title: 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‑chl-oro‑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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Poznan University                     of Medical Sciences, Poznan 60‑781, Poland.
authors.forename: Tomasz P
authors.initials: TP
authors.lastname: Lehmann
authors.forename: Tomasz
authors.initials: T
authors.lastname: Wrzesiński
authors.forename: Paweł P
authors.initials: PP
authors.lastname: Jagodziński
mesh_terms: D018268:Adrenocortical Carcinoma / Q000378:metabolism / Q000473:pathology; D018931:Antineoplastic Agents, Hormonal / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D019314:Dehydroepiandrosterone Sulfate / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D008939:Mitotane / Q000633:toxicity*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
chemical_list: D018931:Antineoplastic Agents, Hormonal; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D050759:Cyclin-Dependent Kinase Inhibitor p21; C517834:PRKAR1A protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D019314:Dehydroepiandrosterone Sulfate; D008939:Mitotane; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D053148:Caspase 3; D053179:Caspase 7; D006854:Hydrocortisone