The human lung carcinoma cell line, H1299, and the mouse colon carcinoma cell line, CT-26, were routinely grown in Dulbecco's modified Eagle medium (GIBCO BRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) in 5% CO2
. The human breast adenocarcinoma cell line, MDA-MB-231, was cultured in DMEM/F-12 1:1 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS in 5% CO2
. The human prostate adenocarcinoma cell line, PC-3, was cultured in RPMI-1640 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS in 5% CO2
. The human microvascular endothelial cell line-1 (HMEC-1) was cultured in MCDB131 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS, 10 ng/ml epidermal growth factor (Becton Dickinson, San Jose, CA), and 1 mg/ml hydrocortisone (Sigma-Aldrich, Inc., St. Louis, MO) in 5% CO2
. Primary human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cord as described [55
] and maintained in medium 199 (GIBCO BRL Life Technologies) supplemented with 20% FBS, 30 μg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), 15 μg/ml heparin (Leo Pharmaceutical Product, Ballerup, Denmark), and 1 mM pyruvate in 5% CO2
Handling of THL
For the in vitro experiments, the culture medium containing THL was prepared as follows. THL (obtained from China-Japan Feida Union Co., Ltd., Hong Kong) was centrifuged to remove insoluble ingredients, and the supernatant was added to the appropriate culture medium to the final concentrations of 0.1, 0.25, 0.5, 0.75 or 1% (v/v). For the in vivo pulmonary metastasis experiment, THL was used directly (without centrifugation before use). For the breast cancer xenograft experiment, THL was centrifuged to remove insoluble ingredients, and the supernatant was diluted with phosphate-buffered saline (PBS) at 1:1 (v/v) ratio.
Preparation of conditioned medium of MDA-MB-231 cancer cells (231-CM)
2 × 106 MDA-MB-231 cells were incubated for 24 h in 5 ml of serum-free medium. The medium was then collected, filtrated to remove cell debris, and stored at -20°C until use. For mouse Matrigel plug assays, the conditioned medium was concentrated 50 fold before use.
Wound healing migration assay
Cancer cells were seeded at a density of 1-5 × 105cells/well in 12-well culture plates and allowed to form a confluent monolayer. The layer of cells was then scraped with a 20-200 μl micropipette tip to create a wound of ~1 mm width. Cells were then washed twice with fresh medium, and replaced with medium containing indicated concentration of THL. After incubation at 37°C for 20 h, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.5% Coomassie Brilliant Blue. Images of the wounds were captures at 0 h and 20 h after scraping at 100-fold magnification and the average distance of the wound was calculated by using Image Pro Plus software (Media Cybernetics, Bethesda, MD). The ability of the cells to close the wound, that is, their motility, was calculated in the following way: (average distance of the wound at 0 h - average distance of the wound at 20 h/average distance of the wound at 0 h) × 100.
Migration and invasion assays
The in vitro cell migration and invasion assays were performed by using a modified Boyden chamber inserted with polyethylene terephthalate filter membrane containing 8-μm pores in 24-well plates (Millipore, Billerica, MA). For cell invasion assays, the filter membranes were coated with Matrigel (30 μg, Becton Dickinson, San Jose, CA).
Cells (1 × 105) suspended in 200 μl of serum-free medium were seeded onto the upper compartment of the transwell chamber. The lower chamber was filled with serum-free medium containing chemoattractants (10% FBS for migration and invasion of cancer cells; 231-CM for migration and invasion of endothelial cells) and various concentrations of THL. After incubation for 6 h (for migration assays) or 24 h (for invasion assays), the medium in the upper chamber was removed and the filters were fixed with 70% ethanol for 10 min. The cells remaining on the upper surface of the filter membrane were then completely removed by wiping with a cotton swab, and the cells on the opposite surface of the filter membrane were stained with 0.5% Coomassie Brilliant Blue for 10 min. The migrated/invaded cells were then visualized and counted from six randomly selected fields (× 200 magnification) under an inverted microscope.
Production of MMPs and uPA by cancer or endothelial cells were analyzed by gelatin and plasminogen-casein zymography, respectively. In MMP gelatin zymography, cells were cultured in serum-free medium with various concentration of THL for 24 h (cancer cells) or 6 h (endothelial cells), and conditioned media were collected, filtrated, and concentrated (~50 fold). Equal amount of conditioned medium samples were mixed with SDS sample buffer containing 2% SDS without β-mercaptoethanol and applied, without boiling, to 7.5% SDS polyacrylamide gels copolymerized with 2 mg/ml gelatin (Sigma-Aldrich, Inc., St. Louis, MO). After electrophoresis, gels were washed for 30 min at room temperature with gentle agitation in renaturing buffer (2.5% Triton X-100 in H2O) to remove SDS. The gels were then equilibrated in developing buffer (40 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM CaCl2) at room temperature with gentle agitation for 30 min. After removing the old developing buffer, the gels were incubated in fresh developing buffer at 37°C overnight. The gels were then stained with 0.5% Coomassie Brilliant Blue and destained. The MMP activities were visualized as clear bands against the blue background of the stained gels. The uPA zymography was performed as described in the MMP gelatin zymography, except that the SDS polyacrylamide gels containing 1 mg/ml casein (MP Biomedicals, Inc., Irvine, CA) and 1 U/ml plasminogen (MP Biomedicals, Inc.) were used.
