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1.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445796

RESUMO

Obesity and metabolic syndrome are associated with cognitive decline and dementia. Palmitic acid (PA) is increased in the cerebrospinal fluid of obese patients with cognitive impairment. This study was therefore designed to examine fatty acid (FA) lipotoxicity in BV2 microglia cells. We found that PA induced time- and dose-dependent decrease in cell viability and increase in cell death without affecting the cell cycle profile and that PA lipotoxicity did not depend on cell surface free fatty acid receptors but rather on FA uptake. Treatment with sulfosuccinimidyl oleate (SSO), an irreversible inhibitor of fatty acid translocase CD36, significantly inhibited FA uptake in BSA- and PA-treated cells and blocked PA-induced decrease in cell viability. Inhibition of ER stress or treatment with N-acetylcysteine was not able to rescue PA lipotoxicity. Our study also showed that unsaturated fatty acids (UFAs), such as linoleic acid (LA), oleic acid (OA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), were not lipotoxic but instead protected microglia against PA-induced decrease in cell viability. Co-treatment of PA with LA, OA, and DHA significantly inhibited FA uptake in PA-treated cells. All UFAs tested induced the incorporation of FAs into and the amount of neutral lipids, while PA did not significantly affect the amount of neutral lipids compared with BSA control.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Microglia/metabolismo , Ácido Palmítico/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Ácidos Graxos não Esterificados/metabolismo , Lipídeos/química , Camundongos
2.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203249

RESUMO

By providing ~70% of the eye's refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells. Here, we review progress in elucidating the roles of ion channels in mediating this important exocytotic response. Much is now known about the modulatory impact of ATP-sensitive potassium channels, nonspecific cation channels and voltage-gated calcium channels. Recently, we discovered that during unremitting extracellular hyperosmolarity, P2X7 receptor/channels also become activated and markedly impair goblet cell viability. However, our understanding of possible adaptive benefits of this P2X7 activation remains limited. In the present study, we utilized high-temporal resolution membrane capacitance measurements to monitor the exocytotic activity of single goblet cells located in freshly excised rat conjunctiva. We now report that activation of P2X7 purinoceptors boosts neural-evoked exocytosis and accelerates replenishment of mucin-filled granules after exocytotic depletion. Thus, P2X7 activation exerts a yin-yang effect on conjunctival goblet cells: the high-gain benefit of enhancing the supply of tear-stabilizing mucin is implemented at the high-risk of endangering goblet cell survival.


Assuntos
Células Caliciformes/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Exocitose/genética , Exocitose/fisiologia , Humanos , Receptores Purinérgicos P2X/genética
3.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299188

RESUMO

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and ß1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and ß1 subunits, mediated via the Ca-activated PKC pathway.


Assuntos
Cálcio/metabolismo , Enterite/enzimologia , Células Epiteliais/enzimologia , Intestinos/enzimologia , Leucotrieno D4/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Enterite/tratamento farmacológico , Enterite/patologia , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos
4.
Methods Mol Biol ; 2319: 69-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331244

RESUMO

There is increasing interest in the study of the mammalian lymphatic system, including the lymphatic endothelial cells (LECs) that make up lymphatic vessels. The ability to isolate primary LECs from tissue of normal and genetically modified mice permits detailed analysis of this unique cell type. Here, we describe a robust protocol for the isolation and in vitro expansion of LECs from mouse lung by antibody-based magnetic separation.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Endoteliais/citologia , Separação Imunomagnética/métodos , Pulmão/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Meios de Cultura/química , Células Endoteliais/metabolismo , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos
5.
Nat Commun ; 12(1): 4222, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244514

RESUMO

The evolutionary transition towards multicellular life often involves growth in groups of undifferentiated cells followed by differentiation into soma and germ-like cells. Theory predicts that germ soma differentiation is facilitated by a convex trade-off between survival and reproduction. However, this has never been tested and these transitions remain poorly understood at the ecological and genetic level. Here, we study the evolution of cell groups in ten isogenic lines of the unicellular green algae Chlamydomonas reinhardtii with prolonged exposure to a rotifer predator. We confirm that growth in cell groups is heritable and characterized by a convex trade-off curve between reproduction and survival. Identical mutations evolve in all cell group isolates; these are linked to survival and reducing associated cell costs. Overall, we show that just 500 generations of predator selection were sufficient to lead to a convex trade-off and incorporate evolved changes into the prey genome.


