The human lung adenocarcinoma cell line A549 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI-1640 (Hyclone, Logan, UT) medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Hyclone) in a 37 °C incubator with 5% CO2. All procedures were carried out using cells were seeded at 70-80% confluence. The cells were more than 95% viable as assessed by trypan blue exclusion.
Female BALB/c nude mice, weighing 17-20 g and 4-6 weeks old, were purchased from Institute of Chinese Academy of Medical Sciences. Before any intervention was initiated, the nude mice were maintained in pathogen-free conditions (55±5% humidity and 23±2℃) for 1 week. The study was approved by Animal Ethics Committee of Qingdao University. Nude mice were injected with 5×106 A549 cells. Tumor size, volume and weight of the mice were calculated daily until the remainder of the experiment. We calculated the tumor volume (V) using the formula: V (mm3) =L×W2/2(W, width of tumor; L, length of tumor).
125I brachytherapy seeds implant
The 125I seeds (4.5 mm long, 0.8 mm diameter) were obtained from Qingdao University Hospital. The energy of 125I was an average from 27.4 to 35.5 keV, and its half-life is about 59.6 days. The 125I is continuously-releasing soft X-ray and low-dose-rate γ-irradiation after decaying into the organs. It is a considerably long of internal radiation, and the brachytherapy dose (93-97%) is depleted in 8-10 months. Once the tumors had reached 300 mm3 in size (about 24 days), mice were randomly divided into 4 groups (n=10/group): sham seed implant group; 125I seed (0.6 mCi) implant group; 125I seed (0.8 mCi) implant group and non-implanted control group. Before cell inoculation, BALB/c nude mice were anesthetized with diethyl ether. The seeds in the form of 18-gauge needles (called Mick-applicator) were directly implanted into the visible tumor of mice. Mice were killed, and tumors from each group were collected and weighed, then fixed with 4% paraformaldehyde (PFA) after 32 days of treatment.
Hematoxylin and eosin (H&E) staining
Tumor tissues were fixed in 4% paraformaldehyde for 24 h. After paraffin imbedding, the tissues were sliced into 4 μm-thick sections. The sections were dehydrated with gradient ethanol, and then stained with hematoxylin for 5 min. After differentiated in 1% hydrochloric acid alcohol for 2s, the sections were then incubated in ammonia water, followed by the staining with eosin. Ultimately, the sections were dehydrated, cleared, mounted with neutral resin, and observed under light microscopy (Olympus, Japan).
Tumor specimens were subjected to a TUNEL assay using the In Situ Cell Death Detection kit (Roche, Basel, Switzerland), according to manufacturer's instructions for detecting apoptosis. As noted above, the fixed tissues were incubated with 100 μl Proteinase K for 30 min at 37°C. Slides were rinsed twice with PBS. Fifty microliter TUNEL reaction mixture was added to the sample in a humid and dark atmosphere at 37℃ and incubated for 60min. Then fifty microliter Converter-POD solution was added to the sample at 37℃ incubated for 30 min. DAB substrate was added to the slides, overlaid with a coverslip and analyzed under light microscope.
The numbers of overall tumor and TUNEL positive cells were quantified in five random sections by a light microscope at magnification of 400X. The apoptotic index was determined as the percentage of TUNEL positive cells to overall tumor cells. Slides with DAB-stained were analyzed by an Olympus BX51TPHD-J11 microscope (Tokyo, Japan). The analysis software (Image Pro Plus, Media Cybernetics, USA) was used for image and data acquisition and analysis.
Caspase-3 and caspase-8 activity test
According to the manufacturer's a protocol, the caspase-3 and caspase-8 detection kits (Beyotime, Shanghai, China) were used to measure the activity of caspase-3 and caspase-8. The treated cell lysates were incubated with lysis buffer on ice for 15 min. Then they were centrifuged (13,000 × g, 4 ◦C, 5 min) and the supernatants were transferred to 96-well plates. Reaction buffer, containing 10 mM DTT was added to each well and the 10µl Ac-DEVD-pNA (2mM) substrate was mixed. The mixture was incubated for 2 h at 37 ◦C，and then protease activity was detected using a fluorescence microplate reader at 450 nm.
Immunohistochemistry for P21, survivin livin and caspase-9
Expression of P21, Survivin, Livin and Caspase-9 was detected by Immunohistochemistry. Three sections were tanked from each xenograft tumors. The main procedures are as follows: after conventional deparaffinization, rehydration, and blocking of endogenous peroxidase activity for 15 min, sections were pretreated for the purpose of antigen retrieval by microwaving, and then washed with PBS. Sections were incubated for 2 h at room temperature with mouse anti-human P21 monoclonal antibody (Zsbio, Beijing, China), rabbit anti-human Caspase-9 monoclonal antibody at a 1:50 dilution (Bioss, Beijing, China), rabbit anti-human Survivin monoclonal antibody (Zsbio, Beijing, China) Company) and rabbit anti-human Livin monoclonal antibody at a 1:50 dilution (Bioss, Beijing, China) respectively. Sections were then washed three times in PBS and incubated for 15 min at room temperature with ready-to-use secondary biotinylated antibodies PV-9000. After that, sections were rinsed with PBS, developed with DAB, counterstained with hematoxylin, cleared with xylene and observed under a light microscope. A negative control was designed by using PBS instead of primary antibody and a known positive section was served as a positive control. All above mentioned procedures were performed in the same conditions.
Two investigators evaluated the IHC-stained tissue sections and photographed representative regions with Kawasaki et al(34) using a microscope. A mean percentage of positive tumor cells was determined in at least five areas at ×400 and assigned to one of five of categories: (a) 0, <5%; (b) 1,5-25%; (c) 2. 25-50%; (d) 3, 50-75%; and (e) 4. >75%. According to cell staining intensity score: (a) cell no color, 0 points; (b) straw colored, 1 point; (c) brownish-yellow, 2 points; (d)tan, 3 points. According to these indicators divided into four, that is negative for the 0-1 points, weak positive 2-3 points, positive 4-5 points, strong positive 6-7 points.
All experiments were performed in three parallels and repeated at least thrice. All data were conducted using SPSS 22.0 software (IBM, Cary, NC, USA). Continuous variables were examined for normality. Results were presented as mean ± standard deviation (SD). Data did pass tests of equal variance. Multiple group comparisons were evaluated by one-way ANOVA. Comparison of means were made by Student-Newman-Keuls test. Differences in proportions were evaluated by chi-square test. Correlations were analyzed by the Spearman rank-correlation coefficient. Difference with P < 0.05 were considered statistically significant.