Cell culture and reagents
Human colorectal cancer HCT-116 cells were obtained from the cell bank of the Chinese Academy of Sciences (CAS) and maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2 humidified atmosphere.
Preparation of YQFS
All crude drugs (defined as a dried, unprocessed plant, and referring to one that was or is an official drug plant or the source of a refined drug substance) of YQFS were purchased from a local herbal medicine market (Xinjiang, China). All components were deposited at the herbarium located in the College of Pharmacy, Xinjiang Medical University. Briefly, Dang-shen, Bai-zhu, Fu-Ling, Gan-cao, Rou-dou-kou and Ba-yue-zha were mixed at a ratio of 4:3:4:3:3:3. YQFS extracts were prepared according to a previously validated method. The mixture (300 g) was homogenized to a fine powder and then extracted twice in a reflux condenser for 2 h with 75% ethanol in a heated water-bath. The pooled extract was filtered to remove debris, and the ethanol was removed by rotary evaporation under reduced pressure. The concentrated extract was then dried by lyophilization to obtain the YQFS extract at a yield of 190 g. According to HPLC analysis of the YQFS extract, the ratio of crude drug to ethanol extract was 1 g/0.633 g. The extract was stored at 4°C, dissolved in distilled water and diluted with physiologic saline for the animal tests.
MTT cell viability assays
Drug sensitivity was determined using the MTT assay. Briefly, cells were trypsinized and plated out into 96 well plates at a density of 3 × 103 cells per well. Cells were cultured overnight and re-fed with fresh medium at various concentrations of YQFS for 24 h. Thereafter, 50 μl 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO) in PBS was added to each well, incubated for 4 h at 37°C and the formazan crystals that formed were dissolved in 150 μl dimethyl sulfoxide. The optical density was recorded at 570 nm on a microplate reader (Bio-Rad, Hercules, CA).
Apoptosis assay in vitro
Cells were seeded in 6-well plates (4 × 105/well). After 24 h, three dose concentrations of YQFS (obtained from the result of YQFS IC10 in the MTT assay) were added. Flow cytometry was used to detect apoptosis by determining the relative amount of Annexin V-FITC-positive-PI-negative cells, as previously described. Unstained cells, cells stained with Annexin V-FITC alone, and cells stained with propidium iodide alone were used as controls. Singly stained cells were used to adjust electronic compensation on FL1 and FL2 channels.
Apoptosis assay in vivo
Dual staining for a-SMA and TUNEL was undertaken in representative liver sections to localize apoptotic hepatic stellate cells (HSCs). The sections were blocked with Dako double stain blocking solution for 3 min. The primary antibody for a-SMA, in a dilution of 1:1000, was added and incubated for 30 min at room temperature. Histofine labeled polymer (Nichirei, Tokyo, Japan) was added for 20 min at room temperature. The sections were then incubated with the substrate chromogen nitro-blue tetrazolium (NBT) for 5 min. The number of apoptotic HSCs were counted by viewing specimens under the microscope. The apoptosis index was defined as the number of apoptotic cells in every hundred cells counted.
Wound healing assay
Migration assays were performed following a standard protocol wound repair assay. Briefly, cells were cultured in standard conditions, as described above, to 70%-80% confluency after treatment with YQFS. The monolayers were incubated and wounded in a line across the well with a standard pipette tip. The wounded monolayers were washed twice with phosphate-buffered saline (PBS) and incubated with serum-containing medium. The rate of wound closure was measured and photographed over 24 h. This allows imaging of both wound edges using the 10× objective.
Western blot analysis
Whole cell lysate for SDS-PAGE and western blot analysis for ERK expression was prepared as previously reported. To prepare the lysates from dissected in vivo tumors, samples were snap frozen in liquid nitrogen immediately after sacrificing the animals and stored at −80°C. The lysate was incubated on ice in immunoprecipitation assay buffer for 2 h before being homogenized using a mortar and pestle. The homogenized sample was centrifuged and the supernatant was collected and stored at −80°C. Protein quantification and western blotting for ERK were done as previously reported. Equal loading was confirmed with β-actin (0.1 μg/mL, Sigma Chemical). Densitometric analysis was done using Scion Imaging software (Scion Corporation), using total ERK or β-actin as a control for each sample.
