Annexin V-enhanced green fluorescent protein (EGFP)/propidium iodide (PI) apoptosis detection kit, cell cycle detection kit, DAPI, bicinchoninic acid assay kit, and cell lysis assay kit containing protease and phosphatase inhibitors were purchased from Nanjing Keygen Biotech, Co., Ltd. 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and trypan blue dye were purchased from Beyotime Institute of Biotechnology. In situ cell death detection TUNEL kit was purchased from Roche Diagnostics GmbH. 3-Methyladenine (3-MA) and rapamycin were purchased from Selleck Chemicals. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (Merck KGaA). Cycloheximide (CHX) was purchased from MedChem Express. Rabbit monoclonal antibodies against β-actin (cat. no. 4970), γ-H2AX (cat. no. 9718), caspase-3 (cat. no. 9662), cleaved caspase-3 (cat. no. 9664), LC3 (cat. no. 3868), CHOP (cat. no. 5554) and Ki-67 (cat. no. 9027) were obtained from Cell Signaling Technology, Inc. Rabbit polyclonal antibodies against p62 (cat. no. 18420) and Grp78/Bip (cat. no. 11587) were obtained from ProteinTech Group, Inc. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. G-21234) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (cat. no. A-11008) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.).

¹²⁵I radioactive seeds (0.8 mCi, model 6711) were kindly provided by Shanghai Xinke Pharmaceutical, Co., Ltd. The ¹²⁵I seed irradiation model used in the present study was designed according to previous studies (29,30), and was designed to provide a relatively homogeneous dose distribution in vitro. This model consisted of a lower irradiation plane and an upper treatment plane (the bottom of a 35-mm polystyrene dish). The height between the irradiation plane and treatment plane was 6 mm. In the irradiation plane, 8 seeds with the same activity were equidistantly spaced around a 35-mm diameter circumference, and a ninth seed was confined to the center. The initial dose rate in the treatment plane was 2.7 cGy/h, and the exposure time for delivering cumulative radiation doses of 2, 4, 6 or 8 Gy were 74, 151, 230 and 312 h, respectively. For the cells in the control group, sham seeds (without radioactivity) were used.

The human ESCC cell lines Eca-109 and KYSE-150 were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were maintained in RPMI 1640 medium (Hyclone; GE Healthcare), containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Hyclone; GE Healthcare), in a 5% CO₂ atmosphere at 37°C. In certain experiments, the cells were incubated with 0.5 mM 3-MA, 2 µM CHX or 5 mM NAC.

After 2, 4, 6 or 8 Gy irradiation, cells were cultured without radiation for 8–10 days to form colonies. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then stained with 0.1% crystal violet solution for 20 min at room temperature. The planting efficiency (PE) was defined as the ratio of colony number to inoculating cell number, and the survival fraction (SF) was calculated as follows: SF = PE (irradiated group)/PE (control group) ×100%. Cell survival curves were fitted using a multi-target single-hit model: SF=1-(1-e^D/D0)^N.

After 48 h of the indicated treatments, the cells were suspended and stained with trypan blue dye for 3 min at room temperature. Cell viability was determined using a trypan blue exclusion assay and observed under a light microscope (magnification, ×100; Eclipse TS100; Nikon Corporation).

Cells were collected 48 h after irradiation. For cell cycle analysis, cells were fixed with 70% ethanol overnight at 4°C and then stained with PI/ribonuclease A for 30 min at room temperature in the dark. For apoptosis analysis, the cells were stained with Annexin V-EGFP and PI for 20 min at room temperature in the dark. Samples were analyzed by flow cytometry (NovoCyte 2060R, ACEA Biosciences, Inc.) using NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.).

After 48 h of the indicated treatments, the cells were incubated with 10 µM DCFH-DA for 30 min at 37°C in the dark. Subsequently, the cells were washed with PBS, and then collected for flow cytometry analysis.

