Non-oxidized and oxidized flaxseed orbitides differently induce HepG2 cell apoptosis: involvement of cellular uptake and membrane death receptor DR4

BACKGROUND: Flaxseed orbitides have health-promoting properties, particularly potent anti-cancer activity. However, flaxseed orbitides containing a methionine structure, such as [1-9-N alpha C]-linusorb B2 (CLB), are easily oxidized to sulfoxide ([1-9-N alpha C],[1-Rs,Ss-MetO]-linusorb-B2 (CLC)) and sulfone ([1-9-N alpha C], [1-MetO]-linusorb B2 (CLK)), with CLC having less anti-cancer ability than CLB. It is unclear why oxidized flaxseed orbitides are less effective against cancer than non-oxidized flaxseed orbitide. RESULTS: Non-oxidized ([1-9-N alpha C]-linusorb-B3 (CLA) and CLB) and oxidized (CLC and CLK) flaxseed orbitides were found to significantly upregulate the levels of pro-apoptotic proteins, including Bax/Bcl-2, CytoC, caspase-3, and caspase-8, in a dose-dependent manner, with non-oxidized flaxseed orbitides being more effective than oxidized flaxseed orbitides. Mechanically, the cellular absorption of non-oxidized flaxseed orbitides was higher than that of oxidized flaxseed orbitides. Moreover, the significant fluorescence quenching of DR4 protein by flaxseed orbitides (especially non-oxidized orbitides) indicated the formation of a DR4-orbitide complex. Molecular docking demonstrated that non-oxidized orbitides could easily dock into the active cavity of DR4 protein. Further blocking DR4 significantly reduced the ability of non-oxidized flaxseed orbitides to stimulate caspase-3 expression, whereas oxidized flaxseed orbitides retained this ability. CONCLUSION: Non-oxidized flaxseed orbitides are more effective against cancer than oxidized flaxseed orbitides due to higher cellular uptake and activation of the DR4-mediated death receptor signaling pathway. (c) 2024 Society of Chemical Industry.