3.2. Pharmacodynamics and pharmacological effects
The main effect of mitotane in ACC is to decrease steroid production,
such as androgens (DHEA) and cortisol, and induce tumor cellular death
[6,10]. The current recommendation is to maintain the plasma level
higher than 7 mg L-1 (>21.8 µM) to
produce anti-steroidogenic effects and between 14–20 mg
L-1 (43.7–62.5 µM) for antitumor effects [30,57].
The major findings on the mechanism of action of mitotane were firstly
reported in dogs and mice, besides reports suggesting that rodents were
more insensitive to antisecretory mitotane effects [25,44,42]. The
mechanism of action of mitotane takes place mainly in adrenal
mitochondria and can be divided into four major categories: (i)
inhibition of steroidogenesis, (ii) endoplasmic reticulum (ER) stress,
(iii) cell death, and (iv) others (Fig. 2)
Inhibition of steroidogenesis
It is known that the rate-limiting step in steroidogenesis is the
transport of cholesterol from the outer mitochondrial membrane to the
inner mitochondrial membrane through the enzyme CYP11A1 (Fig.
3). Cholesterol reaches CYP11A1 via the transduceosome, a multiple
protein complex in the mitochondrial membrane, that contains the
translocator protein/peripheral-type benzodiazepine receptor or sigma 1
receptor (TSPO), steroidogenic acute regulatory protein (StAR), ATPase
family AAA domain-containing protein 3A (ATAD3A), and voltage-dependent
anion channel (VDAC) [58]. Interestingly, incubation with TSPO
inhibitor in combination with mitotane significantly potentiated the
antitumor and antisecretory actions of mitotane in H295R cells [59].
In addition, the expression of protein kinase A regulatory subunit
(PRKAR1A ), which is involved in the activation of
steroidogenesis, was reduced following 24 h of treatment with mitotane
[46]. This confirms that mitotane binds specifically to CYP11A1.
Many years ago, it was suggested that mitotane produced reactive
metabolites, such as DDAC, via mitotane β-hydroxylation through CYP450,
which could covalently bind to mitochondrial macromolecules [44,60].
Later, this hypothesis was confirmed when mitotane was found to bind to
the mitochondria in bovine and dog adrenals and to phospholipids and
proteins in the liver and lung of mice [45]. Mitotane labeled with14C was found to bind to the zona fasciculata /zona reticularis in adrenocortical tumor and mouse adrenals,
which was partially reversed by metyrapone, a known inhibitor of CYP11B1
[33,34]. These data suggest that CYP11B1 may also result in the
formation of reactive species. CYP11B1 is localized in the mitochondrial
inner membrane and is involved in the conversion of progesterone to
cortisol in the adrenal cortex (NCBI database gene). In addition,
CYP21A2, CYP17A1, hydroxy-delta-5-steroid dehydrogenase 3 beta- and
steroid delta-isomerase type 1 (HSD3B1), and type 2 (HSD3B2) were also
found to be decreased in cells after incubation with mitotane
[46,61]. The inhibition of these enzymes results in reduction in
aldosterone, cortisol, DHEA, testosterone, and estradiol production,
reducing ACC symptoms.
Considering that the aforementioned CYPs are steroidogenic enzymes
localized mainly in the adrenals, it may explain partially why mitotane
has local effect on adrenal tissue. However, until now, there is a lack
of studies that assessed the DDAC bound to macromolecules in human
adrenal and liver cells, to support this hypothesis. In addition, the
involvement of other reactive intermediates such as epoxides and free
radicals should also be considered in elucidating the mechanisms of
mitotane action [45].
The adrenocortical cell line is more sensitive to mitotane
(EC50 = 18.1 µM) whereas the EC50 was
3-fold lower in HeLa, HepG2, HEK293, and IMR32 cells [62]. This
supports the idea that mitotane specifically affects the steroidogenesis
pathways in adrenocortical cells. However, it is still unclear whether
mitotane inhibits a key enzyme, such as CYP11A1, resulting in reductions
in several steroidogenic enzymes; inhibits a steroidogenic regulatory
gene, such as SREBF (see below); or if mitotane affects all the
steroidogenic enzymes simultaneously. Additional studies evaluating the
chemical reaction between the intermediate metabolite DDAC and adrenal
mitochondrial compounds are still needed to assess if it occurs with
specific enzymes.
Endoplasmic reticulum (ER) stress