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Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non-small cell lung cancers. We use a nonablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, antitumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, nonablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells and results in a subpopulation of higher-intensity CAR-expressing T cells with high coexpression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell antitumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer-two thoracic cancers for which radiotherapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of "sandwich" cell therapy for solid tumors that combines sequential metastatic site-targeted radiation and CAR T cells-a regional solution to overcome barriers to systemic delivery of CAR T cells.
Assuntos
Mesotelioma Maligno , Mesotelioma , Humanos , Mesotelina , Imunoterapia Adotiva/métodos , Proteínas Ligadas por GPI , Receptores de Antígenos de Linfócitos T , Mesotelioma/radioterapia , Mesotelioma Maligno/tratamento farmacológico , Receptores de Quimiocinas , Quimiocinas , Linhagem Celular TumoralRESUMO
The limited efficacy of chimeric antigen receptor (CAR) T cell therapy for solid tumors necessitates engineering strategies that promote functional persistence in an immunosuppressive environment. Herein, we use c-Kit signaling, a physiological pathway associated with stemness in hematopoietic progenitor cells (T cells lose expression of c-Kit during differentiation). CAR T cells with intracellular expression, but no cell-surface receptor expression, of the c-Kit D816V mutation (KITv) have upregulated STAT phosphorylation, antigen activation-dependent proliferation and CD28- and interleukin-2-independent and interferon-γ-mediated co-stimulation, augmenting the cytotoxicity of first-generation CAR T cells. This translates to enhanced survival, including in transforming growth factor-ß-rich and low-antigen-expressing solid tumor models. KITv CAR T cells have equivalent or better in vivo efficacy than second-generation CAR T cells and are susceptible to tyrosine kinase inhibitors (safety switch). When combined with CD28 co-stimulation, KITv co-stimulation functions as a third signal, enhancing efficacy and providing a potent approach to treat solid tumors.
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Interleucina-2 , Proteínas Proto-Oncogênicas c-kit , Linfócitos T , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva , Interleucina-2/farmacologia , Interleucina-2/metabolismo , Receptores Proteína Tirosina Quinases , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
INTRODUCTION: In solid tumor immunotherapy, less than 20% of patients respond to anti-programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) agents. The role of transforming growth factor ß (TGFß) in diverse immunity is well-established; however, systemic blockade of TGFß is associated with toxicity. Accumulating evidence suggests the role of crosstalk between TGFß and PD-1/PD-L1 pathways. AREAS COVERED: We focus on TGFß and PD-1/PD-L1 signaling pathway crosstalk and the determinant role of TGFß in the resistance of immune checkpoint blockade. We provide the rationale for combination anti-TGFß and anti-PD-1/PD-L1 therapies for solid tumors and discuss the current status of dual blockade therapy in preclinical and clinical studies. EXPERT OPINION: The heterogeneity of tumor microenvironment across solid tumors complicates patient selection, treatment regimens, and response and toxicity assessment for investigation of dual blockade agents. However, clinical knowledge from single-agent studies provides infrastructure to translate dual blockade therapies. Dual TGFß and PD-1/PD-L1 blockade results in enhanced T-cell infiltration into tumors, a primary requisite for successful immunotherapy. A bifunctional fusion protein specifically targets TGFß in the tumor microenvironment, avoiding systemic toxicity, and prevents interaction of PD-1+ cytotoxic cells with PD-L1+ tumor cells.