Assuntos
Evolução Biológica , Chlamydomonas reinhardtii/fisiologia , Modelos Biológicos , Animais , Sobrevivência Celular/fisiologia , Comportamento Predatório , Rotíferos/fisiologia
6.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299300

RESUMO

Erythropoietin (EPO) is a glycoprotein cytokine known for its pleiotropic effects on various types of cells and tissues. EPO and its receptor EPOR trigger signaling cascades JAK2/STAT5, MAPK, and PI3K/AKT that are interconnected and irreplaceable for cell survival. In this article, we describe the role of the MAPK and PI3K/AKT signaling pathways during red blood cell formation as well as in non-hematopoietic tissues and tumor cells. Although the central framework of these pathways is similar for most of cell types, there are some stage-specific, tissue, and cell-lineage differences. We summarize the current state of research in this field, highlight the novel members of EPO-induced PI3K and MAPK signaling, and in this respect also the differences between erythroid and non-erythroid cells.


Assuntos
Eritropoese/fisiologia , Eritropoetina/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Neoplasias/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores da Eritropoetina/fisiologia , Transdução de Sinais
7.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203429

RESUMO

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo-deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60-65%) showed discordant results in vitro and in vivo-similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors.


Assuntos
RNA Helicases DEAD-box/metabolismo , Hepatócitos/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , RNA Helicases DEAD-box/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transcriptoma/genética
8.
Mol Cell ; 81(14): 2914-2928.e7, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34107307

RESUMO

Molecular chaperones assist with protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and, in response, abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics to investigate the ribosome-associated chaperone network in E. coli and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL, and ClpB contribute increasingly when DnaK is deficient. Surprisingly, misfolding because of defects in co-translational chaperone function or amino acid analog incorporation results in recruitment of the non-canonical release factor RF3. RF3 recognizes aberrant NCs and then moves to the peptidyltransferase site to cooperate with RF2 in mediating chain termination, facilitating clearance by degradation. This function of RF3 reduces the accumulation of misfolded proteins and is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Biossíntese de Proteínas/fisiologia , Aminoácidos/metabolismo , Sobrevivência Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Peptidil Transferases/metabolismo , Ligação Proteica/fisiologia , Dobramento de Proteína , Proteômica/métodos , Proteostase/fisiologia , Ribossomos/metabolismo
9.
J Alzheimers Dis ; 82(3): 1315-1332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34151811

RESUMO

BACKGROUND: Palmitic acid (PA) promotes brain pathologies including Alzheimer's disease (AD)-related proteins, neuroinflammation, and microglial activation. The activation of neurons and microglia via their Fc gamma receptors (FcγRs) results in producing inflammatory cytokines. OBJECTIVE: To investigate the expression of FcγRs, FcγR signaling proteins, AD-related proteins, proinflammatory cytokines, and cell viability of neurons and microglia in association with PA exposure as well as the effects of FcγR blockade on these parameters in response to PA. METHODS: 200 and 400µM PA-conjugated BSA were applied to SH-SY5Y and HMC3 cells for 24 h. For FcγR blockage experiment, both cells were exposed to FcγR blocker before receiving of 200 and 400µM of PA-conjugated BSA for 24 h. RESULTS: PA significantly increased AD-related proteins, including Aß and BACE1, as well as increasing TNFα, IL-1ß, and IL-6 in SH-SY5Y and HMC3 cells. However, the p-Tau/Tau ratio was only increased in SH-SY5Y cells. These results were associated with an increase in FcγRs activation and a decrease in cell viability in both cell types. FcγRs blockage diminished the activation of FcγR in SH-SY5Y and HMC3 cells. Interestingly, blocking FcγRs before PA exposure reduced the increment of AD-related proteins, proinflammatory cytokines caused by PA. FcγRs blocking also inhibits cell death for 23%of SH-SY5Y cells and 64%of HMC3 cells, respectively. CONCLUSION: These findings suggest that PA is a risk factor for AD via the increased AD-related pathologies, inflammation, FcγRs activation, and brain cell death, while FcγR blockage can alleviate these effects.


