Animals and Chemicals
Female C57BL/6 mice (H-2Kb and I-Ab), 5 to 6 weeks of age, were purchased from National Cancer Institute (Frederick, Maryland, USA) and kept in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, Maryland, USA). Animals were used in compliance with institutional animal health care regulations, and all animal experimental procedures were approved by the Johns Hopkins Institutional Animal Care and Use Committee. Apigenin was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
Murine Tumor Cell Line
The production and maintenance of TC-1 cells  and E7-specific T cells  have been described previously. Luciferase-expressing TC-1 cells (TC-1-luc) were generated by transducing TC-1 cells with retrovirus containing luciferase pLuci-thy1.1 and flow cytometry sorting following the protocol described previously. Briefly, In order to generate a retrovirus containing luciferase, a pLucithy1.1 construct expressing both luciferase and thy1.1 was made. Firefly luciferase was amplified by PCR from pGL3-basic (Promega) using the 5' primer CGGAGATCTATGGAAGACGCCAAAAAC and the 3' primer CGGGTTAACTTACGGCGATCTTTCC. The amplified luciferase cDNA was inserted into the Bgl II and Hpa I sites of the bicistronic vector pMIG-thy1.1. Both luciferase and thy1.1 cDNA are under the control of a single promoter element and separated by an internal ribosomal entry site (IRES). The pLuci-thy1.1 was transfected into Phoenix packaging cell line and the virion-containing supernatant was collected 48 h after transfection. The supernatant was immediately treated using a 0.45-mm cellulose acetate syringe filter (Nalgene, Rochester, New York, USA) and used to infect TC-1 in the presence of 8 mg/ml Polybrene (Sigma, St Louis, Missouri, USA). TC-1-luc cells were sorted using preparative flow cytometry of stained cells with thy1.1 antibody (BD, Franklin Lakes, New Jersey, USA). Both TC-1 cell line and TC-1-luc cell line were maintained in RPMI 1640, supplemented with 10% (v/v) fetal bovine serum, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids, and 0.4 mg/ml G418 at 37°C with 5% CO2. On the day of tumor challenge, TC-1 cells were harvested by trypsinization, washed twice with 1× Hanks buffered salt solution, and finally resuspended in 1× Hanks buffered salt solution to the designated concentration.
In Vitro Cytotoxicity Assay
Luciferase-expressing TC-1 cells  in medium were seeded into a 24-well round-bottom plate (1 × 105 cells/well). Twenty-four hours later, apigenin alone or in combination with E7-specific T cells were added into each well. Apigenin was dissolved in dimethyl sulfoxide (DMSO) and mixed with fresh medium to achieve the desired concentration of 0.2%. This concentration of DMSO did not alter cell growth . Cells treated with solvent DMSO (0.1%) alone were used as controls. E7-specific cytotoxic T lymphocytes from the spleens of tumor bearing mice immunized with the DNA vaccine served as effector cells and were added in the amount of 1 × 106 cells/well. TC-1 cells expressing luciferase were used as target cells. After incubation, D-luciferin (potassium salt; Xenogen Corp.) was added to each well at 150 μ g/ml in media 5 min before imaging. Bioluminescence imaging was taken at baseline, 24, 48, and 72 hours using the IVIS Imaging System Series 200 (Xenogen, Cranbury, New Jersey, USA). An integration time of 10 s was used for luminescence image acquisition. CTL-mediated killing was assessed using bioluminescence-imaging systems quantifying the decrease of luminescence from baseline.
