Abstract
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.
Highlights
Mitotane (o,p’DDD) is the only drug approved for the treatment of metastatic adrenocortical carcinoma (ACC) [1] but its molecular mechanism of action still remains to be elucidated
It has been previously demonstrated that mitotane has a marked mitochondrial impact [3, 4] but the drug induces endoplasmic reticulum (ER) damages resulting in ER stress as recently shown [5]
We previously showed by immunocytochemistry that mitotane induces a fragmentation of the mitochondrial network and demonstrated that this was accompanied by a specific inhibition of respiratory chain complexes I and IV activities [3]
Summary
Mitotane (o,p’DDD) is the only drug approved for the treatment of metastatic adrenocortical carcinoma (ACC) [1] but its molecular mechanism of action still remains to be elucidated. Identification of mitotane molecular targets would help therapeutic management of ACC patients. We previously reported that therapeutic concentrations of o,p’DDD (50 μM, corresponding to approximately 14 mg/L in plasma) impaired the expression of several steroidogenic enzymes and induced a selective and marked inhibition of mitochondrial respiratory chain complexes I and IV and a fragmentation of the mitochondrial network in human adrenocortical H295R cells [3]. Subsequently showed that mitotane inhibited the expression of voltage-dependent anion channel (VDAC), a protein anchored in the outer mitochondrial membrane [4]
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