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1.
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
2.
Food Funct ; 12(9): 3978-3991, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977989

RESUMO

Tyrosinase is considered a molecular marker of melanoma, and few natural antitumor drugs targeting tyrosinase have been identified. In this study, proanthocyanidins (PAs) were isolated from the leaves of Photinia × fraseri and their structures were characterized by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the effects of antityrosinase activity were investigated. The results showed that the basic structural units of PAs are composed of catechin and epicatechin and that oligomer is the main component. PAs exhibited better antityrosinase activity via chelation of copper ions and by disturbing o-quinone production. Furthermore, analyses of the cell cycle, apoptosis rate, and regulation of melanin protein expression revealed preliminarily that PAs could affect melanin production by downregulating microphthalmia transcription factor (MITF) expression and by inhibiting the activities of tyrosinase and tyrosinase related protein 1 (TRP-1), leading to cell cycle arrest and apoptosis of melanoma cells. Collectively, our study demonstrated that PAs are potential tyrosinase inhibitors and have good antimelanoma effects. These findings provide a theoretical support for the application of tyrosinase inhibitors and for further drug development.


Assuntos
Apoptose , Ciclo Celular/efeitos dos fármacos , Melanoma Experimental/patologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Photinia/química , Proantocianidinas/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Levodopa/química , Levodopa/metabolismo , Melaninas/biossíntese , Melaninas/genética , Melanoma Experimental/enzimologia , Melanoma Experimental/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Estrutura Molecular , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Ácido Periódico , Folhas de Planta/química , Proantocianidinas/química , Proantocianidinas/isolamento & purificação
3.
J Clin Invest ; 131(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33945506

RESUMO

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.


Assuntos
Cromatina/enzimologia , Melanoma Experimental/enzimologia , Melanoma/enzimologia , Sirtuínas/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Cromatina/genética , Melanoma/genética , Melanoma/patologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sirtuínas/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
4.
Molecules ; 26(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917985

RESUMO

Morin is a well-known flavonoid, and has been reported to have various properties, such as anti-cell death, antioxidant, and anti-inflammatory properties. Although studies on the biochemical and biological actions of morin have been reported, the melanin biosynthesis effects and molecular mechanisms are unknown. In this study, we first found that morin has the effect of enhancing melanin biosynthesis in B16F10 mouse melanoma cells, and analyzed the molecular mechanism. In this study, we examined the effects of morin on the melanin contents and tyrosinase activity, as well as the protein expression levels of the melanogenic enzymes TRP-1, TRP-2, and microphtalmia-associated transcription factor (MITF) in B16F10 mouse melanoma cells. Morin showed no cytotoxicity in the concentration range of 5-100 µM, and significantly increased the intracellular tyrosinase activity and melanin contents. In mechanism analysis, morin increased the protein expression of TRP-1, TRP-2, and MITF associated with melanogenesis. Furthermore, morin increased phosphorylated ERK and p38 at the early time, and decreased phosphorylated ERK after 12 h. The results suggest that morin enhances melanin synthesis through the MAPK signaling pathways in B16F10 mouse melanoma cells.


Assuntos
Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melaninas/biossíntese , Melanoma Experimental/enzimologia , Melanoma Experimental/metabolismo , Agaricales/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/química , Camundongos , Modelos Biológicos , Monofenol Mono-Oxigenase/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia
5.
Biomed Res Int ; 2021: 6642973, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33778075

