DOI: 10.14457/tu.the.2016.1473
title: Growth arrest and caspase-dependent apoptosis induced by 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene derived from Dioscorea membranacea Pierre in human lung adenocarcinoma A549 cells

description: An active compound of Dioscorea membranacea Pierre called 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) has been shown to possess the selective antiproliferative effect against human lung large cell carcinoma COR-L23 cells. In this study, an adequate amount of HMP was isolated from D. membranacea Pierre using column chromatography on silica gel 60 as the stationary phase, and the column was eluted by gradient elution in increasing order of polarity. The isolated compound was determined to be HMP by comparing its spectral data of proton nuclear magnetic resonance (1H NMR) with those of previously isolated compound and was further tested for cell-type-specific cytotoxicity in two main types of lung cancer cell lines including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using the sulforhodamine B (SRB) assay. The results showed that the validated HMP exhibited the most antiproliferative and cytotoxic effects on adenocarcinoma cell line A549, one of the three main subtypes of NSCLC. Therefore, the aims of this study were to comprehensively investigate the antiproliferative and cytotoxic effects through cell cycle arrest and apoptosis in human lung carcinoma A549 cells. In the present study, the antiproliferative and cytotoxic effects of HMP were analyzed by the SRB assay. Cell division, cell cycle distribution, membrane asymmetry changes and intracellular ROS generation were each performed with different fluorescent dyes including carboxyfluorescein succinimidyl ester (CFSE), propidium iodide (PI), annexin V-FITC double staining and 2´,7´-dichlorofluorescein (DCF), respectively, and they were then analyzed by flow cytometry. Cell cycle- and apoptosis-related mRNA and proteins levels were measured by real-time PCR and western blot analyses, respectively. The nuclear morphology of apoptotic cells stained with 4’, 6-diamidino-2-phenylindole (DAPI) and DNA fragmentation were detected by fluorescence microscopy and gel electrophoresis, respectively. The results showed that HMP exerted strong antiproliferative (represented as IC50 = 9.37 µM and TGI = 54.81 µM) and cytotoxic effects (represented as LC50 = 94.01 µM) in A549 cells with the highest selectivity index as compared with the human lung fibroblast cell line MRC-5. Treatment of A549 cells with HMP induced a rapid arrest of cell division and halted the cell cycle at G2/M phase through down-regulation of the expression levels of G2/M regulatory proteins cdc25C, cdk1 and cyclin B1. Moreover, HMP treatment induced early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic cells in the sub-G1 phase of the cell cycle and concurrent activation of caspase-3, whose activity was completely abolished with pan-caspase inhibitor Z-VAD-fmk. Indeed, the active form of caspase-3 was detected, and its actions were supported by the results of cleavage of its target PARP and morphological alterations of apoptotic cell death such as nuclear condensation, DNA fragmentation with accompanying DNA ladder formation. In addition, HMP significantly increased the Bax/Bcl-2 mRNA and protein ratios of proapoptotic, especially at 72 h of incubation, leading to subsequent caspase-9 activation and further indicating the induction of the intrinsic apoptotic pathway. Also, HMP induced apoptosis via the extrinsic pathway by causing the proteolytic cleavage of Bid. This study, furthermore, demonstrated that HMP could generate excessive intracellular ROS, which was confirmed using ROS scavenger NAC. This inhibitor was used to further study the involvement of ROS in HMP-induced apoptosis. In conclusion, this is the first molecular evidence of HMP that exerted its anticancer actions through the induction of G2/M cell cycle arrest as well as the intrinsic and extrinsic apoptotic pathways in A549 cells. These data support the potential role of HMP as a cell-cycle arrest and apoptosis-inducing agent for treatment of NSCLC and the use of D. membranacea Pierre in Thai traditional herbal remedies for cancer treatment.