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1.
AAPS PharmSciTech ; 22(3): 115, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33763814

RESUMO

Nanomedicine is a highly demanded discipline. Liposomes have seen an increased attention due to their physicochemical properties that allow them to act as nanocarriers of drugs and also of radioisotopes that can be used to diagnose and treat cancer. In order to obtain a novel permeability cancer imaging agent based on 99mTc-labeled liposomes, we describe microwave-assisted synthesis of stearyl 6-(benzylidenehydrazinyl) nicotinamide lipid, which was included in two formulations: nanometric hydrazinonicotinic acid (HYNIC) liposome and its PEGylated coated analogue, HYNIC-PEG liposome. Radiolabeling with 99mTc via stearyl 6-(benzylidenehydrazinyl) nicotinamide was found to be easy, reproducible, and stable, revealing high radiochemical purity (94 ± 1.7%) for both liposomal formulations. Biodistribution at 4 h and 24 h and scintigraphic images at 4 h were performed in normal and melanoma-bearing C57BL/6 mice. Biodistribution studies at 4 h showed tumor uptake of 99mTc-HYNIC liposome and 99mTc-HYNIC-PEG liposome (1.1 ± 0.6 and 2.5 ± 0.4, respectively) and also at 24 h p.i. (1.8 ± 0.5 and 3.0 ± 1.1, respectively). Scintigraphic images showed appreciable tumor uptake in melanoma tumor-bearing mice with both liposomal formulations. Our results show that 99mTc stearyl 6-(benzylidenehydrazinyl) nicotinamide liposomes can be used as diagnostic noninvasive in vivo tumor-targeting agents capable of evaluating tumor permeability and development who can be used in personalized chemotherapy planning.


Assuntos
Melanoma Experimental/metabolismo , Niacinamida/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/farmacocinética , Animais , Linhagem Celular Tumoral , Lipossomos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/administração & dosagem , Niacinamida/química , Permeabilidade/efeitos dos fármacos , Cintilografia/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Tecnécio/administração & dosagem , Tecnécio/química , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia
2.
Toxicol Appl Pharmacol ; 418: 115497, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33744277

RESUMO

Medical reports indicate a prevalence of pain in 50% of patients with cancer. In this context, this article investigated the antinociceptive activity of α-PHE using in vivo Sarcoma-180-induced hypernociception in mice to detail its mechanism(s) of antinociception under different conditions of treatment and tumor progression. Firsty, in vitro cytotoxic action was assessed using melanoma B-16/F-10 and S-180 murine cells and colorimetric MTT assays. For in vivo studies, acute treatment with α-PHE (6.25, 12.5, 25 and 50 mg/kg orally by gavage) was performed on the 1st day after S-180 inoculation. Subacute treatments were performed for 8 days starting on the next day (early protocol) or on day 8 after S-180 inoculation (late protocol). For all procedures, mechanical nociceptive evaluations were carried out by von Frey's technique in the subaxillary region peritumoral tissue (direct nociception) and in right legs of S-180-bearing mice (indirect nociception). α-PHE showed in vitro cytotoxic action on B-16/F-10 and S-180 (CI50 values of 436.0 and 217.9 µg/mL), inhibition of in vivo tumor growth (ranging from 47.3 to 82.7%) and decreased direct (peritumoral tissue in subaxillary region) and indirect (right leg) mechanical nociception in Sarcoma 180-bearing mice with early and advanced tumors under acute or subacute conditions of treatment especially at doses of 25 and 50 mg/kg. It improved serum levels of GSH as well as diminished systemic lipid peroxidation, blood cytokines (interleukin-1ß, -4, -6, and tumor necrosis factor-α). Such outcomes highlight α-PHE as a promising lead compound that combines antinociceptive and antineoplasic properties. Its structural simplicity make it a cost-effective alternative, justifying further mechanistic investigations and the development of pharmaceutical formulations. Moreover, the protocols developed and standardized here make it possible to use Sarcoma-180 hypernociception model to evaluate the capacity of new antinociceptive molecules under conditions of cancer-related allodynia.


