Modulation of iron homeostasis by broccoli (Brassica oleracea L.) sprouts extract in AML12 cells

Sulforaphane (SFN) is a bioactive compound that modulates the action of nuclear factor erythroid 2 (Nrf2), which activates genes related to iron homeostasis. Broccoli is a vegetable known for its antioxidant and anti-inflammatory effects, mainly attributed to its SFN content. SFN is highly concentrated in broccoli sprouts. The aim of this study was to investigate the effects of broccoli sprout extracts in cellular iron metabolism. After germination, SFN concentrations of sprouts aqueous extracts were determined by HPLC. AML12 cells were treated with SFN at 2 and 50μM and with sprouts aqueous extracts of three (BSE- 3D; at 2 and 50μM SFN) and seven days (BSE-7D; at 2 and 50μM SFN). The expression of genes and proteins related to iron metabolism (hepcidin, ferroportin (FPN), ferritin (FTH1), divalent metal transporter 1 (DMT1) and transferrin receptor (TFR2)) was evaluated by qPCR, Western Blotting or immunocytochemistry. Treatments at the concentration of 50 μM of SFN and BSE-3D decreased FPN expression compared to the control treatment, while it increased ferritin expression. The increased iron accumulation, indicated by ferritin levels, may explain the reduced expression of DMT1, TFR2, and hepcidin genes. Immunocytochemistry images corroborated with these results showing a reduction in the expression of ferroportin in these two groups. ELISA analysis was also done to assess hepcidin secretion and significative differences were not seen among treatments. Reactive oxygen species (ROS) production, observed by fluorimetry, was increased by SFN and BSE-3D (50 μM) treatments. This suggests that both treatments effectively regulated the molecular mechanisms that favor intracellular iron storage with accumulation of ROS, thus leading to decrease in cell viability. The increase in ferritin and the accumulation of ROS may be critical factors in the treatment of diseases, as it suggests an increase in intracellular iron and a decrease in cell viability.