Hescot Ségolène, Amazit Larbi, Lhomme Marie, Travers Simon, DuBow Anais, Battini Stephanie, Boulate Geoffrey, Namer Izzie Jacques, Lombes Anne, Kontush Anatol, Imperiale Alessio, Baudin Eric, Lombes Marc
INSERM UMR-S 1185, Le Kremlin-Bicêtre, France.
Endocrine Oncology, Gustave Roussy, Villejuif, France.
Oncotarget. 2017 Jul 4;8(66):109924-109940. doi: 10.18632/oncotarget.18968. eCollection 2017 Dec 15.
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 . This constitutes a potential and promising pharmacological strategy for patients with adrenocortical carcinoma.
米托坦（邻对滴滴滴）是肾上腺皮质癌最有效的药物，它会聚集在线粒体中并影响线粒体功能。为了探究米托坦作用的分子机制并确定其潜在靶点，研究人员在人肾上腺皮质H295R细胞中采用了代谢组学和脂质组学方法以及成像分析，从而确定线粒体相关膜功能障碍是米托坦的关键影响。通过核磁共振波谱对细胞内能量代谢物的研究表明，米托坦以时间和浓度依赖性方式显著降低天冬氨酸含量，同时增加谷氨酸含量。这种变化很可能与先前描述的米托坦诱导的呼吸链缺陷有关。对细胞内和线粒体内磷脂的脂质组学研究表明，米托坦处理显著降低了磷脂酰丝氨酸/磷脂酰乙醇胺的比率，这表明位于线粒体相关膜中的磷脂酰丝氨酸脱羧酶功能异常。通过蛋白质印迹分析评估，米托坦可显著降低线粒体相关膜蛋白磷脂酰丝氨酸脱羧酶、动力相关蛋白1（DRP1）、ATP酶家族AAA结构域蛋白3A（ATAD3A）或外周型苯二氮䓬受体（TSPO）的表达水平。高分辨率去卷积显微镜和定量分析表明，米托坦处理显著改变了内源性线粒体相关膜的完整性，并减少了线粒体与内质网相互作用的程度。最后，我们发现，嵌入线粒体相关膜中的胆固醇转运体TSPO的药理学抑制剂PK11195与米托坦在诱导线粒体相关膜破坏、凋亡以及抑制类固醇分泌方面具有协同作用。总之，我们的结果表明，米托坦处理的H295R细胞中存在线粒体相关膜功能障碍，并且TSPO抑制可显著增强米托坦的抗肿瘤和抗分泌作用。这为肾上腺皮质癌患者构成了一种潜在且有前景的药理学策略。
