Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
RESEARCH ARTICLE
Open Access
Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells Pintusorn Hansakul1*, Kalaya Aree2, Sermkiat Tanuchit3 and Arunporn Itharat4
Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Dioscoreanone, Dioscorea membranacea, Antiproliferative, Apoptosis, Caspase activation
Background Lung cancer is a leading cause of cancer-related deaths worldwide [1], 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 * Correspondence: [email protected] 1 Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand Full list of author information is available at the end of the article
viral infection are known to increase the risk of this cancer type, the mechanisms involved in lung cancer formation remain largely unknown to date [2]. 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 [1]. 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 [3]. Herbal medicines in the treatment of
© 2014 Hansakul et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
cancer as an alternative therapy have long been an important source for such agents, some of which are currently used in clinical practice [4]. 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 [5]. Over the years, apoptosis has emerged as the major mechanism by which anticancer agents act to eliminate cancer cells [6]. Recent clinical data have revealed that anticancer drugs that restore deregulated apoptosis or apoptosisrelated signaling pathways in cancer cells can significantly improve patient survival for patients who have contracted various advanced cancer diseases [7]. In this context, several plant compounds have been proven, in a more specific manner, to possess promising anticancer activity through their apoptosis-inducing effects [8]. Dioscoreanone (DN) is 1, 4-Phenanthraquinone (Figure 1) isolated from the ethanolic extract of dried rhizomes of Dioscorea membranacea Pierre [9], which has long been used in various traditional Thai herbal remedies for cancer and inflammatory diseases. Previous studies have shown the selective anticancer and anti-inflammatory activities of this herbal plant [10,11], providing the scientific support for its traditional uses. Moreover, DN was demonstrated to exert selective cytotoxic effects against human lung and breast cancer cell lines, but not against normal cells [9]. However, the molecular mechanisms underlying its cytotoxicity have not yet been explored.
Page 2 of 12
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 apoptosisinvolved mechanisms underlying the anticancer activity of DN in human lung adenocarcinoma A549 cells.
Methods Dioscoreanone preparation
Rhizomes of Dioscorea membranacea Pierre ex Prain & Burkill were extracted with 95 % ethanol to obtain crude extract as previously mentioned [11]. 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 [10]. The structure of DN (Figure 1) was confirmed by comparing its structure with previously reported 1H- and 13C-NMR spectral data [9]. 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. Cell culture
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), CORL23 (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
Figure 1 Structure of dioscoreanone (DN).
Antiproliferative effects of DN were measured by the SRB assay. Briefly, cells were seeded in a 96-well plate containing 100 μl of culture medium. Cell numbers are indicated in the bracket (A549 = 1 × 103 cells/well; COR-L23 and NCI-H226 = 3 × 103 cells/well; NCIH1688 and MRC-5 = 2 × 104 cells/well). On the following day, one plate of each cell line was fixed in situ with 100 μl of 10% (w/v) cold trichloroacetic acid (TCA), and it was stained with 0.4% (w/v) sulforhodamine B (SRB) in 1% acetic acid solution as a means of representing the cell number at the time of adding DN (zero hour). One hundred microliters of DN was added to the tested plates to obtain final concentrations of 0.35, 3.5, 17.5 and 35 μM, and these plates were incubated for additional 72 h at 37°C with 5% CO2. At the end of 72 h,
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
the medium was removed, and the cells were gently fixed with 10% (w/v) cold TCA. The cell numbers were estimated indirectly by staining the total cellular protein content of each well with 0.4% (w/v) SRB. The optical density (O.D.) was measured at 492 nm. The percentage of cell survival for each concentration was calculated using the following formula: % cell survival ¼ ½ðT − T0 Þ=ðC − T0 Þ 100; T≥T0 or ¼ ½ðT − T0 Þ= T0 100⋯⋯; T < T0 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).
