Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium-dependent, non-D2 receptor-dependent, manner

Abstract

Dopamine D2 -like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood-brain barrier. Despite a plethora of data reporting anticancer effects for D2 R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D2 receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D2 antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D2 receptors (D2 R) on these cells. Although several chemotypes of D2 R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D2 R antagonist-induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D2 R. Instead, cytotoxicity was associated with a rapid, high-magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D2 R antagonists involves calcium signaling mechanisms, not D2 R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Trial registration: ClinicalTrials.gov NCT02525692.

Keywords: antipsychotic; calcium signaling; dopamine; glioblastoma.

Conflict of interest statement

We report no conflicting interests for this study.

© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

Figures

FIGURE 1

D2R antagonists induce cytotoxicity in glioma cell lines. A, 50% growth inhibitory concentrations (IC50s) for a panel of D2R antagonists of varying chemotype were determined at 48 hours in a panel of glioma cell lines. IC50s were determined via curve‐fitting of MTT concentration response curves generated from 100 nmol/L to 100 µmol/L. These values are presented as a heat map for ease of comparison. Best fit curves were fitted with GraphPad Prism. B, Cell lines showed sensitivity to these compounds at micromolar concentrations, whereas they typically have nanomolar affinities (Ki) for the D2R. A table of hD2R affinities for tested ligands 9 , 10 is presented, along with the fold disparity between observed IC50 concentrations (from A) and Ki. Cytotoxic activity typically is observed only at concentrations of compounds which would be expected to be well beyond fully saturating at the D2R and show high nonselective binding to many other receptors (A and B). C, 4 hours MTT concentration response curve for thioridazine in U87MG, showing cytotoxicity within 4 hours. D, Cell counts for U87MG cells under treatment with 10 µmol/L D2R antagonists. Cells were plated at 20 000 cells/well in 24 well plates and incubated for 24 hours before treatment (treatment at t = 0 hour). Data shown are representative of three independent experiments. ****P < .0001 via two‐way ANOVA with Dunnett's post hoc

FIGURE 2

D2R antagonists induce apoptosis in glioma cell lines. A, Representative plots are presented for annexin V/7‐AAD flow cytometry, measuring apoptotic induction in U251MG cells under treatment with 0, 10, 30, or 100 µmol/L thioridazine for 24 hours. Induction of apoptosis in U87MG cells via treatment with 10 µmol/L thioridazine is time B, and concentration (C) dependent. Flow cytometric experiments were carried out within 1 hour of harvest on a FACSCanto10 flow cytometer, using the PE and Cy‐5 fluorescence channels, gating for whole single cells. Data workup was performed with FlowJo v10. D, 24 hours BrdU incorporation assays were carried out using ELISA methods in U87MG, U251MG, and A‐172 cell lines according to standard protocols. Proliferation (D) is not significantly affected by D2R antagonist treatment until concentrations at which widespread cell death is seen by flow cytometry (A‐C)

FIGURE 3

Dopamine D2 receptor is present and signals in glioma cell lines. A, Western blots indicate the presence of D2R in both mature, glycosylated states (~80 kDa) and immature, lower molecular weight species (~47 kDa) in untreated glioma cell lines (quantified in Figure S1). B, qrt‐PCR for DRD2 and GAPDH transcripts were carried out in untreated glioma cell lines using standard TaqMan probes. Standard deviations for GAPDH expression were less than 0.085. C, 100‐1000 nmol/L D2R agonist sumanirole for 2 hours reduces forskolin‐stimulated cytoplasmic cAMP levels in U87MG, with a IC50 of 250 nmol/L, compared to previously reported EC50 values of 17‐75 nmol/L for cell based assays in D2R expressing CHO cells. 31 Data are representative of 2‐3 independent experiments in triplicate. *P < .05; ****P < .001 via two‐way ANOVA with Dunnett's post hoc

FIGURE 4

Selective D2R antagonist, remoxipride, does not induce cytotoxicity in glioma cell lines. A, Cells were treated with 1 nmol/L to 100 µmol/L remoxipride for 48 hours to generate an MTT concentration response curve. No IC50 could be calculated from generated data due to lack of effect at tested concentrations, but it would be greater than 100 µmol/L. B, At 12‐24 hours, 10 µmol/L remoxipride does not significantly affect the induction of apoptosis by thioridazine as seen by annexin V/7‐AAD flow cytometry. Data are representative of three or more independent experiments in triplicate for MTT and BrdU experiments, while flow cytometry experiments are representative of three independent experiments of 5000‐10 000 cells. Two‐way ANOVA with Sidak's post hoc was used to analyze significance. ns, not significant via two‐way ANOVA with Sidak's post hoc

