RESUMO
Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.
Assuntos
Glucose , L-Lactato Desidrogenase , Microambiente Tumoral , Animais , Camundongos , Glucose/metabolismo , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/imunologia , Humanos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/imunologia , Transportador de Glucose Tipo 1/genética , Linhagem Celular Tumoral , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Glicólise/efeitos dos fármacos , Feminino , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Imunoterapia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
Epigenetic modifications to DNA and chromatin control oncogenic and tumor-suppressive mechanisms in melanoma. Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by Ezh2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models, we further investigated the role of pathways downstream of Ezh2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of a wild-type or Ezh2Y641F epigenetic state. We found that the Atg7 locus is controlled by multiple Ezh2 and Stat3 binding sites, Atg7 expression is dependent on Stat3 expression, and that deletion of Atg7 slows down melanoma cell growth in vivo, but not in vitro. Atg7 deletion also results in increased CD8 + T cells in Ezh2Y641F melanomas and reduced myelosuppressive cell infiltration in the tumor microenvironment, particularly in Ezh2WT melanomas, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.
Assuntos
Proteína 7 Relacionada à Autofagia , Proteína Potenciadora do Homólogo 2 de Zeste , Fator de Transcrição STAT3 , Animais , Fator de Transcrição STAT3/metabolismo , Camundongos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Regulação Neoplásica da Expressão Gênica , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Epigênese Genética , Linhagem Celular Tumoral , Humanos , Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismoRESUMO
Squalene (SQ) is a well-known antioxidant and anti-inflammatory agent that provides promising anti-aging and UV-protective roles on human skin. However, its strong hydrophobic nature, accompanied by issues such as poor solubility and limited tissue permeation, has created challenges for scientists to investigate its untapped potential in more complex conditions, including cancer progression. The present study assessed the potent anti-metastatic properties of a newly synthesized amphiphilic ethylene glycol SQ derivative (SQ-diEG) in melanoma, the most fatal skin cancer. In vitro and in vivo experiments have discovered that SQ-diEG may exert its potential on melanoma malignancy through the mitochondria-mediated caspase activation apoptotic signaling pathway. The potent anti-metastatic effect of SQ-diEG was observed in vitro using highly proliferative and aggressive melanoma cells. Administration of SQ-diEG (25 mg/kg) significantly decreased the tumor burden on the lung and inhibited the metastasis-associated proteins and gene markers in B16F10 lung colonization mice model. Furthermore, global gene profiling also revealed a promising role of SQ-diEG in tumor microenvironment. We anticipated that the amphiphilic nature of the SQ compound bearing ethylene glycol oligomers could potentially augment its ability to reach the pathology site, thus enhancing its therapeutic potential in melanoma.
Assuntos
Melanoma , Esqualeno , Animais , Camundongos , Esqualeno/química , Esqualeno/farmacologia , Humanos , Linhagem Celular Tumoral , Melanoma/patologia , Melanoma/tratamento farmacológico , Camundongos Endogâmicos C57BL , Apoptose/efeitos dos fármacos , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Metástase Neoplásica , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Éteres/farmacologia , Éteres/química , Proliferação de Células/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/químicaRESUMO
Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8+ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber's role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome-lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8+ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape.
