RESUMEN
Ultraviolet A (UVA) irradiation induces various changes in cell biology. The objective of this study was to determine the effect of vanillin on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). UVA-antagonizing mechanisms of vanillin were also examined. The results revealed that vanillin attenuated UVA-induced reduction of the proliferative potential and stemness of hAMSCs evidenced by increased proliferative activity in BrdU incorporation assay and upregulation of stemness-related genes (OCT4, NANOG and SOX2) in response to vanillin treatment. UVA-induced reduction in mRNA level of hypoxia-inducible factor (HIF)-1α was significantly recovered by vanillin. In addition, the antagonizing effect of vanillin on UVA was found to be mediated by reduced production of PGE2 through inhibiting JNK and p38 MAPK. Taken together, these findings showed that vanillin could improve the reduced stemness of hAMSCs induced by UVA. The effect of vanillin is mediated by upregulating HIF-1α via inhibiting PGE2-cAMP signaling. Therefore, vanillin might be used as an antagonizing agent to mitigate the effects of UVA.
Asunto(s)
Tejido Adiposo/patología , Antioxidantes/farmacología , Benzaldehídos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Mesenquimatosas/patología , Rayos Ultravioleta , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/efectos de la radiación , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Ensayo de Inmunoadsorción Enzimática , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de la radiación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Células Tumorales CultivadasRESUMEN
Angiogenesis is crucial for tumor growth and metastasis. Blocking this process is, therefore, a potentially powerful approach for the treatment of cancer. Human apolipoprotein(a) kringle V (rhLK8) is an angiogenesis inhibitor and is currently under development as an anti-cancer therapeutic. However, a relatively short in vivo half-life limits its widespread clinical use. This study was performed to evaluate whether fusion of an Fc domain to rhLK8 can extend plasma half-life. RhLK8-Fc fusion protein was expressed in CHO DG44 cells as a dimer and was readily purified by protein G affinity chromatography. The anti-angiogenic activity of rhLK8-Fc was similar to that of rhLK8, as determined by migration and tube formation assays with endothelial cells in vitro and a chorioallantoic membrane assay in vivo. Pharmacokinetic profiles in mice after single intravenous administration of rhLK8 or rhLK8-Fc showed that Fc fusion significantly increased the elimination half-life (t(½)) and the systemic exposure (AUC(inf)) of the protein, in parallel with a significant decrease in total clearance (CL). These data suggest that Fc fusion to rhLK8 is a powerful strategy for extending the plasma half-life of rhLK8 without affecting its anti-angiogenic activity, and could thus improve the clinical applicability of rhLK8.
Asunto(s)
Inhibidores de la Angiogénesis/farmacocinética , Apolipoproteínas A/farmacocinética , Fragmentos Fc de Inmunoglobulinas/sangre , Fragmentos de Péptidos/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Apolipoproteínas A/química , Apolipoproteínas A/farmacología , Células CHO , Movimiento Celular/efectos de los fármacos , Cricetinae , Cricetulus , Semivida , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.
