Abstract 4887: Curcuminoid CLEFMA induces autophagy-associated apoptosis in lung cancer

Abstract

Abstract CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is a novel curcuminoid discovered in our laboratory. Earlier we showed that CLEFMA induces oxidative stress-dependent cell death in lung adenocarcinoma H441 cells. Although apoptosis is the desired mechanism for chemotherapy-induced cell death, the associated pathways are often defective in lung cancers resulting in high levels of chemoresistance. Therefore, drugs that induce alternate modes of cell death, such as autophagy, may be beneficial. Drug-induced hyperinduction of autophagy is classified as type 2 programmed cell death. In most circumstances, autophagy parallels apoptosis, and such composite cell death is described as “autophagy-associated apoptosis”. Whether autophagy plays a synergistic or antagonistic role in overall cell death response to anticancer drugs is an important focus in cancer research. In this study, we have investigated the interaction between autophagy and apoptosis in CLEFMA mediated cell death. Methods: The human non-small cell lung carcinoma (H441) cell line was treated with CLEFMA (10 µM) and the molecular mechanisms of cell death were investigated. Growth inhibition was assessed by MTT or hexosaminidase assay. Apoptosis was measured by flow cytometry of annexin V-stained cells, RT-PCR and immunoblotting for apoptosis-related biomarkers. Induction of autophagy was established by electron microscopy and immunobloting for LC3B, ATG, beclin1, AKT and mTOR. Autophagy was inhibited by 3-methyladenine (3-MA 1 mM) as well as chloroquine and E64D+pepstatin combination. The tumor suppressive efficacy of CLEFMA was demonstrated in H441 xenograft mice intraperitoneally receiving 0, 5 and 10 µg/day of CLEFMA for 32 days. The mice were imaged for F-18-fluorodeoxyglucose uptake in tumor tissue. Results: CLEFMA suppressed the viability of H441 cell line in a dose-dependent manner (IC50 = 6.25 µM). It induced cell cycle arrest at G2/M phase with accompanying accumulation of p21 protein. Caspase 3/7 immunoblotting and annexin V flow cytometry confirmed the induction of apoptosis. At the same time, we observed induction of LC3BII and suppression of AKT/mTOR phosphorylation. The suppression of autophagy significantly increased CLEFMA-induced cell death and increased caspase cleavage. In the xenograft model, 5 and 10 µg CLEFMA significantly reduced the tumor volume by 72% and 96.2%, respectively; the tumor uptake of F-18-fluorodeoxyglucose was also reduced. Conclusions: We demonstrate the efficacy of CLEFMA as a potent tumor suppressive agent. The mechanistic results suggest that autophagy is a survival mechanism in CLEFMA-treated lung cancer cells and it antagonizes apoptosis. 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 4887. doi:1538-7445.AM2012-4887