Cell line culture and establishment of xenografts in nude mice
The human gallbladder carcinoma cell line GBC-SD was purchased from the Cell Bank of the Chinese Academic of Sciences (Shanghai). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium (Gibco BRL, Carlsbad, CA) supplemented with fetal bovine serum (10%; Gibco BRL), glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cells used in drug treatments or to inject mice were grown until the exponential growth phase.
Exponential-phase cells were trypsinized, washed with RPMI 1640, and suspended in saline solution to obtain a concentration of 5 × 106 cells/0.1 ml. Subsequently, 0.1 ml cell suspension was injected into each nude mouse (Experimental Animal Center, Chinese Academic of Science, Shanghai), subcutaneous in the axilla bilaterally. Formation of a tumor with a diameter of 5 mm was observed approximately 1 week after cell inoculation.
Cell cycle analysis
GBC-SD cells were plated in six-well plates (Corning, New York, NY). Cells floating in the medium were combined with the adherent cell layer, which was then trypsinized. Cells were treated with SST (75 μg/ml; Serono Co., Switzerland) for 12, 24, 72, and 120 h and subsequently, 5 × 105 cells were washed, pelleted, and then incubated with 2 mg/ml RNase A in phosphate-buffered saline (PBS, 200 μl) and 0.1 mg/ml propidium iodide in 0.6% nonidet P-40 in PBS (200 μl) on ice for 30 min. Samples were immediately analyzed by flow cytometry using a fluorescence acquired cell sorter (FACS Calibur, Becton Dickinson Co., Franklin Lakes, NJ). The DNA content histograms were analyzed to determine the cell cycle phase distribution of the gallbladder cancer cells using the CELLQUEST software (Becton Dickinson).
Cell viability assay
Cells from monolayer-grown GBC-SD cultures were harvested and dispensed in 96-well culture plates in 200 μl medium at a concentration of 105 cells per well. Cells were divided into three groups: DOX-use-only group, co-use-group (co-treatment with DOX (Sigma Chemicals, St.-Louis, MO) and SST), and control group. In the control group, PBS was added to the cells. DOX was applied in the following gradient concentrations: 0, 3.33, 6.66, 13.32, 19.98, and 33.30 μg/ml and the intracellular uptake was determined . In the co-use group, cells were first treated with 75 μg/ml SST for 24 h followed by the addition of DOX in the gradient concentrations mentioned above. The cell viability was measured after 48 h of incubation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [13, 14]. Ten microliter MTT (5 mg/ml in PBS) was added to each well. After 4 h incubation at 37°C, supernatants were removed and replaced by 150 μl dimethyl sulfoxide. After formazan solubilization, the optical density at 590 nm (OD590) of each well was measured using an automated microplate reader (Bio-Rad Model 550, Microplate Reader, Hercules, CA). The cell viability was calculated using the following equation: cell viability = mean OD of one group/mean of the control × 100%
Xenograft growth inhibition assay
Twenty-five nude mice bearing a xenograft were randomly divided into five groups: DOX-use-only group (2 mg/kg DOX), SST-use-only group (1 mg/kg SST), co-use group 1 (2 mg/kg DOX plus 1 mg/kg SST), co-use group 2 (2 mg/kg DOX plus 2 mg/kg SST), and the control group. The use and care of animals follwed the guidelines of the Ethical Committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. SST was injected daily, intraperitoneal, from the first day until the ninth day and DOX was injected every other day, intraperitoneal, from the second day until the eighth day (4 times). Those injections were combined in case of the co-use-group. Twenty-four days after the injection of mice with gallbladder cancer cells, tumors were harvested and the wet weight was determined.
Because of the limited number of samples, the Wilcoxson rank sum test, a non-parametric method that allows to test independent samples, was applied to determine whether a given mean was significantly different from a respective control (P < 0.05). The SPSS software for Windows (version 13.0) was utilized to perform all the statistical analyses and the significance level (αvalue) was set at 0.05.