Lactofermented beverage from broccoli leaves induces calcium dysregulation and ER-mitochondrial dysfunction in colorectal cancer cells: A proteomic perspective

Functional foods have emerged as promising agents in cancer prevention due to their bioactivity and safety, with the aim of inhibiting or reversing carcinogenesis. This study investigates the cytotoxic effects and molecular mechanisms of a lactofermented beverage (LB) obtained from broccoli leaves on HT29 colon adenocarcinoma cells. Phytochemical characterisation revealed high levels of hydroxycinnamic acids, flavonoids, and organosulphur compounds. Proteomic characterisation of LB-response in epithelial cells revealed the alteration in diverse cell death pathways and stress responses. LB treatment resulted in elevated cytosolic Ca²⁺ levels, inducing mitochondrial stress, decreasing membrane potential, and increasing reactive oxygen species. The pro-oxidant environment triggersendoplasmic reticulum (ER) stress via lipid oxidation and unfolded protein response (UPR). In addition, LB treatment activated autophagy, antioxidant proteins (AKR1C1/2), and proteasome pathways to counteract cellular stress. Inhibition of these relevant pathways resolved ER stress and autophagy as cell resistance to LB treatment and necroptosis as the earliest cell death mechanism. In conclusion, our findings suggest that a broccoli-based lactofermented beverage, through the valorisation of agro-industrial by-products, targets several cell death mechanisms with anti-carcinogenic potential in colon cancer cells. Further studies are needed to validate if this beverage can be considered as a future functional food.