125I Low Dose Rate Radiation Increases Radiotherapy Sensitivity on Esophageal Cancer Cells via Multiple Mechanisms

Objective: To determine the biological effectiveness of iodine 125 ( 125 I) radioactive seeds continuous low dose rate radiation on the human esophageal cancer cell line KYSE150 compare to single dose radiation and explore its potential cellular mechanisms. Method: Three groups of KYSE150 cells were explored: control group, single dose irradiation (SDR) and 125 I seeds continuous low dose rate irradiation group ( 125 I-CLDR). Dose-survival curves was obtained by colony formation assay. MTT proliferation assay was used to measure KYSE150 cell vibility. KYSE150 cell apoptosis was analysis by Annexin V-FITC/PI staining, meanwhile, cell cycle analysis was performed by ow cytometry. Bim, Bcl-2, caspase-3 and cleaved caspase-3 protein expression were measured by western blotting to vertify the apoptosis level, as well as CyclinB1 protein expression which values the fuction of G2/M check point.Cell morphology changes were observated under phase contrast microscope. Endoplasmic reticulum stress (ER stress) was measured by GPR78/Bip1 and PERK changes in gene expression, which was detected by Real-time PCR. Reactive oxygen species(ROS) changes and mitochondria were meaured by ow cytometry. ATP detection kit was used to measured ATP level afer two modes of irradiation. DNA-Pks, Ku70 and Ku80 expression were measured by western blotting to represent the damage of DNA damage and repair capabilities. Acridine orange (AO) staining was used to detect level of autophagy and quantitative was measured by ow cytometry. LC3-II, ATG5, Beclin1, p-Akt, p-mTOR, mTOR and p-S6 proteins expression were detectd by western blotting. Results: KYSE150 cells were more radiosensitive to 125I-CLDR than SDR. Two modes of irradiation could both inhibit the proliferation vability of KYSE150 cells, while the inhibition effects in 125I-CLDR was signicantly stronger compared with SDR. Compared with SDR, 125I-CLDR showed more proportions of the early and late apoptosis rate as well as cells at G2/M phase. Apoptosis related proteins, such as Bim, caspase-3 and cleaved caspase-3, were elevated, while CyclinB1 expression was decreased in 125I-CLDR group. Cells became bigger and grainy in 125I-CLDR group, meanwhile, ROS levels and ER stress signal was elevated except ATP concentration. Cells stained by AO was anaysised by ow cytometry, results showed that red: green ratio in 125I-CLDR group increased as well as LC3 and ATG5 protein expression detected by western blotting. p-Akt, p-mTOR and p-S6 protein expression were decreased to some extent in 125I-CLDR group cells, while total mTOR protein showed no signicant changes. Conclusion: Our results conrmed that 125 I-CLDR could strong inhibite KYSE150 cancer cells. Furthermore, we revealed that cell cycle arrest and apoptosis were not the only fate after 125I-CLDR, autophagy might be another important choice which mTOR pathway might be involed in. These ndings can support the application of esophageal stent loaded with 125I seeds in clinic.


Introduction
Esophageal cancer is one of the most aggressive gastrointestinal cancers and it remains a global health concern with a dismal prognosis and an estimated 5-year survival rate of approximately 10-15% [1,2].
Dysphagia is one of the main clinical symptoms and prognostic factors [3]. Available palliative options for dysphagia are esophageal stent placement and radiotherapy. In general, intraluminal brachytherapy of high dose rate can relieve dysphagia and improve the quality of patients' life, but other severe symptoms such as esophageal stula, perforation and bleeding. Self-expanding esophageal stent can effectively relieve the patients' clinical symptoms, however, it is no therapeutic effect, esophageal recurrence and narrowness cannot be avoided [3,4]. Due to limited availability of intra-luminal brachytherapy and self-expanding esophageal stent in clinical practice, 125 I radioactive seeds loaded with esophageal stent exhibits the advantages of both options and the signi cant clinical effect [5]. Although there are several reports on the biological effects of continuous low dose rate radiation, the different effectiveness of variant dose rate irradiation on the human esophageal cancer cell lines and the relevant cellular mechanisms is still largely unidenti ed. The aim of the present study is to determine the direct biological effectiveness of single dose radiation (SDR) and 125 I continuous low dose rate radiation ( 125 I-CLDR) on human esophageal cancer cell line KYSE150 in vitro, and we also aimed to unravel the underlying cellular and molecular mechanisms behind distinct modes of irradiation.
The sensitivity of cancer to radiotherapy is affected by many factors. Activation of DNA damage response (DDR) pathways is considered an important factor affecting resistance to radiotherapy [6]. Loss of cell-cycle checkpoint responses may result in increased sensitivity, particularly if the checkpoint controls the G2 transition. Overexpression of cyclin D, which shortens the duration of the G1 transition, is associated with radiation resistance, perhaps by inhibiting apoptosis [7]. ROS are the effector molecules of radiation, and increase of ROS production is known to enhance radioresponse [8]. The role of autophagy in radiotherapy sensitivety has been paid more and more attention. However, the role of autophagy in radiotherapy sensitivity in tumor cells remains controversial [9]. In the radiotherapy of esophageal cancer studies have shown that autophagy promotes the survival of esophageal cancer to resist tumor radiation therapy [10], and recent studies have found that autophagy can promote the death of cancer cells and increase their radiotherapy sensitivity [11]. What's more, although the common upstream signaling pathways have been found which is linked to apoptosis and autophagy [12], the potential regulatory mechanisms have not been clearly understood.
Therefore, in the present study, different biological effects of single dose radiation (SDR) and 125 I continuous low dose rate radiation ( 125 I-CLDR) on human esophageal cancer cell line KYSE150 were explored in vitro. Moreover, we analyzed the inhibitory mechanisms of 125 I-CLDR to cancer cell proliferation, including analysis of the cell cycle, apoptosis, ER stress, mitochondrial function and autophagy in human esophageal cancer cell line KYSE150 in vitro. Given the established roles of the Akt-mTOR signaling pathway and ROS as some of the early changes linked to apoptosis and autophagy, we further examined their potential involvement in the inhibitory effects induced by 125 I-CLDR. This work highlights the great potential clinical applications of 125 I-CLDR in the treatment of esophageal cancer.

