Tumor Biol. DOI 10.1007/s13277-014-2744-9
RESEARCH ARTICLE
Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells Maryam Hamzeloo-Moghadam & Mahmoud Aghaei & Faranak Fallahian & Seyyed Mehdi Jafari & Masoumeh Dolati & Mohammad Hossein Abdolmohammadi & Sima Hajiahmadi & Somayeh Esmaeili
Received: 1 June 2014 / Accepted: 14 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was M. Hamzeloo-Moghadam Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran M. Aghaei : S. Hajiahmadi Department of Clinical Biochemistry School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran F. Fallahian (*) : M. Dolati : M. H. Abdolmohammadi Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran e-mail: [email protected] S. M. Jafari Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran M. Hamzeloo-Moghadam : S. Esmaeili Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran S. M. Jafari Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran
noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy. Keywords Sesquiterpene lactone . Britannin . Apoptosis . Breast cancer . MCF-7 . MDA-MB-468
Introduction Breast cancer is the most common type of cancer and the leading cause of cancer mortality among women, either in developed or developing countries [1]. There are conventional strategies for breast cancer treatment, including surgery, radiotherapy, chemotherapy, and hormone therapy. Major issues concerning conventional anticancer chemotherapy are the occurrence of side effects induced by the non-specific targeting of both normal and cancer cells [2]; therefore, there has been growing interest in the use of natural products from various sources such as plants, animals, and microorganisms with more potent anticancer properties and reduced adverse effects [3–5]. Compounds of herbal origin have played an important role in cancer therapy due to their cytotoxic and apoptosis-inducing activities [6, 7]. Plant-derived anticancer drugs are much more effective and do not have large side effect consequences compared to synthetic drugs. The first study on anticancer agents of plant origin was carried out on vinca
Tumor Biol.
alkaloids [8]. Classic examples of plant-derived anticancer drugs that are currently in clinical use include vinblastine, vincristine, paclitaxel, and camptothecin [6, 9]. Phytochemicals exert their tumor inhibitory effect through several mechanisms. These include increased antioxidants and anti-inflammatory activity, modulation of cellular signaling pathways, and altering gene expression to inhibit cell proliferation and/or induce apoptosis [10]. The plant family of Asteraceae is well known for their plenty bioactive compounds and diverse biological activities, i.e., anticancer, antibacterial, and anti-inflammatory properties. It comprises about 100 species distributed in Asia, Europe, and Africa [11]. Sesquiterpene lactones are a large and structurally diverse group of plant secondary metabolites, being most prevalent in the Asteraceae, where they can be found almost ubiquitously [12]. Sesquiterpene lactones are categorized into the following major classes: germacranolides, eudesmanolides, eremophilanolides, guaianolides, pseud oguaiano lide s, a nd hypocre te nolid es [ 1 3]. Sesquiterpene lactones have exhibited significant inflammatory and anticancer properties, which render them promising anticancer drugs [13–15]. Parthenolide, artemisinin, and tehranolide are examples of such compounds and have already reached cancer clinical trials [13]. In our previous investigation, sesquiterpene lactone britannin isolated from Inula aucheriana DC has been shown to exhibit anticancer effects in a variety of cell lines [16]. The ability to induce apoptosis is an important property of a candidate anticancer drug, which discriminates between anticancer drugs and toxic compounds. The current study was therefore carried out to address the issue if britannin is able to induce apoptosis in the breast cancer cell lines MCF-7 and MDA-MB-468 and to determine the underlying mechanism of its anticancer effects.
