The human TE-1 ESCC cell line was obtained from the Shanghai GeneChem Co., Ltd. and was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Biological Industries) and 100 U/l penicillin sodium/100 U/l streptomycin sulfate (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were incubated in a 95% air/5% CO2 humidified incubator at 37°C for 24, 48 and 72 h. Cells were exposed to 6 MV–X-rays radiation with a linear accelerator source (Varian Medical Systems) at a cumulative dose of 0–8 Gy at a fixed dose rate of 200 cGy/min at room temperature. The 0 Gy group was used as the control group.

An MTT assay was used to assess cell viability. Cells in the logarithmic growth phase were plated at a density of 4×10³ cells per well into 96-well plates in complete medium and cultured overnight at 37°C. The cells were then subjected to various treatments (siHMGB1, radiation or both) for 24, 48 and 72 h at 37°C. The remaining medium was discarded from the wells, 20 µl MTT and 100 µl medium were added to each well, and cells were incubated at 37 °C for an additional 4 h. Subsequently, the supernatant was discarded, and the reaction was stopped with 150 µl dimethyl sulfoxide for 10 min to dissolve MTT. Finally, the absorbance value was quantified at a wavelength of 490 nm on a microplate reader, and each experiment was repeated independently at least three times. Cell viability was calculated using the following formula: Mean optical density of treated cells/mean optical density of control cells.

HMGB1 and RAGE gene expression specific siRNA fragments and one scrambled shRNA (negative control) were designed and synthesized by Shanghai GenePharma Co., Ltd., according to the manufacturer's protocols. The sequences of the siRNAs were as follows: si-HMGB1 forward, 5′-GCUCAAGGAGAAUUUGUAATT-3′ and reverse, 5′-UUACAAAUUCUCCUUGAGCTT-3′; si-RAGE forward, 5′-GCCGGAAAUUGUGAAUCCUTT-3′ and reverse, 5′-AGGAUUCACAAUUUCCGCCTT-3′; and scrambled shRNA forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′. A total of TE-1 cells (4×10⁵) were seeded into 6-well plates for 16 h and then transfected with 100 pmol siRNA in serum-free medium for 8 h at 37°C. Negative control siRNA (20 µM) and siHMGB1/siRAGE (20 µM) were transfected into cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After that, the medium was replaced with serum-supplemented medium for 24 h at 37°C. The time interval between transfection and subsequent experimentation was 48 h. Negative controls using non-transfected cells and empty-vector were performed in parallel and then cells were harvested for analysis of protein expression.

A total of 1×10³ cells were seeded into 6-well plates in triplicate overnight. The transfection subsection of cells was performed as previously described. After radiation (0, 2, 4, 6 or 8 Gy), the cells were incubated for 8 days with 5% CO₂ at 37 °C to allow the formation of colonies. Subsequently, colonies were washed with PBS, and subsequently fixed for 20 min with 70% ethanol at room temperature and stained for 20 min with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature. Colonies containing >50 cells were counted under a light microscope (×200 magnification; Olympus Corporation). The surviving fraction was calculated as the ratio of the plating efficiency of the treated cells to that of control cells. The sensitization enhancement ratio (SER) was calculated as the mean inactivation dose in the control group divided by that in the treated group.

Apoptosis was detected using an Annexin V-phycoerythrin and 7-amino-actinomycin D (Annexin V-PE/7-AAD) apoptosis kit (Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.). Cells (4×10⁵) were plated overnight in 6-well plates, transfected with or without siHMGB1 for 24 h, and then cultured for 24 h after radiation (4 Gy). A total of 1×10⁶ cells/ml of treated cells in each group were collected and double-stained using Annexin V-PE/7-AAD for 15 min at 25°C. Next, cell apoptosis was quantified by flow cytometry (FACSCalibur; BD Biosciences) within 1 h, and the results were analyzed using the FlowJo software (version 7.6.1; Tree Star, Inc.).

Total RNA from each group of cultured TE-1 cells was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was dissolved with RNase-free water and stored at −80°C. RNA concentrations were detected by NanoDrop spectrophotometry (Thermo Fisher Scientific, Inc.). cDNA synthesis and its reaction condition were performed with a Prime-Script™ RT Reagent kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. RT-qPCR was carried out using a Stratagene Mx3000P™ Real-Time PCR System (Agilent Technologies, Inc.) and SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd.) with the following primers (Generay Biotech Co., Ltd.): NADPH oxidase (NOX)1 forward, 5′-CAAGGCCACTGACATCGT-3′ and reverse, 5′-CAGATTACCGTCCTTATTCCTA-3′; NOX5 forward, 5′-GATGACCCACCCAATAAGAC-3′ and reverse, 5′-GCCTCTGGTTCCCTCACTT-3′; and GAPDH forward, 5′-TCAACGGATTTGGTCGTATTG-3′ and reverse, 5′-TGGGTGGAATCATATTGGAAC-3′. GAPDH was used as the internal control. The following thermocycling conditions were used for amplification: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 20 sec and extension at 72°C for 15 sec. Data analysis was performed using the comparative Cq method as previously described (19).

