RESUMEN
Centella asiatica (C. asiatica) is a pharmacological plant in South Asia. It has been demonstrated that C. asiatica extracts containing various pentacyclic triterpenes exert healing effects, especially wound healing and collagen synthesis in skin. However, there are few studies on the effect of C. asiatica extracts on stress-induced premature senescence (SIPS). To determine whether H(2) O(2) -induced senescence is affected by C. asiatica extracts, we performed senescence analysis on cultured human dermal fibroblasts (HDFs). We also analysed whole gene expression level using microarrays and showed that 39 mRNAs are differentially expressed in H(2) O(2) -induced HDFs with and without treatment with C. asiatica extracts. These genes regulate apoptosis, gene silencing, cell growth, transcription, senescence, DNA replication and the spindle checkpoint. Differential expression of FOXM1, E2F2, MCM2, GDF15 and BHLHB2 was confirmed using semi-quantitative PCR. In addition, C. asiatica extracts rescued the H(2) O(2) -induced repression of replication in HDFs. Therefore, the findings presented here suggest that C. asiatica extracts might regulate SIPS by preventing repression of DNA replication and mitosis-related gene expression.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Dermis/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Triterpenos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Centella , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Replicación del ADN/efectos de los fármacos , Regulación hacia Abajo/genética , Factor de Transcripción E2F2/genética , Fibroblastos/metabolismo , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Perfilación de la Expresión Génica , Factor 15 de Diferenciación de Crecimiento/genética , Proteínas de Homeodominio/genética , Humanos , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Extractos Vegetales , Reacción en Cadena de la Polimerasa , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
PURPOSE: Corneal endothelial cells in humans do not replicate to any meaningful extent. Diminishing density of the cell monolayer with age and in the disease states is a major cause of loss of corneal transparency. This study was conducted to test the hypothesis that overexpression of the transcription factor E2F2 results in replication in nonproliferating human corneal endothelial cells. METHODS: Whole human corneas were incubated for 2 hours in a solution of recombinant E1(-)/E3(-) adenovirus incorporating cDNA encoding E2F2 and green fluorescent protein (GFP) under control of a bidirectional promoter and subsequently maintained in ex vivo culture. Control specimens were incubated with an identical virus bearing the GFP sequence only, or virus-free medium. Efficiency of gene transfer and localization was examined by fluorescence microscopy. En face confocal microscopy of the corneal endothelial surface was used to image recombinant E2F2 expression. 5-bromodeoxyuridine (BrdU) incorporation was used to examine progression to the S phase. Changes in density of the corneal endothelium were quantified by specular microscopy and counting of trypan-blue-stained cells. Apoptosis was tested with a TUNEL assay. RESULTS: Recombinant proteins were expressed predominantly in the endothelium and in a high proportion of endothelial cells in the first week after exposure to virus, diminishing thereafter. Compared with the control, transduction with E2F2 resulted in progression from the G(1) to the S phase in a significant number of cells and in increased cell density. Apoptosis was not found to any significant extent. CONCLUSIONS: Overexpression of the transcription factor E2F2 in nonmitotic human corneal endothelial cells results in short-term expression, cell-cycle progression, and increased monolayer cell density.