HY253, a novel decahydrofluorene analog, from Aralia continentalis, induces cell cycle arrest at the G(1) phase and cytochrome c-mediated apoptosis in human lung cancer A549 cells

Abstract

Aim of the study: In the course of our screening for novel modulators on cell cycle progression and apoptosis as anticancer drug candidates, we isolated a novel compound HY253 with the molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis. This study was designed to evaluate the detailed mechanisms of cell cycle arrest and the apoptotic induction of HY253 in human lung cancer A549 cells. Materials and methods: To investigate the effects of HY253 on cell cycle progression in A549 cells, we measured DNA content of A549 cells treated with 35 mu M of HY253 using flow cytometric analysis. Furthermore, TUNEL assay was used to examine apoptotic induction in A549 cells treated with 70 mu M of HY253 for 24 and 48 h. The effects of HY253 on apoptosis-associated and cell cycle regulatory proteins in A549 cells were examined using Western blot analysis. Results: The flow cytometric analysis revealed an appreciable G(1) phase arrest in A549 cells treated with 35 mu M of HY253. This HY253-induced G(1) phase arrest is associated with decreased expression of cyclin D and up-regulation of p21(CIP1), via p53 phosphorylation at Ser-15, which resulted in increased hypophosphorylated pRb in A549 cells. Furthermore, TUNEL assay and Western blot analysis revealed an appreciable apoptotic induction in A549 cells treated with 70 mu M of HY253 for 48 h. This apoptotic induction in HY253-treated A549 cells is also associated with cytochrome c release from mitochondria which in turn resulted in the activation of caspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). Conclusions: These results demonstrate that HY253, a novel antiproliferative compound isolated from the roots of Aralia continentalis, induces cell cycle arrest at the G(1) phase and apoptosis in A549 cells. Based on these results, we suggest that HY253 may be a potent cancer chemotherapeutic candidate for use in treating human lung cancer cells via up-regulation and activation of p53 gene. (C) 2010 Elsevier Ireland Ltd. All rights reserved.