The powder form of an EOD (Catalog number: CA04-019) was obtained from the plant extract bank at the Korean Research Institute of Bioscience and Biotechnology (KRIBB) in Daejeon, Korea. The powder was then immersed in ethanol, sonicated for 15 minutes, and extracted for 72 hours. The extract was filtered through non-fluorescent cotton and evaporated under reduced pressure by using a rotary evaporator (N-1000SWD, Eyela, Japan) at 45°C. The condensed extract was then lyophilized using a Modul Spin 40 dryer (Biotron Corporation, Calgary, Canada) for 24 hours. The final yield of lyophilized powder (EOD) was 12.3%. The EOD was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL and stored at 4°C as a stock solution, which was later diluted with medium to the desired concentration prior to use. The HT-29 human colorectal adenocarcinoma cells were obtained from the American type culture collection (Rockville, MD) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2-mM glutamine, 100 μg/mL of penicillin, and 100 μg/mL of streptomycin in a 5% CO2/95% relative humidity (RH) atmosphere at 37°C.
To investigate cell viability, we used a 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HT-29 cells were seeded into the wells of 12-well plates and cultured in RPMI 1640 for 72 hours. The MTT solution [100 μL, 5 mg/mL in phosphate buffer solution (PBS)] was then added to each well, and the plates were incubated for 4 hours at 37°C. After the supernatants had been removed, they were gently shaken with 200 μL of DMSO (Jersey Lab Supply, Livingston, USA) for 30 minutes, and the absorbance was measured at 570 nm by using a microtiter plate reader (SpectraMax 190; molecular Devices Corporation, Sunnyvale, USA).
Propidium iodine (PI) staining was used to investigate cell cycle disruption, as previously described [11, 12]. About 1 × 106 cells were placed in an e-tube, and 700 μL of ice-cold fixation buffer (ethyl alcohol) was slowly added with vortexing. Tubes were sealed with parafilm, incubated at 4°C overnight, and spun for 3 minutes at 106 g and 4°C, after which the supernatants were aspirated and discarded. Cell pellets were resuspended in 200 μL of PI staining solution (2 μL of PI @ 5 mg/mL and 2 μL of RNase in 196 μL of PBS), spun at 20817 g for 5 s, and left for 30 minutes in the dark at room temperature. Samples were analyzed using a fluorescence- activated cell sorter (FACScan; Becton-Dickinson, Mountain View, USA) at λ = 488 nm and Cell-Quest software (Becton-Dickinson, Franklin Lakes, USA). The deoxyribonucleic acid (DNA) content distributions of normally growing cells are characterized by two peaks, that is, the G1/G0 and G2/M phases, where the former represents normal functioning and the resting state of the cell cycle and has a high diploid DNA content, whereas the latter G2/M phase has a low diploid content and also contains tetraploid DNA. On the other hand, cells in the sub-G1 phase, which have the least DNA content, are termed hypodiploid, and represent DNA fragmentation .
Mitochondrial membrane depolarization was evaluated by using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescence probe, according to the manufacturer’s instructions (Molecular Probes, Eugene, USA). HT-29 cells were labeled with 2 μM JC-1 for 30 minutes at 37°C and were then analyzed at an excitation wavelength 488 nm by using flow cytometry with 530/30 or 585/42 nm bypass emission filters. Cells not exhibiting red fluorescence were regarded to manifest mitochondrial membrane depolarization.
Caspase-3 and -9 assay kits (Cellular Activity Assay Kit Plus) were purchased from BioMol (Plymouth, USA). After experimental treatments, cells were centrifuged (1000 g, 4°C, 10 minutes), washed with PBS, resuspended in ice cold cell lysis buffer, and incubated on ice for 10 minutes. Samples were centrifuged at 10000 g (4°C, 10 minutes), and supernatants were removed. Supernatant samples (10 μL) were then incubated with 50 μL of substrate (400-lM Ac-DEVD-pNA) in 40 μL of assay buffer at 37°C. The absorbance at 405 nm was read at several time-points. The pNA concentrations in the samples were determined using a standard plot of absorbance versus pNA concentration. The pan-caspase inhibitor (zVAD-fmk, Calbiochem, Millipore Corporation, Billerica, USA) was used to validate the assay method.
Intracellular reactive oxygen species (ROS) generation was determined using carboxy-H2DCFDA [5-(and-6)-carboxy- 20,70-dichlorodihydrofluorescein diacetate; Molecular Probes, Eugene, USA]. Briefly, after treatments, cells were treated with 100 μM carboxy-H2DCFDA in culture medium, incubated at 37°C for 30 minutes, and washed with PBS. Fluorescence was measured using a FACScan (Becton-Dickinson, Mountain View, California, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Unless otherwise indicated, results are expressed as means ± standard deviations (SDs) of experiments performed in triplicate. Statistical analysis was performed using a paired Student’s t-test. Statistical significance was accepted for P values < 0.05.