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Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells
© Hansakul et al.; licensee BioMed Central Ltd. 2014
Received: 6 January 2014
Accepted: 16 October 2014
Published: 24 October 2014
Dioscoreanone (DN) isolated from Dioscorea membranacea Pierre has been reported to exert potent cytotoxic effects against particular types of cancer. The present study was carried out to investigate the cytotoxicity of DN against a panel of different human lung cancer cell lines. The study further examined the underlying mechanisms of its anticancer activity in the human lung adenocarcinoma cell line A549.
Antiproliferative effects of DN were determined by SRB and CFSE assays. The effect of DN on cell cycle distribution was assessed by flow cytometric analysis. Apoptotic effects of DN were determined by sub-G1 quantitation and Annexin V-FITC/PI flow cytometric analyses, as well as by changes in caspase-3 activity and relative levels of Bax and Bcl-2 mRNA.
DN exerted antiproliferative and cytotoxic effects on all three subtypes of non-small cell lung cancer (NSCLC) cells, but not on small cell lung cancer (SCLC) cells and normal lung fibroblasts. DN slowed down the cell division and arrested the cell cycle at the G2/M phase in treated A549 cells, leading to a dose- and time- dependent increase of the sub-G1 population (apoptotic cells). Consistently, early apoptotic cells (AnnexinV +/PI-) were detected in those cells that were treated for 24 h and increased progressively over time. Moreover, the highest activity of caspase-3 in DN-treated A549 cells was detected within the first 24 h, and pretreatment with the general caspase inhibitor z-VAD-fmk completely abolished such activity and also DN-induced apoptosis in a dose-dependent manner. Additionally, DN increased the Bax/Bcl-2 ratio in treated A549 cells with time, indicating its induction of apoptosis via the mitochondrial pathway.
This study reveals for the first time that the anticancer activity of DN was induced through regulation of the Bcl-2 family protein-mediated mitochondrial pathway and the subsequent caspase-3 activation in A549 cancer cells, thus supporting its potential role as a natural apoptosis-inducing agent for NSCLC.
KeywordsDioscoreanone Dioscorea membranacea Antiproliferative Apoptosis Caspase activation
Lung cancer is a leading cause of cancer-related deaths worldwide, and it can be divided into two histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for almost 80% of lung cancer cases and consists mainly of adenocarcinoma, squamous cell and large cell carcinoma. Although many factors, e.g., tobacco smoke, ionizing radiation and viral infection are known to increase the risk of this cancer type, the mechanisms involved in lung cancer formation remain largely unknown to date. Despite improvements in tumor response to chemotherapy, the overall survival rate for lung cancer patients in advanced or late stages of the disease is still low and has not improved significantly in recent past decades. Because of the mutation heterogeneity of lung cancer cells associated with acquired drug resistance, the search for novel anticancer agents with an enhanced specificity for cancer cells remains an urgent need in order to increase the potency of chemotherapy. Herbal medicines in the treatment of cancer as an alternative therapy have long been an important source for such agents, some of which are currently used in clinical practice. In recent years, plant-derived compounds have been extensively screened to explore their potential for the development of new anticancer drugs.
Exploring the precise molecular mechanisms involved in actions of anticancer agents has become an important approach for anticancer drug evaluation and development. Among these molecular mechanisms, apoptosis is a highly regulated process of cell death that serves to eliminate heavily damaged cells without injuring surrounding healthy cells, and its dysregulation underlies numerous pathological conditions including cancer. Over the years, apoptosis has emerged as the major mechanism by which anticancer agents act to eliminate cancer cells. Recent clinical data have revealed that anticancer drugs that restore deregulated apoptosis or apoptosis-related signaling pathways in cancer cells can significantly improve patient survival for patients who have contracted various advanced cancer diseases. In this context, several plant compounds have been proven, in a more specific manner, to possess promising anticancer activity through their apoptosis-inducing effects.
In the present study, we first examined dose–response growth inhibitory and cytotoxic effects of DN on lung cancer cells including NSCLC and SCLC versus normal human lung fibroblasts. We further investigated apoptosis-involved mechanisms underlying the anticancer activity of DN in human lung adenocarcinoma A549 cells.
Rhizomes of Dioscorea membranacea Pierre ex Prain & Burkill were extracted with 95 % ethanol to obtain crude extract as previously mentioned. This plant was collected from Amphoe Patue, Chumphorn Province. Authentication of plant material was carried out at the herbarium of the Department of Forestry, Bangkok, Thailand (Voucher number SKP A062041305). Dioscoreanone (DN) was isolated from the crude ethanolic extract using previously described methods. The structure of DN (Figure 1) was confirmed by comparing its structure with previously reported 1H- and 13C-NMR spectral data. The stock solution of DN was prepared in DMSO at a concentration of 35 mM. The final concentration of DMSO was at or below 0.1% in all experiments.
