Expression Levels of ALA Dehydratase as a Marker of ALA-PDT Efficacy

Abstract

Accelerated synthesis of protoporphyrinIX (PpIX) following ALA pre‐treatment followed by light irradiation is the principle of ALA‐PDT. Several limiting enzymes were suggested to control PpIX accumulation and PDT efficacy, among them porphobilinogen deaminase (PBGD) and ferrochelatase. Here we reveal the centrality of ALA dehydratase (ALAD) activity in predicting ALA‐PDT efficacy. Silencing of ALAD expression and activity was carried out in leukemic cells using shRNA plasmid transfection or Pb2+ intoxication. ALAD activity, porphyrin synthesis and mitochondrial activity were determined versus PDT efficacy.In K562 ALAD‐silenced cells, ALAD activity and expression were reduced and as a result, PpIX synthesis was almost abolished. Following ALA treatment and irradiation, ALAD‐silenced cells depicted normal mitochondrial activity, in contrast to control and non‐silencing transfected cells where accumulated PpIX and irradiation caused ROS formation and mitochondrial damage. Morphological analysis by scanning electron microscopy (SEM) of ALA‐PDT treated cells showed no morphological changes in ALAD‐silenced cells, while controls exhibited cell deformations and lysis. Annexin V‐FITC/PI staining as well as LDH‐L leakage testing showed that membrane integrity was undamaged following ALA‐PDT in ALAD silenced cells. Pb2+ treatment in MEL cells impaired ALAD activity and reduced PpIX synthesis but to a lesser extent.In conclusion, we show that a dramatic reduction in PpIX accumulation following down regulation of ALAD expression prevents an efficient PDT. Thus, ALAD has a major role in regulating PpIX synthesis and ALA‐PDT therapeutic outcome. Monitoring ALAD expression or activity in various tumors may be useful as prognostic tool to predict PDT efficacy.