The rhizomes of B. rotunda were purchased from Puchong Market, Selangor Darul Ehsan, Malaysia. The material was identified by a botanist at the Faculty of Science, University Putra Malaysia, where a voucher specimen was deposited (BR-R11-01). The isolation and identification of boesenbergin A has been previously reported by us in detail
Cell culture conditions
Estrogen receptor positive cells (MCF-7) and cervical cancer cells (Hela) were obtained from ATCC (USA). Human T4-lymphoblastoid cell line CEMss were obtained from NIH AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH: USA). The cell lines were grown at 37°C in a humidified CO2 incubator with 5% CO2 in RPMI-1640 (Sigma, MO, USA) supplemented with 10% fetal bovine serum (Invitrogen Corp., Auckland, N.Z.).
Adherent cells (1× 106 cells/ml) were grown in 96-well plates overnight, whereas CEMss cells (1× 106 cells/ml) were plated directly into 96-well plates on the drug treatment day. Boesenbergin A was dissolved in dimethylsulfoxide (DMSO) and media. The final concentration of DMSO was 0.1% (v/v). Different concentrations of the sample were prepared with serial dilution. Dimethylsulfoxide (0.1%) was used as a control. The toxicity profiles of the compound were assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microculture tetrazolium viability assay as described previously
. Thereafter, various concentrations of compound (with a maximum of 100 μg/ml) were plated out in triplicate. Each plate included untreated cell controls and a blank cell-free control. After 72 h of incubation, MTT (5 mg/ml) was added to each well and the plates incubated for a further 4 h before removal of the media. DMSO was then added into each well to solubilize the formazan crystals. The absorbance was read at wavelength of 595 nm using a microtitre plate reader (Labsystems iEMS Reader MF). The percentage cellular viability was calculated with the appropriate controls taken into account. The concentration which inhibited 50% of cellular growth (IC50 value) was determined. All experiments were carried out in triplicate (Figure
The inhibitory rate of cell proliferation was calculated by the following formula: Growth inhibition = (OD control – OD treated) / OD control X 100. The cytotoxicity of sample on cancer cells was expressed as IC50 values (the drug concentration reducing the absorbance of treated cells by 50% with respect to untreated cells). The determined IC50 value was used for many of the subsequent experiments.
Cytotoxicity of boesenbergin A on proliferated primary human blood lymphocytes
The ability of boesenbergin A to act selectively on cancer cells especially leukemia was evaluated by comparing the cytotoxicity of this compound towards primary human blood lymphocytes. Briefly, blood was collected into a cell preparation tube containing sodium citrate (BD Vacutainer®, New Jersey, USA). After collection, tube was stood upright for 20 min at room temperature to allow it to equilibrate and was then centrifuged at 1200xg for 20 min. Mononuclear cells and platelets underneath the plasma layer were collected using a pipette and transferred into a 15 ml centrifuge tube. The cells were washed twice with PBS and cultured in complete Quantum PBL media with phytohemagglutinin (PAA, Pasching, Austria) containing 10% FBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2 atmosphere. Primary human blood lymphocytes (1 × 106 cells/ml) were treated at various concentrations of boesenbergin A in triplicate and cell viability was measured using MTT assay after 48 h of incubation. The cell lines used as normal cells were human peripheral blood lymphocytes obtained from normal healthy donors after informed consent was given. This project was approved by the medical research ethics committee (founded in 2002) of the medical Faculty of UPM at a meeting on April 12, 2012 (UPM 2564/004/12).
Microscopic observation of cellular morphology using phase contrast inverted microscope
This analysis examined whether apoptosis may be implicated in mediating cell death amongst CEMss cells elicited by boesenbergin A. CEMss cells at a concentration of 1 × 106 cells/ml were cultured in RPMI 1640 (PAA, Cölbe, Germany) medium containing 10% FBS was seeded into a 25 ml culture flask (TPP, Trasadingen, Switzerland) and treated with boesenbergin A (8 μg/ml) at different time periods (24, 48 and 72 h). The morphological appearance of treated cells was compared with the untreated control by using a normal inverted microscope post-treatment
. CEMss cells were treated with the compound for 24, 48 and 72 h. Untreated cells served as the negative control.
