Investigation of the cytotoxic and synergistic in vitro effect of fluoxetine-temozolomide combination on human glioblastoma cells

Glioblastoma multiforme (GBM) is a highly aggressive and destructive type of cancer affecting the central nervous system and remains extremely difficult to treat. In this study, the effects of the antidepressant agent fluoxetine (FLT) and the alkylating chemotherapeutic agent temozolomide (TMZ) were evaluated on human glioblastoma (T98-G) and human embryonic kidney (HEK-293) cell lines. To determine effective dose levels in the cell lines, the MTT assay was performed, and IC₅₀ values were determined based on triplicate experiments. Apoptotic effects of the agents were assessed via Annexin V-FITC staining followed by flow cytometry analysis. The impact on cell migration was examined using the wound healing assay, with microscopic imaging at 0, 12, 24, 36, and 48 hours. Autophagic vesicle formation was analyzed using Monodansylcadaverine (MDC) staining, and mitochondrial membrane potential was evaluated using Tetraetilbenzimidazolcarbosiayniniodid (JC-1) dye. Quantitative analysis was conducted using ImageJ software. The IC₅₀ value of FLT was found to be 36 µM/ml for T98-G cells and 320 µM/ml for HEK-293 cells. These values remained stable at both 24 and 48 hours, and therefore constant concentrations were used in all experiments. In T98-G cells, late apoptosis was found to be the predominant form of cell death following treatment with FLT, TMZ, and their combination, while in HEK-293 cells, early apoptosis was more prominent. In combination treatments, 36.4% late apoptotic cells were observed in T98-G and 16.5% early apoptotic cells in HEK-293. In both cell lines, FLT and TMZ, particularly in combination, significantly inhibited cell migration. The most substantial inhibition was observed in the group where each agent was applied at 50% of its IC₅₀ dose. At 24 hours, FLT, TMZ, and their combination led to significant autophagy induction and mitochondrial dysfunction compared to the control group (p<0.01–p<0.0001). At 48 hours, significant autophagic activity persisted only in FLT and TMZ single-agent groups, while statistical significance was lost in the combination group. In contrast, JC-1 analysis showed that all treatments at 48 hours resulted in significant mitochondrial dysfunction (p<0.0001), indicating that the combination therapy may enhance mitochondrial damage and trigger cell death. In the HEK-293 cell line, reliable statistical data from MDC and JC-1 analyses could not be obtained due to cell clustering. FLT and TMZ, both individually and in combination, were shown to activate apoptotic pathways, mitochondrial dysfunction, and autophagy in the T98-G glioblastoma cell line, as well as significantly inhibit cell migration. These effects were most pronounced in the combination treatment group. Overall, the findings suggest that FLT, when used in combination with chemotherapeutic agents, may serve as a potential adjuvant agent in glioblastoma therapy.