The human GC cell lines SGC-7901 and MNK-45 were obtained from the Cell Collection of the Chinese Academy of Sciences (Shanghai, China). These two cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a 5% CO₂ incubator. BaP (Sigma-Aldrich, cat no. B1760), resveratrol (RSV; Sigma-Aldrich, cat no. V90038), ERK1/2 inhibitor U0126 (Cell Signaling, cat no. 9903S) were dissolved in dimethyl sulfoxide (DMSO) and diluted with RPMI-1640 to obtain the desired concentration. Final DMSO concentrations in cultures were ≤0.1%.

The cell proliferation was measured using Cell Counting Kit-8 (CCK-8) assay (Dojindo). Cells were exposed to various concentrations of BaP (i.e., 0.05, 0.1, 0.5, 1, 5 μM) and the vehicle (DMSO) for 48 h in 96-well plates. After treatment, 10 μl of CCK-8 was added to the medium and the cells were incubated for another 3 h. The optical density was measured at 450 nm using a microplate reader (Bio-Tek, USA).

Migration assay, SGC-7901 and MNK-45 cells in serum-free medium were plated in the upper compartment of 24-well Transwell hanging cell culture insert with 8-μm micro-porous membranes (Millipore) in 24-well plates. The lower compartments of the plates were filled with RPMI-1640 containing 10% FBS. After incubated for 16 h with BaP, cells on the upper surface of the membrane were removed. Cells that migrated to the lower surface were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet. The stained cells were counted under a microscope.

The invasion assay was performed in a same way as the migration assays, except that the Transwell inserts were coated with Matrigel mixture (BD Biosciences) prior to plating of the cells and the cells were incubated for 48 h.

Total RNA was extracted from cells with TRIzol reagent (Takara, cat no. 9108). RNA concentration and purity were measured with microplate reader at A260 and A260/280, respectively. The Prime-Script™ RT-PCR kit (Takara, cat no. RR047A) was used to reverse-transcribe RNA into cDNA. qRT-PCR amplifications were performed with the Applied Biosystems 7500/7500 Fast Real-Time PCR Software (Applied Biosystems) using the SYBR^® Premix Ex Taq™ II (cat no. RR820A). Reactions were carried out in a 20-μl reaction volume following the manufacturer's instructions. The thermal cycle conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. GAPDH was used as an internal control. The primer sequences were: CYP1A1, forward, 5′-CCATGTCGGCCACGGAGTT-3′ and reverse, 5′-ACAGTGCCAGGTGCGGGTT-3′; MMP9, forward, 5′-ACG CACGACGTCTTCCAGTA-3′, and reverse, 5′-CCACCTGG TTCAACTCACTCC-3′ ; C-myc, forward, 5′-TACAACACCCG AGCAAGGAC-3′ and reverse, 5′-AGCTAACGTTGAGGG GCATC-3′; GAPDH: forward, 5′-GCACCGTCAAGGCTGA GAAC-3′, and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The data were gathered on ABI 7500 One-Step Plus system.

Western blot analyses were conducted as previously described (31). After every specific treatment, cells were washed with precooled PBS and lysed on ice with 1 mM phenylmethanesulfonyl fluoride and RIPA buffer (Beyotime, cat no. P0033) containing protease inhibitors phosphatase inhibitors for the extraction of cellular protein. The supernatant was collected and stored in aliquots at −80°C until analysis by western blot analysis. The protein concentration was measured by BCA protein assay system, protein samples were loaded into each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidenefluoride (PVDF) membranes. The blots were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies against CYP1A1 (Abcam ab126828, 1:1,000), pERK (Abcam ab76165, 1:500), ERK (Abcam ab36991, 1:2,000), MMP9 (Abcam ab137867, 1:1,000) and c-myc (Abcam ab32072, 1:10,000). GAPDH (Bioworld Ap0063, 1:5,000) was used as a loading control. After being washed in TBST three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room temperature. The specific protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate and imaged using a VersaDoc imaging system (Bio-Rad).

The data are shown as the means ± standard deviation (SD). Statistical analysis was performed using the SPSS 19.0 (IBM, Armonk, NY, USA), and P<0.05 was considered to indicate statistically significant differences. The data were analyzed using ANOVA followed by Dunnett or Bonferroni t-test for multiple comparisons.

