RESUMO
Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/fisiologia , Osteopontina/antagonistas & inibidores , Animais , Comunicação Autócrina , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Inativação Gênica , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Transplante de Neoplasias , Osteopontina/metabolismo , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/metabolismoRESUMO
Most of the proposed extracellular biomineralization processes include the secretion of proteins that interact with mineral ions and/or mineral surfaces. Typically these proteins are acidic or have acidic domains that interact with multivalent cations in the extracellular environment. We propose that most acidic, Ca(2+)-binding proteins challenge the cell's mechanisms for trafficking through the endoplasmic reticulum (ER) lumen due to lumenal mM calcium that cause them to form large aggregates. We have recently shown that >95% of the DSPP mutations that cause non-syndromic genetic dentin diseases start their dominant negative affects by failing to rapidly exit the ER likely by forming complexes that cannot be normally trafficked to the Golgi. The complexes of mutant DSPP then capture more (severe disease) or less (mild disease) of the DSPP translated from the normal allele. After searching genomic databases as well as the published literature, we found the IleProVal (IPV)-like motif at the predicted amino terminus of many acidic proteins made in the mineralizing as well as non-mineralizing tissues of many species including vertebrates, echinoderms, mollusks, and yeast. While we often focused on acidic proteins reported associated with mineralizing structures, proteins associated with hormones and their storage/secretion, digestion, blood functions, as well as milk and other secreted fluids started with variations of the motif. Our hypothesis is that the IPV-like motif interacts with a highly conserved cargo receptor in the ER that efficiently traffics the acidic proteins out of the organelle before they can form harmful aggregates in the Ca(2+)-rich lumen.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/fisiologia , Retículo Endoplasmático/metabolismo , Motivos de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Humanos , Mutação/genética , Transporte ProteicoRESUMO
Families with nonsyndromic dentinogenesis imperfecta (DGI) and the milder, dentin dysplasia (DD), have mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. Because loss of a single Dspp allele in mice (and likely, humans) causes no dental phenotype, the mechanism(s) underling the dominant negative effects were investigated. DSPP mutations occur in three classes. (The first class, the mid-leader missense mutation, Y6D, was not investigated in this report.) All other 5' mutations of DSPP result in changes/loss in the first three amino acids (isoleucine-proline-valine [IPV]) of mature DSPP or, for the A15V missense mutation, some retention of the hydrophobic leader sequence. All of this second class of mutations caused mutant DSPP to be retained in the rough endoplasmic reticulum (rER) of transfected HEK293 cells. Trafficking out of the rER by coexpressed normal DSPP was reduced in a dose-responsive manner, probably due to formation of Ca2+-dependent complexes with the retained mutant DSPP. IPV-like sequences begin many secreted Ca2+-binding proteins, and changing the third amino acid to the charged aspartate (D) in three other acidic proteins also caused increased rER accumulation. Both the leader-retaining A15V and the long string of hydrophobic amino acids resulting from all known frameshift mutations within the 3'-encoded Ca2+-binding repeat domain (third class of mutations) caused retention by association of the mutant proteins with rER membranes. More 5' frameshift mutations result in longer mutant hydrophobic domains, but the milder phenotype, DD, probably due to lower effectiveness of the remaining, shorter Ca2+-binding domain in capturing normal DSPP protein within the rER. This study presents evidence of a shared underlying mechanism of capturing of normal DSPP by two different classes of DSPP mutations and offers an explanation for the mild (DD-II) versus severe (DGI-II and III) nonsyndromic dentin phenotypes. Evidence is also presented that many acidic, Ca2+-binding proteins may use the same IPV-like receptor/pathway for exiting the rER.
