Caspase-3-Dependent Apoptosis of Citreamicin ε-Induced HeLa Cells Is Associated with Reactive Oxygen Species Generation

Citreamicins, members of the polycyclic xanthone family, are promising antitumor agents that are produced by Streptomyces species. Two diastereomers, citreamicin epsilon A (1) and B (2), were isolated from a marine-derived Streptomyces species. The relative configurations of these two diastereomers were determined using NMR spectroscopy and successful crystallization of citreamicin epsilon A (1). Both diastereomers showed potent cytotoxic activity against HeLa (cervical cancer) and HepG2 (hepatic carcinoma) cells with IC50 values ranging from 30 to 100 nM. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed that citreamicin epsilon A (1) induced cellular apoptosis, and Western blot analysis showed that apoptosis occurred via activation of caspase-3. The 2,7-dichlorofluorescein diacetate assay indicated that citreamicin epsilon substantially increased the intracellular concentration of reactive oxygen species (ROS). To confirm the hypothesis that citreamicin epsilon induced apoptosis through an increase in the intracellular ROS concentration, the oxidized products, oxicitreamicin epsilon A (3) and B (4), were obtained from a one-step reaction catalyzed by Ag2O. These products, with a reduced capacity to increase the intracellular ROS concentration, exhibited a significantly weakened cytotoxicity in both HeLa and HepG2 cells compared with that of citreamicin e A (1) and B (2).