Five cell lines were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA), namely three subtypes of human non-small cell lung cancer (NSCLC) consisting of A549 (adenocarcinoma), COR-L23 (large cell carcinoma), and NCI-H226 (squamous cell carcinoma); human small cell lung cancer (SCLC) in the form of NCI-H1688; and human normal lung fibroblasts as MRC-5. All cancer cell lines were maintained in RPMI-1640 supplemented with 10% FBS, and MRC-5 cells were cultured in MEM supplemented with 10% FBS, 1 mM nonessential amino acid, and 1% sodium pyruvate at 37°C in 5% CO2 incubator.
Cell proliferation assay
where T is the average O.D. of cells treated with DN for 72 h; T0 is the average O.D. at 0 h; C is the average O.D of cells treated with media only for 72 h. Based on the formula, the percentage of cell survival can be greater than zero, zero or less than zero. Dose–response curves were plotted as percentage of cell survival versus DN concentration. The concentrations of DN required for 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% loss of cells (lethal concentration, LC50) relative to the untreated cells were obtained by interpolating dose–response curves with cubic spline using GraphPad Prism 4.0 Software (GraphPad Software, Inc., USA).
CFSE proliferation assay
Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) is a cell-tracking dye used to label cells for examining their proliferative activity. It diffuses into the cytoplasm where its acetate groups are cleaved to yield a highly fluorescent derivative (CFSE) that is retained in the cell. A profile of sequential halving of CFSE fluorescence intensity with each generation can be monitored, allowing the visualization of the number of rounds of cell division. Inhibition of cell division by any substance can thus be traced through changes in CFSE profile. In the assay, detached A549 cells were labeled with 10 mM carboxyfluorescein-succinimidyl ester (CFSE) (BD bioscience, USA) at 37°C for 20 min in the dark and washed twice with PBS containing 10% FBS (FACS buffer) to remove excess CFSE. Cells were plated at 1 × 105 cells/well in 24-well plates and incubated at 37°C with 5% CO2. At least 12 h after seeding, 30 μM dioscoreanone was added into each well, and the cells were further incubated for 24, 48 and 72 h. At the appropriate point in time, cells were detached, washed twice, re-suspended in FACS buffer, and analyzed immediately using a FACSCalibur flow cytometer (Becton Dickinson, USA). Numbers of cell division as well as two parameters, e.g. proliferation index and precursor frequency, were analyzed using ModFit LT 3.2 program (Verity Software House, USA). Proliferation index was calculated as the sum of cells in all generations divided by the number of original parent cells; this index is useful for determining antiproliferative effects of DN on a population of cells. On the other hand, precursor frequency was calculated as the percentage of cells in the original parent population that proliferated in response to DN.
Cell cycle analysis
The percentage of cells in sub-G1 (apoptotic cells), G1, S and G2/M phases was determined by DNA flow cytometry. Briefly, A549 cells were plated at 1 × 105 cells/well in 24-well plates and incubated with 15 μM and 30 μM DN for 24, 48 and 72 h at 37°C with 5% CO2. After treatment, cells were collected by trypsinization, fixed gently (drop by drop) in 80% ethanol, and then stored at -20°C overnight. Then, cells were washed with phosphate-buffered saline (PBS) and stained with 0.5 ml PI/RNase staining buffer (BD bioscience, USA) for 30 min at room temperature in the dark. These stained cells were collected using a FACSCalibur flow cytometer (Becton Dickinson, USA) and analyzed for cell cycle phases with ModFit LT 3.2 Software (Verity Software house, USA). Cell cycle distribution was also determined in DN-treated cells after pretreatment for 1 h with the pancaspase inhibitor z-VAD-fmk, which was added to final concentrations of 2.5 and 25 μM.
Annexin-V/PI double staining assay
Flow cytometry was used to discriminate between intact and apoptotic cells. A549 cells were stained with fluorescein isothiocyanate (FITC) labeled annexinV that binds to membrane phosphatidylserine and with propidium iodide (PI) that binds to cellular DNA according to the manufacturer’s instructions (BD bioscience, USA). Briefly, A549 cells were plated at 1 × 105 cells/well in 24-well plates. After exposure to 15 μM and 30 μM DN for 24, 48 and 72 h, cells were trypsinized, washed with cold PBS, and resuspended in 100 μl of binding buffer containing 5 μl of FITC Annexin V and 5 μl of PI. Then cells were gently vortexed and incubated for 20 min at room temperature in the dark. Four hundred microliters of binding buffer was added to each tube. Cells were then collected using a FACSCalibur flow cytometer (Becton Dickinson, USA) and analyzed with CellQuest Software (BD bioscience, USA).
