Advertisement

d,l-Methadone does not improve radio- and chemotherapy in glioblastoma in vitro

  • Henry Oppermann
  • Martina Matusova
  • Annegret Glasow
  • Johannes Dietterle
  • Rainer Baran-Schmidt
  • Karsten Neumann
  • Jürgen Meixensberger
  • Frank GaunitzEmail author
Original Article

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.

Keywords

d,l-Methadone Glioblastoma Temozolomide X-irradiation Standard therapy 

Notes

Acknowledgements

We like to thank Astrid Birnbaum (Department of Pathology, Medical Clinic Dessau) for determining the methylation of the MGMT promotor.

Compliance with ethical standards

Conflict of interest

The authors report no conflicts of interest in this work.

Informant consent

All patients provided written informed consent according to German law as confirmed by the local committee (#144-2008) in accordance with the 1964 Helsinki declaration and its later amendments.

Supplementary material

280_2019_3816_MOESM1_ESM.jpg (21.9 mb)
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)
280_2019_3816_MOESM2_ESM.jpg (3.3 mb)
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)
280_2019_3816_MOESM3_ESM.jpg (3.3 mb)
Supplementary material 3 (JPG 3347 KB)
280_2019_3816_MOESM4_ESM.jpg (2 mb)
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)
280_2019_3816_MOESM5_ESM.jpg (249 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)