Material and methods
Cell culture The human breast cancer cell lines MCF-7 and MB-MDA468 and the normal human fibroblast cell line AGO1522 were obtained from the National Cell Bank of Iran (NCBI). The cells were grown in RPMI 1640 supplemented with 10 % fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin, and were maintained at 37 °C in a humidified incubator with 5 % CO2. Isolation and purification of britannin Isolation and purification of britannin was carried out as described previously [16]. Cell viability assay with MTT reduction Cell viability was determined using the MTT assay, as described previously [17]. Briefly, MCF-7, MDA-MB-468, and AGO1522 normal cells were seeded at 5×103 cells in each well of a 96-well plate. After overnight incubation to allow cell attachment, the RPMI 1640 in each well was replaced with media containing various concentrations of britannin and incubated for 48 h. Afterwards, 20 μl of MTT (5 mg/ml in PBS) was added to each well and the cells were incubated for another 4 h at 37 °C. Active mitochondria in live cells reduce MTT to crystalline purple blue formazan. DMSO was added to each well to dissolve the insoluble formazan, and the absorbance values were read using a microplate reader (BioRad, Hercules, CA, USA) at 570 nm. Stock solutions of compounds were prepared in dimethyl sulfoxide, and the solvent was added to the control cultures in all experiments. The final concentration of vehicle (dimethyl sulfoxide) was 0.1 %. Data were collected from several experiments, and the percentage of cell growth inhibition was determined by comparison with the dimethyl sulfoxide-treated control cells.
Chemical reagents RPMI 1640, trypsin-EDTA, phosphate-buffered saline (PBS), penicillin, and streptomycin were purchased from Gibco (Rockville, USA). Annexin V-FITC apoptosis detection kit, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (Munich, Germany). Caspase-3 and caspase-9 colorimetric assay kits were obtained from R&D Systems Co. (Minneapolis, USA). Mouse monoclonal anti-p53, anti-Bcl-2, anti-Bax, anti-cytochrome c antibodies, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent reactive oxygen species detection kit was obtained from Marker Gene Technologies (MGT, Inc., USA).
Quantitative analysis of apoptotic cells by annexin V/PI staining Apoptotic cell death induced by britannin was quantified by flow cytometry using the annexin V-FITC kit, according to the manufacturer’s protocol. Briefly, cells were plated to a density of 3×105 per well in a six-well plate and incubated with or without the indicated concentration of the tested compound for 48 h. Floating cells as well as residual attached cells were collected and washed twice with PBS. The cell pellets were resuspended in 500 μl of 1× binding buffer at a concentration of 1×106 cells/ml. Five microliters of annexin V-fluorescein isothiocyanate (FITC) and 5 μl of PI were added to the cell suspension. After a 10-min incubation at room temperature, stained samples were examined using a FACSCalibur flow
Tumor Biol.
cytometer (USA). Analysis was performed by the software supplied in the instrument. Western blot analysis Cells were harvested at 4 °C in a lysis buffer (20 mM Tris– HCl (pH 7.5), 0.5 % Nonidet P-40, 0.5 mM PMSF, 100 mM b-glycerol 3-phosphate, and 0.5 % protease inhibitor cocktail) and disrupted by sonication and centrifuged (14,000 rpm, 10 min, 4 °C). Protein concentration was determined using BCA protein assay kit (Pierce, TEMA Ricerca S.R.L., Bologna, Italy). Protein samples (30–50 μg) were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5 % non-fat dry milk in PBS containing 0.1 % Tween-20 (PBST) for 1 h at room temperature, the membranes were incubated with mouse monoclonal antibodies overnight at 4 °C. The membranes were washed with PBST and incubated with corresponding secondary antibodies for 1 h at room temperature. Membranes were again washed with PBST and visualized by enhanced chemiluminescence (ECL) detection reagent (Amersham Corp., Arlington Heights, IL). GAPDH was used as a control for normalization. Measurement of intracellular reactive oxygen species Intracellular changes in reactive oxygen species (ROS) was evaluated using the MGT live cell fluorescent ROS detection kit as described previously [18]. Briefly, cells were plated to a density of 25×103 per well in a 96-well plate and incubated with different concentrations of britannin for 48 h. After treatment, cells were further incubated with 20 μM 2′,7′dichlorofluorescin diacetate in Hanks’ balanced salt solution (HBSS) at 37 °C for 30 min in the dark. Subsequently, cells were harvested and washed with HBSS and analyzed for DCF fluorescence by a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments). Mitochondrial membrane potential (ΔΨm) analysis The mitochondrial membrane potential (ΔΨm) was estimated using a lipophilic cationic JC-1 probe. JC-1 exhibits potentialdependent fluorescence emission. JC-1 accumulates in the mitochondrial matrix under the influence of ΔΨm and gives red fluorescence; mitochondrial depolarization leads to a loss of JC-1 aggregates and an increase in green fluorescent JC-1 monomers. MCF-7 and MDA-MB-468 cells were plated in black clear-bottom 96-well plates. After treatment with different concentrations of britannin for 48 h, cells were loaded with JC-1 by replacing the culture medium with 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (40 mM, pH 7.4) containing 4.5 g/l glucose (high-
