The EC9706 human esophageal squamous cell carcinoma cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Beijing, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 µg streptomycin/ml (all purchased from Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂, and were sub-cultured every three days.

Autophagy inhibitor 3-methyladenine (3-MA) was obtained from Sigma-Aldrich. Anti-microtubule-associated protein light chain 3 (LC3; cat. no. sc-16755), anti-beclin-1 cat. no. sc-48381), anti-vascular endothelial growth factor (VEGF; cat. no. sc-1836), anti-cleaved caspase-3 (cat. no. sc-22171-R), anti-cleaved caspase-9 (cat. no. sc-56073), anti-cleaved poly(ADP ribose) polymerase (PARP; cat. no. sc-23461-R), anti-proliferating cell nuclear antigen (PCNA; cat. no. sc-56), anti-Ki-67 (cat. no. sc-15402), anti-B-cell lymphoma 2 (Bcl-2; cat. no. sc-7382), anti-Bcl-2-associated X protein (Bax; cat. no. sc-6236) and anti-CD31 (cat. no. sc-71873) antibodies were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-actin antibody, and goat anti-rabbit and goat anti-mouse immunoglobulin (Ig)G (cat. no. sc-66931) secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

The exponentially growing cells were exposed at room temperature and irradiated with a Varian 600 CD X-ray linear accelerator (Varian Medical Systems, Inc., Palo Alto, CA, USA), at a dose rate of 2.5 Gy/min.

The exponentially growing cells were exposed at room temperature and a total dose of 6 Gy radiation was delivered in three fractions over three days. In the experimental study groups, 5 or 10 mM 3-MA was added to the cells for 2 h prior to irradiation. Six hours after the treatments, all of the cells were detached by trypsinization (Sigma-Aldrich) and the number of viable cells was counted. For the colony formation assay, the cells were seeded into six-well plates containing Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and incubated for 14 days. The cells were fixed with ethanol (Cusabio Biotech Co., Ltd., Wuhan, China) and stained using 0.5% crystal violet (Sigma-Aldrich), while only colonies containing ≥50 cells were considered surviving colonies. The sensitizing enhancement ratio (SER) was calculated, according to the D₀ values (dose of radiation producing a 37% survival rate), using the following formula: SER=D_{0 untreated cells}/D_{0 treated cells}.

Flow cytometry was performed following DNA staining with propidium iodide (PI; Santa Cruz Biotechnology, Inc.), according to the manufacturer’s instructions. Briefly, the cells were harvested, washed with phosphate-buffered saline (PBS) and treated with 100 mg/ml RNase A (Santa Cruz Biotechnology, Inc.) for 30 min at room temperature. The cells were then stained with PI (1 mg/ml) solution at 4°C and incubated in the dark for 30 min. The cell cycle distribution was evaluated using a BD FACSArray™ Bioanalyzer system (BD Biosciences, San Jose, CA, USA).

The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Santa Cruz Biotechnology, Inc.) was used to assess the rate of cell apoptosis. Briefly, the untreated and treated cells were seeded in six-well plates and incubated for 24 h. The cells were then harvested, washed twice in PBS and stained with Annexin V-FITC and PI, according to the manufacturer’s instructions. Annexin V binds to apoptotic cells with exposed phosphatidylserine (early apoptosis), whereas PI labels cells with membrane damage (late apoptosis). The resulting fluorescence was detected by flow cytometry using CellQuest™ version 5.2.1 (BD Biosciences) analysis software.

Protein extraction and tissue sample homogenization were performed as previously described (18,19). The cells were scraped into ice-cold PBS and centrifuged at 400 × g for 5 min at 4°C. The pelleted cells were then lysed in 50 ml boiling SDS solution (Sigma-Aldrich) and centrifuged at 4,350 × g for 5 min. Membrane protein was extracted from the resulting supernatant using Mem-PER Eukaryotic Membrane Protein Extraction kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein concentrations were measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) using bovine serum albumin (Santa Cruz Biotecnology, Inc.) as a standard. An equal amount of protein (8 µg) was separated using 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skimmed milk (Cusabio Biotech Co., Ltd.) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-LC3-I/II (diluted 1:2,000), anti-beclin-1 (diluted 1:800), anti-VEGF (diluted 1:400), anti-caspase-3 (diluted 1:400), anti-caspase-9 (diluted 1:500), anti-PARP (diluted 1:400), anti-PCNA (diluted 1:1,000), anti-Ki-67 (diluted 1:800), anti-Bax (diluted 1:1,000) and anti-Bcl-2 (diluted 1:1,000). The membranes were then incubated with horseradish peroxidase-conjugated IgG secondary antibodies for 2 h at 37°C. The immunoreactive bands were visualized using an enhanced chemiluminescence system (Pierce Biotechnology, Inc.). To quantify equal loading, the membranes were re-probed with a primary antibody targeting β-actin.

