Endocrine Abstracts

The primary treatment of adrenocortical carcinoma (ACC) is surgery and the adrenolytic mitotane. In order to test the effect of everolimus, nilotinib, imatinib and zoledronic Acid/ZOL in combination with mitotane were used ACC cell line NCI-H295R, in monolayer (2D) and spheroid (3D) cell cultures. The cytotoxicity promoted by treatments was analyzed by MTS and Cell Death assays. These assays were also performed intending of use them to test drugs in tumor cell cultures from patients. In monolayer, Everolimus (10 μM) reduced cell viability in 44±0.10% (P≤0.001) after 72 h and apoptosis. Combination with mitotane 10 μM resulted in a significant inhibition of the cell growth, apoptosis and necrosis after 24 h (58±0.13%-P≤0.001). Zol (10 μM) induced apoptosis and promoted cell viability decreased only at the maximal concentration tested (10 μM), after 72 h (28±0.09%, P≤0.01). Combined with mitotane (10 μM), ZOL (5 μM) increased the cytotoxicity and promoted necrosis and apoptosis. Nilotinib decreased viability in 50±0.02%(P≤0.001) after 72 h, at the minimal dose tested (1 μM). However, in combination with Mitotane (10 μM) the inhibition was obtained after 24 h. Imatinib (5 μM) decreased the cell growth (18±0.08%-P≤0.01) after 72 h. In 2D cell culture, Nilotinib was more potent than Imatinib. In contrast, the spheroids cell culture showed a lower response than monolayer, and required higher drugs concentrations. Mitotano (30 μM) and Everolimus (10 μM) suppressed cell proliferation (50±0.01%, P≤0.001) after 72 h, with necrosis evidence. Mitotano30 μM and Nilotinib5 μM inhibited cell proliferation (41±0.06% P≤0.001) after 24 h, and showed late apoptosis. There was no difference between the Mitotane used alone and in combination with ZOL. In summary, Nilotinib and Everolimus were more efficient in combination with Mitotane, in both 2D and 3D cell culture. There are differences in the efficacy of the drugs in the 3D cell culture, suggesting the spheroids and MTS assay as important tools for the preclinical evaluation of the cytotoxic effect of anticancer agents.