Reagents
Stock solutions of 10 mM TAM and 1 mM CXB (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO, USA), stored at −20°C, and diluted in fresh medium just before use. For western blot analysis, the following antibodies were used: rabbit monoclonal anti-Bak, anti-CBP, anti-p-MAPK (Thr202/Tyr204), anti-MAPK, anti-p-Akt (Ser473), anti-Akt, anti-p-STAT3 (Tyr705), anti-STAT3, anti-p-Src (Tyr416), anti-Src, anti-p-VEGFR2 (Tyr1175), anti-VEGFR2, anti-p-BAD (Ser136), anti-BAD, anti-COX-2, anti-HIFα, anti-MMP-2, anti-VHL, and anti-PARP (all Cell Signalling Technology, Beverly, MA, USA), mouse monoclonal anti-β-Actin (Sigma Aldrich, St. Louis, MO, USA), and mouse monoclonal anti-Bcl2, mouse monoclonal anti-Bax, and horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The pGL3-VEGFR2-780 plasmid (Addgene plasmid 21307) was kindly provided by Dr. Donald Ingber (Harvard Medical School, Boston, MA, USA), and the pGL3-Basic plasmid was purchased from Promega (Madison, WI, USA). FuGENE HD transfection reagent was purchased from Roche Applied Science (Mannheim, Germany); Opti-MEM I reduced serum medium, TRIzol reagent kit and Coomassie Blue R-250 from Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, USA; Nonidet P-40 lysis buffer, chemiluminescent peroxidase substrate, propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), and sense and antisense VEGFR2 oligo primers from Sigma Aldrich, St. Louis, MO, USA; and pyrogallol and H2O2 from Merck (Whitehouse Station, NJ, USA). Stock solutions of PI, DAPI, and MTT were prepared by dissolving 1 mg of each compound in 1 ml of phosphate-buffered saline (PBS). The solution was protected from light, stored at 4°C, and used within 1 month. Stock concentrations of 10 mg/ml RNaseA (Sigma Aldrich, St. Louis, MO, USA) were prepared and kept at −20°C.
Cell cycle analysis
To determine the cell cycle distribution, 5 × 10
5 MCF7 or MDA-MB-231 cells were plated in 60-mm dishes and treated with their respective half maximal inhibitory concentration (IC
50) values of TAM, CXB, or both for 48 h. After treatment, the cells were collected by trypsinization, fixed in 70% ethanol, and kept at −20°C overnight for fixation. Cells were washed in PBS, resuspended in 1 mL of PBS containing 100 μg/mL RNase and 40 μg/mL PI incubated in the dark for 30 min at room temperature [
22‐
24]. The distribution of cells in the cell-cycle phases were analyzed from the DNA histogram using a FACS Caliber flow cytometer (Becton-Dickinson, San Jose, CA, USA) and CellQuest software (CA, USA).
Wound-closure assay
To assess the effect of TAM and CXB on cell migration, MCF7 and MDA-MB-231 cells (1 × 10
5) were plated in 12-well plates in complete growth medium [
23,
25]. After 24 h of growth, a scratch was made through the confluent cell monolayer using a 200-μl pipette tip, and the cells were treated with the IC
50 values of TAM, CXB, or both in 3 ml of complete medium. At 48 h post-treatment, cells were stained with hematoxylin and eosin. Cells invading the wound line were observed under an inverted phase-contrast microscope using 20×, Leica DMR, Germany. The distance between the two sides of the scratch was measured after the indicated time intervals using Leica QWin software, IL, USA. Each experiment was performed three times with triplicate samples.
Boyden chamber assay
To test the anti-invasive effect of TAM and CXB, 8-μm filters were coated with Matrigel (20 μg per filter) and placed in Boyden chambers. MDA-MB-231 cells (1 × 10
5) suspended in DMEM containing 0.1% bovine serum albumin and treated with IC
50 of TAM, CXB, or both, were added to the top chamber. Conditioned medium from mouse fibroblast NIH/3T3 cells was used as a source of chemoattractant and placed in the bottom compartment of the chamber [
26]. After 24 h incubation at 37°C in a 5% CO
2 atmosphere, cells that migrated to the lower surface of filters were detected with traditional staining with hematoxylin and eosin. Cells were counted in five fields of each well under inverted phase-contrast microscope using 20×, Leica DMR, Germany.
Gelatin zymography
Supernatants from MCF7 and MDA-MB-231 cells (5 × 10
4 cells per well, six wells per plate) treated with TAM, CXB, or both for 48 h were collected for matrix metalloproteinase (MMP) activity analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. A total of 1.2 mg/ml gelatin was prepolymerized on a 10% polyacrylamide gel as a substrate. Electrophoresis was carried out at 4°C. The gel was washed with renaturation buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and 2.5% Triton X-100), which was followed by incubation with a developing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl
2, 0.02% NaN
3, and 1 μM ZnCl
2) at 37°C for 16 h and staining with Coomassie Blue R-250, as described previously [
27]. The stained bands are observed through a gel doc system (Bio-Rad). Densitometric analysis of stained bands was performed by ImageMaster 2D Platinum 7.0 Software (GE Healthcare Life Sciences, NJ, USA).
