Elsevier

Phytomedicine

Volume 53, February 2019, Pages 124-133
Phytomedicine

Original Article
Dioscin-6′-O-acetate inhibits lung cancer cell proliferation via inducing cell cycle arrest and caspase-dependent apoptosis

https://doi.org/10.1016/j.phymed.2018.09.033Get rights and content

Abstract

Background

Lung cancer is the leading cause of global cancer-related mortality. Dioscin-6′-O-acetate (DA), a novel natural steroidal saponin, was firstly isolated from the rhizomes of Dioscorea althaeoides R. Knuth. Until now, there were no studies on its pharmacological activities.

Purpose

Here, we investigated the growth inhibitory effect and explored the underlying molecular mechanisms of DA against lung cancer cells.

Methods/study designs

NSCLC H460, H1299, H520 cells and SCLC H446 cells were treated with DA. To display the cytotoxic effects and possible mechanism of DA on these cells, MTT assay, flow cytometry and western blot analysis were carried out.

Results

Our results showed that DA exerted strong anti-proliferative activity against lung cancer cells in a concentration- and time-dependent manner. Flow cytometry demonstrated DA induced the cell cycle arrest at S-phase (NCI-H460, NCI-H1299, NCI-H520) or G1-phase (NCI-H446), caused cellular apoptosis, generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential. Western blotting analysis showed DA treatment increased the levels of caspase 3, 8, 9, Bax, p21, p53, phosphorylated JNK and p38 MAPK and markedly decreased the expression of Bcl-2, p-ERK, p-PI3K, p-AKT and NF-κB. Blockade of caspases with Z-VAD-FMK converted apoptosis-related proteins. Suppression of p53 with pifithrin-α (PFT) attenuated cell cycle-related protein. Inhibition of ROS with N-acetyl-cysteine (NAC) adjusted apoptosis-related proteins and phosphorylated MAPK and PI3K, as well as NF-κB.

Conclusion

Overall, our study indicated that DA suppressed lung cancer cells proliferation via inducing cell-cycle arrest and enhancing caspase-dependent apoptosis, at least partly, through ROS-mediated PI3K/AKT, MAPK and NF-κB signaling pathways.

Introduction

Lung cancer is the most-frequent cancer type and the leading cause of cancer-related death worldwide (El Gaafary et al., 2017, Liu et al., 2016, Siegel et al., 2016). Non-small cell lung cancer (NSCLC), including squamous carcinoma, adenocarcinoma and large cell carcinoma, accounts for about 85% of all lung cancer cases (Liu et al., 2016), while small cell lung cancer (SCLC) as another type of lung cancer, is the most aggressive lung cancer form (Liu et al., 2017). Despite progress in lung cancer diagnosis and treatment with target cancer therapies, the patient outcome remains unsatisfactory. The 5-year survival is only about 12% in NSCLC and 6% in SCLC (Liu et al., 2017). Thus, new drugs or strategies are urgently required for both NSCLC and SCLC patients.

Recently, natural compounds have raised increasing attention for their potential use in oncotherapy due to their high efficiency and low toxicity. Dioscorea plants as important agricultural crops grown in tropical and temperate regions in Asia, Africa, and Oceania, contain large amounts of steroidal saponins and exert good antitumor activity (Chan and Ng, 2013, Chien et al., 2012, Hsu et al., 2011, Tong et al., 2012). Dioscin-6′-O-acetate (DA), as a novel derivative of steroidal saponin dioscin, was firstly isolated from the rhizomes of Dioscorea althaeoides R. Knuth (Dioscoreaceae) (Li et al., 2016). Its deacetylation analogue, dioscin existing in most of Dioscorea plants showed anticancer effects against human lung cancer cells (A549, NCI-H446 and NCI-H460) through blocking cell cycle progression, induction of DNA damage, and activation of mitochondrial signal pathway (Wei et al., 2013). It induced cell apoptosis and autophagy via inhibiting PI3K/AKT/mTOR and activating ERK and JNK signaling pathways in A549 and NCI-H1299 cells (Hsieh et al., 2013). However, there were still no studies on the pharmacological activities of DA. The aim of the present work was to investigate the cytotoxic effects and possible mechanisms of DA on four kinds of human lung cancer cell lines including large cell lung cancer NCI-H460, adenocarcinoma NCI-H1299, squamous cell carcinoma NCI-H520 and small cell lung cancer NCI-H446.

Section snippets

Reagents

Dioscin-6′-O-acetate (DA), was isolated by our laboratory (Li et al., 2016) with the purity >98% (1H and 13C NMR Spectra see Figs. S1 and S2, HPLC chromatogram see Fig. S3). Cell cycle analysis, reactive oxygen species (ROS), apoptosis analysis, senescence β-galactosidase staining, and nuclear and cytoplasmic protein extraction kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT), hoechst 33258,

Effects of DA on viability and morphological changes of lung cancer cells

The viability of four lung cancer cells after exposure to different concentrations of DA and dioscin (Fig. 1A, 0-10.0 µM) was tested by MTT assay. Just as dioscin, DA significantly suppressed the viability of four lung cancer cells in a concentration- and time-dependent manner (Fig. 1B and C). However, DA caused less growth arrest in the normal lung BEAS-2B cells than in the four lung cancer cells. Based on the half-maximal inhibitory concentration (IC50) values at 24 h (IC50H520 < IC50H446

Discussion

As a leading cause of global cancer-related mortality, lung cancer has the greatest annual burden among all cancers. The development of new drugs to treat lung cancer is urgently needed. As a natural steroidal saponin from Dioscorea althaeoides R. Knuth, the cytotoxicity of DA on human lung cancer H460, H1299, H520 (NSCLC) and H446 (SCLC) cells were investigated in this study for the first time.

Cell cycle is an effective target for cancer treatment. Dioscin blocked cell cycle at S phase in

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was financially supported by grants No. 81373904, 81673535 and 81173487 from the National Natural Science Foundation of China.

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