Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration

Abstract

In this work, two neolignans - dehydrodieugenol (1) and dehydrodieugenol B (2) - were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC₅₀ > 500 μM) whereas compound 2 displayed IC₅₀ values of 169 and 174 μM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu²⁺, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO₄)₂·6H₂O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu²⁺. However, the combination of compound 1 and Cu²⁺ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC₅₀ values of 165 and 204 μM, respectively. The activity of compound 1 and Cu²⁺ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu²⁺ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu²⁺ compared with untreated cells (p > 0.0001) - 18.7 ng of Cu²⁺/mg of proteins and 0.37 ng of Cu²⁺/mg of protein, respectively. Conversely, the concentration of Cu²⁺ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu²⁺/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu²⁺ induces cell death through copper homeostasis dysregulation.

Keywords: Breast cancer; Copper; Cytotoxicity; Neolignans.