Preparation of drugs
Gefitinib was kindly provided by AstraZeneca China. SF injection was purchased from Ya-an San-jiu pharmaceutical co., LTD (Sichuan, China), and QKL injection and QKL oral solution were purchased from Ming-xing pharmaceutical co., LTD (Guangzhou, China). The component herbs of SF decoction used in the in vivo experiments, prepared Radix Aconiti Carmichaeli and Radix Ginseng, were purchased from Kang-mei pharmaceutical co., LTD (Guangzhou, China). Seventy-five gram of Radix Ginseng and 150 g prepared Radix Aconiti Carmichaeli were mixed and first soaked in 1000 ml water for 30 minutes, and then boiled for 90 minutes. The liquid was filtered through a piece of medical gauze and the drugs were boiled once more with 800 ml water for 90 minutes. The liquid was filtered again and mixed with that from the first boiling. The solution was concentrated into 250 ml in the rotary evaporator (IKA®RV 10 Basic), with a concentration of 0.9 g/ml crude drug, and then stored at -80˚C until use.
Reagents
4,5-dimethylthiazol-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from MP Biomedicals (California, USA). Annexin V/ propidium iodide (PI) apoptosis Kit was purchased from MultiSciences(Lianke)Biotech Co., Ltd (Hangzhou, China). BCA protein assay kit was purchased from Thermo Fisher Scientific, Inc ( ML, USA). Rabbit anti-human EGFR, phospho-EGFR (p-EGFR), AKT, p-AKT, ERK and p-ERK monoclonal antibodies (mAb) and horseradish peroxidase ༈HRP༉ conjugated anti-rabbit antibody were purchased from Cell Signaling Technology, Inc (MA, USA). Electro-Chemi-Luminescence (ECL) reagent was purchased from Millipore Corporation (MA, USA).
Cell Culture
Human A549 NSCLC cells were obtained from the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). H1975 cells were obtained from the Cell Line Bank of the Macao University of Science and Technology (Macao, China). Cells were maintained in RPMI-1640 medium (Gibco; ThermoFisher Scientific, Inc., MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 0.5% penicillin-streptomycin sulfate (Gibco; Thermo Fisher Scientific, Inc.), and incubated at 37 ̊C with 5% CO2.
Cell Viability Assay
Human A549 cell was used in the in vitro experiments. MTT assay was used to measure cell viability. Briefly, cells were plated in 96-well culture plates at the density of 5 × 103 per well in complete medium. After 24 hours of incubation, cells were treated with gefitinib (20–100 µM), QKL injection and SF injection (0.6%-1.0%) alone or combination for 48 and 72 hours. Then the cells were incubated with 110 µl of 5 mg/ml MTT at 37˚C for 4 h. After removing the medium, 150 µl dimethylsulfoxide (DMSO, Sigma, MO, USA) was added to each well. After shaking for 10 min, the absorbance at 570 nm was measured spectrophotometrically using a microplate reader (VICTOR X5,PerkinElmer). Each condition was duplicated in 6 wells. After removing the maximum and minimum absorbance to calculate the average, the cellular viability in each condition was expressed as the percentage of the average absorbance to that of control cells.
Apoptosis Assay
Apoptotic cell death was determined by the Annexin V/PI apoptosis Kit according to the manufacturer’s instructions. Briefly, cells were plated in 6-well culture plates at a density of 1.5 × 106 cells/mL in complete medium. After 24 hours of incubation, cells were treated with gefitinib, QKL and SF injection alone or combination, with the concentration and exposure time according to the MTT assay results. After removing the medium, cells were trypsinized with EDTA free trypsin solution (Gibco; Thermo Fisher Scientific, Inc.), harvested and then resuspended in 500 µl Binding Buffer (1×) with 5 µl Annexin V-FITC and 10 µl PI. After incubation for 5 min at room temperature in the dark, the samples were analyzed with flow cytometer (Beckman Coulter FC500).