A total of 48 h after irradiation, the cells were collected and fixed in 2.5% glutaraldehyde phosphate buffer overnight at 4°C, washed with PBS, and then fixed in 1% osmium tetroxide for 2 h at 4°C. Subsequently, the fixed cells were washed with PBS, dehydrated in 50–100% ethanol, and then embedded in epoxy resin. After polymerization for 48 h at 60°C, the ultrathin sections were stained with uranyl for 15 min and lead acetate for 10 min both at room temperature, and examined under a transmission electron microscope (H-7650; Hitachi, Ltd.).

Cells were cultured on coverslips in 35-mm dishes. Following irradiation and culturing for 48 h, cells were fixed with ice-cold methanol for 5 min at −20°C and subsequently blocked in 5% BSA for 1 h at room temperature. Fixed cells were incubated with primary antibodies (LC3, 1:200; γ-H2AX, 1:200) overnight at 4°C, washed with PBS, and then incubated with Alexa Fluor 488-conjugated secondary antibodies (1:500) for 1 h at room temperature. Nuclei were stained with DAPI for 10 min at room temperature and the cells were examined under a fluorescence microscope (magnification, ×200; BX53, Olympus Corporation).

Cells were lysed in the ice-cold lysis buffer, containing freshly added protease and phosphatase inhibitors. Cell lysates were centrifuged for 15 min at 16,000 × g at 4°C, and the concentration of protein supernatants was determined using a bicinchoninic acid assay. The protein supernatants were boiled in loading buffer for 5 min, loaded on an SDS-gel. resolved by SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked in 5% BSA in Tris-buffered saline solution containing 0.05% Tween-20 (TBST) for 1 h at room temperature, incubated with the indicated primary antibodies (β-actin, 1:1,000; γ-H2AX, 1:1,000; caspase-3, 1:1,000; cleaved caspase-3, 1:1,000; LC3, 11,:000; p62, 1:2,000; Grp78/Bip, 1:4,000; CHOP, 1:1,000) overnight at 4°C, washed with TBST, and then incubated with the HRP-conjugated secondary antibody (1:3,000) for 1 h at room temperature. Antibody-antigen complexes were detected using an enhanced chemiluminescence kit (Tiangen Biotech, Co., Ltd.) and a light capture system (Clinx Science Instruments, Co., Ltd.). Densitometry analysis was performed using ImageJ version 1.51 (National Institutes of Health). β-actin was used as a loading control.

Eca-109 or KYSE-150 cells (5×10⁶ in 0.1 ml PBS) were injected subcutaneously into the left axilla of female BALB/c nude mice. Tumor volumes were measured every three days using the following formula: Volume = length × width² × 0.5. When the average tumor volume reached 300 mm³ (after ~12 days for Eca-109 cells or 18 days for KYSE-150 cells), the mice were randomized into two groups (n=6 per group): Control group and the ¹²⁵I seed group. For mice in the ¹²⁵I seed group, an 18-gauge needle was punctured into the center of the tumor mass, and then the ¹²⁵I seed (0.8 mCi) was implanted using a Mick applicator. For the control group, only tumor puncture was performed. All mice were euthanized 15 days after treatment, and the cumulative dose in the ¹²⁵I seed group was ~20 Gy. Tumor specimens were harvested and weighed, and then processed for histological analysis.

Tumor specimens were fixed in 4% paraformaldehyde for 48 h at room temperature and embedded in paraffin. Tumor sections (4 µm thickness) were subsequently used for hematoxylin and eosin (H&E) staining, TUNEL assay, and immunohistochemistry staining. TUNEL assay was performed according to the manufacturers protocol. Immunohistochemistry was performed as described previously (23) using primary antibodies against Ki-67 (1:400; overnight at 4°C). HRP-conjugated secondary antibody (1:1,000; 30 min at room temperature) and diaminobenzidine were used for detection. Frozen tumor sections were used for detection of ROS using DCFH-DA staining, as described above. After incubation with DCFH-DA, tumor sections were counter stained with DAPI for 10 min at room temperature.

All experiments involving mice were performed in accordance with the guidelines on animal care and experiments on laboratory animals of the Center of Experimental Animals, Southeast University (Nanjing, China), and were approved by the Animal Experimental Ethics Committee of Southeast University (approval no. 20190225004). Humane endpoints included mice exhibiting poor physical conditions, confined activities affected by the tumor, tumor sizes >2.0 cm in diameter (4.0 cm³), and severe tumor ulceration, necrosis or infection.