RESUMO

Introduction: The aminopeptidase N (APN/CD13) receptor plays an important role in the neoangiogenic process and metastatic tumor cell invasion. Clinical and preclinical studies reported that bestatin and actinonin are cytotoxic to APN/CD13-positive tumors and metastases due to their APN/CD13-specific inhibitor properties. Our previous studies have already shown that 68Ga-labeled NGR peptides bind specifically to APN/CD13 expressing tumor cells. The APN/CD13 specificity of 68Ga-NGR radiopharmaceuticals enables the following of the efficacy of antiangiogenic therapy with APN/CD13-specific inhibitors using positron emission tomography (PET). The aim of this in vivo study was to assess the antitumor effect of bestatin and actinonin treatment in subcutaneous transplanted HT1080 and B16-F10 tumor-bearing animal models using 68Ga-NODAGA-c(NGR). Materials and Methods: Three days after the inoculation of HT1080 and B16-F10 cells, mice were treated with intraperitoneal injection of bestatin (15 mg/kg) or actinonin (5 mg/kg) for 7 days. On the 5th and 10th day, in vivo PET scans and ex vivo biodistribution studies were performed 90 min after intravenous injection of 5.5 ± 0.2 MBq68Ga-NODAGA-c(NGR). Results: Control-untreated HT1080 and B16-F10 tumors were clearly visualized by the APN/CD13-specific 68Ga-NODAGA-c(NGR) radiopharmaceutical. The western blot analysis also confirmed the strong APN/CD13 positivity in the investigated tumors. We found significantly (p ≤ 0.05) lower radiopharmaceutical uptake after bestatin treatment and higher radiotracer accumulation in the actinonin-treated HT1080 tumors. In contrast, significantly lower (p ≤ 0.01) 68Ga-NODAGA-c(NGR) accumulation was observed in both bestatin- and actinonin-treated B16-F10 melanoma tumors compared to the untreated-control tumors. Bestatin inhibited tumor growth and 68Ga-NODAGA-c(NGR) uptake in both tumor models. Conclusion: The bestatin treatment is suitable for suppressing the neoangiogenic process and APN/CD13 expression of experimental HT1080 and B16-F10 tumors; furthermore, 68Ga-NODAGA-c(NGR) is an applicable radiotracer for the in vivo monitoring of the efficacy of the APN/CD13 inhibition-based anticancer therapies.


Assuntos
Acetatos , Antígenos CD13 , Radioisótopos de Gálio , Compostos Heterocíclicos com 1 Anel , Melanoma Experimental , Imagem Molecular , Proteínas de Neoplasias , Oligopeptídeos , Compostos Radiofarmacêuticos , Acetatos/farmacocinética , Acetatos/farmacologia , Animais , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/metabolismo , Radioisótopos de Gálio/farmacocinética , Radioisótopos de Gálio/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacocinética , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Masculino , Melanoma Experimental/diagnóstico por imagem , Melanoma Experimental/enzimologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia
6.
Nat Immunol ; 22(2): 193-204, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398181

RESUMO

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.


Assuntos
Linfócitos T CD8-Positivos/enzimologia , Neoplasias do Colo/enzimologia , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/enzimologia , Melanoma Experimental/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Estabilidade Enzimática , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Microambiente Tumoral
7.
Immunology ; 163(1): 74-85, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33421118

RESUMO

Dendritic cells (DCs) play an important role in linking innate and adaptive immunity. DCs can sense endogenous and exogenous antigens and present those antigens to T cells to induce an immune response or immune tolerance. During activation, alternative splicing (AS) in DCs is dramatically changed to induce cytokine secretion and upregulation of surface marker expression. PTBP1, an RNA-binding protein, is essential in alternative splicing, but the function of PTBP1 in DCs is unknown. Here, we found that a specific deficiency of Ptbp1 in DCs could increase MHC II expression and perturb T-cell homeostasis without affecting DC development. Functionally, Ptbp1 deletion in DCs could enhance antitumour immunity and asthma exacerbation. Mechanistically, we found that Pkm alternative splicing and a subset of Ifn response genes could be regulated by PTBP1. These findings revealed the function of PTBP1 in DCs and indicated that PTBP1 might be a novel therapeutic target for antitumour treatment.


