Identifying mitotane-induced mitochondria-associated membranes dysfunctions: metabolomic and lipidomic approaches
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Ségolène Hescot1,2, Larbi Amazit1,3, Marie Lhomme4, Simon Travers1, Anais DuBow1, Stephanie Battini5, Geoffrey Boulate1, Izzie Jacques Namer5,6, Anne Lombes7, Anatol Kontush4,8, Alessio Imperiale5,6, Eric Baudin1,2 and Marc Lombes1,9
1INSERM UMR-S 1185, Le Kremlin-Bicêtre, France
2Endocrine Oncology, Gustave Roussy, Villejuif, France
3Institut Biomédical de Bicêtre, UMS-32, Le Kremlin Bicêtre, France
4ICANalytics, UMR-ICAN 116, University Pierre et Marie Curie, Paris, France
5ICube, UMR 7357, University of Strasbourg/CNRS and FMTS, Faculty of Medicine, Strasbourg, France
6Biophysics and Nuclear Medicine, University Hospital of Strasbourg, Strasbourg, France
7INSERM UMRS 1016, Institut Cochin, Paris, France
8Assistance Publique, Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpétrière, Paris, France
9Assistance Publique, Hopitaux de Paris, Hopital Bicêtre, Department of Endocrinology, Le Kremlin-Bicêtre, France
Marc Lombes, email: [email protected]
Keywords: adrenocortical carcinoma, mitotane, mitochondria-associated membranes, molecular target, lipidomics
Received: November 14, 2016 Accepted: June 18, 2017 Published: July 04, 2017
Mitotane (o,p’DDD), the most effective drug in adrenocortical carcinoma, concentrates into the mitochondria and impacts mitochondrial functions. To address the molecular mechanisms of mitotane action and to identify its potential target, metabolomic and lipidomic approaches as well as imaging analyses were employed in human adrenocortical H295R cells allowing identification of Mitochondria-Associated Membranes dysfunction as a critical impact of mitotane. Study of intracellular energetic metabolites by NMR spectroscopy showed that mitotane significantly decreased aspartate while concomitantly increased glutamate content in a time- and concentration-dependent manner. Such alterations were very likely linked to the previously described, mitotane-induced respiratory chain defect. Lipidomic studies of intracellular and intramitochondrial phospholipids revealed that mitotane exposure markedly reduced the phosphatidylserine/phosphatidylethanolamine ratio, indicative of a dysfunction of phosphatidylserine decarboxylase located in Mitochondria-Associated Membranes. Expression levels of Mitochondria-Associated Membranes proteins phosphatidylserine decarboxylase, DRP1, ATAD3A or TSPO were greatly reduced by mitotane as assessed by western blot analyses. Mitotane exposure markedly altered endogenous Mitochondria-Associated Membranes integrity and reduced the magnitude of mitochondria and the endoplasmic reticulum interactions as demonstrated by high resolution deconvolution microscopy and quantification. Finally, we showed that PK11195, a pharmacological inhibitor of the cholesterol translocator TSPO, embedded in Mitochondria-Associated Membranes, exerts a synergetic effect with mitotane in inducing Mitochondria-Associated Membranes disruption, apoptosis and in inhibiting steroid secretion. Altogether, our results demonstrate Mitochondria-Associated Membranes dysfunction in H295R cells treated with mitotane and that TSPO inhibition significantly potentiates mitotane antitumoral and antisecretory actions in vitro. This constitutes a potential and promising pharmacological strategy for patients with adrenocortical carcinoma.
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