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.