Effects of copper overload on mitochondrial parameters in GBM-1, U-87 MG, and C6 glioma cell lines

Abstract Over the years, there has been growing interest in developing new therapies for glioblastoma, and copper compounds have emerged as promising therapeutic targets due to their antitumoral properties. In this context, this study aimed to investigate the effects of high copper levels on cell viability, mitochondrial physiology, PINK1 content, and the expression of PGC-1α and TFAM in distinct glioma cell lines, mainly considering the heterogeneity of tumoral cells. GBM-1, U-87 MG, and C6 cells CuSO 4 exposure (0–1200 µM) was performed for 24 h, and cell viability was assessed using the MTT reduction and Neutral Red (NR) assays. NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase activities, and the mitochondrial membrane potential (ΔΨm) were measured to analyze mitochondrial physiology. PINK1 was evaluated by immunofluorescence, while PGC-1α and TFAM expression were assessed by real-time RT-PCR in U-87 MG and C6 cells. Copper exposure reduced cell viability in the cell lines (MTT and NR), except in U-87 MG with the NR assay. High copper levels decreased succinate dehydrogenase activity in GBM-1, U-87 MG, and C6 cells. Cytochrome c oxidase activity was decreased in the C6 cell line. Moreover, reductions in ΔΨm and increases in PINK1 immunostaining were observed across the three cell lines. Finally, an increase in PGC-1α mRNA was observed in C6 cells, and TFAM expression has risen in U-87 MG. In conclusion, high copper levels decrease glioma cell viability in vitro, and mitochondrial dysfunction contributes to this effect, resulting in PINK1 and TFAM or PGC-1α increase as compensatory mechanisms to copper cytotoxicity. Graphical abstract Exposure to high copper levels (CuSO 4 ) promotes mitochondrial dysfunction in glioma cells, including loss of mitochondrial membrane potential (ΔΨm), and impairment of respiratory chain activity, besides increasing PINK1. Indeed, U-87 MG cells display increased TFAM expression, and C6 cells exhibit increased PGC-1α expression, consistent with activation of mitochondrial biogenesis pathways. Created in BioRender.com