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1.
Cancer Immunol Immunother ; 69(4): 569-580, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31980915

RESUMO

BACKGROUND: The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells. DNA vaccines fusing MIP-3α to melanoma-associated antigens have shown improved efficacy and immunogenicity in the B16F10 mouse melanoma model. Here, we report that the combination of type-I interferon therapy (IFNα) with 5-Aza-2'-deoxycitidine (5Aza) profoundly enhanced the therapeutic efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine. METHODS: Beginning on day 5 post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given 2 days later. 5Aza was given intraperitoneally at 1 mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry, respectively. RESULTS: Adding IFNα and 5Aza treatments to mice vaccinated with MIP-3α-Gp100-Trp2 leads to reduced tumor burden and increased median survival (39% over vaccine and 95% over controls). Tumor lysate expression of CCL19 and CCR7 were upregulated ten and fivefold over vaccine, respectively. Vaccine-specific and overall CD8+ TILs were increased over vaccine (sevenfold and fourfold, respectively), as well as the proportion of TILs that were CD8+ (twofold). CONCLUSIONS: Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers an alternative to classic and dendritic cell vaccines. Combining this approach with IFNα and 5Aza treatment significantly improved vaccine efficacy. This improved efficacy correlated with changes in chemokine gene expression and CD8+ TIL infiltration and was dependent on the presence of all therapeutic components.


Assuntos
Vacinas Anticâncer/imunologia , Decitabina/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Melanoma Experimental/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Quimiocina CCL19/genética , Quimiocina CCL19/imunologia , Metilação de DNA/efeitos dos fármacos , Decitabina/administração & dosagem , Células Dendríticas/citologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Interferon-alfa/administração & dosagem , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Receptores CCR7/genética , Receptores CCR7/imunologia
2.
Int J Cancer ; 146(5): 1409-1420, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31702822

RESUMO

Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAFV600E mutation and concomitant PTEN loss in order to characterize alterations in tumor-infiltrating effector immune cells when tumors become resistant to BRAFi. We found that BRAFi-sensitive tumors displayed a pronounced inflammatory milieu characterized by high levels of cytokines and chemokines accompanied by an infiltration of T and NK cells. The tumor-infiltrating effector cells were activated and produced high levels of IFN-γ, TNF-α and granzyme B. When tumors became resistant and progressively grew, they reverted to a low immunogenic state similar to untreated tumors as reflected by low mRNA levels of proinflammatory cytokines and chemokines and fewer tumor-infiltrating T and NK cells. Moreover, these T and NK cells were functionally impaired in comparison to their counterparts in BRAFi-sensitive tumors. Their effector cell function could be restored by additional peritumoral treatment with the TLR7 agonist imiquimod, a clinically approved agent for nonmelanoma skin cancer. Indeed, resistance to BRAFi therapy was delayed and accompanied by high numbers of activated T and NK cells in tumors. Thus, combining BRAFi with an immune stimulating agent such as a TLR ligand could be a promising alternative approach for the treatment of melanoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral/transplante , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Imiquimode/farmacologia , Imiquimode/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Camundongos , Mutação , Células T Matadoras Naturais , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo
3.
BMC Res Notes ; 12(1): 648, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590685

RESUMO

OBJECTIVES: Mycobacterium indicus pranii (MIP) is an atypical mycobacterium species with potent antitumor efficacy. Macrophages and dendritic cells (DCs) are antigen-presenting cells, playing key roles in the activation of antitumor immunity. We have previously shown the potent activation of macrophages and DCs by MIP, which is mediated by MyD88-TLR2 signaling axis. In the present study, we further examined the role of MyD88 and TLR2 in MIP-mediated tumor regression. RESULTS: Wild-type and MyD88-/- mice were implanted with B16F10 tumor cells, treated with MIP or phosphate-buffered saline (PBS) and monitored for tumor growth. As expected, MIP therapy led to significant tumor regression in wild-type mice. However, antitumor efficacy of MIP was lost in MyD88-/- animals. Both PBS-treated (control) and MIP-treated MyD88-/- mice developed tumors with comparable volume. Since MyD88 relays TLR engagement signals, we analyzed the antitumor efficacy of MIP in TLR2-/- and TLR4-/- mice. It was observed that MIP therapy reduced tumor burden in wild-type and TLR4-/- mice but not in TLR2-/- mice. Tumor volume in MIP-treated TLR2-/- mice were comparable with those in PBS-treated wild-type animals. These results implicated the MyD88-TLR2 signaling axis in the antitumor efficacy of MIP.