Tumor xenograft animal model
Male athymic nude mice (NCr-nu) were purchased from Sino-British SIPPR/BK lab Animal Ltd., Co (Shanghai, China, license No. SCXK 2010–0002) and maintained under specific pathogen-free conditions for the studies. All animal protocols were approved by the Institutional Animal Use and Care Committee. All experiments and animal care protocols were approved by the Shanghai Medical Experimental Animal Care Commission in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation. The mice used in these experiments were 8–12 weeks old.
HCT116 cells were grown in culture and then detached by trypsinization, washed, and resuspended in HBSS. Next, 0.2 mL of the resuspended cells (1.0 × 106) were subcutaneously injected into the athymic nude mice to initiate tumor growth. Tumors were allowed to reach an average size of 100 mm3, after which mice were randomized into 4 groups (n = 10 per group). Mice in group 1 were administered distilled water daily, which served as a vehicle control. Mice in group 2 were given 5-fluorouracil (5-FU) intraperitoneally, every 2 days, at a dosage of 0.5 mg/kg, half the maximum tolerated dose (MTD) of 5-FU, as previously described. Mice in groups 3,4 and 5 received YQFS at a daily dose of 200, 400 or 800 mg/kg respectively, by intragastric administration for 19 days. In the clinical practice of Chinese herbal medicine, YQFS is usually prescribed at a daily dose of 400 mg herbal materials. When this human dose was converted into an animal dose (at an extraction yield of 2.5%, for a person weighing 60 kg, and a conversion factor of 12.33 between humans and mice), it was equivalent to the middle dose (400 mg extract/kg) used in this study.
Body weight and tumor growth were measured every 2 days. Tumor growth was determined by measuring the major (L) and minor (W) diameter with a caliper. The tumor volume was calculated according to the following formula: tumor volume = π/6 × L × W2. At the end of experiment, the animals were anesthetized with pentobarbital, and the tumor tissue was removed and weighed.
Gelatin zymography of MMP-2/9 activity
All tumor groups were plated onto 12-well culture plates and made quiescent by incubation in serum-free DMEM/F-12 for 24 h. The culture medium was collected and centrifuged at 10 000 × g for 5 min at 4°C to remove cell debris. MMP-2/9 expression was analyzed as previously described. The gel analysis function in ImageJ (http://rsbweb.nih.gov/ij/) was used to quantify protease activity bands by densitometry. Values are reported in Relative Intensity Units (RIU).
Quantitative RT-PCR analysis
For RNA isolation, tumors were homogenized and suspended, and RNA was extracted using the RNAspin Mini Kit (GE Healthcare, Waukesha, WI, USA) according to the manufacturer’s instructions. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed using oligo-dT primers and the Superscript Amplification System (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Life Technologies). The PCR amplification program consisted of an initial polymerase activation at 94°C for 5 min, followed by 35 cycles at 94 C for 30 s, 59.5°C for 40 s and 72 C for 30 s for VEGF. Amplification of GAPDGH, a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent GAPDGH mRNA levels. Oligonucleotide primers for VEGF and GAPDGH were as follows: Oligonucleotide sequence (5’-3’) of ERK1/2 (188 bp), F: 5’-CCTGCTGGACCGGATGTTA-3’, R: 5’-GTCTCTTGGAAGATCAGCTC-3’, Oligonucleotide sequence (5’-3’) of GAPDH (306 bp), F: 5’-ACCCACTCCTCCACCTTTGA-3’, R: 5’-CTGTTGCTGTAGCCAAATTCGT-3’. mRNA expression was determined by real-time PCR using the TaqMan method as previously described.
All values were expressed as the mean ± S.D. and analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test using SPSS version 13.0 software; a p-value of less than 0.05 was considered significant.