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.

Biochemical mechanism of the o,p’-DDD effect on the adrenal cortex

“…The level of caspase-3 and -7 increased in the cells. The increased caspase-3 and -7 activities in these cells were also shown by other researchers [28].Theinfluen ceofo,p'-DDD on apoptotic DNA fragmentation in vitro was analyzed in our work [29]. Western blot analysis showed that o,p'-DDD reduced the expression of the mitochondrial anion channel VDAC in the outer membrane, increased the expressionofcaspase-3butdidnotaffectthelevelofsuc-cinate dehydrogenase (complex II) in the mitochondriainnermembrane [23].Theeffectofo,p'-DDD on respiratory chain was studied on cultured H295R and SW13 cells derived from human adrenocortical carcinoma [31].…”

“…Three steroidogenesis regulatory genes hSd3B1, hSd3B2 and CYP21A2 were underexpressed, and four genes GdF15, ALdh1L2, TRIB3 and SERPINE2 were overexpressed. A decrease in the mRNA level of cytochromes CYP11A1 and CYP17A1 in H295R cells in the presence of o,p'-DDD was shown in the study [28]. o,p'-DDD was found to cause a decrease in the level of mRNA protein kinase A regulatory subunit (PRKAR1A).…”

Rak kory nadnercza (ACC) — przegląd piśmiennictwa i doświadczenia własne

“…Z ostatnich badań przedklinicznych wiadomo, że mitotan redukuje przeżywal-ność komórek NCIH295R (ludzka linia komórek ACC) poprzez indukcję apoptozy spowodowaną zwiększe-niem aktywności kaspazy 3 oraz kaspazy 7. Badanie to wykazało, że mitotan znacząco hamuje ekspresję genów biorących udział w steroidogenezie, sekrecji kortyzolu oraz DHEAS [47]. Dokładny mechanizm tego działania w komórkach ludzkich wciąż jest badany.…”

“…This study indicated that mitotane markedly inhibited the expression of genes involved in steroidogenesis, secretion of cortisol, and DHEAS [47]. In humans, the exact mechanism of its action remains to be fully elucidated.…”

Management of adrenocortical carcinoma: a consensus statement of the Italian Society of Endocrinology (SIE)

“…Mitotane is an isomer of the insecticide DDT with adrenolytic activity. Mitotane inhibits gene expression of a number of enzymes involved in the steroidogenetic pathway in the mitochondria of steroidogenic cells decreasing both plasma and urine hormone levels [135][136][137]. Mitotane produces focal degeneration of the zona fasciculata and the zona reticularis, while the effects on the glomerular area are relatively scarce [138].…”