X-ray irradiation induced Disabled-2 gene promoter de-methylation enhances radiosensitivity of non-small-cell lung carcinoma cells

A total of 137 lung cancer tissue samples, including 63 squamous cell carcinoma and 74 adenocarcinoma cases, and the corresponding normal lung tissues were obtained randomly from patients who underwent surgery at the First Affiliated Hospital of China Medical University between 2009 and 2011. The follow-up study lasted for 60 months (4–60 months, average follow-up time 30.65 months). Patients included in the study ranged from 32 to 82 years of age (mean, 59 years), and were composed of 77 males and 60 females. These tumors showed different degrees of differentiation and were classified as well (n = 30), moderately (n = 67), or poorly (n = 40) differentiated. 65 cases exhibited lymph node metastasis. The tumor stage was classified according to the TNM classification system of the International Union Against Cancer as stage I (57 cases), stage II (52 cases), and stage III (28 cases). The study was conducted according to the regulations stipulated by the institutional review board at the China Medical University and complied with Declaration of Helsinki.

Sections were immunostained with polyclonal rabbit anti-Dab2 antibody (1400; Abcam Biotechnology Inc., Cambridge, MA), at 4 °C overnight. Antibodies were detected by the streptavidin-peroxidase method. The immunostained slides were reviewed by 2 pathologists who were blinded to the clinical data. Five fields of view per slide were randomly examined at 400× magnification. The positive rate of each case was obtained by calculating the percentage of positively stained cells on each slide. Percentages were scored as (1) 1 to 25%, (2) 26 to 50%, (3) 51 to 75%, or (4) 76 to 100%. The intensity of Dab2 staining was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The two scores were multiplied to give a final score from 0 to 12. The overall expression of Dab2 in these tumors was then defined as low expression (< 5) and high expression (≥5).

Four cell lines of NSCLC, including LTEP-a-2 (LTE, adenocarcinoma), and NCI-H460 (H460, large cell carcinoma), obtained from the ATCC (Manassas, VA, USA); SPC-A-1 (SPC, adenocarcinoma) and LK2 (LK, squamous carcinoma) cell lines were obtained from the Cell Bank of the Chinese Academy (Shanghai, China). Plastic flasks with lung cancer cells were treated with X-ray irradiation using a linear accelerator (Primus, Siemens, Germany) with a dose of 2Gy and 4Gy. X-ray irradiation was delivered soon after the cell density reached 70–80%, and a non-irradiated lung cancer cell line was used as control. After irradiation, the cells were harvested at the appropriate time points and stored in a freezer (− 80 °C) before being processed for further analysis.

The genomic DNA from lung cancer cells either treated or untreated with X-ray irradiation was isolated by using a DNA extraction kit (TIANGEN BIOTECH BEIJING CO., LTD) according to the manufacturer’s instructions. DNA methylation kit was used to treat DNA samples (ZYMO RESEARCH Inc., USA). The methylation status of the region was examined by bisulfite sequencing PCR (BSP) using primers as follows: 5′-ATTTTTGGGGAGTTTTTAGTTG-3′ (forward) and 5′-ATGAGATTTTGTGTTTGGGGAA-3′ (reverse). PCR products were then separated by 3% agarose gel electrophoresis and were then cut from gels for purification using the E.Z.N.A Gel Extraction Kit (Omega Bio-tek, Doraville, GA). The purified products were subsequently cloned into pMD 19-T cloning vector (Takara), and at last, 6 clones were subjected to DNA sequencing (Wanleibio, Shenyang, China) after bacterial amplification. The AMF was defined as the value of mean methylation frequency the 23 CpG sites.

Real-time PCR (Fluorescent dye method, ABI, 7900HT) was performed to evaluate the transcripts of Dab2. The experiments were performed according to the manufactures’ instructions (SYBR® Premix Ex Taq™, Takara Bio) using PCR primers as follows: 5’-CTCGCTTCACGGAGGCAATAG-3′ (forward) and 5’-AGCAAACCTCAGTACCAGTGGACA-3′ (reverse). The experiment was repeated three times, and a mean value was obtained.

Antibodies against Dab2 (ab33441, 1:800, Abcam), Axin (C76H11, 1:500, cell signaling), β-catenin (562,505, 1:800, BD), cyclin D1 (H-295, 1:500, Santa), MMP-7 (sc-30,071, 1:500, Santa), c-myc (D3N8F, 1:800, cell signaling), and β-actin (14C10, 1:1000, cell signaling) were used in this study. The protein bands were visualized using ECL (Pierce, Rockford, IL, USA) and quantified using the DNR Bio-Imaging System. A mean value was calculated by three times repeated experiment.

