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