Methadone-mediated sensitization of glioblastoma cells is drug and cell line dependent

Bodo Haas,Bernd Kaina,Sanja Jermar,Niels Eckstein,Sandra Weickhardt,Janine Ciftcioglu

doi:10.1007/s00432-020-03485-3

Abstract

Purposed,l-methadone (MET), an analgesic drug used for pain treatment and opiate addiction, has achieved attention from oncologists and social media as possible chemoensitizing agent in cancer therapy, notably brain cancer (glioblastoma multiforme, GBM). MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR). Here, we extended this work and quantified the toxic effect of MET in comparison to other opioids alone and in combination with doxorubicin and the clinically more relevant alkylating drug temozolomide (TMZ), using a set of GBM cell lines and primary GBM cells.MethodsMOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone and in combination with anticancer drugs was assessed by MTT assays. Concentration effect curves were fitted by nonlinear regression analysis and IC50 values were calculated. Apoptosis and necrosis rates were determined by annexin V/propidium iodide (PI)-flow cytometry.ResultsMET alone was cytotoxic in all GBM cell lines and primary GBM cells at high micromolar concentrations (IC50 ~ 60–130 µM), observed both in the metabolic MTT assay and by quantifying apoptosis and necrosis, while morphine and oxycodone were not cytotoxic in this concentration range. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death-inducing effects of MET are not MOR-dependent. We recorded doxorubicin and TMZ concentration- response curves in combination with fixed MET concentrations. MET enhanced doxorubicin-induced cytotoxicity in only one cell line, and in primary cells it was observed only in a particular MET concentration range. In all assays, MET was not effective in sensitizing cells to TMZ. In two cell lines, MET even decreased the cell's sensitivity to TMZ.ConclusionMET was found to be cytotoxic in GBM cells in vitro only at high, clinically not relevant concentrations, where it was effective in inducing apoptosis and necrosis. Sensitizing effects were only observed in combination with doxorubicin, but not with TMZ, and are dependent on cell line and the applied drug concentration. Therefore, our findings do not support the use of MET in the treatment of GBM in combination with TMZ, as no sensitizing effect of MET was observed.

Highlights

Grade IV glioma is the most aggressive form of brain cancer with the highest incidence among adults (Louis et al 2016; Siegel et al 2020)
Previous studies indicate that d,lmethadone (MET), an analgesic drug used for pain treatment and opiate addiction (Krantz and Mehler 2004; Parsons et al 2010), increases apoptosis of leukemia cells and the cytotoxic effects of the topoisomerase II-inhibitor doxorubicin (Friesen et al 2008, 2013; Singh et al 2011)
We demonstrate the expression of MOR in commonly used GBM cell lines (U87, U251, U373) and primary GBM cells isolated from a patient biopsy by Western blotting (Fig. 1a) and immunocytochemistry (Fig. 1b), confirming and extending the finding that MOR is expressed in a wide range of GBM cells to a similar extent

Summary

Introduction

Grade IV glioma (glioblastoma multiforme, GBM) is the most aggressive form of brain cancer with the highest incidence among adults (Louis et al 2016; Siegel et al 2020). Previous studies indicate that d,lmethadone (MET), an analgesic drug used for pain treatment and opiate addiction (Krantz and Mehler 2004; Parsons et al 2010), increases apoptosis of leukemia cells and the cytotoxic effects of the topoisomerase II-inhibitor doxorubicin (Friesen et al 2008, 2013; Singh et al 2011). The same group showed that the opioid has the potential to enhance apoptosis induced by doxorubicin in GBM cells (Friesen et al 2014). CAMP displays pro- and anti-apoptotic effects depending on cell type (Insel et al 2012), raising the question if all GBM cells respond to MET treatment. It was shown that MET increases intracellular doxorubicin levels probably by inhibiting P-glycoproteins (P-gp) in GBM cells (Friesen et al 2014). Data published by others indicate no sensitizing effect of MET on various doxorubicin-treated canine tumor cells (Cueni et al 2020)

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