125I seed irradiation induces up-regulation of the genes associated with apoptosis and cell cycle arrest and inhibits growth of gastric cancer xenografts

Animal model

The human NCI-N87 cells (3x10 ⁶/mouse) were subcutaneously injected into right dorsal flank of each BALB/c-nu/nu nude mouse. After 1–2 weeks of implantation with tumor cells, when tumors reached ~20-30 mm 3, the animals were randomized into control and treatment groups (24 animals per group). The 125I seeds (0.9 mCi) were injected into mice in treatment group through 18-gauge needles, while ghost seed were injected into the mice in control group.The tumor size was measured using calipers and the tumor volume was estimated by the formula: tumor volume (mm3) = (L x W 2) × 1/2, where L is the length and W is the width of the tumor. Tumor volumes and body weights were monitored every 3 days over the course of treatment. The tumor weight was measured when the mouse was sacrificed. Mice were sacrificed after 28 days of treatments and tumors were removed and fixed in 10% neutral buffered formalin for histologic and immunohistochemical analyses. All animal procedures were carried out with the approval of the Animal Ethics Committee of Kunming Medical College.

Histological analysis of tumors

Tumors were embedded in paraffin, sectioned at 5 μm, and stained with H&E (Sigma Aldrich, St. Louis, Missouri, USA). The mitotic index and apoptotic index were assessed by quantitative morphometric analysis of proliferating cell nuclear antigen (PCNA) expression and in situ terminal transferase-mediated fluorescein deoxy-UTP nick end labeling (TUNEL), two established markers of proliferation and apoptosis. For PCNA localization, formalin-fixed, paraffin embedded sections were incubated for 30 min with a mouse monoclonal anti-PCNA (Nova Castra Laboratories, Newcastle Upon Tyne, UK) at a 1:100 dilution. A peroxidase -conjugated antibody to mouse IgG (Abcam Inc., Cambridge, MA, USA) was applied followed by diaminobenzidine (Sigma Aldrich,St. Louis, Missouri, USA) to localize PCNA in the sections. DNA fragmentation was assessed by TUNEL, using the Apoptag Peroxidase In situ Apoptosis Detection Kit (Serologicals Corp., Norcross, Ga, USA). PCNA- or TUNEL-positive cells were quantified in 40 randomly selected high-power fields (x 200) of each tissue section.

RNA extraction

Total RNA was retracted from tumors using Trizol reagent (Life Technologies Inc., Gaithersburg, Maryland, USA) according to manufacturer’s instructions. Total RNA from each sample was quantified by the NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE, USA) and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA from one tumor from each mouse (6 tumors per group) was used for qRT-PCR analysis, whereas total RNA from tumors from four mice per group (12 tumors per group) was pooled for each microarray hybridization.

Microarray analysis

Microarray analysis of whole-genome gene expression profiling was performed using Human 12 x 135 K Gene Expression Array (Roche Applied Science, Indianapolis, IL, USA). Double-strand cDNA (ds-cDNA) was synthesized from 5 μg of total RNA using a SuperScript ds-cDNA synthesis kit (Life Technologies Inc., Gaithersburg, Maryland, USA) in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (Roche Applied Science, Indianapolis, IL, USA). Microarrays were then hybridized with Cy3 labeled ds-cDNA in a hybridization chamber (Roche Applied Science, Indianapolis, IL, USA). After hybridization and washing, the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA). Then, the data files were imported into Agilent GeneSpring Software (Agilent Technologies, Santa Clara, CA, USA) for analysis. NimbleScan software’s implementation of robust multichip average offers quantile normalization and background correction. The six gene summary files were imported into Agilent GeneSpring Software for further analysis. Genes that have values greater than or equal to lower cutoff of 50.0 in all samples were chosen for data analysis. The microarray experiment was independently repeated in triplicate for each sample group. Differentially expressed genes were identified through Fold-change and T-test screening. GO analysis and Pathway analysis were performed using the standard enrichment computation method.

Real-time polymerase chain reaction (PCR)

DNase-treated total RNA extracted from each tumor sample was reverse transcribed using the Transcriptor 1st Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). Real-time PCR was performed for quantitative analysis using SYBR green dye (TaKaRa, Tokyo, Japan) on the ABI-Prism 7900HT system (Applied Biosystems, Foster City, CA, USA) according to the protocols recommended by the manufacturer. Cycling parameters: pre-denaturation 1 min, 95°C; denaturation 15 s, 95°C; annealing 15 s, 60 °C; extension 45 s, 72°C, 40 cycles; final extension 5 min, 70°C. The fold change was calculated using the 2 -ΔΔCt method, presented as the fold-expression change in irradiated tumors relative to control tumors after normalization to the endogenous control, GAPDH. All experiments were carried out in triplicate technically. All primers are listed in Additional file 1: Table S1.

