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The antiproliferative effect of the bioaccessible fractions (BFs) of four hydroponic Brassicaceae microgreens (broccoli, kale, mustard and radish) was evaluated on colon cancer Caco-2 cells vs. normal colon CCD18-Co cells after 24 h treatment with BFs diluted 1:10 v/v in cell culture medium. Their bioactivity was compared with the digestion blank, while the colon cancer chemotherapeutic drug 5-fluorouracil was used as a positive control. Cell viability (mitochondrial enzyme activity assay (MTT test) and Trypan blue test) and mechanisms related to antiproliferative activity (cell cycle, apoptosis/necrosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, Ca2+ and glutathione (GSH) intracellular content) were studied. All microgreen BFs increased ROS and decreased GSH, altering the redox status and causing mitochondrial membrane dissipation followed by a general cell cycle arrest in G2/M and apoptotic cell death via a Ca2+-independent mechanism. As a result, the antioxidant bioactive compounds present in these microgreen species reduced the proliferation of tumoral cells (10 to 12.8% -MTT or 20 to 41.9% -Trypan blue), showing lesser effects with broccoli microgreens, in line with their lower ascorbic acid content and total antioxidant capacity. Therefore, the daily intake of microgreens within a balanced diet could be a preventive nutritional strategy to reduce the burden of chronic degenerative diseases such as colon cancer. [ABSTRACT FROM AUTHOR]