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1.
Mater Sci Eng C Mater Biol Appl ; 128: 112331, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474882

RESUMO

A totally biodegradable mixed system made up of phospholipids and zein was developed in order to effectively improve the photostability of all-trans retinoic acid (ATRA) preserving its pharmacological properties. Photon correlation spectroscopy showed that the formulation obtained using phospholipon 85G and zein at a ratio of 7:3 w/w was characterized by an average diameter of less than 200 nm, a narrow size distribution and a significant time- and temperature-dependent stability. The use of specific cryoprotectants such as mannose and glucose favoured the long-term storage of the nanocarriers after the freeze-drying procedure. The nanoparticles increased the stability of the ATRA against photochemical degradation with respect to the free drug and its antitumor effect was preserved as a consequence of the cell uptake of the colloidal systems. The results demonstrate the potential of the proposed hybrid nanosystems to provide a high level of stabilization for sensitive and labile antitumor compounds.


Assuntos
Nanopartículas , Zeína , Portadores de Fármacos , Tamanho da Partícula , Fosfolipídeos , Tretinoína/farmacologia
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1065-1070, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362483

RESUMO

OBJECTIVE: To investigate the expression of peptidylarginine deiminase 4 (PADI4) during the process of differentiation into granulocyte of NB4 cells induced by all-trans-retinoic acid (ATRA) and whether PADI4 is involved in the inflammatory cytokines expression. METHODS: Granulocyte differentiation model of NB4 cells induced by ATRA was established. The cell morphology changes were observed by Wright-Giemsa staining. The expression of cell differentiation marker CD11b was analyzed by flow cytometry. The mRNA and protein expression of PADI4 was detected by RT-PCR and Western blot, respectively. The expression of tumor necrosis factor (TNF) α and interleukin (IL) 1ß was analyzed by ELISA, and also examined with the knockdown of PADI4 expression by siRNA. RESULTS: After NB4 cells induced by ATRA, the cytoplasm increased and the ratio of nuclear to cytoplasmic was reduced. Nuclear dented, and rod-shaped nucleus, lobulated phenomenon increased (P<0.05). Flow cytometry analysis results showed that the cell surface molecule CD11b expression increased (P<0.01). RT-PCR and Western blot showed the expression of PADI4 increased at both transcriptional and translational levels during the process of the differentiation. ELISA showed TNF-α and IL-1ß secretion increased in differentiated macrophages, while they could be inhibited by PADI4-specific siRNA. CONCLUSION: During the differentiation into granulocyte of NB4 cells induced by ATRA, PADI4 expression increased. Furthermore, PADI4 appeared to play a critical role in inflammatory cytokines secretion.


Assuntos
Diferenciação Celular , Citocinas/metabolismo , Leucemia Promielocítica Aguda , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Linhagem Celular Tumoral , Granulócitos , Humanos , Tretinoína/farmacologia
3.
Viruses ; 13(8)2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34452533

RESUMO

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Proteína DEAD-box 58/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Receptores Imunológicos/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos
4.
PLoS One ; 16(8): e0256141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407143

RESUMO

SARS-CoV-2 requires serine protease, transmembrane serine protease 2 (TMPRSS2), and cysteine proteases, cathepsins B, L (CTSB/L) for entry into host cells. These host proteases activate the spike protein and enable SARS-CoV-2 entry. We herein performed genomic-guided gene set enrichment analysis (GSEA) to identify upstream regulatory elements altering the expression of TMPRSS2 and CTSB/L. Further, medicinal compounds were identified based on their effects on gene expression signatures of the modulators of TMPRSS2 and CTSB/L genes. Using this strategy, estradiol and retinoic acid have been identified as putative SARS-CoV-2 alleviation agents. Next, we analyzed drug-gene and gene-gene interaction networks using 809 human targets of SARS-CoV-2 proteins. The network results indicate that estradiol interacts with 370 (45%) and retinoic acid interacts with 251 (31%) human proteins. Interestingly, a combination of estradiol and retinoic acid interacts with 461 (56%) of human proteins, indicating the therapeutic benefits of drug combination therapy. Finally, molecular docking analysis suggests that both the drugs bind to TMPRSS2 and CTSL with the nanomolar to low micromolar affinity. The results suggest that these drugs can simultaneously target both the entry pathways of SARS-CoV-2 and thus can be considered as a potential treatment option for COVID-19.


