Effect of a bispidinone analog on mitochondria-mediated apoptosis in HeLa cells

Abstract

The present study was carried out to investigate the effect of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) analogs on the in vitro growth of human cervical carcinoma (HeLa) cells. A series of 11 bispidinone analogs was synthesized with substituents, e.g., fluoro/methyl/ethyl/isopropyl/thiomethyl/methoxy groups, at various positions. These compounds were synthesized to identify which substituent and position induced the strongest cytotoxic effect in cancer cells. Among these synthetics, analog 9, which contains methoxy groups, had the most significant cytotoxic effect on HeLa cells, and its IC50 value was less than 13 µM. A WST-8 assay also showed that analog 9 inhibited the proliferation of HeLa cells. By using DNA content analysis, we found that analog 9 induced sub-G1 and G1 phase arrest in a time-dependent manner. A [3H]-thymidine incorporation assay suggested that analog 9 inhibited DNA replication in HeLa cells. On performing light microscopy, morphological changes such as cellular shrinkage and disruption, which are apoptotic features, were observed in HeLa cells. Annexin V/propidium iodide double staining and rhodamine-123 staining showed that analog 9 induced apoptosis and disrupted the intracellular mitochondrial membrane potential in HeLa cells. The western blot analysis results suggested that analog 9 induced mitochondria-mediated apoptosis. In addition, we have shown that analog 9 may play a role in the Fas signaling apoptotic pathway.