Assuntos
Neoplasias , Fator de Crescimento Transformador beta , Humanos , Fator de Crescimento Transformador beta/metabolismo , Antígeno B7-H1 , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Transdução de Sinais , Microambiente TumoralRESUMO
Mesothelioma is an aggressive cancer of the serous membranes with poor prognosis despite combination therapy consisting of surgery, radiotherapy, and platinum-based chemotherapy. Targeted therapies, including immunotherapies, have reported limited success, suggesting the need for additional therapeutic targets. This study investigates a potential new therapeutic target, gC1qR/HABP1/p32 (gC1qR), which is overexpressed in all morphologic subtypes of mesothelioma. gC1qR is a complement receptor that is associated with several cellular functions, including cell proliferation and angiogenesis. In vitro and in vivo experiments were conducted to test the hypothesis that targeting gC1qR with a specific gC1qR monoclonal antibody 60.11 reduces mesothelioma tumor growth, using the biphasic mesothelioma cell line MSTO-211H (MSTO). In vitro studies demonstrate cell surface and extracellular gC1qR expression by MSTO cells, and a modest 25.3 ± 1.8% (n = 4) reduction in cell proliferation by the gC1qR blocking 60.11 antibody. This inhibition was specific for targeting the C1q binding domain of gC1qR at aa 76-93, as a separate monoclonal antibody 74.5.2, directed against amino acids 204-218, had no discernable effect. In vivo studies, using a murine orthotopic xenotransplant model, demonstrated an even greater reduction in MSTO tumor growth (50% inhibition) in mice treated with the 60.11 antibody compared to controls. Immunohistochemical studies of resected tumors revealed increased cellular apoptosis by caspase 3 and TUNEL staining, in 60.11 treated tumors compared to controls, as well as impaired angiogenesis by decreased CD31 staining. Taken together, these data identify gC1qR as a potential new therapeutic target against mesothelioma with both antiproliferative and antiangiogenic properties.
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OBJECTIVE: Aggregation and modification of LDLs (low-density lipoproteins) promote their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of agLDL (aggregated LDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse. Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites, such as cholesterol, promotes foam cell formation, a process that drives atherogenesis. Furthermore, there is accumulating evidence for the involvement of TLR4 (Toll-like receptor 4) and its adaptor protein MyD88 (myeloid differentiation primary response 88) in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the lysosomal synapse and foam cell formation. Approach and Results: Using bone marrow-derived macrophages from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment, and foam cell formation. Using siRNA (small interfering RNA), pharmacological inhibition and knockout bone marrow-derived macrophages, we implicate SYK (spleen tyrosine kinase), PI3K (phosphoinositide 3-kinase), and Akt in agLDL catabolism using the lysosomal synapse. Using bone marrow transplantation of LDL receptor knockout mice with TLR4 knockout bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL. CONCLUSIONS: We present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the lysosomal synapse, catabolism of agLDL, and lipid accumulation in vitro and in vivo.
Assuntos
Aorta Torácica/metabolismo , Aterosclerose/metabolismo , Líquido Extracelular/metabolismo , Células Espumosas/metabolismo , Lipoproteínas LDL/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Aorta Torácica/patologia , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Células Espumosas/patologia , Immunoblotting , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.
Assuntos
Actinas/química , Ceramidas/farmacologia , Lipoproteínas LDL/metabolismo , Multimerização Proteica/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Ceramidas/química , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Espumosas/citologia , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Técnicas de Inativação de Genes , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Placa Aterosclerótica/metabolismo , Estrutura Quaternária de Proteína , Células RAW 264.7RESUMO
Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M-containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein-coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.
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Endotélio Vascular/efeitos dos fármacos , Hipertensão/prevenção & controle , Lisofosfolipídeos/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Esfingosina/análogos & derivados , Animais , Apolipoproteínas M/metabolismo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Lipoproteínas HDL/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , Receptores Fc/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologiaRESUMO
RATIONALE: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. OBJECTIVE: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. METHODS AND RESULTS: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. CONCLUSIONS: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.
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Anafilaxia/metabolismo , Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Homeostase/fisiologia , Lisofosfolipídeos/deficiência , Esfingosina/análogos & derivados , Anafilaxia/patologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esfingosina/deficiênciaRESUMO
TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1.
Assuntos
Apoptose , Inflamação/patologia , NF-kappa B/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pele/patologia , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Psoríase/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Transport of oxygen by red blood cells (rbc) is critical for life and embryogenesis. Here, we determined that provision of the lipid mediator sphingosine 1-phosphate (S1P) to the systemic circulation is an essential function of rbc in embryogenesis. Mice with rbc-specific deletion of sphingosine kinases 1 and 2 (Sphk1 and Sphk2) showed embryonic lethality between E11.5 and E12.5 due to defects in vascular development. Administration of an S1P1 receptor agonist to pregnant dams rescued early embryonic lethality. Even though rbc-specific Sphk1 Sphk2-KO embryos were anemic, the erythropoietic capacity of hematopoietic stem cells (HSCs) was not impaired, suggesting that rbc can develop in the absence of sphingosine kinase activity. Indeed, transplantation of HSCs deficient for Sphk1 and Sphk2 into adult mice produced rbc that lacked S1P and attenuated plasma S1P levels in recipients. However, in adult animals, both rbc and endothelium contributed to plasma S1P. Together, these findings demonstrate that rbc are essential for embryogenesis by supplying the lysophospholipid S1P, which regulates embryonic vascular development via its receptors.