Pulmonary metastasis assay
All animal experiments in this study were performed following the Guidelines for Animal Experiments in National Taiwan University and were approved by the Institutional Animal Care and Use Committee in College of Medicine, National Taiwan University (IACUC Approval No: 20060184). Balb/c female mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University (Taipei, Taiwan) and given food and water ad libitum. The mice were oral fed with either THL or water (200 μl; twice a day) throughout the experimental duration. On Day 8 of treatment, the mice were injected intravenously (via tail veins) with 2 × 105 mouse CT-26 colon cancer cells to establish pulmonary metastasis. Mice were killed 15 days after tumor cell injection and the metastatic nodules on the surface of the lungs were counted. The lungs were fixed with formalin. Thin sections were stained with hematoxylin and eosin. Representative fields (at × 40 or × 100 magnification) for each group were photographed.
Tube formation assay
Tube formation of HUVEC and HMEC-1 endothelial cells on Matrigel was performed as described [56
]. Matrigel (200 μl) was added to 24-well plates and allowed to solidify for 30 min at 37°C. Endothelial cells (5 × 104
cells/well) suspended in 500 μl of complete medium or 231-CM containing various concentration of THL were seeded on the solidified Matrigel. After incubation for 5 h, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet in 20% methanol. Randomly chosen fields were photographed at × 100 magnification and the closed networks of vessel-like tubes were counted.
Mouse Matrigel plug assay
Female NOD-SCID mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University and housed in pathogen-free condition. The mice were subcutaneously injected with 500 μl of Matrigel containing concentrated 231-CM (10 μl), heparin (10 U) and either THL (5 μl) or water (5 μl). Fourteen days later, mice were killed and the Matrigel plugs were removed. To quantitate the formation of functional blood vessel, the amount of hemoglobin was measured using the Drakin's reagent kit (Sigma-Aldrich, St Louis, MO).
Human MDA-MB-231 breast cancer xenograft model
Female NOD-SCID mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University and housed in pathogen-free condition throughout the experimental duration. Mice were given free access to commercial rodent chow and water. MDA-MB-231 cancer cells (3 × 106, suspended in 100 μl of PBS) were injected subcutaneously into both flanks of the mouse. One week after tumor cell inoculation, the mice were randomly divided into two groups. The weight of the mice in these two groups was similar at this time point. One group was intraperitoneally injected with 100 μl of PBS-diluted THL [1:1 (v/v) dilution] once a day until the end of the experiment (It should be noted that the dosage of THL was optimized in our preliminary experiments and that the THL dose used here showed no toxic effect to the mice). The other group was administrated with PBS using similar protocol as described above. The mouse body weight and tumor size were measured at different time points following tumor implantation, and the tumor volume was calculated according to the following formula: 1/2 (Length × Width2). The tumors were removed at Day 36 after tumor implantation, photographed, and weighed. The tumors were snap-frozen in liquid nitrogen for immunohistochemical (IHC) and TUNEL analyses.
IHC and TUNEL analyses
Cryostat sections of frozen tumors were fixed with 4% paraformaldehyde, washed with PBS, and the endogenous peroxidase activity was blocked with Dako Dual endogenous enzyme block (Dako, Glostrup, Denmark). After washing with PBS, the sections were blocked with 5% FBS in PBS. To detect CD31-positive stained microvessels, the sections were probed with rat anti-CD31 antibody (Becton Dickinson, San Jose, CA), and then incubated with horseradish peroxidase-conjugated secondary antibody by using Rat on Mouse HRP-Polymer Kit (Biocare Medical, Concord, CA). Following color development by using Dako DAB reagent (Dako), the nuclei were stained with hematoxyline. The sections were then sealed with glycerol-gelatin (Sigma-Aldrich, Inc., St. Louis, MO) for microscopic observation. Randomly chosen fields were photographed at × 200 magnification and the number of CD31-positive stained blood vessels was counted. For TUNEL assay, the cryostat sections were fixed in 4% paraformaldehyde, washed with PBS and permeated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate in PBS). The sections were then labeled with TUNEL reaction mixture according to the protocol provided by the manufacturer (Roche Applied Science, Mannheim, Germany) to detect apoptotic cells. Following TUNEL reaction, the sections were rinsed three times with PBS, and incubated in Hoechest 33258 solution to label nuclear DNA. The sections were then sealed with mounting medium (Sigma-Aldrich, Inc.) and subjected to fluorescence microscopy. Randomly chosen fields were photographed at × 200 magnification and the number of TUNEL-positive cells was counted.
Data are present as the mean ± SD. The significance of the difference between groups was evaluated with the Student's t-test, p < 0.05 was considered significant.