Assuntos
Microglia/metabolismo , Neurônios/metabolismo , Ácido Palmítico/toxicidade , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Inibidores Enzimáticos/toxicidade , Humanos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos
10.
AAPS PharmSciTech ; 22(5): 192, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34184160

RESUMO

Neurodegenerative diseases like Alzheimer's disease require treatment where it is essential for drug to reach brain. Nose to brain delivery of drugs enables direct transport to brain bypassing blood brain barrier. Imatinib mesylate, an anti-cancer agent, was found to have potential anti-Alzheimer's activity and thus repurposed for the same. However, the drug has severe side effects, poor brain bioavailability which may hinder effective treatment of Alzheimer's disease. In the current work, imatinib mesylate-loaded liposomes were prepared with particle size below 150 nm with sustained drug release up to 96 h. The liposomal drug formulation was compared with plain drug solution for cytotoxicity on N2a cells and did not show any kind of toxicity at concentrations up to 25 µg/mL. The nanocarrier formulation was then evaluated for brain deposition by nose to brain administration in comparison with drug solution in rats. The liposomes effectively improved the brain deposition of drug in brain from formulation compared to pure drug solution as indicated by AUC from in vivo experiments. These results indicate that the nose to brain delivery of liposomal imatinib mesylate improved the drug deposition and residence time in brain compared to drug solution administered through oral and intranasal routes.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Desenvolvimento de Medicamentos/métodos , Mesilato de Imatinib/administração & dosagem , Mesilato de Imatinib/farmacocinética , Administração Intranasal , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Mesilato de Imatinib/síntese química , Lipossomos , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley
11.
AAPS PharmSciTech ; 22(5): 191, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34169366

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but it is limited by systemic toxicities, poor bioavailability, rapid clearance, and instability in vivo. Nanoparticles (NPs) may overcome these limitations and provide a mechanism for passive targeting of tumors. This study aimed to develop GM-CSF-loaded PLGA/PLGA-PEG NPs and evaluate them in vitro as a potential candidate for in vivo administration. NPs were created by a phase-separation technique that did not require toxic/protein-denaturing solvents or harsh agitation techniques and encapsulated GM-CSF in a more stable precipitated form. NP sizes were within 200 nm for enhanced permeability and retention (EPR) effect with negative zeta potentials, spherical morphology, and high entrapment efficiencies. The optimal formulation was identified by sustained release of approximately 70% of loaded GM-CSF over 24 h, alongside an average size of 143 ± 35 nm and entrapment efficiency of 84 ± 5%. These NPs were successfully freeze-dried in 5% (w/v) hydroxypropyl-ß-cyclodextrin for long-term storage and further characterized. Bioactivity of released GM-CSF was determined by observing GM-CSF receptor activation on murine monocytes and remained fully intact. NPs were not cytotoxic to murine bone marrow-derived macrophages (BMDMs) at concentrations up to 1 mg/mL as determined by MTT and trypan blue exclusion assays. Lastly, NP components generated no significant transcription of inflammation-regulating genes from BMDMs compared to IFNγ+LPS "M1" controls. This report lays the preliminary groundwork to validate in vivo studies with GM-CSF-loaded PLGA/PEG-PLGA NPs for tumor immunomodulation. Overall, these data suggest that in vivo delivery will be well tolerated.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/síntese química , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Poliésteres/síntese química , Polietilenoglicóis/síntese química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Composição de Medicamentos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacocinética , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Poliésteres/administração & dosagem , Poliésteres/farmacocinética , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinética
12.
Nat Commun ; 12(1): 4006, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183658