All experiments were performed in triplicate. Data are expressed as the mean ± standard deviation. A Students t-test or one-way ANOVA followed by a post-hoc Tukeys test was used to detect differences. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS version 23.0 (IBM, Corp.).

To investigate the anti-cancer effects of ¹²⁵I seed radiation, Eca-109 and KYSE-150 cells were irradiated at different radiation doses (up to 8 Gy). As shown in Fig. 1A, the effects on cell proliferation were increased as the cumulative dose administered was increased. The SF values of 8 Gy were 0.04±0.01 for Eca-109 cells and 0.02±0.01 for KYSE-150 cells (P=0.099). In Eca-109 cells, the number of multinucleated giant cells (MNGCs) increased following irradiation, and cytoplasmic vacuolation was observed. The cytoplasmic vacuoles were primarily observed in MNGCs, and the number as well as the size of the vacuoles increased over time (Fig. 1B). In KYSE-150 cells, extensive cytoplasmic vacuolation was observed following irradiation, and the number of vacuolated cells increased in a dose-dependent manner (Fig. 1C).

Radiation-induced DNA damage serves an important role in the anti-cancer effects of ¹²⁵I seed radiation. Histone H2AX localizes to the site of DNA double stranded breaks, where it is phosphorylated to γ-H2AX. The phosphorylated γ-H2AX further recruits DNA damage repair factors to repair the damaged DNA double strands (31). For the purpose of investigating cellular DNA damage and repair, nuclear γ-H2AX foci and the protein expression levels of γ-H2AX were observed 48 h after irradiation (Fig. 2A and B). The results suggested that ¹²⁵I seed radiation significantly induced DNA damage and repair in both Eca-109 and KYSE-150 cells, even at a low cumulative dose of 2 Gy. Progression of the cell cycle is delayed until the damaged DNA is repaired (31), thus cell cycle distribution was assessed (Fig. 2C). As shown in Fig. 2D, ¹²⁵I seed radiation significantly increased the proportion of cells in the G2/M phase and decreased the proportion of cells in the G0/G1 phase, in a dose-dependent manner. Furthermore, the degree of G2/M cell cycle arrest following irradiation was higher in KYSE-150 cells compared with the Eca-109 cells.

In previous studies, apoptosis was considered the major mode of cell death induced by ¹²⁵I seed radiation in various types of cancer cells (8,13,14). An annexin V/PI assay was used to investigate apoptosis induced by ¹²⁵I seed radiation in both cell lines. As shown in Fig. 3A and B, the apoptotic rates of Eca-109 and KYSE-150 cells increased as the cumulative dose increased, peaking at 6 Gy and declining at 8 Gy. The apoptotic rates of KYSE-150 cells were relatively low compared with the Eca-109 cells. The maximum apoptotic rate was 7.07±0.81% for KYSE-150 cells and 17.70±1.61% for Eca-109 cells (P=0.001). However, the proportion of necrotic cells (EGFP-/PI+) in KESY-150 cells increased along with the increase in cumulative dose. These results suggest that apoptosis is not the major mode of cell death induced by ¹²⁵I seed radiation in KESY-150 cells. To assess caspase activation, the levels of caspase-3 and cleaved caspase-3 were determined by western blot analysis. In Eca-109 cells, increase in cleavage of caspase-3 was consistent with the results obtained from the Annexin V/PI assay (Fig. 3C). In the KYSE-150 cells, caspase-3 cleavage was observed with 2 Gy radiation, and the levels decreased at higher doses (Fig. 3D). These results suggest that cell death induced by ¹²⁵I seed radiation in Eca-109 cells was primarily through caspase-dependent apoptosis, whereas in KYSE-150 cells, both apoptosis and non-apoptotic (primary) modes were responsible.