Caspase-3 activity assay
Activity of caspase-3 was detected using the CaspACE™ Assay System (Promega, USA). Briefly, A549 cells were plated at 1 × 105 cells/well in 24-well plates. After exposure to 30 μM DN for 24, 48 and 72 h, control or treated cells were lysed in 50–100 μl of cold lysis buffer by sonication, and the mixture was incubated on ice for another 20 min. The cell lysates were centrifuged at 15,000 × g for 20 min at 4°C, and the supernatant fraction was collected. The assay was performed in a total volume of 100 μl in 96-well plates. Cell extracts with an equal amount of protein (50–100 μg of total protein) were added to each reaction containing caspase assay buffer and specific colorimetric substrate (DEVD-pNA) for caspase-3, and the mixture was incubated at 37°C for another 4 h according to the manufacturer’s protocol. The absorbance was measured at 405 nm. Caspase-3 activity was also measured in cell extracts treated with DN after pretreatment for 1 h with the pancaspase inhibitor z-VAD-fmk, which was added to a final concentration of 25 μM.
Real-time Quantitative PCR Analysis
After exposure to 30 μM DN for 24, 48 and 72 h, total RNA was isolated from A549 cells using the total RNA extraction kit (Real Biotech Corporation, Taiwan). Two hundred and fifty nanograms of input total RNA was converted to single-stranded cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Bcl-2 and Bax gene expression analyses were performed using the Applied Biosystems (ABI) StepOne™ and StepOne Plus™ Real-Time PCR System (The Applied Biosystems, USA) as well as commercially available primer/probe sets, which are pre-designed FAM™ dye-labeled TaqMan® MGB (minor groove binder) probe and primer sets (inventoried Taqman® Gene Expression Assays) for human BCL2, BAX and GAPDH genes. The thermal cycling parameters were one cycle of 50°C for 2 min, one cycle of 95°C for 10 min, 40 amplification cycles of 95°C for 15 sec, and 60°C for 1 min. Relative quantification of gene expression was performed using the comparative threshold (CT) method according to the 2-ΔΔCT method as described by the manufacturer. The CT values of target genes (Bcl2 and Bax) in each sample set (ΔCT test sample) were normalized to those of GAPDH. The ΔΔCT is calculated by the formula ΔΔCT = ΔCT test sample – ΔCT control. ΔΔCT was converted to fold changes in mRNA gene expression relative to control by the equation 2 -ΔΔCT. The fold changes are presented as the mean ± SD.
All experiments were performed independently at least three times, and the results were expressed as the mean ± SD. Statistically significant differences among the groups were analyzed by one-way analysis of variance (ANOVA), followed by post-hoc analysis. p value <0.05 was considered statistically significant (SPSS 16.0 for Windows).
The antiproliferative and cytotoxic effects of DN in several lung cancer cell lines
Cytotoxic effects of dioscoreanone (DN) on human lung cell lines
Lung cell type
Normal lung fibroblast
0.016 ± 0.005
0.297 ± 0.008
11.4 ± 1.3
19.6 ± 0.7
3 subtypes of NSCLC
0.016 ± 0.001
0.058 ± 0.012
2.800 ± 0.400a
6.2 ± 0.7a
10.3 ± 0.3a
15.7 ± 1.3a
Squamous cell carcinoma
0.017 ± 0.001
4.898 ± 0.480a
20.672 ± 0.436a
5.7 ± 1.2a
9.8 ± 0.3a
13.6 ± 1.2a
Large cell carcinoma
0.017 ± 0.001
3.450 ± 0.167a
17.725 ± 0.084a
3.7 ± 0.9a
8.0 ± 1.4a
18.9 ± 2.5a
0.012 ± 0.001
20.560 ± 0.147a
15.2 ± 2.1
21.1 ± 2.4
DN exhibited a significant growth-inhibitory effect in all three subtypes of NSCLC cell lines with GI50 values ranging from 3.7 to 6.2 μM, as compared to NCI-H1688 and MRC-5 cells. At high concentrations of DN, the representatives of each subtype of NSCLC - A549, COR-L23 and NCI-H226 cell lines also showed the negative values of cell survival presented as LC50, thus indicating a net loss of cancer cells. Altogether, these data revealed that DN exerted selective antiproliferative activity (growth inhibition) and cytotoxic activity (cell death induction) against these NSCLC cancer cells.
In contrast to DN, paclitaxel showed a very strong antiproliferative activity with ~140-1270-fold higher GI50 and TGI values in all tested cell lines (except NCI-1688). However, paclitaxel appeared to exert selective growth inhibitory effects on NSCLC cancer cells but high cytotoxicity on human normal lung fibroblasts. Therefore, DN has potential as a promising anticancer agent because of its selective antiproliferative and cytotoxic activities without harming normal cells. Several studies described below were performed to provide insights into the molecular mechanism(s) underlying the anticancer activity of DN.
