Synergistic Combination of Perphenazine and Temozolomide Suppresses Patient-Derived Glioblastoma Tumorspheres
Academic Background
Glioblastoma (GBM) is a highly malignant primary brain tumor. Despite current standard treatments such as surgical resection, radiotherapy, and chemotherapy, the prognosis remains extremely poor, with a median survival of only 14.6 months. Traditional treatments often fail to completely eradicate the tumor and are prone to recurrence. Therefore, finding new treatment strategies has become an urgent priority. In recent years, drug repurposing has emerged as a promising research direction, leveraging drugs already approved for other diseases to treat glioblastoma, thereby reducing development time and costs.
The expression and role of dopamine receptors (DR) in glioblastoma have gradually gained attention. Studies have shown that dopamine receptor D2 (DRD2) and D3 (DRD3) are highly expressed in glioblastoma cells, and activation of DRD2 enhances tumor stemness and invasive capabilities. Therefore, dopamine receptor antagonists are considered a potential therapeutic target. Perphenazine (Per) is a dopamine receptor D2/D3 antagonist widely used to treat schizophrenia and bipolar disorder. Previous studies have shown that Per exhibits anticancer effects in skin cancer and leukemia, but its efficacy in glioblastoma has not been thoroughly investigated.
This study aims to explore the therapeutic effects of combining Perphenazine with the standard chemotherapy drug Temozolomide (TMZ) in glioblastoma, particularly the synergistic effects in patient-derived glioblastoma tumorspheres (TS).
Source of the Paper
This paper was co-authored by Jun Pyo Hong, Ran Joo Choi, Jin-Kyoung Shim, and others, from multiple research institutions including the Brain Tumor Center at Severance Hospital, Yonsei University College of Medicine, and the Department of Biomedical Sciences at Yonsei University. The paper was published in 2025 in the journal Neuro-Oncology under the title “Synergistic Combination of Perphenazine and Temozolomide Suppresses Patient-Derived Glioblastoma Tumorspheres.”
Research Process
1. Culture and Treatment of Glioblastoma Tumorspheres
The study first obtained tumor tissue samples from 55 glioblastoma patients and isolated six different glioblastoma tumorspheres (TS13-64, TS15-88, TS18-48, TS19-137, TS19-65, and TS19-156). These tumorspheres were used for subsequent in vitro and in vivo experiments. The tumorspheres were cultured in TS complete medium at 37°C.
2. CRISPR/Cas9-Mediated DRD2/3 Knockout
To investigate the role of DRD2/3 in glioblastoma, the research team used CRISPR/Cas9 technology to knockout DRD2/3 in TS13-64 cells. GFP-positive knockout cells were sorted using flow cytometry, and a significant reduction in DRD2/3 protein expression was confirmed. The results showed that DRD2/3 knockout significantly inhibited the proliferation and ATP production of TS13-64 cells.
3. In Vitro Combination Therapy with Perphenazine and Temozolomide
The research team evaluated the effects of Perphenazine (Per) and Temozolomide (TMZ) alone and in combination on glioblastoma tumorspheres in vitro. Through MTT and ATP assays, it was found that combination therapy significantly reduced cell viability and ATP levels in tumorspheres. Additionally, flow cytometry, Western blot, and RNA sequencing results showed that combination therapy significantly increased the apoptosis rate of tumorspheres and downregulated the expression of proteins and mRNAs associated with stemness and invasiveness.
4. Three-Dimensional Invasion Assay
To assess the impact of combination therapy on the invasive ability of tumorspheres, the research team conducted a three-dimensional invasion assay. Tumorspheres were implanted in a collagen matrix and treated with Per, TMZ, or a combination for 72 hours. The results showed that combination therapy significantly inhibited the invaded area of tumorspheres.
5. Mouse Orthotopic Xenograft Model
The research team further evaluated the in vivo efficacy of Perphenazine and Temozolomide combination therapy in a mouse orthotopic xenograft model. TS13-64 or TS19-156 cells were implanted into the brains of nude mice, and the mice were treated with Per (20 mg/kg) and TMZ (7.5 mg/kg) alone or in combination. MRI image analysis showed that combination therapy significantly reduced tumor volume. Kaplan-Meier survival analysis indicated that combination therapy significantly prolonged the survival of the mice.
Key Findings
High Expression of DRD2/3 in Glioblastoma: RNA sequencing data revealed that DRD2 and DRD3 expression was significantly higher in glioblastoma tissues compared to normal brain tissues.
DRD2/3 Knockout Inhibits Tumorsphere Proliferation: CRISPR/Cas9-mediated DRD2/3 knockout significantly reduced the proliferation and ATP production of TS13-64 cells.
Synergistic Effect of Combination Therapy: The combination of Perphenazine and Temozolomide significantly reduced cell viability and ATP levels in glioblastoma tumorspheres and induced a higher apoptosis rate.
Inhibition of Stemness and Invasiveness: Combination therapy significantly downregulated the expression of proteins and mRNAs associated with stemness and invasiveness, such as SOX2, Nestin, ZEB1, and Snail.
In Vivo Therapeutic Efficacy: In the mouse orthotopic xenograft model, combination therapy significantly reduced tumor volume and prolonged the survival of the mice.
Conclusion
This study demonstrates that the combination of Perphenazine and Temozolomide exhibits significant synergistic anticancer effects both in vitro and in vivo. The combination therapy inhibits the DRD2/3 signaling pathway, reducing the proliferation, stemness, and invasiveness of glioblastoma tumorspheres while inducing apoptosis. This research provides a new strategy for glioblastoma treatment, particularly for patients who are resistant to traditional therapies.
Research Highlights
Application of Dopamine Receptor Antagonists: This is the first systematic evaluation of the anticancer effects of Perphenazine in glioblastoma, validating its synergistic effects with Temozolomide.
Patient-Derived Tumorsphere Model: The use of patient-derived glioblastoma tumorspheres as a research model closely mirrors clinical reality.
Comprehensive In Vitro and In Vivo Validation: The efficacy of combination therapy was comprehensively validated through in vitro experiments and a mouse orthotopic xenograft model.
Research Value
The scientific value of this study lies in revealing the potential mechanisms of Perphenazine and Temozolomide combination therapy in glioblastoma and providing new insights for clinical treatment. This combination therapy has the potential to become a standard treatment for glioblastoma in the future, especially for patients resistant to traditional therapies. Additionally, this study provides strong evidence for the application of drug repurposing in cancer treatment.
Other Valuable Information
The research team also found that the combination of Perphenazine and Temozolomide significantly increased the expression of γH2AX, indicating that the combination therapy enhanced the anticancer effects of TMZ by inducing DNA damage. Furthermore, the team explored other potential mechanisms of Perphenazine in glioblastoma cells, such as the regulation of the ERK/MAPK signaling pathway.
Through this study, we have deepened our understanding of the pathogenesis of glioblastoma and provided new directions for future treatment strategies.