FLP ointment was supplied from Pharmaceutical preparation center of Guang’anmen Hospital, China Academy of Chinese Medical Sciences. (No.(98)JWY DF-1657) (Beijing, China). Cyclophosphamide (CTX) was supplied by Shanxi Powerdone Pharmaceutics Co. (Shanxi, China). Antibodies: NF-κB (p65), E-cadherin, N-cadherin, and MMP9, were purchased from Abcam (Cambridge, MA, USA). β-actin was purchased from Cell Signaling Technology (Danvers, MA, USA). ELISA kits for IL6, IL1β, and TNF-α were purchased from R&D systems (Minneapolis, MN, USA).
Preparation of FLP ointment
The herbs used in FLP ointment are the roots of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao (Huang-Qi), Panax quinquefolium L. (Xi-Yang-Shen), Ophiopogon japonicas (Thunb.) Ker-Gawl. (Mai-Dong), Glehnia littoralis Fr. Schmidt ex Miq. (Bei-Sha-Shen), Agrimonia pilosa Ledeb. (Xian-He-Cao), Polygonum bistorta L. (Quan-Shen), Patrinia villosa (Thunb.) Juss. (Bai-Jiang-Cao), Panax notoginseng (Burk.) F.H. Chen (San-Qi), Fritillaria cirrhosa D. Don (Chuan-Bei-Mu), Glycyrrhiza uralensis Fisch. (Gan-Cao), Cordycrps sinensis(Berk.) Sacc. (Dong-Cong-Xia-Cao) and the fruits of Prunus persica (L.) Batsch (Tao-Ren) and Prunus armeniaca L. var ansu Maxim. (Xing-Ren). All those herbs were from Guang’anmen Hospital according to the original proportion, and decocted twice with 8-fold volume of distilled water for 1 hour. The decoction were collected, filtered, merged and concentrated to 2 g/mL (equivalent to crude herb materials), and stored at 4°C for oral using.
All mouse experiments were agreed to and regulated by the Ethical Committee of China Academy of Chinese Medical Sciences. Pathogen free male C57BL/6 mice were purchased from Vital River Company (Beijing, China) at 6-weeks of age and were maintained in a temperature- and humidity-controlled animal facility with a 12-h light/dark cycle. Animals were given free access to water and are monitored closely for any clinical signs of poor health throughout the study. All the animal experiments were conducted in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals. After one-week acclimation, mice were randomly sorted into 5 treatment groups of 10 animals: Non-tumor-bearing (NT), Vehicle control (PBS) Fei-Liu-Ping ointment treated (FLP), cyclophosphamide treated (CTX), and FLP + CTX treated. Xenografts were performed under anesthesia by injecting 2 × 106 Lewis lung cancer cells in 0.1 ml volume into the right flank of the mouse. The following day, mice were treated according to group. FLP mice were treated orally with 100 mg/kg FLP by gavaging each day, for 21 days. CTX group mice were given three IP injections of CTX at 60 mg/kg, on day 2, 7, and 14 following tumor injection. FLP + CTX group mice were given a combination of CTX and FLP treatments described above. The vehicle control group received PBS gavaging each day, for 21 days. Mice were euthanized by CO2 asphyxiation on day 21 and tumor tissues were collected, measured and weighed. Serum from each mouse was also collected into 1.5 ml sterilized micro-tubes and kept on ice for 40 minutes, then centrifuged at 4°C at 3000 RPM for 15 minutes, and stored at -80°C for further analysis.
Tumor inhibition rate
Tumor inhibition rate was calculated as follows: Tumor inhibition rate = (1- average weight of tumors in treatment group/average weight of tumors in control group) × 100%.
Paraffin embedded tumor tissue was sectioned at 5 μm, onto glass microscope slides and subjected to immunohistochemical analysis. Briefly, tissue sections were deparaffinized in xylene and rehydrated in graded ethanol. Sections were then subjected to antigen retrieval and blocked for 1 hour in normal serum. Sections were incubated in primary antibody solution at the following dilutions, NF-κB, 1:50; N-cadherin, 1:100; E-cadherin, 1:200; MMP9, 1:200, overnight. This was followed by incubation in a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 hour. Sections were visualized with 3, 3’diaminobenzidine (DAB) (Millipore, Billerica, MA, USA) and lightly counterstained with hematoxylin. Expression of positive cells was performed using image analysis software ImageProPlus 4.5 (Diagnostic Instruments, USA).
Western blot analysis
Western blot analysis of protein isolated from tumor tissue was performed as follows. Briefly, 100 mg tumor tissue from each sample was homogenized in 1 ml radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo scientific, Waltham, MA, USA). Protein concentration was calculated using standard bovine serum albumin (BSA) curve. Approximately 30 μg of protein was loaded and fractionated by SDS-PAGE and transferred to PVDF membrane and probed with the following primary antibodies at the indicated dilution. NF-κB (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), MMP9 (1:500), and β-actin (1:5000) followed by a horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal was visualized by adding ECL detection reagent (Amersham Life Science, Piscataway, NJ, USA).