Assuntos
Analgésicos/farmacologia , Antineoplásicos/farmacologia , Dor do Câncer/tratamento farmacológico , Monoterpenos Cicloexânicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Sarcoma 180/tratamento farmacológico , Animais , Dor do Câncer/etiologia , Dor do Câncer/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Glutationa/metabolismo , Mediadores da Inflamação/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Limiar da Dor , Sarcoma 180/complicações , Sarcoma 180/metabolismo , Sarcoma 180/patologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas
3.
Methods Mol Biol ; 2265: 385-406, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704729

RESUMO

Metastatic melanoma is one of the most aggressive types of cancers, diffused worldwide and with a significant percentage of lethality. The employment of animal models to test therapeutic strategies against melanoma growth and metastatic spread is of key relevance for cancer biologists. In this regard, the count of metastatic foci in murine lung tissue is one of the recognized methods to monitor macrometastases of melanoma. Here, we illustrate a clonogenic assay method to detect with high sensitivity the presence of single melanoma cells (micrometastases) at the pulmonary level when metastatic foci are still not detectable in the tissue. This method allows for high precision detection and quantification of melanoma metastatic spread to the lung at early stages.


Assuntos
Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Ensaio Tumoral de Célula-Tronco , Animais , Linhagem Celular Tumoral , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica
4.
Methods Mol Biol ; 2265: 407-416, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704730

RESUMO

Melanin exists in the most of melanoma lesions. Melanin plays an important role in melanoma progression, metastasis, therapy response, and the overall survival of patients. Therefore, melanin is a critical target for melanoma diagnosis and therapy. Many melanin targeting probes, such as radioisotope-labeled benzamide analogs, have been developed for melanoma diagnosis using positron emission tomography (PET). The N-(2-(diethylamino)-ethyl)-18F-5-fluoropicolinamide (18F-P3BZA) probe is one of the benzamide analogs and has been preliminarily tested for clinical diagnosis of melanoma in our recent studies. It has shown high specificity and favorable in vivo performance for PET of melanoma. Herein, we describe the detailed synthesis protocol of 18F-P3BZA and PET/CT imaging procedure for animal models and patients.


Assuntos
Radioisótopos de Flúor , Melaninas/metabolismo , Melanoma Experimental , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Animais , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacologia , Melanoma Experimental/diagnóstico , Melanoma Experimental/metabolismo , Camundongos , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacologia
5.
Methods Mol Biol ; 2265: 417-425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704731

RESUMO

Cancer mortality rates are primarily a result of cancer metastasis. Recent advances in microscopy technology allow for the imaging of circulating tumor cells (CTCs) as they extravasate (exit) blood vessels, a key step in the metastasis process. Here, we describe the use of intravital microscopy techniques to image and isolate both extravasating melanoma CTCs and the extravasation-participating endothelial cells. These techniques can be used as a means to study cancer metastasis and as a screening tool for anticancer therapeutics.


Assuntos
Células Endoteliais , Microscopia Intravital , Melanoma Experimental , Células Neoplásicas Circulantes , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia
6.
Methods Mol Biol ; 2265: 645-654, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704745

RESUMO

Expression of chimeric antigen receptors can redirect T cell specificity and allow for MHC-independent recognition of melanoma associated antigens. Retroviral transduction is used to express chimeric antigen receptor constructs in murine CD4+ and CD8+ T cells. Here we describe the production of retroviral supernatants and the activation, transduction, expansion, and selection of murine T cells expressing the chimeric-PD1 receptor. This protocol can be modified for any murine chimeric antigen receptor construct.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Melanoma Experimental , Receptores de Antígenos Quiméricos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia
7.
Nat Commun ; 12(1): 1394, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654093

RESUMO

N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.


Assuntos
Adenosina/análogos & derivados , Reprogramação Celular , Macrófagos/metabolismo , Macrófagos/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , RNA Neoplásico/metabolismo , Adenosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células , Citocinas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Neoplasias Pulmonares/secundário , Metilação , Metiltransferases/metabolismo , Camundongos Knockout , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral
8.
Arch Biochem Biophys ; 700: 108774, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33548212

RESUMO

Homoharringtonine (HHT), an approved anti-leukemic alkaloid, has been reported effectively in many types of tumor cells. However, its effect on melanoma cells has not been investigated. And the anti-melanoma mechanism of HHT is still unknown. In this study, we detected the effects of HHT on two melanoma cell lines (A375 and B16F10) and on the A375 xenograft mouse model. HHT significantly inhibited the proliferation of melanoma cells as investigated by the CCK8 method, cell cloning assay, and EdU experiment. HHT induced A375 and B16F10 cells DNA damage, apoptosis, and G2/M cell cycle arrest as proved by TdT-mediated dUTP Nick-End Labeling (TUNEL) and flow cytometry assay. Additionally, the loss of mitochondrial membrane potential in HHT-treated cells were visualized by JC-1 fluorescent staining. For the molecule mechanism study, western blotting results indicated the protein expression levels of ATM, P53, p-P53, p-CHK2, γ-H2AX, PARP, cleaved-PARP, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, Aurka, p-Aurka, Plk1, p-Plk1, Cdc25c, CDK1, cyclin B1, and Myt1 were regulated by HHT. And the relative mRNA expression level of Aurka, Plk1, Cdc25c, CDK1, cyclin B1, and Myt1 were ascertained by q-PCR assay. The results in vivo experiment showed that HHT can slow down the growth rate of tumors. At the same time, the protein expression levels in vivo were consistent with that in vitro. Collectively, our study provided evidence that HHT could be considered an effective anti-melanoma agent by inducing DNA damage, apoptosis, and cell cycle arrest.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Mepesuccinato de Omacetaxina/farmacologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Melanoma Experimental , Animais , Apoptose , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Proteínas de Neoplasias/biossíntese
9.
Nat Immunol ; 22(2): 179-192, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462452

