Epidemiological studies indicate that NOCs are positively associated with stomach cancer, therefore, the human gastric cancer SGC7901 cell line was used in the present study. The SGC7901 cell line was established from an untreated patient with progressive adenocarcinoma of the stomach. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. The SGC7901 cells stably transfected with the vector and BRCA2 siRNA (siBRCA2) were cultured in DMEM containing 200 μg/ml of G418 (Invitrogen, Carlsbad, CA, USA). The NDEA, NDELA, NDPA and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). All chemicals and solvents were of the highest grade commercially available. The NOCs were dissolved in sterile DMSO (0.1%) and freshly prepared each time prior to use. Logarithmically growing SGC7901 cells were treated with NOCs at appropriate concentrations where indicated.

The comet assay (Trevigen, Inc., Gaithersburg, MD, USA) was performed as described previously, using neutral conditions to detect the DSBs (33). In brief, the cells were harvested, washed with ice-cold PBS and combined with molten LMP agarose, then 75 μl (500–1,000 cells) was immediately added to the comet slide. Subsequent to being hardened, the slides were incubated for 30 min in lysis solution at 4°C, then rinsed with 1X Tris/borate/EDTA prior to electrophoresis for 60 min at 30 V. The slides were rinsed with distilled H₂O, placed in 70% ethanol for 10 min and then air-dried. To visualize the DNA, 50 μl of a 1:1,000 dilution of SYBR Green (Molecular Probes, Invitrogen) in PBS was added to each slide. The slides were visually scored using fluorescence microscopy (Leica DMS 4000B; Leica, Mannheim, Germany). The comet tail to head ratios or tail lengths were determined using the software package ‘Comet Assay II’ (Perceptive Instruments, Haverhill, Suffolk, UK). A minimum of 50 cells per experiment were analyzed. All the experiments were performed at least three times independently and in triplicate.

The cells were harvested and lysed with a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl₂, 100 μg/ml phenylmethylsulfonyl fluoride and 1% Triton X-100 for 30 min on ice. Total cellular extracts (50 μg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with specific primary antibodies, followed by incubation with IRDye680-conjugated secondary antibodies (Rockland, Inc., Gilbertsville, PA, USA). Detection was performed using an Odyssey IR imaging system (LI-COR Biotechnology, Lincoln, NE, USA). The following antibodies were used for the immunoblotting studies: mouse anti-γ-H2AX (serine 139; Upstate, Charlottesville, VA, USA), mouse anti-BRCA2 (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-RAD51 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin (Sigma).

The cells were plated onto coverslips and treated with 1X IC₅₀ concentrations of NDEA, NDELA and NDPA (0.76, 1.09 and 0.39 mM, respectively) for 1 h. The cells were then fixed and stained with monoclonal anti-γ-H2AX (Upstate). Subsequent to being stained with Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (Invitrogen), the slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) containing 5 ng/ml 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). The staining images were captured using fluorescence microscopy (Leica DMS 4000B) and a Spot digital camera (Spot Imaging Solutions, Sterling Heights, MI, USA).

To determine the cytotoxicity and IC₅₀ concentrations of NDEA, NDELA and NDPA, a clonogenic survival assay was performed on 60-mm cell culture dishes as described previously (34). The SGC-7901 cells were treated with various concentrations of NDEA, NDELA or NDPA for 1 h, followed by drug-free incubation for 10 days. The colonies were stained with crystal violet and counted if ≥50 cells were present. The IC₅₀ concentration was calculated as the concentration of NDEA, NDELA or NDPA that killed 50% of the untreated control colonies.

BRCA2 siRNA (SiBRCA2) oligos containing the target sequences of 5′-AAGACACGCTGCAACAAAGCA-3′ were designed and synthesized. Following annealing, the double-stranded siBRCA2 fragment was inserted into a pSilencer 2.1-U6-neo vector (Ambion, Austin, TX, USA) and transfected into the SGC7901 cells with Lipofectamine-2000 (Invitrogen). The pSilencer 2.1-U6-neo vector containing a scrambled sequence (Ambion) was transfected as a nonspecific control. Stable cell lines were established by performing selection in a medium containing G418.

Images of 50 randomly selected cells were evaluated per treatment and the test was performed three times. The Student’s t-test was used to provide the statistical comparisons and P<0.05 was considered to indicate a statistically significant difference.

First, the effects of NDEA, NDELA and NDPA on the DNA damage in the SGC7901 cells were examined. DNA damage was evaluated using the comet assay under neutral electrophoresis conditions to predominantly detect the DSBs. Logarithmically growing SGC-7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 15, 30 and 60 min. The comet assays revealed that NDEA, NDELA and NDPA induced apparent DNA damage in the SGC7901 cells as evidenced by the presence of DNA comet tails (Fig. 1A). Analysis of the tail to head ratio, which reflected the degree of DNA damage in the cells, showed that each of the three compounds induced a time-dependent increase in the extent of the DNA damage (Fig. 1B).

