Ketoconazole Treatment Decreases the Viability of Immortalized Pituitary Cell Lines Associated with an Increased Expression of Apoptosis-Related Genes and Cell Cycle Inhibitors.

Abstract

Ketoconazole, which was initially developed as an antifungal agent, is a potent inhibitor of adrenal steroidogenesis and has therefore been used in the management of Cushing's disease. Surprisingly, the reduction of cortisol levels during ketoconazole treatment is not accompanied by the expected elevation in plasma adrenocorticotrophic hormone (ACTH) at the loss of negative cortisol feedback from corticotrophic cells, suggesting a direct effect of ketoconazole on these cells. To characterize the direct effects of ketoconazole, we evaluated its invitro effect on cell viability using the pituitary tumoural cell lines AtT-20 (which secretes ACTH), GH3 (which secretes growth hormone and prolactin) and αT3.1 (which secretes α-subunit) and we also determined the expression levels of genes involved in apoptosis and DNA replication by the quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also evaluated ACTH levels in AtT-20 cells during ketoconazole treatment. We observed a ketoconazole concentration-dependent decrease in pituitary cell viability and reduced ACTH levels in AtT-20 cells after removal of the drug. We also observed increased expression of cell death receptors (e.g. Fas, tumour necrosis factor receptor) and caspases (e.g., caspase-6, caspase-7, caspase-9), suggesting activation of the apoptosis pathway. In addition, we observed increased gene expression of the cell cycle inhibitors p21 and p27 in GH3 cells and increased expression of p21 in αT3.1 cells. In conclusion, our findings suggest that ketoconazole significantly reduces cell viability in a concentration-dependent manner in pituitary tumour cell lines and is associated with an increase in apoptosis- and cell cycle regulation-related gene expression.