Silencing of insulin-like growth factor-1 receptor enhances the radiation sensitivity of human esophageal squamous cell carcinoma in vitro and in vivo

The human esophageal cancer cell lines Eca-109 and TE-1 were obtained from the American Type Culture Collection (Manassas, Virginia, United States). The cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin (Sigma-Aldrich, St. Louis, Missouri, United States). The cells were passaged every two to three days to maintain exponential growth prior to experimental usage and were maintained in 5% CO₂ at 37°C.

Eca-109 and TE-1 cells were transfected with 100 nM IGF-1r siRNA or a negative control vector (Qiagen, Lafayette, Colorado, United States) using Lipofectamine™ 2000 transfection reagent (Invitrogen, Carlsbad, California, United States) after the above cells were grown to between 75 and 85% confluency to obtain a higher transfection efficiency. After eight hours of transfection, the cell culture medium was replaced with DMEM. The IGF-1r gene targeting sequences were as follows: 5ʹ-ATTGAGGAGGTCACAGAGAAC-3ʹ and 5ʹ-TTCATATCCTGTTTTGGCCTG-3ʹ.

After being subjected to IGF-1r siRNA transfection, which was performed as described above, both the Eca-109 and TE-1 cells received radiation treatment with γ-irradiation at a single dose of 4 Gy/min every three days in the presence or absence of IGF-1r siRNA.

Western blotting was used to detect IGF-1r expression after IGF-1r siRNA transfection for 72 hours to evaluate the siRNA transfection efficiency in Eca-109 and TE-1 cells. At 24 hours after transfection, the medium was changed to serum-free medium. After a 72-hour transfection period, the cells were harvested, and cell lysates prepared in a buffer containing 0.1 M NaCl, 1 mM EDTA (ethylenediaminetetra-acetate, pH 8.0), 0.01 M Tris-HCl (pH 7.6), 1% (w/v) NP-40 (Nonidet P-40, octylphenoxy- polyethoxyethanol), 1% (w/v) Triton X-100, and 100 mg/ml PMSF(phenylmethanesulfonyl fluoride) (Sigma-Aldrich, St. Louis, Missouri, United States). Total protein was quantified by a Lowry protein concentration assay (Suolai, Beijing, China) after centrifugation at 12,000 × g for 60 minutes at 4°C. Equal amounts of protein were separated by SDS-PAGE and electrically transferred onto a PVDF (polyvinylidene fluoride) membrane. After blocking, the membrane was incubated overnight at 4°C with a primary antibody against IGF-1r (1:1,000; Santa Cruz Biotechnology, Santa Cruz, California, United States), followed by horseradish peroxidase-conjugated secondary antibody. The enhanced chemiluminescence system ECL-Plus (Suolai, Beijing, China) was used to detect the immunopositive bands, and the blot was stripped and re-probed using an antibody against β-actin (Sigma-Aldrich, St. Louis, Missouri, United States).

In the next experiments, cell proliferation was evaluated by the method of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Briefly, Eca-109 and TE-1 cells were cultured in triplicate in 96-well plates at a density of 5 × 10³ cells/well. The cells were transfected with IGF-1r siRNA and received the following irradiation treatment as described above. Cells in each treatment group were harvested by trypsinization, and the cell growth was analyzed by a Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, Vermont, United States).

An animal model with subcutaneous tumor xenografts was established by injecting Eca-109 and TE-1 cells into the left dorsal flank (1 × 10⁵ cells per animal) of female nude mice (between six and eight-weeks-old) (Shandong University, Jinan, China). After 14 days, the above animals received the irradiation treatment in the presence or absence of IGF-1r siRNA transfection. Student’s t-test was used to determine the statistical significance of the therapeutic effects. All procedures were approved by the Animal Ethics Committee of Shandong University (QL-2012JMK-231).

Western blotting was used to test the expression level of IGF-1r to evaluate the transfection efficiency of the IGF-1r siRNA. It was confirmed that the expression of IGF-1r decreased by approximately 33% and 46% in the Eca-109 and TE-1 cells, respectively, after IGF-1r siRNA transfection for 72 hours. In addition, a difference in IGF-1r expression between Eca-109 and TE-1 cells was also observed, with IGF-1r expression being much higher in TE-1 cells than in Eca-109 cells (Figure 1A).

