Ku80 correlates with neoadjuvant chemotherapy resistance in human lung adenocarcinoma, but reduces cisplatin/pemetrexed-induced apoptosis in A549 cells

Lung cancer patients consulted in the Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University from September 2013 to September 2016 were examined by bronchoscopy. 110 patients with pathological diagnosis as lung adenocarcinoma with clinical classification stage IIIA were recruited for this study. No patient received chemotherapy, radiotherapy or gene-targeted treatments. All patients and their relatives provided the written informed consent, and the research was approved by the Ethical Committee of our institution. Tumor tissues were collected through bronchoscopic biopsy. After immunohistochemistry, patients were divided into two groups according to the Ku80 protein abundance (high and low expression). Two groups received same first-line neoadjuvant chemotherapy, with cisplatin 75 mg/m² combined with pemetrexed 500 mg/m² adjusted in terms of their creatinine clearance [9].

Tissues were fixed by 4% paraformaldehyde overnight, embedded in paraffin and cut in 5 μm for experiments. Tissue sections were deparaffinized, hydrated, and heated in a steamer for antigen retrieval. The tissue samples were then incubated overnight with rabbit anti-human Ku80 monoclonal antibody (Thermo Fisher Scientific, Fremont, CA, USA) at 1:500 dilution, followed by incubation with goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Fremont, CA, USA). The sections were washed extensively and incubated with DAB Quanto (Thermo Fisher Scientific, Fremont, CA, USA) for 5 min. The sections were stained, fixed, and visualized. Each section was examined by two pathological experts independently who were blinded to the clinical data. The immunohistochemical score (IHS) was obtained by combination of proportion score (average percentage of the five fields) with intensity score of the stained tumor cells. Staining intensity was divided into 3 level: 0 (negative), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The proportion of the tumor cells were as follow: 0 = 0 ~ 5%, 1 = 5 ~ 10%, 2 = 11 ~ 49% and 3 ≥ 50%. The final IHS ranged from 4 to 9 was considered positive staining [10].

Total RNA was extracted by using Trizol reagent (Invitrogen, USA) from tumor samples, and was subjected to reverse transcribed to cDNA according to manufacture’ instruction. M-MLV reverse transcriptase (Takara, Otsu, Japan) was used for cDNA synthesis. Real-time PCR reaction was proceeded using SyBR-Green in a LightCycler ® 480 Real-Time PCR system (Roche Diagnostics, Indianapolis, IN, USA). The primers sequences for Ku80 were (F) 5′-ACGATTTGGTACAGATGGCACTc-3′, (R) 5′-GCTCCTTGAAGACGCACAGTTT-3′. The β-actin primer sequences used were (F) 5′-TGGAGAAAATCTGGCACCAC-3′, (R) 5′-GGTCTCAAACATGATCTGG-3′. The relative mRNA expression was normalized to amplification of β-actin gene.

Chest CT scan was used to evaluate the response to chemotherapy after 2 cycles of therapy protocol. The result of therapy was classified by RECIST guideline (Version1.1) [11]. Complete Response (CR) was defined as all target lesions disappeared. The short axis of any enlarged lymph nodes must have reduction to <10 mm. Partial Response (PR) was defined as the sum of lesions diameters decreased at least 30 percentages. When either of these criteria met, this patient was classified in response group.

Lung adenocarcinoma A549 cell line was purchased from Academia Sinica (Shanghai, China). Cells were growing as a monolayer in cell culture flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium enriched with 10% fetal bovine serum (FBS) and antibiotics (1% penicillin/streptomycin) maintained in humidified incubators with 5% CO₂ at 37 °C. Cells with logarithmic growth phase were used for experiment.

To knock down Ku80 expression, A549 cells were transfected with lentiviral vector including specific shRNA (specific sequence was: 5′-CTTTAACAACTTCCTGAAA-3′) and scrambled shRNA lentivirus (A549kd and NCkd, respectively). Meanwhile, to upregulate Ku80 gene expression, A549 cells were infected with lentiviruses contained full-length cDNA of Ku80 and the control lentiviral vector (A540oe and NCoe, respectively). The lentiviral vector green fluorescent protein (GFP) expressed in all lentiviruses was used to evaluate the transduction efficiency. All lentiviral vectors were purchased from the Genechem (Shanghai, China). The lentivirus transfection was performed in A549 cell line with MOI of 20:1 according to the manufacture’s instruction supplemented with 6 μg/mL polybrene. After 24 h, replacing medium containing vector by complete medium.

Protein of cells was extracted with RIPA lysis buffer plus phenylmethysulfonyl fluoride (PMSF) (Zhongshan Biotech, China) according to the manufacturer’s instruction. The protein concentration was measured by the Bio-Rad protein assay reagent (Bio-Rad). 40 μg of protein were mixed with 4x loading Buffer (Beyotime, China), which was loaded into each lane of 10% poly-acrylamide gel electrophoresis. Protein samples were transferred to PVDF membranes after electrophoresis. Membranes were blocked by incubation in TBS containing 0.1% Tween-20, supplement with 5% skim milk. PVDF membranes were then incubated overnight at 4 °C with mouse anti-human Ku80 (1:1,000 dilution, monoclonal; Abcam, Cambridge, UK), cleaved caspase 3 (1:1,000, polyclonal; Cell Signaling Technology, Bedford, MA, USA) or β-actin (1:10,000, monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. The membranes were then incubated with goat anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (1:5000, Zhongshan Biotech, China) for 1 h at room temperature after washing by TBST. Protein bands were visualized using enhanced chemiluminescence (ECL) detection system LAS-4000 MINI System (GE, USA), and densitometry of each band was analyzed by the ImageJ software.