Autophagy is characterized by the formation of autophagosomes and autolysosomes, which manifest as cytoplasmic vacuoles (27). In the present study, extensive cytoplasmic vacuoles were observed following irradiation, particularly in KYSE-150 cells (Fig. 1B and C). Therefore, whether ¹²⁵I seed radiation induced autophagy in Eca-109 and KYSE-150 cells was further investigated. During autophagy, LC3-I is converted to LC3-II through lipidation, thus the levels of LC3, and the ratio of LC3-II to LC3-I are frequently used as indicators of autophagy (20,24,32). A total of 48 h after 6 Gy irradiation, the cells were stained with an anti-LC3 antibody and observed under a fluorescent microscope. As shown in Fig. 4A, the number of LC3 punctuations per cell increased notably following irradiation. Subsequently, western blot analysis was performed to determine the ratio of LC3-II to LC3-I in cells following exposure to 2, 4, 6 or 8 Gy radiation. The ratio of LC3-II to LC3-I in cells increased significantly at 4 Gy in both cell lines, and then is decreased slightly in Eca-109 cells at higher does, but maintained in KYSE-150 cells (Fig. 4B). Theoretically, the increased levels of LC3-II may have been the result of augmented formation of autophagosomes or impaired maturation of autophagosomes, which can block the degradation of LC3-II (24). To determine autophagic degradation activity, the protein levels of p62, a ubiquitin-binding protein which is selectively degraded in autolysosomes (18), was analyzed by western blot analysis. The levels of p62 decreased in both cell lines following irradiation (Fig. 4B), suggesting that ¹²⁵I seed radiation induced autophagic flux in Eca-109 and KYSE-150 cells.

To further investigate the roles of autophagy in cell death induced by ¹²⁵I seed radiation, the effect of 3-MA, an autophagy inhibitor, were assessed. Given that the apoptotic rates of both cell lines peaked at 6 Gy, the cells were pretreated with 0.5 mM 3-MA for 2 h and then exposed to 6 Gy radiation. Western blot analysis demonstrated that the ratios of LC3-II to LC3-I decreased following 3-MA treatment in both cell lines (Fig. 4C), suggesting that 3-MA attenuated ¹²⁵I seed radiation-induced autophagy. Annexin V/PI assay demonstrated that 3-MA improved ¹²⁵I seed radiation-induced apoptosis in both cell lines (Fig. 4D). Given the higher degree of non-apoptotic cell death in KYSE-150 cells, cell viability was assessed in these cells. As shown in Fig. 4E, 3-MA further decreased cell viability of irradiated Eca-109 and KYSE-150 cells, but did not affect the cell viability of unirradiated cells. These results suggest that in Eca-109 and KYSE-150 cells, autophagy serves a protective role in ¹²⁵I seed radiation-induced cell death.

3-MA did not reduce the percentage of vacuolated cells in both irradiated and unirradiated cells (Fig. 4F). Additionally, immunofluorescence images stained with an anti-LC3 antibody showed that certain vacuoles did not co-localize with the LC3 punctuations (Fig. 5A), suggesting that these vacuoles did not originate from autophagosome/autolysosomes. These findings demonstrate that autophagy is not wholly responsible for cytoplasmic vacuolation in either cell line following ¹²⁵I seed irradiation.

Paraptosis results in cytoplasmic vacuolation, and the number and size of vacuoles increase over time during paraptosis (21,26). In the irradiated KYSE-150 cells, the cytoplasmic vacuoles enlarged in size gradually, the nuclei were located peripherally, and subsequently, death-associated cellular detachment occurred (Fig. 5B). To a lesser degree, similar phenomena were also observed in irradiated Eca-109 cells, particularly in MNGCs (Fig. 1B). Therefore, the ultrastructure of mitochondria and ER by TEM were observed. Compared with the unirradiated controls, irradiated cells exhibited swollen mitochondria and ER, and also consequent vacuolation in both cell lines (Fig. 5C). Given that ER vacuolization is typically associated with persistent ER stress (33), ER stress was assessed by measuring the protein expression levels of the ER stress markers, Grp78/Bip and CHOP. Elevated levels of Grp78/Bip and CHOP were detected in both Eca-109 and KYSE-150 cells following irradiation, and persistent elevation of CHOP was observed in KYSE-150 cells (Fig. 5D). Studies have shown that de novo protein synthesis is required for cytoplasmic vacuolation in paraptosis, and CHX, a protein synthesis inhibitor, inhibits paraptosis (21). Therefore, KYSE-150 cells were pre-treated with CHX (2 µM) for 2 h prior to 4 Gy irradiation. The results showed that CHX effectively attenuated cytoplasmic vacuolation in irradiated cells (Fig. 5E). Taken together, these results suggest that paraptosis is a key mechanism of cell death induced by ¹²⁵I seed radiation in KYSE-150 cells, and paraptosis is partially responsible for ¹²⁵I seed radiation induced cell death in Eca-109 cells.

