Tangeretin (98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Tangeretin was dissolved in dimethylsulfoxide (DMSO) for all in vitro experiments. DMSO, RPMI-1640 medium, fetal calf serum (FBS), Tween-20, sodium dodecyl sulfate (SDS), phenylmethylsulphonyl fluoride (PMSF), trypsin and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Notch-1 siRNA and miR-410 mimics were obtained from Dharmacon (Chicago, IL, USA). The Notch-1 cDNA plasmid was purchased from OriBioGene Biological Inc. (Shanghai, China). Deionized water was purified by a Milli-Q water purification system (Millipore, Milford, MA, USA). All other reagents were of analytical grade and obtained from Nanjing Chemical Reagent Co. (Nanjing, China).

GC cell lines (MGC80-3, AGS, SGC7901 and MKN45) and normal gastric mucosa GES-1 cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and were cultured in RPMI-1640 supplemented with 10% FBS. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Radiosensitivity was examined by colony formation assay. Cells were exposed to increasing doses of irradiation (2, 4, 6 and 8 Gy) and tangeretin for 24 h. Irradiation was produced with a 4-MeV electron beam accelerator (Elekta, Sweden). After being washed with fresh medium, cells were allowed to grow for 14 days to form colonies, and cells were then fixed with 4% formaldehyde (Sigma). Fixed cells were then stained with 0.5% crystal violet (Sigma) for 15 min, and the colonies were counted. D0 values were calculated by a multitarget-single hit model.

Athymic male nude mice weighing ~20 g (SLARC Laboratory Animal Center, Shanghai, China) were used and maintained under standard pathogen-free conditions. SGC7901 cells were harvested and injected subcutaneously into the nude mice. After the onset of tumor development, tangeretin [30 mg/kg dissolved in 1 ml of phosphate-buffered saline (PBS) containing 0.1% DMSO/nude mice] was administered intraperitoneally for 3 weeks. After the mice were anesthetized, the tumors were exposed to radiation with 2 Gy 5 times a week for 3 weeks. After 3 weeks of administration, all mice were sacrificed and the tumors were subjected to further analysis. The numbers of lung metastatic lesions were counted under a microscope (Zeiss Axio Observer A1; Zeiss, Germany).

Cells were extracted in extraction buffer. Equal amounts of protein extracts were separated on 10% polyacrylamide gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Invitrogen, Carlsband, CA, USA) with a semi-dry blot system. After blocking, the membranes were incubated with each primary antibody (Nanjing Bioworld Biotech Co., Nanjing, China) and then with IgG.

After co-treatment with tangeretin (10 and 30 μM) and radiation (8 Gy) for 24 h, the cells were then placed in the upper chamber of a Transwell coated with BD Matrigel™. Medium with 10% FBS was placed in the lower chamber. After 18 h, the cells that had migrated to the lower surface were photographed and quantified in 5 fields at a magnification of ×100 under a microscope (Zeiss Axio Observer A1).

Cells were seeded in 24-well plates and grown until reaching confluency. In each well, a scratch was made with a 1-ml pipette tip. The cells were washed thrice with PBS to remove detached cells. Then the cells were treated with irradiation (8 Gy) and tangeretin (10 and 30 μM), and images of the wound area were captured at 0 and 18 h under a microscope (Zeiss Axio Observer A1).

After exposure to irradiation (8 Gy) for 24 h, immunofluorescence assay was performed to examine EMT biomarkers in the SGC7901 cells. After fixation and permeabilization, E-cadherin and N-cadherin were measured following staining with anti-E-cadherin and anti-N-cadherin antibodies. Nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma Chemical Co.). The stained cells were observed with a fluorescence inverted microscope (Zeiss Axio Observer A1).

Total RNA was harvested using miRNeasy Mini kit (Qiagen, Copenhagen, Denmark) according to the manufacturer’s instructions (23,24). The profiles of samples were generated using Agilent Human miRNA Microarray V3 (Agilent Technologies, Inc., Santa Clara, CA, USA). The scanned images were imported into GenePix Pro 6.0 software (Axon Instruments, Molecular Devices Corp., Foster City, CA, USA) for data extraction. Real-time PCR was carried out to confirm the miRNA expression profiling with U6 as the internal reference gene.

Data are expressed as the mean ± SD from 3 independent experiments. Group results were analyzed using one-way analysis of variance (ANOVA). Two groups were analyzed by the Student’s t-test. P-value <0.05 was considered to indicate a statistically significant result.

Cytotoxicity was detected by MTT assay. Cells were treated with tangeretin (0–100 μM) for 24 h. Tangeretin caused a cytotoxicity effect on the SGC7901 cells in a dose-dependent manner. Tangeretin did not affect cell viability until reaching concentrations of 30 μM (Fig. 1A). The non-cytotoxic concentrations used for the present study were identified, to ensure that the radiosensitizing effect of tangeretin was not caused by a direct cytotoxic effect.

A colony forming assay was carried out to explore whether tangeretin increased radiosensitivity in the SGC7901 cells. Cell survival curves measured by clonogenic survival assay are illustrated in Fig. 1B. Tangeretin considerably increased radiation-induced cell clonogenic death. D0 of the tangeretin plus radiation group was significantly increased (P=0.01), compared with the radiation alone group (Fig. 1C).