Assuntos
Catepsina B/genética , Catepsina L/genética , Estradiol/farmacologia , Genômica/métodos , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Tretinoína/farmacologia , Catepsina B/química , Catepsina L/química , Bases de Dados Genéticas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos
5.
Cancer Sci ; 112(9): 3645-3654, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34288263

RESUMO

CD38 expression on myeloma cells is a critical factor affecting the early response to the anti-CD38 antibody daratumumab. However, factors affecting CD38 expression in untreated multiple myeloma are not fully elucidated. In this study, we found that CD38 expression was significantly lower in myeloma patients with the translocation t(11;14)-associated immature plasma cell phenotype, and particularly in those expressing B-cell-associated genes such as PAX5 and CD79A. CD138, a representative marker of plasmacytic differentiation, was also significantly lower in these patients, suggesting that CD38 expression may be associated with the differentiation and maturation stages of myeloma cells. Furthermore, the BCL2/BCL2L1 ratio, a response marker of the BCL2 inhibitor venetoclax, was significantly higher in patients with the immature phenotype expressing B-cell-associated genes. The BCL2/BCL2L1 ratio and CD38 expression were significantly negatively correlated. We also confirmed that patients with translocation t(11;14) expressing B-cell-associated genes were indeed less sensitive to daratumumab-mediated direct cytotoxicity but highly sensitive to venetoclax treatment in ex vivo assays. Moreover, all-trans-retinoic acid, which enhances CD38 expression and induces cell differentiation in myeloma cells, reduced B-cell marker expression and the BCL2/BCL2L1 ratio in myeloma cell lines, leading to reduced efficacy of venetoclax. Venetoclax specifically induces cell death in myeloma with t(11;14), although why patients with translocation t(11;14) show BCL2 dependence is unclear. These results suggest that BCL2 dependence, as well as CD38 expression, are deeply associated with the differentiation and maturation stages of myeloma cells. This study highlights the importance of examining t(11;14) and considering cell maturity in myeloma treatment strategies.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Translocação Genética/genética , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos B/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Tretinoína/farmacologia
6.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199197

RESUMO

In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.


Assuntos
Comunicação Celular , Cisplatino/efeitos adversos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Junções Comunicantes/metabolismo , Ototoxicidade/metabolismo , Transdução de Sinais , Células A549 , Animais , Comunicação Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Colforsina/farmacologia , Colforsina/uso terapêutico , Conexina 26/metabolismo , Junções Comunicantes/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/tratamento farmacológico , Perda Auditiva/prevenção & controle , Humanos , Camundongos , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/patologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico
7.
Int J Nanomedicine ; 16: 4251-4276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211271

RESUMO

Aim: Retinyl palmitate (RP), the most stable vitamin A derivative, is used to treat photoaging and other skin disorders. The need to minimize the adverse effects of topical drug administration has led to an enhanced interest in loading RP on ethosomes for topical drug delivery. The aim of the current study was to prepare and compare the performance of RP decorated ethosomal hydrogel with tretinoin cream in the treatment of acne vulgaris as an approach to improve drug efficacy and decrease its side effects. Methods: RP-loaded ethosomes were prepared using the injection sonication technique. A Box-Behnken design using Design Expert® software was used for the optimization of formulation variables. Particle size, zeta potential (ZP), entrapment efficiency percent (EE%), % drug release, and permeation over 24 h of different formulations were determined. The optimal formulation was incorporated into a hydrogel. Finally, the efficacy and tolerability of the optimized RP ethosomal hydrogel were clinically evaluated for acne treatment using a split-face comparative clinical study. Results: The optimized ethosomal RP showed particle size of 195.8±5.45 nm, ZP of -62.1±2.85 mV, EE% of 92.63±4.33%, drug release % of 96.63±6.81%, and drug permeation % of 85.98 ±4.79%. Both the optimized RP ethosomal hydrogel and tretinoin effectively reduced all types of acne lesions (inflammatory, non-inflammatory, and total lesions). However, RP resulted in significantly lower non-inflammatory and total acne lesion count than the marketed tretinoin formulation. Besides, RP-loaded ethosomes showed significantly improved tolerability compared to marketed tretinoin with no or minimal skin irritation symptoms. Conclusion: RP ethosomal hydrogel is considerably effective in controlling acne vulgaris with excellent skin tolerability. Therefore, it represents an interesting alternative to conventional marketed tretinoin formulation for topical acne treatment.