Quantification of apoptosis using propidium iodide and acridine orange double staining
Boesenbergin A-induced cell death in CEMss cells was quantified using propidium iodide (PI) and acridine orange (AO) double staining according to standard procedures and examined under a fluorescence microscope (Lieca attached with Q-Floro Software). Briefly, treatment was carried out in a 25 ml culture flask (Nunc). CEMss cells were plated at a concentration of 1 × 106 cells/ml and treated with boesenbergin A (8 μg/ml). The cells were incubated in 5% CO2 atmosphere at 37°C for 24, 48 and 72 h and then spun down at 200 g for 10 min. The supernatant was discarded and the cells were washed twice using PBS after centrifuging at 200 g for 10 min to remove the remaining media. Ten microliters of fluorescent dyes containing AO (10 μg/ml) and PI (10 μg/ml) were added into the cellular pellet at equal volumes of each. The freshly stained cell suspension was dropped into a glass slide and covered by cover slip. Slides were observed under a UV-fluorescence microscope within 30 min before the fluorescence colour started to fade. The percentages of viable, early apoptotic, late apoptotic and secondary necrotic cells were determined in more than 200 cells. Acridine orange (AO) and propidium iodide (PI) are intercalating nucleic acid specific fluorochromes which emit green and orange fluorescences respectively, when they are bound to DNA. Of the two, only AO can cross the plasma membrane of viable and early apoptotic cells. The criteria for identification are as follows: (a) viable cells appear to have a green nucleus with an intact structure; (b) early apoptotic cells exhibit a bright-green nucleus showing condensation of chromatin in the nucleus; (c) late apoptotic cells show dense orange areas of chromatin condensation; (d) secondary necrotic cells appear to have an orange intact nucleus
. This assay provides a useful quantitative evaluation and was carried out in triplicate.
Flow cytometric analysis of DNA cell cycle
CEMss cells at concentration of 1 × 106 cells/ml were cultured in RPMI 1640 (PAA) medium containing 10% FBS seeded in to 25 ml culture flask (TPP) and treated with boesenbergin A (8 μg/ml) at different time periods (24, 48 and 72 h). After incubation, the cells were spun down by centrifugation at 200 g for 10 min. The supernatant was discarded and the pellet was washed with PBS twice to remove any remaining media. To restore the integrity, fixation of the cell population for flow cytometery analysis was performed. Briefly, cell pellets were fixed by mixing 500 μl of 70% cold ethanol and 250 μl of cell suspension and kept at −20°C overnight. The cells were then spun down at 200 g for 10 min and the ethanol was decanted. After washing twice with PBS, the cells were resuspended in PBS. Twenty microliters of RNase A (10 μg/ml) and 2 μl of PI (2.5 μg/ml) were added and the fixed cells were kept in the dark on ice for 30 min. Propidium iodide has the ability to bind to RNA molecules and hence RNase enzyme was added in order to allow PI to bind directly to DNA. The DNA content of cells was then analyzed using a flow cytometer (BD FACSCanto™ II). The fluorescence intensity of the subG1 cell fraction represents the apoptotic cell population.
Annexin V assay
CEMss cells (1 × 106 cells/ml) were exposed to boesenbergin A (8 μg/ml) for 24, 48 and 72 h and the annexin V assay performed using an annexin V:FITC assay kit (ABD Serotec, UK). Briefly the treated cells were centrifuged for 10 min at 200 g to remove the media. After that, PBS was added to wash the cells and the same process was repeated twice. Then 5 μl annexin V:FITC was added to 195 μl of the cell suspension binding buffer, which was prepared by diluting the binding buffer 1:4 in distilled water (50 ml binding buffer +150 ml distilled water). The suspension was then mixed and incubated for 10 min in the dark at room temperature. The cells were then washed and resuspended in 190 μl prediluted binding buffer. Then 10 μl of the PI solution was added to the cell suspension and the sample was analyzed using a flow cytometer (BD FACSCanto™ II).
