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1.
Nat Commun ; 12(1): 5733, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593794

RESUMO

In addition to increasing the expression of programmed death-ligand 1 (PD-L1), tumor cells can also secrete exosomal PD-L1 to suppress T cell activity. Emerging evidence has revealed that exosomal PD-L1 resists immune checkpoint blockade, and may contribute to resistance to therapy. In this scenario, suppressing the secretion of tumor-derived exosomes may aid therapy. Here, we develop an assembly of exosome inhibitor (GW4869) and ferroptosis inducer (Fe3+) via amphiphilic hyaluronic acid. Cooperation between the two active components in the constructed nanounit induces an anti-tumor immunoresponse to B16F10 melanoma cells and stimulates cytotoxic T lymphocytes and immunological memory. The nanounit enhances the response to PD-L1 checkpoint blockade and may represent a therapeutic strategy for enhancing the response to this therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Portadores de Fármacos/química , Exossomos/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Compostos de Benzilideno/farmacologia , Compostos de Benzilideno/uso terapêutico , Linhagem Celular Tumoral/transplante , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Exossomos/imunologia , Exossomos/metabolismo , Feminino , Ferroptose/imunologia , Humanos , Ácido Hialurônico/química , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Memória Imunológica , Ativação Linfocitária/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
2.
Life Sci ; 284: 119930, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34480938

RESUMO

AIMS: Silk fibroin (SF), a natural product from silkworms, has been used to promote anti-inflammation, induce wound healing, and reduce melanin production. However, the underlying regulatory mechanism of SF on melanin production remains unknown. The aim of this study was to investigate the distinct regulatory mechanism of SF in B16 melanoma cells by applying quantitative proteomic approach. MATERIALS AND METHODS: B16 melanoma cells were treated with PBS, KA or SF for 48 h, respectively. Cell viability, melanin content, and tyrosinase activity were examined. A label-free quantitative proteomic approach was utilized to investigate the regulatory mechanism of SF. The differentially expressed proteins and their related biological processes were subsequently identified by bioinformatics methods. Furthermore, the identified differentially expressed proteins were validated by western blot. KEY FINDINGS: Both SF and KA were able to suppress the melanin synthesis of B16 melanoma cells without appreciable toxicity; yet, SF had a distinct effect on mushroom tyrosinase activity in vitro. Moreover, quantitative proteomic approach identified 141 proteins differentially expressed only in SF/Con group. Bioinformatic analysis of these proteins revealed that oxidation-reduction process, RNA processing, fatty acid degradation, as well as melanin biosynthetic process were enriched with SF treatment. The proteins associated with melanin biosynthetic process, including microphthalmia-associated transcription factor (MITF) and tyrosinase, were down-regulated in SF group, which was confirmed by western blot. SIGNIFICANCE: SF inhibited melanin synthesis in B16 melanoma cells via down-regulation of MITF and tyrosinase expression, which provides a rationale for future utilization of SF.


Assuntos
Fibroínas/farmacologia , Melaninas/biossíntese , Melanoma Experimental/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Proteômica , Animais , Melanoma Experimental/patologia , Camundongos , Pironas/farmacologia , Reprodutibilidade dos Testes
3.
Nat Commun ; 12(1): 5329, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504078

RESUMO

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Melanócitos/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/química , Actinas/genética , Animais , Sítios de Ligação , Bovinos , Citoesqueleto/ultraestrutura , Gelsolina/química , Gelsolina/genética , Gelsolina/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Melanócitos/citologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Modelos Moleculares , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citologia , Timo/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
4.
Int J Mol Sci ; 22(17)2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34502483