Methyl-DNA immunoprecipitation and microarray hybridization

Genomic DNA from tumors from six mice in the control group was pooled for Methyl-DNA immunoprecipitation (MeDIP) experiment. MeDIP was performed as described previously [12]. Briefly, Genomic DNA was sonicated to produce random fragments in size of 200–600 bp. Four micrograms of fragmented DNA was used for a standard MeDIP assay as described. After denaturation at 95°C for 10 min, immunoprecipitation was performed using 10 μg monoclonal antibody against 5-methylcytidine in a final volume of 500 μL IP buffer (10 mmol/L sodium phosphate, pH 7.0), 140 mmol/L NaCl, 0.05% Triton X-100) at 4°C for 2 h. Immunoprecipitated complexes were collected with Dynabeads Protein A and M-280 sheep anti-mouse IgG (Roche Diagnostics GmbH, Mannheim, Germany) at 4°C for 12 h, washed with 1 × IP buffer for 4 times, treated with Proteinase K at 50°C for 4 h, and purified by phenol-chloroform extraction and isopropanol precipitation. Immunoprecipitated methylated DNA was labeled with Cy5 fluorophere and the input genomic DNA was labeled with Cy3 fluorophere. Labeled DNA from the enriched and the input pools was combined (1–2 μg) and hybridized to a NimbleGen HG18 CpG promoter Array (Roche Diagnostics GmbH, Mannheim, Germany), which contained all well-characterized RefSeq promoter regions [from −800 bp to +200 bp transcription start sites (TSSs)]. Array was then washed and scanned with Axon GenePix 4000B microarray scanner. After normalization, raw data was input into SignalMap software (Roche Diagnostics GmbH, Mannheim, Germany) to observe and evaluate the methylation peaks. A customized peak-finding algorithm provided by NimbleGen was applied to analyze methylation data from MeDIP-microarray as previously described. The algorithm was used to perform the modified Kolmogorov-Smirnov test on several adjacent probes using sliding windows to predict enriched regions across the array.

MeDIP-quantitative PCR assay

A MeDIP assay, combined with qPCR, was used to evaluate quantitatively the methylation status of candidate genes in the tumors derived from the control and 125I treatment groups. MeDIP was performed as described above. Purified DNA from the immunoprecipitated DNA complexes and from input DNA was analyzed by qRT-PCR on an Applied Biosystems 7900 Real- Time PCR System. The experiment was performed in triplicate. The relative changes in the extent of gene methylation were determined by measuring the amount of detected genes in immunoprecipitated DNA after normalization to the input DNA. The primer sequences are listed in Additional file 1: Table S1.

Statistical analysis

The results of the animal experiments and real-time PCR were analyzed using SPSS 13.0 software. (SPSS Inc., Chicago, IL, USA) All data were plotted as mean ± standard deviation. Student’s t-test was used to compare values between two independent groups. Differences were considered to be significance when p < 0.05.

Effect of 125I seed irradiation on tumor morphology of gastric cancer

To investigate the effect of 125I irradiation on the histology of NCI-N87 xenografts, tumor sections taken from mice in the control and 125I treatment groups were stained with H&E. As shown in Figure 2, the histologic appearance of tumors in the control group was quite different from that in the 125I treatment group. In the control group, the cancer cells were densely arranged with large darkly-stained nuclei and obvious karyokinesis. In the treatment group, large necrotic regions were observed around the 125I seed. The cancer cells adjacent to the necrotic region were loosely arranged with condensed nuclei and reduced eosinophilic cytoplasm. These results indicated that 125I seed implantation caused growth inhibition of cancer cells in NCI-N87 xenografts.

Effect of 125I seed irradiation on cell apoptosis and mitosis of gastric cancer

To quantitatively compare the mitotic and apoptotic index of tumors treated with 125I seed irradiation, immunostainings for PCNA and TUNEL assays were performed. As shown in Figure 3A, the number of PCNA- positive cells in the 125I treatment group was obviously less than that of control group. And the mitotic index was significantly decreased in irradiated tumors as compared to the tumors in the control group Figure 3B). In contrast to the mitotic index, 125I-irradiated tumors showed increased numbers of apoptotic cells with condensed and irregularly shaped nuclei, staining positively for TUNEL Figure 3 C). the apoptotic index was significantly increased in the 125I treatment group as compared to the control group Figure 3D).

Identification of genes induced by 125I seed irradiation

To independently confirm the microarray results, real-time RT PCR was performed on samples from BALB/c mice that had been exposed to the same experimental conditions that were used in microarray assay. The relative expression levels of six genes—BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A—were assayed in irradiated and non-irradiated tumors. There was a close correlation between microarray data and qRT-PCR data Figure 4), indicating the accuracy of our microarray data and the significant induction in the expression of selected genes following irradiation.

Collectively, these data indicated that many critical molecules and pathways associated with apoptosis and cell cycle arrest were activated by 125I seed irradiation in NCI-N87 xenografts, thereby highlighting their important roles in 125I irradiation-induced inhibition of tumor growth.

DNA methylation analysis of 125I-irradiation induced genes

Aberrant DNA hypermethylation is commonly associated with cancer. The Dnmt1 DNA methyltransferase is responsible for maintenance of the DNA methylation pattern. Consistent with previous study [11], significant decrease of DNMT1 expression was observed in our array data, and this result was validated via the real time RT-PCR Figure 5A). These data suggest that DNA demethylation might be involved in 125I-induced tumor suppression. Because promoter demethylation is associated with gene re-activating, we focused our attention on the 125I irradiation-induced genes by coupling global gene expression and methylation profiles. The genes with promoter hypermethylation in the non-irradiated tumors were indentified with MeDIP-chip analysis (Additional file 5: Table S5). Among them, we identified 20 genes whose expression was significantly upregulated in the irradiated tumors as compared to the non-irradiated tumors (Table 2). Thus, we speculated that the expression levels of these 20 genes might be modulated via the promoter demethylation induced by 125I irradiation. Notably, several of these genes were associated with apoptosis or cell cycle arrest, such as BNIP3, WNT9A and GSG2. To confirm our hypothesis, methylation status of these three genes was examined with MeDIP-PCR assay in the treatment and control groups. As shown, BNIP3 and WNT9A in 125I treatment group displayed lower levels of methylation status compared with control group (P < 0.05), which decreased to 50.9% and 41.0%, respectively Figure 5B). Meanwhile, the expression levels of BNIP3 and WNT9A were significantly upregulated in the treatment group Figure 5 C). These data indicated that some apoptosis- and cell cycle-related genes could be activated by the demethylation of their promoters, which were induced by 125I seed irradiation.