Anticancer Properties of the Antipsychotic Drug Chlorpromazine and Its Synergism With Temozolomide in Restraining Human Glioblastoma Proliferation In Vitro

Abstract

The extremely poor prognosis of patients affected by glioblastoma (GBM, grade IV glioma) prompts the search for new and more effective therapies. In this regard, drug repurposing or repositioning can represent a safe, swift, and inexpensive way to bring novel pharmacological approaches from bench to bedside. Chlorpromazine, a medication used since six decades for the therapy of psychiatric disorders, shows in vitro several features that make it eligible for repositioning in cancer therapy. Using six GBM cell lines, three of which growing as patient-derived neurospheres and displaying stem-like properties, we found that chlorpromazine was able to inhibit viability in an apoptosis-independent way, induce hyperdiploidy, reduce cloning efficiency as well as neurosphere formation and downregulate the expression of stemness genes in all these cell lines. Notably, chlorpromazine synergized with temozolomide, the first-line therapeutic in GBM patients, in hindering GBM cell viability, and both drugs strongly cooperated in reducing cloning efficiency and inducing cell death in vitro for all the GBM cell lines assayed. These results prompted us to start a Phase II clinical trial on GBM patients (EudraCT # 2019-001988-75; ClinicalTrials.gov Identifier: NCT04224441) by adding chlorpromazine to temozolomide in the adjuvant phase of the standard first-line therapeutic protocol.

Keywords: antipsychotic drugs (APDs); cancer stem cells (CSC); clinical trials; drug repurposing and repositioning; drug synergism; glioblastoma; neurospheres.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Matteoni, Matarrese, Ascione, Buccarelli, Ricci-Vitiani, Pallini, Villani, Pace, Paggi and Abbruzzese.

Figures

Figure 1

CPZ reduces cell viability in GBM cells. (A). Representative dose–response curves of all cell lines treated with CPZ for 48 h are shown. GBM anchorage-dependent cell lines are represented by black lines, while RPE-1 by a green line and neurospheres by red ones. (B). Table showing μM CPZ concentrations corresponding to the IC30 and IC50 calculated for each cell line. Values are expressed as mean ± SE.

Figure 2

CPZ induces alterations of the cell cycle and hyperdiploidy in GBM cells. Anchorage-dependent GBM cell lines [panels (A) and (B)] or neurospheres [panels (C) and (D)] underwent treatment with CPZ at the calculated IC30 for 48 h. Flow cytometry analysis of the cell cycle. Left panels (A), (C). Histograms obtained in a representative experiment. Numbers represent the percentage of cells in the different phases of the cell cycle or hyperdiploid cells. Right panels (A), (C). Bar graphs showing mean ± SE of data obtained from three independent experiments. Left panels (B), (D). Representative micrographs of the analysis of nuclear morphology performed after cell staining with Hoechst 33258. Right panels (B), (D). Bar graphs showing the quantification of hyperdiploid cells performed by counting at least 50 cells from 10 different fields observed with a 40× objective. Data are reported as mean ± SE of data obtained from three independent experiments. Statistical significance is referred toward the Control (*** p < 0.001).

Figure 3

CPZ reduces cloning efficiency in GBM cells. (A). Colony-forming assay. Anchorage-dependent cells were treated with increasing doses of CPZ, as indicated. At the end of the experiment, cells were stained and counted. The results are reported in the histogram on the far right of the panel along with statistical significance referred toward the Control (** p < 0.01; *** p < 0.001). (B). Neurosphere formation assay. Cells were treated with increasing doses of CPZ, as indicated. Of note, TS#83 neurospheres displayed a partial anchorage-dependent growing capability. At the end of the experiment, neurospheres were photographed under an inverted microscope using a 4× objective.