Chorioallantoic Membrane (CAM) assay
To determine the in vivo anti-angiogenic activity of TAM and CXB, a CAM assay was performed as described previously with some modifications [
28]. Two day-old fertilized eggs were incubated at 37°C in 60–70% relative humidity. After 5 d of incubation, a 1- to 2-cm
2 window was opened and a sterile round filter paper (5-mm in diameter; Whatman qualitative filter papers, Sigma-Aldrich, St. Louis, MO, USA) containing serum-free medium alone or supplemented with VEGF, TAM, CXB, or both TAM and CXB (at IC
50 concentrations) was applied onto the CAM of each embryo. After 2 d of incubation, the upper eggshell was removed, and capillaries within 2.5 mm around the filter paper were observed and photographed under a stereomicroscope (Olympus, SZX16, USA). Neovascularization around the disk was quantitated by determining the number of angiogenic vessels within the CAM around the disk.
For the capillary-like tube formation assay, growth factor-depleted Matrigel from BD Pharmingen, San Jose, CA, USA was applied to a 96-well tissue culture plate (50 μl per well). After polymerization of the Matrigel at 37°C for 1 h, human umbilical vein endothelial cells (HUVECs) (Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, USA) starved of serum for 2 h were harvested by using trypsin/EDTA, washed with assay medium, and seeded at a density of 7.5 × 10
3 cells per well (final volume 500 μl) on the polymerized Matrigel in the presence or absence of 30 ng/ml VEGF along with TAM, CXB, or both [
29,
30]. Plate was incubated at 37°C, 5% CO
2 for 24 h, then the medium was aspirated and cells were fixed in 10% neutral buffered formalin. Tube formation was observed for 24 h, representative pictures were taken at 10× magnifications under a stereomicroscope (Olympus, SZX16, USA) and tubes were counted in five random fields.
Transfection studies
MCF7 and MDA-MB-231 cells were plated in 60-mm petri dishes at a density of more than 4 × 10
5 per plate in DMEM supplemented with 10% fetal bovine serum. After being allowed to grow for 16–20 h, cells were starved for 6 h with 2% fetal bovine serum. Confluent cells (70–80%) were transiently transfected with 5 μg of pGL3-VEGFR2-780 plasmid with 7.5 μl of FuGENE HD transfection reagent in 100 μl of Opti-MEM I reduced serum medium according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany) [
31]. After 24 h of transfection, the mix was replaced with complete medium containing TAM, CXB, both, or neither for 24 h and then lysed in luciferase lysis buffer (Sigma) [
32,
33]. Luciferase activity was measured with a luminometer (Varian cary eclipse, Palo Alto, CA, USA) and a luciferase assay kit (Sigma) and was normalized to β-galactosidase activity. All luciferase experiments were done in triplicate and repeated three times. Data is presented as means ± SD.
Measurement of VEGF levels
To measure VEGF levels, MCF7 and MDA-MB-231 cells (5 × 10
5 cells per well, six wells per plate) were plated and incubated under culture conditions overnight, and the medium was replaced by serum-free culture conditioned medium. TAM, CXB, or both were added to the culture, and the medium was collected at 72 h [
10]. VEGF levels were measured using a VEGF enzyme-linked immunosorbent assay (ELISA) kit (DVE00, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density at 570 nm of each well was measured using an automated microplate reader (model 550, Bio-Rad, Hercules, CA, USA).
Animal studies
Tumor response to CXB and TAM was studied using S180 tumor bearing female Swiss albino mouse model. Our study was approved by the Department of Biotechnology (DBT), INDIA under the project number: E-1/MMSMST/12, at Indian Institute of Technology Kharagpur, INDIA and the mice were maintained in accordance with the institute animal ethical committee (IAEC) guidelines approved by Indian Council of Medical Research (ICMR), New Delhi. The mice were housed and acclimatized in a pathogen-free environment at our institute’s animal facility for 1 week prior to injection with mouse S180 sarcoma cells. Exponentially growing S180 cells were harvested and a tumorigenic dose of 2.5 × 10
6 cells was injected intraperitoneally into 6- to 7-week-old female Swiss albino mouse [
24,
35,
36]. Tumors were allowed to grow in the mouse for 7 d, when the animals were randomly assigned into one of four treatment groups (5 mice per group). The control group received 1% polysorbate resuspended in deionized water. The other three groups were treated with CXB (3.7 mg/kg body weight), TAM (2 mg/kg body weight), or CXB plus TAM (2 and 1 mg/kg body weight, respectively) intraperitoneally on alternative days for 2 weeks. The doses were selected based on previous experiments [
37,
38]. Mouse body weight was measured before the treatment injections were given and on the 7
th and 14
th day of treatment. On 15
th day, the animals were euthanized using chloroform and their liver and kidney tissues were collected for enzymatic assays. Spleens were collected and cultured for a splenocyte surveillance study. Furthermore, S180 cells were collected from the site of treatment injections for in vivo and ex vivo cell cycle phase distribution studies.
Measurement of antioxidative enzyme activity
Parts of mouse liver and kidney tissues were homogenized in 0.1 M Tris buffer (pH 7.0), and the homogenate was centrifuged at 4000
g for 20 min. The supernatant was immediately assayed for catalase (CAT) and superoxide dismutase (SOD). Determination of CAT activity was performed at room temperature in a 1-ml mixture containing clear cell lysate, 100 mM phosphate buffer (pH 7.0), and 10 mM of H
2O
2[
40]. The decomposition of H
2O
2 is followed directly by a decrease in absorbance at 240 nm spectrophotometrically using Perkin Elmer Lambda45. CAT activity was expressed in micromoles of H
2O
2 consumed per minute per milligram of protein.
Total SOD was determined using the pyrogallol assay, based on the competition between pyrogallol oxidation by superoxide radicals and superoxide dismutation by SOD [
41], and spectrophotometrically read at 420 nm using Perkin Elmer Lambda45. SOD activity was expressed in units per minute per milligram of protein.