Patients and samples
Tumor samples from resection specimens were collected from patients with primary lung adenocarcinomas between January 1998 and July 2003, who underwent general thoracic surgery at the Second Hospital of Jilin University. The study was approved by the Ethics Committee of the Second Hospital of Jilin University (Changchun, China) and all patients gave informed consent. All excised tissues were frozen immediately in liquid nitrogen and then stored at −80 °C. Patient medical records were reviewed to obtain tumor staging, pathology, and survival information. The pathologic diagnosis of the resected tumors was based on the World Health Organization histological classification of tumors of the lung. The post-operative disease stage was performed according to the International Union against Cancer's tumor-node-metastasis (TNM) classification. All 106 patients underwent radical surgery. Patients with preoperative chemotherapy or radiotherapy treatment or with evidence of other malignancies were excluded. No patients received gene-targeted therapy during the follow-up period. Eighty-six patients received appropriate chemotherapy or radiotherapy as needed. Among them, 66 patients received more than three cycles of cisplatin-based chemotherapy. Platinum sensitivity, as a measure of treatment response, was defined as no disease progression or relapse during or within 6 months after chemotherapy. The median clinical follow-up time was 38.5 months (range: 7–60 months). Overall survival was defined as the time from the diagnosis to death from any cause. Progression-free survival was defined as the time from the diagnosis to progressive disease, relapse or death from any cause, whichever occurred first. Cases lost to follow-up and deaths caused by conditions other than lung adenocarcinoma were regarded as censored data in the survival analysis.
Paraffin-embedded tissue sections of primary lung adenocarcinoma and the adjacent normal lung tissues were used for immunohistochemical studies. Sections from paraffin-embedded tumors were incubated overnight with mouse anti-human Ku80 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution, followed by incubation with goat anti-mouse secondary antibody (Pierce, Rockford, IL, USA). Immunohistochemical evaluation was performed by two pathologists without knowledge of the clinical and pathological characteristics of these patients. The average optical density and the average positive area ratio from five nonoverlapping, randomly selected fields per section with high-magnification (400×) were examined and counted. Average optical density reflected the positive intensity of area expressing Ku80 protein and equaled to average optical density of positive stained area. The positive area ratio reflected the scope of area with positive Ku80 expression and was calculated as (the total positive area per unit area/the total cells per unit area) × 100%. In the case of nuclear staining of Ku80, the percentage of positive cells was determined and divided into three groups: nuclear staining in less than 25% of cells (weak), nuclear staining in ≥25% of tumor cells and ≤50% of tumor cells (low) or nuclear staining in >50% of tumor cells (high).
Cell lines and transfection
The human lung adenocarcinoma cell line A549 and its cisplatin-resistant variant A549/DDP were cultured as previously described. Small interfering RNA (siRNA) sequences targeting human Ku80 and a non-target sequence were constructed by Genechem (Genechem, Shanghai, China). The Ku80 siRNA (si-Ku80) were designed with the following sequences as previously described[18, 19]: sense 5'GGAUGGAGUUACUCUGAUUTT3', antisense 5'AAUCAGAGUAACUCCAUCCTT3'. The non-target siRNA (Scramble) sequences were as follows: sense 5'UUCUCCGAACGUGUCACGUTT3', antisense 5'ACGUGACACGUUCGGAGAATT3'. Transfection with siRNA was performed using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s protocol. Briefly, A549/DDP cells were seeded into six-well plates at the density of 2 × 105 cells/well, and the cells grew to 50-70% confluent in the next day. Then the cells were transfected with 100 pmol si-Ku80 or si-Scramble by using 10 μl LipofectAMINE 2000 (Invitrogen). For the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and flow cytometry analysis, the transfected cells were treated with cisplatin for 24 h. The cells were harvested 48 h after transfection.
Cell viability assay
The MTT staining kit (Sigma-Aldrich, St. Louis, MO) was used to determine cell viability. A549/DDP cells were plated into 96-well plates (1 × 104/well) for 24 h and then treated with various concentrations of cisplatin for 24 h. Next the cells were treated with 0.5 g/l MTT solution for 4 h. The medium was removed, and 100 μl of dimethylsulphoxide was added to each well. The formazan dye crystals were solubilized for 15 min and the optical density was measured using a microplate reader (Bio-Rad, Richmond, CA) at a wavelength of 570 nm. All experiments were performed in triplicate.
Flow cytometry analysis of apoptosis
After treatment for the defined time, A549/DDP cells were trypsinized and collected, washed, and stained using the Annexin-V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). The samples were subjected to a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Annexin-V-FITC(−)/PI(−) was used to indicate cells that had survived, Annexin-V-FITC(+)/PI(−) was used to indicate cells in the early stage of apoptosis, and Annexin-V-FITC(+)/PI(+) was used to indicate cells in the late stage of apoptosis or necrosis.
Reverse-transcriptase PCR analysis
Total RNA were isolated from cultured cells or tumor samples by using Trizol (Invitrogen, USA) according to the manufacturer’s instruction. Complementary DNA (cDNA) was synthesized by reverse transcription of 1 μg RNA samples with SuperScript pre-amplification system (Promega, Madison, MI). One tenth of the reverse transcribed RNA was used in PCR reaction. The primer sequences were as follows: GAPDH forward 5′ - GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′- GAAGATGGTGATGGGATTTC′ (product 300 bp); Ku80 forward 5′-ACGATTTGGTACAGATGGCACT−3′ and reverse 5′-GCTCCTTGAAGACGCACAGTTT −3′ (product 497 bp). RT-PCR products were separated by electrophoresis on 1.5% agarose gel containing ethidium bromide.
Western blot analysis
Total protein was isolated from culture cells or tumor samples and subjected to western blotting analysis as previously described. Equal amounts of protein (40 μg) as determined by the Protein Assay Kit (Bio-Rad, Hercules, CA) was separated by 12% PAGE and transferred onto nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk diluted in buffer (10 mM Tris–HCl, 100 mM NaCl and 0.1% Tween 20) for 1 h at room temperature. The membranes were then incubated with primary antibodies at 1: 1000 dilution for Ku80, cleaved-PARP, cleaved-Caspase 3, or β-actin (Abcam, MA, USA), followed by incubation with Horseradish peroxidase-conjugated secondary antibodies (Thermo, Waltham, USA) at 1: 2000 dilution for 1 h at room temperature. The protein bands were detected by an enhanced chemiluminescene kit (Pierce, Rockford, USA). Protein levels were quantified by densitometry using Quantity One software (Bio-Rad).
The data were presented as mean ± standard deviation. All statistical analysis was performed using SPSS.17.0 software (SPSS, Chicago, IL, USA). The paired-samples Wilcoxon signed rank test was used to compare the expression of Ku80 between tumor and adjacent normal tissues. A 2-fold difference between control and test was considered the cut-off point to define high or low expression. Comparisons between treatments were made using one-way ANOVA for multiple group comparisons and differences between treatments were examined with a Tukey test. The correlation between Ku80 expression and clinic pathologic features was examined using the Pearson’s Chi-squared test. Overall survival and progression-free survival were calculated using the Kaplan–Meier method and log-rank tests. A 2-tailed P value of less than 0.05 was defined as statistical significance.