It has previously been shown that the MOR is expressed in GBM cell lines and primary cells [9, 15]. Here, we demonstrate 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 that MOR is expressed in a wide range of GBM cells to a similar extend.
We next asked whether MET is cytotoxic in these cells in MTT experiments including the A172 cell line in which beneficial effects of MET on doxorubicin-induced cytotoxicity has been reported [9]. We obtained very similar IC50 values in all GBM cells in the range from 62 to 130 µM after 72-hour treatment. MET displayed a steep concentration response in all cells (Hill slopes ranging from − 3.3 to -4.4). The lowest IC50 of 62 µM was obtained for A172 cells (Fig. 2a). Annexin V/propidium iodide staining of cell lines treated with the previously determined IC50 MET concentrations revealed that approximately 50% of cell deaths account for apoptosis and necrosis, respectively (Fig. 2b). In order to test if MET-induced cytotoxicity is mediated via MOR, we co-treated A172, U373 and primary cells with the MOR antagonist naloxone and MET. Naloxone was not capable of abolishing MET-induced cytotoxicity (Fig. 2c-e). Contrarily, naloxone even significantly increased MET toxicity in A172 and primary cells (Fig. 2f), indicating that MET does not require the MOR for its cytotoxic action.
To further proof these findings, we treated cells with other MOR agonists used in clinical practice for pain management such as morphine and oxycodone. Strikingly, both compounds showed very weak cytotoxicity in supra-therapeutic concentrations in a subset of GBM cell lines and primary cells (Fig. 3a-d). Determined IC50 values were in the millimolar range or could not be established due to to low cytotoxicity (Fig. 3e).
Friesen et al have previously shown that various MET concentrations (e.g. 10, 3, 1 µg/mL corresponding to ~ 30, 10, 3 µM, respectively) were capable of sensitizing A172 cells towards a fixed doxorubicin concentration (0.3 µg/mL = 0.5 µM) in apoptosis assays. In order to determine a concentration relationship, we treated cells with ascending doxorubicin concentrations combined with 10 µM MET, a concentration which itself is not cytotoxic but close to what might be reached in plasma of patients. MTT concentration-response curves overlapped in both tested GBM cell lines and in primary GBM cells (Fig. 4a-c); respective IC50 values showing no significant differences are displayed in Fig. 4d. However, it has to be noted that in A172 cells at 0.3 µM doxorubicin, combination treatment with 10 µM MET reduced cell viability as compared to doxorubicin treated cells alone (red circle in Fig. 4a). Interestingly, this is close to the concentrations tested by Friesen et al. (0.5 µM doxorubicin combined with 3–30 µM MET) where effects on apoptosis were observed.
Next, we repeated this experiment with previously determined IC50 concentrations of MET (Fig. 2a). High dose MET treatment (60 µM) resulted in a slight left shift of the doxorubicin concentration-response curve in A172 cells (Fig. 5a), while curves overlapped in U87 cells at 100 µM MET (Fig. 5b). Also in primary GBM cells curves largely overlapped independently of treatment (Fig. 5c). Statistical analysis revealed that MET treatment significantly reduced doxorubicin IC50 values in A172 and primary GBM cells indicating a sensitization (Fig. 5d). Looking closer at the curves of primary GBM cells reveals that both curves overlap at most data points except at 1 µM (~ 0.5 µg/mL) doxorubicin (red circle in Fig. 5c), which explains the difference in IC50 values and indicates that a potential synergism of MET and doxorubicin highly depends on cell line and applied drug concentrations.
Doxorubicin is not used in the first-line treatment for GBM. Therefore, we studied the effect of MET on the toxicity of TMZ, which is the clinically relevant treatment option. We treated four GBM cell lines and primary GBM cells with ascending TMZ concentrations in combination with 10 µM MET and performed MTT assays. In A172 cells MET even reduced sensitivity towards TMZ (Fig. 6a) while in all other cells no effect was observed (Fig. 6b-d). The only statistical difference in IC50 values was obtained for A172 cells (Fig. 6e) confirming the negative impact of MET on TMZ treatment in this cell line. Similar results were obtained when we treated cells with an IC50 MET concentration in combination with TMZ (Fig. 7). At this high MET concentrations both, A172 and U373 cell lines, respectively, were less sensitive to TMZ.