RESUMO
Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.
Assuntos
Apigenina/farmacologia , Proteínas ELAV/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Neoplasias Induzidas por Radiação/prevenção & controle , Neoplasias Cutâneas/prevenção & controle , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Trombospondina 1/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Quimioprevenção/métodos , Feminino , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Pelados , Camundongos Endogâmicos BALB C , Neoplasias Induzidas por Radiação/irrigação sanguínea , Neoplasias Induzidas por Radiação/metabolismo , Neovascularização Patológica , Pele/irrigação sanguínea , Pele/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/metabolismo , Raios UltravioletaRESUMO
BACKGROUND: Obesity, particularly visceral adiposity, confers a worse prognosis for prostate cancer (PCa) patients, and increasing periprostatic adipose (PPA) tissue thickness or density is positively associated with more aggressive disease. However, the cellular mechanism of this activity remains unclear. Therefore, in this pilot study, we assessed the functional activity of PPA tissue secretions and established a biochemical profile of PPA as compared to subcutaneous adipose (SQA) tissues from lean, overweight and obese PCa patients. METHODS: Adipose tissues were collected from PCa patients undergoing surgical prostate removal. Tissues were analyzed by histologic and magnetic resonance (MR) techniques. Explant tissue culture secretions were used in proliferation assays on PCa and endothelial cells. RESULTS: PPA secretions obtained from obese patients were significantly more pro-proliferative in both PCa and endothelial cells as compared to PPA obtained from lean or overweight men and SQA tissues. Consistent with this, PPA microvessel density was increased, and the T2 relaxation time was decreased, compared to SQA tissues, and we observed a modest, inverse correlation between the T2 and tumor stage. Moreover, the ratio of unsaturated to saturated fatty acids, obtained using MR spectroscopy, showed a modest, inverse correlation with Gleason score. CONCLUSIONS: These pilot data show that PPA stimulates PCa cell proliferation and angiogenesis and that obesity intensifies this activity, thus generating a mechanistic hypothesis to explain the worse prognosis observed in obese PCa patients. Our pilot study also shows that MR technology may be useful in further elucidating the relationship between obesity and PCa progression.
Assuntos
Tecido Adiposo/patologia , Células Endoteliais/patologia , Obesidade/complicações , Próstata/patologia , Neoplasias da Próstata/patologia , Tecido Adiposo/metabolismo , Índice de Massa Corporal , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Obesidade/patologia , Projetos Piloto , Prognóstico , Neoplasias da Próstata/complicações , Técnicas de Cultura de TecidosRESUMO
Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.
Assuntos
Proliferação de Células , Colo/metabolismo , Neoplasias do Colo/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos , Proteínas de Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/patologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mucosa Intestinal/patologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Proteínas de Neoplasias/genética , Perilipina-2 , Regiões Promotoras Genéticas , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. METHODS: In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. RESULTS: EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). CONCLUSIONS: These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.
Assuntos
Adipócitos Brancos/patologia , Biomarcadores Tumorais/deficiência , Carcinoma Ductal Pancreático/metabolismo , Fatores de Crescimento Neural/deficiência , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Serpinas/deficiência , Adipócitos Brancos/metabolismo , Adiposidade , Animais , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proteínas do Olho , Marcadores Genéticos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Mutação , Invasividade Neoplásica , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Pigment epithelium-derived factor (PEDF) is important for maintaining the normal extracellular matrix. We hypothesized that the initiation of pancreatic fibrosis is dependent on the loss of PEDF. Pancreatic PEDF expression was assessed in wild-type mice fed either a control or ethanol diet using an intragastric feeding model. Pancreatitis responses were elicited with either a single episode or a repetitive cerulein-induced (50 µg/kg, 6 hourly i.p. injections) protocol in wild-type and PEDF-null mice. Quantitative real-time PCR and immunoblotting were performed to assess fibrogenic responses. In wild-type animals, PEDF expression increased with pancreatitis and was more pronounced in mice fed ethanol. Compared with wild-type mice, α-smooth muscle actin staining and expression levels of fibrogenic markers (eg, transforming growth factor-ß1, platelet-derived growth factor, collagen I, and thrombospondin-1) were higher in PEDF-null mice at baseline. Sirius red staining revealed more fibrosis in PEDF-null versus wild-type pancreas 1 week after pancreatitis. Differences in tissue fibrosis resolved with longer recovery periods. PEDF overexpression suppressed thrombospondin-1 levels in vitro. Ethanol feeding and experimental pancreatitis increased PEDF expression in wild-type mice. PEDF-null mice, however, demonstrated enhanced early fibrotic responses compared with wild-type mice with pancreatitis. These findings indicate that PEDF acts as a compensatory antifibrotic cytokine in pancreatitis.
