., barrier height and barrier crossing rates) are good indicators of ordering drugs by permeability. Extending the analysis to a 2D Markov model provides a physical description of the membrane crossing mechanism.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Simulação de Dinâmica Molecular , Clorpromazina/química , Clorpromazina/farmacologia , Desipramina/química , Desipramina/farmacologia , Domperidona/química , Domperidona/farmacologia , Cinética , Labetalol/química , Labetalol/farmacologia , Bicamadas Lipídicas/química , Loperamida/química , Loperamida/farmacologia , Estrutura Molecular , Propranolol/química , Propranolol/farmacologia , Termodinâmica , Verapamil/química , Verapamil/farmacologia
15.
Sci Rep ; 8(1): 13763, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30213984

RESUMO

Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington's disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.


Assuntos
Meduloblastoma/tratamento farmacológico , Domínios Proteicos/efeitos dos fármacos , Proteínas Repressoras/química , Animais , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clorpromazina/química , Clorpromazina/farmacologia , Clorprotixeno/química , Clorprotixeno/farmacologia , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Sertralina/química , Sertralina/farmacologia
16.
J Photochem Photobiol B ; 188: 50-59, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30216760

RESUMO

Bombyx mori silk extracts, derived from the cocoon degumming process of draw and dye silk in the textile industry, are mainly composed of sericin protein. To add value to the Thai silk extracts, and hence the silk industry, a simple enrichment process was recently developed and the enriched silk extracts were then applied in nano-cosmeceutical products and nano-delivery systems. In this study, the protective effect of Thai silk extracts from three different strains of Bombyx mori on the drug-induced phototoxicity was evaluated in vitro using chlorpromazine (CPZ), a commonly used antipsychotic drug, as a representative phototoxic drug. The human epidermal A431 cell line and reconstructed human epidermis (RhE) model were used as the in vitro skin model. The silk extracts significantly improved the viability of A431 cells after CPZ exposure and ultraviolet A (UVA) irradiation, as shown by the significantly increased CPZ and UVA IC50 values and the decreased proportion of apoptotic cells. The protective effect of these silk extracts against the CPZ-induced UVA-phototoxicity in A431 cells was associated with the attenuation of intracellular oxidative stress via an increased intracellular glutathione level. Likewise, the silk extracts exhibited a protective effect on the CPZ-induced UVA-phototoxicity in the RhE model, in terms of an improved tissue viability and attenuation of the released inflammatory cytokine, interleukin-1α. These findings support the potential usefulness of silk extracts in novel applications, especially in the protection of drug-induced phototoxicity.


Assuntos
Epiderme/efeitos dos fármacos , Substâncias Protetoras/química , Seda/química , Raios Ultravioleta , Animais , Bombyx/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Clorpromazina/farmacologia , Citocinas/metabolismo , Epiderme/patologia , Epiderme/efeitos da radiação , Glutationa , Humanos , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Seda/metabolismo , Pele Artificial , Tailândia
17.
J Alzheimers Dis ; 65(4): 1109-1124, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30103329

RESUMO

Amyloid-ß (Aß) peptides, Aß40, Aß42, and recently Aß25 - 35, have been directly implicated in the pathogenesis of Alzheimer's disease (AD). We have previously shown that all three peptides decrease neuronal viability, but Aß40 also promotes synaptic disassembling. In this work, we have studied the effects of these peptides on astrocytes in primary culture and found that the three Aß peptides were internalized by astrocytes and significantly decreased astrocyte viability, while increasing ROS production. Aß peptide internalization is temperature-dependent, a fact that supports the idea that Aß peptides are actively endocytosed by astrocytes. However, inhibiting caveolae formation by methyl-beta-cyclodextrin or by silencing caveolin-1 with RNA interference did not prevent Aß endocytosis, which suggests that Aß peptides do not use caveolae to enter astrocytes. Conversely, inhibition of clathrin-coated vesicle formation by chlorpromazine or by silencing clathrin with RNA interference significantly decreased Aß internalization and partially reverted the decrease of astrocyte viability caused by the presence of Aß. These results suggest that Aß is endocytosed by clathrin-coated vesicles in astrocytes. Aß-loaded astrocytes, when co-incubated with non-treated astrocytes in separate wells but with the same incubation medium, promoted cell death in non-treated astrocytes; a fact that was associated with the presence of Aß inside previously unloaded astrocytes. This phenomenon was inhibited by the presence of chlorpromazine in the co-incubation medium. These results suggest that astrocyte may perform Aß transcytosis, a process that could play a role in the clearance of Aß peptides from the brain to cerebrospinal fluid.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Astrócitos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Transcitose/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Antipsicóticos/farmacologia , Encéfalo/citologia , Caveolina 1/genética , Caveolina 1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorpromazina/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Temperatura Ambiente , Transfecção , beta-Ciclodextrinas/farmacologia
18.
PLoS Negl Trop Dis ; 12(8): e0006685, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30092029