LK and SPC cell lines were used in Colony Formation assays. The process was performed as follows: 600 cells were grown in a 60 mm dish with culture medium. The cells were treated with 4Gy irradiation, 4Gy irradiation and Dab2 siRNA transfection, 4Gy irradiation and Axin siRNA transfection respectively, and untreated cells were used as a control group. After 24 h of incubation, the cells were then continuously cultured until visible colonies were formed (14 days). Only those containing ≥50 cells were counted. The rate of colony formation was indicated by the ratio of the number of clones over the number of seeded cells. The experiment was repeated three times, and a mean value was calculated. Matrigel cell invasion assay: LK and SPC cell lines were used and the groups were classified as mentioned in the Colony Formation assay. In each upper chamber, 5 × 10⁵ cells were grown in serum-free culture medium. The lower chambers were filled with medium containing 10% fetal calf serum. After incubation for 24 h, the cells that migrated through the pores were fixed with methanol for 30 min and stained with hematoxylin. For each filter, the number of cells was counted microscopically in 5 random fields under 200× magnification. A mean value was calculated by three times repeated experiment.

Four-week-old male BALB/c nude mice were obtained from the animal facility (Shanghai Slake Experimental Animal Co., Ltd.). The mice were handled in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health [26]. The protocol was approved by the Committee on the Ethics of Animal Experiments of the China Medical University and complied with Declaration of Helsinki. The mice were randomly divided into 4 groups (5 mice in each group, weight 17.34–22.58 g). Each mouse was inoculated subcutaneously in the right axilla with 5 × 10⁶ human lung cancer cells suspended in 0.2 ml sterile PBS. The large dimension (L) and short dimension (W) of the subcutaneous nodules were measured with vernier calipers every week. The tumor volume was calculated by the following formula: V=W² × L× π/6, before plotting into the growth curve for each group. Five weeks after inoculation, the mice were sacrificed and the tumor nodules from each mouse were completely excised and measured. The rate of tumor growth inhibition (%) was calculated according to the formula: (mean tumor weight of control group-mean tumor weight of X-ray irradiation group)/mean tumor weight of control group× 100%.

SPSS version 22 for Windows was utilized to analyze the data. Survival probability was analyzed by Kaplan-Meier analysis. The relationship between Dab2 expression and lung cancer patients’ clinical and pathological characteristic was analyzed by the Chi-Squared test. The Student’s t test and Mann–Whitney U test were used to evaluate statistical difference of other experimental data.

Immunohistochemical staining for Dab2 revealed low expression in 97 cases and high expression in 40 cases. Low expression of Dab2 is not correlated with the patients’ age (P = 0.8692), gender (P = 0.5747) or histological classification (P = 0.5991), but has a close relationship with the differentiation (P < 0.0001), TNM stage (P = 0.006194) and lymph node metastasis (P = 0.00004, Table 1). The above results indicate that high Dab2 expression is negatively correlated with the progression of lung cancer (Fig. 1a). A survival analysis shows that the patients with high Dab2 expression have a better outcome than those patients with reduced Dab2 expression (P < 0.05, Fig. 1b).

A 353 bp sequence was assessed for average methylation frequency (AMF) in this study. This segment contains 23 CpG sites and includes a sequence of 92 bp upstream and 262 bp downstream of the transcription start site was presented (Fig. 2a) [12]. BSP shows the AMF of lung cancer cell lines as following: LK 47.83% (66/138), SPC 10.14% (14/138), H460 28.26% (39/138) and LTE 34.78% (48/138). Based on the AMF, LK and SPC were chosen as hypermethylated and hypomethylated cell lines for further studies, respectively (Fig. 2b, left column). BSP demonstrated that after 2Gy X-ray treatment (Fig. 2b, middle column), the AMF of the promoter regions of the Dab2 gene in LK cells was down-regulated significantly (P < 0.05), both at 24 h 16.16% (24/138) and 48 h 8% (11/138), compared to LK cells without X-ray treatment 47.83% (66/138). After 4Gy X-ray treatment, the AMF of the promoter regions of the Dab2 gene in LK cells was even more significantly down-regulated 7.97% (11/138) at 24 h and 2.9% (4/138) at 48 h, respectively (P < 0.05). We also identified down-regulation of methylation levels after irradiation in SPC cells, with 2.17% (3/138) at 24 h and 1.45% (2/138) at 48 h after 2Gy irradiation; and 2.17% (3/138) at 24 h and 0.72% (1/138) at 48 h after 4Gy irradiation (Fig. 2b, right column), compared to the non-irradiated SPC cells 10.14% (P < 0.05).

