Two human gastric cancer cell lines, AGS and NCI-N87, obtained from the American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator with 5% CO₂ and 95% air at 37°C. For treatment, Tax was obtained from Shanghai Huicheng Technology, Ltd. The AGS and NCI-N87 cells were treated with increasing concentrations of Tax (1, 3, 10, 30 and 100 µM) for 48 h. In addition, the aryl hydrocarbon receptor (AhR) agonist SB203580 (10 µM; Sigma-Aldrich; Merck KGaA) was applied for further treatment.

Cell viability was assessed using a CCK-8 assay (Beyotime Institute of Biotechnology). The AGS and NCI-N87 cells were cultured in 96-well plates (1×10⁴ cells/well) for 24 h, and were then treated with various concentrations of Tax (1, 3, 10, 30 and 100 µM) for a further 48 h. Then, 10 µl CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 3 h. The absorbance at 450 nm was detected using a microplate reader (ELx800; BioTek Instruments, Inc.). The results are presented as a relative percentage of the untreated control cells.

The cell proliferative ability was assessed using a colony formation assay. Cells were seeded into six-well plates at a density of 500 cells/well. After being subjected to the Tax (100 µM) treatment with or without SB203580 (10 µM) treatment, cells were incubated in a 5% CO₂ incubator at 37°C for 2 weeks. Thereafter, the cells were fixed with methanol for 10 min at room temperature and stained with 0.5% crystal violet for a further 10 min at room temperature. Images were captured under a light microscope (magnification, ×10), and colonies containing >50 cells were counted.

The cell migratory ability was assessed using a wound-healing assay. The cells were re-suspended with serum-free medium and added to 24-well plates for a 24-h incubation at 37°C. Upon reaching 100% confluency, a scratch was subsequently generated in the cell monolayer using a sterile micropipette tip. The cells were then incubated in serum-free medium containing Tax (100 µM) with or without SB203580 (10 µM) at 37°C for 24 h. The wound width at 0 and 24 h was captured using a light microscope (magnification, ×100).

The cell invasive ability was assessed using a Transwell assay with a 24-well Transwell plate with pore size of 8-µm (EMD Millipore) precoated with BD Matrigel (BD Biosciences) at 37°C for 1 h. The cells were suspended in serum-free medium and added to the upper chamber (3×10⁴ cells/well) of the 24-well Transwell plate, followed by a 24-h incubation of Tax (100 µM) with or without SB203580 (10 µM). Complete medium containing 10% FBS was added to the lower chamber. Following 24 h of incubation at 37°C, the non-invasive cells were removed using cotton swabs, and the invasive cells were fixed with 100% methanol for 10 min and stained with 0.1% crystal violet for 20 min at room temperature. Images were captured under a light microscope (magnification, ×100).

Cells were lysed using RIPA lysis (Wuhan Boster Biological Technology, Ltd.) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). A BCA assay was used to determine the protein concentration. Equal amounts of protein (30 µg/lane) were separated by a 12% SDS-PAGE gel and transferred to PVDF membranes (EMD Millipore). Subsequently, the membranes were blocked in 5% skimmed milk for 2 h at room temperature, and then incubated with corresponding primary antibodies against matrix metalloproteinase (MMP)2 (1:1,000; product code ab92536), MMP9 (1:1,000; product code ab38898), E-cadherin (1:1,000; product code ab231303), Zonula occludens-1 (ZO-1; 1:1,000; product code ab96587), N-cadherin (1:5,000; product code ab76011), Snail (1:1,000; product code ab180714), AhR (1:1,000; product code ab108518), cytochrome P450 1A1 (CYP1A1; 1:1,000; product code ab126887), Ki67 (1:1,000; product code ab16667), proliferating cell nuclear antigen, (PCNA; 1:1,000; product code ab92552) and GAPDH (1:1,000; product code ab8245) from Abcam at 4°C overnight. On the second day, the membranes were washed three times and incubated with HRP-conjugated goat anti-mouse (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or goat anti-rabbit (1:2,000; product code ab97051; Abcam) antibodies for 2 h at room temperature. Bands were exposed by an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology) and analyzed using ImageJ software version 1.50 (National Institutes of Health).

A total of 24 male 6-week-old BALB/c null nude mice (22±2 g) were obtained from HFK Bioscience Co., Ltd. and housed at the Animal Care Facility of West China Hospital, Sichuan University (Chengdu, China) under a controlled temperature (22±°C) and humidity (55±5%), with a 12-h light/dark cycle and free access to water and food. Prior to the operation, all mice were acclimatized for 1 week. Tumor xenografts in mice were established by injecting 1×10⁶ AGS or NCI-N87 cells subcutaneously into the right flank region. After 5 days, the mice were randomly assigned into two groups (n=6 for each group) and intraperitoneally injected with 25 mg/kg Tax twice weekly or an equal volume of saline, respectively. During this period, the tumor size and body weight of the mice were observed and recorded every 3 days. The allowed maximum diameter of the tumors was 1.5 cm. At the end of the experiment (the 21st day), all the 24 mice were sacrificed by cervical dislocation under deep anesthesia (sodium pentobarbital intraperitoneal injection, 50 mg/kg). After the cessation of the heartbeat and respiratory arrest of the mice was confirmed, the tumors were collected for measuring the weight and size, and frozen at -80°C for use in subsequent western blot analysis. All animal experiments were performed in accordance with the Care and Use of Laboratory Animals established by the US National Institutes of Health (10), and were approved by the Ethics Committee of West China Hospital, Sichuan University (approval no. 2021-05).