FIGURE 5

D2R is not involved in cytotoxic responses to D2R antagonists. A, 48 hours MTT concentration response curves were generated from 10 nmol/L to 100 µmol/L for thioridazine in U87MG cells in the presence of 0, 10, 50, or 100 µmol/L ropinirole, a D2R agonist. The presence of ropinirole did not significantly alter thioridazine sensitivity. B, Cotreatment with 30 µmol/L sumanirole, a D2R agonist, did not significantly reduce the induction of apoptosis in U87MG cells treated with 0.3‐100 µmol/L thioridazine for 24 hours and analyzed via annexin V/&‐AAD flow cytometry. C, Cotreatment with 30 µmol/L PHNO, a D2R agonist, did not significantly alter sensitivity of U87MG cells to 0.3‐100 µmol/L pimozide according to 48 hours MTT concentration response curves. D, Knockdown of DRD2 by ~50% (Figure S3D) in U87MG cells via stable lentiviral transduction reduces cell proliferation slightly, but does not significantly affect response to 10 µmol/L thioridazine as measured by cell counts. E, 48 hours MTT concentration response curves are presented for U87MG‐DRDOE and U87MG‐DRD2KD cells under treatment with 0.3‐100 µmol/L thioridazine. No significant differences in response were noted from parental cells. F, 48 hours concentration response curves for U87MG, U87MG‐DRDOE and U87MG‐DRD2KD cells treated with 0.1‐100 µmol/L remoxipride. No significant sensitivity is noted in any cell line used. Data are representative of two independent experiments carried out in triplicate and is analyzed by two‐way ANOVA with Sidak's post hoc. ****P < .0001 via two‐way ANOVA with Sidak's post hoc vs control for series of matched color.; #P < .0001 for matched treatment pairs via two‐way ANOVA with Sidak's post hoc

FIGURE 6

D2R antagonists, other than remoxipride, alter calcium signaling in glioma cell lines. A, U87MG cells were treated with 0.1‐100 µmol/L thioridazine for 48 hours in the presence or absence of 3 µmol/L fendiline, a nonspecific calcium channel blocker and calmodulin inhibitor. MTT concentration response curves suggest a significant sensitization to thioridazine in cells co‐treated with fendiline. Values shown are normalized to vehicle treated cells and represent three independent experiments. B, 10 µmol/L thioridazine induces a strong and sustained increase in cytosolic calcium release as measured by single cell imaging of U87MG cells preloaded with 2 µmol/L Fura‐2 AM for 30 minutes. This increase is due to both ER calcium release (as shown by the increase in the absence of extracellular calcium, in red) and calcium influx from the extracellular space (2 mmol/L CaCl2, in blue). Unlike the positive control, 2 µmol/L thapsigargin (Figure S4A), the cell is unable to clear this signal for a significant amount of time. Traces are presented as the ratio of fluorescence at 340 and 380 nm. C, Thioridazine increases cytosolic calcium signal in a concentration dependent manner in the presence of 0 or 2 mmol/L CaCl2 as measured by Fura‐2 AM fluorescence (shown as maximum ratio of fluorescence at 340 and 380 nm) of U87MG cells preloaded with Fura‐2 AM for 30 minutes as measured by FLIPR assay. D, Maximal calcium responses of selected compounds as measured by Fura‐2 AM FLIPR assay are presented. All compounds induced increases in cytosolic [Ca++] in the presence of 2 mmol/L CaCl2 except for remoxipride. E, Remoxipride does not affect cytoplasmic calcium levels as measured by single cell imaging with Fura‐2 AM as in (B). F, Maximal calcium responses are not significantly altered by overexpression or knockdown of DRD2 in FLIPR imaging of Fura‐2 AM as in C. A is representative of three experiments in triplicate, while single cell imaging experiments are representative of three independent experiments with at least two cover slips and 40 cells. FLIPR assays are representative of at least two independent experiments carried out in quadruplicate. *P < .05; **P < .01; ***P < .005; ****P < .0001 via two‐way ANOVA with Sidak's post hoc

FIGURE 7

Calcium signaling is involved in thioridazine‐induced cytotoxicity. A, Pretreatment of 4000 U87MG cells for 30 minutes with 0‐5 µmol/L BAPTA‐AM concentration‐dependently reduced cytoplasmic calcium signal from 2 µmol/L thapsigargin exposure in U87MG cells via FLIPR assay with Fura‐2 AM preloaded cells. B, 4 hours MTT response for 0‐10 µmol/L thioridazine under cotreatment with 0‐3 µmol/L BAPTA‐AM. C, Pretreatment with 1‐5 µmol/L BAPTA‐AM for 20 minutes reduces the induction of apoptosis by 10 µmol/L thioridazine in U87MG cells via 24 hours cell counts. D, Representative images from CEPIAmt2 and CEPIAer stained U87MG cells treated with 10 µmol/L thioridazine for 0‐15 minutes. E, Quantification of CEPIA staining in U87MG under treatment with 10 µmol/L thioridazine. Results are presented as the ratio of fluorescence over initial fluorescence (F/F0). Similar results were seen with 2 µmol/L thapsigargin (Figure S4C). F, JC‐1 staining for mitochondrial potential indicates a concentration dependent loss of mitochondrial potential within 15 minutes under treatment with 0‐100 µmol/L thioridazine. CCCP, a protonophore, serves as a positive control. 10 µmol/L remoxipride had no effect on mitochondrial membrane potential. G, Release of cytochrome c is induced by treatment with 10‐100 µmol/L thioridazine for 4 hours, as measured by flow cytometry with staining for cytochrome c, to equivalent or greater levels than the positive control, 30 µmol/L etoposide. Experiments were analyzed via two‐way ANOVA with Dunnett's post hoc. *P < .05; **P < .01; ***P < .005; ****P < .0001 via two‐way ANOVA with Sidak's post hoc