Generally, Dab2 mRNA level was up-regulated in hypermethylated LK cells but not in hypomethylated SPC cells (Fig. 3c, P < 0.05). Real-time PCR results demonstrated that the Dab2 mRNA level was significantly up-regulated at 24 h and 48 h after 2Gy and 4Gy irradiation in LK cells (Fig. 2c, P < 0.05). The result is similar with the change of AMF in LK cells; however, no increased in Dab2 mRNA was observed after X-ray treatment in SPC cells, and there was a slight decreased of the Dab2 mRNA level (Fig. 2c). These results indicate that X-ray irradiation could up-regulate Dab2 mRNA level in cells with hypermethylation of the Dab2 gene promoter, but not in cells with hypomethylation of the Dab2 gene promoter.

We examined the protein levels of DNMTs (including DNMT1, 3a and 3b) at 24 h after 4Gy X-ray irradiation in LK and SPC cell lines. We noted that DNMT1 and DNMT3b were both significantly down-regulated in each cell line (Fig. 3, P < 0.05), but DNMT3a was not down-regulated. It is known that methyl CpG binding protein 2 (MeCP2) binds to DNA methyl groups and recruits histone deacetylase (HDAC), resulting in histone deacetylation, chromatin condensation and consequently, transcriptional inactivation of genes. Therefore, we examined the expression of MeCP2 and demonstrated a decrease in MeCP2 protein in LK and SPC cells, (Fig. 3a and b, P < 0.05). These results indicate that X-ray irradiation may down-regulate DNMT1, DNMT3b and MeCP2, and thus lead to transcriptional activation of the Dab2 gene in hypermethylated lung cancer cells.

It has been reported that Dab2 can stabilize Axin and attenuate the classic Wnt/β-catenin signaling pathway. Axin is a negative regulator of the classic Wnt pathway, while β-catenin is the key positive regulator of the Wnt pathway. Cyclin D1 and MMP-7 are important downstream factors of the Wnt pathway which correlate with cell proliferation and invasion [10‐14, 17‐19, 27]. We noted that Dab2 was up-regulated in LK cells after X-ray treatment, but the expression of β-catenin, cyclin D1 and MMP-7 were down-regulated significantly (Fig. 4a and b, P < 0.05); Dab2 siRNA or Axin siRNA transfection were performed to identify whether X-ray induced Dab2 expression could inhibit the classic Wnt pathway. Combination of X-ray irradiation with Dab2 siRNA demonstrated only slight increased Dab2 expression while there was a slight decrease in Axin expression compared to X-ray irradiation alone. β-catenin, cyclin D1 and MMP-7 all demonstrated increased expression with combination of X-ray irradiation and Dab2 siRNA (Fig. 4a and b, P < 0.05). These results indicate that X-ray irradiation leads to the inhibition of the Wnt pathway via increased Dab2 expression in LK cells. Interestingly, a similar effect was observed with the combination of X-ray irradiation and Axin siRNA (Fig. 4a and b).

For SPC cells, X-ray irradiation showed almost no effect on the expression of Dab2 and other factors. Combination of X-ray irradiation and Dab2 siRNA lead to significantly decreased levels of Dab2 and Axin, while the expression of β-catenin, cyclin D1 and MMP-7 all significantly increased (Fig. 4c and d, P < 0.05). Combination of X-ray irradiation and Axin siRNA has almost no effect on Dab2 levels but lead to significantly decreased levels of Axin, while again the expression of β-catenin, cyclin D1 and MMP-7 all significantly increased (Fig. 4c and d, P < 0.05). These results indicate that X-ray irradiation in SPC cell lines does not induce up-regulation of Dab2, and that Dab2 knockdown leads to increased Wnt pathway signaling via the inhibition of Axin. Overall, the results in LK and SPC cells suggest that X-ray irradiation inhibits the classic Wnt pathway via increased expression of Dab2, which is probably the result of de-methylation of the Dab2 gene promoter due to the irradiation.