RESUMO

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias do Colo/metabolismo , Metabolismo Energético , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Linhagem da Célula , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Glucose/metabolismo , Glicólise , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-17/metabolismo , Metabolismo dos Lipídeos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Técnicas de Cultura de Órgãos , Fenótipo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplante , Timo/imunologia , Carga Tumoral
10.
Nat Immunol ; 22(2): 205-215, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398183

RESUMO

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Mitocôndrias/metabolismo , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Células HEK293 , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/imunologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Hipóxia Tumoral
11.
Nat Cell Biol ; 23(1): 75-86, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33420490

RESUMO

Nutrient availability is central for T-cell functions and immune responses. Here we report that CD8+ T-cell activation and anti-tumour responses are strongly potentiated by the non-essential amino acid Asn. Increased Asn levels enhance CD8+ T-cell activation and effector functions against tumour cells in vitro and in vivo. Conversely, restriction of dietary Asn, ASNase administration or inhibition of the Asn transporter SLC1A5 impairs the activity and responses of CD8+ T cells. Mechanistically, Asn does not directly alter cellular metabolic fluxes; it instead binds the SRC-family protein tyrosine kinase LCK and orchestrates LCK phosphorylation at Tyr 394 and 505, thereby leading to enhanced LCK activity and T-cell-receptor signalling. Thus, our findings reveal a critical and metabolism-independent role for Asn in the direct modulation of the adaptive immune response by controlling T-cell activation and efficacy, and further uncover that LCK is a natural Asn sensor signalling Asn sufficiency to T-cell functions.


Assuntos
Asparagina/farmacologia , Linfócitos T CD8-Positivos/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/prevenção & controle , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Melanoma Experimental/prevenção & controle , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais , Células Tumorais Cultivadas , Tirosina/metabolismo , Quinases da Família src/metabolismo
12.
Carbohydr Polym ; 255: 117393, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436222

RESUMO

In this paper, a novel redox-responsive nanoparticles has been designed for targeted delivery of docetaxel (DTX). Chondroitin sulfate (CS) was used to construct the nanoparticles due to the ability of tumor targeting through binding with CD44 receptor that overexpresses on the surfaces of various tumor cells. A redox-responsive small-molecular DTX prodrug was prepared through modifying with cystamine containing disulfide bonds (Cys-DTX). Then the DTX prodrug was grafted to the CS to construct the amphiphilic polymer (CS-ss-DTX). Further, Cys-DTX/CS-ss-DTX nanoparticles were formed by self-assembly of amphiphilic polymer and incorporation of free Cys-DTX prodrug. This category of nanosized DTX delivery system was expected for not only exhibiting high permeability and cytotoxicity of Cys-DTX prodrug, but also targeting transportation of encapsulated redox-responsive Cys-DTX prodrug. According to results of related researches on physicochemical properties and biological evaluation, the novel redox-responsive Cys-DTX/CS-ss-DTX nanoparticles increased amount of DTX released from the nanoparticles in reductive environment, improved permeability in tumor tissues, enhanced cytotoxicity and decreased side effects compared with free DTX. All of these results showed that this kind of Cys-DTX/CS-ss-DTX nanoparticles were worthy of being expectation in tumor chemotherapy in future.