Assuntos
Autofagia , Benzilisoquinolinas , Melanoma , Evasão Tumoral , Autofagia/efeitos dos fármacos , Animais , Camundongos , Linhagem Celular Tumoral , Humanos , Evasão Tumoral/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos Endogâmicos C57BL , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Cisteína EndopeptidasesRESUMO
Focused on the newly secreted tumorous exosomes during melanoma immunotherapy, this work has pioneered an ultra-sensitive spatiotemporal-specific exosome detection strategy, leveraging advanced exosomal membrane engineering techniques. The proposed strategy harnesses the power of amplified lanthanide luminescence signals on these exosomes, enabling precise and real-time monitoring of the efficacy of melanoma immunotherapy. The methodology comprises two pivotal steps. Initially, Ac4ManNAz-associated metabolic labeling is employed to evolve azide groups onto the membranes of newly secreted exosomes with remarkable selectivity. These azide groups serve as versatile clickable artificial tags, enabling the precise identification of melanoma exosomes emerging during immunotherapy. Subsequently, lanthanide-nanoparticle-functionalized polymer chains are controllably grafted onto the exosome surfaces through click chemistry and in situ Fenton-RAFT polymerization, serving as robust signal amplifiers. When integrated with time-resolved fluorescence detection, this strategy yields detection signals with an exceptionally high signal-to-noise ratio, enabling ultra-sensitive detection of PD-L1 antigen expression levels on the spatiotemporal-specific exosomes. The detection strategy boasts a wide linear concentration range spanning from 1.7 × 104 to 1.7 × 109 particles/mL, with a remarkable theoretical detection limit of 1.28 × 103 particles/mL. The remarkable enhancements in detection sensitivity and accuracy facilitate the evaluation of the efficacy of immunotherapeutic interventions in the mouse B16 melanoma model, notably revealing a substantial disparity in PD-L1 levels between immunotherapy-treated and untreated groups (P < 0.01) and further emphasizing the cumulative therapeutic effect that intensifies with repeated treatments (P < 0.001).
Assuntos
Exossomos , Imunoterapia , Elementos da Série dos Lantanídeos , Exossomos/química , Exossomos/metabolismo , Animais , Camundongos , Elementos da Série dos Lantanídeos/química , Melanoma/terapia , Melanoma/metabolismo , Melanoma/imunologia , Melanoma/patologia , Luminescência , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/terapia , Melanoma Experimental/patologia , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Nanopartículas/químicaRESUMO
T cell inhibitory mechanisms prevent autoimmune reactions, while cancer immunotherapy aims to remove these inhibitory signals. Chronic ultraviolet (UV) exposure attenuates autoimmunity through promotion of poorly understood immune-suppressive mechanisms. Here we show that mice with subcutaneous melanoma are not responsive to anti-PD1 immunotherapy following chronic UV irradiation, given prior to tumor injection, due to the suppression of T cell killing ability in skin-draining lymph nodes. Using mass cytometry and single-cell RNA-sequencing analyzes, we discover that skin-specific, UV-induced suppression of T-cells killing activity is mediated by upregulation of a Ly6ahigh T-cell subpopulation. Independently of the UV effect, Ly6ahigh T cells are induced by chronic type-1 interferon in the tumor microenvironment. Treatment with an anti-Ly6a antibody enhances the anti-tumoral cytotoxic activity of T cells and reprograms their mitochondrial metabolism via the Erk/cMyc axis. Treatment with an anti-Ly6a antibody inhibits tumor growth in mice resistant to anti-PD1 therapy. Applying our findings in humans could lead to an immunotherapy treatment for patients with resistance to existing treatments.
Assuntos
Antígenos Ly , Linfócitos T CD8-Positivos , Imunoterapia , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos Ly/metabolismo , Antígenos Ly/imunologia , Camundongos , Imunoterapia/métodos , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/patologia , Feminino , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/patologia , Mitocôndrias/metabolismo , Melanoma/imunologia , Melanoma/terapia , Interferon Tipo I/metabolismoRESUMO
In this paper, three new iridium(III) complexes: [Ir(piq)2(DFIPP)]PF6 (piq = deprotonated 1-phenylisoquinoline, DFIPP = 3,4-difluoro-2-(1H-imidazo[4,5-f][1,10]phenenthrolin-2-yl)phenol, 3a), [Ir(bzq)2(DFIPP)]PF6 (bzq = deprotonated benzo[h]quinoline, 3b), and [Ir(ppy)2(DFIPP)]PF6 (ppy = deprotonated 1-phenylpyridine, 3c), were synthesized and characterized. The complexes were found to be nontoxic to tumor cells via 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide (MTT) assay. Surprisingly, its liposome-entrapped complexes 3alip, 3blip, and 3clip on B16 cells showed strong cytotoxicity (IC50 = 13.6 ± 2.8, 9.6 ± 1.1, and 18.9 ± 2.1 µM). Entry of 3alip, 3blip, and 3clip into B16 cells decreases mitochondrial membrane potential, regulates Bcl-2 family proteins, releases cytochrome c, triggers caspase family cascade reaction, and induces apoptosis. In addition, we also found that 3alip, 3blip, and 3clip triggered ferroptosis and autophagy. In vivo studies demonstrated that 3blip inhibited melanoma growth in C57 mice with a high inhibitory rate of 83.95%, and no organic damage was found in C57 mice.