The loss of the epithelial phenotype marker, E-cadherin, is obvious in the EMT process. Features of the mesenchymal phenotype, such as augmented formation of pseudopodia and the appearance of spindle-shaped cells, were observed at 24 h post-irradiation (data not shown). To determine whether this alteration correlates with EMT, we examined EMT-specific markers by immunofluorescence assay and western blotting. As shown in Fig. 2A and B, radiation induced a decrease in E-cadherin and promoted the expression of the mesenchymal marker N-cadherin in the SGC7901 cells. Similar results were observed in the immunofluorescence assay (Fig. 2C). These data suggest that radiation induced EMT in the human GC SGC7901 cells.

Tangeretin suppressed radiation-induced EMT, as revealed by a reduced reduction in expression of vimentin and N-cadherin and an increase in expression of E-cadherin (Fig. 3A). The EMT process is also accompanied with enhanced cell invasion and migration. We further evaluated the invasive and migratory properties of SGC7901 cells via Matrigel Transwell and wound-healing assays. Radiation accelerated the rate of wound closure (Fig. 3B). Likewise, the acquisition of increased invasive ability induced by irradiation was prohibited by tangeretin treatment (Fig. 3C). These data indicate that tangeretin diminished the radiation-induced responses in SGC7901 cells.

Evidence has proven the essential role of the Notch-1 pathway in the EMT process (25). To uncover the molecular mechanism responsible for radiation-induced EMT, the expression of the Notch pathway in irradiated cells was examined. After treatment with tangeretin for 24 h, the upregulation of Notch-1, Jagged1/2, Hes-1 and Hey-1 expression levels in the irradiated cells was almost blocked. However, radiation or/and tangeretin co-incubation failed to influence the expression of Notch-2/3 (Fig. 4A). Moreover, western blot assays showed that there was no significant change in Notch-1 expression in the gastric mucosa GES-1 cells after exposure to irradiation (Fig. 4B).

After binding to Jagged or δ ligands, the Notch receptor is activated and goes through a succession of cleavages. The final cleavage depends on the γ-secretase protease complex, which contains 4 catalytic subunits such as PEN2, presenilin 1, APH1 and nicastrin (26), causing the translocation of Notch into the nucleus (27). We then tested the effect of tangeretin on the activation of Notch-1 signaling. Tangeretin did not influence the protein level of the 4 catalytic subunits, suggesting that tangeretin only suppressed the expression rather than the activation of Notch-1 (Fig. 4C).

To further demonstrate the essential role of Notch-1, the Notch-1 gene was knocked down by siRNA. Notch-1 siRNA transfection downregulated the protein levels of Notch-1, Hey-1, Hes-1, Snail1 and Twist1 in the irradiated SGC7901 cells, accompanied with a decline in invasive ability. After 24 h post-transfection, tangeretin treatment inhibited both Notch-1 activity and cell invasion to a more significant degree in the irradiated SGC7901 cells than tangeretin treatment alone (Fig. 5A and B). We then explored the relationship between tangeretin and Notch-1 with the overexpression plasmid of Notch-1. Overexpression of Notch-1 overrode the inhibitory effect of tangeretin on EMT (Fig. 5C). Accordingly, we hypothesized that tangeretin prevented EMT probably via suppressing Notch-1 expression.

Radiation regulates miRNA expression, and in turn affects tumor progression. As demonstrated here, in SGC7901 cells exposed to tangeretin, enhanced expression of miR-143, miR-200c and miR-410 was noted when compared to the control cells. Moreover, the relative change in miR-410 expression was greater in the tangeretin plus irradiation group, than the radiation alone group (Fig. 6A). In addition, tangeretin promoted the expression of miR-410 and miR-503 in tumor tissue lysates from nude mice (Fig. 6B). These results demonstrated that tangeretin elevated miR-410 expression both in vitro and in vivo.

We then compared miR-410 expression between GC cell lines (MGC80-3, AGS, SGC7901 and MKN45) and the normal gastric mucosa GES-1 cells. miR-410 expression was reduced in the GC cell lines compared to the GES-1 cells. MGC80-3 and AGS cell lines were derived from non-metastatic tissues, and SGC7901 and MKN45 cell lines were from metastatic tissues. Expression of miR-410 was higher in the MGC80-3 and AGS cells than those derived from metastatic tissues (P<0.05, Fig. 7A).

Since miRNAs are crucial modulators of EMT and Notch-1 signaling, we further investigated the effect of miR-410 on the EMT of GC cells subjected to radiation. Enhanced miR-410 expression and weakened Notch-1 expression were observed in the irradiated-SGC7901 cells after miR-410 mimics were transfected into the cells (Fig. 7B). Overexpression of miR-410 also reduced EMT-specific marker expression and invasion in the irradiated cells, indicating that miR-410 is closely associated with EMT (Fig. 7C). In addition, miR-410 expression was upregulated after the knockdown of Notch-1 by siRNA in the irradiated SGC7901 cells, and these functions were similar to the effect by tangeretin (Fig. 7D). In summary, miR-410 plays an essential role in the biologic function of tangeretin in EMT.

Body weight partly reflects a healthy condition. A decrease in body weight was observed in the group exposed to radiation for 3 weeks. Tangeretin considerably alleviated radiation-induced weight loss in nude mice after 3 weeks of administration. During the period of administration, mice in the tangeretin + radiation group were in better physical condition and exhibited higher weight gain, compared with the radiation group (Fig. 8A).

Following treatment with tangeretin + radiation for 3 weeks, the tumor sizes were smallest among all the groups at the observation endpoint (Fig. 8B). The incidence of pulmonary metastasis in the radiotherapy and control groups was 100 and 50% (6/6 vs. 3/6, P<0.05; Fig. 8C). Compared with the radiation group, the tangeretin + radiation group had considerably attenuated lung metastases, with metastatic rates of 16.67% (1/6 vs. 6/6, P<0.05).