PO-454 Biological activities of novel brassinosteroid analogues in breast cancer cells

Abstract

Introduction Brassinosteroids (BS) are plant steroid hormones playing an important roles in various physiological processes and being of particular interest because of their antiproliferative activities towards human cancer cells. The study was dedicated to the synthesis of novel BS analogues and to the analysis of their effects on breast cancer (BC) cells and normal epithelium. Material and methods We developed an approach for the synthesis of novel secasterol derivatives with variations in the B cycle and side chain. Six new compounds, BS2-BS7, were obtained. Biological experiments were performed on the MCF-7 BC and MCF-10A normal breast epithelium cell cultures. The cytotoxicity was assessed by MTT test. Dock6 and Autodock Vina were used for molecular docking. Binding energies were evalueted using MM_PBSA approach on the 15 ns trajectories of MD simulation (Gromacs 5.1). Reporter assays were performed in steroid-free conditions. The MCF-7 BC cells were transfected with plasmids containing sequences for ERα- and AP-1-driven luciferase expression together with β-galactosidase plasmid to control the transfection efficacy. After transfection cells were treated with BS or vehicle control; then in 9–24 hour the cells were collected for subsequent analysis of luciferase and β-galactosidase activities. ERα expression was measured by immunoblotting. Results and discussions Evaluation of BS toxicity against MCF-7 revealed that compounds with cholestane/ergostane based side chain inhibited cell growth with IC50 values of 12.7–23.6 μmol. (22 R ,23 R ,24 R )−22,23-Dihydroxy-24-methyl-B-homo-7-oxa-5α-cholest-2-en-6-one ( BS4 ) was chosen as the lead compound with low toxicity in MCF-10A normal epithelium and high activity against BC. ERα was found to be a presumable target of BS4 . BS/ERα complex structures were obtained using molecular docking software. The best BS4 conformation was found to match 17β-estradiol conformation in ERα ligand-binding domain and had the best binding energy values during MD simulation compared with BS2 analogue. Reporter assays showed that compound BS4 decreased (up to 47%) 17β-estradiol-mediated activation of ERα transcription; moreover it also reduced ERα expression. BS4 was found to downregulate ERα–related transcription factor, AP-1. Conclusion Anticancer activities of novel compound BS4 look promising and require further studies to investigate their complex effects on breast cancer cells. The study was carried out with financial support by RFBR (project No. 17-54-04054 Bel_mol_a) and BRFFR (project No. X17PM-040).