Assuntos
Antineoplásicos , Apoptose , Complexos de Coordenação , Irídio , Lipossomos , Irídio/química , Irídio/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Camundongos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos Endogâmicos C57BL , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
T cell-based immunotherapies are a promising therapeutic approach for multiple malignancies, but their efficacy is limited by tumor hypoxia arising from dysfunctional blood vessels. Here, we report that cell-intrinsic properties of a single vascular component, namely the pericyte, contribute to the control of tumor oxygenation, macrophage polarization, vessel inflammation, and T cell infiltration. Switching pericyte phenotype from a synthetic to a differentiated state reverses immune suppression and sensitizes tumors to adoptive T cell therapy, leading to regression of melanoma in mice. In melanoma patients, improved survival is correlated with enhanced pericyte maturity. Importantly, pericyte plasticity is regulated by signaling pathways converging on Rho kinase activity, with pericyte maturity being inducible by selective low-dose therapeutics that suppress pericyte MEK, AKT, or notch signaling. We also show that low-dose targeted anticancer therapy can durably change the tumor microenvironment without inducing adaptive resistance, creating a highly translatable pathway for redosing anticancer targeted therapies in combination with immunotherapy to improve outcome.
Assuntos
Pericitos , Animais , Pericitos/imunologia , Pericitos/metabolismo , Pericitos/patologia , Camundongos , Humanos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Imunoterapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/patologia , Fenótipo , Melanoma/imunologia , Melanoma/terapia , Melanoma/patologia , Melanoma/tratamento farmacológico , Linhagem Celular Tumoral , Tolerância Imunológica/efeitos dos fármacosRESUMO
Traditional fungi ß-glucan commonly possesses high molecular weight with poor water solubility, which remains significant challenge in the drug development and medical application. Water-soluble ß-glucan with high molecular weight (dHSCG) of 560 kDa, low molecular weight (dLSCG) of 60 kDa, and sulfated derivative (SCGS) with a molecular weight of 146 kDa and sulfate degree at 2.04 were obtained through well-controlled degradation and sulfated modification from Saccharomyces cerevisiae in this study. The structural characteristics were confirmed as ß-1,3/6-glucan by FT-IR and NMR spectroscopy. Carbohydrate microarrays and surface plasmon resonance revealed distinct and contrasting binding affinities between the natural ß-glucans and sulfated derivatives. SCGS exhibited strong binding to FGF and VEGF, while natural ß-glucan showed no response, suggesting its potential as a novel antitumor agent. Moreover, SCGS significantly inhibited the migration rate of the highly metastatic melanoma (B16F10) cells. The lung metastasis mouse model also demonstrated that SCGS significantly reduced and eliminated the nodules, achieving an inhibition rate of 86.7% in vivo, with a dramatic improvement in IFN-α, TNF-α, and IL-1ß levels. Through analysis of protein content and distribution in lung tissues, the anti-tumor and anti-metastasis mechanism of SCGS involves the regulation of degrading enzymes to protect extracellular matrix (ECM), as well as the reduction of angiogenic factor release. These findings provide a foundation for exploring the potential of SCGS in the development of new anti-tumor and anti-metastasis drugs and open up a new field in cancer research.
Assuntos
Antineoplásicos , Saccharomyces cerevisiae , Solubilidade , beta-Glucanas , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , beta-Glucanas/química , beta-Glucanas/farmacologia , Água/química , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Sulfatos/química , Movimento Celular/efeitos dos fármacos , HumanosRESUMO
Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 µM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes.
