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1.
Polymers (Basel) ; 16(2)2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38257023

RESUMO

Acute myeloid leukemia carrying FMS-like tyrosine kinase receptor-3 (FLT3) mutations is a fatal blood cancer with a poor prognosis. Although the FLT3 inhibitor gilteritinib has recently been approved, it still suffers from limited efficacy and relatively high nonresponse rates. In this study, we report the potentiation of gilteritinib efficacy using nanocomplexation with a hyaluronic acid-epigallocatechin gallate conjugate. The self-assembly, colloidal stability, and gilteritinib loading capacity of the nanocomplex were characterized by reversed-phase high-performance liquid chromatography and dynamic light scattering technique. Flow cytometric analysis revealed that the nanocomplex efficiently internalized into FLT3-mutated leukemic cells via specific interactions between the surface-exposed hyaluronic acid and CD44 receptor overexpressed on the cells. Moreover, this nanocomplex was found to induce an eradication of the leukemic cells in a synergistic manner by elevating the levels of reactive oxygen species and caspase-3/7 activities more effectively than free gilteritinib. This study may provide a useful strategy to design nanomedicines capable of augmenting the therapeutic efficacy of FLT3 inhibitors for effective leukemia therapy.

2.
Nat Commun ; 14(1): 563, 2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36732506

RESUMO

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Linfócitos T , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Modelos Animais de Doenças
3.
J Nanobiotechnology ; 20(1): 481, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36384529

RESUMO

BACKGROUND: Currently available anti-leukemia drugs have shown limited success in the treatment of acute myeloid leukemia (AML) due to their poor access to bone marrow niche supporting leukemic cell proliferation. RESULTS: Herein, we report a bone marrow-targetable green tea catechin-based micellar nanocomplex for synergistic AML therapy. The nanocomplex was found to synergistically amplify the anti-leukemic potency of sorafenib via selective disruption of pro-survival mTOR signaling. In vivo biodistribution study demonstrated about 11-fold greater bone marrow accumulation of the nanocomplex compared to free sorafenib. In AML patient-derived xenograft (AML-PDX) mouse model, administration of the nanocomplex effectively eradicated bone marrow-residing leukemic blasts and improved survival rates without noticeable off-target toxicity. CONCLUSION: This study may provide insights into the rational design of nanomedicine platforms enabling bone marrow-targeted delivery of therapeutic agents for the treatment of AML and other bone marrow diseases.


Assuntos
Catequina , Leucemia Mieloide Aguda , Camundongos , Animais , Humanos , Medula Óssea , Catequina/farmacologia , Micelas , Sorafenibe , Distribuição Tecidual , Leucemia Mieloide Aguda/tratamento farmacológico , Modelos Animais de Doenças , Chá
4.
NPJ Vaccines ; 7(1): 121, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271019

RESUMO

Hepatitis B Virus (HBV) is a hepadnavirus that is the principal pathogen underlying viral liver disease in human populations. In this study, we describe the isolation and characterization of a fully human monoclonal antibody for HBV. This HuMab was isolated by a combinatorial screen of the memory B-cell repertoire from an acute/recovered HBV-infected patient. Lead candidate selection was based upon strong binding and neutralizing activity for live HBV. We provide a detailed biochemical/biophysical, and subclass characterization of its specificity and affinity against all of the principal HBV genotypes combined with a functional analysis of its in vitro activity. We also demonstrate its potential as a prophylaxis/therapy in vivo using human liver chimeric mouse models for HBV infection. These data have important implications for our understanding of natural human immunity to HBV and suggest that this potentially represents a new antibody-based anti-viral candidate for prophylaxis and/or therapy for HBV infection.