Assuntos
Asma/enzimologia , Células Dendríticas/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Pulmão/enzimologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/enzimologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Neoplasias Cutâneas/enzimologia , Linfócitos T/metabolismo , Processamento Alternativo , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Homeostase , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Knockout , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/imunologia , Evasão Tumoral , Microambiente Tumoral
8.
Molecules ; 25(21)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153234

RESUMO

In a previous study, EphB4 was demonstrated to be a positive regulator of A375-melanoma growth but a negative regulator of tumor vascularization and perfusion. To distinguish between EphB4 forward and ephrinB2 reverse signaling, we used the commercially available EphB4 kinase inhibitor NVP-BHG712 (NVP), which was later identified as its regioisomer NVPiso. Since there have been reported significant differences between the inhibition profiles of NVP and NVPiso, we compared the influence of NVP and NVPiso on tumor characteristics under the same experimental conditions. Despite the different inhibitory profiles of NVP and NVPiso, the comparative study conducted here showed the same EphB4-induced effects in vivo as in the previous investigation. This confirmed the conclusion that EphB4-ephrinB2 reverse signaling is responsible for increased tumor growth as well as decreased tumor vascularization and perfusion. These results are further substantiated by microarrays showing differences between mock-transfected and EphB4-transfected (A375-EphB4) cells with respect to at least 9 angiogenesis-related proteins. Decreased expression of vascular endothelial growth factor (VEGF), angiotensin 1 (Ang-1), and protein kinase B (Akt/PKB), together with the increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta-2 (TGF-ß2), is consistent with the impaired vascularization of A375-EphB4 xenografts. Functional overexpression of EphB4 in A375-EphB4 cells was confirmed by activation of a variety of signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT), rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen activated protein kinase kinase (Ras/Raf/MEK), and nuclear factor kappa-B (NFkB) pathways.


Assuntos
Proliferação de Células/efeitos dos fármacos , Melanoma Experimental , Proteínas de Neoplasias , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor EphB4/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Front Immunol ; 11: 576310, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133093

RESUMO

Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.


Assuntos
Adenocarcinoma/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias do Colo/enzimologia , Melanoma Experimental/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Neoplasias Cutâneas/enzimologia , Macrófagos Associados a Tumor/enzimologia , Imunidade Adaptativa , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Antígenos de Diferenciação/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Humanos , Imunidade Inata , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células THP-1 , Carga Tumoral , Microambiente Tumoral , Macrófagos Associados a Tumor/imunologia
10.
J Immunother Cancer ; 8(2)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33037113

RESUMO

BACKGROUND: kalirin RhoGEF kinase (KALRN) is mutated in a wide range of cancers. Nevertheless, the association between KALRN mutations and the pathogenesis of cancer remains unexplored. Identification of biomarkers for cancer immunotherapy response is crucial because immunotherapies only show beneficial effects in a subset of patients with cancer. METHODS: We explored the correlation between KALRN mutations and antitumor immunity in 10 cancer cohorts from The Cancer Genome Atlas program by the bioinformatics approach. Moreover, we verified the findings from the bioinformatics analysis with in vitro and in vivo experiments. We explored the correlation between KALRN mutations and immunotherapy response in five cancer cohorts receiving immune checkpoint blockade therapy. RESULTS: Antitumor immune signatures were more enriched in KALRN-mutated than KALRN-wildtype cancers. Moreover, KALRN mutations displayed significant correlations with increased tumor mutation burden and the microsatellite instability or DNA damage repair deficiency genomic properties, which may explain the high antitumor immunity in KALRN-mutated cancers. Also, programmed cell death 1 ligand (PD-L1) expression was markedly upregulated in KALRN-mutated versus KALRN-wildtype cancers. The increased antitumor immune signatures and PD-L1 expression in KALRN-mutated cancers may favor the response to immune checkpoint blockade therapy in this cancer subtype, as evidenced in five cancer cohorts receiving antiprogrammed cell death protein 1 (PD-1)/PD-L1/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) immunotherapy. Furthermore, the significant association between KALRN mutations and increased antitumor immunity was associated with the fact that KALRN mutations compromised the function of KALRN in targeting Rho GTPases for the regulation of DNA damage repair pathways. In vitro and in vivo experiments validated the association of KALRN deficiency with antitumor immunity and the response to immune checkpoint inhibitors. CONCLUSIONS: The KALRN mutation is a useful biomarker for predicting the response to immunotherapy in patients with cancer.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Imunoterapia/métodos , Mutação , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genômica/métodos , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Neoplasias/enzimologia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/imunologia
11.
Molecules ; 25(10)2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32443441

RESUMO

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 µM) and diphenolase (IC50 = 3.8 µM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 µM-1 min-1 and k4 = 0.003 min-1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 µM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 µM.