RESUMO

We examined the effects of ALOS4, a cyclic peptide discovered previously by phage library selection against integrin αvß3, on a human melanoma (A375) xenograft model to determine its abilities as a potential anti-cancer agent. We found that ALOS4 promoted healthy weight gain in A375-engrafted nude mice and reduced melanoma tumor mass and volume. Despite these positive changes, examination of the tumor tissue did not indicate any significant effects on proliferation, mitotic index, tissue vascularization, or reduction of αSMA or Ki-67 tumor markers. Modulation in overall expression of critical downstream αvß3 integrin factors, such as FAK and Src, as well as reductions in gene expression of c-Fos and c-Jun transcription factors, indirectly confirmed our suspicions that ALOS4 is likely acting through an integrin-mediated pathway. Further, we found no overt formulation issues with ALOS4 regarding interaction with standard inert laboratory materials (polypropylene, borosilicate glass) or with pH and temperature stability under prolonged storage. Collectively, ALOS4 appears to be safe, chemically stable, and produces anti-cancer effects in a human xenograft model of melanoma. We believe these results suggest a role for ALOS4 in an integrin-mediated pathway in exerting its anti-cancer effects possibly through immune response modulation.


Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Life Sci ; 284: 119915, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34453947

RESUMO

Age spots are a significant phenotypic marker of aging formed by lipofuscin. Melanin is another skin pigment molecule responsible for skin aging. The present study aims to investigate the relationship between melanin production and lipofuscin synthesis in normal mouse melanoma cell line B16F1 cells and Tyrosinase (TYR) gene knockout cells. TYR gene KO cells were successfully developed using CRISPR/Cas9 system and confirmed by Sanger DNA sequencing analysis. Furthermore, the melanin production and lipofuscin formation were validated through RT-PCR and Western blot analysis. The expression levels of gene microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosine-related protein-1 (TRP-1), tyrosine-related protein-2 (TRP-2), and antioxidant proteins such as methionine sulfoxide reductase A (MSRA), Catalase and Glutathione reductase (GR) related to melanogenesis was found to be decreased in TYR gene KO cells compared with normal cells. Moreover, lipofuscin formation was increased in TYR gene KO cells compared to normal cells. Therefore, the above findings suggest that melanin production and lipofuscin formation could be linked by the TYR gene in melanocytes.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Lipofuscina/metabolismo , Melaninas/biossíntese , Melanócitos/enzimologia , Monofenol Mono-Oxigenase/genética , Animais , Antioxidantes/metabolismo , Sequência de Bases , Sobrevivência Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Raios Ultravioleta
6.
J Immunol ; 207(6): 1662-1671, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34417261

RESUMO

The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W in encoded protein lymphoid tyrosine phosphatase) is present in 5-10% of the North American population and is strongly associated with numerous autoimmune diseases. Although much research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its proautoimmune allele have in tumor immunity is poorly defined. To interrogate the role this allele may have in the antitumor immune response, we used CRISPR/Cas9 to generate mice in which the ortholog of lymphoid tyrosine phosphatase, PEST domain-enriched protein (PEP), is mutated at position 619 to produce the relevant proautoimmune mutation (R619W). Results of this study show that mice homozygous for this alteration (PEP-619WW) resist tumor growth as compared with wild-type mice. Consistent with these results, tumors from PEP-619WW mice have more CD45 infiltrates containing more activated CD8 T cells and CD4 T cells. In addition, there are more conventional dendritic cell type 1 (cDC1) cells and fewer myeloid-derived suppressor cells in tumors from PEP-619WW mice. Interestingly, the tumor-infiltrating PEP-619WW cDC1 cells have decreased PD-L1 expression compared with cDC1 cells from PEP-wild-type mice. Taken together, our data show that the proautoimmune allele of Ptpn22 drives a strong antitumor response in innate and adaptive immune cells resulting in superior control of tumors.


Assuntos
Alelos , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/imunologia , Imunidade , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/imunologia , Animais , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Feminino , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias/métodos , Fenótipo , Neoplasias Cutâneas/patologia
7.
Nat Commun ; 12(1): 4734, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354077

RESUMO

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.