The Apoptotic DNA Ladder Detection kit (CHEMICON International Inc., CA, USA) was used for DNA extraction from cells. Briefly, CEMss cells treated with boesenbergin A (16 μg/ml) were collected at 24 and 48 h post treatment. The cells were washed with PBS and the cells were spun down by centrifugation at 500 × g for 5 min. After removal of the supernatant, the cells were lysed by the addition of 40 μl of TE lysis buffer and gentle pipetting, followed by the addition of 5 μl of Enzyme A (RNase A) into the crude lysate and incubated at 37°C for 10 min. Then 5 μl of Enzyme B (Proteinase K) was added and the lysate was further incubated at 50°C for 30 min. Then 5 μl of ammonium acetate solution and 50 μl of isopropanol were added and mixed well and the suspension was kept at −20°C for 10 min. The sample was then centrifuged 16000 × g for 10 min at to precipitate the DNA. After washing the DNA pellet with 70% ice cold ethanol, the air dried pellet was dissolved in 30 μl of DNA suspension buffer. For detecting the DNA ladder, the extracted DNA samples were run on 1% agarose gel in tris–acetic acid–EDTA buffer. After electrophoresis, the gel was stained with ethidium bromide (Gibco BRL, Paisley, Scotland), visualized with a UV light transilluminator (UVP, Upland, CA, USA) and photographed.
Caspase-3/7, -8 and -9 activity assay
Assays of caspase-3/7, -8 and =-9 was performed using the Caspase-Glo Assay kit (Promega, WI, USA). CEMss cells were plated and treated with boesenbergin A (8 μg/ml) and incubated for 24, 48 and 72 h in 96 well white plates. After allowing the cells to equilibriate at room temperature, 50 μl of Caspase-Glo® reagent was added to each well containing 50 μl of blank, negative control cells and treated cells in culture medium. The contents of the plate were gently mixed using a plate shaker at 100 g for 30 sec. It was then incubated at room temperature for 30 min in the dark. Readings were taken every 10 min for 3 h using a luminescence microplate reader (Infinite M200PRO, Tecan, Männedorf, Switzerland).
Detection of mitochondrial membrane potential (Δψm)
Rhodamine 123 (Rh123) is a fluorescent cationic dye that binds to polarized mitochondrial membrane and accumulates as aggregates in the mitochondria of normal cells. Rh123 was prepared in ethanol as a 5 mg/ml stock solution. CEMss cells were treated with 8 μg/ml boesenbergin A for 24, 48 and 72 h. At the end of the reaction time, the cells were harvested and washed twice in cold PBS, then resuspended in Rh123 (2 μg/ml) for 30 min in the dark. The Rh123 staining intensity was captured using a fluorescence microscope. Intensity of Rh 123 is directly related to mitochondrial membrane potential. The percentage of rhodamine negative cells gives the percentage collapse of Mitochondria Membrane Permeability.
Human apoptosis proteome profiler array
To investigate the pathways by which boesenbergin A induces apoptosis, we performed a determination of apoptosis-related proteins using the Proteome Profiler Array (RayBio® Human Apoptosis Antibody Array Kit, RayBiotech, GA, USA), according to the manufacturer’s instructions. Briefly, the cells where treated with boesenbergin A. Three hundred micrograms of protein from each sample were incubated with the human apoptosis array overnight. The apoptosis array data was quantified by scanning the membrane on a Biospectrum AC ChemiHR 40 (UVP, Upland, CA, USA) and analysis of the array image file was performed using image analysis software according to the manufacturer’s instructions.
Western blot analysis
CEMss cells were seeded in 12-well plates and treated with boesenbergin A (8 μg/ml) at 3, 6, 12 and 24 h. The total protein of the cells was extracted with cell lysis buffer (50 mM Tris–HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM PMSF). Forty micrograms of protein extract was separated by 10% SDS-PAGE, transferred to a polyvinylidenedifluoride (PVDF) membrane (Bio-Rad), blocked with 5% nonfat milk in TBS-Tween buffer 7 (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween20) for 1 h at room temperature, incubated with the appropriate antibody overnight at 4°C and then incubated with horseradish peroxidase conjugated secondary antibody for 30 min at room temperature. The bound antibody was detected with peroxidase-conjugated anti-rabbit antibody (1:10000) or anti-mouse antibody (1:10000) followed by chemiluminescence (ECL System) and exposed by autoradiography. The following primary antibodies β-actin (1:10000), Bcl2 (1:1000), Bax (1:1000), HSP70 (1:1000), were purchased from Santa Cruz Biotechnology, Inc, (California, USA).
Data is reported as the mean ± SD of three replicates. The independent t-test and ANOVA were used for comparisons with P < 0.05 considered to be significant. All statistical analyses were performed using the SPSS software (Release 18, SPSS Inc, Chicago, IL, USA).