Assuntos
Autofagia , Ceramidas , Melaninas , Melanoma Experimental , Melanossomas , MicroRNAs , Transdução de Sinais , Serina-Treonina Quinases TOR , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Melanossomas/metabolismo , Ceramidas/metabolismo , Melaninas/metabolismo , Melaninas/biossíntese , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/genética , Linhagem Celular Tumoral , alfa-MSH/metabolismo , Melanócitos/metabolismo , Melanócitos/efeitos dos fármacosRESUMO
BACKGROUND: Melanoma is a highly aggressive form of skin cancer. The existence of cancer stem cells (CSCs) and tumor immune evasion are two major causes of melanoma progression, but no effective treatment has been found at present. Astragalus polysaccharide (APS) is a principal active component derived from Astragalus membranaceus, showing anti-tumor effects in various tumors including melanoma. However, the underlying mechanism is still unclear. METHODS: The regulation of APS on self-renewal ability and CSC markers expression in melanoma stem cells (MSCs) was measured by tumor sphere formation and tumorigenicity assays, RT-qPCR, and western blot. Flow cytometry was conducted to evaluate the activation of immune system by APS in melanoma mice. Further, the mechanism was explored based on PD-L1 overexpression and knock-down B16 cells. RESULTS: APS attenuated the tumor sphere formation of MSCs in vitro as well as the tumorigenicity in vivo. It also decreased the expression of CD133, BMI1 and CD47. Based on the PD-L1 overexpression and knock-down B16 cells, it was confirmed that APS inhibited the induction of MSCs by down-regulating PD-L1 expression. Meanwhile, APS increased the infiltration of CD4+ and CD8+T cells in tumor tissues because of its inhibitory effect on PD-L1. CONCLUSIONS: APS inhibited MSC induction and overcame tumor immune evasion through reducing PD-L1 expression. This study provided compelling evidence that APS could be a prospective therapeutic agent for treating melanoma.
Assuntos
Antígeno B7-H1 , Melanoma Experimental , Células-Tronco Neoplásicas , Polissacarídeos , Antígeno B7-H1/metabolismo , Animais , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/imunologia , Camundongos , Polissacarídeos/farmacologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Evasão Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Melanoma/imunologia , Astrágalo/química , Evasão da Resposta ImuneRESUMO
Melanoma has gained considerable attention due to its high mortality and morbidity rate worldwide. The currently available treatment options are associated with several limitations such as nonspecificity, drug resistance, easy clearance, low efficacy, toxicity-related issues, etc. To this end, nanotechnology has garnered significant attention for the treatment of melanoma. In the present manuscript, we have demonstrated the in vitro and in vivo anticancer activity of silver nitroprusside nanoparticles (abbreviated as AgNNPs) against melanoma. The AgNNPs exhibit cytotoxicity against B16F10 cells, which has been investigated by several in vitro experiments including [methyl 3H]-thymidine incorporation assay, cell cycle and apoptosis analysis by flow cytometry, and ROS generation through DCFDA, DHE, and DAF2A reagents. Further, the internalization of nanoparticles was determined by ICPOES analysis, while their colocalization was analyzed by confocal microscopy. Additionally, JC-1 staining is performed to examine mitochondrial membrane potential (MMP). Cytoskeleton integrity was observed by phalloidin staining. Expression of different markers (Ki-67, cytochrome c, and E-cadherin) was checked using an immunofluorescence assay. The in vivo therapeutic efficacy of AgNNPs has been validated in the melanoma model established by inoculating B16F10 cells into the dorsal right abdomen of C57BL/6J mice. The intraperitoneal administration of AgNNPs reduced melanoma growth and increased the survivability of tumor-bearing mice. The in vivo immunofluorescence studies (Ki-67, CD31, and E-cadherin) and TUNEL assay support the inhibitory and apoptotic nature of AgNNPs toward melanoma, respectively. Furthermore, the various signaling pathways and molecular mechanisms involved in anticancer activity are evaluated by Western blot analysis. These findings altogether demonstrate the promising anticancer potential of AgNNPs toward melanoma.