Assuntos
Displasia da Dentina/metabolismo , Dentinogênese Imperfeita/metabolismo , Retículo Endoplasmático Rugoso/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Genes Dominantes/genética , Proteínas Mutantes/classificação , Proteínas Mutantes/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Western Blotting , Cromogranina A/metabolismo , Displasia da Dentina/genética , Dentinogênese Imperfeita/genética , Proteínas da Matriz Extracelular/química , Mutação da Fase de Leitura/genética , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Camundongos , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Fosfoproteínas/química , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Sialoglicoproteínas/químicaRESUMO
Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor-C (VEGF-C) and VEGF-D, we studied the effect of the angioregulatory growth factor angiopoietin-2 (Ang-2) on PDAC progression. Ang-2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie-2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang-2 was subject to autocrine/paracrine TGF-ß stimulation (2-fold induction, P=0.0106) acting on the -61- to +476-bp element of the human Ang-2 promoter. In turn, Ang-2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang-2, but not Ang-1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7- to 9.1-fold, P<0.01), which was prevented by sequestration of Ang-2 via coexpression of soluble Tie-2. Notably, elevated circulating Ang-2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang-2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang-2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.
Assuntos
Adenocarcinoma/patologia , Angiopoietina-2/metabolismo , Metástase Linfática/fisiopatologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Idoso , Angiopoietina-2/sangue , Angiopoietina-2/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Linfangiogênese/fisiologia , Masculino , Camundongos , Camundongos SCID , Neoplasias Experimentais , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismoRESUMO
Secreted Frizzled-related proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic ß-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1, and Naked cuticle-1 homolog (NKD1). These results support sFRP2's role as an enhancer of Wnt/ß-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Wnt/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Transcrição Gênica , Proteínas Wnt/antagonistas & inibidores , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMO
The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP(MQDeltaIE) during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (< or = 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.
Assuntos
Proteína Morfogenética Óssea 1/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos , Animais , Produtos Biológicos , Dentina/metabolismo , Proteínas da Matriz Extracelular , Glicoproteínas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Fosfoproteínas , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Sialoglicoproteínas , Syzygium/metabolismoRESUMO
Dentin matrix protein-1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN) are three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) co-expressed/secreted by skeletal and active ductal epithelial cells. Although etiological mechanisms remain unclear, DMP1 is the only one of these three genes currently known to have mutations resulting in human disease, and yet it remains the least studied. All three contain the highly conserved integrin-binding tripeptide, RGD, and experiments comparing the cell attachment and haptotactic migration-enhancing properties of DMP1 to BSP and OPN were performed using human skeletal (MG63 and primary dental pulp cells) and salivary gland (HSG) cells. Mutation of any SIBLING's RGD destroyed all attachment and migration activity. Using its alphaVbeta5 integrin, HSG cells attached to BSP but not to DMP1 or OPN. However, HSG cells could not migrate onto BSP in a modified Boyden chamber assay. Expression of alphaVbeta3 integrin enhanced HSG attachment to DMP1 and OPN and promoted haptotactic migration onto all three proteins. Interchanging the first four coding exons or the conserved amino acids adjacent to the RGD of DMP1 with corresponding sequences of BSP did not enhance the ability of DMP1 to bind alphaVbeta5. For alphaVbeta3-expressing cells, intact DMP1, its BMP1-cleaved C-terminal fragment, and exon six lacking all post-translational modifications worked equally well but the proteoglycan isoform of DMP1 had greatly reduced ability for cell attachment and migration. The sequence specificity of the proposed BMP1-cleavage site of DMP1 was verified by mutation analysis. Direct comparison of the three proteins showed that cells discriminate among these SIBLINGs and among DMP1 isoforms.