RESUMO

MYCN activation is a hallmark of advanced neuroblastoma (NB) and a known master regulator of metabolic reprogramming, favoring NB adaptation to its microenvironment. We found that the expression of the main regulators of the molecular clock loops is profoundly disrupted in MYCN-amplified NB patients, and this disruption independently predicts poor clinical outcome. MYCN induces the expression of clock repressors and downregulates the one of clock activators by directly binding to their promoters. Ultimately, MYCN attenuates the molecular clock by suppressing BMAL1 expression and oscillation, thereby promoting cell survival. Reestablishment of the activity of the clock activator RORα via its genetic overexpression and its stimulation through the agonist SR1078, restores BMAL1 expression and oscillation, effectively blocks MYCN-mediated tumor growth and de novo lipogenesis, and sensitizes NB tumors to conventional chemotherapy. In conclusion, reactivation of RORα could serve as a therapeutic strategy for MYCN-amplified NBs by blocking the dysregulation of molecular clock and cell metabolism mediated by MYCN.


Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Lipogênese/fisiologia , Camundongos , Regiões Promotoras Genéticas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
AAPS PharmSciTech ; 22(5): 177, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34128106

RESUMO

Fluvastatin (FLV) is known to inhibit the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), which is over-expressed in various cancers. FLV has been reported to decrease cancer development and metastasis. However, because of low bioavailability, extensive first-pass metabolism and short half-life of FLV (1.2 h), it is not appropriate for clinical application. Therefore, FLV-loaded emulsomes were formulated and optimized using Box-Behnken experimental design to achieve higher efficiency of formulation. Antitumor activity of optimized FLV-loaded emulsomes was evaluated in prostate cancer cells using cell cytotoxicity, apoptotic activity, cell cycle analysis, and enzyme-linked immunosorbent assay. The FLV-loaded emulsomes exhibited a monodispersed size distribution with a mean particle size less than 100 nm as measured by zetasizer. The entrapment efficiency was found to be 93.74% with controlled drug release profile. FLV-EMLs showed a significant inhibitory effect on the viability of PC3 cells when compared to the free FLV (P < 0.0025). Furthermore, FLV-EMLs showed significant arrest in G2/M and increase in percentage of apoptotic cells as compared to free FLV. FLV-EMLs were more effective than free FLV in reducing mitochondrial membrane potential and increase in caspase-3 activity. These results suggesting that FLV-EMLs caused cell cycle arrest which clarifies its significant antiproliferative effect compared to the free drug. Therefore, optimized FLV-EMLs may be an effective carrier for FLV in prostate cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Citotoxinas/farmacologia , Portadores de Fármacos/farmacologia , Fluvastatina/farmacologia , Neoplasias da Próstata , Antineoplásicos/síntese química , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citotoxinas/síntese química , Preparações de Ação Retardada/síntese química , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/síntese química , Fluvastatina/síntese química , Humanos , Masculino , Células PC-3 , Tamanho da Partícula
14.
Nat Commun ; 12(1): 3997, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183666

RESUMO

Despite obesity being a predisposing factor for pancreatic ß-cell dysfunction and loss, the mechanisms underlying its negative effect on insulin-secreting cells remain poorly understood. In this study, we identify an islet-enriched long non-coding RNA (lncRNA), which we name ß-cell function and apoptosis regulator (ßFaar). ßFaar is dramatically downregulated in the islets of the obese mice, and a low level of ßFaar is necessary for the development of obesity-associated ß-cell dysfunction and apoptosis. Mechanistically, ßFaar promote the synthesis and secretion of insulin by upregulating islet-specific genes Ins2, NeuroD1, and Creb1 through sponging miR-138-5p. In addition, using quantitative mass spectrometry, we identify TRAF3IP2 and SMURF1 as interacting proteins that are specifically associated with ßFaar. We demonstrate that SMURF1 ubiquitin ligase activity is essential for TRAF3IP2 ubiquitination and activation of NF-κB-mediate ß-cell apoptosis. Our experiments provide direct evidence that dysregulated ßFaar contributes to the development of obesity-induced ß-cell injury and apoptosis.


