Background: 
Adrenocortical carcinoma (ACC) is an orphan disease and has a poor prognosis in advanced stages. In ~60% of patients, there is clinical excess of adrenal steroid hormones. Mitotane is the single approved drug for treatment of ACC and used both in an adjuvant setting and in metastatic disease but associated with frequent and in part severe side effects, especially nausea, gastrointestinal disorders and vertigo. The exact mechanism of action is not fully understood. In addition, the drug has a small therapeutic range and unfavorable pharmacokinetics; therapy is monitored by serum-levels of mitotane. Therefore, a more specific treatment would be desirable. Recently, it has been discovered that mitotane inhibits the Sterol-O-Acyl Transferase 1 (SOAT1). SOAT1 is located in specialized endoplasmatic reticulum (ER) membrane domains and functions to form cholesteryl esters from cholesterol. The current concept is that through the inhibition of SOAT, accumulation of cholesterols in the tumor cells induces their cell death. However, a few studies suggested mitochondrial effects of mitotane, but its mechanisms are far from being resolved.

Aim: 

To assess in more detail the impact of mitotane and other SOAT1-inhibitors on mitochondrial function and cellular functions in different tissues.

Methods and Results: 

We determined respiration on mitochondria isolated from mouse heart, brain or kidney using a Clark electrode and pyruvate/malate (0.5 M) as complex I substrates. Respiration was determined in the absence (state 2) and presence of increasing concentrations of ADP (0.03-1mM; state 3 respiration) and in the presence of ADP plus oligomycin (to block complex IV of the respiratory chain; state 4). Mitotane as well as the SOAT inhibitors ATR-101 and AZD 3988 (1 – 100 µM) had no effect on respiration in the absence of ADP (state 2) or in the presence of oligomycin (state 4), indicating that these compounds do not damage the inner mitochondrial membrane. However, ADP-stimulated state 3 respiration was inhibited by all compounds in a concentration-dependent way. In contrast, Sandoz 58-035, a SOAT inhibitor without killing efficiency in ACC cells, did not inhibit respiration. Interestingly, in isolated, field-stimulated ventricular murine cardiac myocytes, mitotane did not affect the NAD(P)H/NAD(P)⁺ and FADH₂/FAD redox pools even at concentrations which inhibit respiration. Also, cytosolic Ca²⁺ levels and sarcomere shortening were not affected by mitotane at therapeutically relevant concentrations. In contrast, in an ACC tumor cell line (NCI - H295), administration of mitotane increased cytosolic calcium concentrations with a comparable effect as endothelin-1.  

Conclusion: 

The SOAT-inhibitors Mitotane, ATR-101 and AZD 3988 are inhibitors of mitochondrial respiration at concentrations at which anti-tumor effects in ACC have been observed in vitro and which are attained in vivo. In contrast, the SOAT inhibitor Sandoz 58-035, which does not inhibit respiration, neither reduces tumor growth. This indicates that inhibition of mitochondrial respiration rather than SOAT inhibition per se is required for antitumor efficacy of these drugs. Further research will elucidate the underlying reasons for cell specificity of these effects to explain why heart function is maintained despite tumor killing efficacy.