3.1. Reagents
MMC was purchased from Hisun Pharmaceutical Co., Ltd (Zhejiang, China), dissolved in normal sodium as a 1 mM/L stock solution and stored at 4 °C in the dark. The anti-LMP1 antibody Fab was supplied by Key Laboratory of Ministry of Health, Nanjing Medical University (Jiangsu, China). VEGF antibody was obtained from Sigma (St. Louis, MO, USA). Second antibody was purchased from ZhongShan Goldbridge Co., Ltd (Shanghai, China). Annexin V-FITC apoptosis detection kit was purchased from Biouniquer Technology Co., Ltd (Shanghai, China).
3.2. Cell Culture
Human nasopharyngeal carcinoma cells HNE2-LMP1 (LMP1 positive) was purchased from Xiangya Central Laboratory (Hunan, China) and cultured in RPMI-1640 medium (Gibco, San Francisco, CA, USA) supplemented with 10% fetal calf serum (Gibco, San Francisco, CA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). The cell culture was maintained at 37 °C with 5% CO2 in a humidified atmosphere.
3.3. In Vivo Tumor Xenograft Model
Male 3-week-old BALB/C nude mice with a body weight of approximately 20 g were purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China), and maintained in standard vinyl cages with air filter tops in a filtered laminar air flow room at 25 °C on a 12 h light/dark cycle; water and food were autoclaved and provided. The experimental design for mice model was shown in . For tumor establishment, 5 × 106 HNE2 cells/mL were washed twice with PBS and injected subcutaneously in a volume of 0.1 mL into the flank of mice. After inoculation, tumor-bearing mice were divided randomly into 5 treatment groups (8 mice per group) and treatment initiated when the xenograft solid tumors reached a volume of about 100 mm3. Each mouse was injected intraperitoneally on day 1, 4, 7, 10 with different drugs as follows: group I: MMC alone (2 mg/kg); group II: Fab alone (4 mg/kg); group III: MMC (2 mg/kg) + Fab (4 mg/kg); group IV: MMC (1 mg/kg) + Fab (4 mg/kg); and group V: phosphate buffered solution (PBS) as negative control. The animal studies were conducted in accordance with public Health Service policy and approved by the Animal Care and Use Committee of Nanjing Medical University. After xenograft transplantation, mice bearing tumors were observed and tumor size was measured once every 3 days with vernier caliper. The tumor volume in each animal was estimated according to the formula: tumor volume (mm3) = L × W2/2 (where L was the length and W was the width) with the final measurement taken on day 33. At the same time, the body weight of each animal was measured once every 3 days. At the end of the experiments (on day 33), the animals were anaesthetized by CO2 and killed. Tumors from each animal were removed, measured and weighed individually. Inhibition rate of tumor growth was calculated by the following formula: Inhibition rate (%) = (tumor weight of control group – tumor weight of experimental group)/tumor weight of control group × 100%. The tumor tissues were also dissected and collected for further examination.
3.4. Annexin V/PI Assay for Apoptosis
Tumor tissues from the mice in five groups were excised and suspended, respectively. Apoptosis of tumor cells was assessed by measuring membrane redistribution of phosphatidilserine using an Annexin V-FITC apoptosis detection kit (Biouniquer Technology, Shanghai, China) according to the manufacturer’s protocol. Tumor cell suspensions were prepared and washed with PBS, adjusted to a concentration of 1 × 106 cells/mL, then resuspended in 250 μL of binding buffer and stained with staining solution containing Annexin V/FITC and PI. The cells were analyzed using a FACScan flow cytometer (Becton-Dickinson Immunocytometry System), and the acquired data were analyzed. Additional exposure to propidium iodide (PI) made it possible to distinguish early apoptotic cells (Annexin-positive and PI-negative) from late apoptotic cells (Annexin- and PI- positive). All experiments were performed at least in triplicate.
3.5. Immunohistochemistry
The tumors from five groups were excised and then paraffin wax-embedded at Department of Pathology, Nanjing Medical University (Jiangsu, China). Sections (5 μm) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. Endogenous peroxidase activity was blocked by hydrogen peroxidase (3%) in Tris-buffered saline (TBS) for 30 min. Then the sections were boiled for 10 min in citrate buffer for antigen retrieval. Nonspecific binding was blocked by incubation with 5% goat serum in TBS for 15 min. Next the sections were incubated with VEGF antibody in TBS containing 1% bovine serum albumin at 37 °C for 1 h, then washed with TBS and incubated with EnVision goat anti-mouse/horseradish peroxidase antibody (1:2000) for at 37 °C for 1 h. The color was developed in diaminobenzidine solution and counterstained with Mayer’s hematoxylin. Four fields in each slide were randomly selected and counted, the percentage of positive staining was determined by two clinical pathologists independently using immunohistochemistry score (IHS) [
30]. When a conclusion differed, the final decision was made by consensus. IHS was determined by the evaluation of both staining density and intensity. The percentage of positive tumor cells was scored as follows: 1 (0–10% positive cells), 2 (11–50% positive cells), 3 (51–80% positive cells), 4 (81–100% positive cells); and the intensity of staining was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Multiplication of the intensity and the percentage scores gave rise to the ultimate immunoreactivity score: 0–1 (negative), 2–3 (weak), 4–6 (moderate), and 8–12 (strong).
3.6. Statistical Analysis
All data were expressed as mean ± SD and analyzed with SPSS 18.0 statistic software (SPSS Inc, Chicago, IL, USA). Group differences in tumor weight and tumor growth inhibition rate were analyzed by a one-way ANOVA, and post hoc multiple comparison was performed with the LSD method. For all tests, the significance level for statistical analysis was set at P < 0.05.
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