Silencing of ALA dehydratase affects ALA-photodynamic therapy efficacy in K562 erythroleukemic cells
Silencing of ALA dehydratase affects ALA-photodynamic therapy efficacy in K562 erythroleukemic cells
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DOI：
10.1039/b9pp00007k
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发表时间：
2009-01-01
影响因子：
3.1
通讯作者：
Malik, Zvi
中科院分区：
文献类型：
--
作者：
Feuerstein, Tamar;Schauder, Avital;Malik, Zvi
肿瘤细胞合成原卟啉IX(PPIX)是ALA光动力疗法(PDT)成功的关键。在ALA处理过程中，影响PPIX积累的两个关键酶是胆红素原脱氨酶(PBGD)和铁络合酶。在这里，我们发现，特定的shRNA下调ALA脱水酶(ALAD)的表达和活性，导致K562红白血病细胞PpIX合成显著减少。ALA-PDT后的光灭活效果与ALAD沉默和PpIX细胞水平直接相关。ALA-PDT后，ALAD沉默细胞的MTT代谢率比对照细胞高60%，表明沉默细胞中线粒体受到保护。扫描电子显微镜对ALA-PDT处理的细胞的形态分析显示，ALAD沉默的细胞没有形态变化，而对照组则表现为细胞变形和裂解。Annexin V-FITC/PI染色和乳酸脱氢酶L漏出量检测显示，ALA-PDT后的细胞膜完整性保持完好，未受损伤。我们得出的结论是，尽管ALAD在细胞中以丰富的水平存在，但在调节PpIX合成和ALA-PDT结果方面具有重要的和有限的作用。
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.