References

  1. 1.
    Ostrom QT, Gittleman H, Xu J et al (2016) CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2009–2013. Neuro-Oncology 18(suppl_5):v1–v75.  https://doi.org/10.1093/neuonc/now207 CrossRefGoogle Scholar
  2. 2.
    Stupp R, Mason WP, van den Bent MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996CrossRefGoogle Scholar
  3. 3.
    Stupp R, Taillibert S, Kanner A et al (2017) Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: a randomized clinical trial. JAMA 318(23):2306–2316.  https://doi.org/10.1001/jama.2017.18718 CrossRefGoogle Scholar
  4. 4.
    Hübner J, Hartmann M, Wedding U et al (2017) Methadon in der Onkologie: “Strohhalmfunktion” ohne Evidenz. Dtsch Arztebl International 114(33–34):A–A1530Google Scholar
  5. 5.
    Winkler E (2017) Methadon gegen Krebs—auch eine Frage für die Ethik und Theorie der Medizin. Ethik Med 29(4):269–272.  https://doi.org/10.1007/s00481-017-0460-x CrossRefGoogle Scholar
  6. 6.
    Friesen C, Hormann I, Roscher M et al (2014) Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma. Cell Cycle 13(10):1560–1570.  https://doi.org/10.4161/cc.28493 CrossRefGoogle Scholar
  7. 7.
    Onken J, Friesen C, Vajkoczy P et al (2017) Safety and tolerance of d,l-methadone in combination with chemotherapy in patients with glioma. Anticancer Res 37(3):1227–1235.  https://doi.org/10.21873/anticanres.11438 CrossRefGoogle Scholar
  8. 8.
    Brawanski K, Brockhoff G, Hau P et al (2018) Efficacy of d,l-methadone in the treatment of glioblastoma in vitro. CNS Oncol.  https://doi.org/10.2217/cns-2018-0006 Google Scholar
  9. 9.
    Oppermann H, Dietterle J, Purcz K et al (2018) Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts. Cancer Cell Int 18:111.  https://doi.org/10.1186/s12935-018-0611-2 CrossRefGoogle Scholar
  10. 10.
    Arai H, Ikota H, Sugawara K-i et al (2012) Nestin expression in brain tumors: its utility for pathological diagnosis and correlation with the prognosis of high-grade gliomas. Brain Tumor Pathol 29(3):160–167.  https://doi.org/10.1007/s10014-012-0081-5 CrossRefGoogle Scholar
  11. 11.
    Schiffer D, Giordana MT, Germano I et al (1986) Anaplasia and heterogeneity of GFAP expression in gliomas. Tumori 72(2):163–170CrossRefGoogle Scholar
  12. 12.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675CrossRefGoogle Scholar
  13. 13.
    Merz F, Gaunitz F, Dehghani F et al (2013) Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments. Neuro-Oncology 15(6):670–681.  https://doi.org/10.1093/neuonc/not003 CrossRefGoogle Scholar
  14. 14.
    Oppermann H, Alvanos A, Seidel C et al (2018) Carnosine influences transcription via epigenetic regulation as demonstrated by enhanced histone acetylation of the pyruvate dehydrogenase kinase 4 promoter in glioblastoma cells. Amino Acids.  https://doi.org/10.1007/s00726-018-2619-2 Google Scholar
  15. 15.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B (Methodol) 57(1):289–300Google Scholar
  16. 16.
    Ferrer-Alcón M, La Harpe R, García-Sevilla JA (2004) Decreased immunodensities of micro-opioid receptors, receptor kinases GRK 2/6 and beta-arrestin-2 in postmortem brains of opiate addicts. Brain Res Mol Brain Res 121(1–2):114–122.  https://doi.org/10.1016/j.molbrainres.2003.11.009 CrossRefGoogle Scholar
  17. 17.
    Saify K, Saadat M (2016) Expression levels of OPRM1 and PDYN in human SH-SY5Y cells treated with morphine and methadone. Life Sci 150:39–41.  https://doi.org/10.1016/j.lfs.2016.02.078 CrossRefGoogle Scholar
  18. 18.
    Eap CB, Buclin T, Baumann P (2002) Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence. Clin Pharmacokinet 41(14):1153–1193.  https://doi.org/10.2165/00003088-200241140-00003 CrossRefGoogle Scholar
  19. 19.
    Lugo RA, Satterfield KL, Kern SE (2005) Pharmacokinetics of methadone. J Pain Palliat Care Pharmacother 19(4):13–24.  https://doi.org/10.1080/J354v19n04_05 CrossRefGoogle Scholar
  20. 20.
    Milroy CM (2000) Methadone deaths: a toxicological analysis. J Clin Pathol 53(4):277–281.  https://doi.org/10.1136/jcp.53.4.277 CrossRefGoogle Scholar
  21. 21.
    Kreye G, Masel E-K, Hackner K et al (2018) Methadon als antitumortherapie: Schwindel, Hoffnung oder Risiko?: Eine Serie von Kasuistiken und kurzer Überblick über aktuelle Literatur sowie Empfehlungen der Fachgesellschaften (Methadone as anticancer treatment: hype, hope, or hazard?: a series of case reports and a short review of the current literature and recommendations of the societies). Wien Med Wochenschr 168(7–8):159–167.  https://doi.org/10.1007/s10354-018-0623-5 CrossRefGoogle Scholar
  22. 22.
    Tacar O, Sriamornsak P, Dass CR (2013) Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol 65(2):157–170.  https://doi.org/10.1111/j.2042-7158.2012.01567.x CrossRefGoogle Scholar
  23. 23.
    Michalska M, Schultze-Seemann S, Kuckuck I et al (2018) Impact of methadone on cisplatin treatment of bladder cancer cells. Anticancer Res 38(3):1369–1375.  https://doi.org/10.21873/anticanres.12360 Google Scholar
  24. 24.
    Brüggen M-C, Mangana J, Irmisch A et al (2018) Methadone-Not a magic bullet in melanoma therapy. Exp Dermatol 27(6):694–696.  https://doi.org/10.1111/exd.13543 CrossRefGoogle Scholar
  25. 25.
    Maneckjee R, Minna JD (1992) Nonconventional opioid binding sites mediate growth inhibitory effects of methadone on human lung cancer cells. Proc Natl Acad Sci USA 89(4):1169–1173CrossRefGoogle Scholar
  26. 26.
    Renner C, Seyffarth A, Arriba S de et al (2008) Carnosine inhibits growth of cells isolated from human glioblastoma multiforme. Int J Pept Res Ther 14:127–135.  https://doi.org/10.1007/s10989-007-9121-0 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of NeurosurgeryUniversity Hospital LeipzigLeipzigGermany
  2. 2.Department of RadiooncologyUniversity of LeipzigLeipzigGermany
  3. 3.Department of PathologyMedical Clinic DessauDessauGermany

Personalised recommendations