The effects of mitotane and 1α,25-dihydroxyvitamin D3 on Wnt/beta-catenin signaling in human adrenocortical carcinoma cells

Purpose Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), however, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/ beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1 alpha,25(OH)(2)D-3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling. Methods and results Multiple concentrations of mitotane and 1 alpha,25(OH)(2)D-3, individually or in combination, were tested on H295R cells for 24-96 h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/timedependent manner for both mitotane and 1 alpha,25(OH)(2)D-3. In addition, a combination of clinically sub-therapeutic concentrations of mitotane with 1 alpha,25(OH)(2)D-3, had an additive anti-proliferative effect (Combination Index =1.02). In a wound healing assay, individual treatments of both mitotane and 1 alpha,25(OH)(2)D-3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways. Conclusion Our results show an additive effect of mitotane and 1 alpha,25(OH)(2)D-3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/betacatenin pathways.