Clofazimine targets human glioblastoma cells by cytochrome C translocation and PI3K/AKT/mTOR survival axis suppression

Glioblastoma multiforme (GBM) remains the most aggressive and lethal form of primary brain cancer, characterised by high rates of recurrence and resistance to conventional therapies. This study investigates the anti-neoplastic potential of clofazimine against the human glioblastoma U251 cell line. Our results demonstrate that clofazimine significantly reduces cell viability and induces apoptosis in a dose-dependent manner. Flow cytometric analysis using Annexin V/PI and JC-1 staining revealed that clofazimine triggers apoptosis and substantial mitochondrial membrane depolarisation (65.9%). Clofazimine promotes the intrinsic apoptotic pathway by facilitating the translocation of cytochrome c from the mitochondria to the cytosol. Furthermore, clofazimine inhibited invasive and migratory capabilities of U251 cells. These phenotypic changes were driven by the potent suppression of the PI3K/AKT/mTOR survival signalling axis, with phosphorylation levels of PI3K, AKT, and mTOR reduced by approximately 50%, 48%, and 44%, respectively. The subsequent activation of cleaved caspase-3 further confirmed the commitment to programmed cell death. Molecular docking also suggested strong binding affinities between clofazimine and PI3K. Collectively, these findings provide compelling evidence that clofazimine acts as a multi-targeted agent that disrupts survival signalling and mitochondrial integrity, offering a promising therapeutic strategy for the treatment of glioblastoma.