glucose medium) or 1.5 g/l glucose (low-glucose medium), 0.65 % NaCl, and 2.5 mM JC-1 for 30 min at 37 °C, then washed once with HEPES buffer. Fluorescence was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments) that allows for the sequential measurement of each well at two excitation/emission wavelength pairs, 490/ 540 and 540/590 nm. Changes in the ratio between the measured red (590 nm) and green (540 nm) fluorescence intensities indicate changes in mitochondrial membrane potential. Measurement of caspase activity Caspase-3 and caspase-9 activity was assessed according to the manufacturer’s instructions for the Caspase Colorimetric Assay Kit (R&D Systems). Briefly, cells after treatment for 48 h were harvested and lysed in lysis buffer on ice for 10 min and then centrifuged at 10,000g for 1 min. After centrifugation, the supernatants were incubated with caspase-3 and caspase-9 substrates in reaction buffer. Samples were incubated in a 96-well flat-bottom microplate at 37 °C for 1 h. The amount of p-nitro aniline released was measured using a microplate reader (Bio-Rad) at a wavelength of 405 nm. Analysis of nuclear morphology To observe cells undergoing apoptosis, Hoechst 33258 staining was performed. Hoechst 33258 is a cell-permeant DNA-binding dye that brightly stains the condensed chromatin of apoptotic cells. Briefly, after treatment with britannin at its IC50 concentration, cells were fixed with 4 % paraformaldehyde for 30 min at room temperature and then washed twice with PBS. Hoechst 33258 (50 ng/ml) was added to the fixed cells, incubated for 30 min at 37 °C in the dark, and then washed with PBS. Cells were examined by fluorescence microscopy. Statistical analysis The results of quantitative studies are reported as mean±SD. IC50 value was determined using GraphPad Prism5 software. All experiments were repeated at least three times. To compare data, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used. In all cases, P
RESEARCH ARTICLE
Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells Maryam Hamzeloo-Moghadam & Mahmoud Aghaei & Faranak Fallahian & Seyyed Mehdi Jafari & Masoumeh Dolati & Mohammad Hossein Abdolmohammadi & Sima Hajiahmadi & Somayeh Esmaeili
Received: 1 June 2014 / Accepted: 14 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was M. Hamzeloo-Moghadam Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran M. Aghaei : S. Hajiahmadi Department of Clinical Biochemistry School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran F. Fallahian (*) : M. Dolati : M. H. Abdolmohammadi Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran e-mail: [email protected] S. M. Jafari Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran M. Hamzeloo-Moghadam : S. Esmaeili Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran S. M. Jafari Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran
noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy. Keywords Sesquiterpene lactone . Britannin . Apoptosis . Breast cancer . MCF-7 . MDA-MB-468
Introduction Breast cancer is the most common type of cancer and the leading cause of cancer mortality among women, either in developed or developing countries [1]. There are conventional strategies for breast cancer treatment, including surgery, radiotherapy, chemotherapy, and hormone therapy. Major issues concerning conventional anticancer chemotherapy are the occurrence of side effects induced by the non-specific targeting of both normal and cancer cells [2]; therefore, there has been growing interest in the use of natural products from various sources such as plants, animals, and microorganisms with more potent anticancer properties and reduced adverse effects [3–5]. Compounds of herbal origin have played an important role in cancer therapy due to their cytotoxic and apoptosis-inducing activities [6, 7]. Plant-derived anticancer drugs are much more effective and do not have large side effect consequences compared to synthetic drugs. The first study on anticancer agents of plant origin was carried out on vinca
Tumor Biol.