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Eritrócitos/metabolismo , Lisofosfolipídeos/fisiologia , Neovascularização Fisiológica , Esfingosina/análogos & derivados , Animais , Desenvolvimento Embrionário , Eritropoese , Feminino , Genes Letais , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/fisiologiaRESUMO
Sphingosine 1-phosphate (S1P), a lipid mediator produced by sphingolipid metabolism, promotes endothelial cell spreading, vascular maturation/stabilization, and barrier function. S1P is present at high concentrations in the circulatory system, whereas in tissues its levels are low. This so-called vascular S1P gradient is essential for S1P to regulate much physiological and pathophysiological progress such as the modulation of vascular permeability. Cellular sources of S1P in blood has only recently begun to be identified. In this review, we summarize the current understanding of S1P in regulating vascular integrity. In particular, we discuss the recent discovery of the endothelium-protective functions of HDL-bound S1P which is chaperoned by apolipoprotein M.
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Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Endotélio Vascular/citologia , Humanos , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Sphingosine kinases (Sphks), which catalyze the formation of sphingosine 1-phosphate (S1P) from sphingosine, have been implicated as essential intracellular messengers in inflammatory responses. Specifically, intracellular Sphk1-derived S1P was reported to be required for NFκB induction during inflammatory cytokine action. To examine the role of intracellular S1P in the inflammatory response of innate immune cells, we derived murine macrophages that lack both Sphk1 and Sphk2 (MΦ Sphk dKO). Compared with WT counterparts, MΦ Sphk dKO cells showed marked suppression of intracellular S1P levels whereas sphingosine and ceramide levels were strongly up-regulated. Cellular proliferation and apoptosis were similar in MΦ Sphk dKO cells compared with WT counterparts. Treatment of WT and MΦ Sphk dKO with inflammatory mediators TNFα or Escherichia coli LPS resulted in similar NFκB activation and cytokine expression. Furthermore, LPS-induced inflammatory responses, mortality, and thioglycolate-induced macrophage recruitment to the peritoneum were indistinguishable between MΦ Sphk dKO and littermate control mice. Interestingly, autophagic markers were constitutively induced in bone marrow-derived macrophages from Sphk dKO mice. Treatment with exogenous sphingosine further enhanced intracellular sphingolipid levels and autophagosomes. Inhibition of autophagy resulted in caspase-dependent cell death. Together, these data suggest that attenuation of Sphk activity, particularly Sphk2, leads to increased intracellular sphingolipids and autophagy in macrophages.
Assuntos
Autofagia , Inflamação/enzimologia , Lisofosfolipídeos/biossíntese , Macrófagos Peritoneais/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Animais , Caspases/genética , Caspases/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Lisofosfolipídeos/genética , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/biossíntese , Esfingosina/genéticaRESUMO
HuR, also known as Elavl1, is an RNA-binding protein that regulates embryonic development, progenitor cell survival, and cell stress responses. The role of HuR in angiogenesis is not known. Using a myeloid-specific HuR knock-out mouse model (Elavl1Mø KO), we show that HuR expression in bone marrow-derived macrophages (BMDMs) is needed to maintain the expression of genes enriched in AU-rich elements and U-rich elements in the 3'-UTR. In addition, BMDMs from Elavl1Mø KO mice also showed alterations in expression of several miRNAs. Interestingly, computational analysis suggested that miR-200b, which is up-regulated in Elavl1Mø KO BMDMs, interacts with myeloid mRNAs very close to the HuR binding sites, suggesting competitive regulation of gene expression. One such mRNA encodes vascular endothelial growth factor (VEGF)-A, a major regulator of angiogenesis. Immunoprecipitation of RNA-protein complexes and luciferase reporter assays indicate that HuR antagonizes the suppressive activity of miR-200b, down-regulates miR-200b expression, and promotes VEGF-A expression. Indeed, Vegf-a and other angiogenic regulatory transcripts were down-regulated in Elavl1Mø KO BMDMs. Interestingly, tumor growth, angiogenesis, vascular sprouting, branching, and permeability were significantly attenuated in Elavl1Mø KO mice, suggesting that HuR-regulated myeloid-derived factors modulate tumor angiogenesis in trans. Zebrafish embryos injected with an elavl1 morpholino oligomer or miR-200b mimic showed angiogenesis defects in the subintestinal vein plexus, and elavl1 mRNA rescued the repressive effect of miR-200b. In addition, miR-200b and HuR morpholino oligomer suppressed the activity of a zVEGF 3'-UTR luciferase reporter construct. Together, these studies reveal an evolutionarily conserved post-transcriptional mechanism involving competitive interactions between HuR and miR-200b that controls angiogenesis.