Assuntos
Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Técnicas de Reprogramação Celular/métodos , Quimiocina CXCL13/genética , Quimiocina CXCL13/imunologia , Feminino , Vetores Genéticos , Interleucina-33/deficiência , Interleucina-33/genética , Interleucina-33/imunologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Vírus da Coriomeningite Linfocítica/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Estromais/imunologia , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/imunologia
8.
Cancer Sci ; 112(10): 4013-4025, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252226

RESUMO

Although the role of bromodomain-containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP-activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin-related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c-Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.


Assuntos
Apoptose/fisiologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Melanoma/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Respiração Celular/efeitos dos fármacos , Dinaminas/efeitos dos fármacos , Dinaminas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo
9.
J Immunol ; 207(2): 720-734, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34261667

RESUMO

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.


Assuntos
Criopreservação/métodos , Citometria de Fluxo/métodos , Leucócitos Mononucleares/imunologia , Melanoma Experimental/patologia , Células Mieloides/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Imunofenotipagem/métodos , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Pandemias , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia
10.
Int J Nanomedicine ; 16: 4661-4674, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262274

RESUMO

Purpose: Gold nanoparticles (AuNPs) are widely studied as radiosensitizers, but their radiosensitization in carbon ion radiotherapy is unsatisfactory. There is a lack of in vivo data on the radiosensitization of AuNPs under carbon ion irradiation. This study focused on the radiosensitization effect of AuNPs in the mouse melanoma cell line B16-F10 in vitro and in vivo. Materials and Methods: 11-mercaptoundecanoic acid (11-MUA)-coated gold (Au) nanoparticles (mAuNPs) formulations were prepared and characterized. To verify the radiosensitization effect of mAuNPs, hydroxyl radicals were generated in aqueous solution, and the detection of intracellular reactive oxygen species (ROS) and clone survival were carried out in vitro. The tumor growth rate (TGR) and survival of mice were analyzed to verify the radiosensitization effect of mAuNPs in vivo. The apoptosis of tumor cells was detected, and the expression of key proteins in the apoptosis pathway was verified by immunohistochemistry. Results: The intracellular ROS level in B16-F10 cells was enhanced by mAuNPs under carbon ion irradiation. The sensitization rate of mAuNPs was 1.22 with a 10% cell survival rate. Compared with irradiation alone, the inhibitory effect of mAuNPs combined with carbon ion irradiation on tumor growth was 1.94-fold higher, the survival time of mice was prolonged by 1.75-fold, and the number of apoptotic cells was increased by 1.43-fold. The ratio of key proteins Bax and Bcl2 in the apoptosis pathway was up-regulated, and the expression of caspase-3, a key executor of the apoptosis pathway, was up-regulated. Conclusion: In in vivo and in vitro experiments, mAuNPs showed radiosensitivity to carbon ion irradiation. The sensitization effect of mAuNPs on mice tumor may be achieved by activating the mitochondrial apoptosis pathway and increasing tumor tissue apoptosis. To our best knowledge, the present study is the first in vivo evidence for radiosensitization of mAuNPs in tumor-bearing mice exposed to carbon ion irradiation.


Assuntos
Ácidos Graxos/química , Ouro/química , Radioterapia com Íons Pesados , Nanopartículas Metálicas/química , Compostos de Sulfidrila/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Melanoma Experimental/patologia , Melanoma Experimental/radioterapia , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Resultado do Tratamento , Carga Tumoral
11.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208566

RESUMO

Regarding the increased incidence and high mortality rate of malignant melanoma, practical early-detection methods are essential to improve patients' clinical outcomes. In this study, we successfully prepared novel picolinamide-benzamide (18F-FPABZA) and nicotinamide-benzamide (18F-FNABZA) conjugates and determined their biological characteristics. The radiochemical yields of 18F-FPABZA and 18F-FNABZA were 26 ± 5% and 1 ± 0.5%, respectively. 18F-FPABZA was more lipophilic (log P = 1.48) than 18F-FNABZA (log P = 0.68). The cellular uptake of 18F-FPABZA in melanotic B16F10 cells was relatively higher than that of 18F-FNABZA at 15 min post-incubation. However, both radiotracers did not retain in amelanotic A375 cells. The tumor-to-muscle ratios of 18F-FPABZA-injected B16F10 tumor-bearing mice increased from 7.6 ± 0.4 at 15 min post-injection (p.i.) to 27.5 ± 16.6 at 3 h p.i., while those administered with 18F-FNABZA did not show a similarly dramatic increase throughout the experimental period. The results obtained from biodistribution studies were consistent with those derived from microPET imaging. This study demonstrated that 18F-FPABZA is a promising melanin-targeting positron emission tomography (PET) probe for melanotic melanoma.


