The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells

Abstract

Mitotane, also known as o,p'-DDD or (RS)-1-chloro-2-[2,2-dichloro-1-(4- chlorophenyl)-ethyl]-benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24-h mitotane treatment on viability of the ACC cell line, NCI-H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase-3 and -7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450 (CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor β1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24-h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI-H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased caspase-3 and -7 activities. Since p21 and cMyc mRNA levels were stable in the presence of mitotane, the mechanism by which caspase-3 and -7 are induced remains unknown.