Cancer cell proliferation and metastasis are key indices of GC advancement and the major cause of death from the disease (32–34). Therefore, we explored whether BaP promotes the proliferation and metastasis of GC cells. After being treated with BaP, SGC-7901 and MNK-45 cells reached a faster growth rate as demonstrated by the CCK-8 cell proliferation assay (Fig. 1), The proliferation rate increased with higher dose of BaP, showing a dose-dependent effect of BaP on GC cell growth.

We also assessed the effects of BaP exposure on GC cell migration and invasion, the essential activities that allow tumor cells to spread to multiple organs through blood and lymph circulation. Transwell migration assay showed cell numbers were significantly increased on the lower surface in BaP-exposed SGC-7901 and MNK-45 cells, which indicated cell migration was enhanced by BaP treatment (Fig. 2). Similarly, BaP-treated SGC-7901 and MNK-45 cells also showed an increased ability of invasion (Fig. 3).

MMP9 is one of the type IV collagenase/gelatinases that can degrade extracellular matrix (ECM) components and is widely associated with tumor invasion and metastasis (35). A previous study suggested that BaP induced MMP9 expression in breast cancer cells (28). To determine if BaP has the same effect on MMP9 expression in GC cells, we examined the expression of MMP9 in BaP-treated SGC-7901 and MNK-45 cells using RT-PCR and western blotting. Similar to previous reports on breast cancer (28), our results clearly showed that BaP treatment enhanced MMP9 expression in a dose-dependent manner in GC cells (Fig. 4A and C).

C-myc, a classic oncogene, plays an important role in cancer initiation and maintenance (36). When activated in GC and other cancer cells, c-myc stimulates unlimited proliferation and growth of these cells (37–41). We therefore investigated whether c-myc is induced by BaP treatment in GC cells. As shown in Fig. 4B and 4C, the expression of c-myc was increased following BaP exposure at the mRNA and protein levels.

Previous studies have confirmed that BaP is an exogenous ligand of AhR (17), and in GC cells the AhR pathway can be activated by another AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (42). To verify whether AhR pathway can also be activated by BaP in GC cell lines, the expression of a classic target gene of AhR pathway, cytochrome P450 1A1 (CYP1A1), was detected by RT-PCR and western blotting (43). As shown in Fig. 5A, the expression of CYP1A1 mRNA and protein were elevated in both SGC-7901 and MNK-45 cell lines after BaP treatment. In addition, to clarify whether this BaP-induced CYP1A1 expression is AhR-dependent, AhR pathway was inhibited by an AhR antagonist RSV (42,44). As shown in Fig. 5B, RSV could reverse BaP-induced CYP1A1 expression. These data indicated that BaP can activate AhR pathway in GC cells.

The ERK pathway was also demonstrated to take part in BaP-induced cancer development (28). Western blot analysis showed that pERK protein expression in SGC-7901 and MNK-45 cells were induced by BaP treatment (Fig. 6A). Furthermore, U0126, a known ERK inhibitor, significantly blocked BaP-induced ERK activation (Fig. 6B).

To date, little is known about the impact of BaP on intracellular signal transduction in GC. As shown above, we demonstrated that BaP promoted the expression of MMP9 and c-myc and activated AhR and ERK pathways. Thus, we explored the potential relationship between the two oncogenes and these activated pathways. To elucidate whether the AhR activation really linked to MMP9 and c-myc overexpression, AhR antagonist RSV was used to block the AhR pathway. In the presence of RSV, the overexpression of MMP9 and c-myc induced by BaP were attenuated in SGC-7901 and MNK-45 cells (Fig. 7A, B, E), indicating that AhR pathway mediated the upregulation of MMP9 and c-myc expression by BaP.

In addition, we investigated the role of ERK pathway in the regulation of MMP9 and c-myc expression in BaP-treated GC cells. As shown in Fig. 7C, D and E, inhibition of ERK signaling by U0126 significantly attenuated BaP-induced MMP9 and c-myc expression at the mRNA and protein level. Therefore, the ERK pathway appears to participate in mediation of MMP9 and c-myc expression.