Mitotane inhibited the function of sterol-O -acyl transferase 1 (SOAT1) in H295R cells which highly express this enzyme [62]. SOAT1 is an intracellular protein located in the ER that promotes the formation of fatty acid-cholesterol esters (NCBI database gene). The suppression of SOAT1 by mitotane led to an accumulation of free cholesterol and fatty acids, which explains why patients had an increase in cholesterol level throughout mitotane treatment [63]. This increase in free cholesterol ester and fatty acid levels induced ER stress promoting downregulation of sterol regulatory element-binding transcription factor 1 (SREBF) [62].SREBF is a gene that stimulates the transcription of steroid-regulated genes (NCBI database gene); thus, the inhibition of steroid production induced by mitotane may be due to the downregulation of SREBF . As ER stress persisted, it induced the expression of protein kinase R-like endoplasmic reticulum kinase (PERK) which phosphorylated the eukaryotic initiation factor 2α phosphorylation (eIF2α) and activated the C/EBP homologous protein (CHOP) expression. The upregulation of CHOP activated the intrinsic apoptosis pathway, leading to cellular death [62]. Therefore, the inhibition of theSREBF gene through ER stress may underlie one of the mechanisms by which mitotane induces the activity of caspases and the reduction in steroid production on ACC. In this line, a novel SOAT1 inhibitor (ATR-101, PD132301-2, nevanimibe HCl) was demonstrated to induce ER stress and apoptosis in adrenal cells [64]. Thus, a recent multicenter phase 1 study was conducted to assess the safety and pharmacokinetics (PK) of nevanimibe in adults with metastatic ACC. Although no patients experienced a complete or partial response, 27% of the patients had stable disease. However, given that the expected exposure levels necessary for an apoptotic effect could not be achieved with the maximum feasible dose, the current formulation of nevanimibe had limited efficacy in patients with advanced ACC [65].
Cell death
The attachment of the DDAC to adrenal cells can either induce oxidative stress and cellular death by apoptosis and/or necroptosis. It was also found that the decrease in adrenal cell viability was due to increase in caspase 3/7 activity, which induced cellular apoptosis [46]. Moreover, the apoptosis was also due to inhibition of mitochondrial respiratory chain complexes I and IV in H295R cells by inducing cytochrome c oxidase defect [66]. It has been observed that mitotane (30 and 50 µM) induced mitochondrial morphological alterations in human tumor adrenocortical cells (H295R and SW13), including membrane disruption, affecting respiratory chain enzymes, such as succinate dehydrogenase and the voltage-dependent anion channel, resulting in inhibition of tumor cell proliferation and induction of cellular apoptosis [66,67]. Despite the evidence that adrenal cortical cells were also susceptible to ferroptosis dependent on steroid pathways, mitotane did not induce this form of cell death in ACC cells [68].
Other mechanisms
In addition to inhibition of steroidogenesis, ER stress, and apoptosis, mitotane also alters other pathways that need further investigations. Mitotane modulates proteins involved in cellular metabolism (NADPH), stress response (peroxiredoxin I, II, and VI and heat shock protein 27), cytoskeleton structure (tubulin-b isoform II and profilin-1), and tumorigenesis (prohibitin, heterogenous nuclear ribonucleoprotein A2/B1, and cathepsin D). These proteins represent good targets for the development of strategies that can directly inhibit ACC growth [69]. Moreover, mitotane inhibited the expression of transforming growth factor β1 (TGF-β) gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. However, it appeared to be independent of CYP11A1 or CYP11B1 inhibition [46].
Despite the evidence that mitotane affects the aforementioned pathways, the molecular mechanisms of acquired mitotane resistance are currently unknown. Thus, a recent study established an in vitro model of mitotane resistance in ACC using the HAC-15 cell line. The findings showed an absence of mitochondrial damage and increased in intracellular free cholesterol levels, downregulation of adrenal steroids production, and regulation of extracellular-signal-regulated kinase (ERK), apoptotic cell clearance, and response to xenobiotics in mitotane-treated resistant cells, compared to that in nonresistant controls. The study pointed at the changes in lipoprotein and lipid homeostasis which collectively may contribute to the resistant phenotype. Although this model might help develop strategies to overcome mitotane resistancein vitro , further studies should be performed using in vivo models of mitotane resistance before conducting clinical trials [70].
Toxicity
Mitotane concentrations above 20 mg L-1 are highly toxic. The oral LD50 of mitotane were reported to be 17, 5, 4, and 5 g in humans, guinea pigs, mice, and rats, respectively (NIH-TOXNET; ChemIDplus). Even though plasma mitotane levels are maintained below 20 mg L-1 for ACC treatment, patients with plasma levels < 15 mg L-1 can experience several toxic effects, probably due to the variability in CYP activity among patients.
The most common side effects of mitotane are unspecific, such as those involving the gastrointestinal, nervous, and endocrine systems, and can vary among patients. The unspecific gastrointestinal symptoms include diarrhea, nausea, vomiting, and anorexia, which can evolve to mucositis [71,21,10]. These symptoms were reported in 78% of patients who received daily doses of 2 g or more [72]. At higher doses, mitotane affected the central nervous system and produced neuromuscular manifestations, including ataxia, speech disturbance, confusion, somnolence, depression, decrease memory, muscle tremors, polyneuropathy, and dizziness [72]. Mitotane increased hepatic production of sex hormone binding globulin (i.e human corticosteroid-binding globulin [CBG], sex hormone binding globulin [SHBG], thyroxine-binding globulin [TBG], and vitamin D binding protein) and cortisol binding globulin, which increased total serum levels of gonadal steroids and cortisol. Thus, the androgen-synthesis reducing effect of mitotane and a relative increase in SHBG may cause gynecomastia and primary hypogonadism [71,10,2]. Mitotane treatment may also increase bilirubin levels and skin rashes [37,71].
An increase in cholesterol levels was also documented during mitotane administration. Mitotane’s inhibition of CYP450, an enzyme involved in cholesterol metabolites formation, may result in an increase in mevalonic acid and oxysterols responsible for downregulating hepatic cholesterol synthesis [37]. However, the exact effect of the increase in the activity of the mevalonate pathway during mitotane treatment is not well explored. Mitotane also influenced the production of thyroid hormones, leading to a decrease in the levels of thyroid-stimulating hormone and free thyroxine, which must be monitored and replaced if necessary [71]. Other manifestations included impotence, thrombocytopenia, anemia, and increases in hepatic enzyme levels [71,10]. Recently, dyspnea was also reported in a female patient [73]. In general, adverse events were reversible after the cessation of mitotane treatment [71]. Because mitotane may effectively decrease the synthesis of all steroids, it is mandatory that clinicians consider the need to prescribe glucocorticoid and mineralocorticoid replacements. This concern is more urgent in tumors presenting with high levels of glucocorticoids, suppression of the hypothalamus-pituitary-adrenal-axis, and potential acute or chronic adrenal insufficiency. Mitotane may aggravate adrenal cortex suppression. Taken together, mitotane toxicity may limit tolerability, and it may be necessary to discontinue treatment.
New perspectives for mitotane treatment
To improve the current therapeutic options for ACC with less toxic effects, the development of new formulations containing mitotane or new compounds containing its metabolites should be an attractive option. Below we discuss two potential options for ACC treatment.