Mitotane induces mitochondrial membrane depolarization and apoptosis in thyroid cancer cells

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria-dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC-133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0-100 mu M). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC-1 staining and by western blot analysis using an antibody against caspase-3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit beta (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 mu M for 24 h) decreased the viability of FTC-133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone gamma H2AX, indicating DNA damage, and to caspase-3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro-apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine-protein kinase B-raf gene and proto-oncogene tyrosine-protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)-derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.