RESUMO
The mechanism of transition of ductal carcinoma in situ (DCIS) to invasive cancer is elusive but recently changes in the myoepithelial cells (MECs) have been implicated. The aim of this study is to investigate the changes in gene profile of MECs in DCIS that could compromise their tumor suppressor function leading to promotion of tumor progression. Immuno-laser capture microdissection (LCM) was used to isolate MECs from normal and DCIS breast tissues followed by whole genome expression profiling using Affymetrix HGU-133 plus2.0 arrays. The data were analyzed using Bioconductor packages then validated by using real-time quantitative polymerase chain reaction and immunohistochemistry. Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer and cell death networks, with the Wnt/B-catenin pathway as the top canonical pathway. Validation revealed a 71.4% correlation rate with the array results. Most dramatic was upregulation of Fibronectin 1 ( FN1 ) in DCIS-associated MECs. Immunohistochemistry analysis for FN1 on normal and DCIS tissues confirmed a strong correlation between FN1 protein expression by MECs and DCIS ( P <0.0001) and between high expression level and presence of invasion ( P =0.006) in DCIS. Other validated alterations in MEC expression profile included upregulation of Nephronectin and downregulation of parathyroid hormone like hormone ( PTHLH ), fibroblast growth factor receptor 2 ( FGFR2 ), ADAMTS5 , TGFBR3 , and CAV1 . In vitro experiments revealed downregulation of PTHLH in DCIS-modified MECs versus normal lines when cultured on Fibronectin matrix. This is the first study to use this in vivo technique to investigate molecular changes in MECs in DCIS. This study adds more evidences to the molecular deviations in MECs toward tumor progression in DCIS through upregulation of the tumor-promoting molecules that may lead to novel predictive and therapeutic targets.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal não Infiltrante , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Células Epiteliais/metabolismo , Feminino , Fibronectinas/metabolismo , Humanos , Imuno-HistoquímicaRESUMO
Tumor cells exhibit altered lipid metabolism compared with normal cells. Cell signaling kinases are important for regulating lipid synthesis and energy storage. How upstream kinases regulate lipid content, versus direct targeting of lipid-metabolizing enzymes, is currently unexplored. We evaluated intracellular lipid concentrations in prostate and breast tumor spheroids, treated with drugs directly inhibiting metabolic enzymes fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), diacylglyceride acyltransferase (DGAT), and pyruvate dehydrogenase kinase (PDHK), or cell signaling kinase enzymes PI3K, AKT, and mTOR with lipidomic analysis. We assessed whether baseline lipid profiles corresponded to inhibitors' effectiveness in modulating lipid profiles in three-dimensional (3D) growth and their relationship to therapeutic activity. Inhibitors against PI3K, AKT, and mTOR significantly inhibited MDA-MB-468 and PC3 cell growth in two-dimensional (2D) and 3D spheroid growth, while moderately altering lipid content. Conversely, metabolism inhibitors against FASN and DGAT altered lipid content most effectively, while only moderately inhibiting growth compared with kinase inhibitors. The FASN and ACC inhibitors' effectiveness in MDA-MB-468, versus PC3, suggested the former depended more on synthesis, whereas the latter may salvage lipids. Although baseline lipid profiles did not predict growth effects, lipid changes on therapy matched the growth effects of FASN and DGAT inhibitors. Several phospholipids, including phosphatidylcholine, were also upregulated following treatment, possibly via the Kennedy pathway. As this promotes tumor growth, combination studies should include drugs targeting it. Two-dimensional drug screening may miss important metabolism inhibitors or underestimate their potency. Clinical studies should consider serial measurements of tumor lipids to prove target modulation. Pretherapy tumor classification by de novo lipid synthesis versus uptake may help demonstrate efficacy.
Assuntos
Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células/métodos , Inibidores Enzimáticos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Acetil-CoA Carboxilase/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Feminino , Humanos , Masculino , Fosfolipídeos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismoRESUMO
Alzheimer's disease (AD) is an irreversible and highly progressive neurodegenerative disease. Clinically, patients with AD display impairments in episodic and spatial memory. However, the underlying neuronal dysfunctions that result in these impairments remain poorly understood. The hippocampus is crucial for spatial and episodic memory, and thus we tested the hypothesis that abnormal neuronal representations of space in the hippocampus contribute to memory deficits in AD. To test this hypothesis, we recorded spikes from place cells in hippocampal subfield CA1, together with corresponding rhythmic activity in local field potentials, in the 3xTg AD mouse model. We observed disturbances in place cell firing patterns, many of which were consistent with place cell disturbances reported in other rodent models of AD. We found place cell representations of space to be unstable in 3xTg mice compared to control mice. Furthermore, coordination of place cell firing by hippocampal rhythms was disrupted in 3xTg mice. Specifically, a smaller proportion of place cells from 3xTg mice were significantly phase-locked to theta and slow gamma rhythms, and the theta and slow gamma phases at which spikes occurred were also altered. Remarkably, these disturbances were observed at an age before detectable Aß pathology had developed. Consistencies between these findings in 3xTg mice and previous findings from other AD models suggest that disturbances in place cell firing and hippocampal rhythms are related to AD rather than reflecting peculiarities inherent to a particular transgenic model. Thus, disturbed rhythmic organization of place cell activity may contribute to unstable spatial representations, and related spatial memory deficits, in AD. © 2017 Wiley Periodicals, Inc.
