Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). We have found that mitotane leads to endoplasmic reticulum stress and decreased viability in ACC cells. It is not known by which downstream mechanisms cell death is induced by mitotane.
Aim: To characterize the mechanisms underlying cell death resulting from mitotane treatment in ACC cells.
Methods: Lipid peroxidation in the ACC cell line NCI-H295R was measured with BODIPY 581/591 C11 and cell viability quantified by CellTiter Glo assay. Combination treatment with pan caspase inhibitor zvad-fmk, inhibitors of necroptosis (necrostatin-1) and ferroptosis (liproxstatin, deferoxamine) and ferroptosis activator RSL3 in three ACC cell lines was applied. Marker protein expression was assessed by immunoblotting.
Results: Mitotane induced caspase3 cleavage but caspase inhibition was unable to rescue ACC cells from mitotane-induced cell death. Necroptosis markers were unchanged after mitotane treatment, while lipid peroxides accumulated after mitotane treatment in a concentration dependent manner. Deferoxamine prevented mitotane induced cell death by up to 40%. In line with this finding, synergism of mitotane and RSL3 was observed. RSL3 showed cytotoxicity at nanomolar concentrations in ACC cells (EC50: NCI-H295R: 240 nM, CU-ACC1: 814 nM, CU-ACC2: 14.5 nM) but not in cells of non-adrenal origin.
Conclusion: Mitotane-induced cell death involves caspase action. Iron-dependent accumulation of lipid peroxides are hallmarks of ferroptosis. In addition, synergism of mitotane and RSL3 suggests relevance of this novel pathway but detailed mechanisms require further Investigation.
18 - 21 May 2019
European Society of Endocrinology