Assuntos
Apoptose/genética , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Obesidade/patologia , RNA Longo não Codificante/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Diabetes Mellitus Tipo 2/patologia , Humanos , Insulina/genética , Secreção de Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , MicroRNAs/genética , NF-kappa B/metabolismo , Obesidade/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
15.
FASEB J ; 35(7): e21706, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34160104

RESUMO

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality rates. The pathological features of AKI are tubular injury, infiltration of inflammatory cells, and impaired vascular integrity. Pyruvate kinase is the final rate-limiting enzyme in the glycolysis pathway. We previously showed that pyruvate kinase M2 (PKM2) plays an important role in regulating the glycolytic reprogramming of fibroblasts in renal interstitial fibrosis. The present study aimed to determine the role of PKM2 in fibroblast activation during the pathogenesis of AKI. We found increased numbers of S100A4 positive cells expressing PKM2 in renal tissues from mice with AKI induced via folic acid or ischemia/reperfusion (I/R). The loss of PKM2 in fibroblasts impaired fibroblast proliferation and promoted tubular epithelial cell death including apoptosis, necroptosis, and ferroptosis. Mechanistically, fibroblasts produced less hepatocyte growth factor (HGF) in response to a loss of PKM2. Moreover, in two AKI mouse models, fibroblast-specific deletion of PKM2 blocked HGF signal activation and aggravated AKI after it was induced in mice via ischemia or folic acid. Fibroblast proliferation mediated by PKM2 elicits pro-survival signals that repress tubular cell death and may help to prevent AKI progression. Fibroblast activation mediated by PKM2 in AKI suggests that targeting PKM2 expression could be a novel strategy for treating AKI.


Assuntos
Injúria Renal Aguda/metabolismo , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Piruvato Quinase/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Fibrose/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necroptose/fisiologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia
16.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065598

RESUMO

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.


Assuntos
Tecido Adiposo/fisiologia , Medula Óssea/fisiologia , Gelatina/química , Folículo Piloso/fisiologia , Hidrogéis/química , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Tecido Adiposo/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Regeneração Óssea/fisiologia , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Expressão Gênica/fisiologia , Folículo Piloso/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Modelos Animais , Suínos , Tecidos Suporte/química
17.
AAPS PharmSciTech ; 22(5): 171, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34100170

RESUMO

Macrophages act as a cellular reservoir in HIV infection. Elimination of HIV from macrophages has been an unfulfilled dream due to the failure of drugs to reach them. To address this, we developed CD44 receptor-targeted, novel hyaluronic acid (HA)-coated nanostructured lipid carriers (NLCs) of efavirenz via washless layer-by-layer (LbL) assembly of HA and polyallylamine hydrochloride (PAH). NLCs were subjected to TEM analysis, size and zeta potential, in vitro release and encapsulation efficiency studies. The uptake of NLCs in THP-1 cells was studied using fluorescence microscopy and flow cytometry. The anti-HIV efficacy was evaluated using p24 antigen inhibition assay. NLCs were found to be spherical in shape with anionic zeta potential (-23.66 ± 0.87 mV) and 241.83 ± 5.38 nm particle size. NLCs exhibited prolonged release of efavirenz during in vitro drug release studies. Flow cytometry revealed 1.73-fold higher uptake of HA-coated NLCs in THP-1 cells. Cytotoxicity studies showed no significant change in cell viability in presence of NLCs as compared with the control. HA-coated NLCs distributed throughout the cell including cytoplasm, plasma membrane and nucleus, as observed during fluorescence microscopy. HA-coated NLCs demonstrated consistent and significantly higher inhibition (81.26 ± 1.70%) of p24 antigen which was 2.08-fold higher than plain NLCs. The obtained results suggested preferential uptake of HA-coated NLCs via CD44-mediated uptake. The present finding demonstrates that HA-based CD44 receptor targeting in HIV infection is an attractive strategy for maximising the drug delivery to macrophages and achieve effective viral inhibition.