Assuntos
Melanoma Experimental/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Micobactérias não Tuberculosas/imunologia , Receptor 2 Toll-Like/imunologia , Carga Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/microbiologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Micobactérias não Tuberculosas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Carga Tumoral/genética
4.
Immunology ; 158(2): 136-149, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31515801

RESUMO

Immune-checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti-Toll-like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell-surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti-TLR4 mAb in T-cell-mediated antitumour immunity. The anti-TLR4 mAb induced the activation of antigen-specific T-cells in adoptive transfer studies. The growth of ovalbumin (OVA)-expressing tumours was significantly suppressed by administration of OVA and the anti-TLR4 mAb in combination, but not individually. The antitumour effect of anti-PD-1 mAb was enhanced in mice administered with OVA plus the anti-TLR4 mAb. The OVA-specific IFN-γ-producing CD8 T-cells were induced by administration of OVA and the anti-TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T-cells. The inflammatory response to the anti-TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF-κB activation and production of TNF-α, IL-6 and IL-1ß. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti-TLR4 mAb) plus OVA induced no or less-marked activation of OVA-specific T-cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti-TLR4 mAb induces potent CD8 T-cell-dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti-TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.


Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Imunização , Imunoterapia/métodos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , Ovalbumina/administração & dosagem , Cultura Primária de Células , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
Int Immunopharmacol ; 75: 105763, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325728

RESUMO

Emodin is a Chinese herb-derived compound that exhibits a variety of pharmacological benefits. Although emodin has been shown to inhibit growth of cancer cells, its antineoplastic function is incompletely understood. CD155 is a member of poliovirus receptor-related (PRR) family of adhesion molecules; it is constitutively expressed on many tumor cell lines and tissues and has diverse functions. CD155 has been reported to mediate activation of T cells via CD226 or inhibition of T cells via T-cell immunoreceptor with Ig and ITIM domains (TIGIT). In addition, CD155 may play a critical role through non-immunological mechanisms in cancer. In this study, we tested the ability of emodin to modulate CD155 expression in cancer cells. We found that emodin significantly decreased the expression of CD155 in tumor cells and inhibited tumor cell proliferation and migration, and induced cell-cycle arrest at G2/M phase. The tumor inhibitory effects of emodin were lost with CD155 knockdown. Furthermore, emodin was used to treat mice bearing B16 melanoma. It was shown that emodin attenuated tumor growth accompanied by suppressing CD155 expression. Therefore, we propose that emodin could inhibit tumor growth, and the antineoplastic properties of emodin are at least partially CD155 dependent. Our study provides new insights into the mechanisms by which emodin inhibits tumor growth.


Assuntos
Antineoplásicos/farmacologia , Emodina/farmacologia , Melanoma Experimental/genética , Receptores Virais/genética , Neoplasias Cutâneas/genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Emodina/uso terapêutico , Feminino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos
6.
Int J Med Sci ; 16(4): 529-536, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31171904

RESUMO

Epithelial-mesenchymal transition (EMT), which involves the dramatic reorganization of the cytoskeleton, is a crucial initiating step in tumor invasion and metastasis. Protein 4.1B is a membrane-cytoskeleton cross-linker that plays an important role in tumor progression and metastasis; however, the functional roles of 4.1B in melanoma remain unclear. In this study, we aimed to investigate the effect and underlying mechanism of 4.1B on melanoma cells. Our results demonstrated that 4.1B expression was downregulated in murine B16 and B16-F10 melanoma cell lines. Ectopic 4.1B expression significantly inhibited the migration of melanoma cells and pulmonary metastasis. We further investigated the possible mechanism underlying the effect of 4.1B on EMT. The results showed that ectopic 4.1B expression altered the expression of representative EMT markers (E-cadherin, vimentin and N-cadherin), and inhibited the expression of three important transcription factors (Slug, Snail, and Twist) related to EMT in melanoma cells. Moreover, the expression of integrin α5, ß3 and matrix metalloproteinase 9 (MMP-9), which is known to regulate cell adhesion, migration and invasion, were suppressed. In conclusion, our data indicate that 4.1B is an important regulator during EMT progression in melanoma cells, which may present a potential target for the prevention and treatment of melanoma.


