RESUMEN
A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A(165)), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Animales , Anticuerpos Monoclonales , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Pollos , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Colágeno/metabolismo , Combinación de Medicamentos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Femenino , Fibronectinas/química , Humanos , Laminina/metabolismo , Neoplasias/irrigación sanguínea , Molécula L1 de Adhesión de Célula Nerviosa/química , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Oligopéptidos/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Secuencias Repetitivas de Aminoácido , Solubilidad/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Antibodies directed against the L1 cell adhesion protein inhibit growth of SKOV3ip human ovarian cancer cells in vitro and in vivo. Responses of SKOV3ip cells in vitro to anti-L1 mAb chCE7 and Genistein were investigated. Genistein potentiated the anti-proliferative and pro-apoptotic effects of chCE7 in SKOV3ip cells. A combination of mAb chCE7 and Genistein strongly reduced the sensitivity of p44/42 (Erk1,2) kinase, Src kinase and Akt kinase to extracellular stimulation with serum, Epidermal Growth Factor and Hepatocyte Growth Factor. The observed synergy of antibodies directed against L1 with Genistein could lead to a new therapeutic option for ovarian cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Genisteína/farmacología , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Neoplasias Ováricas/patología , Apoptosis/inmunología , Western Blotting , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN , Sinergismo Farmacológico , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Familia-src Quinasas/metabolismoRESUMEN
The modification of proteins by chemical methods is well-established, however usually difficult to control. In this paper, we describe the posttranslational modification of different IgGs via the Lys or Gln side chains catalyzed by bacterial and human tissue transglutaminase (BTGase and TG2). For proof of concept, different IgG1s (commercial bovine IgG1, and L1CAM targeting chCE7 and chCE7 aglycosylated) were enzymatically functionalization with different fluorescent TGase substrates based on the CY3 analogue Dy547. The optimal reaction conditions were determined in order to assess the two enzymes. The efficiency of the enzymatic method was also compared with a standard chemical method employing a reactive NHS ester of Dy547. Three new TGase substrates were synthesized for this study including Lys-substrate 1 useful for BTGase and TG2 and two Gln-substrates tailor-made for BTGase (substrate 2) and TG2 (substrate 3). Of the two TGases tested, BTGase incorporated Lys-substrate 1 more efficiently than TG2. On the other hand, both enzymes reacted equally efficiently with the corresponding Gln-substrates 2 and 3. Reproducible labeling could be achieved in a broad concentration "window" of the substrates (up to 400 microM) without the risk of overlabeling of chCE7 or chCE7 aglycosylated. The biological activities of the functionalized antibodies were unaltered as shown by in vitro antigen affinity measurements and cell internalization experiments using confocal laser scanning microscopy. A maximum label-to-protein ratio of approximately 1 was achieved with chCE7 aglycosylated and substrate 1 using BTGase. It is important to recognize that the enzymatic activity of TGases enables the stable functionalization of proteins via the side chains of Gln, which is not possible by any chemical method available today. In addition, we could prove that the enzymatic modification of all antibodies occurred selectively at the heavy chain whereas the chemical method led to labeling of both the heavy and the light chains.