Assuntos
Portadores de Fármacos/administração & dosagem , HIV-1/efeitos dos fármacos , Receptores de Hialuronatos , Macrófagos/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Alcinos/administração & dosagem , Alcinos/síntese química , Alcinos/metabolismo , Benzoxazinas/administração & dosagem , Benzoxazinas/síntese química , Benzoxazinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclopropanos/administração & dosagem , Ciclopropanos/síntese química , Ciclopropanos/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/fisiologia , Humanos , Receptores de Hialuronatos/metabolismo , Lipídeos/administração & dosagem , Lipídeos/síntese química , Macrófagos/metabolismo , Nanoestruturas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/metabolismo , Células THP-1
18.
Anticancer Res ; 41(6): 2781-2793, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083268

RESUMO

BACKGROUND/AIM: This study explored the mechanisms of the allogeneic graft versus leukemia effect in acute lymphoblastic leukemia (ALL) cells by examining whether they change gene expression in the post-transplant environment containing cytokines and the immunosuppressant cyclosporine, and if such changes affect ALL cell survival. MATERIALS AND METHODS: RNASeq was used to assess leukemia global gene expression and flow cytometry to measure ALL survival in the presence of T cells, NK cells, cytokines, and cyclosporine. RESULTS: A total of 4,805 genes were differentially expressed. Gene set enrichment analysis demonstrated up-regulation of biological processes related to cytokine responses, control of viral infection, and regulation of leukocyte function including proliferation. Down-regulated genes were related to mesenchymal tissue morphogenesis. ALL cells exposed to cytokines and cyclosporine retained susceptibility to T and NK cell killing, and also exhibited increased cell death without exposure to killer cells. CONCLUSION: A significant portion of the graft versus leukemia effect may be mediated by cytokines and cyclosporine.


Assuntos
Sobrevivência Celular/genética , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adulto , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criança , Ciclosporina/farmacologia , Citocinas/sangue , Citocinas/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transplante Homólogo
19.
Toxicol Appl Pharmacol ; 423: 115576, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34000264

RESUMO

Metastatic breast cancer is a prevalent life-threatening disease. Paclitaxel (PTX) is widely used in metastatic breast cancer therapy, but the side effects limit its chemotherapeutic application. Multidrug strategies have recently been used to maximize potency and decrease the toxicity of a particular drug by reducing its dosage. Therefore, we have evaluated the combined anti-cancerous effect of PTX with tested natural compounds (andrographolide (AND), silibinin (SIL), mimosine (MIM) and trans-anethole (TA)) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue dye exclusion assay, proliferating cell nuclear antigen (PCNA) staining, network pharmacology, molecular docking, molecular dynamics (MD) and in vivo chick chorioallantoic membrane (CAM) angiogenesis assay. We observed a reduction in the IC50 value of PTX with tested natural compounds. Further, the network pharmacology-based analysis of compound-disease-target (C-D-T) network showed that PTX, AND, SIL, MIM and TA targeted 55, 61, 56, 31 and 18 proteins of metastatic breast cancer, respectively. Molecular docking results indicated that AND and SIL inhibited the C-D-T network's core target kinase insert domain receptor (KDR) protein more effectively than others. While MD showed that the binding of AND with KDR was stronger and more stable than others. In trypan blue dye exclusion assay and PCNA staining, AND and SIL along with PTX were found to be more effective than PTX alone. CAM assay results suggested that AND, SIL and TA increase the anti-angiogenic potential of PTX. Thus, natural compounds can be used to improve the anti-cancer potential of PTX.


Assuntos
Antineoplásicos Fitogênicos/metabolismo , Produtos Biológicos/metabolismo , Neoplasias da Mama/metabolismo , Paclitaxel/metabolismo , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Produtos Biológicos/administração & dosagem , Produtos Biológicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Embrião de Galinha , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Simulação de Acoplamento Molecular/métodos , Paclitaxel/administração & dosagem , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Resultado do Tratamento
20.
Front Immunol ; 12: 639572, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34012434

RESUMO

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.


Assuntos
Bioimpressão/métodos , Técnicas de Cultura de Células/métodos , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Quimiocinas/genética , Replicação do DNA/genética , Expressão Gênica/genética , Humanos , Hidrogéis/química , Leucemia Linfocítica Crônica de Células B/genética , Impressão Tridimensional , Tecidos Suporte/química
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