Assuntos
Doença de Alzheimer/fisiopatologia , Hipocampo/fisiopatologia , Células de Lugar/fisiologia , Percepção Espacial/fisiologia , Potenciais de Ação/fisiologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Eletrodos Implantados , Ritmo Gama/fisiologia , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos da Linhagem 129 , Camundongos Transgênicos , Células de Lugar/patologia , Comportamento Espacial/fisiologia , Ritmo Teta/fisiologia , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
BACKGROUND: Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets. RESULTS: Using functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival. CONCLUSIONS: Our data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment.
RESUMO
A majority of breast cancers are driven by estrogen via estrogen receptor-α (ERα). Our previous studies indicate that hypoxia-inducible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. However, whether ERα is implicated in the direct regulation of HIF-1α and the role of HIF-1α in endocrine therapy response are unknown. In this study we found that a subpopulation of HIF-1α targets, many of them bearing both hypoxia response elements and estrogen response elements, are regulated by ERα in normoxia and hypoxia. Interestingly, the HIF-1α gene itself also bears an estrogen response element, and its expression is directly regulated by ERα. Clinical data revealed that expression of the HIF-1α gene or a hypoxia metagene signature is associated with a poor outcome to endocrine treatment in ERα(+) breast cancer. HIF-1α was able to confer endocrine therapy resistance to ERα(+) breast cancer cells. Our findings define, for the first time to our knowledge, a direct regulatory pathway between ERα and HIF-1α, which might modulate hormone response in treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transdução de Sinais , Tamoxifeno/uso terapêutico , Transcrição Gênica/fisiologiaRESUMO
A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.
Assuntos
Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Ácidos Graxos/metabolismo , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia , Células MCF-7 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Estresse FisiológicoRESUMO
Genetic ablation of endothelial focal adhesion kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularization. Here we show that reduced stromal FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumour growth in vivo. Our results highlight a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neovascularização Patológica/enzimologia , Animais , Proliferação de Células , Separação Celular , Sobrevivência Celular , Células Endoteliais/patologia , Heterozigoto , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Proteínas Mutantes/metabolismo , Neoplasias/patologia , Neovascularização Patológica/patologia , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Tela Subcutânea/patologia , Talina/metabolismo , Carga Tumoral , Vinculina/metabolismoRESUMO
Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking ß3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvß3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in ß3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.
Assuntos
Neovascularização Fisiológica/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Integrina alfaVbeta3/deficiência , Integrina alfaVbeta3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/deficiência , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5-72 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Perfilação da Expressão Gênica , Neovascularização Patológica/genética , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Bevacizumab , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/irrigação sanguínea , Carcinoma Ductal de Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Microscopia Confocal , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Interferência de RNA , Transplante Heterólogo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
INTRODUCTION: Activation of the hypoxia response pathway is a feature of many tumours and is one of the key mechanisms associated with tumour growth, chemoresistance and radioresistance. The major component of the hypoxia response pathway is the heterodimeric transcription factor, hypoxia-inducible factor (HIF), which is upregulated in many human cancers. Therefore, HIF is an attractive therapeutic target and several strategies have been developed to target it. AREAS COVERED: Approaches used in targeting the hypoxia response pathway are discussed. Reviewed are agents that target upstream, directly and downstream of HIF, as well as some of the challenges in HIF-targeted therapy. EXPERT OPINION: Many of the therapeutic agents that are in clinical use inhibit downstream HIF target genes, but ideally a molecule specific to HIF will have a more potent effect in inhibiting multiple HIF pathways. However, many anti-HIF molecules have multiple targets, which may increase non-specific cytotoxicity. In addition, many anti-HIF agents cannot discriminate between the different isoforms of HIF-α. So, it is important to assess whether targeting both HIF-1α and HIF-2α or each subunit selectively will provide better therapeutic effects.
Assuntos
Antineoplásicos/farmacologia , Hipóxia/metabolismo , Neoplasias/tratamento farmacológico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenho de Fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , Regulação para CimaRESUMO
Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling-all without the need for cellular dissociation-thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6-14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.
