Dataset Information

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

ABSTRACT:

Purpose

D,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.

Methods

MOR 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.

Results

MET 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.

Conclusion

MET 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.

SUBMITTER: Haas B

PROVIDER: S-EPMC7872955 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Publications

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

Haas Bodo B Ciftcioglu Janine J Jermar Sanja S Weickhardt Sandra S Eckstein Niels N Kaina Bernd B

Journal of cancer research and clinical oncology 20201214 3

<h4>Purpose</h4>D,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 ...[more]

PMID: 33315125

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Project description:Methadone is an analgesic drug used for pain treatment and heroin substitution. Recently, methadone has been proposed to be useful also for cancer therapy, including glioblastoma multiforme (GBM), the most severe form of brain cancer, because experiments on cultured glioma cells treated with doxorubicin showed promising results. Doxorubicin, however, is not used first-line in GBM therapy. Therefore, we analyzed the cytotoxic effect of methadone alone and in combination with temozolomide, a DNA-alkylating drug that is first-line used in GBM treatment, utilizing GBM-derived cell lines and a human fibroblast cell line. We show that methadone is cytotoxic on its own, inducing apoptosis and necrosis, which was observed at a concentration above 20 µg/mL. Methadone was similar toxic in isogenic MGMT expressing and non-expressing cells, and in LN229 glioblastoma and VH10T human fibroblasts. The apoptosis-inducing activity of methadone is not bound on the opioid receptor (OR), since naloxone, a competitive inhibitor of OR, did not attenuate methadone-induced apoptosis/necrosis. Administrating methadone and temozolomide together, temozolomide had no impact on methadone-induced apoptosis (which occurred 3 days after treatment), while temozolomide-induced apoptosis (which occurred 5 days after treatment) was unaffected at low (non-toxic) methadone concentration (5 µg/mL), and at high (toxic) methadone concentration (20 µg/mL) the cytotoxic effects of methadone and temozolomide were additive. Methadone is not genotoxic, as revealed by comet and ?H2AX assay, and did not ameliorate the genotoxic effect of temozolomide. Further, methadone did not induce cellular senescence and had no effect on temozolomide-induced senescence. Although methadone was toxic on senescent cells, it cannot be considered a senolytic drug since cytotoxicity was not specific for senescent cells. Finally, we show that methadone had no impact on the MGMT promoter methylation. Overall, the data show that methadone on glioblastoma cells in vitro is cytotoxic and induces apoptosis/necrosis at doses that are above the level that can be achieved in vivo. It is not genotoxic, and does not ameliorate the cell killing or the senescence-inducing effect of temozolomide (no synergistic effect), indicating it has no impact on temozolomide-induced signaling pathways. The data do not support the notion that concomitant methadone treatment supports temozolomide-based chemotherapy.

Project description:Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α v activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α v activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α v -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α v knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α v in GBM cells.

Dataset Information

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

Purpose

Methods

Results

Conclusion

Publications

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

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