The tumor specimens were dissected and fixed in 4% paraformaldehyde at 37°C for 48 h. Then, the tissues were paraffin-embedded, and sectioned into 4-µm-thick slices. The slices were deparaffinized, rehydrated and subjected to antigen retrieval. Subsequently, the slices were blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min, and incubated with anti-Ki67 antibody (1:200; product code ab16667; Abcam) at 4°C overnight. After washing with PBS, the slices were incubated with HRP-conjugated goat anti-rabbit antibody (1:1,000; product code ab97051; Abcam). Slices were counterstained with hematoxylin for 2 min at room temperature and visualized with DAB (ZSJQ-BIO) under a light microscope (magnification, ×200).

SPSS 17.0 software (SPSS, Inc.) was used for statistical analysis and data are presented as the mean ± SD. All data were determined from at least three independent experiments. A Student's unpaired t-test was performed for comparisons between two groups, and a one-way AVONA followed by a Tukey's post hoc test was performed for comparisons among more than two groups. P<0.05 was considered to indicate a statistically significant difference.

To examine the antitumor effects of Tax, two gastric cancer cell lines, AGS and NCI-N87, were treated with various concentrations of Tax (1, 3, 10, 30 and 100 µM). It was observed that treatment of the gastric cancer cells with Tax suppressed cell viability in a concentration-dependent manner (Fig. 1A and B). In subsequent experiments, 100 µM Tax was selected to treat the AGS and NCI-N87 cells. A colony formation assay was performed to detect the changes in colony formation following Tax treatment. As revealed in Fig. 1C, Tax treatment decreased the number of cell colonies formed compared with the control group in both AGS and NCI-N87 cells. These results indicated that Tax had the ability to hinder cell proliferation.

After demonstrating the inhibitory effects of Tax on the cell proliferative ability, the present study then determined whether Tax suppressed the cell migratory and invasive abilities. In a wound-healing assay, Tax treatment led to a lower 'healing' ability at 24 h in both the AGS and NCI-N87 cells. In addition, a Transwell assay revealed that Tax hindered the invasive ability of not only the AGS cells, but also the NCI-N87 cells (Fig. 2A-C). Moreover, the downregulated protein expression levels of MMP2 and MMP9 upon Tax treatment further demonstrated the role of Tax in gastric cancer (Fig. 2D). Furthermore, Tax treatment increased the protein expression levels of E-cadherin and ZO-1, and reduced the protein expression levels of N-cadherin and Snail (Fig. 2E), indicating a potential inhibitory role of Tax in epithelial-mesenchymal transition (EMT) in gastric cancer. Therefore, these results indicated that Tax impeded the migratory and invasive abilities of gastric cancer cells by regulating EMT.

To elucidate the potential mechanisms underlying the protective role of Tax in gastric cancer, the effects of Tax on the AhR/CYP1A1 signaling pathway were examined. As revealed in Fig. 3A, Tax treatment significantly decreased the protein expression levels of AhR and CYP1A1 in AGS cells. A similar trend was observed in NCI-N87 cells (Fig. 3B). Therefore, these results indicated that Tax suppressed the activation of the AhR/CYP1A1 signaling pathway.

Subsequently, to identify whether the protective role of Tax in gastric cancer was mediated via AhR/CYP1A1 signaling, the effects of the AhR agonist, SB203580, on Tax-treated gastric cancer cells were examined. Firstly, SB203580 was revealed to increase the protein expression levels of AhR and CYP1A1 in Tax-treated AGS or NCI-N87 cells (Fig. 4A and B). Subsequently, a series of cell biological behaviors were examined, as aforementioned. On the one hand, the addition of SB203580 increased the viability of AGS and NCI-N87 cells, and increased the colony formation number compared with Tax treatment alone (Fig. 4C-E). On the other hand, the hindered migratory and invasive abilities of Tax were partly restored by SB203580, which was further verified by the upregulated protein expression levels of MMP2 and MMP9 in the SB203580 + Tax group (Fig. 5A-D). Furthermore, SB203580 diminished the expression levels of E-cadherin and ZO-1, whereas it elevated the expression levels of N-cadherin and Snail in Tax-treated AGS and NCI-N87 cells (Fig. 5E). Therefore, these results indicated that the inhibition of AhR/CYP1A1 signaling partly attenuated the effects of Tax on gastric cancer cells.

Finally, the antitumor effects of Tax were further examined in vivo. A mouse tumor model was established by injecting AGS or NCI-N87 cells subcutaneously into the right flank region. Following sacrifice, the tumors were removed and weighed. As revealed in Fig. 6A and B, compared with the controls, the size and weight of tumors from mice injected with AGS cells and treated with Tax were significantly decreased. During the process of tumor growth, tumor size was recorded every 3 days. The curve presented in Fig. 6C illustrates a continuous inhibition of tumor size by Tax treatment. The body weights of mice were also monitored every three days, but there were no significant differences between these two groups (Fig. 6D). Similar results were obtained from mice injected with NCI-N87 cells (Fig. 6E-H). These results demonstrated that Tax suppressed the growth of gastric cancer in vivo. In addition, immunohistochemical analysis revealed that Tax treatment greatly reduced the expression level of Ki67 of tumor tissues from mice injected with AGS or NCI-N87 cells (Fig. 7A and B). The protein expression levels of Ki67, PCNA, MMP2 and MMP9 in the tumor tissues were markedly reduced by Tax treatment (Fig. 7C and D). Concurrently, the protein expression levels of E-cadherin and ZO-1 were upregulated, whereas the protein expression levels of N-cadherin and Snail were downregulated by Tax treatment (Fig. 7C and D); these results were consistent with those obtained in vitro. Furthermore, AhR/CYP1A1 signaling was reduced, as evidenced by the significantly downregulated protein expression levels of AhR and CYP1A1 following Tax treatment (Fig. 7E and F). Therefore, the in vivo experiments further demonstrated the antitumor activity of Tax in gastric cancer and its potential mechanisms of action.