Assuntos
Radioisótopos de Flúor , Melanoma Experimental/diagnóstico por imagem , Niacinamida , Ácidos Picolínicos , Compostos Radiofarmacêuticos , Animais , Linhagem Celular Tumoral , Radioisótopos de Flúor/química , Melaninas/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Niacinamida/química , Ácidos Picolínicos/química , Tomografia por Emissão de Pósitrons , Ligação Proteica , Compostos Radiofarmacêuticos/química , Distribuição Tecidual
12.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299073

RESUMO

Theasinensin A (TSA) is a major group of catechin dimers mainly found in oolong tea and black tea. This compound is also manufactured with epigallocatechin gallate (EGCG) as a substrate and is refined after the enzyme reaction. In previous studies, TSA has been reported to be effective against inflammation. However, the effect of these substances on skin melanin formation remains unknown. In this study, we unraveled the role of TSA in melanogenesis using mouse melanoma B16F10 cells and normal human epidermal melanocytes (NHEMs) through reverse transcription polymerase chain reaction (RT-PCR), Western blotting analysis, luciferase reporter assay, and enzyme-linked immunosorbent assay analysis. TSA inhibited melanin formation and secretion in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 cells and NHEMs. TSA down-regulated the mRNA expression of tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and Tyrp2, which are all related to melanin formation in these cells. TSA was able to suppress the activities of certain proteins in the melanocortin 1 receptor (MC1R) signaling pathway associated with melanin synthesis in B16F10 cells: cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), protein kinase A (PKA), tyrosinase, and microphthalmia-associated transcription factor (MITF). We also confirmed α-MSH-mediated CREB activities through a luciferase reporter assay, and that the quantities of cAMP were reduced by TSA in the enzyme linked immunosorbent assay (ELISA) results. Based on these findings, TSA should be considered an effective inhibitor of hyperpigmentation.


Assuntos
Benzopiranos/farmacologia , AMP Cíclico/metabolismo , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Fenóis/farmacologia , Animais , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Fosforilação , Transdução de Sinais
13.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299130

RESUMO

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.


Assuntos
Cisplatino/toxicidade , Frutas/química , Macrófagos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Moraceae/química , Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia , Animais , Antineoplásicos/toxicidade , Apoptose , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Macrófagos/patologia , Melanoma Experimental/induzido quimicamente , Melanoma Experimental/patologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Substâncias Protetoras/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299131

RESUMO

The use of immune checkpoint inhibitors has revolutionized the treatment of melanoma patients, leading to remarkable improvements in the cure. However, to ensure a safe and effective treatment, there is the need to develop markers to identify the patients that would most likely respond to the therapies. The microenvironment is gaining attention in this context, since it can regulate both the immunotherapy efficacyand angiogenesis, which is known to be affected by treatment. Here, we investigated the putative role of the ECM molecule EMILIN-2, a tumor suppressive and pro-angiogenic molecule. We verified that the EMILIN2 expression is variable among melanoma patients and is associated with the response to PD-L1 inhibitors. Consistently, in preclinical settings,the absence of EMILIN-2 is associated with higher PD-L1 expression and increased immunotherapy efficacy. We verified that EMILIN-2 modulates PD-L1 expression in melanoma cells through indirect immune-dependent mechanisms. Notably, upon PD-L1 blockage, Emilin2-/- mice displayed improved intra-tumoral vessel normalization and decreased tumor hypoxia. Finally, we provide evidence indicating that the inclusion of EMILIN2 in a number of gene expression signatures improves their predictive potential, a further indication that the analysis of this molecule may be key for the development of new markers to predict immunotherapy efficacy.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Glicoproteínas/fisiologia , Inibidores de Checkpoint Imunológico/farmacologia , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Microambiente Tumoral/imunologia , Animais , Antígeno B7-H1/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205035

RESUMO

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.