Assuntos
Acne Vulgar/tratamento farmacológico , Diterpenos/administração & dosagem , Hidrogéis/química , Hidrogéis/farmacologia , Ésteres de Retinil/administração & dosagem , Administração Cutânea , Adulto , Animais , Diterpenos/efeitos adversos , Diterpenos/química , Diterpenos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Feminino , Humanos , Hidrogéis/efeitos adversos , Masculino , Tamanho da Partícula , Estudos Prospectivos , Ratos Wistar , Ésteres de Retinil/efeitos adversos , Ésteres de Retinil/química , Ésteres de Retinil/farmacocinética , Absorção Cutânea/efeitos dos fármacos , Testes de Irritação da Pele , Tretinoína/administração & dosagem , Tretinoína/farmacologia
8.
Eur Rev Med Pharmacol Sci ; 25(11): 4174-4184, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34156699

RESUMO

Currently, the COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, represents the greatest global health threat. Most people infected by the virus present mild to moderate respiratory symptoms and recover with supportive treatments. However, certain susceptible hosts develop an acute respiratory distress syndrome (ARDS), associated with an inflammatory "cytokine storm", leading to lung damage. Despite the current availability of different COVID-19 vaccines, the new emerging SARS-CoV-2 genetic variants represent a major concern worldwide, due to their increased transmissibility and rapid spread. Indeed, it seems that some mutations or combinations of mutations might confer selective advantages to the virus, such as the ability to evade the host immune responses elicited by COVID-19 vaccines. Several therapeutic approaches have been investigated but, to date, a unique and fully effective therapeutic protocol has not yet been achieved. In addition, steroid-based therapies, aimed to reduce inflammation in patients with severe COVID-19 disease, may increase the risk of opportunistic infections, increasing the hospitalization time and mortality rate of these patients. Hence, there is an unmet need to develop more effective therapeutic options. Here, we discuss the potential use of natural immunomodulators such as Thymosin α1 (Tα1), all-trans retinoic acid (ATRA), and lactoferrin (LF), as adjunctive or preventive treatment of severe COVID-19 disease. These agents are considered to be multifunctional molecules because of their ability to enhance antiviral host immunity and restore the immune balance, depending on the host immune status. Furthermore, they are able to exert a broad-spectrum antimicrobial activity by means of direct interactions with cellular or molecular targets of pathogens or indirectly by increasing the host immune response. Thus, due to the aforementioned properties, these agents might have a great potential in a clinical setting, not only to counteract SARS-CoV-2 infection, but also to prevent opportunistic infections in critically ill COVID-19 patients.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/imunologia , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/imunologia , Humanos , Fatores Imunológicos/farmacologia , Lactoferrina/imunologia , Lactoferrina/farmacologia , Lactoferrina/uso terapêutico , Tretinoína/imunologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico
9.
Mol Cell Biochem ; 476(10): 3601-3612, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34024029

RESUMO

Neuroblastoma (NB) is the most common malignant extra cranial solid tumors in children. It has been well established that retinoic acid (RA) inhibits proliferation of neuroblastoma (NB) by blocking cells at G1 phase of the cell cycle. Clinically, RA has been successfully used to treat NB patients. However, the precise mechanism underlying the potent action of RA-treated NB is not fully explored. In this work, we carried out a gene expression profiling by RNA sequencing on all-trans retinoic acid (ATRA)-treated NB cells. Cancer-related pathway enrichment and subsequent protein-protein interaction (PPI) network analysis identified fibronectin 1 (FN1) as one of the central molecules in the network, which was significantly upregulated during ATRA treatment. In addition, we found that although downregulation of FN1 had no significant effects on either cell proliferation or cell cycle distributions in the presence or absence of ATRA, it increased cell migration and invasion in NB cells and partially blocked ATRA-induced inhibition of cell migration and invasion in SY5Y NB cells. Consistent with this finding, FN1 expression levels in NB patients positively correlate with their overall survivals. Taken together, our data suggest that FN1 is a potential target for effective ATRA treatment on NB patients, likely by facilitating ATRA-induced inhibition of cell migration and invasion.


Assuntos
Movimento Celular/efeitos dos fármacos , Fibronectinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Tretinoína/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Fibronectinas/genética , Humanos , Invasividade Neoplásica/genética , Proteínas de Neoplasias/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética
10.
Stem Cell Res Ther ; 12(1): 291, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001245

RESUMO

BACKGROUND: Glucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA). METHODS: Bone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein-DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis. RESULTS: ATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARß) was elevated. RARß inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARß can regulate the expression of Serpina3n. CONCLUSION: ATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.