Upon the induction of a DSB, the histone variant, H2AX, is rapidly phosphorylated (γ-H2AX) and forms discrete nuclear foci. γ-H2AX foci formation also allows the sensitive detection of DSBs (16,18,35,36). To further confirm that NOCs induce the generation of DSBs, the SGC-7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 1 h and then immunofluorescently stained using γ-H2AX antibodies. An examination of the results showed that the NOC treatment led to the formation of γ-H2AX (Fig. 1C), thus indicating the presence of DSBs. Immunoblotting analysis further demonstrated that NDEA, NDELA and NDPA induced the expression of γ-H2AX in a time-dependent manner (Fig. 1D).

The comet assay and the data on the expression of γ-H2AX demonstrated that the three compounds, NDEA, NDELA and NDPA, were able to induce a time-dependent increase in the extent of DNA damage. These observations indicated that these three NOCs similarly induced the generation of DSBs. As with the varying carcinogenic potentials, it was unclear whether these NOCs induced similar DNA repair. We hypothesized that the repair mechanism of these NOC-induced DSBs may be different. To examine this hypothesis, the SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA (0.76 mM), NDELA (1.09 mM) or NDPA (0.39 mM) for 1 h, followed by a 12-h drug-free incubation to allow DNA repair. The formation and resolution of the DNA damage were then analyzed using the comet assay. Following a drug-free incubation of 12 h, the tail to head ratio of the comet DNA in the cells treated with NDELA (P<0.05) or NDPA (P<0.01) was observed to be significantly reduced (Fig. 2A and B). However, there was no apparent reduction in the tail to head ratio of the comet DNA in the cells treated with NDEA (P>0.05). The levels of γ-H2AX expression were also studied and it was observed that following a drug-free incubation of 12 h, there was a significant reduction in the expression of γ-H2AX in the SGC7901 cells treated with NDELA or NDPA (Fig. 2C). However, there was no clear change in the γ-H2AX levels in the cells treated with NDEA. These observations suggested that the DSBs induced by NDELA or NDPA were more effectively repaired than those induced by NDEA.

In order to determine whether HR was involved in the repair of NOC-induced DSBs, the expression levels of BRCA2 and RAD51, two key proteins in HR (4,8,20), were observed in response to the NOC treatment. The SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA, NDELA and NDPA for 15 and 60 min. The expression of BRCA2 and RAD51 was reduced in the SGC7901 cells treated with NDEA (Fig. 3A). However, the expression levels of BRCA2 and RAD51 were notably upregulated in a time-dependent manner in the cells treated with NDELA or NDPA (Fig. 3A). The results suggest that the BRCA2-RAD51-mediated HR pathway may be significant in the DNA damage repair induced by NDELA and NDPA.

The role of BRCA2 in the repair of NOC-induced DNA damage was further investigated. A BRCA2-targeted siRNA was designed (as described in the Materials and methods) and stably transfected into the SGC7901 cells. The effectiveness of the siBRCA2 construct in knocking down the endogenous BRCA2 level was demonstrated by western blotting. As shown in Fig. 3B, the expression of BRCA2 was effectively knocked down using BRCA2 siRNA transfected into the SGC7901 cells. The control group consisted of SGC7901 cells stably transfected with the control vector plasmid. The vector- and siBRCA2-transfected SGC7901 cells were treated with 1X IC₅₀ concentrations of NDEA, NDELA or NDPA for 1 h, followed by a 12-h drug-free incubation. Similar to the cells treated with NDEA (Fig. 3C), the level of γ-H2AX expression in the BRCA2-knockdown cells treated with NDELA (Fig. 3D) or NDPA (Fig. 3E) was not reduced to the level of the vector cells following the drug-free incubation. The results showed that in the BRCA2-knockdown cells, the DNA damage induced by NDELA or NDPA was not repaired as effectively as in the vector cells. This also suggested that BRCA2 contributes to the variations in DNA repair in response to these NOC-induced DSBs.

Based on the previous observations that BRCA2 is significant for the repair of DNA damage induced by NOCs, we hypothesized that BRCA2 may affect the sensitivity to NOCs. To determine whether BRCA2 affects the sensitivity to NDEA, NDELA and NDPA, a clonogenic survival assay was performed. The vector- and siBRCA2-transfected SGC7901 cells were treated with various concentrations of NDEA, NDELA or NDPA for 1 h, followed by drug-free incubations. When the IC₅₀ concentrations were compared, the results showed that the cells with the endogenous knock down of BRCA2, caused by RNA interference, exhibited a >2-fold increase in sensitivity to NDELA (Fig. 4B) or NDPA (Fig. 4C) compared with the vector cells. No significant change was observed in the sensitivity to NDEA (Fig. 4A). These results show that BRCA2 confers sensitivity to NOCs.