Assuntos
Antineoplásicos , Apoptose , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos Endogâmicos C57BL , Nitroprussiato , Prata , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , Nitroprussiato/farmacologia , Nitroprussiato/química , Apoptose/efeitos dos fármacos , Prata/química , Prata/farmacologia , Proliferação de Células/efeitos dos fármacos , Tamanho da Partícula , Teste de Materiais , Melanoma/tratamento farmacológico , Melanoma/patologia , Nanopartículas Metálicas/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/metabolismoRESUMO
Melanin naturally exists in organisms and is synthetized by tyrosinase (TYR); however, its over-production may lead to aberrant pigmentation and skin conditions. Loquat (Eriobotrya japonica (Thunb.) Lindl.) flowers contain a variety of bioactive compounds, while studies on their suppressive capabilities against melanin synthesis are limited. Loquat flower isolate product (LFP) was obtained by ethanol extraction and resin purification, and its inhibitory efficiency against TYR activity was investigated by enzyme kinetics and multiple spectroscopy analyses. In addition, the impact of LFP on melanin synthesis-related proteins' expression in mouse melanoma B16 cells was analyzed using Western blotting. HPLC-MS/MS analysis indicated that LFP was composed of 137 compounds, of which 12 compounds, including flavonoids (quercetin, isorhamnoin, p-coumaric acid, etc.) and cinnamic acid and its derivatives, as well as benzene and its derivatives, might have TYR inhibitory activities. LFP inhibited TYR activity in a concentration-dependent manner with its IC50 value being 2.8 mg/mL. The inhibition was an anti-competitive one through altering the enzyme's conformation rather than chelating copper ions at the active center. LFP reduced the expression of TYR, tyrosinase-related protein (TRP) 1, and TRP2 in melanoma B16 cells, hence inhibiting the synthesis of melanin. The research suggested that LFP had the potential to reduce the risks of hyperpigmentation caused by tyrosinase and provided a foundation for the utilization of loquat flower as a natural resource in the development of beauty and aging-related functional products.
Assuntos
Eriobotrya , Flores , Melaninas , Melanoma Experimental , Monofenol Mono-Oxigenase , Extratos Vegetais , Animais , Monofenol Mono-Oxigenase/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Camundongos , Melaninas/biossíntese , Melaninas/metabolismo , Flores/química , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Eriobotrya/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/químicaRESUMO
BACKGROUND: As a medicinal and food homologous plant, Rosa damascena is not only highly ornamental, but also rich in a variety of active ingredients such as polyphenols and flavonoids. It is widely used in cosmetics, food and pharmaceutical industries. OBJECTIVE: To study the in vitro efficacy of Rosa damascena solid state fermentation liquid (RDF) and water extract (RDE). METHODS: Firstly, the effect of RDF and RDE on the proliferation rate of B16F10 cells was detected by CCK-8 method, and the melanin content was measured by sodium hydroxide lysis method to evaluate the whitening effect of them. Finally, the antioxidant, anti-wrinkling and soothing effects of RDF and RDE were evaluated by biochemical methods in vitro. RESULTS: RDF and RDE within a certain concentration range (0.05%-0.5%) had no effect on the proliferation of B16F10 cells. Compared with Rosa damascena extract (RDE), RDF showed significant effects on bleaching, antioxidant, anti-wrinkling and soothing, among which 0.5% RDF showed the best effect. CONCLUSION: Both RDF and RDE at a certain concentration have effect on skin care in vitro, but the effect of RDF is more significant than that of RDE.