Intracellular ROS levels were detected in living cells using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Beyotime Institute of Biotechnology), which is converted by ROS into the fluorescent product 2′,7′-dichlorofluorescein (10). A total of 2×10⁶ cells at 37°C were transfected with or without siHMGB1 for 24 h and subsequently irradiated with 4-Gy irradiation for 12 h. Subsequently, the cells were isolated and incubated with 10 µM DCFH-DA diluted in serum-free medium for 25 min at 37°C in a dark humidified incubator, which was added directly to the cell culture media to a final concentration of 10 µg/ml. After treatment, the cells were washed three times with PBS at room temperature for a total of 5 min and suspended in serum-free medium. Subsequently, the ROS levels were measured immediately via flow cytometry (FACSCalibur; BD Biosciences) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Serum-free medium was used as a negative control. Fluorescence data were analyzed using the FlowJo software (version 7.6.1; Tree Star, Inc.).

Cells at 37°C were transfected with siHMGB1 for 24 h prior to irradiation with 4 Gy X-rays for 1 h. Cells were harvested with trypsin and washed with ice-cold PBS twice. Subsequently, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with Tris-buffered saline containing 0.25% Triton X-100 on ice for 15 min, followed by final resuspension containing 5% BSA (Sigma-Aldrich; Merck KGaA) in PBS for 1 h at 25°C. Next, the cells were incubated with an anti-phospho-histone H2AX (Ser139) monoclonal antibody (dilution, 1:400 in PBS; cat. no. 9718; Cell Signaling Technology, Inc.) as the primary antibody overnight at 4°C. The cells were then washed with PBS three times at room temperature and incubated with secondary antibody Cy3-conjugated goat anti-rabbit IgG (1:200; cat. no. GB21303; Wuhan Servicebio Technology Co., Ltd.) for 1 h at 25°C and then washed with PBS twice. Phospho-γH2AX (p-γH2AX) levels were measured by flow cytometry (FACSCalibur; BD Biosciences). Untreated cells were used as the negative control. Data were analyzed using the FlowJo software (version 7.6.1; Tree Star, Inc.).

Cells were treated as aforementioned with ionizing radiation, siRNA or a combination of the two treatments, which were divided into the following groups: TE-1, negative control, 4-Gy, siHMGB1, siRAGE, 4-Gy+siHMGB1, 4-Gy+siRAGE. Cells were collected and washed twice with PBS. The cellular protein was extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C, according to the manufacturer's protocol, and total cellular protein concentrations were determined with a BioMate 3S (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Subsequently, 5 µg protein/lane for each sample was separated by electrophoresis on a 10–15% SDS-polyacrylamide gel, and then electronically transferred onto a polyvinylidene fluoride membrane (EMD Millipore). After blocking for 1 h at 37°C with Tris-buffered saline containing 1% Tween-20 (TBST) or 5% BSA, the membranes were incubated with the primary antibody at 4°C overnight. The following primary antibodies were diluted in PBS at 1:500 and purchased from Cell Signaling Technology, Inc.: ERK1/2 (cat. no. 9194S), p-ERK1/2 (cat. no. 4370T), JNK (cat. no. 9252S), p-JNK (cat. no. 9251S), p38 (cat. no. 9212S), p-p38 (cat. no. 9216S), RAGE (cat. no. 42544S), HMGB1 (cat. no. 3935S), β-actin (cat. no. 12620S), caspase-3 (cat. no. 14220S) and cleaved-poly(ADP-ribose) polymerase (PARP; cat. no. 9185S). Subsequently, the membranes were washed three times with TBST and incubated for 1 h at 37°C with the anti-rabbit secondary antibody conjugated to horseradish peroxidase (dilution, 1:5,000 in TBST; cat. no. 7076; Cell Signaling Technology, Inc.). After washing three times with TBST, the bands were detected using Pierce ECL Plus Western Blotting substrate (Thermo Fisher Scientific, Inc.) and scanned using a FluorChem FC3 imaging system (ProteinSimple). The images were semi-quantified with AlphaView software (version 3.4.0; ProteinSimple), normalized to β-actin (standard control) and expressed as the fold change compared with the control.

Data are presented as the mean ± SEM and were derived from ≥3 independent experiments. Unpaired Student's t-test was used to compare differences between two groups (Fig. 1B). Multiple group comparisons of the means were carried out by one-way ANOVA followed by Tukey's post hoc test. SPSS v20.0 (IBM Corp.) and GraphPad Prism 8.02 (GraphPad Software, Inc.) were used to perform the analysis. P<0.05 was considered to indicate a statistically significant difference.