Assuntos
Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Metaloproteinase 9 da Matriz/genética , Melanoma Experimental/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica
7.
Int J Med Sci ; 16(4): 602-606, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31171912

RESUMO

Hyperpigmentation is a type of pigmentary disorder induced by overexpression of melanin content activated severe esthetic problems as melasma, freckle, ephelides, lentigo and other forms on human skin. Several whitening agents have restricted use because of their side effects or stability such as kojic acid, ascorbic acid and hydroquinone can act as cytotoxic substance which associated to dermatitis and skin cancer. To find for the safe substance, this study aimed to find for the ability of several components in Sucrier banana peel (SBP) extracts to inhibit melanogenesis process through p38 signaling pathway in B16F10 mouse melanoma cells. Tyrosinase activity and the cellular melanin content were dose dependent manner decreasing after SBP treatment. Furthermore, SBP decreased the expression of melanogenesis relate protein as microphthalmia-associated transcription factor (MITF) and tyrosinase protein after 24 hours incubation with α-melanocyte stimulating hormones (MSH) stimulating. The findings demonstrated that SBP contained an effective agent for hyperpigmentation inhibitor through p38 signaling pathways without any effect to ERK pathway, and subsequent down-regulate MITF expression and tyrosinase enzyme family production.


Assuntos
Hiperpigmentação/tratamento farmacológico , Melaninas/biossíntese , Melanoma Experimental/tratamento farmacológico , Musa/química , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melaninas/antagonistas & inibidores , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Monofenol Mono-Oxigenase/genética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , alfa-MSH/farmacologia
8.
Anticancer Res ; 39(5): 2307-2315, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092422

RESUMO

BACKGROUND: Several studies have highlighted hyperthermia's ability to enhance the effectiveness of radiation and chemotherapy in various in vitro and in vivo cancer models. MATERIALS AND METHODS: In vivo murine models of malignant melanoma and colon carcinoma were utilized for demonstrating hyperthermia's therapeutic effectiveness by examining levels of caspase 3, COX-2 and phospho-H2A.X (Ser139) as endpoints of apoptosis, proliferation and DNA damage respectively. RESULTS: Hyperthermia induced in vitro cytotoxicity in malignant melanoma (B16-F10) and colon carcinoma (CT26) cell lines. In addition, it reduced post-in vitro proliferation and suppression of tumor growth by inducing the expression of caspase-3 and phospho-H2A.X (Ser139) while reducing the expression of COX-2 in both murine cancer models. CONCLUSION: Hyperthermia can exert therapeutic effectiveness against melanoma and colon carcinoma by inhibiting a number of critical cellular cascades including apoptosis, proliferation and DNA damage.


Assuntos
Neoplasias do Colo/terapia , Hipertermia Induzida , Melanoma Experimental/terapia , Melanoma/terapia , Animais , Apoptose/efeitos da radiação , Carcinoma/patologia , Carcinoma/terapia , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Histonas/genética , Humanos , Melanoma/patologia , Melanoma Experimental/genética , Camundongos
9.
Nat Commun ; 10(1): 2157, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089138

RESUMO

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , MicroRNAs/metabolismo , Neoplasias Cutâneas/imunologia , Fatores de Transcrição/metabolismo , Transferência Adotiva/métodos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Senescência Celular/genética , Senescência Celular/imunologia , Epigênese Genética/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Complexo Repressor Polycomb 2/imunologia , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
10.
Cancer Immunol Immunother ; 68(7): 1121-1132, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31134297

RESUMO

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.


