Abstract 256: Involvement of ERK and ROS in CLEFMA-induced apoptosis in lung cancer cells

Abstract

Abstract CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is novel synthetic molecule with potent growth inhibitory activity in human cancer cells. Our microarray gene expression analysis suggested that it induces oxidative stress in lung adenocarcinoma H441 cells but not in normal lung fibroblasts. That oxidative stress is a triggering mechanism for both apoptotic and autophagic cell death has been supported by several recent reports. Compared to normal cells, the cancer cells are more vulnerable to oxidative stress-induced cell death because of their high metabolic activity, increased likelihood of electron escape during oxidative phosphorylation and overburdened antioxidant system. In this study, we tested our hypothesis that CLEFMA-induced apoptotic cell death was dependent on the accompanying increase in oxidative stress in H441cells. Methods: Inhibition of cell growth by CLEFMA (10 µM) was estimated by MTT or hexosaminidase assay. ROS production, cell cycle and apoptotic cell population were assessed by flow cytometry. The phosphorylation levels of extracellular signal-regulated kinase (ERK1/2), p38 and JNK1/2 were examined by Western blotting. N-acetylcysteine (NAC, 5 mM) and PD98059 (5 mM) were used as ROS scavenger and ERK1/2 inhibitors, respectively. The molecular markers of apoptosis were analyzed by RT-PCR and immunobloting. Results: NCI-DTP program confirmed anticancer activity of CLEFMA in 60 human cancer cell lines. CLEFMA-treated H441 cells showed typical molecular signatures of apoptosis- upregulated pro-apoptotic and downregulated anti-apoptotic genes. There was time-dependent activation of caspases 3/7, PARP cleavage and BAX; the expression of anti-apoptotic XIAP1 and BCL-xL was reduced, whereas pro-apoptotic p53 was increased. Since BID was not activated, it appears that CLEFMA works through intrinsic apoptosis pathway. CLEFMA also induced sustained activation of ERK, p38 and JNK. The pharmacologic inhibition of ERK, but not that of p38 and JNK, resulted in decreased anti-proliferative activity of CLEFMA. ERK inhibition with PD98059 abrogated CLEFMA-induced apoptosis. The sustained ERK activation was found to be associated with the inhibition of dual specificity phosphatase, DUSP6, expression. Treatment with ROS scavenger NAC abrogated CLEFMA-induced cell death, ERK activation and pro-apoptotic biomarkers, and reinstated DUSP6 expression. Conclusion: From the results of this study, we conclude that CLEFMA-induces apoptosis by enhancing oxidative stress in the cancer cells, and that the accompanying prolonged ERK activation is required for CLEFMA-induced H441 cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 256. doi:1538-7445.AM2012-256