Background: Glioblastoma (GBM) is the most aggressive subtype of malignant glioma. Current therapeutic options remain limited, highlighting the need for the development of novel compounds capable of improving the efficacy of standard treatments. This study aimed to evaluate the biological activity of a series of pyrazolone-based hydrazone compounds (TPPs) in vitro GBM cell lines. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylethan-1-one and tested on two human GBM cell lines, T98 and U87. The cytotoxic effects were evaluated via MTT assay. The most active compound was further investigated at IC₅₀ and IC₂₅ concentrations to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨ_m) changes. Additional assays included colony formation, cell cycle analysis, and evaluation of DNA damage and apoptosis markers. Results: TPP25 exhibited the highest activity with IC₅₀ values of 11.01 μM (95% CI: 10.42 to 11.64) and 13.12 μM (95% CI: 10.23 to 16.87) on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization, along with a significant reduction in colony formation. Cell cycle analysis revealed accumulation in the sub-G0 phase, consistent with increased cell death, supported by propidium iodide uptake. Furthermore, the results suggest the involvement of an apoptotic-like mechanism as supported by Annexin V positivity, γ-H2AX upregulation and transient caspase-3 activation. Conclusions: TPP25 demonstrates significant in vitro cytotoxicity, likely driven by a pro-apoptotic mechanism. This profile positions it as a potential lead compound for further preclinical evaluation, supporting its future transition into in vivo GBM models.

Background: The prognosis of glioblastoma (GBM) patients remains dismal due to chemoresistance and ineffective therapies. This study investigates the anti-tumor potential of combining minor phytocannabinoids, cannabinol (CBN) or cannabigerol (CBG), with melatonin (MLT). Methods: Cytotoxicity, synergy (Bliss model), and selectivity were evaluated in U87, T98, and U251 GBM lines and normal fibroblasts. Mechanisms of damage were characterized via Western blot (γ-H2AX), flow cytometry (DCFDA, JC-1, MitoBright , COX IV, BODIPY), and confocal analysis. Results: CBN-MLT and CBG-MLT regimens exerted potent, synergistic cytotoxicity while sparing healthy fibroblasts. The combinations induced necrotic cell death characterized by severe double-strand DNA damage. In T98 and U251 cells, this was driven by an early accumulation of intracellular ROS, which triggered mitochondrial depolarization, loss of organelle mass, and extensive lipid peroxidation. CBN combinations consistently achieved better results than CBG-based treatments. Conversely, U87 cells delayed this oxidative-mitochondrial cascade, despite a drop in late-stage cytotoxicity. Conclusions: This study provides a strong basis for combining minor cannabinoids and MLT to treat GBM. By a selective, p53-independent necrotic mechanism driven, these findings evidenced a potential novel therapeutic strategy against GBM.

The prognosis of glioblastoma (GBM) patients remains dismal due to chemoresistance. Repurposing of natural and endogenous compounds, such as the pineal hormone melatonin (MLT) and minor phytocannabinoids like cannabinol (CBN) or cannabigerol (CBG), represents a promising strategy. This study investigates the cytotoxic potential of combining these phytocannabinoids with MLT, evaluating their efficacy both alone and synergistically with temozolomide (TMZ) to overcome drug resistance. To achieve this, cytotoxicity, synergy (Bliss model), and selectivity were evaluated in U87, T98, and U251 GBM lines and normal astrocytes. Mechanisms of damage were characterized via Western blot (γH2AX and PARP-1), flow cytometry using fluorescent dyes/probes (DCFDA, JC-1, MitoBright, BODIPY, PI, and Annexin-V), or the protein marker COX IV and confocal analysis. The results demonstrated that CBN-MLT and CBG-MLT regimens exerted synergistic cytotoxicity while sparing healthy astrocytes. Notably, combining these regimens (U87: MLT 0.3 mg/mL + CBN 25 µM; MLT 0.2 mg/mL + CBG 15 µM. T98: MLT 0.7 mg/mL + CBN 25 µM; MLT 0.6 mg/mL + CBG 30 µM. U251: MLT 0.4 mg/mL + CBN 20 µM; MLT 0.5 mg/mL + CBG 35 µM) with TMZ significantly enhanced chemotherapeutic efficacy, overcoming baseline effects of TMZ in these cell lines. The combinations induced necrotic cell death characterized by severe double-strand DNA damage. This was driven by an early accumulation of intracellular ROS, which triggered mitochondrial depolarization, loss of organelle mass, and lipid peroxidation. CBN combinations consistently triggered more robust biochemical alterations than CBG-based treatments. Taken together, this study provides a strong preclinical basis for utilizing minor cannabinoids combined with MLT in GBM management. Crucially, this co-treatment emerges as a promising approach to potentiate TMZ efficacy, offering a novel and potentially effective therapeutic strategy to counter GBM resilience.

Background: Glioblastoma (GBM) is the most aggressive subtype of malignant glioma. Current therapeutic options remain limited, highlighting the need for the development of novel compounds capable of improving the efficacy of standard treatments. This study aimed to evaluate the biological activity of a series of pyrazolone-based hydra-zone compounds (TPP) against GBM. Methods: The eight TPPs were synthesized by a nucleophilic addition reaction of different substituted hydrazines with the 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylethan-1-one, and tested on two human GBM cell lines, T98 and U87. The cytotoxic effects were evaluated via MTT assay. The most active compound was further investigated at IC₅₀ and IC₇₅ concentra-tions, to evaluate mechanisms of cellular damage, including reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔѰm) changes. Additional assays included colony formation, cell cycle analysis, and evaluation of DNA damage and apoptosis markers. Results: TPP25 exhibited the highest activity with IC₅₀ values of 11 and 13 µM on T98 and U87 lines, respectively. Treatment induced early ROS production and mitochondrial depolarization within 24 h, along with a significant reduction in colony formation. Cell cycle analysis revealed sub-G0 accumulation, consistent with increased cell death, confirmed by propidium iodide uptake. TPP25 also increased phosphorylated ɣ-H2A.X and cleaved Caspase-3 expression. Conclusions: TPP25 emerges as a promising candidate for GBM treatment, demonstrating significant cytotoxic activity.