It has been reported that oxidative stress induced by single high-dose radiation results in apoptosis and autophagy (17). Thus, the effects of ROS on cell death induced by ¹²⁵I seed radiation were assessed. Firstly, 48 h after 4 Gy irradiation, the cells were labeled with the intracellular ROS probe, DCFH-DA, and analyzed by flow cytometry. The results showed that ¹²⁵I seed radiation increased the levels of intracellular ROS in both Eca-109 and KYSE-150 cells. KYSE-150 cells had higher basal levels of ROS compared with Eca-109 cells (P<0.001). The increase in ROS levels were more prominent in KYSE-150 cells compared with Eca-109 cells (fold change, 3.13±0.34 vs. 2.00±0.39, respectively, P=0.020; Fig. 6A). Subsequently, cells were pretreated with 5 mM NAC, an ROS scavenger, 4 h prior to 4 Gy irradiation. The results showed that NAC reduced the accumulation of intracellular ROS induced by ¹²⁵I seed radiation in both cell lines (Fig. 6B). Western blot analysis demonstrated that NAC decreased the levels of the autophagy indicator, the ratio of LC3-II to LC3-I, and ER stress markers, Grp78/Bip and CHOP, in irradiated Eca-109 and KYSE-150 cells (Fig. 6C). Furthermore, as shown in Fig. 6D, NAC attenuated ¹²⁵I seed radiation-induced apoptosis in Eca-109 cells (P=0.002), but did not significantly attenuate apoptosis in KYSE-150 cells (P=0.695). As ¹²⁵I seed radiation killed KYSE-150 cells primarily through paraptosis, the changes in cell viability and cytoplasmic vacuolation were assessed. The results showed that NAC attenuated ¹²⁵I seed radiation-induced decreases in cell viability in both cell lines (Fig. 6E). Furthermore, for both irradiated cell lines, the percentage of vacuolated cells decreased significantly following NAC treatment (Fig. 6F). Taken together, these results suggest that ¹²⁵I seed radiation-induced increases in ROS levels are critical for autophagy, apoptosis and paraptosis in Eca-109 and KYSE-150 cells.

The in vivo effects of ¹²⁵I seed radiation using both Eca-109 and KYSE-150 human ESCC xenograft mouse models were assessed. The ¹²⁵I seed was implanted into the tumor mass of each mouse, which received continuous low-dose-rate irradiation for 15 days with a cumulative dose of ~20 Gy. Tumor growth was significantly reduced after 9 days of ¹²⁵I seed irradiation (Fig. 7A), and the weights and volumes of harvested tumors were significantly lower in the ¹²⁵I seed group compared with the control group (Fig. 7B and C). To evaluate oxidative stress in vivo, the levels of ROS were measured by staining the tissue with DCFH-DA. Increased levels of ROS were observed in the ¹²⁵I seed group (Fig. 7D). H&E staining showed cellular vacuolation was present in tissue sections obtained from the irradiated tumor mass (Fig. 7E). Furthermore, ¹²⁵I seed radiation resulted in apoptosis (TUNEL assay, Fig. 7F) and reduced proliferation (immunohistochemistry for Ki-67, Fig. 7G) in irradiated tumor tissues. The results show that ¹²⁵I seed radiation induced ROS generation and DNA damage, initiated cell apoptosis and vacuolation-related cell death (potentially via paraptosis), and reduced cell proliferation and tumor growth in vivo.