Assuntos
Células-Tronco Mesenquimais , Serpinas , Proteínas de Fase Aguda , Animais , Diferenciação Celular , Células Cultivadas , Dexametasona/farmacologia , Fibroblastos , Humanos , Camundongos , Osteogênese , Ratos , Tretinoína/farmacologia , Microtomografia por Raio-X
11.
Methods Mol Biol ; 2311: 9-23, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033074

RESUMO

The use of primary mammalian neurons derived from embryonic central nervous system tissue is limited by the fact that once terminally differentiated into mature neurons, the cells can no longer be propagated. Transformed neuronal-like cell lines can be used in vitro to overcome this limitation. However, several caveats exist when utilizing cells derived from malignant tumors. In this context, the popular SH-SY5Y neuroblastoma cell line and its use in in vitro systems is described. Originally derived from a metastatic bone tumor biopsy, SH-SY5Y (ATCC® CRL-2266™) cells are a subline of the parental line SK-N-SH (ATCC® HTB-11™). SK-N-SH were subcloned three times; first to SH-SY, then to SH-SY5, and finally to SH-SY5Y. SH-SY5Y were deposited to the ATCC® in 1970 by June L. Biedler. Three important characteristics of SH-SY5Y cells should be considered when using these cells in in vitro studies. First, cultures include both adherent and floating cells, both types of which are viable. Few studies address the biological significance of the adherent versus floating phenotypes, but most reported studies utilize adherent populations and discard the floating cells during media changes. Second, early studies by Biedler's group indicated that the parental differentiated SK-N-SH cells contained two morphologically distinct phenotypes: neuroblast-like cells and epithelial-like cells (Ross et al., J Natl Cancer Inst 71(4):741-747, 1983). These two phenotypes may correspond to the "N" and "S" types described in later studies in SH-SY5Y by Encinas et al. (J Neurochem 75(3):991-1003, 2000). Cells with neuroblast-like morphology are positive for tyrosine hydroxylase (TH) and dopamine-ß-hydroxylase characteristic of catecholaminergic neurons, whereas the epithelial-like counterpart cells lacked these enzymatic activities (Ross et al., J Natl Cancer Inst 71(4):741-747, 1983). Third, SH-SY5Y cells can be differentiated to a more mature neuron-like phenotype that is characterized by neuronal markers. There are several methods to differentiate SH-SY5Y cells and are mentioned below. Retinoic acid is the most commonly used means for differentiation and will be addressed in detail.


Assuntos
Neurobiologia , Neuroblastoma/patologia , Neurogênese , Neurônios/patologia , Biomarcadores/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Criopreservação , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Tretinoína/farmacologia
12.
Methods Mol Biol ; 2311: 25-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033075

RESUMO

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Neuroblastoma/metabolismo , Neurogênese , Neurônios/patologia , Feocromocitoma/patologia , Teratocarcinoma/patologia , ADP Ribose Transferases/farmacologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Técnicas de Cultura de Células , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Neurogênese/efeitos dos fármacos , Crescimento Neuronal , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Fenótipo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Ratos , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Transfecção , Tretinoína/farmacologia
13.
Methods Mol Biol ; 2311: 39-49, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033076

RESUMO

This chapter describes the culture and propagation of murine embryonic stem cells, F9 and P19, and strategies for differentiation of these stem cells into neurons. Additional techniques are described for obtaining enriched populations of mature neurons from P19 cells and differentiation of F9 cells into serotonergic or catecholaminergic neurons. The protocols described herein can be used for dissection of the pathways such as gliogenesis and neurogenesis that are involved in differentiation of pluripotent stem cells such as F9 and P19 into glial cells or terminally differentiated neurons.