A549 human adenoma cell line was obtained from cell resource center of the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. Cells were cultured in RPMI1640 with 10% FBS, 100U/ml penicillin and 100 mg/ml streptomycin at 37°C in humidified air with 5% CO2. THP-1 human monocyte cell line was obtained from cell resource center of the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences and cultured in RPMI1640 with 10% FBS at 37°C in humidified air with 5% CO2.
To study the effects of Fei-Liu-Ping (FLP) ointment in vitro that closely resembles the serum bioavailability of FLP treatment in vivo, we utilized serum pharmacology previously described in
[13–16]. Briefly, Wistar rats, a strain of albino laboratory rats that are often used to study safety and efficacy testing, were subjected to saline, FLP, or CTX treatment. Animals received either oral gavage of saline treatment, or FLP treatment at a low, medium or high dose (50, 100, 200 mg/kg, respectively), or IP injections of CTX (30 mg/kg) twice a day for three consecutive days. On the third day, serum was collected from the animals, filtered through a 0.2 μm cellulose acetate membrane. The serum collected was used for the treatments in all downstream in vitro assays and listed as follows; NRS (normal rat serum, saline treated), FLPL (FLP low dose), FLPM (FLP medium dose), FLPH (FLP high dose).
In-vitro system of inflammatory microenvironment
THP-1 cells were seeded on 6-well plates at 1 × 106 cells/well. The THP-1 monocyte cells were converted to macrophages using Phorbol 12-Myristate 13 acetate (PMA) purchased from Sigma Aldrich (Shanghai, China) at a final concentration of 320nM and incubated at 37°C with 5% CO2 for 48 hours to allow for maximal conversion of the THP-1 monocytes to THP-1 macrophages. All the experiments were performed in polystyrene with Transwell inserts with a pore size of 0.4 μm from BD Biosciences (San Diego, CA USA) using serum free medium. The co-culture experiments consisted of adding an insert containing confluent THP-1 macrophages to cultured A549 cells that are grown in the bottom compartment of the plate. The THP-1 macrophages were never in direct contact with the A549 cells. The cultures were exposed to either vehicle control or various FLP treatments. Cultured A549 without the addition of THP-1 macrophages was used as an experimental control. After 24 hours, the medium was recovered and stored at -80°C for further analysis.
ELISA detection of inflammatory cytokines
ELISA analysis was performed according to the manufacturers’ instructions and analyzed using a spectrometer reading at an absorbance of 450 nm with 570 nm used as the reference wavelength (Synogen4, Gene company Ltd).
Cell proliferation assay
A549 cells were incubated in 96-well plates in the presence of FLP or CTX treatments for 48 hours. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) purchased from Sigma-Aldrich (Shanghai, China) was added for 4 h at the final concentration of 0.5 mg/ml. Subsequently, the culture medium was removed and after dissolving the formazan crystals in DMSO, plates were read immediately at 570 nm using an absorbance plate reader (Bio- Rad, CA, USA). Cell survival was defined as the growth of treated cells compared with untreated cells.
Scratch wound assay
A549 cells were grown to confluence on a 24-well dish in serum-free medium. A single stripe (500 μM wide) was scraped on the cell-coated surface with a disposable plastic pipette tip. Cells were treated with FLP or CTX and incubated for 24 hours at 37°C with 5% CO2. Migration was analyzed using light microscopy with Image-Pro Plus Version 6.0.
Cell invasion was determined using Matrigel invasion chambers purchased from BD Biosciences (San Diego, CA, USA) and according to the manufacturers’ instructions. Briefly, 1 × 104 A549 cells were seeded into the upper chamber in serum free medium separated by a permeable membrane. The bottom chamber contained 10% serum in the presence or absence of FLP or CTX. Serum-free medium in the bottom chamber was used as a control. After 24 hours the cells in the membrane was fixed, stained and counted using light microscopy.
After pre-treatment with FLP or CTX, A549 cells were fixed for 20 minutes using 4% formaldehyde in PBS. The cells were blocked in 1% bovine serum albumin with 0.02% Triton X-100 for 1 hour and then incubated with primary antibodies, NF-κB (p65) (1:200), E-cadherin (1:200), N-cadherin (1:200), MMP9 (1:100), MMP2 (1:100), overnight at 4°C followed by a 1 hour incubation in Alex Fluor594 secondary antibody (1:400) and counterstained with DAPI. Molecular DevicesMetaXpress® Image Acquisition and Analysis Software were used for analysis of translocation of NF-κB p65, E-cadherin, N-cadherin, MMP2 and MMP9.
All data is presented as the mean ± standard deviation. The significance of the difference between groups was evaluated by one-way repeated-measures analysis of variance (ANOVA) and multiple comparisons with Prism 5.0 software. Student’s t-test for paired observation was used where appropriate. P < 0.05 were considered to be statistically significant.