Assuntos
Curcumina/farmacologia , Hiperpigmentação/tratamento farmacológico , Melaninas/biossíntese , Melanoma Experimental/tratamento farmacológico , Adrenomedulina/genética , Animais , Curcumina/análogos & derivados , Curcumina/química , Endotelina-1/genética , Humanos , Hiperpigmentação/metabolismo , Hiperpigmentação/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Melaninas/antagonistas & inibidores , Melanócitos/efeitos dos fármacos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanossomas/efeitos dos fármacos , Melanossomas/genética , Camundongos , Fagocitose/genética , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
16.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298956

RESUMO

Cucurbit[7]uril (CB[7]) is a molecular container that may form host-guest complexes with platinum(II) anticancer drugs and modulate their efficacy and safety. In this paper, we report our studies of the effect of CB[7]-oxaliplatin complex and the mixture of CB[7] and carboplatin (1:1) on viability and proliferation of a primary cell culture (peripheral blood mononuclear cells), two tumor cell lines (B16 and K562) and their activity in the animal model of melanoma. At the same time, we studied the impact of platinum (II) drugs with CB[7] on T cells and B cells in vitro. Although the stable CB[7]-carboplatin complex was not formed, the presence of cucurbit[7]uril affected the biological properties of carboplatin. In vivo, CB[7] increased the antitumor effect of carboplatin, but, at the same time, increased its acute toxicity. Compared to free oxaliplatin, its complex with CB[7] shows a greater cytotoxic effect on tumor cell lines B16 and K562, while in vivo, the effects of the free drug and encapsulated drug were comparable. However, in vivo studies also demonstrated that the encapsulation of oxaliplatin in CB[7] lowered the toxicity of the drug.


Assuntos
Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Carboplatina/farmacologia , Imidazóis/farmacologia , Fatores Imunológicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Piridinas/farmacologia , Animais , Feminino , Humanos , Células K562 , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos
17.
Dokl Biochem Biophys ; 498(1): 199-202, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34189650

RESUMO

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Epitopos/imunologia , Melanoma Experimental/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Feminino , Melanoma Experimental/etiologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL
18.
Cell Death Dis ; 12(7): 643, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162829

RESUMO

Among the principal causative factors for the development of complications related to aging is a diet rich in fats and sugars, also known as the Western diet. This diet advocates numerous changes that might increase the susceptibility to initiate cancer and/or to create a tissue microenvironment more conducive to the growth of malignant cells, thus favoring the progression of cancer and metastasis. Hypercaloric diets in general lead to oxidative stress generating reactive oxygen species and induce endoplasmic reticulum stress. Our results demonstrate that mice bearing tumors fed with a Western diet presented bigger tumor mass with increased insulin sensitivity in these tissues. Several markers of insulin signaling, such as AKT phosphorylation and mTOR pathway, are promoted in tumors of Western diet-fed animals. This process is associated with increased macrophage infiltration, activation of unfolded protein response pathway, and initiation of epithelial-mesenchymal transition (EMT) process in these tumor tissues. Summing up, we propose that the Western diet accelerates the aging-related processes favoring tumor development.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Dieta Ocidental/efeitos adversos , Transição Epitelial-Mesenquimal , Mediadores da Inflamação/metabolismo , Melanoma Experimental/metabolismo , Neoplasias Cutâneas/metabolismo , Resposta a Proteínas não Dobradas , Fatores Etários , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Fatores de Tempo , Carga Tumoral , Microambiente Tumoral , Resposta a Proteínas não Dobradas/genética
19.
Exp Cell Res ; 406(1): 112633, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34089726

