2,4,6,8-(3)-Tetranitrophenyl-3,7-diazabicyclo[3.3.1] nonan-9-one (B16), a bispidinone analog, was synthesized to investigate its effects on cell viability, the cell cycle, and apoptotic pathways in HeLa human cervical cancer cells. B16 decreased the percentage of viable cells in WST-8 assays, and morphological changes associated with apoptotic cell death were observed, including cell shrinkage and disruption. Annexin V-FITC/PI dual staining assays showed that B16 significantly increased the

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... y apoptosis of HeLa cells after 24 h of treatment. Moreover, DNA content analysis and [ 3 H]-thymidine incorporation assays showed that B16 induced S-phase cell cycle arrest and inhibited DNA replication after 24 h of treatment. Following treatment with 25 µM of B16, an increase in reactive oxygen species and a decrease in mitochondrial membrane potential were observed by flow cytometry. In addition, the expression levels of caspase cascade and Bcl-2 family proteins determined by western blotting suggested that the induction of apoptosis by B16 was associated with a caspase-and mitochondrial-dependent pathway in HeLa cells. In conclusion, B16 induced early apoptosis and S-phase cell cycle arrest in HeLa cells via a caspase-and mitochondrial-dependent pathway.