Furthermore, the IGF-1r siRNA transfection efficiency was also evaluated with green fluorescence analysis using a fluorescence microscope (OLYMPUS, Tokyo, Japan). The results showed that the level of green fluorescence was greatly increased, which confirmed that effective transfection was obtained after 72 hours of siRNA transfection both in Eca-109 and TE-1 cells (Figure 1B).

To evaluate the effect of radiotherapy on cell proliferation in the presence or absence of IGF-1r siRNA transfection in vitro, the cell proliferation of Eca-109 and TE-1 cells was determined in the following experiment. The control group was treated with IGF-1r siRNA or with radiation alone. The controls were compared to cells receiving a combination of γ-irradiation therapy of 4 Gy/min and IGF-1r siRNA transfection for 72 hours. Both radiation and IGF-1r siRNA can effectively inhibit cell proliferation alone, with radiation reducing the cell counts by 42.1% and 45.1% and IGF-1r siRNA reducing them by 36.5% and 43.1% in Eca-109 and TE-1 cells, respectively, compared to the untreated group (P <0.05, n = 6). For the group receiving the combination therapy, the inhibition of cell proliferation was also more effective than that in the control group for both Eca-109 and TE-1 cells (P <0.001, n =6). After radiotherapy, the number of Eca-109 cells decreased by approximately 67.3% in the presence of IGF-1r siRNA transfection, and there was a 78.9% reduction in the TE-1 cells after radiation (Figure 2). In the TE-1 cells, the inhibition of cell proliferation after IGF-1r siRNA transfection was much higher than in the Eca-109 cells (P <0.01, n =6); this difference may result from the different expression levels of IGF-1r in these two cell lines. These results also show that the inhibition of cell proliferation after IGF-1r siRNA or radiation treatment alone was lower than in the combination treated group.

To test the effect of IGF-1r knockdown on Eca-109 and TE-1 cell apoptosis, flow cytometry was used to investigate the percentage of apoptotic cells. The percentage of apoptosis was 11.2 ± 0.8% after irradiation alone in Eca-109 cells. In the combination group, 16.2 ± 1.1% apoptotic Eca-109 cells were observed after irradiation coupled with IGF-1r siRNA treatment. In the TE-1 cells, 13.1 ± 1.1% apoptotic cells were observed after irradiation alone, and 20.3 ± 2.1% apoptotic cells were observed in the presence of IGF-1r siRNA treatment following irradiation, respectively (Figure 3). This result also suggests that some differences in apoptosis were observed between the Eca-109 and TE-1 cells (P <0.01, n =6), and these data were also consistent with the results that showed a lower expression of IGF-1r in Eca-109 cells than in TE-1 cells. Furthermore, it was clear that the percentage of apoptotic cells increased significantly after combination treatment compared to the cells that only received radiation (P <0.01, n =6).

The therapeutic efficacy of combination treatment was also investigated through in vivo tumor xenograft studies after injecting Eca-109 and TE-1 cells. The tumor volumes and survival rates were evaluated 14 days after cell injection in the different treated groups. The tumor volumes were 3.4 ± 0.2 cm³ in the Eca-109 group and 3.8 ± 0.1 cm³ in the TE-1 group after treatment with phosphate buffered saline (negative control group). After radiotherapy alone, the tumor volume decreased significantly both in the Eca-109 (2.1 ± 0.1 cm³) and TE-1 cells (2.4 ± 0.1 cm³) (P <0.05, n =8). In the group that received IGF-1r siRNA treatment alone, the tumor volume was also reduced (2.8 ± 0.1 cm³ for Eca-109 cells; 3.1 ± 0.1 cm³ for TE-1 cells) (P <0.05, n =8). Notably, it was clear that the tumors were much smaller after irradiation in the presence of IGF-1r siRNA transfection than in any single treatment group (1.1 ± 0.2 cm³ in the Eca-109 group; 0.8 ± 0.1 cm³ in the TE-1 group) (P <0.001, n =8) (Figure 4A). In addition, the survival rates of the tumor-bearing mice were also calculated after different therapeutic strategies. The results showed that the combination therapy significantly enhanced the survival rate compared to the groups with a single treatment (Figure 4B). These in vivo results confirmed that IGF-1r silencing can increase the radiation sensitivity of ESCC tumors in this established tumor model.