Synergistic combination of perphenazine and temozolomide suppresses patient-derived glioblastoma tumorspheres

Abstract

Background: Glioblastoma (GBM), a primary malignant brain tumor, has a poor prognosis, even with standard treatments such as radiotherapy and chemotherapy. In this study, we explored the anticancer effects of the synergistic combination of perphenazine (PER), a dopamine receptor D2/3 (DRD2/3) antagonist, and temozolomide (TMZ), a standard treatment for GBM, in patient-derived human GBM tumorspheres (TSs).

Methods: The biological effects of the combination of PER and TMZ in GBM TSs were assessed by measuring cell viability, ATP, stemness, invasiveness, and apoptosis. Changes in protein and mRNA expression were analyzed using western blotting and RNA sequencing. Co-administration of PER and TMZ was evaluated in vivo using a mouse orthotopic xenograft model.

Results: The Severance dataset showed that DRD2 and DRD3 expressions were higher in tumor tissues than in the tumor-free cortex of patients with GBM. DRD2/3 knockout by CRISPR/Cas9 in patient-derived human GBM TSs inhibited cell growth and ATP production. The combined treatment with PER and TMZ resulted in superior effects on cell viability and ATP assays compared to those in single treatment groups. Flow cytometry, western blotting, and RNA sequencing confirmed elevated apoptosis in GBM TSs following combination treatment. Additionally, the combination of PER and TMZ downregulated the expression of protein and mRNA associated with stemness and invasiveness. In vivo evaluation showed that combining PER and TMZ extended the survival period of the mouse orthotopic xenograft model.

Conclusions: The synergistic combination of PER and TMZ has potential as a novel combination treatment strategy for GBM.

Keywords: DRD2/3; glioblastoma; perphenazine; temozolomide; tumorsphere.

Figure 1.

Expression of DRD2/3 in GBM and the DRD2/3 inhibitory effects following combined treatment with PER and TMZ in GBM TSs. (A) The expression levels of DRD2/3 in GBM tumor tissues (n = 62) and tumor-free cortex tissues (n = 13) were determined using Severance RNA-Seq data. DRD2/3 is highly expressed in GBM tumors compared to the tumor-free cortex. (B) Western blotting of DRD2/3 expression in TS13-64 following knockout by CRISPR/Cas9. (C) Quantification of DRD2 and DRD3 protein expression relative to GAPDH for the DRD2/3 double knockout in TS13-64. (D) Cell viability and ATP levels were measured in TS13-64 after DRD2/3 knockout. DRD2/3 loss of function reduced cell proliferation in TS13-64. (E) Western blotting of dopamine receptor protein expression following combined treatment with PER (5 μM) and TMZ (250 μM). PER single treatment and combined treatment of PER and TMZ decreased DRD2/3 protein expression levels.

Figure 2.

Cytotoxic and synergistic effects of combined treatment with PER and TMZ in GBM TSs. (A) Cell viability and ATP levels were measured after combined treatment with PER (5 and 10 μM) and TMZ (250 μM). The combined treatment reduced the cell viability and ATP production of GBM TSs. (B) Bliss synergy score for combined treatment with PER and TMZ. Combined treatment showed excellent synergistic effects in TS13-64 (15 points) and TS15-88 (31 points). (C) Apoptotic rates were measured after combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment induced higher apoptosis in GBM TSs compared to the single treatments of PER or TMZ. (D) Western blotting and (E) RNA-Seq analysis of apoptosis-related markers following combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment increased the expression of pro-apoptosis-related markers and, decreased the expression of anti-apoptosis-related markers.

Figure 3.

Effects of combined treatment with PER and TMZ on stemness in GBM TSs. (A) Neurosphere formation was evaluated following combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment reduced the sphere formation ability and size of the GBM TSs. (B) Western blotting and (C) RNA-Seq analysis of stemness-related markers after combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment decreased the protein and mRNA expression levels of the stemness-related markers.

Figure 4.

Effects of combined treatment with PER and TMZ on invasiveness in GBM TSs. (A) A 3D invasion assay of the collagen matrigel matrix environment was conducted following combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment inhibited the invasion of implanted GBM TSs. (B) Western blotting and (C) RNA-Seq analysis of invasiveness-related markers following the combined treatment with PER (5 μM) and TMZ (250 μM). The combined treatment decreased the protein and mRNA expression levels of the invasiveness-related markers.

Figure 5.

Therapeutic responses in a mouse orthotopic xenograft model. For in vivo evaluation, MGMT unmethylation GBM TS (TS13-64) or MGMT methylation GBM TS (TS19-156) injected mice were administrated with PER (20 mg/kg, p.o.) and/or TMZ (7.5 mg/kg, i.p.). (A) MR images of TS13-64 injected mice at 4 weeks of modeling. (B) Tumor volume was measured after the combined administration of PER and TMZ in TS13-64 injected mice. The combined administration suppressed tumor growth more than the single administration of PER or TMZ. (C) Overall Kaplan-Meier survival analysis after administration with PER and/or TMZ in TS13-64 injected mice. The survival period of mice was prolonged in the combined administration group. (D) Immunohistochemical analysis of Zeb1 in TS13-64 injected mice. (E) The zeb1-positive cells were quantitatively counted after sacrificing the mice. Zeb1-positive cell infiltration was inhibited by the combined administration of PER and TMZ. (F) Graphical summary of this study. PER targeting DRD2/3 inhibits dopamine signaling to the nucleus, and TMZ causes DNA damage simultaneously. The combination of the above effects resulted in the effective induction of apoptosis and suppression of cell proliferation, stemness, and invasiveness, eventually leading to robust GBM TS growth inhibition.