Assuntos
Antioxidantes , Proliferação de Células , Fermentação , Extratos Vegetais , Rosa , Rosa/química , Extratos Vegetais/farmacologia , Camundongos , Animais , Proliferação de Células/efeitos dos fármacos , Antioxidantes/farmacologia , Higiene da Pele/métodos , Água/química , Envelhecimento da Pele/efeitos dos fármacos , Melaninas , Linhagem Celular Tumoral , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologiaRESUMO
Ganoderma lucidum, a member of the Basidiomycetes family, is attracting attention for its medicinal potential due to its biological activity and the presence of numerous bioactive compounds. Although it is known that extracts of this mushroom inhibit melanin production, there are few reports on a single substance associated with this effect. In this study, we identified ganodermanontriol (GT), a novel compound from G. lucidum, that effectively inhibited melanin biosynthesis in B16F10 cells. GT inhibits melanin production by suppressing the expression of cellular tyrosinase proteins and microphthalmia-related transcription factor (MITF). Furthermore, GT affects the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and mitogen-activated protein kinase (MAPK) signaling molecules, which are involved in melanogenesis in B16F10 cells. Finally, the biosynthesis of GT and other substances by G. lucidum was evaluated using HPLC analysis. Thus, this study revealed the mechanism by which GT in G. lucidum inhibits melanin production in B16F10 cells, and these findings will contribute to promoting the potential use of this mushroom in the future.
Assuntos
Sistema de Sinalização das MAP Quinases , Melaninas , Reishi , Melaninas/biossíntese , Melaninas/metabolismo , Animais , Camundongos , Reishi/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Linhagem Celular Tumoral , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Fosforilação/efeitos dos fármacos , Fator de Transcrição Associado à Microftalmia/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
CD27 belongs to the tumor necrosis factor receptor superfamily and acts as a co-stimulatory molecule, modulating T and B cell responses. CD27 stimulation enhances T cell survival and effector functions, thus providing opportunities to develop therapeutic strategies. The current study aims to investigate the role of endogenous CD27 signaling in tumor growth and metastasis. CD8 + T cell-specific CD27 knockout (CD8Cre-CD27fl) mice were developed, while global CD27 knockout (KO) mice were also used in our studies. Flow cytometry analyses confirmed that CD27 was deleted specifically from CD8 + T cells without affecting CD4 + T cells, B cells, and HSPCs in the CD8Cre-CD27fl mice, while CD27 was deleted from all cell types in global CD27 KO mice. Tumor growth and metastasis studies were performed by injecting B16-F10 melanoma cells subcutaneously (right flank) or intravenously into the mice. We have found that global CD27 KO mice succumbed to significantly accelerated tumor growth compared to WT controls. In addition, global CD27 KO mice showed a significantly higher burden of metastatic tumor nests in the lungs compared to WT controls. However, there was no significant difference in tumor growth curves, survival, metastatic tumor nest counts between the CD8Cre-CD27fl mice and WT controls. These results suggest that endogenous CD27 signaling inhibits tumor growth and metastasis via CD8 + T cell-independent mechanisms in this commonly used melanoma model, presumably through stimulating antitumor activities of other types of immune cells.
Assuntos
Linfócitos T CD8-Positivos , Melanoma Experimental , Transdução de Sinais , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral , Animais , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Here we report a brand-new bioactive polymer featuring sulfonium moieties that exhibits the capability of inducing immunogenic cell death (ICD) for anticancer therapy. The optimized polysulfonium presents a wide spectrum of potent anticancer activity and remarkable selectivity. In-depth mechanistic studies reveal that the polymer exerts its cytotoxic effects on cancer cells through a membrane-disrupting mechanism. This further initiates the release of a plethora of damage-associated molecular patterns, effectively triggering ICD and resulting in systemic anticancer immune responses. Notably, the compound demonstrated significant efficacy in suppressing tumor growth in the B16-F10 melanoma tumor model. Furthermore, it exhibits robust immune memory effects, effectively suppressing tumor recurrence and metastasis in both the rechallenge model and the lung metastatic tumor model. To the best of our knowledge, the study represents the pioneering exportation of cationic polysulfoniums, showcasing not only their remarkable safety and efficacy against primary tumors but also their unique ability in activating long-term immune memory.
