Pharmacological Modulation of 5-ALA-Induced PpIX in GBM Cells

Glioblastoma (GBM) is one of the most frequent and most devastating brain tumors. We have previously shown that expression of different status of epidermal growth factor receptor (EGFR) in GBM cell lines reduces 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence by influencing the rate limiting enzyme heme oxygenase-1 (HO-1).¹ We hypothesized that 5-ALA-induced PpIX fluorescence can be pharmacologically influenced by adding different drugs.

U87MG, U87wtEGFR, U87vIII (GBM cell lines) having different EGFR expression status were exposed to exogenous 5-ALA (1 mM) and different pharmacological conditions such as exposition to DFO (iron chelator of Fe2+), SnPP (HO-1 inhibitor), genistein (ABC transporter G2 inhibitor), and stimulation with EGF. Cell lines were exposed to 5-ALA and PpIX fluorescence was monitored over time in the cells and in the cell culture medium (CM). After 24 hours, the medium was removed and PpIX washout was measured. As regards the treatments, cells were incubated with EGF (10 ng/mL) for 18 hours and in the last 4 hours cells are co-treated with 5-ALA. Whereas the treatments with 5-ALA plus DFO and/or SnPP and Genistein were performed incubating cells for 8 hours. All samples were analyzed with the microplate reader.

5-ALA-induced fluorescence was observed in U87MG (low EGFR expression), U87wtEGFR cells (EGFR overexpression), and in U87vIII (EGFR overexpression/EGFRvIII+). We observed a significant increase in PpIX in all the cell lines between 8 and 24 hours of 5-ALA treatment. At the same time, we noticed that GBM cells start to release PpIX in the CM. After removal of the 5-ALA stimulation, there was a significant reduction in PpIX level, in particular in the first hour. Whereas we saw an increased amount of PpIX in the CM at the same time. After 24 hours, the whole amount of PpIX was secreted by all the cell lines. On the contrary, treatment of U87MG cells with EGF lead to reduced cellular fluorescence, by promoting HO-1 transcription and expression. Remarkably, inhibition of HO-1 activity by SnPP treatment was able to restore the fluorescence in all cell lines. We observed a major increase in PpIX fluorescence in U87vIII with respect to the other cells when we treat with drugs.

This approach could be used to determine the optimal time point for the PpIX visualization after 5-ALA induction. Moreover, it could be very interesting to test different combination of these drugs to better improve the accumulation of PpIX in GBM tumor cells and their visualization during the surgery.

Acknowledgment: This study is supported by Lega Ticinese contro il Cancro, Fondazione Gabriel Charitable, and Fondo Scientifico Neurochirurgia.

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