LS4-P-16 Microscopy techniques to evaluate the cytotoxic effect induced by copper complexes on glioblastoma cells

Introduction: Glioblastoma (GBM) is a highly proliferative and poorly differentiated primary brain tumor. The current standard of care includes surgery, radiotherapy, and systemic therapy, mainly based on temozolomide (TMZ) chemotherapy. However, despite recent advances in these multimodal approaches, the prognosis for GBM patients remains poor, primarily due to the development of TMZ resistance and tumor recurrence. Therefore, there is an urgent need to identify less toxic and more effective therapeutic alternatives. In this context, metal-based compounds have emerged as promising pharmaceutical agents for cancer diagnosis and treatment. Among them, copper complexes are particularly interesting candidates, as copper is an essential element involved in cellular growth and development, and metal-based therapeutics using endogenous metals may exhibit reduced toxicity. Methods: In this study, the bis(pyrazol-1-yl)acetic acid was conjugated to the bioactive molecule amantadine and used as supporting ligand for the synthesis of novel Cu(I) and Cu(II) complexes. The lipophilic triphenylphosphine (PPh₃) and the hydrophilic 1,3,5-triaza-7-phosphaadamantane (PTA) were used as co-ligands to stabilize copper in its +1 oxidation state and to modulate the solubility of the related complexes. The antitumor activity of the synthesized Cu(I) and Cu(II) complexes was evaluated against U87 and LN18 human glioblastoma cell lines. Cell viability studies were performed by MTT assay. The most promising cytotoxic effects were observed for the Cu(I) complexes containing the PPh3 co-ligands. Apoptosis induction and cell cycle analysis were evaluated by flow cytometry. Moreover, to explore the mechanism of action and the morphological alterations associated with cell death, Scanning Electron Microscopy (SEM), Fluorescence Microscopy (IF), and Confocal Laser Scanning Microscopy (CLSM) were employed. Results: Viability assays in U87 and LN18 glioblastoma cell lines showed that the copper complexes with PPh₃ ligands, owing to their increased lipophilicity, exhibited the strongest cytotoxic activity. Notably, their micromolar IC₅₀ values were lower than those of cisplatin. Treatment with these copper compounds at their respective IC50 concentrations led to an increase in the percentage of Annexin V-positive cells and caused alterations in cell cycle profiles. These cytometry results suggest the induction of apoptotic cell death. According to cytotoxicity studies, SEM and FI micrographs acquired 24 hours after treatment with the copper compounds showed that both showed a reduced number of adherent cells and morphological features indicative of cellular distress, such as rounding and alterations in cytoskeletal architecture. Discussion and conclusions: These preliminary results suggest that copper complexes, particularly those bearing PPh₃ ligands, may be promising as potential therapeutic agents for glioblastoma. To overcome the potential systemic toxicity, further studies are needed to develop nanoformulations capable of delivering these compounds selectively to tumor cells.