Human colon cancer cell HT-29 (ATCC® Number:HTB-38™) was obtained from the American Type Culture Collection (ATCC, USA) and cultured in RPMI 1640 (Hyclone, USA) supplemented with fetal bovine serum (10%), penicillin (100 units/ml) and streptomycin (100 mg/ml) (Hyclone, USA) in a humidified incubator (37°C) containing 95% air and 5% CO2. Trypsin (Hyclone, USA) was used for trypsination.
Preparation of TXL
Tian Xian Liquid (TXL) (Batch number: L2-171040) was provided by China-Japan Feida Union Company Ltd. and stored away from light at 4°C. TXL was diluted and incorporated into the cell culture medium RPMI 1640. Residues were removed by filtration.
Cell proliferation was assessed in vitro with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) according to the manufacturer's protocol (Roche, USA). HT-29 cells (10000 per well) were incubated in triplicates in a 96-well plate. TXL was serially diluted with RPMI1640 and the final concentrations were 0.25, 0.5, 1, 2 and 5%. The plates were incubated with or without TXL for 24 and 48 hours. At the end of the incubation, cells were exposed to MTT (10 μL, 5 mg/mL in phosphate-buffered saline) in culture medium for four hours at 37°C. The supernatant was removed and 150 μL DMSO (Sigma, USA) was added to dissolve the formazan crystals. The absorbance was measured at 595 nm with an ELISA plate reader (Bio-Rad, USA).
DAPI (Sigma, USA) (4' 6-diamidino 2-phenylindole)-stained nuclei were observed with fluorescence microscopy. HT-29 cells (70-80% confluent) in 24-well uncoated plates were exposed to 0.5% and 1% TXL for 24 hours respectively. Cells were fixed with 4% paraformaldehyde for 30 minutes and incubated with 1 μg/mL DAPI solution for 30 minutes in the dark. Stained cells were imaged under a fluorescence microscope (Carl Zeiss, Germany).
Assessment of apoptosis by determination of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'tetraethylbenzimidazolylcarbocyanine iodide (JC-1) according to the manufacturer's protocol (Biotium, USA). After trypsinization and centrifugation (500× g)(Eppendorf, Germany) for ten minutes at room temperature, the pellets of cell culture with or without TXL were re-suspended in RPMI 1640 medium (1 ml), stained with 5 mg/ml JC-1 for 30 minutes at 37°C in the dark, washed twice in phosphate buffered saline (PBS) and re-suspended in 0.5 ml PBS. Δψm depletion was observed under a fluorescence microscope. A green filter was used for green-fluorescent monomer at depolarized membrane potentials and a red filter for orange-fluorescent J-aggregate at hyperpolarized membrane potentials.
To measure the quantitative change of mitochondrial potential, we applied JC-1 with fluorescence plate reader. Briefly, cells (1 × 105) in 100 μl culture medium/well were seeded in black 96-well plate (Nunc, Denmark) and treated with TXL (0.15, 0.3, 0.6, 1.25 and 2.5%). After 24 and 48 hours incubation, JC-1 (5 μg/ml) was added for the last 30 minutes of treatment. Cells were washed twice with PBS to remove unbound dye. The concentration of retained JC-1 dye was measured (490 nm excitation/600 nm emission) with a luminescence spectrometer (PerkinElmer, USA).
The HT29 cells were incubated with increasing concentrations of TXL (0, 0.5%, 0.75%, 1%) for 48 hours. For the time-course experiment, HT29 were treated with 1% TXL for 12, 24, or 48 hours. Cellular levels of cleaved caspase-3, 9 (Cell Signaling Technology, USA) Bax/Bcl-2 cytochrome C and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, USA) were determined by Western blot. Lysates were prepared from 1 × 107 cells by dissolving cell pellets in 100 μl of lysis buffer. Lysates were centrifuged (Eppendorf, Germany) at 18000× g for 15 minutes and the supernatant was collected. The protein concentration was estimated with the Bio-Rad protein assay kit (Bio-Rad, USA) using bovine serum albumin as a standard. Sample proteins were resolved by 10% sodium dodecylsulfate polyacrylamide gel (Bio-Rad, USA) electrophoresis and then electrophoretically transferred to PVDF membrane (Millipore, USA) and blocked with 5% BSA (Sigma, USA). Subsequently the primary antibodies caspase-9, cleaved caspase3, Bax, Bcl-2, cytochrome C and GAPDH were added. After overnight incubation at 4°C the blots were washed, exposed to HRP-conjugated corresponding secondary antibodies for one hour and finally were visualized by ECL Advanced Solution (GE Healthcare Life Sciences, USA). Digital images were captured by Gel Doc™ gel documentation system (Bio-Rad, USA) and intensity was quantified using Quantity-One software version 4.62(Bio-Rad, USA).
In vivo tumor-growth inhibition studies
The experiment was approved by the Department of Health, Hong Kong SAR, China and the Committee on the Use of Live Animals in Teaching and Research (CULATR) of Li Ka Shing Faculty of Medicine, University of Hong Kong. Six-week-old female nude mice were purchased from the Laboratory Animal Unit, University of Hong Kong and kept under sterile conditions in accordance with the institutional guidelines of animal care. The HT-29 carcinoma was established in nude mice by injecting the suspensions of HT-29 (1 × 106 cells per animal)  cells subcutaneously into the right flank of each animal. When the tumors became palpable (size: 18 mm3) after xenografting, mice were divided into three groups (n = 8) by a random numbered table: (1) Control group orally administered with 200 μl PBS); (2) 5-fluorouracil (5-FU) (Choongwae, Korea) group (injected intraperitoneally with 5-FU, 30 mg per kg of body weight) three times a week [9, 10]; (3) TXL group (orally administered with 200 μl TXL daily for 14 days. To evaluate the antitumor activity of TXL, we measured the tumor volume with a digital caliper six times every week (from day1 to day 6 and from day 8 to day 14) and calculated using the formula: (longest diameter) × (shortest diameter)2 × 0.5. The body weights of all animals were recorded throughout the experiment to assess drug toxicity.
Data were presented as mean and standard deviation (SD). When one-way ANOVA showed significant differences among groups, Tukey's post hoc test was used to determine the specific pairs of groups that were statistically different. A level of P < 0.05 was considered statistically significant. Analysis was performed with the software SPSS version 16.0 (SPSS Inc, USA).