Assuntos
Aorta/fisiologia , Neovascularização Fisiológica/fisiologia , Técnicas de Cultura de Tecidos , Animais , Aorta/citologia , Movimento Celular , Proliferação de Células , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia de Contraste de FaseRESUMO
Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hipóxia/metabolismo , Integrina alfa3beta1/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Citometria de Fluxo , Hipóxia/genética , Hipóxia/patologia , Imuno-Histoquímica , Integrina alfa3beta1/genética , Masculino , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.
Assuntos
Vasos Sanguíneos/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vasos Linfáticos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Separação Celular/métodos , Células Cultivadas , Embrião de Mamíferos , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Galactosídeos/metabolismo , Deleção de Genes , Imuno-Histoquímica , Indóis/metabolismo , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , RNA Interferente Pequeno/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transfecção , beta-Galactosidase/metabolismo , Proteínas rac1 de Ligação ao GTP/análise , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Integrina alfaVbeta3/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Receptores de Vitronectina/uso terapêutico , Animais , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: Hypoxia-inducible factor-alpha (HIF-alpha) is a transcription factor that regulates the response to hypoxia. HIF-alpha protein is found at high levels in many cancers, and the redox protein thioredoxin-1 (Trx-1) increases both aerobic and hypoxia-induced HIF-alpha. Therefore, Trx-1 and HIF-alpha are attractive molecular targets for novel cancer therapeutics. EXPERIMENTAL DESIGN: We investigated whether two novel anticancer drugs AJM290 and AW464 (quinols), which inhibit Trx-1 function, can inhibit the HIF pathway. RESULTS: Treatment of several cancer cell lines with AJM290 or AW464 prevented the hypoxia-induced increase of vascular endothelial growth factor (VEGF) at subtoxic concentrations. AJM290 and AW464 also decreased VEGF in pVHL mutant renal cell carcinoma cells that constitutively overexpress HIF-alpha protein. They surprisingly up-regulated HIF-alpha expression in breast cancer cell lines in normoxia and hypoxia as well as in pVHL mutant cells. In the MDA-MB-468 breast cancer cell line, the compounds inhibited RNA and protein expression of the HIF-alpha target genes, carbonic anhydrase IX, VEGF, and BNIP3, concordantly with HIF-alpha up-regulation. Both compounds specifically inhibited HIF-alpha-dependent induction of hypoxia regulatory element-luciferase and HIF-1alpha hypoxia regulatory element-DNA binding. To analyze the HIF-1alpha domain inhibited by AJM290, we transfected cells with plasmids expressing a fusion protein of Gal linked to HIF-1alpha or HIF-1alpha COOH-terminal transactivation domain (CAD) with a Gal4-responsive luciferase reporter gene. AJM290 inhibited both the full-length HIF-1alpha and HIF-1alpha CAD transcriptional activity. CONCLUSIONS: AJM290 and AW464 are inhibitors of HIF-1alpha CAD transcription activity and DNA binding, but they also inhibit degradation of HIF, in contrast to other Trx inhibitors.
Assuntos
Benzotiazóis/farmacologia , Cicloexanonas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Indóis/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Sulfonas/farmacologia , Tiorredoxinas/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leupeptinas/farmacologia , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hypoxia-inducible factor-alpha (Hif-alpha) plays an important role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Therefore, Hif-alpha is an attractive target for development of novel cancer therapeutics. We have generated Chinese hamster ovary cells, which stably express luciferase reporter construct under the control of a hypoxia response element to screen 15,000 compounds. We identified 40 compounds that inhibited hypoxic up-regulation of luciferase, and the top 30 compounds were further screened in a secondary assay using MDA-468 breast cancer cell line. Eight compounds were shown to inhibit VEGF expression in hypoxic cells at subtoxic concentrations. Three top putative Hif inhibitors, DJ12, DJ15, and DJ30, were chosen for further analysis. Transient transfection of cells with hypoxia-regulated luciferase reporter plasmids further validated that these compounds inhibit hypoxia up-regulated genes. All three compounds failed to inhibit Hif-1alpha protein levels but they did inhibit induction of downstream targets of Hif-alpha under hypoxia. Two of the three compounds were cell type specific, whereas compound DJ12 inhibited VEGF at subtoxic levels in breast cancer cell lines MDA-468 and ZR-75, melanoma cell line MDA-435, and pVHL mutant renal cancer cell lines RCC4 and 786-0. Compound DJ12 down-regulated mRNA of downstream targets of Hif-alpha, and significantly inhibited Hif-1alpha transactivation activity by blocking Hif-1alpha hypoxia response element-DNA binding. Our cell-based approach and deconvolution of the inhibitory effect of DJ12 has identified a novel compound that targets the hypoxia pathway by inhibiting Hif-alpha-inducible transcription.
