MATERIALS AND METHODS

Cell culture

Human colorectal carcinoma HT-29 and SW620 cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the Roswell Park Memorial Institute medium (RPMI; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, 100 units/mL penicillin and 2 mmol/L L-glutamine.


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Cell viability assay

Cell viability was determined using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT).20 Briefly, after culturing cells in 96-well plates for 24 h, they were treated with various concentrations of 3D for 24 h. Freshly prepared 10 μL of 5 mM MTT solution was added to the cells and was further incubated for 2 h at 37°C in 5% CO2. In all, 100 μL of dimethyl sulfoxide (DMSO) was added and mixed in each well to dissolve the formazan crystal formed in the reaction of MTT at the time of incubation. The absorbance of the product was measured at 540 nm using a microplate reader. The experiments were performed in triplicates for each condition and the mean and standard deviation (SD) values of three independent experiments were provided.

Cytotoxicity assay using xCELLigence system

Optimal seeding concentration for proliferation of HT-29 cells was determined. Briefly, HT-29 cells (5,000 cells in 150 μL medium/well) were seeded in 16-well plates (E-plate 16; ACEA Biosciences Inc., San Diego, CA, USA) following the xCELLigence Real-Time Cell Analyzer (RTCA)-DP instrument manual as provided by the manufacturer’s protocol. After 24 h, 3D (5, 10, 20 μM) was added and incubation continued for another 48 h. Baseline cell indices were calculated for at least two measurements from three replicate experiments.

Apoptosis

Control and 3D-treated cells were cultured in six-well plates for 24 h. Cells were harvested and washed twice with ice-cold PBS. Cells were resuspended in Annexin-binding buffer and incubated with Annexin V-fluorescein isothiocyanate (5 μL) and propidium iodide (1 μL) at room temperature for 15 min and then analyzed by flow cytometry on FACSCalibur (BD Biosciences, San Jose, CA, USA).

Western blot

Whole cell lysates were prepared using radioimmunoprecipitation assay buffer (RIPA) lysis buffer as described.20 Total protein was extracted by RIPA lysis buffer (Boston Bio Products, Boston, MA, USA), and concentration was determined using Bradford protein reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of soluble proteins were loaded and electrophoresed on 4%–20% of Mini-Protean TGX precast gels (Cat No 456-1094; Bio-Rad Laboratories Inc.) and subsequently transferred to trans-blot turbo 0.2 μm nitrocellulose transfer membrane using trans-blot turbo transfer system (Bio-Rad Laboratories Inc.). The membranes were blocked in 5% skimmed milk in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature and were incubated overnight with the following primary antibodies at 4°C: anti-cytochrome c (1:200; Abcam, Cambridge, MA, USA), anti-poly-ADP-ribose polymerase (PARP, 1:200; BioVision, San Farncisco, CA, USA), anti-p53, anti-Bax, anti-Bcl2, anti-BclxL, anti-cyclin D1, anti-survivin, anti-JAK2, anti-STAT3 (1:1,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-Phospho-JAK2 and anti-Phospho-STAT3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:10,000; Sigma-Aldrich Co., St Louis, MO, USA). The membranes were washed twice with PBST and incubated with horseradish peroxisome-conjugated secondary antibodies (1:3,000; Santa Cruz Biotechnology Inc.) at room temperature for 1 h. After two PBST washes, reactivity was detected using chemiluminescence by Clarity Western ECL substrate (Bio-Rad Laboratories Inc.). Membranes were developed using C-DiGit Blot Scanner (LI-COR, Hamburg, Germany). β-actin was used as an internal loading control.

Cytochrome c measurement

Briefly, cells were treated with 3D for 24 h, and harvested cells were incubated in 1× cytosolic extraction buffer. The homogenate was centrifuged at 10,000× g for 10 min. The cytosolic extracts were immunoblotted for cytochrome c.

ROS measurement by flow cytometry

Cells were pretreated with different concentrations of compound 3D for 24 h. Cells were then treated with 2′,7′-dichlorodihydrofluorescein diacetate (c-H2DCFDA, 5 μM) for 15 min at 37°C to assess hydrogen peroxide (H2O2)-mediated oxidation to fluorescent compound DCF.20 Fluorescence of oxidized DCF was measured using flow cytometry (FACSCalibur) at an excitation wavelength of 480 nm and an emission wavelength of 525 nm.

Measurement of mitochondrial membrane potential

Cells were treated with 3D (10 μM) for 24 h and then were incubated with rhodamine 123 (25 ng/mL; Molecular Probes, Eugene, OR, USA) in PBS for 15 min at 37°C. Rhodamine 123-positive populations were monitored using flow cytometry.20

Caspase activity assay

Caspase activity assay was determined using Caspase Colorimetric Protease Assay Sampler Kit for measuring caspase-3, -6, -8 and -9 (KHZ1001; Thermo Fisher Scientific). In brief, control and treated cells were harvested and resuspended in 50 μL cold cell lysis buffer with incubation on ice for 10 min. Cytosolic fraction was extracted by centrifuging at 10,000× g for 1 min. In all, 50 μg of cytosolic extract was loaded into 96-well plates, followed by addition of 50 μL of reaction buffer containing 10 mM of dithiothreitol. A total of 5 μL of caspase substrate was added and incubated at 37°C for 2 h. Plate was read at 400 nm on the microplate reader.

Cell viability assay for combination studies

The effect of 3D and 3D/doxorubicin (Dox) combination on cell proliferation was measured by MTT assay. The cells were seeded in 96-well plates and incubated overnight. Cells were treated with different concentrations of Dox alone or in combination with 3D (10 μM) for 24 h. Freshly prepared 10 μL of 5 mM MTT solutions were added to the cells, and the cells were further incubated for 2 h at 37°C in 5% CO2 incubator. In all, 100 μL of DMSO was added in each well to dissolve formazan crystal formed in the reaction of MTT at the time of incubation. The absorbance of the product was measured at 540 nm using a microplate reader.

Statistical analysis

Data were presented as mean ± SD values. Statistical analysis was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). The mean values between the control and treated groups were compared using Student’s t-test. P≤0.05 was considered to indicate a statistically significant difference.