Assuntos
Células-Tronco Embrionárias Murinas/patologia , Células-Tronco Neurais/patologia , Neurogênese , Neurônios/patologia , Teratocarcinoma/patologia , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Teratocarcinoma/metabolismo , Tretinoína/farmacologia
14.
Nat Struct Mol Biol ; 28(6): 521-532, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34045724

RESUMO

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Totipotentes/citologia , Tretinoína/fisiologia , Acitretina/farmacologia , Animais , Massa Celular Interna do Blastocisto/citologia , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Redes Reguladoras de Genes/genética , Genes Reporter , Isotretinoína/farmacologia , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Piperazinas/farmacologia , Pirazóis/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Totipotentes/efeitos dos fármacos , Transcrição Genética , Tretinoína/antagonistas & inibidores , Tretinoína/farmacologia
15.
J Alzheimers Dis ; 82(2): 485-491, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34057078

RESUMO

Neuroblastoma cell line SH-SY5Y, due to its capacity to differentiate into neurons, easy handling, and low cost, is a common experimental model to study molecular events leading to Alzheimer's disease (AD). However, it is prevalently used in its undifferentiated state, which does not resemble neurons affected by the disease. Here, we show that the expression and localization of amyloid-ß protein precursor (AßPP), one of the key molecules involved in AD pathogenesis, is dramatically altered in SH-SY5Y cells fully differentiated by combined treatment with retinoic acid and BDNF. We show that insufficient differentiation of SH-SY5Y cells results in AßPP mislocalization.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fator Neurotrófico Derivado do Encéfalo , Diferenciação Celular/fisiologia , Neurônios/fisiologia , Tretinoína , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular Tumoral , Humanos , Microscopia Intravital/métodos , Modelos Biológicos , Neuroblastoma , Estresse Oxidativo , Proteólise , Tretinoína/metabolismo , Tretinoína/farmacologia
16.
J Virol ; 95(15): e0012721, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011542

RESUMO

Small-molecule drugs inhibiting BK polyomavirus (BKPyV) represent a significant unmet clinical need in view of polyomavirus-associated nephropathy or hemorrhagic cystitis, which complicate 5% to 25% of kidney and hematopoietic cell transplantations. We characterized the inhibitory activity of acitretin on BKPyV replication in primary human renal proximal tubular epithelial cells (RPTECs). Effective inhibitory concentrations of 50% (EC50) and 90% (EC90) were determined in dilution series measuring BKPyV loads, transcripts, and protein expression, using cell proliferation, metabolic activity, and viability to estimate cytotoxic concentrations and selectivity indices (SI). The acitretin EC50 and EC90 in RPTECs were 0.64 (SI50, 250) and 3.25 µM (SI90, 49.2), respectively. Acitretin effectively inhibited BKPyV replication until 72 h postinfection when added 24 h before infection until 12 h after infection, but decreased to <50% at later time points. Acitretin did not interfere with nuclear delivery of BKPyV genomes, but it decreased large T-antigen transcription and protein expression. Acitretin did not inhibit the initial round of BKPyV replication following transfection of full-length viral genomes, but it affected subsequent rounds of reinfection. Acitretin also inhibited BKPyV replication in human urothelial cells and in Vero cells, but not in COS-7 cells constitutively expressing Simian virus 40 (SV40) large T antigen. Retinoic acid agonists (all-trans retinoic acid, 9-cis retinoic acid [9-cis-RA], 13-cis-RA, bexarotene, and tamibarotene) and the RAR/RXR antagonist RO41-5253 also inhibited BKPyV replication, pointing to an as-yet-undefined mechanism. IMPORTANCE Acitretin selectively inhibits BKPyV replication in primary human cell culture models of nephropathy and hemorrhagic cystitis. Since acitretin is an approved drug in clinical use reaching BKPyV-inhibiting concentrations in systemically treated patients, further studies are warranted to provide data for clinical repurposing of retinoids for treatment and prevention of replicative BKPyV-diseases.


Assuntos
Acitretina/farmacologia , Antivirais/farmacologia , Vírus BK/crescimento & desenvolvimento , Retinoides/farmacologia , Tretinoína/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antígenos Virais de Tumores/biossíntese , Antígenos Virais de Tumores/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Cistite/tratamento farmacológico , Cistite/virologia , Genoma Viral/genética , Células HEK293 , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/virologia , Testes de Sensibilidade Microbiana , Infecções por Polyomavirus/tratamento farmacológico , Tretinoína/análogos & derivados , Infecções Tumorais por Vírus/tratamento farmacológico , Células Vero
17.
J Pharm Sci ; 110(8): 3047-3060, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33933435

