Endocrine Abstracts

Background: Mitotane, 1,1-dichloro-2-(o chlorophenyl) -2- (p-chloro phenyl) ethane (o,p′-DDD) is a compound which represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. Nevertheless the biological mechanism induced by this treatment in this cancer remains unknown. Recently, there has been a strong interest in applying proteomics to foster a better understanding of disease processes, mechanism of action and new pharmacological targets of drugs.

Aim: In this study, we describe the effects of mitotane on growth, steroidogenesis, cell cycle and proteomic profile on H295R cells, a characteristic model of ACC able to produce all the adrenocortical steroids, either in total cell extract or in mitochondria-enriched fraction after drug treatment.

Materials and methods: H295-R cells were treated with o,p′-DDD 10–5 M final concentration. The progesterone, testosterone, cortisol and aldosterone levels in the culture medium were determined by electrochemiluminescence (ECLIA) and immunoenzymatic assay. Flow cytometry cell cycle analysis was evaluated using FACScan cytofluorimeter. Total protein extracts and mithocondria-enriched fraction were employed on two-dimensional gel electrophoresis. Spots of interest were identified by peptide mass fingerprint on a Voyager-DE MALDI-ToF mass spectrometer.

Results: We confirmed that mitotane inhibited steroid secretion (Fig. 1) but not significantly influence cell growth (Fig. 2) and not induce perturbation of cell cycle (Fig. 3). Proteomic approach allowed us to identify a total of 18 proteins showing the characteristic expression changes in adrenocortical H295R cells treated with mitotane (Fig. 4). The proteins involved in drug response can be divided in different functional classes including: energetic metabolism, stress response, cytoskeleton structure and tumorigenesis (Fig. 5–6). In particular, a perturbation of the electron transport toward the steroidogenic cytochrome P450 enzyme systems was evidenced.