Cell culture
The human esophageal cancer cell lines KYSE150 was obtained from Chines academy of medical sciences Tumor Cell Bank (Beijing, China) and cultured in Roswell Park Memorial Institute (RPMI 1640, HyClone) containing 10% fetal bovine serum (FBS) (HyClone), 10units/mL penicillin, and 10mg/mL streptomycin and maintained in humidi ed incubators at 37°C and 5% CO 2 .
X-ray and 125 I seeds irradiation The irradiation method was performed as previously described [13]. Brie y, Liner accelerators producing 6 MV X-ray beams were provided by the 306th Hospital of the People's Liberation Army (Beijing, China). The dose rate was 400 cGy/min and the source-to-skin distance (SSD) was 100 cm. The surface of the culture dishes was covered by 1.5 cm thickness packing materials. Radiation dose were 0, 2, 4, 6 and 8Gy. An inhouse model for in vitro iodine-125 seed irradiation was developed for this study. The activity of the single seed used in this study was 92.5 MBq (2.5 mCi), which translates to an initial dose rate of 2.77 cGy/h to model cells. The exposure times for delivering doses of 2, 4, 6 and 8Gy were 73.7, 154.6, 245.8 and 345.1 hours (i.e., 3.07, 6.44, 10.24 and 14.38 days), respectively [14,15]. The absorbed dose was equivalent to SDR treatment.

Colony formation assay
Colony formation assay was used to detect the radiation sensitivity of KYSE150 cells after different modes irradiation. Different numbers of cells were plated into 6-well plates depending on the doses of radiation: 0Gy (100), 2Gy (200), 4Gy (800), 6Gy (1600) and 8Gy (3200). And, the transfected cells were subjected to radiation at the above indicated doses. Then, the cells were cultured for 14 days to form colonies, and next, stained with methylene blue. Those colonies (≥50 cells) were then counted. The experimental data was tted to the single-hit multi-target model to make the survival curves of different groups.
MTT proliferation assay MTT proliferation assay was used to measure KYSE150 cell viability after two modes irradiation on the doses of radiation: 0Gy, 1Gy, 2Gy and 4Gy, after cell culture incubation (24, 48 and 72h), 10μl MTT (dissolved in RPMI-1640, 5mg/mL) was added to well, and plates were further incubated at37 °C for 4h. Finally, the purple formazan crystals were dissolved by adding 100μl acid-isopropanol (0.04N HCI in isopropanol) into each well. Absorbance was then measured at 550nm against reference wavelength of 630nm and stimulation index (SI) was determined. Te SI is expressed with average OD value in the test group divided by average OD value in negative controls. Cell proliferation activity (%) = (each dose point) Absorbance value/(0 Gy) Absorbance value.
Cell cycle analysis Cells after two modes irradiation on the doses of 4Gy for 72h, were harvested and washed with PBS for 3 times and then xed in pre-cooled 70% ethanol overnight at -20°C. Fixed cells were then pelleted through centrifugation, washed with PBS and incubated with 20 ug/mL RNase A (Sigma-Aldrich) in PBS at 37°C for 30 min. Cells were then analyzed on a BD FACS Calibur (BD bioscience, San Jose, CA, USA) after stained with 10 mg/ml PI (Sigma-Aldrich) in 0.1% Triton X-100. Cell cycle distribution was analyzed with the ModFit LT software (Verity Software House, Topsham, ME). Cellular apoptosis assay Cellular apoptosis was evaluated by FITC-conjugated Annexin V/propidium iodide (PI) staining followed by ow cytometry analysis.