Assuntos
DNA/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ativação Transcricional/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células CHO , Processos de Crescimento Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Luciferases/biossíntese , Luciferases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Cellular iron is needed for cell survival and hydroxylation of hypoxia-inducible factor-1alpha (HIF-alpha) by prolyl hydroxylases (PHD). One mechanism of iron uptake is mediated by the cell surface transferrin receptor (TfR). Because iron is required for cell growth and suppression of HIF-alpha levels, we tested the effects of the two anti-TfR monoclonal antibodies (mAb) E2.3 and A27.15 on growth of breast cancer cells and induction of HIF-alpha and hypoxia-regulated genes. Treatment with both mAbs together synergistically inhibited cell proliferation in a dose-responsive manner by up to 80% following 8 days of exposure, up-regulated HIF-1alpha and HIF transcription targets, down-regulated TfR expression, and down-regulated cellular labile iron pool by 60%. Because combined treatment with anti-TfR mAbs resulted in the up-regulation of the hypoxia pathway, which may increase tumor angiogenesis, we analyzed the effects of ascorbate on cell viability and HIF-1alpha levels in cells treated with both anti-TfR mAbs together, as ascorbate has been shown to be required by PHD enzymes for full catalytic activity. Ascorbate at physiologic concentrations (25 micromol/L) suppressed HIF-1alpha protein levels and HIF transcriptional targets in anti-TfR mAb-treated cells but did not suppress the antiproliferative effect of the mAbs. These results indicate that the addition of ascorbate increased the activity of the PHD enzymes in down-regulating HIF but not the proliferation of iron-starved anti-TfR mAb-treated cells. The use of anti-TfR mAbs and ascorbate in inhibiting both cell proliferation and HIF-1alpha and angiogenesis under normoxic conditions may be of therapeutic use.
Assuntos
Anticorpos Monoclonais/farmacologia , Ácido Ascórbico/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Receptores da Transferrina/antagonistas & inibidores , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ferro/metabolismo , Receptores da Transferrina/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVES: P-glycoprotein (P-gp) is a major cause of multidrug resistance (MDR) in acute myelogenous leukemia (AML) and is thought to contribute to the failure of chemotherapy. Zosuquidar trihydochloride (Z.3HCL) is a potent and selective inhibitor of P-gp which rapidly and effectively inhibits drug efflux. DESIGN AND METHODS: The aim of this study was to evaluate the clinical effects of Z.3HCL and determine its influence on P-gp activity. Sixteen AML patients were entered into a phase 1 dose ranging clinical trial of Z.3HCL, co-administered intravenously with daunorubicin and cytosine arabinoside (ARA-C). Clinical outcomes, toxicity abd adverse events were assessed. P-gp function was analyzed by flow cytometry. In vitro cytotoxicity was studied using the MTT assay. RESULTS: Eleven patients achieved a complete remission and one a partial remission with a median survival of 559 (range 38-906) days. Non-hematologic grade 3 and 4 toxicities were seen in 4 patients. Z.3HCL infusion was associated with rapid inhibition of Rh123 efflux in CD56+ cells in 16/16 patients and in CD33+ cells from 6/10 patients. The median inhibition was 95% for CD56+ cells and 85.25% for CD33+ cells was significantly elevated in 6/16 patients. The median IC50, using a MTT assay for daunorubicin, decreased significantly between Z.3HCL modulated and unmodulated cells (n=11,153 and 247 ng/mL respectively, p=0.01). INTERPRETATION AND CONCLUSIONS: The modulator Z.3HCL is a specific inhibitor of P-gp efflux and can be given safely to patients with AML in combination with induction doses of conventional cytotoxic drugs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Dibenzocicloeptenos/administração & dosagem , Leucemia Promielocítica Aguda/tratamento farmacológico , Quinolinas/administração & dosagem , Dor Abdominal/induzido quimicamente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Diarreia/induzido quimicamente , Dibenzocicloeptenos/efeitos adversos , Dibenzocicloeptenos/toxicidade , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Quinolinas/efeitos adversos , Quinolinas/toxicidade , Indução de RemissãoRESUMO
We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.
Assuntos
Apoptose , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Quinonas/farmacologia , Vidarabina/análogos & derivados , Antibióticos Antineoplásicos/farmacologia , Antígenos CD34/biossíntese , Benzoquinonas , Western Blotting , Células da Medula Óssea/citologia , Separação Celular , Clorambucila/farmacologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Proteínas de Choque Térmico HSP70/biossíntese , Humanos , Concentração Inibidora 50 , Lactamas Macrocíclicas , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Rifabutina/farmacologia , Linfócitos T/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Vidarabina/farmacologia , Proteína-Tirosina Quinase ZAP-70RESUMO
We have studied the actions of tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super-additive apoptosis induction in approximately half of the isolates. Molecular evidence of super-additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super-additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro-apoptotic form of the BCL-2 family member BID. Our data suggest that co-administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.