LK and SPC cells were treated with 4Gy X-ray irradiation, 4Gy X-ray irradiation and Dab2 knockdown, 4Gy X-ray irradiation and Axin knockdown, respectively, with non-irradiated cells serving as a control. For LK cells, colony formation rate was 68.89% ± 15.83% for the control group (Fig. 5a, a) and 7.2% ± 4.2% for X-ray irradiation alone (Fig. 5a, b), but combination of X-ray irradiation and Dab2 siRNA lead to reversal of the deceased colony formation rate observed with X-ray irradiation alone (54.11% ± 9.6%, Fig. 5a, c, P < 0.05). A similar effect was observed for the combination of X-ray irradiation and Axin siRNA (59.22% ± 14.73%, Fig. 5a, d, P < 0.05). In contrast, X-ray irradiation was less effective in SPC cells with the efficacy of colony formation being 65.67% ± 9.54% for the control group and 23.67% ± 8.51% for X-ray irradiation alone (Fig. 5a, e and f), respectively. Interestingly, more remarkable reduction of colony formation could be seen in LK cells than that in SPC cells after irradiation (P < 0.05). Combination of X-ray irradiation and Dab2 siRNA in SPC cells resulted in reversal of the deceased colony formation rate observed with X-ray irradiation alone (54.44% ± 15.74%, Fig. 5a, g), as well as the combination use of X-ray irradiation and Axin siRNA (53.78% ± 12.19%, Fig. 5a, h). These results suggest that X-ray irradiation significantly reduced the proliferation of lung cancer cells with hypermethylation of the Dab2 gene promoter, but is less effective in lung cancer cells with hypomethylation of the Dab2 gene promoter. Dab2 or Axin knockdown significantly decreased the ability of X-ray irradiation to reduce the proliferative activity of lung cancer cells.

We then assessed what effect X-ray irradiation has using the transwell cell invasive assay performed with LK and SPC cell lines and the same group classifications. After X-ray irradiation alone, the number of invasive cells of the LK cell line was significantly decreased compared to the control group (Fig. 5c a and b; 165 ± 30 VS 47 ± 10, P < 0.05), however, combination of X-ray irradiation and Dab2 siRNA or Axin siRNA reversed the effect (Fig. 5c c and d, 135 ± 40 and 139 ± 24, respectively). For the SPC cell line, X-ray irradiation alone also demonstrated a significant decrease in the number of invasive cells compared to the control group (Fig. 5c e and f, 172 ± 40 VS 83 ± 18, P < 0.05), but the effect was less than that of the LK cells (Fig. 5c b and f, and Fig. 5d b and f, P < 0.05). Combination of X-ray irradiation and Dab2 siRNA or Axin siRNA also reversed the effect in SPC cells (Fig. 5c, g and h, 144 ± 45 and 140 ± 52, P < 0.05). These results indicate that X-ray irradiation leads to decreased invasiveness in lung cancer cells with hypermethylation of the Dab2 gene promoter.

LK and SPC cells, after 4Gy X-ray irradiation, were inoculated into nude mice in the right axilla, respectively. Non-irradiated cells served as control group. After 2 weeks, tumor nodule formation was observed and their volumes were measured weekly thereafter. The xenograft tumors were completely excised at 5 weeks (Fig. 6a). The results showed a remarkable reduction of the average tumor volume after comparing non-irradiated LK cells with the 4Gy treatment group (Fig. 6a, a and b, 1.7 ± 0.26 cm³ VS 0.53 ± 0.15 cm³, P < 0.05), while in SPC cells, X-ray irradiation was less effective after comparing non-irradiated SPC cells with the 4Gy treatment group (Fig. 6a c and d, 1.56 ± 0.21 cm³ VS 1.05 ± 0.17 cm³, P < 0.05). The average weight of the tumors were markedly reduced in LK cells receiving X-ray irradiation, from 1.68 ± 0.41 g to 0.43 ± 0.07 g (Fig. 6b, P < 0.05); however, irradiation was less effective in weight reduction in the SPC xenograft tumors (from 1.57 ± 0.33 g to 0.89 ± 0.25 g, P < 0.05). Comparison of the reduction rate of tumor weight in LK and SPC cells with X-ray irradiation (74.4% VS 43.31%, Fig. 6b, P < 0.05), demonstrates that LK cells with hypermethylation of the Dab2 gene promoter are more sensitive to X-ray treatment than SPC cells with hypomethylation of the Dab2 gene promoter. The average tumor size of LK and LK with 4Gy irradiation treatment at 2, 3, 4 and 5 weeks showed that the growth of the LK cell line was significantly reduced (Fig. 6c, P < 0.05), and although X-ray treatment reduced the growth of SPC cells, the effect was less significant (Fig. 6d, P < 0.05). Western blot was performed to evaluate the expression of Dab2, Axin and other Wnt factors in LK and SPC xenograft tumors with or without X-ray treatment. The result showed that average Dab2 and Axin expression were significantly up-regulated in irradiated LK xenograft tumors comparing with un-treated LK xenograft tumors (Fig. 6e, P < 0.05), and the expression of β-catenin, c-myc, MMP-7 and cyclinD1 were down-regulated in LK xenograft tumors with irradiation (Fig. 6e, P < 0.05), but no significant change was identified in SPC xenograft tumors with or without irradiation (Fig. 6f). Overall, X-ray irradiation demonstrated suppression of tumor growth in both cell lines, although the extent of suppression in LK cells was much more prominent than in SPC cells.