Viola Giampietro, Fortunato Elena, Cecconet Laura, Del Giudice Laura, Dall'Acqua Francesco, Basso Giuseppe
Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy.
Toxicol Appl Pharmacol. 2008 Feb 15;227(1):84-96. doi: 10.1016/j.taap.2007.10.004. Epub 2007 Oct 11.
Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21(Waf1/Cip1), a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21(Waf1/Cip1) paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21(Waf1/Cip1) induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.
尽管有强有力的证据表明补骨脂素加紫外线A光疗法（PUVA疗法）具有高效性，但其作用机制尚未完全阐明。在本研究中，我们在人角质形成细胞系（NCTC - 2544）中评估了两种线性补骨脂素衍生物8 - 甲氧基补骨脂素（8 - MOP）和5 - 甲氧基补骨脂素（5 - MOP）的作用，这两种衍生物在PUVA疗法中广泛使用，同时还评估了两种角型衍生物当归素（ANG）和4,6,4'-三甲基当归素（TMA）。所有衍生物光诱导细胞死亡，其中TMA是最具活性的化合物。细胞周期分析表明，这四种衍生物在照射后24小时诱导细胞周期停滞在G1期，随后是大量细胞凋亡。G1期停滞与p21（Waf1/Cip1）表达增加相关，p21是一种与细胞周期阻滞和细胞凋亡相关的蛋白质。此外，用5 - MOP、8 - MOP和ANG处理NCTC - 2544导致p53激活，但TMA未导致p53激活，且p53在丝氨酸15处发生磷酸化。p21（Waf1/Cip1）水平与p53蛋白染色模式平行，表明p53激活与p21（Waf1/Cip1）诱导相关。与p53激活同时，补骨脂素诱导线粒体去极化、细胞色素c释放、线粒体活性氧生成以及caspase - 3和 - 9激活。因此，这些结果强烈表明p53激活以及线粒体下游凋亡机制诱导的必要性。