Characterization of apoptotic cell death
The in vitro apoptotic effects of apigenin on TC-1 cells were evaluated using two-colored fluorescence using a phycoerythrin conjugated-Annexin V and 7-AAD apoptosis kit (BD PharMingen, San Diego, CA) following the manufacturer's instructions. Briefly, TC-1 cells were co-incubated with the indicated concentrations of apigenin, and DMSO (0.1% v/v) was used as negative controls. At 6 and 48 hr post-incubation, TC-1 cells were harvested and washed twice in cold PBS and resuspended in binding buffer at a concentration of 1 × 106/ml. The cells were then exposed to the labeling solutions phycoerythrin conjugated-Annexin V and 7-AAD for 15 min. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software (BD Biosciences, Mountain View, California, USA). Both early apoptotic (annexin V-positive and 7-AAD-negative) and late apoptotic (annexin V- negative and 7 AAD-positive) cells were included in the analysis.
Plasmid DNA Constructs and Preparations
DNA fragment encoding M. tuberculosis Hsp70 was obtained from pKS70. For the generation of Hsp70-expressing plasmid (pcDNA3-hsp70), the hsp70 was subcloned from pKS70 plasmid into the unique Bam HI and Hin dIII cloning sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, California, USA) downstream of the cytomegalovirus promoter . The generation of HPV-16 E7-expressing plasmid (pcDNA3-E7) and the E7-hsp70 chimera DNA (pcDNA-E7-hsp70) has been described previously .
DNA-coated gold particles were prepared, and gene gun particle-mediated DNA vaccination was performed, according to a protocol described previously . Gold particles coated with DNA vaccines (1 μg DNA/bullet) were delivered to the shaved abdominal regions of mice by using a helium-driven gene gun (Bio-Rad Laboratories Inc., Hercules, Calif.) with a discharge pressure of 400 lb/in2. C57BL/6 mice (5 per group) were immunized with 2 μg of the DNA vaccine and received a booster dose 7 days after the first vaccination.
Intracytoplasmic Cytokine Staining and Flow Cytometry Analysis
Pooled splenocytes from mice treated with the various treatment regiments were harvested either 14 days (for primary immune response) and 42 days (for memory immune response) after tumor challenge. For the memory recall experiment, the vaccinated mice were re-vaccinated twice with E7-HSP70 DNA vaccine, 60 days after the last vaccination to generate the memory recall response. Ten days after the recall vaccination, splenocytes were harvested and incubated for 20 h with 1 μg/ml of E7 peptide containing an MHC class I epitope (aa49-57, RAHYNIVTF) in the presence of GolgiPlug (BD Pharmingen, San Diego, California, USA) . The stimulated splenocytes were then washed once with FACScan buffer and stained with phycoerythrin-conjugated monoclonal rat anti-mouse CD8a (clone 53.6.7). Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instruction (BD Pharmingen, San Diego, CA, USA). Intracellular IFN-γ was stained with FITC-conjugated rat anti-mouse IFN-γ. All antibodies were purchased from BD Pharmingen. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software (BD Biosciences, Mountain View, California, USA).
In Vivo Tumor Treatment Experiments
C57BL/6 mice were divided into four groups (control, apigenin only, E7-hsp70 only, and apigenin with E7-hsp70). All mice were inoculated with TC-1 cells (5 × 104) s.c. over right leg. For the control group, mice were regularly followed after TC-1 implantation without specific treatment. For the apigenin only group, 3 days after TC-1 implantation, each mouse was given i.p. apigenin (25 mg/kg) and continued for 10 days. For the E7-hsp70 only group, 3 days after TC-1 implantation, each mouse was vaccinated with E7-hsp70 2 μg via gene gun first, and was boosted 7 d later. For the apigenin with E7-hsp70 group, after TC-1 implantation, each mouse received the same vaccination schedule as the E7-hsp70 only group, and the same i.p. apigenin schedule as the apigenin only group.
Statistical analysis was performed using Prism 3.0 software (GraphPad, San Diego, California, USA). Observed differences in apoptosis between different concentrations of apigenin were evaluated using student's t test. Differences in IFN-γ expression in CD8+ cells were measured using Pearson's chi-square test. Survival curves were plotted using Kaplan-Meier method and compared by log-rank test. For all analyses p < 0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD) unless otherwise specified.