alkaloids [8]. Classic examples of plant-derived anticancer drugs that are currently in clinical use include vinblastine, vincristine, paclitaxel, and camptothecin [6, 9]. Phytochemicals exert their tumor inhibitory effect through several mechanisms. These include increased antioxidants and anti-inflammatory activity, modulation of cellular signaling pathways, and altering gene expression to inhibit cell proliferation and/or induce apoptosis [10]. The plant family of Asteraceae is well known for their plenty bioactive compounds and diverse biological activities, i.e., anticancer, antibacterial, and anti-inflammatory properties. It comprises about 100 species distributed in Asia, Europe, and Africa [11]. Sesquiterpene lactones are a large and structurally diverse group of plant secondary metabolites, being most prevalent in the Asteraceae, where they can be found almost ubiquitously [12]. Sesquiterpene lactones are categorized into the following major classes: germacranolides, eudesmanolides, eremophilanolides, guaianolides, pseud oguaiano lide s, a nd hypocre te nolid es [ 1 3]. Sesquiterpene lactones have exhibited significant inflammatory and anticancer properties, which render them promising anticancer drugs [13–15]. Parthenolide, artemisinin, and tehranolide are examples of such compounds and have already reached cancer clinical trials [13]. In our previous investigation, sesquiterpene lactone britannin isolated from Inula aucheriana DC has been shown to exhibit anticancer effects in a variety of cell lines [16]. The ability to induce apoptosis is an important property of a candidate anticancer drug, which discriminates between anticancer drugs and toxic compounds. The current study was therefore carried out to address the issue if britannin is able to induce apoptosis in the breast cancer cell lines MCF-7 and MDA-MB-468 and to determine the underlying mechanism of its anticancer effects.
Material and methods
Cell culture The human breast cancer cell lines MCF-7 and MB-MDA468 and the normal human fibroblast cell line AGO1522 were obtained from the National Cell Bank of Iran (NCBI). The cells were grown in RPMI 1640 supplemented with 10 % fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin, and were maintained at 37 °C in a humidified incubator with 5 % CO2. Isolation and purification of britannin Isolation and purification of britannin was carried out as described previously [16]. Cell viability assay with MTT reduction Cell viability was determined using the MTT assay, as described previously [17]. Briefly, MCF-7, MDA-MB-468, and AGO1522 normal cells were seeded at 5×103 cells in each well of a 96-well plate. After overnight incubation to allow cell attachment, the RPMI 1640 in each well was replaced with media containing various concentrations of britannin and incubated for 48 h. Afterwards, 20 μl of MTT (5 mg/ml in PBS) was added to each well and the cells were incubated for another 4 h at 37 °C. Active mitochondria in live cells reduce MTT to crystalline purple blue formazan. DMSO was added to each well to dissolve the insoluble formazan, and the absorbance values were read using a microplate reader (BioRad, Hercules, CA, USA) at 570 nm. Stock solutions of compounds were prepared in dimethyl sulfoxide, and the solvent was added to the control cultures in all experiments. The final concentration of vehicle (dimethyl sulfoxide) was 0.1 %. Data were collected from several experiments, and the percentage of cell growth inhibition was determined by comparison with the dimethyl sulfoxide-treated control cells.
Chemical reagents RPMI 1640, trypsin-EDTA, phosphate-buffered saline (PBS), penicillin, and streptomycin were purchased from Gibco (Rockville, USA). Annexin V-FITC apoptosis detection kit, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (Munich, Germany). Caspase-3 and caspase-9 colorimetric assay kits were obtained from R&D Systems Co. (Minneapolis, USA). Mouse monoclonal anti-p53, anti-Bcl-2, anti-Bax, anti-cytochrome c antibodies, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent reactive oxygen species detection kit was obtained from Marker Gene Technologies (MGT, Inc., USA).
Quantitative analysis of apoptotic cells by annexin V/PI staining Apoptotic cell death induced by britannin was quantified by flow cytometry using the annexin V-FITC kit, according to the manufacturer’s protocol. Briefly, cells were plated to a density of 3×105 per well in a six-well plate and incubated with or without the indicated concentration of the tested compound for 48 h. Floating cells as well as residual attached cells were collected and washed twice with PBS. The cell pellets were resuspended in 500 μl of 1× binding buffer at a concentration of 1×106 cells/ml. Five microliters of annexin V-fluorescein isothiocyanate (FITC) and 5 μl of PI were added to the cell suspension. After a 10-min incubation at room temperature, stained samples were examined using a FACSCalibur flow
Tumor Biol.
