In vitro mass production, chemical modification, and cytotoxicity of shikonin derivatives on breast cancer cells

Abstract

Echium italicum L. is found to produce shikonin and its natural derivatives such as acetylshikonin that has cytotoxic activity on cancer cells. Thus, in vitro mass production of this plant is important for the overproduction of derivatives with the usage of hairy root cultures in bioreactor together with the inclusion of elicitors to the in vitro proliferation medium. Hence, the aims of the study concern the usage of recent SETISTM temporary im-mersion bioreactor systems with the application of different immersion times for the induced production of shikonin derivatives from hairy root cultures, synthesis of novel shikonin derivatives with imidazole-and indole-substituted compounds, and determination of cytotoxic activity of these new derivatives by investigation of their anti-proliferative influence on breast cancer cell lines. Within the various tested conditions, the highest prolif-eration of hairy roots was obtained with the inclusion of methyl jasmonate (MeJa) as an elicitor and immersion of cultures for 12 min for every 6 h. Two shikonin derivatives, acetylshikonin (ASK) and mixture of isovaleryl shikonin and 2-n-methyl-butyryl shikonin (IVS+MBS) were isolated from the extracts and characterized by HPLC, UV-Vis, mass spectroscopy, 1H and 13C NMR and FT-IR spectroscopy techniques. Among them, isolated acetylshikonin was modified with the inclusion of the bis-imidazole-(C34) and indole-(C36) substituted com-pounds. Cytotoxic activities of acetylshikonin and its novel modified ASK derivatives were evaluated on three breast cancer cell lines (MCF-7, SKBR-3, and MDA-MB-231). Overall results demostrated that both modified shikonin molecules had a more significant anti-proliferative effects on breast cancer cells than purified acetyl-shikonin from hairy roots of Echium italicum in the first 24 h. Moreover, all tested molecules, except 50 & mu;M in 48 h, did not show any cytotoxic activity against breast epithelial cells (MCF-12A) at 24 h and 48 h.