Assuntos
Antígenos de Neoplasias/imunologia , Imunofenotipagem , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Animais , Linhagem Celular Tumoral/transplante , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Análise Serial de Tecidos , beta Catenina/genética
11.
Science ; 364(6439): 485-491, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048490

RESUMO

Tumors with mismatch repair deficiency (MMR-d) are characterized by sequence alterations in microsatellites and can accumulate thousands of mutations. This high mutational burden renders tumors immunogenic and sensitive to programmed cell death-1 (PD-1) immune checkpoint inhibitors. Yet, despite their tumor immunogenicity, patients with MMR-deficient tumors experience highly variable responses, and roughly half are refractory to treatment. We present experimental and clinical evidence showing that the degree of microsatellite instability (MSI) and resultant mutational load, in part, underlies the variable response to PD-1 blockade immunotherapy in MMR-d human and mouse tumors. The extent of response is particularly associated with the accumulation of insertion-deletion (indel) mutational load. This study provides a rationale for the genome-wide characterization of MSI intensity and mutational load to better profile responses to anti-PD-1 immunotherapy across MMR-deficient human cancers.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Imunoterapia/métodos , Instabilidade de Microssatélites , Neoplasias/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos/uso terapêutico , Variação Genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Proteína 2 Homóloga a MutS/genética , Mutação , Resultado do Tratamento
12.
Oncol Rep ; 42(1): 414-424, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115580

RESUMO

MicroRNA­21 (miR­21) is a potential therapeutic target for melanoma. Whether miR­21 inhibitor affects the anti­cancer activity of doxorubicin assisted by c(RGDyK)­modified liposome (DLN) in melanoma and the underlying mechanisms are largely unknown. In this study, in vitro and animal models were used to explore the effect of DLN combined with miR­21 inhibitor on melanoma cells. The data demonstrated that treatment with 5 µl DLN (final concentration of doxorubicin 5 mg/ml) for 72 h effectively inhibited melanoma cell growth (~75% inhibition). The experiments were then divided into five groups: Control group, vector group, DLN group, miR­21 inhibitor group and miR­21 inhibitor + DLN group. Compared with the control group, DLN (5 µl) or miR­21 inhibitor significantly reduced migration and invasion of melanoma cells, promoted apoptosis and arrested cells at the G1 phase. Notably, the combined application of DLN with miR­21 inhibitor further promoted the anti­cancer effects (reducing migration and invasion of melanoma cells, promoting apoptosis and arresting cells at G1 phase) compared with individual application of DLN or miR­21 inhibitor. Mechanically, DLN did not function by reducing miR­21 expression, whereas DLN and miR­21 inhibitor downregulated B­cell lymphoma-2 (BCL­2) expression, and facilitated BCL­2­associated X protein (Bax) and P53 expression in melanoma cells. DLN and miR­21 inhibitor together displayed stronger effects on Bcl­2, Bax and P53 expression that each alone. In vivo data further demonstrated that DLN inhibited tumor growth further than a similar dose of doxorubicin only. Furthermore, miR­21 inhibitor and DLN exerted the optimal anti­cancer effect compared with single application of DLN or miR­21 inhibitor. Together, the findings demonstrated miR­21 inhibitor facilitated the anti­cancer activity of DLN in melanoma, and the mechanisms involved Bcl­2, Bax and P53 expression.


Assuntos
Doxorrubicina/administração & dosagem , Melanoma Experimental/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipossomos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , MicroRNAs/genética , Nanopartículas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nano Lett ; 19(5): 2774-2783, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-30943039

RESUMO

While tumor-infiltrating cytotoxic T lymphocytes play a critical role in controlling tumor development, they are generally impotent in an acidic tumor microenvironment. Systemic treatment to neutralize tumor acidity thus holds promise for the reversal of the anergic state of T cells and the improvement of T cell-associated immunotherapy. Herein, we report a proof-of-concept of RNAi nanoparticle-mediated therapeutic reversion of tumor acidity to restore the antitumor functions of T cells and potentiate the checkpoint blockade therapy. Our strategy utilized an in vivo optimized vesicular cationic lipid-assisted nanoparticle, as opposed to its micellar counterpart, to mediate systematic knockdown of lactate dehydrogenase A (LDHA) in tumor cells. The treatment resulted in the reprogramming of pyruvate metabolism, a reduction of the production of lactate, and the neutralization of the tumor pH. In immunocompetent syngeneic melanoma and breast tumor models, neutralization of tumor acidity increased infiltration with CD8+ T and NK cells, decreased the number of immunosuppressive T cells, and thus significantly inhibited the growth of tumors. Furthermore, the restoration of tumoral pH potentiated checkpoint inhibition therapy using the antibody of programmed cell death protein 1 (PD-1). However, in immunodeficient B6/ Rag1 -/- and NOG mice, the same treatment failed to control tumor growth, further proving that the attenuation of tumor growth by tumor acidity modulation was attributable to the activation of tumor-infiltrating immune cells.