cytometer (USA). Analysis was performed by the software supplied in the instrument. Western blot analysis Cells were harvested at 4 °C in a lysis buffer (20 mM Tris– HCl (pH 7.5), 0.5 % Nonidet P-40, 0.5 mM PMSF, 100 mM b-glycerol 3-phosphate, and 0.5 % protease inhibitor cocktail) and disrupted by sonication and centrifuged (14,000 rpm, 10 min, 4 °C). Protein concentration was determined using BCA protein assay kit (Pierce, TEMA Ricerca S.R.L., Bologna, Italy). Protein samples (30–50 μg) were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5 % non-fat dry milk in PBS containing 0.1 % Tween-20 (PBST) for 1 h at room temperature, the membranes were incubated with mouse monoclonal antibodies overnight at 4 °C. The membranes were washed with PBST and incubated with corresponding secondary antibodies for 1 h at room temperature. Membranes were again washed with PBST and visualized by enhanced chemiluminescence (ECL) detection reagent (Amersham Corp., Arlington Heights, IL). GAPDH was used as a control for normalization. Measurement of intracellular reactive oxygen species Intracellular changes in reactive oxygen species (ROS) was evaluated using the MGT live cell fluorescent ROS detection kit as described previously [18]. Briefly, cells were plated to a density of 25×103 per well in a 96-well plate and incubated with different concentrations of britannin for 48 h. After treatment, cells were further incubated with 20 μM 2′,7′dichlorofluorescin diacetate in Hanks’ balanced salt solution (HBSS) at 37 °C for 30 min in the dark. Subsequently, cells were harvested and washed with HBSS and analyzed for DCF fluorescence by a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments). Mitochondrial membrane potential (ΔΨm) analysis The mitochondrial membrane potential (ΔΨm) was estimated using a lipophilic cationic JC-1 probe. JC-1 exhibits potentialdependent fluorescence emission. JC-1 accumulates in the mitochondrial matrix under the influence of ΔΨm and gives red fluorescence; mitochondrial depolarization leads to a loss of JC-1 aggregates and an increase in green fluorescent JC-1 monomers. MCF-7 and MDA-MB-468 cells were plated in black clear-bottom 96-well plates. After treatment with different concentrations of britannin for 48 h, cells were loaded with JC-1 by replacing the culture medium with 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (40 mM, pH 7.4) containing 4.5 g/l glucose (high-
glucose medium) or 1.5 g/l glucose (low-glucose medium), 0.65 % NaCl, and 2.5 mM JC-1 for 30 min at 37 °C, then washed once with HEPES buffer. Fluorescence was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments) that allows for the sequential measurement of each well at two excitation/emission wavelength pairs, 490/ 540 and 540/590 nm. Changes in the ratio between the measured red (590 nm) and green (540 nm) fluorescence intensities indicate changes in mitochondrial membrane potential. Measurement of caspase activity Caspase-3 and caspase-9 activity was assessed according to the manufacturer’s instructions for the Caspase Colorimetric Assay Kit (R&D Systems). Briefly, cells after treatment for 48 h were harvested and lysed in lysis buffer on ice for 10 min and then centrifuged at 10,000g for 1 min. After centrifugation, the supernatants were incubated with caspase-3 and caspase-9 substrates in reaction buffer. Samples were incubated in a 96-well flat-bottom microplate at 37 °C for 1 h. The amount of p-nitro aniline released was measured using a microplate reader (Bio-Rad) at a wavelength of 405 nm. Analysis of nuclear morphology To observe cells undergoing apoptosis, Hoechst 33258 staining was performed. Hoechst 33258 is a cell-permeant DNA-binding dye that brightly stains the condensed chromatin of apoptotic cells. Briefly, after treatment with britannin at its IC50 concentration, cells were fixed with 4 % paraformaldehyde for 30 min at room temperature and then washed twice with PBS. Hoechst 33258 (50 ng/ml) was added to the fixed cells, incubated for 30 min at 37 °C in the dark, and then washed with PBS. Cells were examined by fluorescence microscopy. Statistical analysis The results of quantitative studies are reported as mean±SD. IC50 value was determined using GraphPad Prism5 software. All experiments were repeated at least three times. To compare data, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used. In all cases, P