Figure 4

CPZ downregulates stemness gene expression in GBM cells. (A). Expression of stemness genes in the three stem-like cell lines (neurospheres). (B). Expression of stemness genes in the three anchorage-dependent cell lines. In all cases, determinations were performed via qRT-PCR after 24 h of exposure to CPZ. Histogram values represent the fold-changes referred to the respective value for untreated cells (Control), arbitrarily reported as 1.0 (gray columns on the left of each graph). Statistical significance is referred toward the Control (* p < 0.05; ** p < 0.01; *** p < 0.001). In TS#83 cells and U-251 MG cells, in panel A and in panel B respectively, the amount of OLIG2 mRNA was undetectable in Control as well in CPZ-treated cells.

Figure 5

CPZ synergizes with TMZ in reducing GBM cell viability. Anchorage-dependent GBM cell lines (A) and neurospheres (B). Dose–response effects of CPZ (blue), TMZ (green) and TMZ plus a constant (k) CPZ concentration, indicated for each cell line (red) on percent cell viability (left panels). Histograms show cell viability at selected drug concentrations, as indicated, to highlight the effect of the association of the two drugs (right panels). The effects of TMZ and CPZ combination were considered synergistic when the CI was <0.8 (in red). CPZ k values were 6.0, 6.0, 4.0, 10.0, 8.0 and 5.0 μM for T98G, U-87 MG, U-251 MG, TS#1, TS#83 and TS163 GBM cells, respectively.

Figure 6

CPZ cooperates with TMZ in inducing cell death. Anchorage-dependent GBM cell lines (A) and neurospheres (B) were analyzed after treatment with TMZ (96 h) and CPZ (48 h), or their combination, at the lowest concentrations considered synergistic on the basis of the viability analysis. Left panels. FACS analysis after staining with Calcein-AM (which is retained in the cytoplasm of live cells). Numbers represent the percentage of Calcein-negative cells (dead cells). One representative experiment is shown. Bar graphs below show the results obtained from four independent experiments, reported as means ± SE. Right panels. FACS analysis after double staining with Annexin V/PI. Dot plots from a representative experiment are shown. Numbers represent the percentages of Annexin V-positive cells (bottom right quadrant), Annexin V/PI double positive cells (upper right quadrant), or PI-positive cells (upper left quadrant). Note the high percentage of cells positive for PI in CPZ treated cells. Bar graphs below show results obtained from four independent experiments, reported as means ± SE. ** p < 0.01 and *** p < 0.001 indicate significant differences vs single drug treatments (TMZ or CPZ).

Figure 7

CPZ cooperates with TMZ in reducing GBM cell cloning efficiency. (A). Anchorage-dependent cells T98G, U-87 MG and U-251 MG were exposed to solvent(s) (Control), TMZ, CPZ or their association at the doses indicated, then rinsed and allowed to grow in the absence of drugs for the subsequent 12 d. Cell colonies, after staining with crystal violet (left), were counted, and the values reported as percent colony number in the histogram (right). In these panels, variance among groups was assessed via the Bartlett’s test for equal variances. Statistical analysis among groups was done using the One-way ANOVA test followed by the Tukey’s Multiple Comparison Test (### significance <0.001 vs. Control; ^^^ significance <0.001 vs. CPZ; §§§ significance <0.001 vs. TMZ; § significance <0.05 vs TMZ). (B). Neurospheres TS#1, TS#83, and TS#163 were treated as described and allowed to grow and form spheres for the subsequent 20 d. For each cell line, the four left images illustrate the effect of TMZ, CPZ or both drugs on neurosphere number and size, observed using a 4× objective. The two framed images on the right are enlarged pictures of the respective neurospheres indicated by the red arrows; these neurospheres were randomly chosen due to their dimensions, in order to appreciate their decrease in volume due to the treatment with the drug combo. This suggests a reduced sphere-forming ability in TMZ + CPZ-treated cells. In this panel, no histograms with values and statistical significance are reported, due to the intrinsic difficulty of objectively counting floating neurospheres.