The experiments of the present study followed the Declaration of Helsinki, and were approved by the institutional review board of Zhengzhou University Affiliation Cancer Hospital (Zhengzhou, China). The EC9706 cells (2.0×10⁶ cells) were injected subcutaneously into the lower right side flank of male athymic nude BALB/C-nu/nu mice (5–6 weeks-old; weighing 18–20 g), which were purchased from Keli China Experimental Animal Center (Beijing, China). The care and treatment of the mice were in accordance with institutional guidelines. When the tumor volume reached 100 mm³, six mice/group were treated with 3-MA (30 mg/kg intraperitoneally 1 h prior to radiation) and/or radiation (2 Gy/fraction, five fractions per week to equal a total dose of 20 Gy). The mice were divided into four treatment groups (n=12/group): Untreated, 3-MA alone, radiation alone and radiation combined with 3-MA. At the end of the experiments all of the mice were sacrificed by cervical dislocation. Tumor diameters were measured with calipers and tumor volume (V) was calculating using the following formula for a rotational ellipsoid: V=A×B²/2 (A, axial diameter; B, rotational diameter).

Tumor tissues derived from the four groups were fixed in 10% formalin (Cusabio Biotech Co., Ltd.), embedded in paraffin and cut into 5-µm sections. Hematoxylin and eosin (H&E; Santa Cruz Biotechnology, Inc.) staining was performed on the tumor tissue for general morphological analysis. The samples were assayed for DNA fragmentation [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay] using the in situ Cell Death Detection kit (Roche Molecular Biochemicals, Indianapolis, IN, USA). Briefly, following deparaffinization and dehydration, the tissue sections were incubated in proteinase K (DAKO North America, Inc., Carpinteria, CA, USA) for 15 min, washed with PBS, incubated in equilibration buffer and then in terminal deoxynucleotidyl transferase enzyme solution. The sections were subsequently rinsed in PBS, incubated with streptavidin-peroxidase conjugate (Sigma-Aldrich) and visualized using diaminobenzidine (Sigma-Aldrich), according to the manufacturer’s instructions.

Specific staining for endothelial cells was conducted using the neo-angiogenesis marker CD31. The slides were fixed using cold acetone (Cusabio Biotech Co., Ltd.) for 20 min. Following two washes with PBS, the tissue sections were incubated with 3% hydrogen peroxide (Santa Cruz Biotechnology, Inc.) in methanol for 30 min, in order to block endogeneous peroxidase activity. Primary antibody incubation was conducted at 37°C for 2 h, the slides were then incubated with goat anti-mouse IgG secondary antibody for 30 min, and with streptavidin biotin-peroxidase complex (Santa Cruz Biotechnology, Inc.) for 40 min. Following incubation with diaminobenzidine chromogen (Santa Cruz Biotechnology, Inc.), the tissue sections were re-stained with hematoxylin. Vessel density was determined by counting the number of microvessels per high-power field (Olympus IX 70; Olympus Corporation, Tokyo, Japan).

All values are presented as the mean ± standard error of the mean. Statistical significance was analyzed by one-way analysis of variance with post hoc Dunnett’s test, using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to demonstrate the enhancing effect of autophagy inhibition on radiosensitivity, cells were treated with 3-MA, which is a well-known inhibitor of autophagy in mammalian cells. Cell proliferation and colony formation were investigated in the EC9706 esophageal squamous carcinoma cell line. Treatment with 10 mM 3-MA alone led to a slight inhibition of cell growth. The viability of the cells was decreased in response to radiation, whereas a combination of radiation and 3-MA treatment markedly decreased the number of surviving cells (Fig. 1A). The radiosensitizing potential of 3-MA was ascertained by a clonogenic survival assay, and the SER was calculated based on the D₀ values extrapolated from from the survival curves. The SER reached 1.76 when the cells were treated with a combination of 10 mM 3-MA and ionizing radiation (Fig. 1B). These results indicated that autophagy inhibition exhibits a radiosensitization potential in vitro.

Autophagosome formation and expression of two essential autophagy-associated proteins, LC3 and beclin-1, were detected in the present study, in order to assess autophagy. During autophagy, phosphatidylethanolamine conjugates to the cytosolic form of LC3 (LC3-I), resulting in the formation of LC3-II. The amount of LC3-II is a commonly used indicator of autophagy (20). Beclin-1 participates in the early stages of autophagy, where it promotes the nucleation of the autophagic vesicle and recruits proteins from the cytosol (21). The present study detected an upregulation of beclin-1 protein expression levels and a downregulation of the conversion of LC3-I/II in EC9706 cells 24 h after co-treatment with radiation and 3-MA, thus indicating an increase in autophagic activity (Fig. 2). Following co-treatment of the cells with 3-MA, autophagic activity was downregulated; furthermore, decreased protein expression levels of beclin-1 and LC3-II were observed by western blotting. The expression levels of beclin-1 and LC3-II partly reverted to their original levels when the cells were treated with a combination of radiation and 10 mM 3-MA. These results provided evidence for the effective inhibitory effect of 3-MA on autophagy.

