Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel.
Photochem Photobiol Sci. 2009 Oct;8(10):1461-6. doi: 10.1039/b9pp00007k. Epub 2009 Aug 17.
Synthesis of protoporphyrin IX (PpIX) by malignant cells is essential for the success of ALA-based photodynamic therapy (PDT). Two key enzymes that were described as affecting PpIX accumulation during ALA treatment are porphobilinogen deaminase (PBGD) and ferrochelatase. Here, we show that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells. Photo-inactivation efficacy following ALA-PDT was directly correlated with ALAD-silencing and cellular levels of PpIX. MTT metabolism following ALA-PDT was shown to be 60% higher in ALAD-silenced cells in comparison to control cells, indicating that mitochondria were protected in the silenced cells. Morphological analysis by scanning electron microscopy (SEM) of cells treated by ALA-PDT showed no morphological changes in ALAD-silenced cells, in contrast to controls exhibiting cell deformations and lysis. Membrane integrity following ALA-PDT was kept intact and undamaged in ALAD-silenced cells as examined by Annexin V-FITC/PI staining and LDH-L leakage. We conclude that ALAD, although it is present in the cell at abundant levels, has a major and limiting role in regulating PpIX synthesis and ALA-PDT outcome.
恶性细胞中原卟啉 IX（PpIX）的合成对于 ALA 基光动力疗法（PDT）的成功至关重要。有两种关键酶被描述为影响 ALA 治疗期间 PpIX 积累，即卟胆原脱氨酶（PBGD）和亚铁螯合酶。在这里，我们表明，通过特异性 shRNA 下调 ALA 脱水酶（ALAD）的表达和活性，可显著降低 K562 红白血病细胞中 PpIX 的合成。ALA-PDT 后的光灭活功效与 ALAD 沉默和细胞内 PpIX 水平直接相关。与对照细胞相比，ALA-PDT 后 MTT 代谢在 ALAD 沉默细胞中高 60%，表明沉默细胞中的线粒体得到了保护。ALA-PDT 处理细胞的扫描电子显微镜（SEM）形态分析显示，ALAD 沉默细胞无形态变化，而对照细胞则表现出细胞变形和裂解。ALA-PDT 后，通过 Annexin V-FITC/PI 染色和 LDH-L 渗漏检测，发现 ALAD 沉默细胞中的膜完整性保持完整且未受损。我们得出结论，尽管 ALAD 在细胞中大量存在，但它在调节 PpIX 合成和 ALA-PDT 结果方面具有主要且限制作用。