5.
Int J Mol Sci ; 24(1)2022 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-36613821

RESUMO

Chemoresistance is one of the major challenges for the treatment of acute myeloid leukemia. Epigallocatechin gallate (EGCG), a bioactive polyphenol from green tea, has attracted immense interest as a potential chemosensitizer, but its application is limited due to the need for effective formulations capable of co-delivering EGCG and anti-leukemic drugs. Herein, we describe the formation and characterization of a micellar nanocomplex self-assembled from EGCG and daunorubicin, an anthracycline drug for the first-line treatment of acute myeloid leukemia. This nanocomplex was highly stable at pH 7.4 but stimulated to release the incorporated daunorubicin at pH 5.5, mimicking an acidic endosomal environment. More importantly, the nanocomplex exhibited superior cytotoxic efficacy against multidrug-resistant human leukemia cells over free daunorubicin by achieving a strong synergism, as supported by median-effect plot analysis. The observed chemosensitizing effect was in association with enhanced nucleus accumulation of daunorubicin, elevation of intracellular reactive oxygen species and caspase-mediated apoptosis induction. Our study presents a promising strategy for circumventing chemoresistance for more effective leukemia therapy.


Assuntos
Catequina , Leucemia Mieloide Aguda , Humanos , Daunorrubicina/farmacologia , Apoptose , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/tratamento farmacológico , Catequina/farmacologia , Chá/química
6.
Front Immunol ; 12: 638447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679796

RESUMO

Viral hepatitis particularly Hepatitis B Virus (HBV) is still an ongoing health issue worldwide. Despite the vast technological advancements in research and development, only HBV vaccines, typically given during early years, are currently available as a preventive measure against acquiring the disease from a secondary source. In general, HBV can be cleared naturally by the human immune system if detected at low levels early. However, long term circulation of HBV in the peripheral blood may be detrimental to the human liver, specifically targeting human hepatocytes for cccDNA integration which inevitably supports HBV life cycle for the purpose of reinfection in healthy cells. Although there is some success in using nucleoside analogs or polyclonal antibodies targeting HBV surface antigens (HBsAg) in patients with acute or chronic HBV+ (CHB), majority of them would either respond only partially or succumb to the disease entirely unless they undergo liver transplants from a fully matched healthy donor and even so may not necessarily guarantee a 100% chance of survival. Indeed, in vitro/ex vivo cultures and various transgenic animal models have already provided us with a good understanding of HBV but they primarily lack human specificity or virus-host interactions in the presence of human immune surveillance. Therefore, the demand of utilizing humanized mice has increased over the last decade as a pre-clinical platform for investigating human-specific immune responses against HBV as well as identifying potential immunotherapeutic strategies in eradicating the virus. Basically, this review covers some of the recent developments and key advantages of humanized mouse models over other conventional transgenic mice platforms.


Assuntos
Modelos Animais de Doenças , Vírus da Hepatite B , Hepatite B , Animais , Humanos , Camundongos
7.
Hepatology ; 73(2): 486-502, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32333814

RESUMO

BACKGROUND AND AIMS: Interferon (IFN)-α, composed of numerous subtypes, plays a crucial role in immune defense. As the most studied subtype, IFN-α2 has been used for treating chronic hepatitis B virus (HBV) infection, with advantages of finite treatment duration and sustained virologic response, but its efficacy remains relatively low. This study aimed to screen for IFN-α subtypes with the highest anti-HBV potency and to characterize mechanisms of IFN-α-mediated HBV restriction. APPROACH AND RESULTS: Using cell culture-based HBV infection systems and a human-liver chimeric mouse model, IFN-α subtype-mediated antiviral response and signaling activation were comprehensively analyzed. IFN-α14 was identified as the most effective subtype in suppression of HBV covalently closed circular DNA transcription and HBV e antigen/HBV surface antigen production, with median inhibitory concentration values approximately 100-fold lower than those of the conventional IFN-α2. IFN-α14 alone elicited IFN-α and IFN-γ signaling crosstalk in a manner similar to the combined use of IFN-α2 and IFN-γ, inducing multiple potent antiviral effectors, which synergistically restricted HBV replication. Guanylate binding protein 5, one of the most differentially expressed genes between IFN-α14-treated and IFN-α2-treated liver cells, was identified as an HBV restriction factor. A strong IFN-α-IFN-α receptor subunit 1 interaction determines the anti-HBV activity of IFN-α. The in vivo anti-HBV activity of IFN-α14 and treatment-related transcriptional patterns were further confirmed, and few adverse effects were observed. CONCLUSIONS: A concerted IFN-α and IFN-γ response in liver, which could be efficiently elicited by IFN-α subtype 14, is associated with potent HBV suppression. These data deepen the understanding of the divergent activities of IFN-α subtypes and the mechanism underlying the synergism between IFN-α and IFN-γ signaling, with implications for improved IFN therapy and HBV curative strategies.


