3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT); acridine orange (AO); ethidium bromide (EB); propidium iodide (PI); and cell culture chemicals were purchased from Sigma-Aldrich Chemicals Pvt. Ltd. (Mumbai, India). Curcumin was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Proteinase-K and RNase were purchased from Bangalore Genei (Bangalore, India), and the rest of the chemicals and solvents used were of analytical grade.
The CGNs were collected from Kangchup Hills, Manipur, India (N24°52′10′ E093°46′12′) at an elevation of 1534.66 m above sea level. The specimen was identified by Dr. Biseshwori Thongam, Plant Systematics and Conservation Laboratory, Medicinal, Aromatic and Horticultural Plant Resources Division, Institute of Bioresources and Sustainable Development (IBSD), Manipur, India and by Dr. S.K. Verma, National Bureau of Plant Genetics Resources, Meghalaya, India. A voucher specimen (IBSD/C/102) has been deposited to the IBSD herbarium.
Preparation of CGN extracts
The CGNs were air dried at room temperature and powdered. The powdered needles were then exhaustively extracted successively by soaking (which prevents the loss of biological activity of some heat-sensitive ingredients) in petroleum ether (PE), acetone (ACE), and methanol (MeOH) in order to fractionate the phytochemical constituents. Filtration was performed and the filtrates were concentrated in vacuo using a vacuum rotary evaporator (EYELA, Japan) and finally freeze dried (Thermo, Modulyod). The dried extracts were kept at 4°C until further analysis.
Test sample preparation
Solutions of the test samples for the entire study were prepared in DMSO, except for the PE extract in which the sample was prepared in 1, 4-dioxan.
The experimental cell lines were procured from the National Centre for Cell Science (Pune, India). HeLa and ZR751 cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) and HepG2 was grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% antibiotic antimycotic solution (10,000 U/ml penicillin, 10 mg/ml streptomycin sulfate, and 25 μg/ml amphotericin-B), and maintained at 37°C in a humidified atmosphere with 5% CO2/95% air.
MTT reduction assay
Cytotoxicity analysis was determined using the MTT assay as reported by Mosmann. Cancer cells grown in T-25 culture flasks were harvested by trypsinization, plated at an approximate density of 2 × 104 cells/well in 96-well culture plates, and incubated for 24 h. Next the medium from each well was removed and the cells were washed twice with Dulbecco’s phosphate buffered saline (PBS). The cells were then exposed to increasing concentrations of extract (5–160 μg/ml) for 24 h. After incubation, the contents were replaced with MTT dissolved in serum-free medium (1.2 mM) after which the plates were further incubated for 3 h. The contents were then replaced with equal amounts of DMSO to solubilise the formazan grains formed by viable cells. Finally, the absorbance was read at 570 nm using a multi-well plate reader (Thermo, Multiskan spectrum). The viability percentage was calculated by using the formula below:
ZR751 cells (2 × 104 cells/well) were cultured in 96-well culture plates, treated with or without test samples and incubated for 24 h. Staining was done using DNA-intercalate fluorescent dyes EB and AO, and analyzed under a fluorescence microscope (Nikon, TS 100-F).
DNA fragmentation assay
For laddering experiments, ZR751 cells (2 × 105 cells/well) were cultured in 6-well culture plates, treated with or without test samples and incubated for 24 h. Cells were then harvested, washed with ice-cold PBS (pH 7.2), and centrifuged at 500 g for 6 min at 4°C. The resulting cell pellet was dispersed in 30 μl of lysis buffer (10 mM EDTA; 50 mM Tris HCl, pH 7.8; 1% SDS) by gentle vortexing. 4 μl of proteinase-K (10 μg/μl) was then added to the above mixture, followed by incubation at 37°C for 1 h. Then, 2 μl of RNase (10 μg/μl) was added to the cell lysates, which were further incubated for 1 h at 57°C. After incubation cell lysates were mixed with 4 μl of 6X DNA loading dye and subjected to run at 2% agarose gel electrophoresis. The gel was then stained with ethidium bromide (0.5 μg/ml) and visualized under a gel documentation system (Bio-Rad).
