Effects of mitotane on gene expression in the NCI-H295R cell line

A. Zsippai; D. Szabo; Z. Tombol; P. Szabo; K. Eder; A. Patocs; S. Toth; A. Falus; K. Racz; P. Igaz

Background: The adrenolytic agent mitotane is widely used in the adjuvant treatment of adrenocortical cancer, but its mechanism of action including potential effects on mRNA expression is poorly elucidated.

Objective: To examine mitotane-induced mRNA expression changes in the H295R adrenocortical cancer cell line.

Methods: Various concentrations of mitotane in different treatment periods (24–120 h) have been tested in cell viability assays (MTT colorimetric assay and propidium iodide flow cytometry) and for hormone measurements (cortisol and androstenedione) to select the optimal mitotane concentration effectively inhibiting hormone secretion without affecting cell viability. Total RNA isolated from cultures treated for 48 and 72 hours have been subjected to Agilent 4×44 K microarray platforms. Significantly differentially expressed mRNAs have been validated by quantitative real-time polymerase chain reaction (qRT-PCR).

Results: The mitotane concentration 5×10⁻⁶ M has been selected based on analysis of hormone secretion and cell viability. Altogether 227 genes with significant differences in expression have been found, and the underexpression of 3 genes involved in steroid hormone biosynthesis (3-beta-hydroxysteroid dehydrogenase types 1 and 2 (HSD3B1 and HSD3B2), 21-hydroxylase (CYP21A2)) and 3 significantly overexpressed genes (aldehyde dehydrogenase 1 (ALDH1L2), growth differentiation factor 15 (GDF15) and serpin peptidase inhibitor (SERPINE2)) have been validated relative to the ZNF625 housekeeping gene.

Conclusions: These results demonstrate that the mitotane-induced decrease in adrenocortical hormone secretion is in part mediated at the mRNA level, and mRNA expression changes might be involved in the adrenolytic action of mitotane.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Endocrine Abstracts

P824