RESUMO

BACKGROUND: Dengue is the most prevalent arthropod-borne viral human disease in tropical and subtropical regions, caused by four dengue virus (DENV) serotypes. In spite of the increasing global incidence, no specific antiviral therapy is available. Cells of the mononuclear phagocyte lineage are the main targets either for direct antibody (Ab)-independent or Ab-mediated human DENV infection, usually associated to the severe forms of disease. Since the virus entry may be a convenient therapeutic alternative, this study aimed to investigate the mode of DENV internalization into myeloid cells in the absence and presence of DENV Ab and evaluate the inhibitory activity of diverse biochemical inhibitors of endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: By infectivity assays and quantitative RT-PCR determinations, it was demonstrated that DENV-2 entry into U937 and K562 cells in the absence of Ab was highly inhibited by the early treatment with ammonium chloride, chlorpromazine and dynasore, but it was not affected by methyl-ß-cyclodextrin, indicating that DENV-2 utilizes a low pH-dependent, clathrin- and dynamin-mediated endocytic infectious pathway for the direct entry into both human myeloid cells. To study the Ab-mediated entry of DENV, the experimental conditions for enhancement of infection were established by inoculating immune complexes formed with DENV-2 and the Ab 2H2 or 3H5. The internalization of DENV-2-2H2 or DENV-2-3H5 complexes in both myeloid cells was also dependent on acid pH and dynamin but a differential requirement of the clathrin-mediated endocytic route was observed depending on the FcγR involved in the complex uptake: the infection through FcγRII was dependent on clathrin-coated vesicles whereas the internalization pathway mediated by FcγRI was independent of clathrin. This property was not serotype-specific. CONCLUSIONS/SIGNIFICANCE: DENV entry into myeloid cells in the absence or presence of Ab can be blocked by diverse biochemical inhibitors affecting the cellular factors involved in endocytosis. The identification of the virus-host interactions involved in virus penetration may allow the finding of host-targeted antivirals widely active against diverse pathogenic flaviviruses with similar requirements for virus entry.


Assuntos
Anticorpos Antivirais/fisiologia , Vírus da Dengue/fisiologia , Endocitose/efeitos dos fármacos , Células Mieloides/virologia , Internalização do Vírus/efeitos dos fármacos , Cloreto de Amônio , Sobrevivência Celular , Clorpromazina/farmacologia , Humanos , Hidrazonas/farmacologia , Células K562 , Células Mieloides/efeitos dos fármacos , Células U937 , beta-Ciclodextrinas/farmacologia
19.
J Biol Chem ; 293(41): 15977-15990, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30131338

RESUMO

Thioridazine is an antipsychotic that has been shown to induce cell death and inhibit self-renewal in a broad spectrum of cancer cells. The mechanisms by which these effects are mediated are currently unknown but are presumed to result from the inhibition of dopamine receptor 2 (DRD2). Here we show that the self-renewal of several, but not all, triple-negative breast cancer cell lines is inhibited by thioridazine. The inhibition of self-renewal by thioridazine in these cells is mediated by DRD2 inhibition. Further, we demonstrate that DRD2 promotes self-renewal in these cells via a STAT3- and IL-6-dependent mechanism. We also show that thioridazine induces a G1 arrest and a loss in cell viability in all tested cell lines. However, the reduction in proliferation and cell viability is independent of DRD2 and STAT3. Our results indicate that although there are cell types in which DRD2 inhibition results in inhibition of STAT3 and self-renewal, the dramatic block in cancer cell proliferation across many cell lines caused by thioridazine treatment is independent of DRD2 inhibition.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Autorrenovação Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Tioridazina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/farmacologia , Antagonistas dos Receptores de Dopamina D2/farmacologia , Humanos , Fator de Transcrição STAT3/metabolismo
20.
J Virol Methods ; 259: 66-73, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29890241

RESUMO

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.


Assuntos
Cloreto de Amônio/farmacologia , Antivirais/farmacologia , Clorpromazina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Nodaviridae/efeitos dos fármacos , Cloreto de Amônio/toxicidade , Animais , Antivirais/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/toxicidade , Efeito Citopatogênico Viral , Ensaio de Imunoadsorção Enzimática , Peixes , Testes de Sensibilidade Microbiana , Nodaviridae/crescimento & desenvolvimento , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/análise , Replicação Viral/efeitos dos fármacos
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