Citrus limonoids induce apoptosis in human neuroblastoma cells and have radical scavenging activity

Citrus limonoid glucosides, a family of fruit bioactive compounds, were postulated to have free radical-scavenging and apoptosis-inducing properties against certain types of cancers. Four highly purified limonoid glucosides, limoin 170 D-glucopypranoside (LG), obacunone 170 D-glucopyranoside (OG), nomilinic acid 170 D-glucopyranoside (NAG), and deacetylnomilinic acid 17 beta D-glucopyranoside (DNAG) were tested for superoxide radical (O-2(-))-quenching activity and cytotoxic action against undifferentiated human SH-SY5Y neuroblastoma cells in culture. All 4 scavenged O-2 as measured by inhibition of pyrogallol decomposition in a spectrophotometric assay. Quenching by NAG in particular emulated an equivalent concentration of vitamin C. When added to the medium of SH-SY5Y cells in culture, micromolar amounts of LG and OG, compared with untreated controls, caused a cessation of cell growth and rapid cell death (P < 0.001); NAG and DNAG were better tolerated, but nonetheless toxic as well. Cytotoxicity was related to a concentration- and time-dependent increase in caspase 3/7 activity, suggesting that limonoid glucosides were capable of inducing apoptosis. Arrested cell growth and the induction of apoptosis were confirmed by flow cytometry and DNA fragmentation analysis. Importantly, caspase induction at 12 h correlated with cell survival at 24 h (P = 0.046), suggesting that apoptosis was the primary cause of cell death. We conclude that citrus limonoid glucosides are toxic to SH-SY5Y cancer cells. Cytotoxicity is exerted through apoptosis by an as yet unknown mechanism of induction. Individual limonoid glucosides differ in efficacy as anticancer agents, and this difference may reside in structural variations in the A ring of the limonoid molecule.