ECE2014 Oral Communications Adrenal & Thyroid (5 abstracts)
Mitotane induces endoplasmic reticulum stress triggering apoptosis and decrease of steroid hormone synthesis
Silviu Sbiera 1 , Laura Wiemer 1 , Ellen Leich 3 , Annemarie Gehl 1 , Felix Gardill 1 , Cristina L. Ronchi 1 , Margarita Bala 1 , Andreas Schirbel 4 , Andreas Rosenwald 3 , Bruno Allolio 1 , Martin Fassnacht 2, & Matthias Kroiß 1
1Department of Endocrinology, Internal Medicine I, University Hospital Würzburg, Würzburg, Germany; 2Department of Endocrinology, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, München, Germany; 3Institute of Pathology, University of Würzburg, Würzburg, Germany; 4Department of Nuclear Medicine, University Hospital Würzburg, Würzburg, Germany.
Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). Molecular events leading to cell death in adrenocortical cells are unknown preventing the development of drugs with improved efficacy-toxicity ratio.
Methods: We employed the ACC model cell line NCI-H295 to investigate the effects of mitotane on genome-wide mRNA expression using microarray analysis. Several algorithms were used to delineate signaling pathways triggered by mitotane. Changes of steroid hormone production, apoptosis rate and cholesterol metabolism were measured.
Results: In dose-finding experiments, we confirmed the results of others showing inhibition of cortisol secretion and increased apoptotic rate by mitotane (EC50: 9.4 μM(3.12 mg/l) and 11.9 μM(3.9 mg/l) respectively). Microarray analyses showed profound changes in the expression profile of NCI-H295 cells after 6 h exposure to mitotane (459 and 1867 genes at 50 μM and 100 μM mitotane respectively). Among the top 30 downregulated genes several are implicated in sterol metabolism and steroidogenesis such as LDL-receptor, stearyl coA-desaturase, sterol-responsive element binding factor 1, and the cholesterol exporter ABCG1 (ATP-binding cassette subfamily G member 1). Most upregulated genes play a role in apoptosis such as GDF15, DUSP4, TRIB3, and CHOP. Unsupervised pathway analyses revealed endoplasmic reticulum (ER-) stress response among the pathways most significantly altered by mitotane. Accordingly, we found an increase of XBP1 mRNA-splicing pointing to activation of the most conserved pathway of ER-stress induction. ER-stress inhibitor salubrinal partly reversed mitotane-induced expression of ER-stress marker CHOP while ER-stress inducer thapsigargin increased the mitotane effect. Finally, strong and rapid intracellular accumulation of fluorescently labeled cholesterol was detected in mitotane-treated NCI-H295 cells but not in other cells.
Conclusion: Our data provide strong evidence for a key role of mitotane-induced endoplasmic reticulum stress in its adrenal specific toxicity. We hypothesize that intracellular overload of sterols triggers the canonical ER-stress pathway which eventually leads to increased apoptosis and diminished steroidogenesis.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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