Journal of Neuro-Oncology

, Volume 108, Issue 3, pp 443–450

Phenytoin reduces 5-aminolevulinic acid-induced protoporphyrin IX accumulation in malignant glioma cells

Laboratory Investigation

Abstract

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.

Keywords

Glioblastoma multiforme Fluorescence-guided surgery Photodynamic therapy 5-Aminolevulinic acid Antiepileptic drugs Phenytoin 

Abbreviations

5-ALA

5-Aminolevulinic acid

AEDs

Antiepileptic drugs

DMSO

Dimethyl sulfoxide

FCS

Fetal calf serum

GBM

Glioblastoma multiforme

GSH

Reduced glutathione

GSSG

Oxidized glutathione

LEVE

Levetiracetam

MMP

Mitochondrial membrane potential

PDD

Photodynamic diagnosis

PDT

Photodynamic therapy

PHY

Phenytoin

PpIX

Protoporphyrin IX

RFU

Relative fluorescence units

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Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  1. 1.Department of NeurosurgeryUniversity Hospital Schleswig HolsteinKielGermany
  2. 2.Institute of BiotechnologyZurich University of Applied SciencesWaedenswilSwitzerland
  3. 3.Klinik Am RosenbergHeidenSwitzerland

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