RESUMO
Sialyltransferase-catalyzed membrane protein and lipid glycosylation plays a vital role as one of the most abundant post-translational modifications and diversification reactions in eukaryotes. However, aberrant sialylation has been associated with cancer malignancy and metastasis. Sialyltransferases thus represent emerging targets for the development of small molecule cancer drugs. Herein, we report the inhibitory effects of a recently discovered lithocholic acid derivative FCW393 on sialyltransferase catalytic activity, integrin sialyation, cancer-associated signal transduction, MDA-MB-231 and B16F10 cell migration and invasion, and in in vivo studies, on tumor growth, metastasis, and angiogenesis. FCW393 showed effective and selective inhibition of the sialyltransferases ST6GAL1 (IC50 = 7.8 µM) and ST3GAL3 (IC50 = 9.45 µM) relative to ST3GAL1 (IC50 > 400 µM) and ST8SIA4 (IC50 > 100 µM). FCW393 reduced integrin sialylation in breast cancer and melanoma cells dose-dependently and downregulated proteins associated with the integrin-regulated FAK/paxillin and GEF/Rho/ROCK pathways, and with the VEGF-regulated Akt/NFκB/HIF-1α pathway. FCW393 inhibited cell migration (IC50 = 2.6 µM) and invasion in in vitro experiments, and in in vivo studies of tumor-bearing mice, FCW393 reduced tumor size, angiogenesis, and metastatic potential. Based on its demonstrated selectivity, cell permeability, relatively low cytotoxicity (IC50 = 55 µM), and high efficacy, FCW393 shows promising potential as a small molecule experimental tool compound and a lead for further development of a novel cancer therapeutic.
Assuntos
Movimento Celular , Sialiltransferases , Sialiltransferases/metabolismo , Sialiltransferases/antagonistas & inibidores , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Metástase Neoplásica , Feminino , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Litocólico/farmacologiaRESUMO
Melanotan II (MTII), a synthetic analogue of the alpha-melanocyte stimulating hormone (α-MSH), has been applied for skin tanning in humans. However, the carcinogenic consequence of topical MTII has been equivocal. This study aims to delineate the anti-neoplastic efficacy and mechanism of MTII using the B16-F10 melanoma model in vitro and in vivo. It was found that, despite a lack of influence on proliferation, MTII potently inhibited the migration, invasion, and colony-forming capability of melanoma cells. Moreover, topical MTII application significantly attenuated the tumor progression in mice bearing established melanoma. Histological analysis revealed that MTII therapy induced apoptosis while inhibiting the proliferation and neovaluarization in melanoma tissues. By immunoblot and immunohistochemical analysis, it was found that MTII dose-dependently increased the phosphatase and tensin homolog (PTEN) protein level while reducing PTEN phosphorylation, which resulted in the inhibition of AKT/nuclear factor kappa B (NFκB) signaling. Consistently, MTII treatment inhibited cyclooxygenase II (COX-2) expression and prostaglandin E2 (PGE2) production in melanoma cells. Finally, studies of antibody neutralization suggest that the melanocortin 1 receptor (MC1R) plays a critical role in MTII-induced PTEN upregulation and melanoma suppression. Together, these results indicate that MTII elicits PTEN upregulation via MC1R, thereby suppressing melanoma progression through downregulating COX-2/PGE2 signaling. Hence, topical MTII therapy may facilitate a novel therapeutic strategy against melanoma.
Assuntos
Antineoplásicos/administração & dosagem , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Ciclo-Oxigenase 2/metabolismo , Melanoma Experimental/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Peptídeos Cíclicos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , alfa-MSH/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Dinoprostona/genética , Dinoprostona/metabolismo , Humanos , Imuno-Histoquímica , Melanoma Experimental/metabolismo , Camundongos , Modelos Biológicos , PTEN Fosfo-Hidrolase/genética , Fosforilação , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , Regulação para Cima/efeitos dos fármacos , alfa-MSH/administração & dosagemRESUMO
Hepatoma-derived growth factor (HDGF) participates in tumourigenesis but its role in breast cancer is unclear. We set out to elucidate the expression profile and function of HDGF during breast carcinogenesis. Immunoblot and immunohistochemical studies revealed elevated HDGF expression in human breast cancer cell lines and tissues. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumour recurrence. Gene transfer studies were performed to elucidate the influence of cellular HDGF level on the malignant behaviour and epithelial-mesenchymal transition (EMT) of breast cancer cells. Adenovirus-mediated HDGF over-expression stimulated the invasiveness and colony formation of MCF-7 cells. Moreover, HDGF over-expression promoted breast cancer cell EMT by E-cadherin down-regulation and vimentin up-regulation. Conversely, HDGF knockdown by RNA interference in MDA-MB-231 cells attenuated the malignant behaviour and elicited EMT reversal by enhancing E-cadherin expression while depleting vimentin expression. Because HDGF is a secreted protein, we evaluated the cellular function of recombinant HDGF and found that exogenously supplied HDGF enhanced the invasiveness of breast cancer cells by down-regulating E-cadherin and up-regulating vimentin at transcriptional and translational levels. In contrast, blockade of HDGF secretion with an HDGF antibody inhibited the malignant behaviours and EMT. Finally, exogenous HDGF partially reversed benzyl isothiocyanate (BITC)-induced EMT suppression. HDGF over-expression may exert a prognostic role for tumour metastasis and recurrence in breast cancer by modulating EMT. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/secundário , Adulto , Anticorpos Bloqueadores/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Técnicas de Transferência de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Antígeno Ki-67/metabolismo , Linfonodos/patologia , Metástase Linfática , Mastectomia , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Recidiva Local de Neoplasia , Prognóstico , Proteínas RecombinantesRESUMO
BACKGROUND: Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin, melanocyte-stimulating hormone and the endogenous opioid (ß-endorphin). Our previous studies have indicated that POMC gene delivery inhibited the progression and metastasis of B16-F10 melanoma via the α- melanocyte-stimulating hormone/melanortin-1 receptor (MC-1R) pathway. METHODS: In the present study, the therapeutic efficacy of POMC gene therapy was evaluated in mice bearing established Lewis lung carcinoma (LLC) models both in vitro and in vivo. We also investigated the MC-1R-independent mechanism underlying POMC gene therapy. RESULTS: We found that POMC gene delivery significantly inhibited the growth and colony formation in MC-1R-deficient LLC cells. In addition, POMC gene transfer effectively suppressed the growth of established LLC in mice. The inhibitory mechanisms underlying POMC gene delivery were attibuted to be inhibition of proliferation and the induction of apoptosis. Moreover, POMC gene delivery attenuated tumor ß-catenin signaling by reducing protein levels of ß-catenin and its downstream proto-oncogenes, including cyclin D1 and c-myc. Lastly, POMC gene delivery induced a significant suppression of tumor vasculature. CONCLUSIONS: These results support the existence of an MC-1R-independent pathway for POMC gene therapy, which further expands the therapeutic spectrum of POMC therapy for multiple types of cancer.
Assuntos
Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/terapia , Terapia Genética/métodos , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/uso terapêutico , Transdução de Sinais , Animais , Apoptose , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Proliferação de Células , Progressão da Doença , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/terapia , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , beta Catenina/metabolismoRESUMO
Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fosfatidilinositol 3-Quinase/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Formação de Roseta , Animais , Movimento Celular , Citoesqueleto/genética , Citoesqueleto/metabolismo , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Células NIH 3T3 , TransfecçãoRESUMO
We report the synthesis and evaluation of a series of cell-permeable and N- versus O-selective sialyltransferase inhibitors. Inhibitor design entailed the functionalization of lithocholic acid at C(3) and at the cyclopentane ring side chain. Among the series, FCW34 and FCW66 were shown to inhibit MDA-MB-231 cell migration as effectively as ST3GALIII-gene knockdown did. FCW34 was shown to inhibit tumor growth, reduce angiogenesis, and delay cancer cell metastasis in animal models. Furthermore, FCW34 inhibited vessel development and suppressed angiogenic activity in transgenic zebrafish models. Our results provide clear evidence that FCW34-induced sialyltransferase inhibition reduces cancer cell metastasis by decreasing N-glycan sialylation, thus altering the regulation of talin/integrin/FAK/paxillin and integrin/NFκB signaling pathways.
Assuntos
Neoplasias da Mama/patologia , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Metástase Neoplásica/prevenção & controle , Sialiltransferases/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Catálise , Linhagem Celular Tumoral , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glicoproteínas/metabolismo , Humanos , Integrinas/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Paxilina/metabolismo , Fosforilação , Sialiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Talina/metabolismo , Peixe-ZebraRESUMO
Hypoxia in tumors is known to trigger the pro-survival pathways such as autophagy. Systemic proopiomelanocortin (POMC) gene therapy suppresses melanoma through apoptosis induction and neovascularization blockage. In this study, we investigated the crosstalk between autophagic and apoptotic signaling in POMC-mediated melanoma suppression. By histological and immunoblot analysis, it was shown that POMC-treated melanoma tissues exhibited the prominent LC3 immunostaining, which was correlated with reduced CD31-positive tumor vascularization. Such autophagy induction could be recapitulated in melanoma cells receiving POMC gene delivery and hypoxia-mimicking agent cobalt chloride (CoCl2). We then utilized the POMC-derived peptide α-MSH with CoCl2 to elicit the autophagy as well as apoptosis in cultured melanoma cells. To delineate the role of autophagy during cell death, application of autophagy-inducer rapamycin enhanced, whereas autophagy inhibitor 3-MA attenuated, the α-MSH-induced apoptosis in melanoma cells. Genetic silencing of ATG5, an autophagy regulator, by RNA interference perturbed the α-MSH-induced apoptosis in melanoma cells. Finally, it was delineated that α-MSH stimulated the HIF-1α signaling as well as the expression of BNIP3/BNIP3L, thereby promoting the autophagy and apoptosis in melanoma cells. Therefore, the present study unveiled a unique function of autophagy in promoting cell death during POMC-mediated melanoma suppression via α-MSH/HIF-1α/BNIP3/BNIP3L signaling pathway.
RESUMO
Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, ß-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short-term ocular transgene expression, without causing hepatic injury and immune activation. Therefore, subconjunctivally administered adenovirus may be a promising gene delivery approach for managing anterior and posterior segment eye diseases requiring short-term therapy.
Assuntos
Adenoviridae/genética , Túnica Conjuntiva , Técnicas de Transferência de Genes , Vetores Genéticos , Transgenes , Animais , Técnicas de Transferência de Genes/efeitos adversos , Masculino , RatosRESUMO
Hypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl2), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including α-MSH, ß-MSH, and ACTH, but not γ-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated α-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of α-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.
Assuntos
Terapia Genética , Melanoma Experimental/genética , NADPH Oxidases/genética , Pró-Opiomelanocortina/genética , Animais , Apoptose/genética , Caspase 3/metabolismo , Técnicas de Transferência de Genes , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Pró-Opiomelanocortina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Melanoma/genética , Melanoma/patologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Regulação para Cima/genéticaRESUMO
The prognosis of malignant melanoma is poor due to high incidence of metastasis, underscoring the demand for development of novel therapeutic strategies. Stress hormone pro-opiomelanocortin (POMC) is the precursor for several anti-inflammatory peptides that hold promise for management of cancer-related diseases. The present study evaluated the antimetastatic potential and mechanism of POMC therapy for metastatic melanoma. Adenovirus-mediated POMC gene delivery potently inhibited the invasiveness of human and mouse melanoma cells. Moreover, after induction of lung metastasis, systemic POMC expression significantly reduced the foci formation and neovascularization in lungs. Mechanistic studies revealed that POMC therapy inhibited the epithelial-mesenchymal transition (EMT) of melanoma cells by upregulation of E-cadherin and downregulation of vimentin and α-smooth muscle actin (α-SMA). In addition, microarray analysis unveiled POMC gene transfer reduced the mRNA level of multiple prometastatic factors, including hepatoma-derived growth factor (HDGF). Cell culture and immunohistochemical studies further confirmed that POMC gene delivery significantly decreased the expression of HDGF in melanoma cells and tissues. Despite stimulating the invasion and EMT, exogenous HDGF supply only partially attenuated the POMC-mediated invasion inhibition and EMT change in melanoma cells. Finally, we delineated the contribution of melanocortins to POMC-induced inhibition of invasion, HDGF downregulation, and E-cadherin upregulation. Together, these results indicate that HDGF downregulation participates in POMC-induced suppression of metastasis and EMT in melanoma.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Pró-Opiomelanocortina/genética , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma Experimental/patologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Pró-Opiomelanocortina/administração & dosagemRESUMO
Malignant melanoma is one of the leading causes of cancer mortality worldwide, underlining the need for effective novel therapies. In this study, the therapeutic efficacy and mechanism of systemic pro-opiomelanocortin (POMC) therapy were evaluated in mice bearing established melanoma. Injection of adenovirus encoding POMC (Ad-POMC) led to hepatic POMC overexpression and elevated adrenocorticotropin (ACTH) levels in the circulation. Systemic POMC therapy significantly attenuated the growth of established melanoma and prolonged the survival of tumor-bearing mice. Histological analysis revealed that systemic POMC therapy induced melanogenic differentiation while reducing melanoma growth. In addition, POMC therapy also elicited a significant reduction in the neovascular network of melanoma. Last, we demonstrated that POMC-derived peptides, including ACTH, α-melanocyte-stimulating hormone (α-MSH), and ß-MSH, are involved in POMC-mediated melanogenic differentiation and angiogenesis inhibition. In summary, systemic POMC therapy suppresses melanoma growth via induction of melanogenic differentiation and angiogenesis blockade, thereby demonstrating its potential as a novel treatment modality for melanoma.