RESUMO

Low molecular weight branched polyethylenimines (LMW bPEIs) are almost nontoxic but display poor transfection efficiency due to lack of adequate complexation ability with nucleic acids followed by transportation across the cell membrane. Here, a series of amphiphilic retinoyl-bPEI conjugates (RP-1, RP-2 and RP-3) has been synthesized by allowing the reaction between bPEI (1.8 kDa) and a bioactive and hydrophobic vitamin A metabolite, all-trans-retinoic acid (ATRA), in varying amounts. In aqueous medium, these conjugates self-assembled into core-shell RP nanocomposites with size ranging from ~113-178 nm and zeta potential from ~ +15-35 mV. Evaluation of pDNA complexes of RP nanocomposites revealed that all the complexes exhibited significantly enhanced transfection efficiency without compromising on the cytocompatibility. RP-3/pDNA complex, with the highest content of retinoic acid, exhibited the best transfection efficiency. Further, due to anticancer properties of ATRA, these nanocomposites significantly reduced the viability of cancer cells (HepG2 and MCF-7 cells) without affecting the viability of non-cancerous cells (HEK 293 cells) demonstrating the cell-selective nature of the formulated nanocomposites. The intracellular trafficking and co-localization studies involving RP-3 nanocomposites also showed their higher uptake with intracellular and nuclear accumulation properties. Altogether, the results demonstrate the promising potential of the RP conjugates that can be used in future hepatocellular carcinoma targeted gene delivery applications.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanocompostos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Plasmídeos , Polietilenoimina , Transfecção , Transgenes , Tretinoína/farmacologia
18.
Cell Prolif ; 54(6): e13049, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33960560

RESUMO

OBJECTIVES: Mouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2-cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2-cell embryo-like (2C-like) cells in the chemically defined media remains largely unknown. MATERIALS AND METHODS: We established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C-like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter-positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA-seq. RESULTS: Retinoic acid drives a NELFA (negative elongation factor A)-mediated 2C-like state in mouse ESCs, characterized with 2C-specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs. CONCLUSIONS: We showed that RA has a broader role in 2C-like cells state, not only is one of the upstream regulators of the 2C-like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C-like transition.


Assuntos
Meios de Cultura/farmacologia , Epigênese Genética/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo
19.
Eur J Med Chem ; 220: 113451, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33895500

RESUMO

All-trans-retinoic acid (ATRA) is effective for preventing cancer and treating skin diseases and acute promyelocytic leukaemia (APL). These pharmacological effects of ATRA are mainly mediated by retinoid X receptors (RXRs) and retinoic acid receptors (RARs). This article provides a comprehensive overview of the clinical progress on and the molecular mechanisms of ATRA in the treatment of APL. ATRA can promote the transcriptional activation of differentiation-related genes and regulate autophagy by inhibiting mTOR, which results in anti-APL effects. In detail, the structures, pharmacological effects, and clinical studies of 68 types of ATRA analogues are described. These compounds have excellent antitumour therapeutic potential and could be used as lead compounds for further development and research.


Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tretinoína/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Serina-Treonina Quinases TOR/metabolismo , Tretinoína/análogos & derivados , Tretinoína/química
20.
Aging (Albany NY) ; 13(9): 13179-13194, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33901013

RESUMO

Better understanding of the transcriptional regulatory network in acute promyelocytic leukemia (APL) cells is critical to illustrate the pathogenesis of other types of acute myeloid leukemia. Previous studies have primarily focused on the retinoic acid signaling pathway and how it is interfered with by promyelocytic leukemia/retinoic acid receptor-α (PML/RARα) fusion protein. However, this hardly explains how APL cells are blocked at the promyelocytic stage. Here, we demonstrated that C/EBPα bound and transactivated the promoter of long non-coding RNA NEAT1, an essential element for terminal differentiation of APL cells, through C/EBP binding sites. More importantly, PML/RARα repressed C/EBPα-mediated transactivation of NEAT1 through binding to NEAT1 promoter. Consistently, mutation of the C/EBP sites or deletion of retinoic acid responsive elements (RAREs) and RARE half motifs abrogated the PML/RARα-mediated repression. Moreover, silencing of C/EBPα attenuated ATRA-induced NEAT1 upregulation and APL cell differentiation. Finally, simultaneous knockdown of C/EBPα and C/EBPß reduces ATRA-induced upregulation of C/EBPε and dramatically impaired NEAT1 activation and APL cell differentiation. In sum, C/EBPα binds and transactivates NEAT1 whereas PML/RARα represses this process. This study describes an essential role for C/EBPα in PML/RARα-mediated repression of NEAT1 and suggests that PML/RARα could contribute to the pathogenesis of APL through suppressing C/EBPα targets.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , RNA Longo não Codificante/genética , Tretinoína/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Humanos , Receptor alfa de Ácido Retinoico/genética , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
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