The possible effects of autophagy inhibition on the cell cycle distribution were investigated in the EC9706 cells treated with 3-MA, radiation, or their combination. Treatment of the cells with radiation (6 Gy) alone slightly affected the percentage of cells in each phase. In the presence of 3-MA, there was an increase in the number of EC9706 cells in G₂/M phase by 65.7% and 218.0% following treatment with 5.0 and 10 mM of 3-MA, respectively. These results suggested that treatment with 3-MA resulted in a cell cycle arrest in G₂/M phase, which is important for radiation sensitivity. Concomitant with the G₂/M arrest was an elevation in the sub-G₁ population, which is an indicator of apoptotic cell death (Fig. 3A and B). These results indicated that the effects of autophagy inhibition on radiation sensitization may be attributed to the induction of G₂/M phase arrest and apoptosis.

To determine whether inhibition of autophagy was able to induce apoptosis, Annexin V-FITC and PI staining was conducted. Flow cytometry detected apoptotic cells 24 h after EC9706 cells were treated with radiation. When the irradiated cells were co-treated with 3-MA for 24 h, the number of Annexin V- and Annexin V/PI-positive cells significantly increased, as compared with that of cells treated with radiation alone (Fig. 4A). To further confirm the increase in apoptosis, the protein expression levels of the executioner caspases, caspase-3, caspase-9 and PARP, were determined. In the radiation-treated groups, caspase-3 and PARP were cleaved into their specific active forms, and their activity in the combined treatment group was significantly higher as compared with that in the cells treated with radiation alone (Fig. 4B). These results indicated that radiation-induced autophagy has a protective role in tumor cells against apoptosis, and inhibition of autophagy subsequently enhances the rate of apoptosis in the cells.

To explore other potential mechanisms underlying the positive effects of autophagy inhibition on radiation in esophageal cancer, the role of autophagy in tumor angiogenesis was examined. VEGF is currently regarded as the most potent pro-angiogenic factor (22); therefore, the present study assessed VEGF protein expression levels in EC9706 cells. The protein expression levels of VEGF were reduced in EC9706 cells treated with 3-MA, as compared with those in the untreated cells. Of note, when irradiated cells were co-treated with 3-MA for 24 h, the protein expression levels of VEGF were significantly lower as compared with those in the radiation group (Fig. 5).

The present study also investigated whether inhibition of autophagy was able to affect the tumor response to radiotherapy. The response of the EC9706 xenografts to radiation plus 3-MA was significantly enhanced, as compared with the that of the untreated, 3-MA alone and radiation alone groups (P<0.01, Fig. 6A). Treatment with 3-MA alone reduced the mean tumor volume by 28.2% (2,138.5±247.7 mm³, as compared with 2,977.3±352.2 mm³ in the untreated group), and treatment with radiation alone reduced the mean tumor volume by 68.29% (925.6±127.3 mm³, as compared with 2,977.3±352.2 mm³ in the untreated group). However, co-treatment of 3-MA with radiation resulted in significantly smaller tumors, with the tumor volume reduced by 93.9% (181.7±97.3 mm³, as compared with 2,977.3±352.2 mm³ in the untreated group; P<0.01).

Of note, the in vitro results were concordant with the findings of the in vivo experiments in the present study. A TUNEL assay was used to measure levels of apoptosis, and increased apoptosis was detected in the radiation plus 3-MA treatment group (Fig. 6B and C). The number of TUNEL-positive apoptotic cells per field was 4.1±0.9 in the untreated group, 9.3±2.2 in the 3-MA group, 18.9±3.1 in the radiation group and 31.2±3.9 in the co-treatment group (P<0.05).

Western blot analysis showed that radiation increased the protein expression levels of beclin-1 and LC3-II/LC3-I in the xenografts, whereas treatment with 3-MA inhibited the expression levels of LC3-II and beclin-1 (Fig. 6D). In addition, the protein expression levels of the proliferative markers PCNA and Ki-67 were assessed; in the tumors treated with radiation and 3-MA combined, the levels were decreased to a level that was barely detectable. Furthermore, the expression levels of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 were detected. A significant upregulation in the protein expression levels of Bax and downregulation in the protein expression levels of Bcl-2 was detected in the tumors treated with radiation and 3-MA combined (Fig. 6C).

To investigate the effects of autophagy inhibition on tumor angiogenesis, immunohistochemical staining of frozen tumor tissue was performed using antibodies targeting CD31. Furthermore, angiogenesis within the tumor sections was evaluated by counting the number of microvessels in each section. Inhibition of autophagy by 3-MA suppressed angiogenesis, and mice in the radiation plus 3-MA treatment group exhibited a decreased microvessel density within the tumor, as compared with that in the radiation only group (Fig. 7; P<0.05).