Assuntos
Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/farmacologia , Interferon gama/metabolismo , Animais , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/transplante , Humanos , Interferon-alfa/genética , Interferon-alfa/uso terapêutico , Camundongos , Camundongos Knockout , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resposta Viral Sustentada , Quimeras de Transplante , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
8.
Front Immunol ; 11: 553362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193321

RESUMO

Advancements in science enable researchers to constantly innovate and create novel biologics. However, the use of non-human animal models during the development of biologics impedes identification of precise in vivo interactions between the human immune system and treatments. Due to lack of this understanding, adverse effects are frequently observed in healthy volunteers and patients exposed to potential biologics during clinical trials. In this study, we evaluated and compared the effects of known immunotoxic biologics, Proleukin®/IL-2 and OKT3 in humanized mice (reconstituted with human fetal cells) to published clinical outcomes. We demonstrated that humanized mice were able to recapitulate in vivo pathological changes and human-specific immune responses, such as elevated cytokine levels and modulated lymphocytes and myeloid subsets. Given the high similarities of immunological side effects observed between humanized mice and clinical studies, this model could be used to assess immunotoxicity of biologics at a pre-clinical stage, without placing research participants and/or patients at risk.


Assuntos
Interleucina-2/análogos & derivados , Modelos Imunológicos , Animais , Avaliação de Medicamentos , Feto , Humanos , Interleucina-2/efeitos adversos , Interleucina-2/imunologia , Interleucina-2/farmacologia , Camundongos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia
9.
Front Immunol ; 11: 580968, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013934

RESUMO

Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4+ T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4+ central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4+ T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4+ memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Cirrose Hepática Experimental/etiologia , Cirrose Hepática Experimental/imunologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Animais , Linfócitos T CD4-Positivos/patologia , Citocinas/sangue , Dieta Hiperlipídica/efeitos adversos , Feminino , Células-Tronco Fetais/transplante , Hepatócitos/transplante , Xenoenxertos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Cirrose Hepática Experimental/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Hepatopatia Gordurosa não Alcoólica/patologia
10.
JHEP Rep ; 2(1): 100062, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32039403

RESUMO

BACKGROUND & AIMS: Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206+ macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease. METHODS: Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206+ monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice. RESULTS: Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206+ macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206+ macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV+ patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206+ macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206+ macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice. CONCLUSIONS: While the direct involvement of CD206+ macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease. LAY SUMMARY: Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes.

11.
Cell Death Dis ; 10(3): 225, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837450

RESUMO

Since publication of this paper, the authors have noticed that there were errors in Fig. 2A (the GAPDH of Mel-CV, Mel-CV.S, Mel-RMu and Mel-RMu.S), Fig. 2C (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3F (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3J(the GAPDH of Mel-RMu.S), Fig. 5C (the ERK1/2 of patient#3(post)), and Fig. 5F (the RIP1 of Mel-CV.S and Mel-RMu.S, the GAPDH of Mel-CV and Mel-RMu). As a result of the misfiling of the data during preparation of figures, incorrect images were inadvertently inserted in these figures. The correct figures are given below. The corrections do not alter the conclusions of the paper.

12.
Viruses ; 10(11)2018 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-30453598

RESUMO

The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viral⁻host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism.