Assuntos
Imunoterapia , Melanoma Experimental/tratamento farmacológico , Nanopartículas/administração & dosagem , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Ácidos/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Nanopartículas/química , Microambiente Tumoral/efeitos dos fármacos
14.
Pharmazie ; 74(3): 163-167, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30961683

RESUMO

The natural phytoalexin resveratrol (RES) has exhibited excellent anti-tumor effects on a variety of tumors including malignant melanoma. However, its specific mechanism of anti-melanoma needs to be further explored. It has been reported, that the expression of tumor suppressor gene RUNX3 was lost or substantially decreased in melanoma. Whether RES exerts its anti-tumor effect by regulating the expression of RUNX3 gene in melanoma is worthy of study. In the present study, we found the RUNX3 promoter is hypermethylated and the expression of RUNX3 mRNA and protein are absent in melanoma cells B16F10. After intervention with RES, promoter hypermethylation of RUNX3 in B16F10 cells could be significantly decreased and mRNA and protein expression of it was upregulated in a dose-dependent manner. We further investigated the effects of RES on B16F10 xenograft models. The intervention of RES and treatment of melanoma positive drug dacarbazine (DTIC) both could significantly inhibit tumor growth in xenograft mice, but only RES could upregulate the expression of RUNX3 mRNA and protein in peripheral blood and tumor tissues. Therefore, the upregulation of mRNA and protein expression of RUNX3 resulting from promoter demethylation might be one of the mechanisms of RES inhibiting melanoma. This research has revealed a novel mechanism for RES against melanoma from the epigenetic perspective, which is helpful to improve the understanding of the anti-tumor mechanism of RES and provide new insights for the treatment of melanoma.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/genética , Desmetilação do DNA/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Resveratrol/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Feminino , Crescimento/efeitos dos fármacos , Xenoenxertos , Masculino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
J Immunol ; 202(10): 2843-2848, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30971442

RESUMO

Malignancy increases sepsis incidence 10-fold and elevates sepsis-associated mortality. Advances in treatment have improved survival of cancer patients shortly after sepsis, but there is a paucity of information on how sepsis impacts cancer growth, development, and prognosis. To test this, cecal ligation and puncture surgery was performed on B16 melanoma-bearing mice to show that sepsis has detrimental effects in hosts with advanced tumors, leading to increased mortality. Surprisingly, mice experiencing cecal ligation and puncture-induced sepsis earlier during tumor development exhibited CD8 T cell-dependent attenuation of tumor growth. Sepsis-resistant CD8 tumor-infiltrating T cells showed increased in vivo activation, effector IFN-γ cytokine production, proliferation, and expression of activation/inhibitory PD-1/LAG-3 receptors because of a sepsis-induced liberation of tumor Ags. Sepsis-reinvigorated CD8 tumor-infiltrating T cells were also amenable to (anti-PD-L1/LAG-3) checkpoint blockade therapy, further prolonging cancer-associated survival in sepsis survivors. Thus, sepsis has the capacity to improve tumor-specific CD8 T cell responses, leading to better cancer prognosis and increased survival.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Sepse/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/patologia , Feminino , Interferon gama/genética , Interferon gama/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Sepse/genética , Sepse/microbiologia , Sepse/patologia
16.
Cancer Sci ; 110(6): 1974-1986, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012976

RESUMO

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.


Assuntos
Movimento Celular/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Peptídeos/farmacologia , beta 2-Glicoproteína I/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Homologia de Sequência de Aminoácidos , beta 2-Glicoproteína I/genética , beta 2-Glicoproteína I/metabolismo
17.
ACS Appl Mater Interfaces ; 11(14): 13058-13068, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30888149

