Assessment of cytotoxic effects of new derivatives of pyrazino[1,2-a] benzimidazole on isolated human glioblastoma cells and mitochondria

Aims: Glioblastoma multiforme (GBM) is a highly devastating malignant brain tumor with poor pharmacotherapy. Based on COX-2 inhibitory effects in preventing cancer progression, new pyrazino[1,2-a]benzimidazole derivatives were assessed on isolated human GBM cells.

Main methods: In this study, firstly, primary culture of astrocytes from human GBM samples was prepared and exposed to 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) for finding their half-maximal inhibitory concentration (IC₅₀). In the following, in two phases, cell apoptosis pathway and mitochondrial markers were investigated on GBM and also HEK293 cells (as non-cancerous normal cells).

Key findings: The MTT results represented a remarkable selective cytotoxic effect of both L1 and L2 on GBM cells, and interestingly not on normal cells. After 48 h, IC₅₀ of L1 and L2 were calculated as 13 μM and 85 μM, respectively. Annexin/PI staining showed that L1 and L2 induce apoptosis in GBM cells, and caspase measurement showed that apoptosis occurs through mitochondrial signaling. In the clonogenic assay, GBM cells formed more paraclones and fewer holoclones after treating with L1 and L2. L1 and L2 also selectively enhanced mitochondrial damaged markers, including reactive oxygen species (ROS) formation, and mitochondrial swelling, decreased mitochondrial membrane potential (MMP) and cytochrome c release in isolated cancerous GBM mitochondria.

Significance: Our findings on human primary astrocyte cells illustrated that L1 and L2 compounds, with COX-2 inhibitory effect, through the intrinsic pathway of apoptosis concerning mitochondrial damage enhancement have therapeutic potentials on GBM.