P08.57 Involvement of both the extrinsic and intrinsic apoptotic pathways with clomipramine treatment of human glioblastoma cells under normoxic and hypoxic conditions

AbstractIntroduction:The tricyclic antidepressant clomipramine, used clinically since the 1960’s, has been shown to have selective cytotoxicity against glioma cells in vitro and has also been shown to cross the blood brain barrier. While reports suggest clomipramine acts via the mitochondria, the mechanism of action is not fully understood.Materials and Methods:In this study, 8 human biopsy-derived glioblastoma early passage cell lines were treated with 0–200µM clomipramine over 72hrs. Cell viability was measured using the MTS assay, cellular ATP levels using the Cell Titer Glo assay and capsase activation by Caspase-Glo 3/7, 8 and 9 assays. Immunocytochemistry using Mitotracker Red and cytochrome C antibodies was performed. The JC-1 dye was used to evaluate mitochondrial transmembrane potential and DAPI was used to characterise the cell cycle.Results:Clomipramine reduced cell viability in a dose and time dependent manner with IC50 values in the range of 22–60µM at 24hrs. Compared to controls, following 24 hr treatment with 75µM clomipramine, there was a significant reduction of cellular ATP levels (by approximately 100%, p<0.0001), depolarisation of the mitochondrial transmembrane potential and release of cytochrome C from the mitochondria. The extrinsic apoptotic pathway was activated at 12 hours whereas the intrinsic (mitochondrial-mediated) pathway was activated at 24 hours as shown by elevation of caspase 8, 9 and 3/7 levels. The reduction in cell viability in response to clomipramine was attenuated by the pan-caspase inhibitor Z-VAD-FMK, indicating involvement of caspases in the mechanism of action. With the most significant effects observed with SEBTA-023; 24 hours at 75µM clomipramine cell viability increased by 50% and 67% under hypoxic and normoxic conditions respectively (p<0.0001). The effects on the cell cycle were evaluated and clomipramine treatment led to cell cycle arrest in G2/M phase. Two other tricyclic antidepressants, imipramine and amitriptyline were also examined and clomipramine was deemed to be the most effective inducer of apoptosis.Conclusions:This work provides further insights to the mechanism of action of this class of repurposed drug as a potential adjuvant treatment for GBM.