Assuntos
Proteínas do Olho/fisiologia , Fatores de Crescimento Neural/fisiologia , Pâncreas/patologia , Serpinas/fisiologia , Trombospondina 1/antagonistas & inibidores , Animais , Células Cultivadas , Ceruletídeo/toxicidade , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/deficiência , Pancreatite/induzido quimicamente , Pancreatite/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Mensageiro/metabolismo , Serpinas/deficiência , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 µm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.
Assuntos
Transformação Celular Neoplásica/patologia , Colo/patologia , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Mucosa Intestinal/patologia , Neovascularização Patológica , Adenoma/irrigação sanguínea , Adenoma/induzido quimicamente , Adenoma/patologia , Animais , Azoximetano/toxicidade , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intracellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intraepithelial neoplasms (PanIN), and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC showed intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase colocalized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or short-hairpin RNA targeting the V1E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2's most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens show loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.
Assuntos
Isoenzimas/metabolismo , Metaloproteinases da Matriz/metabolismo , Neoplasias Pancreáticas/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Castration experiments in rodents show that the stromal vasculature is critical to the androgen-mediated prostate growth regulation. However, the role of angiogenesis inhibitors, such as thrombospondin-1 (TSP-1), in this process is unclear. TSP-1 is a multifunctional glycoprotein that can function as a potent angiogenesis inhibitor and an in vivo activator of latent transforming growth factor-beta (TGF-beta) in some tissues. On the basis of these observations, we hypothesized that TSP-1 regulated androgen withdrawal-induced prostate regression and that this process was mediated not only through antiangiogenic activity but also through TGF-beta activation. To test this, we evaluated angiogenic activity in human prostate epithelial and stromal cells treated with androgens and hypoxia in vitro. TSP-1 knockout mice were characterized to investigate the in vivo functions of TSP-1. In vitro, we found that androgens and hypoxia differentially regulated TSP-1 and angiogenic activity. Androgens stimulated normal epithelial cell, but inhibited normal stromal cell, angiogenic activity. Conversely, hypoxia stimulated stromal while inhibiting epithelial activity. Thus, in vivo, net angiogenic activity must reflect cellular interactions. And, we found that media conditioned by epithelial cells grown under normoxic conditions stimulated stromal cell angiogenic activity, and if epithelial cells were grown under hypoxic conditions, stromal activity was further increased. TSP-1 levels, however, were unchanged. In vivo, TSP-1 loss in a mouse model led to prostate epithelial hyperplasia by 3 months of age with only a modest stromal effect. Androgens suppressed TSP-1 as expression increased after castration both in normal mouse prostate and in human prostate cancer tissues. In addition, TSP-1 expression corresponded to increased TGF-beta activation in mouse tissues, specifically in the stromal compartment. These data show a critical role for TSP-1 in prostate epithelial and stromal growth regulation through angiogenic inhibition and activation of latent TGF-beta. Therefore, loss of TSP-1 during tumorigenesis would eliminate two barriers to cancer progression.
Assuntos
Androgênios/deficiência , Neovascularização Fisiológica , Próstata/fisiologia , Trombospondina 1/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinoma/fisiopatologia , Linhagem Celular , Di-Hidrotestosterona , Células Epiteliais/metabolismo , Humanos , Hiperplasia , Hipóxia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Orquiectomia , Fenótipo , Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Células Estromais/fisiologiaRESUMO
Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.