RESUMO

The combination of chemotherapeutic agents with immune stimulating agents for treating degenerative diseases, called chemoimmunotherapy, has emerged as a promising cancer treatment modality. Despite the tremendous potential, chemoimmunotherapy by the combination of drugs and immune stimulators often suffers because of the lack of controlled delivery nanostructures in the microenvironment. To this end, we show that by using pH-responsive smart nanocubes (NCs), cancer cells and tumor-associated immune cells can be precisely targeted with a chemotherapeutic agent (doxorubicin, DOX) and immune stimulating agent (plasmid ovalbumin, pOVA) for enhanced chemoimmunotherapy. The pH-responsive smart NCs protect payloads from nuclease degradation and avoid renal clearance and undergo supersensitive structural change at the extracellular tumor regions that mediate efficient release. Concurrent release of pOVA vaccines encoding tumor-specific antigen laden with polyplexes were loaded on tumor-associated immune cells and produce antigen-specific humoral immune response, whereas DOX enables effective infiltration into the cancer cells and is involved in the eradication of tumor tissues. The amount of anti-OVA IgG1 antibody produced by the intravenous administration of NC formulation was similar to that of free OVA formulation. Importantly, the combined delivery of pDNA and DOX using NCs showed significantly enhanced antitumor efficacy in B16/OVA melanoma tumor xenografts, which remarkably outperforms the monotherapy counterparts. These results suggest that pH-responsive smart NCs laden with pDNA and DOX provide a promising nanostructure for chemoimmunotherapy that simultaneously involves cancer cell killing and stimulates antigen-specific immune response to prevent cancer recurrence.


Assuntos
Sistemas de Liberação de Medicamentos , Melanoma Experimental/terapia , Nanopartículas/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Imunoterapia/métodos , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Nanopartículas/química , Ovalbumina/administração & dosagem , Ovalbumina/química , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nature ; 567(7749): 530-534, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814732

RESUMO

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3-5, in part because they enter a hyporesponsive ('exhausted' or 'dysfunctional') state6-9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19+ tumour-bearing mice. CD8+CAR+ tumour-infiltrating lymphocytes and CD8+ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10-12. CD8+ T cells from humans with cancer or chronic viral infections13-15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias/genética , Neoplasias/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Fatores de Transcrição/metabolismo , Transferência Adotiva , Animais , Antígenos CD19/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Esteroides/deficiência , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/metabolismo , Taxa de Sobrevida , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/deficiência
19.
Int J Cancer ; 145(7): 1946-1957, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30873585

RESUMO

Cancer-associated fibroblasts (CAFs) play a key role in orchestrating the tumor malignant biological properties within tumor microenvironment and evidences demonstrate that CAFs are a critical regulator of tumoral immunosuppression of the T cell response. However, the functions and regulation of CAFs in the expression of programmed death-ligand 1 (PD-L1) in melanoma and colorectal carcinoma (CRC) are not completely understood. Herein, by scrutinizing the expression of α-SMA and PD-L1 in melanoma and CRC tissues, we found that CAFs was positive correlated with PD-L1 expression. Further analyses showed that CAFs promoted PD-L1 expression in mice tumor cells. By detecting a majority of cytokines expression in normal mice fibroblasts and CAFs, we determined that CXCL5 was abnormal high expression in CAFs and the immunohistochemistry and in situ hybridization confirmed that were CAFs which were expressing CXCL5. In addition, CXCL5 promoted PD-L1 expression in B16, CT26, A375 and HCT116. The silencing of CXCR2, the receptor of CXCL5, inhibited the PD-L1 expression induced by CAFs in turn. Functionally, CXCL5 derived by CAFs promoted PD-L1 expression in mice tumor cells through activating PI3K/AKT signaling. LY294002, the inhibitor of PI3K, confirmed that CXCL5 forested an immunosuppression microenvironment by promoting PD-L1 expression via PI3K/AKT signaling. Meanwhile, the B16/CT26 xenograft tumor models were used and both CXCR2 and p-AKT were found to be positively correlated with PD-L1 in the xenograft tumor tissues. The immunosuppressive action of CAFs on tumor cells is probably reflective of them being a potential therapeutic biomarker for melanoma and CRC.


Assuntos
Antígeno B7-H1/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Quimiocina CXCL5/metabolismo , Neoplasias Colorretais/metabolismo , Melanoma Experimental/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma Experimental/genética , Camundongos , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Regulação para Cima
20.
Nat Commun ; 10(1): 818, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778069

RESUMO

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.


Assuntos
Regulação da Expressão Gênica/genética , Técnicas Genéticas , MicroRNAs/genética , Elementos de Resposta , Regiões 3' não Traduzidas , Animais , Antígeno B7-H1/genética , Sistemas CRISPR-Cas , Genes BRCA1 , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Ovalbumina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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