Assuntos
Antineoplásicos , Morte Celular Imunogênica , Polímeros , Animais , Morte Celular Imunogênica/efeitos dos fármacos , Camundongos , Humanos , Linhagem Celular Tumoral , Polímeros/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Compostos de Sulfônio/química , Compostos de Sulfônio/farmacologia , Compostos de Sulfônio/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologiaRESUMO
Cancer immunotherapy has emerged as a promising approach to cancer treatment in recent years. The physical and chemical properties of nanocarriers are critical factors that regulate the immune activation of antigen-presenting cells (APCs) in the tumor microenvironment (TME). Herein, we extensively investigated the behavior of liposome nanoparticles (Lipo-NPs) with different elasticities, focusing on their interaction with immune cells and their transport mechanisms from tumors to tumor-draining lymph nodes (tdLNs). Successfully preparing Lipo-NPs with distinct elastic properties, their varied behaviors were observed, concerning immune cell interaction. Soft Lipo-NPs exhibited an affinity to cell membranes, while those with medium elasticity facilitated the cargo delivery to macrophages through membrane fusion. Conversely, hard Lipo-NPs enter macrophages via classical cellular uptake pathways. Additionally, it was noted that softer Lipo-NPs displayed superior transport to tdLNs in vivo, attributed to their deformable nature with lower elasticity. As a result, the medium elastic Lipo-NPs with agonists (cGAMP), by activating the STING pathway and enhancing transport to tdLNs, promoted abundant infiltration of tumor-infiltrating lymphocytes (TILs), leading to notable antitumor effects and extended survival in a melanoma mouse model. Furthermore, this study highlighted the potential synergistic effect of medium elasticity Lipo-NPs with immune checkpoint blockade (ICB) therapy in preventing tumor immune evasion. These findings hold promise for guiding immune-targeted delivery systems in cancer immunotherapy, particularly in vaccine design for tdLNs targeting and eradicating metastasis within tdLNs.
Assuntos
Elasticidade , Imunoterapia , Lipossomos , Lipossomos/química , Animais , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral/efeitos dos fármacos , Nanopartículas/química , Humanos , Feminino , Melanoma Experimental/terapia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Linhagem Celular TumoralRESUMO
BACKGROUND: Dysregulation of melanogenesis contributes to the development of skin hyperpigmentation diseases, which poses a treatment challenge. Following the establishment of CRTC3 screening methods to explore small molecules inhibiting melanogenesis for the topical treatment of hyperpigmentation diseases, we identified a candidate molecule, semaxanib. OBJECTIVE: To explore the antimelanogenic effects of semaxanib, a vascular endothelial growth factor receptor (VEGFR) 2 inhibitor, for potential applications in hyperpigmentation management and to unravel the role of VEGF signaling in melanocyte biology by investigating mechanism of action of semaxanib. METHODS: Mouse-derived spontaneously immortalized melanocytes, B16F10, and normal human primary epidermal melanocytes cells were treated with semaxanib, and cellular responses were assessed using cell viability assays and melanin content measurements. Molecular mechanisms were investigated using transcriptional activity assays, reverse-transcription polymerase chain reaction, and immunoblotting analysis. In vivo studies were conducted using an epidermis-humanized transgenic mouse model and ex vivo human skin tissues. RESULTS: Semaxanib ameliorated melanin content in cultured melanocytes by downregulating the expression of melanogenesis-associated genes by suppressing the CRTC3/microphthalmia-associated transcription factors. Topical application of semaxanib reduced melanin accumulation in the ultraviolet B-stimulated ex vivo human epidermis and tail of K14-stem cell factor transgenic mice. Mechanistically, the antimelanogenic effect induced by semaxanib was associated with SIK2-CRTC3-MITF rather than VEGF signaling in melanocytes. CONCLUSION: Semaxanib emerges as a promising candidate for the development of therapeutics for hyperpigmentation, potentially working independently of VEGF signaling in human melanocytes.
Assuntos
Melaninas , Melanócitos , Fator de Transcrição Associado à Microftalmia , Transdução de Sinais , Fatores de Transcrição , Fator A de Crescimento do Endotélio Vascular , Animais , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Humanos , Melaninas/biossíntese , Melaninas/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Indóis/farmacologia , Hiperpigmentação/tratamento farmacológico , Camundongos Transgênicos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Células Cultivadas , MelanogêneseRESUMO
BACKGROUND: Melanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored. METHODS: The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays. RESULTS: Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8. CONCLUSION: Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.