The antiproliferative activity of DN in adenocarcinoma A549 cells
The inhibitory effect of DN on cell cycle progression in A549 cells
Annexin V-FITC flow cytometric analysis of apoptotic effects of DN in A549 cells
Effect of DN on caspase-3 activity in A549 cells
Effect of DN on mRNA expression of Bax and Bcl2 proteins in A549 cells
DN potentially exerted dose-dependent antiproliferative (described as GI50 and TGI values) and cytotoxic effects (represented as LC50 values) only on the three subtypes of NSCLC cell lines, but not on the SCLC cell line. The differential sensitivity of these cancer cells to DN could be explained in part because of their distinctive molecular characteristics. Studies on molecular mechanisms of DN in regard to its antiproliferative effect revealed significantly low proliferation index and precursor frequency values in DN-treated A549 cells via G2/M-phase cell cycle arrest. Several anticancer agents have been noted to mediate such G2/M phase arrest in cancer cells through multiple mechanisms: downregulation of CDK1-cyclin A/B complexes, inactivation of Cdc25C activity, and disruption of tubulin polymerization and spindle assembly[15, 16]. In this regard, the effects of DN on the types and levels of these key regulators need to be identified.
In addition to the antiproliferative effect, significant net loss of cell viability in NSCLC cells at high concentrations of DN in the SRB assay indicated its cytotoxic effect. Such data is in accordance with the results obtained by flow cytometric analysis, which determined the presence of apoptotic cells reflected by the sub-G1 DNA peak and early (AnnexinV +/PI-) and late (AnnexinV+/PI+) apoptotic cells in DN-treated A549 cells. In this context, Annexin V is a recombinant PS-binding protein that interacts strongly and specifically with PS residues and is typically used in conjunction with PI to distinguish early from late apoptotic cells. All these findings demonstrated apoptosis-involved mechanisms underlying cytotoxicity of DN for the first time. Although both late apoptotic and necrotic cells are Annexin V and PI-positive, the presence of these cells with early apoptotic cells suggests that such dead cells resulted from an apoptotic process rather than a necrotic process.
The apoptotic caspases appear to be activated in a protease cascade in which the activated apical caspases responding to apoptotic stimuli directly activate the executioner (effector) caspases in a precisely controlled process. Among these effector caspases, caspase-3 plays a central role in the execution phase of both the intrinsic (the mitochondrial) and extrinsic (the death receptor) pathways of apoptosis by cleaving many key cellular proteins, such as poly (ADP ribose) polymerase (PARP), inhibitor of caspase-activated DNase (ICAD), and various other proteins. This cleavage mediates disassembly of the cell into the typical apoptotic morphological changes, such as cell shrinkage, chromatin condensation, membrane blebbing, and DNA fragmentation. In this study, the pancaspase inhibitor z-VAD-fmk, which irreversibly binds to and blocks the cleavage site of the caspases, was able to completely abrogate the DN-induced increase in the sub-G1 cell population and DN-induced activation of caspase-3 in A549 cancer cells. These results strongly indicated that DN-induced apoptosis was executed mainly through a caspase-dependent pathway. In addition to such a pathway, the execution of apoptosis also constitutes a caspase-independent pathway that is mediated by mitochondrial proteins AIF and EndoG.
Pro-apoptotic (e.g. Bax) or anti-apoptotic (e.g. Bcl-2) proteins of the Bcl-2 family, a group of structurally related proteins, play a critical role in regulating the permeability of the outer mitochondrial membrane (OMM). Indeed, an increase in the ratio of Bax/Bcl-2 causes the permeabilization of OMM, leading to a hierarchical release of the apoptogenic proteins such as cytochrome c, Smac/Diablo and Omi/HtrA2, and subsequently resulting in downstream caspase-3 activation[22, 23]. Increases of Bax/Bcl-2 ratio in this study suggest that the susceptibility of DN-treated A549 cells to apoptosis could be modulated, at least in part, through the mitochondrial pathway.
Besides new insights on the anticancer mechanisms of DN, understanding its bioavailability when consumed per ounce (oz) also helps determine the amount of DN to be taken orally in compliance with the DN concentrations examined in this study. However, no such data are available, and in vivo oral bioavailability of DN needs to be further evaluated.
In this study, DN selectively inhibited the growth of A549, COR-L23 and NCI-H226 representative cell lines for each of three subtypes of NSCLC. In adenocarcinoma A549 cells, DN induced G2/M-phase cell cycle arrest and apoptosis, at least in part, via mitochondrial membrane permeabilization mediated by Bax and Bcl-2 proteins, leading to caspase-3 activation. The present study greatly contributes to the understanding of the anticancer activity of DN for the first time and also provides evidence that DN deserves additional evaluation as a natural anticancer agent for human NSCLC.
This work was mainly supported by The Thailand Research Fund (TRF) and partially supported by National Research University Project of Thailand, Office of the Higher Education Commission.
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