RESUMO

Sorafenib is a multi-kinase inhibitor that has been highlighted as a tumor suppressor due to its anti-proliferative and anti-angiogenic properties, whereas the clinical application of Sorafenib is restricted by the side effects it may cause. The past decade has witnessed the development of a series of sorafenib derivatives to improve the clinical performance of sorafenib. Gold nanoparticles (AuNPs) have been widely utilized in drug delivery systems due to their unique properties, including biocompatible nature, simple preparation, and easy surface modification. Herein, this study is aimed to investigate the anti-tumor effects of new sorafenib derivatives-capped gold nanoparticles (AuNPs-New Sor) in tumor formation and metastasis as well as the underlying mechanisms. Initially, new sorafenib derivatives were constructed and combined with AuNPs to form AuNPs-New Sor, and the properties of synthesized AuNPs-New Sor were identified in a mouse model of tumorigenesis. The effect of AuNPs-New Sor on tumor vascular normalization was investigated by assessing vascular permeability and perfusion rate. Next, we evaluated the effect of AuNPs-New Sor on migration and viability of tumor cells and human umbilical vein endothelial cells (HUVECs) as well as on HUVEC angiogenesis in vitro. A melanoma mouse model was further established for in vivo substantiation of the anti-tumor effect of AuNPs-New Sor. According to the results, AuNPs could deliver new sorafenib derivatives into tumor tissues and downregulate the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), thereby suppressing tumor migration, EMT, and angiogenesis in vitro. In addition, AuNPs-New Sor displayed competitive anti-tumor activities in vivo. Taken together, AuNPs-New Sor may attenuate tumor development and angiogenesis through downregulation of EGFR and VEGFR-2.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Nanopartículas Metálicas/química , Neovascularização Patológica/prevenção & controle , Neoplasias Cutâneas/tratamento farmacológico , Sorafenibe/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Antineoplásicos/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Ouro/química , Células HCT116 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Células MCF-7 , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/secundário , Sorafenibe/análogos & derivados , Carga Tumoral/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
20.
Arch Biochem Biophys ; 710: 108978, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34174222

RESUMO

BACKGROUND: Melanoma is an aggressive cancer with a rapidly increasing incidence rate worldwide. Acteoside has been shown to have antitumor effects in multiple human cancers; however, the underlying function and mechanisms of acteoside in melanoma remain unclear. PURPOSE: This study explored the inhibitory effect of acteoside on melanoma and the possible mechanisms. METHODS: Acteoside (15 mg/kg, 30 mg/kg) was administered to mice daily for 21 days. ICI182,780 (0.5 mg/kg) was intraperitoneally injected 30 min before acteoside administration three times a week to evaluate whether the effects elicited by acteoside were mediated via the estrogen receptor. Tumor growth and metabolism, cardiac function, ROS and apoptosis levels in the spleen, serum inflammatory factors, and immune cells in the spleen were monitored. STAT3, p-STAT3, CD31, and survivin levels in tumor tissues were measured via immunofluorescence. Ras, Raf1, STAT3, p-STAT3, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 levels in tumor tissues were determined via Western Blotting. RESULTS: The results showed that acteoside inhibited melanoma growth, alleviated inflammation levels in mice, attenuated ROS and apoptosis levels in the spleen, downregulated the levels of CD31, survivin, Ras, Raf1, p-STAT3, and Bcl-2, and upregulated the levels of ERß, Bax, cleaved caspase-3, and cleaved caspase-9. Moreover, the effect of acteoside was blocked by ICI182,780. CONCLUSION: Acteoside may promote the apoptosis of tumor cells by regulating the ERß-Ras/Raf1-STAT3 signaling axis, thus inhibiting the occurrence and development of melanoma.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Glucosídeos/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Fenóis/uso terapêutico , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Glucosídeos/administração & dosagem , Coração/efeitos dos fármacos , Coração/fisiologia , Humanos , Masculino , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenóis/administração & dosagem , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
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