RESUMEN
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
We previously demonstrated, using the glioblastoma cell line U87MG as an experimental model, that the adenoviral mediated overexpression of the truncated protein HARPΔ111-136 inhibits the proliferation of these cells in vitro as well as tumor growth and angiogenesis in vivo. This study focused on identifying the underlying mechanisms for the observed antitumoral effect. The present study demonstrated that HARPΔ111-136 induced the ATF4/ATF3/CHOP cascade resulting in a strong expression of the proapoptotic protein CHOP, leading to tumor cell apoptosis as demonstrated by PARP cleavage and FACS analysis. siRNA-mediated CHOP gene silencing abolished Ad-HARPΔ111-136 induced apoptosis. Moreover, Ad-HARPΔ111-136 increased the expression of the death receptor DR5 and enhanced U87MG cells sensitivity in vitro to TRAIL a DR5 ligand with subsequent activation of caspase 8. Infection of U87MG cells with Ad-HARPΔ111-136 also enhanced radiation-induced apoptosis. In vivo, the combination of Ad-HARPΔ111-136 and radiation therapy resulted in a striking inhibition (92%) of the growth of U87MG xenografts, resulting from the potent effect on tumor angiogenesis and tumor cell apoptosis as determined by TUNEL analysis. Taken together, our results indicated that the inhibitor HARPΔ111-136 sensitized U87MG cells to apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , ADN Helicasas/biosíntesis , Glioblastoma/patología , Glioblastoma/radioterapia , Fragmentos de Péptidos/biosíntesis , Animales , Apoptosis/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , ADN Helicasas/genética , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Fragmentos de Péptidos/genética , Factor de Transcripción CHOP , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
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.
Asunto(s)
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
Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., alpha5beta1 and alphav, are preferentially expressed on endothelial cells on angiogenesis. Furthermore, metargidin interacts with these integrins via its disintegrin domain. In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated. At concentrations of 2-10 micro g/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen. RDD (10 micro g/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel. To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle. RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting. In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD. Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice. Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Desintegrinas/uso terapéutico , Neoplasias Pulmonares/prevención & control , Melanoma Experimental/prevención & control , Proteínas de la Membrana/uso terapéutico , Metaloendopeptidasas/uso terapéutico , Neovascularización Patológica/prevención & control , Proteínas ADAM , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Escherichia coli/genética , Femenino , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/secundario , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Músculo Esquelético/patología , Proteínas Recombinantes/uso terapéutico , Células Tumorales CultivadasRESUMEN
We describe an approach employing intramuscular plasmid electrotransfer to deliver secretable forms of K1-5 and K1-3-HSA (a fusion of K1-3 with human serum albumin), which span, respectively, five and three of the five kringle domains of plasminogen. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. K1-3-HSA and K1-5, produced from C2C12 muscle cells, were found to inhibit endothelial cell (HMEC-1) proliferation by 30 and 51%, respectively. In vivo, the expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) and could be maintained for at least 3 months. Blood half-lives of 2.1 and 3.7 days were found for K1-5 and K1-3-HSA, respectively. The K1-5 protein was secreted from muscle into blood at a level of 45 ng/ml, which was sufficient to inhibit MDA-MB-231 tumor growth by 81% in nude mice and B16-F10 melanoma cell lung invasion in C57BL/6 mice by 73%. PECAM-1 immunostaining studies revealed modest tumor vasculature in mice expressing K1-5. In contrast, K1-3-HSA, although secreted into blood at much higher level (250 ng/ml) than K1-5, had no effect on tumor growth.