Assuntos
Modelos Animais de Doenças , Camundongos SCID , Viroses/patologia , Viroses/virologia , Animais , Humanos , Camundongos
13.
PLoS Pathog ; 14(10): e1007298, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30286211

RESUMO

Natural killer (NK) cells provide the first line of defense against malaria parasite infection. However, the molecular mechanisms through which NK cells are activated by parasites are largely unknown, so is the molecular basis underlying the variation in NK cell responses to malaria infection in the human population. Here, we compared transcriptional profiles of responding and non-responding NK cells following exposure to Plasmodium-infected red blood cells (iRBCs) and identified MDA5, a RIG-I-like receptor involved in sensing cytosolic RNAs, to be differentially expressed. Knockout of MDA5 in responding human NK cells by CRISPR/cas9 abolished NK cell activation, IFN-γ secretion, lysis of iRBCs. Similarly, inhibition of TBK1/IKKε, an effector molecule downstream of MDA5, also inhibited activation of responding NK cells. Conversely, activation of MDA5 by liposome-packaged poly I:C restored non-responding NK cells to lyse iRBCs. We further show that microvesicles containing large parasite RNAs from iRBCs activated NK cells by fusing with NK cells. These findings suggest that NK cells are activated through the MDA5 pathway by parasite RNAs that are delivered to the cytoplasm of NK cells by microvesicles from iRBCs. The difference in MDA5 expression between responding and non-responding NK cells following exposure to iRBCs likely contributes to the variation in NK cell responses to malaria infection in the human population.


Assuntos
Micropartículas Derivadas de Células/imunologia , Eritrócitos/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Células Matadoras Naturais/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Sistemas CRISPR-Cas , Células Cultivadas , Citoplasma/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Helicase IFIH1 Induzida por Interferon/antagonistas & inibidores , Helicase IFIH1 Induzida por Interferon/genética , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/parasitologia , Ativação Linfocitária , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/isolamento & purificação
14.
Cell Death Dis ; 9(6): 679, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880840

RESUMO

Many recent studies have uncovered the necessary role for the receptor-interacting protein kinase 1 (RIP1) in regulating apoptosis and necrosis that cells undergo in response to various cellular stresses. However, the functional significance of RIP1 in promoting cancer cells survival remains poorly understood. Here, we report that RIP1 was upregulated and contributed to both intrinsic and acquired resistance of melanoma cells to BRAF/MEK inhibitors through activation of NF-κB. Strikingly, Snail1-mediated suppression of CYLD played a crucial role in promoting RIP1 expression upon ERK activation, particularly, in melanoma cells with acquired resistance to BRAF inhibitors. In addition, RIP1 kinase activity was not required for melanoma cells to survive BRAF/MEK inhibition as RIP1 mediated NF-κB activation through its intermediate domain. Collectively, our findings reveal that targeting RIP1 in combination with BRAF/MEK inhibitors is a potential approach in the treatment of the disease.


Assuntos
Apoptose/efeitos dos fármacos , Citoproteção , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Enzima Desubiquitinante CYLD/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Indóis/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacos
15.
J Am Chem Soc ; 140(12): 4244-4252, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29504396

RESUMO

Drug resistance to chemotherapeutics is a recurrent issue plaguing many cancer treatment regimens. To circumvent resistance issues, we have designed a new class of macromolecules as self-contained chemotherapeutic agents. The macromolecular chemotherapeutic agents readily self-assemble into well-defined nanoparticles and show excellent activity in vitro against multiple cancer cell lines. These cationic polymers function by selectively binding and lysing cancer cell membranes. As a consequence of this mechanism, they exhibit significant potency against drug-resistant cancer cells and cancer stem cells, prevent cancer cell migration, and do not induce resistance onset following multiple treatment passages. Concurrent experiments with the small-molecule chemotherapeutic, doxorubicin, show aggressive resistance onset in cancer cells, a lack of efficacy against drug-resistant cancer cell lines, and a failure to prevent cancer cell migration. Additionally, the polymers showed anticancer efficacy in a hepatocellular carcinoma patient derived xenograft mouse model. Overall, these results demonstrate a new approach to designing anticancer therapeutics utilizing macromolecular compounds.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Hepáticas Experimentais/patologia , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/farmacologia , Camundongos , Estrutura Molecular , Nanopartículas/química , Tamanho da Partícula , Relação Estrutura-Atividade
16.
Autophagy ; 11(7): 975-94, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26018731

RESUMO

Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.


Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Melanoma/enzimologia , Melanoma/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Autofagia/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Proteína Beclina-1 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico , Humanos , Melanoma/genética , Proteínas de Membrana , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fatores de Transcrição de Fator Regulador X , Tapsigargina/farmacologia , Fatores de Transcrição/metabolismo , Tunicamicina/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-Box
17.
Cancer Res ; 75(8): 1736-48, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25724678

RESUMO

Although many studies have uncovered an important role for the receptor-binding protein kinase RIP1 in controlling cell death signaling, its possible contributions to cancer pathogenesis have been little explored. Here, we report that RIP1 functions as an oncogenic driver in human melanoma. Although RIP1 was commonly upregulated in melanoma, RIP1 silencing inhibited melanoma cell proliferation in vitro and retarded the growth of melanoma xenografts in vivo. Conversely, while inducing apoptosis in a small proportion of melanoma cells, RIP1 overexpression enhanced proliferation in the remaining cells. Mechanistic investigations revealed that the proliferative effects of RIP1 overexpression were mediated by NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced the proliferation of primary melanocytes, triggering their anchorage-independent cell growth in an NF-κB-dependent manner. We identified DNA copy-number gain and constitutive ubiquitination by a TNFα autocrine loop mechanism as two mechanisms of RIP1 upregulation in human melanomas. Collectively, our findings define RIP1 as an oncogenic driver in melanoma, with potential implications for targeting its NF-κB-dependent activation mechanism as a novel approach to treat this disease.


Assuntos
Transformação Celular Neoplásica/genética , Melanoma/genética , Oncogenes , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Neoplasias Cutâneas/genética , Animais , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Oncotarget ; 5(22): 11237-51, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25365078

RESUMO

Reduction in the expression of the anti-survival BH3-only proteins PUMA and Bim is associated with the pathogenesis of melanoma. However, we have found that the expression of the other BH3-only protein Noxa is commonly upregulated in melanoma cells, and that this is driven by oncogenic activation of MEK/ERK. Immunohistochemistry studies showed that Noxa was expressed at higher levels in melanomas than nevi. Moreover, the expression of Noxa was increased in metastatic compared to primary melanomas, and in thick primaries compared to thin primaries. Inhibition of oncogenic BRAFV600E or MEK downregulated Noxa, whereas activation of MEK/ERK caused its upregulation. In addition, introduction of BRAFV600E increased Noxa expression in melanocytes. Upregulation of Noxa was due to a transcriptional increase mediated by cAMP responsive element binding protein, activation of which was also increased by MEK/ERK signaling in melanoma cells. Significantly, Noxa appeared necessary for constitutive activation of autophagy, albeit at low levels, by MEK/ERK in melanoma cells. Furthermore, it was required for autophagy activation that delayed apoptosis in melanoma cells undergoing nutrient deprivation. These results reveal that oncogenic activation of MEK/ERK drives Noxa expression to promote autophagy, and suggest that Noxa has an indirect anti-apoptosis role in melanoma cells under nutrient starvation conditions.


Assuntos
Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Autofagia/fisiologia , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Melanócitos/enzimologia , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Regulação para Cima
19.
J Invest Dermatol ; 134(2): 488-497, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23921951

RESUMO

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF(V600E) or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF(V600E) into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma.


Assuntos
Adaptação Fisiológica/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição de Fator Regulador X , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box
20.
Carcinogenesis ; 34(2): 237-47, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23087082

RESUMO

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Melanoma/patologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase II como Assunto , Sinergismo Farmacológico , Humanos , Imunoprecipitação , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mutação/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas
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