Assuntos
Inibidores Enzimáticos/química , Fabaceae/química , Melanoma Experimental/tratamento farmacológico , Monofenol Mono-Oxigenase/antagonistas & inibidores , Animais , Ligação Competitiva/efeitos dos fármacos , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/química , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Melaninas/antagonistas & inibidores , Melaninas/biossíntese , Melanoma Experimental/enzimologia , Camundongos , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Tirosina/análogos & derivados , Tirosina/química
12.
Cells ; 9(4)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244541

RESUMO

Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.


Assuntos
Regulação para Baixo , Melanoma Experimental/enzimologia , Melanoma Experimental/metabolismo , Dinâmica Mitocondrial , Esfingomielina Fosfodiesterase/metabolismo , Animais , Modelos Animais de Doenças , Feminino , GTP Fosfo-Hidrolases/metabolismo , Melanoma Experimental/ultraestrutura , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Biogênese de Organelas , Oxirredução
13.
Commun Biol ; 3(1): 196, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332858

RESUMO

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/ß-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/ß-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/ß-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of ß-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome ß-catenin-mediated resistance to immune checkpoint blockade in melanoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Sulfonas/farmacologia , Tanquirases/antagonistas & inibidores , Triazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Tanquirases/metabolismo , Carga Tumoral/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismo
14.
Sci Adv ; 6(10): eaaz0575, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32181355

RESUMO

Although photodynamic therapy (PDT) has been clinically applied tumor hypoxia still greatly restricts the performance of this oxygen-dependent oncological treatment. The delivery of oxygen donors to tumor may produce excessive reactive oxygen species (ROS) and damage the peripheral tissues. Herein, we developed a strategy to solve the hypoxia issue by enhancing the lethality of ROS. Before PDT, the ROS-defensing system of the cancer cells was obstructed by an inhibitor to MTH1, which is a key for the remediation of ROS-caused DNA damage. As a result, both nuclei and mitochondrial DNA damages were increased, remarkably promoting cellular apoptosis. The therapeutic results demonstrated that the performance of PDT can be improved by the MTH1 inhibitor, leading to efficient cancer cell killing effect in the hypoxic tumor. This strategy makes better use of the limited oxygen, holding the promise to achieve satisfactory therapeutic effect by PDT without generating redundant cytotoxic ROS.


Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/genética , DNA de Neoplasias/genética , Inibidores Enzimáticos/farmacologia , Melanoma Experimental/tratamento farmacológico , Monoéster Fosfórico Hidrolases/genética , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/metabolismo , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/síntese química , Composição de Medicamentos/métodos , Inibidores Enzimáticos/química , Feminino , Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Luz , Células MCF-7 , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos Nus , Nanopartículas/administração & dosagem , Nanopartículas/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacocinética , Porfirinas/química , Porfirinas/farmacocinética , Pirimidinas/química , Espécies Reativas de Oxigênio/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Molecules ; 25(6)2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32210078

RESUMO

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing intracellular enzyme that catalyzes the first and rate-determining step of tryptophan metabolism and is an important immunotherapeutic target for the treatment of cancer. In this study, we designed and synthesized a new series of compounds as potential IDO1 inhibitors. These compounds were then evaluated for inhibitory activity against IDO1 and tryptophan 2,3-dioxygenase (TDO). Among them, the three phenyl urea derivatives i12, i23, i24 as showed potent IDO1 inhibition, with IC50 values of 0.1-0.6 µM and no compound exhibited TDO inhibitory activity. Using molecular docking, we predicted the binding mode of compound i12 within IDO1. Compound i12 was further investigated by determining its in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that compound i12 had satisfactory properties in mice, with moderate plasma clearance (22.45 mL/min/kg), acceptable half-life (11.2 h) and high oral bioavailability (87.4%). Compound i12 orally administered at 15 mg/kg daily showed tumor growth inhibition (TGI) of 40.5% in a B16F10 subcutaneous xenograft model and 30 mg/kg daily showed TGI of 34.3% in a PAN02 subcutaneous xenograft model. In addition, the body weight of i12-treated mice showed no obvious reduction compared with the control group. Overall, compound i12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Indolamina-Pirrol 2,3,-Dioxigenase , Melanoma Experimental , Proteínas de Neoplasias , Compostos de Fenilureia , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Compostos de Fenilureia/farmacologia , Relação Estrutura-Atividade
16.
Mol Cancer Ther ; 19(4): 1018-1030, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32024684

RESUMO

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Fator de Transcrição STAT1/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Biol Chem ; 295(11): 3635-3651, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31988240

RESUMO

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.