Western Blot Analysis
The cells were plated in 60 mm dishes at a density of 3.5 × 106cells/ml in complete medium. After 24 hours of incubation, cells were treated with gefitinib, QKL and SF injection alone or combination, with the concentration and exposure time according to the MTT assay results. After the treatment, the cells were washed in PBS and lysed in lysis buffer. Protein concentrations were determined using the BCA protein assay kit. The samples corresponding to 30 µg of protein were boiled for 8–10 min, resolved on an 8% denatured SDS-polyacrylamide gel, and transferred onto a PVDF membrane (Millipore, MA, USA). After blocking non-specific binding sites for 30 minutes using 5% skim milk, the membranes were incubated with rabbit anti-human EGFR, p-EGFR, AKT, p-AKT, ERK and p-ERK monoclonal antibodies for 2 hours at room temperature. After washed with TBST for 3 times, the membranes were incubated with HRP conjugated anti-rabbit antibody for 1 hour at room temperature. After washed with TBST for 3 times, visualization of the protein bands was accomplished using ECL reagent. ImageLab software (version 4.0) was used to calculate the expression of each protein, which was normalized by GAPDH.
Determination of antitumor effect in nude mice.
Female BALB/c nude mice (18–20 g) were obtained from the Laboratory Animal Center of Southern Medical University (Guangzhou, China, License NO. 44002100006205). The animals were kept in the Animal Center of Guangdong Provincial Hospital of Chinese Medicine (License NO. SYXK(yue)2013-0094), under a specific pathogen-free (SPF) condition with a 12 h light/dark cycle and freely accessed autoclaved standard food and water. Human NSCLC cells H1975 (3 × 107/ml) were suspended in RPMI-1640 medium and 0.2 ml of the suspension was subcutaneously inoculated into the right forelimb of nude mice. Tumor growth was measured with the longest diameter (a) and the shortest diameter vertical to a (b). Tumor volume was calculated using the formula, V= ∏ab2/6.
When the tumors reached the size over 150mm3, the mice were randomly divided into 6 groups (n = 10): control (saline solution 0.2 ml), gefitinib (1 mg, in 0.2 ml saline solution), QKL oral solution (0.25 ml), SF decoction (0.2 ml), gefitinib + SF, and gefitinib + QKL. The compounds were administered orally once a day for consecutive 21 days. Tumor volume and body weight of the mice were measured every 3 days during the administration period. The daily dosage of each drug for nude mice (with average weight of 20 g) was obtained based on the daily dosage for humans in clinical and the human-mouse transfer formula: Animal dose = Human dose x (HKm/AKm), where HKm and AKm represent the Km factor of human (37) and mouse (3)12. The daily dosages for humans (with average weight of 60 kg) are 250 mg of gefitinib, 60 ml of QKL oral solution, and 45 g crude drug of SF decoction.
Immunohistochemistry
For immunohistochemical staining, paraffin-embedded tumor tissues were applied. The sections were deparaffinized in xylene and rehydrated with graded alcohol. Hydrogen peroxide (3%) was applied to block endogenous peroxide activity and then boiled in 0.01M citrate buffer (pH 6.0) twice with an autoclave. After blocking non-specific binding sites using normal goat serum (Boster Biological Technology co.ltd, California, USA), tissue sections were incubated with rabbit anti-human EGFR (1:100), p-EGFR (1:100), AKT (1:200), p-AKT (1:50), ERK (1:200) or p-ERK (1:100) monoclonal antibodies at 4℃ overnight. After washed with phosphate buffer saline (PBS) for 3 times, the sections were incubated with HRP conjugated anti-rabbit antibody for 30 minutes at 37℃, and the peroxidase reaction was developed with diaminobenzidine substrate kit (Zhongshan Golden Bridge-Bio, Beijing, China). Hematoxylin (Dingguo Changsheng Biotechnology CO., Ltd, Beijing, China) was then used for nucleus staining. Image-ProPlus 5.0 software was used to calculate the ratio of integrated optical density (IOD) to area (IOD/Area).
Statistics
Statistical analysis was performed using SPSS 19.0 statistical software (SPSS, Inc., Chicago, USA). The in vitro experiments were performed three times, independently. All data were presented as the mean ± standard deviation (SD). Differences between groups were assessed by two-tailed t test, one-way analysis of variance or analysis of variance for repeated measuring data and least significant difference (LSD)-t test was used for multiple comparisons. P < 0.05 was considered to indicate a statistically significant difference. q value method was used to evaluate the combination effect of gefitinib and QKL/SF, and it was calculated using the equation: q = EAB/ (EA + EB- EA × EB), where EA and EB were the inhibition effect of gefitinib and QKL/SF, respectively. EAB represented an observed value of combined effect, and (EA + EB- EA × EB) represented an expected value of combine effect. A q value of 1.15 or more is considered synergism, q < 0.85 as antagonism and the value between 0.85 and 1.1.5 is considered as additive effect13.