Cell lines and cell culture
Human lung cancer cell lines (H1299, A549, H460) and normal cell lines (293, HBE and fibroblast) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured as monolayers in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Reagents and antibodies
Antibodies against TFAP2B, GAPDH, VEGF, PEDF, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against cytochrome-c, PARP, caspase-3/8/9, BAX, and Bcl-2 were purchased from Cell Signaling (Beverly, MA).
Tissue array and immunohistochemistry
The tissue array consisted of 147 formalin-fixed, paraffin-embedded (FFPE) lung adenocarcinomas and corresponding adjacent normal tissues. These tissue samples were previously obtained with informed consent from patients having no anticancer treatment prior to tumor resection. The tissue specimens were histologically examined and classified according to the 2004 World Health Organization classification system. Detailed clinical and pathologic information, including the clinical and pathologic tumor-node-metastasis (TNM) stage, overall survival (OS) duration, and time to recurrence, was available for all cases. The pathological TNM status of all of the lung adenocarcinomas was assessed according to the criteria of the seventh edition of the American Joint Committee on Cancer (2010).
Immunohistochemistry was conducted using Envisionþ Kit/HRP (DakoCytomation). Briefly, slides were immersed in Target Retrieval Solution (pH 9; DakoCytomation) and boiled at 108°C for 15 min in an autoclave for antigen retrieval. The anti-TFAP2B antibody was added to each slide after blocking of the endogenous peroxidase and proteins, and the sections were incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. The substrate-chromogen was added, and the specimens were counterstained with hematoxylin. A negative control was obtained by replacing the primary antibody with normal rabbit IgG.
To evaluate the immunohistochemical staining, two independent observers blinded to the clinicopathologic information performed scoring using light microscopy (magnification 20×). The intensity of TFAP2B staining was semiquantitatively evaluated using the following criteria: strongly positive (scored 2+), dark-brown staining in more than 50% of the tumor cells, completely obscuring the nucleus; weakly positive (scored 1+), any lesser degree of brown staining appreciable in the tumor cell nucleus; absent (scored 0), no appreciable staining in the tumor cells. Cases were accepted as strongly positive if 2 or more investigators independently defined them as such.
Western blot analysis
The proteins in cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA) and electrophoretically transferred to a PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The western blots were probed with specific antibodies, and the protein bands were detected using enhanced chemiluminescence.
Preparation of siRNA or plasmid DC nanoparticles
The TFAP2B siRNA and overexpression plasmid were purchased from Shanghai GenePharma Co. (Shanghai China). The sequence of the human TFAP2B-specific siRNA was 5′-GGA CCA GUC UGU CAU UAA ATT-3′ and 5′-CCC GAA AGA AUA UGC UGU UTT-3′, and the scramble siRNA was 5′-UUC UCC GAA CGU GUC ACG UTT-3′. The siRNA and plasmid were incorporated with additional chemical modifications for superior serum stability in the in vivo applications, and the knockdown efficiency was validated in vitro[28, 29]. For the in vivo delivery of the siRNA and plasmid into tumors, the sequences were first encapsulated into DOTAP-cholesterol (DC) (Avanti Polar, Birmingham, AL, USA) nanoparticles that had validated by many analyses by dissolving in sterilized de-ionized water and then mixing with the nanosomes.
A total of 2 × 105 H1299 cells were seeded into each well of a six-well tissue culture plate (Costar). The next day (when the cells were 70-80% confluent), the culture medium was aspirated, and the cell monolayer was washed with prewarmed sterile phosphate-buffered saline (PBS). The cells were transfected with the siRNA or plasmid at the indicated dose using the DC nanoparticles. The cells were harvested after 48 h of transfection, and western blot analyses or other experiments were performed.
Cell viability assay
A cell proliferation assay was performed using the MTT assay (Roche Diagnosis, Indianapolis, IN). Briefly, cells were plated in 96-well plates (2000 cells/well) in triplicate and treated with siRNA or plasmid. Cell viability was determined after 48 h.
Anchorage-independent colony formation
Cells were transfected with TFAP2B siRNA or plasmid for 24 h, trypsinized, and resuspended as single cells. The cells (8 × 103/ml) were then mixed in 1 ml of 0.3% McCoy’s 5a agar containing 10% FBS. The cultures were maintained in a 37°C/5% CO2 incubator for 14 days. The cell clones were then washed three times with phosphate-buffered saline (PBS), fixed in methanol for 10 minutes, and stained with Crystal Violet for 10 minutes at room temperature. The dye was washed off, and the colonies that contained more than 50 cells were counted.
