The viability of the test cells were checked by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We used non-cancer cells, liver (WRL- 68), and mouse peripheral blood mononuclear cells (PBMCs) as controls.
An ethanol extract of Condurango (Gonolobus condurango) was procured from Boiron Laboratory®, France. Before treatment, the alcohol containing part of the drug was allowed to evaporate by drying at 40°C. The desired amount of dried Condurango was then made soluble by sonication in ice cold Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute medium (RPMI-1640), with care taken not to raise the temperature of the mix. This mix was made fresh before use. The medium without a drug was served as a control.
HeLa cells, human prostate cancer cells (PC3) and human normal liver cells (WRL-68) were procured from the National Centre for Cell Science (NCCS, India), and were kept in a humidified, ─ 37ºC incubator (ESCO Medical, Singapore) maintained at 5% CO2 and ambient O2 levels. Cells were processed and harvested by using 0.025% trypsin-Ethylenediaminetetraacetic acid (EDTA) (Gibco, U.S.A.) in phosphate buffer saline (PBS) solution. PBMCs were isolated from mice by using the conventional ficoll gradient method .
Cells dispensed in 96-well flat bottom microtiter plates (Tarsons, India) at a density of 1 × 103 cells per well were treated with various concentrations of CE (range: 15 to 180 μg/mL) and were allowed to incubate for 24 hours. MTT reagent was purchased from Sigma, U.S.A., and was used according to the manufacturer’s recommendation. Briefly, MTT was added at 10 μM to each well, and the plates were then incubated for a minimum of 2 hours at 37ºC in the dark. The reaction was then stopped, and the color was allowed to develop. The optical density (OD) was taken at 595 nm in a microtiter plate reader (Thermo, U.S.A.). Experiments were performed in triplicate, where each group was six in number.
For the quantitative estimate of the intra-cellular ROS generation in viable cells after drug treatment, cells were fixed in 70% chilled methanol and incubated with RNase-A (Novagen, U.S.A.) at 5 μg/mL. The RNase-A treated cells were then incubated with 10-μM 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) and 5-μM propidium iodide (PI) together for 30 minutes at room temperature in the dark. Then, the fluorescence intensity of the viable cells (PI-positive cells) was measured by using the frequency lavatory-1 higher (FL-1H) filter of a flow cytometer (Becton Dickinson, fluorescence activated cell sorter (FACS) Calibur, BD, U.S.A.). Data were analyzed by using Cyflogic software (CyF, Finland). For the determination of mitochondrial membrane’s depolarization, the cells were stained with rhodamine 123 after harvesting and fixation. Then, the extent of mitochondrial depolarization in the stained cells was measured by using the FACS calibur flow cytometer with the FL-1H filter.
The CE treated (0, 8, 16, 24 hours) cells were fixed in 70% chilled ethanol. The fixed cells were then treated with 10-mM RNase-A for 10 minutes in the dark at 37ºC. The RNase-A-treated cells were then stained with PI (10 μM, Sigma, U.S.A.) for 20 minutes. The fluorescence intensities were determined by using the flow cytometer with a FL-2A filter. Data were analyzed by using CyF software.
For the evaluation of the apoptosis process by using flow cytometry, the treated and the control cells were harvested in PBS and then kept in 5-μg/mL RNase-A for 10 minutes in the dark at room temperature. Then, the cells were incubated in the binding buffer for annexin V assay. The binding buffer was composed of 10-mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES, pH 7.4), 150- mM Sodium chloride (NaCl), 5-mM Potassium chloride (KCl), 1-mM Magnesium chloride (MgCl2) and 1.8-mM Calcium chloride (CaCl2). For the apoptosis analysis, the treated and the control sets were then treated with annexin V and PI according to the manufacturer’s protocol (Santa Cruz Biotechnology, U.S.A.). The fluorescence intensities were determined by using the FACS Calibur, BD flow cytometer with the FL-1H filter for annexin V staining and the FL-2A filter for PI staining.
Total Ribonucleic acid (RNA) was extracted from the drug treated and the controlled sets of HeLa cells by using Trizol reagent according to the manufacturer’s instructions (Hi- Media, India), and the gene expressions were analyzed by using a semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and the Eppendorf Master Cycler (Eppendorf, Germany) . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as housekeeping gene for normalization (Table 1).
To isolate total cellular protein, we washed the treated and the control cells twice with ice cold PBS and we prepared cell lysate by using lysis buffer (10-mM Tris-hydrogen chloride (HCl, pH 7.4), 1-mM MgCl2, 1-mM EDTA, 0.1-mM phenylmethanesulfonyl fluoride (PMSF), 5-mM β-mercaptoethanol, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS), 10% glycerol and a cocktail of protease inhibitors in tablet form from Roche, Switzerland). Cell lysate was cleared by centrifugation at 5,000 g for 20 minutes at 4°C. The required amount of protein lysate was denatured by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer and subjected to 12% electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, U.S.A.), followed by blocking with 3% bovine serum albumin (BSA) (w/v) in Tris-buffered saline and Tween 20 (TBST) (10-mM Tris, 100-mM NaCl, 0.1% Tween 20) for 1 hour. The PVDF membrane was probed with anti-p21, anti-cyclin D1, anti-cyclin- dependent kinase 1 (CDK1), anti-protein kinase B (Akt), anti-pAkt, anti-B-cell lymphoma 2 (Bcl-2), anti-Bcl2 Antagonist X (Bax), anti-Fas, anti-NF-κB, anti-TNF-α and anti-beta actin (Santa Cruz Biotechnology, U.S.A.) overnight at 4°C and then was incubated for three hours with anti-mouse Immunoglobulin G (IgG) secondary alkaline phosphatase antibody (Sigma, U.S.A.) and developed by using a 5-bromo-4-chloro-3-indolyl-phosphate-nitro blue tetrazolium (BCIP-NBT) kit (Merck, U.S.A.). All antibodies were anti-mouse IgGs. The densitometry was quantified by using ‘image J’ software (National Institutes of Health, U.S.A.).
For indirect staining, cells were suspended in ice cold PBS with 10% fetal bovine serum (FBS) and 1% sodium azide. Anti-Fas primary antibody was added at 10 μg/mL; then, the cells were incubated at room temperature for 30 minutes. Cells were then incubated with 2-μg/mL fluorescein isothiocyanate (FITC)-tagged anti-mouse secondary antibody for 20 minutes in the dark. Fluorescence was measured by using flow cytometry with FL-IH filters. Data were analyzed with Cyf software. For the cytochrome c assay, cells were lysed in lysis buffer, and the lysates were transferred at 50 μg of protein per previously coated 96-well plate . The indirect enzyme-linked immunosorbent assay (ELISA) method was performed according to manufacturer’s protocol (Santa Cruz Biotechnology, U.S.A.).
Experiments were performed in triplicate, and statistical analyses were performed by using the one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc tests and SPSS.20 software (IBM, U.S.A.). Results were expressed as means ± standard errors (SEs). P-values of less than 0.05 were considered significant.