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Proteínas ELAV/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Antígeno CD11b/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Deleção de Genes , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Peixe-ZebraRESUMO
BACKGROUND & AIMS: Antiangiogenic agents can sometimes promote tumor invasiveness and metastasis, but little is known about the effects of the antiangiogenic drug sorafenib on progression of hepatocellular carcinoma (HCC). METHODS: Sorafenib was administered orally (30 mg · kg(-1) · day(-1)) to mice with orthotopic tumors grown from HCC-LM3, SMMC7721, or HepG2 cells. We analyzed survival times of mice, along with tumor growth, metastasis within liver and to lung, and induction of the epithelial-mesenchymal transition. Polymerase chain reaction arrays were used to determine the effects of sorafenib on gene expression patterns in HCC cells. We analyzed regulation of HIV-1 Tat interactive protein 2 (HTATIP2) by sorafenib and compared levels of this protein in tumor samples from 75 patients with HCC (21 who received sorafenib after resection and 54 who did not). RESULTS: Sorafenib promoted invasiveness and the metastatic potential of orthotopic tumors grown from SMMC7721 and HCC-LM3 cells but not from HepG2 cells. In gene expression analysis, HTATIP2 was down-regulated by sorafenib. HCC-LM3 cells that expressed small hairpin RNAs against HTATIP2 (knockdown) formed less invasive tumors in mice following administration of sorafenib than HCC-LM3 without HTATIP2 knockdown. Alternatively, HepG2 cells that expressed transgenic HTATIP2 formed more invasive tumors in mice following administration of sorafenib. Sorafenib induced the epithelial-mesenchymal transition in HCC cell lines, which was associated with expression of HTATIP2. Sorafenib regulated expression of HTATIP2 via Jun-activated kinase (JAK) and signal transducer and activator of transcription (STAT)3 signaling. Sorafenib therapy prolonged recurrence-free survival in patients who expressed lower levels of HTATIP2 compared with higher levels. CONCLUSIONS: Sorafenib promotes invasiveness and the metastatic potential of orthotopic tumors from HCC cells in mice, down-regulating expression of HTATIP2 via JAK-STAT3 signaling.
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Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/fisiopatologia , Regulação para Baixo/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/fisiopatologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Hep G2 , Humanos , Janus Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Niacinamida/farmacologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/fisiologia , Sorafenibe , Transplante HeterólogoRESUMO
BACKGROUND: Antiangiogenesis is a promising therapy for advanced hepatocellular carcinoma (HCC), but the effects are difficult to be evaluated. Pazopanib (GW786034B) is a pan-vascular endothelial growth factor receptor inhibitor, the antitumor effects or antiangiogenic effects haven't been investigated in HCC. METHODS: In vitro direct effects of pazopanib on human HCC cell lines and endothelial cells were evaluated. In vivo antitumor effects were evaluated in three xenograft nude mice models. In the subcutaneous HCCLM3 model, intratumoral blood perfusion was detected by contrast-enhanced ultrasonography (CEUS), and serial quantitative parameters were profiled from the time-intensity curves of ultrasonograms. RESULTS: In vitro proliferation of various HCC cell lines were not inhibited by pazopanib. Pazopanib inhibited migration and invasion and induced apoptosis significantly in two HCC cell lines, HCCLM3 and PLC/PRF/5. Proliferation, migration, and tubule formation of human umbilical vein endothelial cells were inhibited by pazopanib in a dose-dependent manner. In vivo tumor growth was significantly inhibited by pazopanib in HCCLM3, HepG2, and PLC/PRF/5 xenograft models. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed. Mean transit time of the contrast media in hotspot areas of the tumors was reversely correlated with intratumoral microvessel density. CONCLUSIONS: Antitumor effects of pazopanib in HCC xenografts may owe to its antiangiogenic effects, and the in vivo antiangiogenic effects could be evaluated by quantitative CEUS.