Cell cycle analysis was carried out using PI staining. ZR751 cells (2 × 105 cells/well) were cultured in 6-well culture plates, treated with or without test samples for 24 h. After incubation, cells were harvested and fixed in ice-cold 70% ethanol overnight at −20°C. Fixed cells were then treated with 0.5 ml of DNA extraction buffer (192 ml of 0.2 M Na2HPO4 with 8 ml of 0.1% Triton X-100 (v/v)) for 5 min at room temperature. DNA was stained with PI (0.02 mM) and incubated for 1 h in the dark. Flow cytometric analysis was then performed using a flow cytometer (BD FACSCaliber).
Assessment of caspase activities
Caspase-3/7, caspase-8 and caspase-9 activity was assayed by measuring the light intensity using an assay kit (Caspase-Glo® 3/7Assay, Caspase-Glo® 8 Assay and Caspase-Glo® 9 Assay, Promega) with some modification. Briefly, ZR751 cells (1 × 105 cells/well) were treated with different concentrations (5–40 μg/ml) of test samples for 24 h. After incubation, cell pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride and protease inhibitor cocktail). Equal amount of protein extract cell lysates were loaded in opaque white 96-well plates and 40 μl of caspase-3/7 or caspase-8 or caspase-9 reaction buffer was added and incubated at room temperature for 1 h before measurement.
RNA extraction and reverse transcriptase PCR
The ZR751 cells (2 × 105 cells/well) were cultured in 6-well culture plates, treated with or without test samples for 24 h. After incubation, total RNA was prepared using an RNeasy kit (Qiagen) and primed with random hexamers to synthesize complementary DNA using superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out in a Mastercycler (Biorad) with specific primers (Additional file1: Table S1). Amplification products obtained by PCR were electrophoretically separated on 1.5% agarose gel, stained with ethidium bromide (0.5 μg/ml) and visualized under gel documentation system (Bio-Rad).
Western blot analysis
Protein expression was carried out as per the standard protocol. Briefly, ZR751 cells (2 × 105 cells/well) were cultured in 6-well culture plates, treated with different concentrations (5–40 μg/ml) of PE extract for 24 h. Cell pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride and protease inhibitor cocktail). Equal amount of protein extract cell lysates were electrophoresed in SDS polyacrylamide gel and then transferred onto PVDF membranes. The membranes were subsequently incubated with primary antibodies of anti-p53, anti-BAX, anti-Bcl2, anti-caspase-3, anti-PARP, anti-cleaved PARP or anti-β-actin at room temperature for 60 min. Antibody recognition was detected with the anti-rabbit or anti-mouse IgG linked to horseradish peroxidase at room temperature for 60 min. Protein bands were visualized on Chemi Doc XRS Imaging System, Bio-Rad after treating with enhanced chemiluminescence reagent.
p53 expression was knocked down using validated silencer TP53 siRNA (Ambion). Sequences were: sense 5′-GUAAUCUCAUGGGACGGAAtt-3′ and antisense 5′- UUCCGUCCCAGUAGAUUACca-3′. For siRNA transfection experiments, ZR751 cells were plated and transfected after 24 h at ~70% confluency by using Lipofectactamine RNAiMAX transfection agent, according to the manufacturer's instructions. After 24 h of transfection, cells were exposed to different concentrations of PE extract for 24 h prior to analysis of protein expression by western blot or cell viability by MTT assay. A non-targeting control siRNA (SilencerH Negative Control-Ambion) was used as transfection control.
Thin layer chromatography
Equal concentration (16 μg/μl) of CGN extracts were loaded on silica gel 60 TLC aluminium sheets. The plates were developed using petroleum ether: ethyl acetate (5:2). The spots were located by exposing the plates to UV light (365 nm).
The values are presented as mean ± SD (standard deviation). Comparision between two groups were done by t–test and multiple comparisons between more than two groups were performed by one-way ANOVA supplemented with Tukey’s HSD test. Values at P < 0.05 were considered to indicate statistical significance.