Assuntos
Antineoplásicos/farmacologia , Sulfatos de Condroitina/química , Docetaxel/farmacologia , Glicoconjugados/farmacologia , Melanoma Experimental/tratamento farmacológico , Pró-Fármacos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/química , Docetaxel/metabolismo , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacologia , Liberação Controlada de Fármacos , Feminino , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Células MCF-7 , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Micelas , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/ultraestrutura , Oxirredução , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos
13.
Nat Immunol ; 22(3): 358-369, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33432230

RESUMO

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Interleucina-2/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Microambiente Tumoral , 5-Hidroxitriptofano/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/genética , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Células MCF-7 , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Triptofano Hidroxilase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cancer Lett ; 496: 1-15, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991950

RESUMO

Vascular normalisation, the process that reverses the structural and functional abnormalities seen in tumour-associated vessels, is also accompanied by changes in leucocyte trafficking. Our previous studies have shown the normalisation effects of the agent CD5-2 which acts to stabilise VE-Cadherin leading to increased penetration of CD8+ T cells but decreased infiltration of neutrophils (CD11b+Gr1hi) into tumour parenchyma. In the present study, we demonstrate that VE-Cadherin stabilisation through CD5-2 treatment of purified endothelial cells (ECs) results in a similar leucocyte-selective regulation of transmigration, suggesting the existence of an endothelial specific intrinsic mechanism. Further, we show by RNA sequencing (RNA-seq)-based transcriptomic analysis, that treatment of ECs with CD5-2 regulates chemokines known to be involved in leucocyte transmigration, including upregulation of CCL2 and CXCL10 that facilitate CD8+ T cell transmigration. Both in vitro and in vivo mechanistic studies revealed that the increased CCL2 expression was dependent on expression of VE-Cadherin and downstream activation of the AKT/GSK3ß/ß-catenin/TCF4 signalling pathway. CD5-2 treatment also contributed to the reorganisation of the cytoskeleton, inducing reorganisation of stress fibres to circumferential actin, which previously has been described as associated with the stabilisation of the endothelial barrier, and amplification of the transcellular migration of CD8+ T cells. Thus, we propose that promotion of endothelial junctional integrity during vascular normalisation not only inhibits vascular leak but also resets the endothelial dependent regulation of immune cell infiltration.


Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Caderinas/metabolismo , Endotélio Vascular/patologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Oligonucleotídeos/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Proliferação de Células , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Pessoa de Meia-Idade , Neutrófilos/imunologia , beta Catenina/genética
15.
PLoS One ; 15(12): e0243565, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332393

RESUMO

Melanin, a pigment synthesized by melanocytes in the skin, resists the damage caused by ultraviolet rays to cells. Citric acid, a well-known food additive, is commonly used as an antioxidant and is an important part of the tricarboxylic acid (TCA) cycle for energy production during cellular metabolism. Here, we aimed to investigate whether the addition of excess citric acid regulates melanin synthesis, and to delineate the underlying mechanism. First, we observed that citric acid exerts opposite redox effects on mouse and human cells. Interestingly, treatment with excess citric acid increased the melanin content in mouse cells but decreased it in human cells. Furthermore, the expression of factors important for melanin synthesis, such as microphthalmia-associated transcription factor (MITF), was also regulated by citric acid treatment-it was promoted in mouse cells and suppressed in human cells. Citric acid also impacted the upstream regulators of MITF, glycogen synthase kinase 3ß (GSK3ß), and ß-catenin. Second, we determined the importance of GSK3ß in the citric acid-mediated regulation of melanin synthesis, using a GSK3ß inhibitor (BIO). To the best of our knowledge, this is the first study to show that citric acid regulates melanin synthesis via the GSK3ß/ß-catenin signaling pathway, and that equal amounts of exogenous citric acid exert opposing effects on mouse and human cells.


Assuntos
Ácido Cítrico/farmacologia , Melaninas/metabolismo , Melanócitos/metabolismo , Animais , Linhagem Celular Tumoral , Ácido Cítrico/metabolismo , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma Experimental/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Pigmentação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo
16.
PLoS One ; 15(8): e0237646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845913

RESUMO

Tumor antigen-primed CD8 T cells differentiate into effector T cells that kill tumor cells rapidly, whereas durable responses of CD8 T cells are required to cope with long-lasting tumor growth. However, it is not well known how persisting CD8 T cells are generated. In this study, we analyzed CD8 T cells primed by antigens in tumor-draining lymph nodes and found that CD8 T cells first differentiated into a CD62L-intermediate (CD62Lint) stage upon antigen stimulation. These cells gave rise to tumor-infiltrating CD62L-CD44high Bcl6- effector T cells and CD62L+CD44highBcl6+ memory-like T cells. Memory-like T cells within the tumor expressed CD127, CXCR3 and had the potential to proliferate significantly when they were transferred into tumor-bearing mice. Bcl6 expression in these T cells was critical because Bcl6-/-CD62L+CD44highCD8T cells within the tumor were defective in expansion after secondary transfer. Taken together, our findings show that CD62L+CD44highBcl6+ cells are generated from highly proliferating CD62Lint T cells and retain high proliferative potential, which contributes to replenishment of effector T cells within the tumor.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/patologia , Memória Imunológica/imunologia , Selectina L/metabolismo , Melanoma Experimental/patologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Diferenciação Celular , Feminino , Selectina L/genética , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Células Tumorais Cultivadas
17.
PLoS One ; 15(8): e0237577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790741

