Title: Antiproliferative Effect of Bioaccessible Fractions of Four Brassicaceae Microgreens on Human Colon Cancer Cells Linked to Their Phytochemical Composition Authors: Beatriz de la Fuente,1 Gabriel López-García,1 Vicent Máñez,2 Amparo Alegría,1 Reyes Barberá,1 and Antonio Cilla 1 Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, Burjassot, 46100 Valencia, Spain CIAM (Centro de Innovación Agronómica_Grupo Alimentario Citrus), Av. dels Gremis, Parcela 28. Pol. Ind. Sector 13, Riba-roja de Túria, 46394 Valencia, Spain Published: April 28, 2020 Read or download the PDF: https://www.mdpi.com/2076-3921/9/5/368Abstract: 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. Introduction: Brassica vegetables represent one of the ten most economically important crops in the global agriculture and markets. Cruciferous vegetables are good sources of fibre, vitamins, and minerals, whilst having a low lipid content; thus, these plant foods have traditionally been recommended in low-fat and heart-friendly diets. In addition, different antioxidant bioactive compounds such as ascorbic acid, tocopherols, carotenoids, polyphenols and glucosinolates have been considered responsible for the prevention of chronic diseases attributed to cruciferous vegetables. In this sense, the consumption of Brassica greens has been associated with a reduced risk of the development of colorectal, stomach, pancreatic, lung, breast and ovarian cancer. A recent meta-analysis from observational studies concluded that the high intake of Brassica vegetables was negatively correlated with gastrointestinal cancer risk. Moreover, The World Cancer Research Fund has pointed out that diets rich in cruciferous vegetables particularly protect against colon, rectum and thyroid cancers. Regarding colon cancer, the third most common cancer worldwide, several epidemiological, experimental and clinical studies have considered vegetables belonging to the Brassicaceae family as one of the protective plant foods for this type of cancer. In addition to the increasing scientific interest surrounding the relationship between diet and cancer, the population’s concerns about the effect of food in cancer prevention have led to the search for healthy products by both consumers and the food industry. Such is the case for the novel consumption of microgreens, which are considered functional food due to their important phytochemical content, which is usually even higher than their mature counterparts. Recently, Choe et al. have reported that microgreens present potential anti-cancer prevention because of their content of vitamins, carotenoids, polyphenols, and glucosinolates. However, as far as we are aware, there is no study in the literature about the bioactivity of Brassica microgreens. In this sense, only a few in vitro studies have shown antiproliferative effects on human colon cancer cell lines using extracts from broccoli sprouts, mature broccoli, kale in the adult stage, mature mustard leaves [and the edible parts (pod and flower) of radishes. Conclusions: The bioaccessible fractions of broccoli, kale, mustard and radish Brassicaceae microgreens showed a statistically significant (p ˂ 0.05) antiproliferative effect in the colon cancer Caco-2 cells, whilst a lower effect was observed in the case of broccoli microgreens, in line with their lower ascorbic acid content and total antioxidant capacity. This in turn indicates that differences in the bioaccessibility of antioxidant bioactive compounds could be a useful tool to select samples with higher bioactivity linked to their phytochemical content in pre-clinical models for subsequent assays in humans. The bioactivity of edible microgreens after the in vitro gastrointestinal digestion process occurred through a concatenation of different events: (i) the alteration of the redox cellular state by ROS increase and GSH decrease (non-dependent on intracellular calcium accumulation); (ii) the arrest of the cell cycle in the G2/M phase and a reduction in the proportion of cells in the G0/G1 phase; (iii) the dissipation of mitochondrial membrane potential; and (iv) the induction of programmed cell death via the mitochondrial pathway (absence of necrosis). It is worth noting that the consumption of fresh Brassica microgreens within a balanced diet could be an effective nutritional strategy in colon cancer prevention and possibly other gastrointestinal and non-gastrointestinal cancers.