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Inibidores da Angiogênese/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Aumento da Imagem , Indazóis , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carga Tumoral/efeitos dos fármacos , Ultrassonografia/métodosRESUMO
PURPOSE: In recent years, anti-angiogenesis drugs have shown promising clinical effects against many tumors, particularly in combination with chemotherapy. Although the combination has become a standard of care for many tumors, the mechanisms of the chemosensitizing activity of anti-angiogenic drugs are not fully understood. Here, we sought to determine if anti-angiogenesis drug bevacizumab could enhance the chemosensitivity of HCC by inhibition of survivin. METHODS: After treatment of human umbilical vein endothelial cells (HUVECs) and hepatocellular carcinoma (HCC) cell line PLC/PRF/5 (PLC) with bevacizumab or/and adriamycin, the direct effects were examined by survival assays, and the expression of Akt, Phospho-Akt and survivin were evaluated by western blot. Tumor growth was observed in a human HCC xenograft nude mouse model treated with different drugs, and the expression of PCNA, CD31 and survivin in tumor tissues were evaluated by means of immunohistochemistry. RESULTS: Bevacizumab enhanced the chemosensitivity of HCC by inhibiting the VEGF-PI3 K/Akt-survivin signaling cascade in endothelial cells. The combination of bevacizumab with adriamycin therapy resulted in better outcomes compared with monotherapy in hepatocellular carcinoma xenografts; bevacizumab significantly inhibited tumor angiogenesis and growth. In addition, bevacizumab reduced survivin expression in tumor tissues, including tumor vascular endothelial cells in vivo, although it did not inhibit survivin expression in tumor cells in vitro. CONCLUSION: These results implicate the bevacizumab-increased efficacy of adriamycin via an inhibition of survivin expression in malignant cells as well as tumor vasculature cells, which provides other insights into the mechanism of enhanced efficacy by combination of VEGF blocker and chemotherapeutic agents.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Bevacizumab , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Células Endoteliais/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Survivina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque, leading to the formation of sphingosine-1-phosphate (S1P), which activates G protein-coupled receptors to regulate vascular and immune cells. The role of S1P receptors in atherosclerosis has not been examined. METHODS AND RESULTS: We tested the hypothesis that S1P receptor-2 (S1PR2) regulates atherosclerosis. Apoe(-/-) S1pr2(-/-) mice showed greatly attenuated atherosclerosis compared with the Apoe(-/-) mice. Bone marrow transplant experiments indicate that S1PR2 function in the hematopoietic compartment is critical. S1PR2 is expressed in bone marrow-derived macrophages and in macrophage-like foam cells in atherosclerotic plaques. Reduced macrophage-like foam cells were found in the atherosclerotic plaques of Apoe(-/-)S1pr2(-/-) mice, suggesting that S1PR2 retains macrophages in atherosclerotic plaques. Lipoprotein profiles, plasma lipids, and oxidized low-density lipoprotein uptake by bone marrow-derived macrophages were not altered by the S1pr2 genotype. In contrast, endotoxin-induced inflammatory cytokine (interleukin [IL]-1ß, IL-18) levels in the serum of S1PR2 knockout mice were significantly reduced. Furthermore, treatment of wild-type mice with S1PR2 antagonist JTE-013 suppressed IL-1ß and IL-18 levels in plasma. CONCLUSIONS: These data suggest that S1PR2 signaling in the plaque macrophage regulates macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.
Assuntos
Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Doenças da Aorta/etiologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Transplante de Medula Óssea , Modelos Animais de Doenças , Endotoxinas , Inflamação/etiologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/sangue , Interleucina-18/sangue , Interleucina-1beta/sangue , Lipídeos/sangue , Lipoproteínas/sangue , Lipoproteínas LDL/sangue , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/genética , Receptores de Esfingosina-1-FosfatoRESUMO
PURPOSE: To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib. EXPERIMENTAL DESIGN: Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma. RESULTS: Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1alpha, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip. CONCLUSIONS: Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect.