RESUMEN
BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance. METHODS: Using RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression. RESULTS: We show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo. CONCLUSIONS: This study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/patología , Neuropilina-1 , Neoplasias de la Próstata Resistentes a la Castración/patología , Antagonistas de Andrógenos , Resistencia a Medicamentos , Diferenciación Celular , Línea Celular TumoralRESUMEN
Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys6-LHRH to target the LHRH receptor. This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.
Asunto(s)
Proteínas Anfibias/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas Anfibias/metabolismo , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Factores Inmunológicos/metabolismo , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dermaseptin-B2 (DRS-B2) is a multifunctional cationic antimicrobial peptide (CAP) isolated from frog skin secretion. We previously reported that DRS-B2 possesses anticancer and antiangiogenic activities in vitro and in vivo. In the present study, we evaluated the antiproliferative activity of DRS-B2 on numerous tumor cell lines, its cell internalization and studies of its molecular partners as well as their influences on its structure. Confocal microscopy using ([Alexa594]-(Cys0)-DRS-B2) shows that in sensitive human tumor cells (PC3), DRS-B2 seems to accumulate rapidly at the cytoplasmic membranes and enters the cytoplasm and the nucleus, while in less sensitive tumor cells (U87MG), DRS-B2 is found packed in vesicles at the cell membrane. Furthermore FACS analysis shows that PC3 cells viability decreases after DRS-B2 treatment while U87 MG seems to be unaffected. However, "pull down" experiments performed with total protein pools from PC3 or U87MG cells and the comparison between the antiproliferative effect of DRS-B2 and its synthetic analog containing all D-amino acids suggest the absence of a stereo-selective protein receptor. Pretreatment of PC3 cells with sodium chlorate, decreases the antiproliferative activity of DRS-B2. This activity is partially restored after addition of exogenous chondroitin sulfate C (CS-C). Moreover, we demonstrate that at nanomolar concentrations CS-C potentiates the antiproliferative effect of DRS-B2. These results highlight the partial implication of glycosaminoglycans in the mechanism of antiproliferative action of DRS-B2. Structural analysis of DRS-B2 by circular dichroism in the presence of increasing concentration of CS-C shows that DRS-B2 adopts an α-helical structure. Finally, structure-activity-relationship studies suggest a key role of the W residue in position 3 of the DRS-B2 sequence for its antiproliferative activity.
Asunto(s)
Proteínas Anfibias/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Neoplasias/tratamiento farmacológico , Secuencia de Aminoácidos , Proteínas Anfibias/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Antineoplásicos/química , Anuros , Línea Celular Tumoral , Humanos , Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Citocinas/metabolismo , Neuropilina-1/metabolismo , Transducción de Señal/fisiología , Animales , Sitios de Unión/genética , Células CHO , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Cricetinae , Cricetulus , Citocinas/genética , Endocitosis/efectos de los fármacos , Endocitosis/genética , Endocitosis/fisiología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Microscopía Fluorescente , Invasividad Neoplásica , Neuropilina-1/genética , Unión Proteica/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. In normal or pathological angiogenesis, angiogenic growth factors activate cognate receptors on endothelial cells. Fibroblast growth factor-2 (FGF-2) and heparin affin regulatory peptide (HARP) are two heparin-binding growth factors and were described for their pro-angiogenic properties on human umbilical endothelial cells (HUVEC). We now show that HARP acts as a mediator of FGF-2's stimulatory effects, since it is able to inhibit the proliferation and migration of HUVEC induced by FGF-2. We demonstrate by ELISA and optical biosensor binding assay that HARP and FGF-2 interact through direct binding. We have adapted a previously developed structural proteomics method for the identification of residues involved in protein-protein interactions. Application of this method showed that two sequences in HARP were involved in binding FGF-2. One was in the C-thrombospondin type 1 repeat (C-TSR-1) domain and the other in the C-terminal domain of HARP. The identification of these regions as mediating the binding of FGF-2 was confirmed by ELISA using synthetic peptides, which are as well mediators of FGF-2-induced proliferation, migration and tubes formation on HUVEC in vitro. These results imply that besides a regulation of the proliferation, migration and angiogenesis of HUVEC by direct interaction of FGF-2 with its receptors, an alternative pathway exists involving its binding to growth factors such as HARP.
Asunto(s)
Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Técnicas Biosensibles , Proteínas Portadoras/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/farmacología , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Neovascularización Fisiológica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.
Asunto(s)
Proteínas Anfibias/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Desnudos , Microscopía Confocal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery of new molecules with potential antitumor activity continues to be of great importance in cancer research. In this respect, natural antimicrobial peptides isolated from various animal species including humans and amphibians have been found to be of particular interest. Here, we report the presence of two anti-proliferative peptides active against cancer cells in the skin secretions of the South American tree frog, Phyllomedusa bicolor. The crude skin exudate was fractioned by size exclusion gel followed by reverse-phase HPLC chromatography. After these two purification steps, we identified two fractions that exhibited anti-proliferative activity. Sequence analysis indicated that this activity was due to two antimicrobial α-helical cationic peptides of the dermaseptin family (dermaseptins B2 and B3). This result was confirmed using synthetic dermaseptins. When tested in vitro, synthetic B2 and B3 dermaseptins inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line by more than 90%, with an EC(50) of around 2-3 µM. No effect was observed on the growth of the NIH-3T3 non-tumor mouse cell line with Drs B2, whereas a slight inhibiting effect was observed with Drs B3 at high dose. In addition, the two fractions obtained after size exclusion chromatography also inhibited PC-3 cell colony formation in soft agar. Interestingly, inhibition of the proliferation and differentiation of activated adult bovine aortic endothelial cells was observed in cells treated with these two fractions. Dermaseptins B2 and B3 could, therefore, represent interesting new pharmacological molecules with antitumor and angiostatic properties for the development of a new class of anticancer drugs.
Asunto(s)
Proteínas Angiostáticas/metabolismo , Proteínas Angiostáticas/farmacología , Antineoplásicos/farmacología , Piel/química , Piel/metabolismo , Proteínas Angiostáticas/análisis , Proteínas Angiostáticas/aislamiento & purificación , Animales , Antineoplásicos/análisis , Antineoplásicos/aislamiento & purificación , Anuros , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Células 3T3 NIH , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit in vitro and in vivo the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop. METHODS: A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. In vitro, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. In vivo, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay. RESULTS: Using pull down experiments, we identified the HARP receptors RPTPß/ζ, ALK and nucleolin as P111-136 binding proteins. In vitro, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice CONCLUSIONS: Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPß/ζ, nucleolin). In vivo, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.
Asunto(s)
Adenocarcinoma/metabolismo , Proteínas Portadoras/farmacología , Citocinas/farmacología , Péptidos/farmacología , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/química , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica/patología , Péptidos/síntesis química , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation.
Asunto(s)
Neoplasias/tratamiento farmacológico , Péptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Ligandos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Terapia Molecular Dirigida/métodos , Neoplasias/metabolismo , Péptidos/farmacocinética , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Distribución Tisular , Ensayos Antitumor por Modelo de Xenoinjerto , NucleolinaRESUMEN
BACKGROUND: Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor. RESULTS: Our results show that P(122-131) inhibits in vitro adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPß/ζ. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk1/2. Finally, P(122-131) not only interacts with RPTPß/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPß/ζ expression with the RNAi technology. CONCLUSIONS: In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPß/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.
Asunto(s)
Proteínas Portadoras/química , Citocinas/química , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , HumanosRESUMEN
BACKGROUND: The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, we recently reported that HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in the athymic nude mice without apparent toxicity. METHODS: The in vivo antitumoral action of HB-19 treatment was assessed on the spontaneous development of melanoma in the RET transgenic mouse model. Ten days old RET mice were treated with HB-19 in a prophylactic setting that extended 300 days. In parallel, the molecular basis for the action of HB-19 was investigated on a melanoma cell line (called TIII) derived from a cutaneous nodule of a RET mouse. RESULTS: HB-19 treatment of RET mice caused a significant delay in the onset of cutaneous tumors, several-months delay in the incidence of large tumors, a lower frequency of cutaneous nodules, and a reduction of visceral metastatic nodules while displaying no toxicity to normal tissue. Moreover, microvessel density was significantly reduced in tumors recovered from HB-19 treated mice compared to corresponding controls. Studies on the melanoma-derived tumor cells demonstrated that HB-19 treatment of TIII cells could restore contact inhibition, impair anchorage-independent growth, and reduce their tumorigenic potential in mice. Moreover, HB-19 treatment caused selective down regulation of transcripts coding matrix metalloproteinase 2 and 9, and tumor necrosis factor-alpha in the TIII cells and in melanoma tumors of RET mice. CONCLUSIONS: Although HB-19 treatment failed to prevent the development of spontaneous melanoma in the RET mice, it delayed for several months the onset and frequency of cutaneous tumors, and exerted a significant inhibitory effect on visceral metastasis. Consequently, HB-19 could provide a novel therapeutic agent by itself or as an adjuvant therapy in association with current therapeutic interventions on a virulent cancer like melanoma.
Asunto(s)
Membrana Celular/metabolismo , Neoplasias Pulmonares/prevención & control , Melanoma/prevención & control , Fragmentos de Péptidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/fisiología , Proteínas de Unión al ARN/antagonistas & inhibidores , Neoplasias Cutáneas/prevención & control , Animales , Western Blotting , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Tasa de Supervivencia , NucleolinaRESUMEN
Glioblastoma is the most common primary brain tumor in human adults. Since existing treatments are not effective enough, novel therapeutic targets must be sought. The heparin-binding growth factor, heparin affin regulatory peptide (HARP), also known as pleiotrophin (PTN), could potentially represent such a target. We have previously shown that a mutant protein, HARPDelta111-136, which lacks HARP's C-terminal 26 amino acids, acts as a dominant negative HARP effector by heterodimerizing with the wild-type growth factor. The aim of our study was to evaluate the potential inhibitory activity of HARPDelta111-136 on the U87 MG human glioblastoma cell line. By overexpressing the truncated form of HARP in stably established clones of U87 MG cells, we observed an inhibition of proliferation under both anchorage-dependent and anchorage-independent conditions. We confirmed these results in an in vivo subcutaneous tumor xenograft model. In addition, we found that HARPDelta111-136 inhibited cell proliferation in a paracrine manner. Analysis of key cellular pathways revealed a decrease of cell adhesion in U87 MG cells that overexpressed the mutant protein, which could explain this inhibitory effect. A replication-defective adenovirus model that encoded HARPDelta111-136 supported a putative antiproliferative role for the truncated protein in vitro and in vivo. Interestingly, HARPDelta111-136 was also able to abolish angiogenic activity in HUVEC proliferation and in a Matrigel plug assay. These results demonstrate that considering its antiproliferative and angiostatic effects, HARPDelta111-136 could be of great interest when used in conjunction with standard treatments.
Asunto(s)
Neoplasias Encefálicas/patología , Proteínas Portadoras/genética , Citocinas/genética , Glioblastoma/patología , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Animales , Apoptosis , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Células CHO , Adhesión Celular , Movimiento Celular , Proliferación Celular , Colágeno/metabolismo , Cricetinae , Cricetulus , Combinación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Técnicas para Inmunoenzimas , Laminina/metabolismo , Ratones , Ratones Desnudos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Proteoglicanos/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.
Asunto(s)
Carcinoma Hepatocelular/patología , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/antagonistas & inhibidores , Glicosaminoglicanos/farmacología , Neoplasias Hepáticas/patología , Unión Competitiva/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/biosíntesis , Glicosaminoglicanos/agonistas , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Invasividad Neoplásica , ARN Interferente Pequeño/farmacología , Células Tumorales CultivadasRESUMEN
Pleiotrophin (PTN) is a 136-amino acid secreted heparin-binding protein that is considered as a rate-limiting growth and an angiogenic factor in the onset, invasion, and metastatic process of many tumors. Its mitogenic and tumorigenic activities are mediated by the COOH-terminal residues 111 to 136 of PTN, allowing it to bind to cell surface tyrosine kinase-linked receptors. We investigated a new strategy consisting in evaluating the antitumor effect of a truncated PTN, lacking the COOH-terminal 111 to 136 portion of the molecule (PTNDelta111-136), which may act as a dominant-negative effector for its mitogenic, angiogenic, and tumorigenic activities by heterodimerizing with the wild-type protein. In vitro studies showed that PTNDelta111-136 selectively inhibited a PTN-dependent MDA-MB-231 breast tumor and endothelial cell proliferation and that, in MDA-MB-231 cells expressing PTNDelta111-136, the vascular endothelial growth factor-A and hypoxia-inducible factor-1alpha mRNA levels were significantly decreased by 59% and 71%, respectively, compared with levels in wild-type cells. In vivo, intramuscular electrotransfer of a plasmid encoding a secretable form of PTNDelta111-136 was shown to inhibit MDA-MB-231 tumor growth by 81%. This antitumor effect was associated with the detection of the PTNDelta111-136 molecule in the muscle and tumor extracts, the suppression of neovascularization within the tumors, and a decline in the Ki-67 proliferative index. Because PTN is rarely found in normal tissue, our data show that targeted PTN may represent an attractive and new therapeutic approach to the fight against cancer.
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Neoplasias de la Mama/patología , Proteínas Portadoras/farmacología , Citocinas/farmacología , Células Endoteliales/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/irrigación sanguínea , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Desnudos , Peso Molecular , Proteínas Mutantes/metabolismo , Neovascularización Patológica/patología , Receptores de Superficie Celular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.
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Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias/patología , Neovascularización Patológica/prevención & control , Péptidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias/irrigación sanguínea , NucleolinaRESUMEN
Pleiotrophin (PTN), is a heparin-dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat-I (TSR-I) motif in its two beta-sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13-39) and P(65-97) corresponding to a part of the N-terminal and C-terminal TSR-I motif of PTN, respectively. P(65-97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor-2 (FGF-2). However, P(65-97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65-97) but not P(13-39) inhibited the binding of PTN and to a lesser extent of FGF-2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a K(d) of 120 nM. These findings suggest that P(65-97), containing amino acids 65-97 of the TSR-I motif of the C-terminal domain of PTN, inhibits the activities of PTN and FGF-2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co-receptor.
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Proteínas Portadoras/química , Citocinas/química , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Péptidos/farmacología , Trombospondinas/química , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/patología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular , Citocinas/antagonistas & inhibidores , Citocinas/genética , Endotelio Vascular/citología , Femenino , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/genética , Heparina/metabolismo , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Células 3T3 NIH , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Trasplante Heterólogo , Venas Umbilicales/citologíaRESUMEN
Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.
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Inhibidores de la Angiogénesis/metabolismo , Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Factor de Crecimiento del Tejido Conjuntivo , Medios de Cultivo Condicionados , Activación Enzimática , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Marcaje Isotópico , Ratones , Células 3T3 NIH , Péptidos/metabolismo , Biosíntesis de Proteínas , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Heparin affin regulatory peptide (HARP) is an 18 kDa heparin-binding protein that plays a key role in tumor growth. We showed previously that the synthetic peptide P(111-136) composed of the last 26 HARP amino acids inhibited HARP-induced mitogenesis. Here, to identify the exact molecular domain involved in HARP inhibition, we investigated the effect of the shorter basic peptide P(122-131) on DU145 cells, which express HARP and its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). P(122-131) was not cytotoxic; it dose-dependently inhibited anchorage-independent growth of DU145 cells. Binding studies using biotinylated P(122-131) indicated that this peptide interfered with HARP binding to DU145 cells. Investigation of the mechanisms involved suggested interference, under anchorage-independent conditions, of P(122-131) with a HARP autocrine loop in an RPTPbeta/zeta-dependent fashion. Thus, P(122-131) may hold potential for the treatment of disorders involving RPTPbeta/zeta.
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ADN Helicasas/fisiología , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , División Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismoRESUMEN
Bisphosphonates are extensively used in the treatment of patients with metastasis-induced osteolysis. The major drawback in the efficacy of all bisphosphonates lies in their high hydrophilic nature, which results in poor membrane permeability and low availability for soft tissues. A reasonable approach to overcome these problems consists in masking one or more ionizable groups of bisphosphonates, notably by esterification of the hydroxyl functions. We have previously shown that the novel non-nitrogen-containing bisphosphonate BP7033 inhibited angiogenesis and growth of primary tumors in nude mice. The present study focuses on the dimethyl-esterified analog of this compound (Me-BP7033). In-vitro, Me-BP7033 inhibited proliferation of human carcinoma A431 cells as well as their invasive activity based on a transwell invasion assay. in-vivo, administration of Me-BP7033 (0.3 mg/kg) twice a week for 5 weeks inhibited the tumor growth of A431 cells xenografted in nude mice by 65%. Immunostaining of endothelial cells (ECs) in tumor sections revealed that Me-BP7033 inhibited the intratumor ECs density by 60%. The in-vivo anti-angiogenic properties of Me-BP7033 were also demonstrated in an in-vivo angiogenesis assay showing that Me-BP7033 reduced the vascular endothelial growth factor-stimulated infiltration of ECs in a Matrigel plug by 70%. In summary, we demonstrated for the first time that a diesterified bisphosphonate exhibited in vivo both anti-tumoral and anti-angiogenic activities with no apparent sign of toxic effects. These new diesterified compounds, which could display enhanced bioavailability and pharmacokinetics, thus represent interesting candidates for therapeutic applications such as cancer treatment.
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Alcoholes Bencílicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Difosfonatos/farmacología , Neovascularización Patológica/tratamiento farmacológico , Animales , Alcoholes Bencílicos/química , Carcinoma de Células Escamosas/irrigación sanguínea , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Difosfonatos/química , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.