Phenytoin reduces 5-aminolevulinic acid-induced protoporphyrin IX accumulation in malignant glioma cells
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Epileptic seizures are among the presenting clinical signs of malignant glioma patients, frequently necessitating treatment with antiepileptic drugs (AEDs). The efficacy of 5-aminolevulinic acid (5-ALA)-based intraoperative fluorescence-guided surgery and photodynamic therapy (PDT) in glioblastoma multiforme (GBM) patients depends on the specific accumulation and total amount of intracellularly synthesized protoporphyrin IX (PpIX) in tumour cells. In this study, we investigated the effect of the AEDs phenytoin (PHY) and levetiracetam (LEVE) on 5-ALA-induced PpIX accumulation in two glioma cell lines (U373 MG and U-87 MG) and primary GBM cells isolated from a human biopsy. After treatment with PHY and LEVE for three days cells were incubated with 1 mM 5-ALA for 4 h and PpIX accumulation was determined by fluorescence measurement. We observed a decrease in PpIX synthesis of up to 55 ± 12 % in primary GBM cells after incubation with phenytoin. This reduction was dose-dependent for all tested cell lines and primary GBM cells. LEVE on the other hand did not alter PpIX concentration in GBM cells. PDT was performed in vitro by irradiating the GBM cells with light doses from 0.5 to 10 J cm−2 at 627 nm after AED and 5-ALA treatment. Although less PpIX accumulated in PHY-treated cells, efficacy of PDT was not affected. We assume that damage to the mitochondrial membrane by PHY inhibits PpIX synthesis in vitro, because we showed mitochondrial dysfunction as a result of reduced mitochondrial membrane potential in PHY-treated cells. No change in glutathione status was observed. To evaluate further the effect of PHY on PpIX fluorescence, and to establish its significance in clinical practice, animal and clinical studies are required, because the results presented here imply PHY may reduce intracellular accumulation of PpIX in patients with high-grade gliomas.
KeywordsGlioblastoma multiforme Fluorescence-guided surgery Photodynamic therapy 5-Aminolevulinic acid Antiepileptic drugs Phenytoin
Fetal calf serum
Mitochondrial membrane potential
Relative fluorescence units
Development of the photodynamic device used in this study was financially supported by the Swiss Innovation Promotion Agency (CTI) and by Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland. The authors thank Professor Herbert Looser from the School of Engineering FHNW in Windisch for constructing the photodynamic device. We are much obliged to Medac GmbH, Wedel, Germany for providing Gliolan® (5-ALA). The authors thank Rebecca Wyer, Marino Angelozzi, and Jenny Pally-Eggenschwiler for their experimental support.
Conflict of interest
The authors declare that they have no conflict of interest.
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