Berlanda Juergen, Kiesslich Tobias, Oberdanner Christian Benno, Obermair Franz Josef, Krammer Barbara, Plaetzer Kristjan
Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
J Environ Pathol Toxicol Oncol. 2006;25(1-2):173-88. doi: 10.1615/jenvironpatholtoxicoloncol.v25.i1-2.100.
Hypericin is a naturally occurring metabolite extracted from Hypericum plants and is regarded as a promising photosensitizing agent for applications in the frame of photodynamic treatment (PDT). This treatment procedure is based on the light-induced formation of reactive oxygen species and subsequent destruction of target cells. We used an in vitro model system consisting of human epidermoid carcinoma cells (A431) and hypericin as a photosensitizer to study the time- and dose-dependent characteristics of hypericin-PDT-based induction of cytotoxicity and apoptotic cell death. The induction of apoptosis by hypericin-PDT was found to follow a strict dose-dependent manner with a transition to necrotic cell death at higher doses. Apoptosis was analyzed by characteristical biochemical and morphological markers (activation of caspases, nuclear fragmentation and membrane blebbing). Time-course analysis of an almost homogenous apoptotic population of cells (at 1.44 J/cm2) showed a rapid increase in nuclear fragmentation and activation of caspases reaching a maximum at 5 hr after irradiation. Using specific caspase substrates, significant activation of caspase-2, -3, -6, and -9 was found. Mitochondrial involvement during hypericin-PDT-induced apoptosis could be proven by a rapid reduction of the mitochondrial membrane potential; interestingly, the level of intracellular adenosine-5'-triphosphate (ATP) remains at control level for up to 6 hr post irradiation suggesting upregulation of glycolysis as a compensating mechanism of energy supply. Our data contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with hypericin.
金丝桃素是一种从金丝桃属植物中提取的天然代谢产物，被认为是一种有前途的光敏剂，可用于光动力治疗（PDT）。该治疗过程基于光诱导产生活性氧物种并随后破坏靶细胞。我们使用了一个体外模型系统，该系统由人表皮样癌细胞（A431）和作为光敏剂的金丝桃素组成，以研究基于金丝桃素-PDT诱导细胞毒性和凋亡性细胞死亡的时间和剂量依赖性特征。发现金丝桃素-PDT诱导的凋亡遵循严格的剂量依赖性方式，在较高剂量下会转变为坏死性细胞死亡。通过特征性的生化和形态学标志物（半胱天冬酶激活、核碎片化和膜泡形成）分析凋亡。对几乎均匀的凋亡细胞群体（在1.44 J/cm2时）进行的时间进程分析表明，核碎片化和半胱天冬酶激活迅速增加，在照射后5小时达到最大值。使用特异性半胱天冬酶底物，发现半胱天冬酶-2、-3、-6和-9有显著激活。金丝桃素-PDT诱导凋亡过程中线粒体的参与可通过线粒体膜电位的快速降低得到证实；有趣的是，细胞内三磷酸腺苷（ATP）水平在照射后长达6小时保持在对照水平，表明糖酵解上调作为能量供应的补偿机制。我们的数据有助于更深入地了解用金丝桃素进行光动力治疗后凋亡性细胞死亡所涉及的过程。