Assuntos
Núcleo Celular/enzimologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Estresse Fisiológico , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Cinética , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Células NIH 3T3 , Mononucleotídeo de Nicotinamida/química , Nicotinamida Fosforribosiltransferase/química , Ligação Proteica , Multimerização Proteica , Transporte Proteico
18.
Mol Cancer Ther ; 19(3): 755-764, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31974273

RESUMO

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8+ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.


Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase/química , Neoplasias do Colo/imunologia , Macrófagos/imunologia , Melanoma Experimental/imunologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Quinase Syk/antagonistas & inibidores , Animais , Apoptose , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Citocinas/metabolismo , Humanos , Tolerância Imunológica , Imunossupressão , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Food Chem Toxicol ; 135: 110993, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31765702

RESUMO

3S, 3'S-Astaxanthin is the most powerful antioxidant to scavenge free radicals in the world. In this study, a 3S, 3'S-astaxanthin biosynthesis pathway was constructed in a probiotic yeast, Kluveromyces marxianus, denoted YEAST, and its bioactive metabolites were extracted for biofunctional assessments. The bio-safety examination was achieved by two animal models as following: First, no significant toxic effects on YEAST groups were found in zebrafish; Second, after feeding YEAST for 4 weeks, the rat-groups showed no visible abnormality, and no significant change of the body weight and blood biochemistry tests. The inhibition of lung metastasis of melanoma cells and the increment of the survival rate were demonstrated by feeding YEAST and injecting the intravenous commercial astaxanthin in vivo rodent model. Based on in vitro assays of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging analysis, ferrous ion chelating ability, reducing power assessment, and mushroom tyrosinase inhibition evaluation, YEAST-astaxanthin showed anti-oxidative and tyrosinase suppressive properties. Taken together, the 3S, 3'S-astaxanthin producing probiotic yeast is safe to be used in the bio-synthesis of functional and pharmaceutical compounds, which have broad industrial applications on cosmetic, food and feed additive and healthcare.


Assuntos
Kluyveromyces/metabolismo , Melanoma Experimental/patologia , Engenharia Metabólica , Metástase Neoplásica/prevenção & controle , Probióticos , Animais , Antioxidantes/farmacologia , Feminino , Masculino , Melanoma Experimental/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Monofenol Mono-Oxigenase/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Xantofilas/química , Xantofilas/metabolismo , Xantofilas/farmacologia , Peixe-Zebra
20.
JCI Insight ; 4(24)2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31751318

RESUMO

As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here, we show that the arginase isoform expressed by T cells, the mitochondrial Arg2, is a cell-intrinsic regulator of CD8+ T cell activity. Both germline Arg2 deletion and adoptive transfer of Arg2-/- CD8+ T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8+ T cell activation, effector function, and persistence. Transcriptomic, proteomic, and high-dimensional flow cytometry characterization revealed a CD8+ T cell-intrinsic role of Arg2 in modulating T cell activation, antitumor cytoxicity, and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8+ T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations, coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells, unveil Arg2 as a potentially new therapeutic target for T cell-based cancer immunotherapies.


Assuntos
Arginase/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Melanoma Experimental/imunologia , Mitocôndrias/enzimologia , Animais , Arginase/genética , Arginina/metabolismo , Linfócitos T CD8-Positivos/enzimologia , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Citotoxicidade Imunológica , Feminino , Deleção de Genes , Humanos , Tolerância Imunológica/imunologia , Memória Imunológica/imunologia , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/imunologia , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/antagonistas & inibidores
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