Page 3 of 12
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
CFSE proliferation assay
Carboxyfluorescein diacetate, succinimidyl ester (CFDASE) 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
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
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
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 (DEVDpNA) 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. Statistical analysis
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 32
11.4 ± 1.3
19.6 ± 0.7
>32
0.016 ± 0.001
0.058 ± 0.012
2.800 ± 0.400a
6.2 ± 0.7a
10.3 ± 0.3a
15.7 ± 1.3a
a
3 subtypes of NSCLC
NCI-H226
Squamous cell carcinoma
0.017 ± 0.001
4.898 ± 0.480
COR-L23
Large cell carcinoma
0.017 ± 0.001
3.450 ± 0.167a
NCI-1688
SCLC
0.012 ± 0.001
a
20.560 ± 0.147
a
a
a
13.6 ± 1.2a
20.672 ± 0.436
5.7 ± 1.2
9.8 ± 0.3
17.725 ± 0.084a
3.7 ± 0.9a
8.0 ± 1.4a
18.9 ± 2.5a
>32
15.2 ± 2.1
21.1 ± 2.4
>32
DN concentrations required for 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% decrease in cell numbers (LC50) relative to the untreated cells as measured by the SRB assay. Data are presented as the mean ± SD of at least 3 independent experiments. a Statistical significance (p
RESEARCH ARTICLE
Open Access
Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells Pintusorn Hansakul1*, Kalaya Aree2, Sermkiat Tanuchit3 and Arunporn Itharat4
Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Dioscoreanone, Dioscorea membranacea, Antiproliferative, Apoptosis, Caspase activation
Background Lung cancer is a leading cause of cancer-related deaths worldwide [1], 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 * Correspondence: [email protected] 1 Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand Full list of author information is available at the end of the article
viral infection are known to increase the risk of this cancer type, the mechanisms involved in lung cancer formation remain largely unknown to date [2]. 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 [1]. 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 [3]. Herbal medicines in the treatment of
© 2014 Hansakul et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
cancer as an alternative therapy have long been an important source for such agents, some of which are currently used in clinical practice [4]. 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 [5]. Over the years, apoptosis has emerged as the major mechanism by which anticancer agents act to eliminate cancer cells [6]. Recent clinical data have revealed that anticancer drugs that restore deregulated apoptosis or apoptosisrelated signaling pathways in cancer cells can significantly improve patient survival for patients who have contracted various advanced cancer diseases [7]. In this context, several plant compounds have been proven, in a more specific manner, to possess promising anticancer activity through their apoptosis-inducing effects [8]. Dioscoreanone (DN) is 1, 4-Phenanthraquinone (Figure 1) isolated from the ethanolic extract of dried rhizomes of Dioscorea membranacea Pierre [9], which has long been used in various traditional Thai herbal remedies for cancer and inflammatory diseases. Previous studies have shown the selective anticancer and anti-inflammatory activities of this herbal plant [10,11], providing the scientific support for its traditional uses. Moreover, DN was demonstrated to exert selective cytotoxic effects against human lung and breast cancer cell lines, but not against normal cells [9]. However, the molecular mechanisms underlying its cytotoxicity have not yet been explored.
Page 2 of 12
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 apoptosisinvolved mechanisms underlying the anticancer activity of DN in human lung adenocarcinoma A549 cells.
Methods Dioscoreanone preparation
Rhizomes of Dioscorea membranacea Pierre ex Prain & Burkill were extracted with 95 % ethanol to obtain crude extract as previously mentioned [11]. 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 [10]. The structure of DN (Figure 1) was confirmed by comparing its structure with previously reported 1H- and 13C-NMR spectral data [9]. 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. Cell culture
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), CORL23 (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
Figure 1 Structure of dioscoreanone (DN).
Antiproliferative effects of DN were measured by the SRB assay. Briefly, cells were seeded in a 96-well plate containing 100 μl of culture medium. Cell numbers are indicated in the bracket (A549 = 1 × 103 cells/well; COR-L23 and NCI-H226 = 3 × 103 cells/well; NCIH1688 and MRC-5 = 2 × 104 cells/well). On the following day, one plate of each cell line was fixed in situ with 100 μl of 10% (w/v) cold trichloroacetic acid (TCA), and it was stained with 0.4% (w/v) sulforhodamine B (SRB) in 1% acetic acid solution as a means of representing the cell number at the time of adding DN (zero hour). One hundred microliters of DN was added to the tested plates to obtain final concentrations of 0.35, 3.5, 17.5 and 35 μM, and these plates were incubated for additional 72 h at 37°C with 5% CO2. At the end of 72 h,
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
the medium was removed, and the cells were gently fixed with 10% (w/v) cold TCA. The cell numbers were estimated indirectly by staining the total cellular protein content of each well with 0.4% (w/v) SRB. The optical density (O.D.) was measured at 492 nm. The percentage of cell survival for each concentration was calculated using the following formula: % cell survival ¼ ½ðT − T0 Þ=ðC − T0 Þ 100; T≥T0 or ¼ ½ðT − T0 Þ= T0 100⋯⋯; T < T0 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).
Page 3 of 12
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
CFSE proliferation assay
Carboxyfluorescein diacetate, succinimidyl ester (CFDASE) 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
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
Hansakul et al. BMC Complementary and Alternative Medicine 2014, 14:413 http://www.biomedcentral.com/1472-6882/14/413
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 (DEVDpNA) 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. Statistical analysis
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 32
11.4 ± 1.3
19.6 ± 0.7
>32
0.016 ± 0.001
0.058 ± 0.012
2.800 ± 0.400a
6.2 ± 0.7a
10.3 ± 0.3a
15.7 ± 1.3a
a
3 subtypes of NSCLC
NCI-H226
Squamous cell carcinoma
0.017 ± 0.001
4.898 ± 0.480
COR-L23
Large cell carcinoma
0.017 ± 0.001
3.450 ± 0.167a
NCI-1688
SCLC
0.012 ± 0.001
a
20.560 ± 0.147
a
a
a
13.6 ± 1.2a
20.672 ± 0.436
5.7 ± 1.2
9.8 ± 0.3
17.725 ± 0.084a
3.7 ± 0.9a
8.0 ± 1.4a
18.9 ± 2.5a
>32
15.2 ± 2.1
21.1 ± 2.4
>32
DN concentrations required for 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% decrease in cell numbers (LC50) relative to the untreated cells as measured by the SRB assay. Data are presented as the mean ± SD of at least 3 independent experiments. a Statistical significance (p