Cell Counting Kit-8 (Dojindo) was used in determining cell toxicity. Initially, A549, A549kd, A540oe cells and their control groups were seeded in 96-well plates at the concentration of 1 × 10⁴ cells (three wells per group), which was incubated in an incubator for 24 h. According to the protocol of first-line neoadjuvant chemotherapy (cisplatin 75 mg/m² combined with pemetrexed 500 mg/m²), we mixed the two drugs as the same proportion to testify the drug efficiency in our in vitro cytotoxicity test. Mixed drugs at 0.125 μM -8 μM were added into a 96-well plate with cells. After 24 h, 10 μl of CCK-8 solution was added to each well for further incubation 2 h in 5% CO₂ at 37 °C. The optical density (OD) was measured by a microplate reader at the 450 nm wavelengths. The cell viability rate was calculated as follow: (the OD value of experimental groups-the OD value of blank groups)/ (the OD value of controls- the OD value of blank groups).

According to CCK8 analysis, A549 cells, A549 with Ku80-silencing cells (A549kd), A549 with Ku80-oversxpression cells (A549oe), and the negative control (NCkd and NCoe) were treated with or without the mixed drugs (0.9 μM), respectively.

Based on the manufacturer’s instructions, an Annexin V-PE/7AAD Apoptosis Detection Kit (BD Biosciences, Woburn, MA, USA) was used to test apoptosis. The cell apoptosis percentages were measured by a FACScan flow cytometer (BD Biosciences, Woburn, MA, USA).

One hundred ten patients of clinical classification were stage IIIA and pathology diagnosed by bronchoscopy examination (Fig. 1). All patients received neoadjuvant chemotherapy based on cisplatin combined with pemetrexed for 2 cycles. 38 patients obtained obviously response to chemotherapy (Fig. 2), while the lesion or metastatic lymph node of other 72 patients did not decrease (Fig. 3) (Table 1). In the 38 cases of tumor tissues from the patients with response to chemotherapy, Ku80 was positive in 7 cases only (18.4%). Among 72 patients with no response to chemotherapy, Ku80 was positive in 69 cases (95.8%), which were significantly higher than Ku80 expressed in the response group. The IHS of Ku80 expression of response group to chemotherapy was 2.079 ± 1.617, while IHS of nonresponse group was 5.597 ± 2.114. The Ku80 expression in the response group was particularly lower compared to nonresponse group (P < 0.05, Fig. 4c). Subsequently, the differences between the response group and nonresponse group in the Ku80 mRNA level were observed. Compared with the nonresponse group, the response group had significantly lower level of Ku80 mRNA expression (p < 0.05, Fig. 4d). The relative mRNA level was 3.612 ± 2.392 in the response group and 7.981 ± 2.684 in the nonresponse group, respectively. Altogether, Ku80 protein and mRNA levels correlates with resistance to neoadjuvant chemotherapy in patients with lung adenocarcinoma.

Cells were transfected with lentiviruses including specific shRNA (A549kd) and full length cDNA to manipulate Ku80 expression (A549oe), and transfected with corresponding nonsense sequence shRNA and empty vector as negative controls (NCkd and NCoe). To evaluate transfection efficacies of viral vectors, phase contrast image of fluorescence microscope was used. As shown in Fig. 5a, after transfection, GFP expression of transfected cells confirmed over 80%, indicating a high transduction efficiency. Western blot analysis showed that the expression of Ku80 was obviously knocked down and upregulated by Ku80 shRNA and full length cDNA, respectively (Fig. 5b and c). No significant difference was observed in the level of Ku80 expression among control lentiviral vector transfected and untransfected cells. These results illustrate that Ku80 cDNA and shRNA effectively manipulate the Ku80 gene expression in A549 cells.

In contrast to the control group, the mixed drugs reduced viability in A549 cells in a dose-dependent manner (Fig. 5d). The IC50 value of mixed drugs against A549 cells’ viability was 0.97 μM at 24 h. Hence, we chose 0.9 μM of mixed drugs to conduct further experiments to avoid the drug cytotoxicity. To confirm the role of Ku80 to predict the resistance to cisplatin combined with pemetrexed in adenocarcinoma, we used lenti-shRNA and lenti-cDNA to knock down and overexpress Ku80, respectively (Fig. 5a and b), then treated cells with different concentration of mixed drugs. Ku80-silencing A549 cells were 2.2- fold sensitive to mixed drugs (IC50 0.451 vs. 0.972) compared to untransfected A549 cells, which Ku80-overexpression A549 cells were 0.304- fold sensitive to mixed drugs (IC50 3.192 vs. 0.972) than untransfected A549 cells (P < 0.05, Fig. 5d) in terms of reduction of viability.

Flow cytometry was used to identify whether Ku80 played a significant role in cell apoptosis caused by cisplatin combined with pemetrexed. Date from flow cytometry analysis displayed that A549 cells treated by mixed drugs underwent apoptosis, which was augmented in A549kd cells (58.9%). However, A549oe cells apoptosis was reduced compared to NCoe and normal A549 cells treated by the same drugs (18.7%) (P < 0.05, Fig. 6a and b). Furthermore, to confirm the relationship between apoptosis and Ku80 expression level, we detected the apoptosis associated protein cleaved caspase-3 by Western blotting. As shown in Fig. 6c and d, A549kd cells exhibited markedly higher levels of cleaved caspase-3 when treated with the mixed drugs, whereas cleaved caspase-3 level of A549oe cells revealed opposite changes. From the above, these results demonstrate that Ku80 causes lung adenocarcinoma cells resistance to apoptosis caused by cisplatin combined with pemetrexed.