A HMGB1 knockdown cell line was constructed to investigate the role of HMGB1 in the radiotherapy of ESCC, and the transfection efficiency was detected using western blot analysis (Fig. 1A). To investigate the effect of HMGB1 knockdown on ionizing radiation in TE-1 cells, an MTT assay was used to detect changes in viability after siHMGB1 transfection at 24, 48 and 72 h in TE-1 cells. As shown in Fig. 1B, siHMGB1 treatment induced a significant decrease in cell viability compared with that of cells without transfection at all time points. TE-1 cell viability after 2-Gy radiation significantly decreased compared with the effects of the non-transfected cells, whereas irradiation at 4 and 8 Gy did not significantly reduce the number of cells compared with the effects of 2-Gy irradiation (Fig. 1C). siHMGB1 treatment significantly decreased cell viability compared with that of cells without transfection after different doses of radiation (2, 4 and 8-Gy). Cell viability was decreased after exposure to radiation in a dose-dependent manner in HMGB1 knockdown cells (Fig. 1C). Furthermore, a clonogenic survival assay was performed to assess whether HMGB1 knockdown had potential radiosensitization activity. As shown in Fig. 1D and E, treatmen of TE-1 cells with siHMGB1 for 24 h prior to radiation led to a survival curve shift compared with that of untreated TE-1 cells, with a SER of 1.62. The present results demonstrated that the viability of TE-1 cells could not be inhibited by increasing the dose of ionizing radiation (2, 4 and 8-Gy), which was probably due to radioresistance. However, HMGB1 knockdown could increase the radiosensitivity of TE-1 cells to radiotherapeutic agents.

The levels of apoptosis of TE-1 cells after radiation were examined using Annexin V-PE and 7-AAD staining to verify whether HMGB1 knockdown enhanced radiation-induced inhibition of cell viability through apoptosis. As shown in Fig. 2A and B, the apoptosis rate of TE-1 cells after exposure to 4-Gy radiation for 24 h was significantly increased compared with that of cells that were not irradiated. However, siHMGB1 treatment significantly increased cell apoptosis after radiation compared with that of cells without transfection. Consistent with these findings, a marked increase in the expression levels of the pro-apoptotic proteins caspase-3 and cleaved PARP was observed after combined treatment of radiation and transfection by western blotting (Fig. 2C). These results demonstrated that the radiosensitizing effect of HMGB1 knockdown may be caused by induction of apoptosis in TE-1 cells.

Radiation can induce accumulation of ROS production, leading to cell damage and apoptosis (26). In the present study, the levels of ROS production after radiation were detected to examine whether HMGB1 knockdown could promote radiation-induced ROS production. Compared with that of TE-1 control cells, ROS production was not significantly altered after irradiation at 4 Gy, whereas siHMGB1 treatment significantly increased the levels of ROS production (Fig. 3A and B). However, HMGB1 knockdown combined with radiation resulted in higher ROS production than either siHMGB1 or radiation alone (Fig. 3A and B). These results demonstrated that the combination of HMGB1 knockdown and radiation caused the upregulation of ROS production, which may promote an increase in apoptosis of TE-1 cells after radiation.

The mRNA expression levels of NADPH oxidases were further detected to examine whether ROS production was induced by HMGB1 knockdown. It was revealed that the mRNA expression levels of NOX1 and NOX5 were significantly increased by HMGB1 knockdown or radiation treatment alone, while HMGB1 knockdown combined with radiation resulted in higher NOX1 and NOX5 mRNA expression compared with radiation alone (Fig. 3C). These results demonstrated that NADPH oxidase may be crucial in the radiation-induced generation of ROS following knockdown of HMGB1.

ROS accumulation has been demonstrated to cause DNA damage and trigger radiation-induced cell apoptosis (27). The levels of p-γH2AX after radiation were examined to investigate whether 4-Gy X-rays could promote HMGB1 knockdown-induced DNA damage. Compared with that of TE-1 control cells, p-γH2AX expression was significantly altered after 4-Gy radiation or siHMGB1 transfection (Fig. 4). However, HMGB1 knockdown combined with radiation resulted in higher p-γH2AX production than either siHMGB1 or radiation alone (Fig. 4). These results demonstrated that the combination of HMGB1 knockdown and radiation was associated with increased DNA damage, which may promote the apoptosis of TE-1 cells after radiation.

The MAPK signaling pathway is an oxidative stress-sensitive signal transduction pathway that is involved in radiation-induced cell apoptosis (12–14). The present study evaluated whether the MAPK signaling pathway also mediated the effects of apoptosis of HMGB1 knockdown during radiation of TE-1 cells. It was revealed that the levels of p-p38/p-38 and p-ERK1/2/ERK1/2 decreased, while p-JNK/JNK increased by HMGB1 knockdown along with radiation compared with the effects produced by either siHMGB1 or radiation alone (Fig. 5A). To determine whether HMGB1 promoted MAPK signaling-mediated radioresistance via RAGE, which is an HMGB1 receptor, TE-1 cells were transfected with siRAGE (Fig. 5B) and treated with radiation alone or transfected with siRAGE and treated with radiation. As shown in Fig. 5C, RAGE knockdown combined with radiation increased p-JNK levels, and decreased p-p38 and p-ERK1/2 levels. Therefore, these results indicated that the MAPK signaling pathway is involved in the process of HMGB1-promoting radioresistance via RAGE in TE-1 cells.