RESUMO

Abnormal skin melanin homeostasis results in refractory pigmentary diseases. Melanogenesis is influenced by gene regulation, ultraviolet radiation, and host epigenetic responses. Steroid receptor RNA activator (SRA), a long noncoding RNA, is known to regulate steroidogenesis and tumorigenesis. However, how SRA contributes to melanogenesis remains unknown. Using RNA interference against SRA in B16 and A375 melanoma cells, we observed increased pigmentation and increased expression of TRP1 and TRP2 at transcriptional and translational levels only in B16 cells. The constitutive phosphorylation of p38 in B16-shCtrl cells was inhibited in cells with knocked down SRAi. Moreover, the melanin content of control B16 cells was increased by SB202190, a p38 inhibitor. Furthermore, reduced p38 phosphorylation, enhanced TRP1 expression, and hypermelanosis were observed in A375 cells with RNA interference. These results indicate that SRA-p38-TRP1 axis has a regulatory role in melanin homeostasis and that SRA might be a potential therapeutic target for treating pigmentary diseases.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Oxirredutases/genética , Fosforilação , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
Arch Biochem Biophys ; 692: 108519, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32763235

RESUMO

Melanoma is a dangerous type of skin cancer that develops from the melanocytes. Activation of p53 in melanoma cells has been validated as a strategy for melanoma therapy. S-Petasin, a dietary sesquiterpene isolated from Petasites japonicus, has been shown to possess multiple biological effects. However, no studies have reported that s-petasin exerted anti-melanoma or inhibited activity in melanoma cells. We investigated the effect of s-petasin in B16F10 cells and A375 cells and the underlying molecular mechanism. S-Petasin exerted a significant anti-proliferation effect on B16F10 cells and A375 cells as measured by the MTT assay and crystal violet staining assay. S-Petasin induced cell apoptosis in B16F10 cells and A375 cells as evidenced by flow cytometry assay and western blot assay. Wound healing assay and transwell cell migration and invasion assay revealed that s-petasin suppressed B16F10 cells and A375 cells migration in vitro. For mechanism study, western blot assay indicated that s-petasin activated the p53 pathway signaling. Furthermore, expression of Bcl-2, Bcl-XL, Bax, MMP-2, MMP-9, p21, CDK4 and cyclin D1 were regulated by s-petasin. Taken together, our data suggest that s-petasin is a novel compound which can induce apoptosis and inhibit cell migration through activation of the p53 pathway signaling in melanoma B16F10 cells and A375 cells.


Assuntos
Apoptose/efeitos dos fármacos , Melanoma Experimental/metabolismo , Sesquiterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Células 3T3-L1 , Animais , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Melanoma Experimental/patologia , Camundongos
19.
Cancer Cell ; 37(6): 786-799.e5, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32516589

RESUMO

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Melanoma Experimental/imunologia , Células Mieloides/imunologia , Sinapses/imunologia , Linfócitos T/imunologia , Animais , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/metabolismo , Receptores CCR2/fisiologia , Receptores CCR7/fisiologia , Sinapses/metabolismo , Sinapses/patologia , Linfócitos T/citologia , Linfócitos T/metabolismo
20.
Food Chem ; 330: 127030, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32535311

RESUMO

This study was aimed at determining the influence of Folium nelumbinis (Lotus leaf) extracts on melanogenesis in vitro models of melanoma cell line. The anticancer activity of four fractions, including petroleum ether (PEE), n-hexane (HE), ethanol (EE), and ethyl acetate (EAE) from F. nelumbinis on B16 cell lines (C57BL/6J melanoma cell), were evaluated after 24 and 48 h treatment. Results showed that PEE as well as volatile-rich fractions of linolenic acid and linolenic acid ethyl ester significantly (p < 0.05) reduced tyrosinase activity and melanin content in B16 melanoma cells model. Meanwhile, PEE and its primarily contained compound triggered apoptosis of B16 cells in a dose-dependent way. These results demonstrated that PEE possessed effective activities against melanin and tyrosinase generations through the induction of apoptosis. Moreover, a relation between the volatile-rich fractions of F. nelumbinis and the anticancer effects was demonstrated as well.


Assuntos
Lotus/química , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Lotus/metabolismo , Melaninas/metabolismo , Melanoma Experimental/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo
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