RESUMO
Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Polifenóis/farmacologia , Própole , Inibidores da Angiogênese/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Polifenóis/química , Própole/química , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
UNLABELLED: Hypoxia-inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF-1 or HIF-2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia-induced cell death we created a stable knockdown of HIF-1alpha and HIF-2alpha in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1alpha enhanced expression of HIF-2alpha and vice versa. Unexpectedly, knockdown of HIF-1alpha or HIF-2alpha increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-X(L) expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1alpha-knockdown cells. Furthermore, an HIF-2alpha-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1alpha-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2alpha-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2alpha-knockdown clones comparable to wildtype cells. CONCLUSION: An HIF-1alpha-knockdown increases HIF-2alpha expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2alpha raises autophagic activity and attenuates apoptosis by enhancing HIF-1alpha expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development.
Assuntos
Apoptose , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Hepatocelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caspase 3/metabolismo , Crescimento Celular , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Macrophages infiltrating solid tumors exhibit a tumor-supporting phenotype and are critical for tumor development. Little is known which tumor-derived signal provokes this phenotype shift and how these signals are interpreted in macrophages to support tumor growth. We used the supernatant of apoptotic cells and noticed transcriptional, nuclear factor of activated T cells-dependent up-regulation of hypoxia-inducible factor (HIF)-1alpha mRNA, subsequent protein expression, and HIF-1 activity. Blocking calcineurin with cyclosporine A attenuated nuclear factor of activated T cells binding during electrophoretic mobility shift assay analysis and circumvented the HIF-1alpha mRNA increase. Knockdown experiments, receptor analysis, and antibody neutralization pointed to sphingosine-1-phosphate and transforming growth factor-beta as the initiators of the HIF-1 response. The use of macrophages from conditional HIF-1alpha knockout mice revealed that macrophages, under the impact of apoptotic cell supernatants, use HIF-1 to produce factors that induce CD31 expression in murine embryonic stem cells. Our study supports the notion that soluble factors produced from apoptotic tumor cells activate the HIF-1 system under normoxia in macrophages to enhance their tumor-promoting capacity by, for example, releasing vascular endothelial growth factor. This shows the importance of HIF-1-elicited responses in regulatory macrophages under normoxia.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisofosfolipídeos/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Esfingosina/análogos & derivados , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Calcineurina/metabolismo , Inibidores de Calcineurina , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunossupressores/farmacologia , Células Jurkat , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Neoplasias/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , RNA Mensageiro/metabolismo , Esfingosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Tumor-associated macrophages (TAMs) constitute major infiltrates of solid tumors and express a marker profile that characterizes alternatively activated macrophages (MФs). TAMs accumulate in hypoxic tumor regions, express high amounts of hypoxia-inducible factor-1 (HIF-1) and contribute to tumor angiogenesis and invasiveness. However, the precise role of HIF-1 on MФ infiltration and phenotype alterations remains poorly defined. Therefore, we cocultured wild type (wt) versus HIF-1α(-/-) MФs with tumor spheroids. Both, wt and HIF-1α(-/-) MФs, infiltrated hypoxic regions of tumor spheroids at equal rates and got alternatively activated. Interestingly, significantly higher amounts of HIF-1α(-/-) MФs expressed the TAM markers CD206 and stabilin-1 compared with wt phagocytes. Stimulation of infiltrated TAMs with lipopolysaccharide (LPS)/interferon-γ revealed a reduced expression of the pro-inflammatory markers interleukin (IL)-6, tumor necrosis factor-α and inducible nitric oxide synthase in HIF-1α(-/-) MФs. Furthermore, HIF-1α(-/-) MФs were less cytotoxic toward tumor cells. Although infiltration of MФs increased the invasive potential of tumor spheroids independently of HIF-1, the ability to stimulate differentiation of stem cells toward CD31-positive cells was triggered by wt but not by HIF-1α(-/-) MФs. Our data suggest that HIF-1α-deficient MФs develop a more prominent TAM marker profile accompanied by reduced cytotoxicity, whereas HIF-1 seems indispensable for the angiogenesis-promoting properties of TAMs.
Assuntos
Polaridade Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Macrófagos/fisiologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/etiologia , Animais , Moléculas de Adesão Celular Neuronais/análise , Linhagem Celular Tumoral , Humanos , Interferon gama/farmacologia , Interleucina-6/biossíntese , Lectinas Tipo C/análise , Lipopolissacarídeos/farmacologia , Receptor de Manose , Lectinas de Ligação a Manose/análise , Camundongos , Invasividade Neoplásica , Neoplasias/patologia , Óxido Nítrico Sintase Tipo II/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Receptores de Superfície Celular/análise , Receptores de Retorno de Linfócitos/análise , Esferoides Celulares/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an anti-inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-alpha, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization.
Assuntos
Inflamação/imunologia , Macrófagos/fisiologia , Neoplasias Experimentais/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Polaridade Celular , Sobrevivência Celular , Feminino , Humanos , Lisofosfolipídeos/biossíntese , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Transplante HeterólogoRESUMO
The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective ?-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.