Abstract
Purpose
Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Median survival of glioblastoma patients under standard therapy including radiotherapy and chemotherapy using temozolomide (TMZ) is 14.6 months. As cell culture experiments combining d,l-methadone with doxorubicin demonstrated an increased reduction of cell viability of glioblastoma cells, the opioid has been discussed as a drug for the treatment of GBM. Despite lack of clinical and experimental evidence that d,l-methadone in combination with standard therapy will be beneficial, an increasing number of tumor patients medicating themselves with d,l-methadone present to the hospitals in Germany.
Methods
As a first step towards understanding whether d,l-methadone may increase the efficacy of standard therapy, we used a cell culture model of primary GBM and fibroblast cell cultures derived from GBM patients. The cultures were treated with different concentrations of d,l-methadone in combination with X-irradiation, TMZ or both. Cell viability was determined by measuring ATP in cell lysates and dehydrogenase activity in living cells.
Results
When only treated with d,l-methadone, 1 µM of the opioid was sufficient to reduce viability of fibroblasts, whereas 10 µM was needed to significantly reduce glioblastoma cell viability. In addition, d,l-methadone did not improve the anti-neoplastic effects of X-irradiation, temozolomide or both.
Conclusions
As d,l-methadone reduces glioblastoma cell viability only when concentrations are used that had been reported to be toxic to patients and as there were no interactions observable combining it with standard therapy, a recommendation for the use of d,l-methadone in glioblastoma therapy cannot be given.
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Acknowledgements
We like to thank Astrid Birnbaum (Department of Pathology, Medical Clinic Dessau) for determining the methylation of the MGMT promotor.
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Supplemental Figure 1. Expression of GFAP and Nestin in the cell cultures used for the experiments
. Glial fibrillary acidic protein (GFAP) and nestin protein expression were analyzed by immunofluorescence (scale bar: 15 µm). Nestin is detected by red fluorescence and GFAP by green fluorescence. Nuclei are visualized by blue fluorescence. Note: GFAP was only present in cells from the culture P0560 cells (P0560 + GFAP). (JPG 22441 KB)
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Supplemental Figure 2a/b. Viability of patient derived fibroblasts and glioblastoma cells under the influence of D,L-methadone, X-irradiation and TMZ. Glioblastoma cells and fibroblast cultures from patients 1 to 6 were treated with different concentrations of D,L-methadone (0 nM, 1 nM, 10 nM, 100 nM, 1 µM, 5 µM, 10 or 30 µM) in combination with X-irradiation (0 or 4 Gy) and/or temozolomide (TMZ; 200 µM). As a measure of viability the amount of ATP in cell lysates (a) and the activity of dehydrogenases in living cells (b) were determined after 144 hours of incubation. Each data point represents the average and standard deviation determined from six independent wells. (JPG 3377 KB)
Supplemental Figure 3: Comparison of the effect of X-irradiation, TMZ and a combination of both treatment modalities at different concentrations of D,L-methadone on glioblastoma cells
. The data presented in Supplemental Figures 2a and 2b was used to calculate the viability of cells treated with X-irradiation, TMZ and a combination of both treatment modalities at different D,L-methadone concentrations compared to cells treated with D,L-methadone of the same concentration alone (set as 100%). (JPG 2025 KB)
Supplemental Figure 4: Viability of patient derived glioblastoma cells with MGMT promoter methylation under the influence of D,L-methadone, X-irradiation and TMZ
. The glioblastoma cell culture P0023 was treated with or without temozolomide (TMZ; 200 µM), various concentrations of D,L-methadone (1, 5, 10 or 30 µM) and a dose of X-irradiation of 0 or 4 Gy. After 144 hours of incubation, cell viability was determined by measuring ATP in cell lysates (a) or by determining dehydrogenase activity of living cells (b). Bars represent the average and standard deviation of the measurements from six independent wells. Viability of cells that did neither received D,L-methadone nor TMZ and were also not irradiated was set to 100 percent viability. Asterisks indicate statistical significance with adjusted p values according to Benjamini and Hochberg between different treatments indicated by the small bars above the panel (*: p < 0.05; ns: not significant). (JPG 249 KB)
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Oppermann, H., Matusova, M., Glasow, A. et al. d,l-Methadone does not improve radio- and chemotherapy in glioblastoma in vitro. Cancer Chemother Pharmacol 83, 1017–1024 (2019). https://doi.org/10.1007/s00280-019-03816-3
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DOI: https://doi.org/10.1007/s00280-019-03816-3