Assuntos
Proteínas da Matriz Extracelular/química , Fosfoproteínas/química , Motivos de Aminoácidos , Aminoácidos/química , Adesão Celular , Linhagem Celular , Movimento Celular , Éxons , Humanos , Integrina alfaVbeta3/química , Mutação , Oligopeptídeos/química , Peptídeos/química , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro-(lymph), angiogenic protein VEGF-D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF-D exclusively in the tumor cells. The cognate receptor VEGFR-3 was detected on blood and lymphatic vessels. By clinicopathological analysis VEGF-D expression was correlated with pT-stage of the primary, lymph node metastasis and lymphangiosis carcinomatosa. Three out of 4 human HCC cell lines expressed and secreted VEGF-D. To approach its biological function, VEGF-D deficient SKHep-1 cells were stably transfected with VEGF-D cDNA and effects on tumor progression were determined in vivo. Compared to mock-transfected controls, subcutaneous tumors derived from VEGF-D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF-D expressing tumors exhibited increased microvessel density and increased abundance of peri- and intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE-1 and/or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR-3 domain blocked VEGF-D-induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF-D expression resulted in an increased rate of intra- and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF-D is involved in growth and lymphatic spread of HCC. Therefore, VEGF-D might represent a therapeutic target in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linfonodos/patologia , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/metabolismo , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Transfecção , Fator D de Crescimento do Endotélio Vascular/genéticaRESUMO
The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Fator D de Crescimento do Endotélio Vascular/genética , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfangiogênese , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte VesicularRESUMO
The expression pattern and functional interaction of proangiogenic factors in human cholangiocellular carcinoma (CCC) have not been fully defined. We therefore investigated the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta 1 as well as their respective receptors in human CCC tumor samples and further analyzed their functional interaction in vitro. Expression of VEGF, TGF-beta 1, and their receptors was examined by immunohistochemistry, in situ hybridization, quantitative competitive reverse transcription-PCR, and ELISA. VEGF promoter analysis and identification of transcription factors involved in promoter regulation were investigated using transient transfection and electrophoretic mobility shift assays. We observed strong expression of VEGF in CCC tumor cells and localization of VEGF receptors 1 and 2 in endothelial cells; in addition, coexpression of TGF-beta 1 and its receptors in tumor cells suggests a possible functional interaction between both cytokines. In vitro studies confirmed a paracrine/autocrine stimulation of VEGF by TGF-beta 1 at a transcriptional level. Additional molecular studies using 5' deletion and mutational analysis of the human VEGF promoter revealed that TGF-beta 1 stimulates VEGF through Sp1-dependent transcriptional activation. These data suggest that overexpression and functional interaction of TGF-beta 1 and VEGF might contribute to the "angiogenic switch" and the malignant phenotype in human CCC.
Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Fatores de Crescimento Endotelial/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Linfocinas/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Sítios de Ligação , Colangiocarcinoma/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento Endotelial/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Regiões Promotoras Genéticas , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta1 , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Interferon alpha (IFN-alpha) has antiangiogenic activity, although the underlying mechanism of action is unclear. Because human neuroendocrine (NE) tumors are highly vascularized and sensitive to IFN-alpha, we investigated whether the therapeutic effects of IFN-alpha result from an inhibition of angiogenesis mediated by a decrease in vascular endothelial growth factor (VEGF) gene expression. METHODS: VEGF gene and protein expression was analyzed in NE tumors by immunohistochemistry and in NE tumor cell lines by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). VEGF promoter-reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential transcription factors. A xenograft nude mouse model (five mice per group) was used to determine the effect of IFN-alpha on tumor growth (NE Bon cells and pancreatic Capan-1 cells) and microvessel density. Liver metastases from eight patients with NE tumors were analyzed for microvessel density, VEGF mRNA content, and VEGF plasma levels before and after initiation of IFN-alpha therapy. RESULTS: NE tumors and cell lines expressed VEGF mRNA and secreted VEGF protein. In vitro, IFN-alpha decreased transcription of VEGF gene expression through an Sp1- and/or Sp3-dependent inhibition of VEGF promoter activity. Compared with vehicle treatment in mice, IFN-alpha inhibited tumor growth by 36% and reduced microvessel density from 56 (95% confidence interval [CI] = 49 to 69) to 37 per x400 Field (95% CI = 32 to 41, P =.015). Patients with NE tumors had lower VEGF plasma levels and reduced VEGF mRNA levels and microvessel density in liver metastasis biopsy material after IFN-alpha treatment. CONCLUSION: IFN-alpha confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from Sp1- and/or Sp3-mediated inhibition of VEGF gene transcription.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Fatores de Crescimento Endotelial/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon-alfa/farmacologia , Linfocinas/metabolismo , Neovascularização Patológica/tratamento farmacológico , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Crescimento Endotelial/genética , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Luciferases/metabolismo , Linfocinas/efeitos dos fármacos , Linfocinas/genética , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , Tumores Neuroendócrinos/irrigação sanguínea , Proteínas da Gravidez/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells. METHODS: pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis. RESULTS: pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis. CONCLUSIONS: Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.