Determination of VEGF and PEDF production by ELISA
H1299 cells were seeded in 96-well plates and treated with TFAP2B siRNA at 100 nM for 48 hours. The VEGF and PEDF levels in the culture media were quantified using a VEGF Immunoassay Kit (968962) (R & D Systems, Minneapolis, MN) and a Chemikine PEDF ELISA Kit (CYT420) (Chemikine, Billerica, MA) according to the manufacturer’s protocols.
H1299 cells were transfected with TFAP2B siRNA. At 48 h after transfection, the cells were harvested by trypsinization and fixed in 70% cold ethanol for 30 minutes, then stained with 5 μl Annexin V-FITC and 5 μl PI (propidium iodide) using an Annexin V-FITC/PI-staining kit (Becton Dickinson, CA, USA). The cells were placed at room temperature for 15 min in the dark and then analyzed using flow cytometry (EPICS XL; Beckman Coulter). Apoptosis was calculated in terms of the FITC-positive cells, and the PI staining was used to perform a cell cycle analysis. The raw data were analyzed using Multicycle for Windows (Beckman Coulter).
Immunofluorescence and confocal microscopy
Cells were seeded in two-well chamber slides at a density of 1 × 105 cells per well. After 48 h, the cells were washed with PBS, fixed with 4% paraformaldehyde solution, and permeabilized with 0.1% Triton X-100. The cells were incubated with a rabbit anti-cytochrome-c antibody and then incubated with a rhodamine-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI), and the cells were examined under a fluorescence microscope.
Tumor growth inhibition by TFAP2B shRNA in a xenograft mouse model
Endothelial-cell tube-formation assay 200 μL of growth factor-reduced Matrigel (BD Biosciences, USA) was pipetted into each well of a 24-well plate and polymerized for 30 min at 37°C. Human umbilical vein endothelial cells were transfected with si-TFAP2B for 24 h, then harvested by trypsin treatment and suspended in conditioned medium. Then 2 × 104 human umbilical vein endothelial cells in 300 μL conditioned medium were added to each well and incubated at 37°C, 5% CO2 for 8 h. The cultures were photographed by microscopy.
To determine the effect of TFAP2B shRNA on lung cancer cell growth in a xenograft model, A549 cells (2 × 106) were inoculated subcutaneously into the flank of nude mice. Once palpable tumors were observed, tumor volume measurements were obtained every 3 days using calipers. The tumor volume was calculated using the following formula: V = (width2 × length)/2. The body weights were also recorded. At two weeks after injection, the mice were randomized into 2 groups (5 mice/group). Group 1 received an injection of In Vivo Ready nonspecific shRNA, and group 2 received an injection of In Vivo Ready TFAP2B shRNA. The DC nanoparticle-encapsulated shRNA duplexes were injected into the tumors using insulin syringes at a concentration of 10 μg of shRNA/50 mm3 of tumor volume. The two groups were treated twice per week for three weeks. Upon termination, the tumors were harvested and weighted. The animal experiments were approved by the Animal Research Committee of Sun Yat-sen University Cancer Center and were performed in accordance with established guidelines.
Evaluation of angiogenesis factors in xenograft tumor tissues
The tumor tissues from the above treated animals were collected and soaked in 10% formalin and then embedded in paraffin for the analysis. The sections were stained with H&E according to standard immunohistochemical procedures. To assess the impact of the TFAP2B shRNA on angiogenesis factors in vivo, the embedded tissues were stained for VEGF and PEDF to investigate angiogenic factor expression. A negative control was obtained by replacing the primary antibody with normal rabbit or mouse IgG. The immunoreactive-positive cells from each of the differently treated tumor tissue sections were measured at 200× magnification using a light microscope. The amount of protein was analyzed by the integral optical density (IOD) using IPP (Image Plus Pro 6.0, Bethesda, MD, USA).The images were examined under a Nikon TC200 fluorescence microscope equipped with a digital camera.
A statistical analysis was performed using the SPSS statistical software package (standard version 16.0; SPSS, Chicago, IL). Strong TFAP2B immunoreactivity was assessed for the association with clinicopathologic variables, such as gender, age, and pathologic tumor-node-metastasis stage using the Pearson Chi-square test. Survival curves were calculated from the date of surgery to the time of death related to NSCLC or to the last follow-up observation. Kaplan–Meier curves were calculated for each relevant variable and for TFAP2B expression; the differences in survival time among the patient subgroups were analyzed by the log-rank test. Univariate and multivariate analyses were performed with the Cox proportional hazard regression model to determine associations between the clinicopathologic variables and cancer-related mortality. First, we analyzed the associations between death and possible prognostic factors, including TFAP2B expression, gender, age, pT classification, and pN classification, taking into consideration one factor at a time. Second, a multivariate analysis was applied using forward (stepwise) procedures.