Cell morphology observation
Cell morphology changes were observed under phase contrast microscope (Olympus IX71, Japan) after two modes of irradiation and photographed (magni cation, ×800), which was analyzed by ow cytometry.
Detection of intracellular reactive oxygen species (ROS) ROS labeled by DCFH-DA probes was measured by ow cytometry in FL1 channel, at the meantime, mitochondria was detected by ow cytometry in FL1 channel and uorescence microscope after Mito-Tracker Green uorescent probe stained.
Detection of ATP ATP detection kit was used to measured ATP level after two modes of irradiation, the detail was followed by protocol. ATP concentration was standardized to nmol/mg after BCA protein RT-qPCR assay of endoplasmic reticulum stress (ER stress) ER stress was measured by GPR78/Bip1 and PERK changes in gene expression, which was detected by Real-time PCR. According to the product manual, TRIzol reagent (Invitrogen) was applied to isolate total RNA from the cell samples. Then, NanoDrop (Thermo Fisher Scienti c, 2000C) was used to measure the concentration of RNAs. Reverse transcription was performed according to standard protocols using a RevertAid™ II First Strand cDNA synthesis Kit (Thermo Fisher Scienti c Inc., Waltham, MA USA). For detection of mRNA, Talent qPCR PreMix (SYBR Green) (TIANGEN, China) was used to detect the mRNA expression. GAPDH was ampli ed as an internal standard. The primers for PCR were as follows: Acridine orange (AO) staining was used to detect autophagy AO staining was used to detect level of autophagy in KYSE150 cells and quantitative was measured by ow cytometry with red: green signal intensity ratio, which was performed as previously described [16]. AO solution with nal concentration of 1g/ml was used to stain adherent cells at 37°C for 15 min, then the buffer solution without phenol red was used to rinse cells for 3 times. Cells was digested with trypin and resuspended in 5% serum PBS, cell concentration was adjusted and detected by ow cytometry. The excitation light was 488 nm, and the emission light of green (510-530nm) and red (640-650nm) corresponded to FL1 and FL3 channels in ow cytometry (Beckman Coulter Epics XL, USA), respectively.
The ratio of FL3 to FL1 represented the level of autophagy.

Western blot and antibodies
The whole-cell lysate was extracted with RIPA lysis buffer (50mM Tris [pH 7.4], 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium uoride, EDTA, from Beyotime Company). Then BCA assay was used to measure the protein concentration. Afterwards, 30 µg protein was loaded onto sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) to separate. After that, proteins on the gels were transferred to polyvinylidene uoride (PVDF, 0.22um, Millipore Company) membrane, and the transferring time was based on the protein molecular weight. Then, the membranes were incubated with primary antibodies (at the appropriate dilution according to the supplementary table 6) in 6 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. Followed that, the membranes were incubated with Anti-rabbit/mouse IgG, HRP-linked antibody (1:2000, Beyotime Company) in 10 ml secondary antibody dilution buffer with gentle agitation for 2 hours at room temperature. After washing three times for 5 min each with 15 ml of Tris Buffered Saline with Tween® 20 (TBST), the membranes were exposed to get the blot images with 1X SignalFire™ ECL Reagent (Millipore Company). anti-caspase-3, anti-Bim, anti-AKT, anti-mTOR, anti-p-S6, anti-p-mTOR, anti-ATG5 ,anti-Beclin-1, anti-DNA-PKcs, anti-Ku80 and anti-Ku70 antibodies were obtained from Cell Signling Technology, Inc (Cell Signaling Technology, MA, USA). Anti-CyclinB1 and anti-Bcl-2 antibodies were obtained from Abcam, Inc (Abcam,USA). Anti-LC3 were obtained from MEDICAL&BIOLOGICAL LABORATIORIES CO, LTD, Inc (MEDICAL&BIOLOGICAL LABORATIORIES CO, LTD, Japan).

Statistics analysis
Each experiment was performed ≥ 3 times and values were expressed as mean ± S.D. Data analysis was done using the GraphPad Prism. The experimental data were analyzed using SPSS software 16.0 (SPSS, Inc., Chicago, IL, USA). Statistical signi cance among different groups was measured by Student's t test or one-way analysis of variance. A P-value < 0.05 was considered to represent a statistically signi cant difference.

Results
KYSE150 cells were more radiosensitive to 125 I-CLDR than SDR.
Two modes of irradiation could both inhibit the proliferation viability of KYSE150 cells, while the inhibition effects in 125 I-CLDR was more signi cantly compared with SDR. After KYSE150 cells were irradiated with 1, 2, 4, 6 and 8 Gy, the survival cells in SDR group and 125 I-CLDR group gradually decreased with the increase of radiation dose, and the survival cells of 125 I-CLDR group at each dose point were lower than SDR group, with statistically signi cant difference (Fig. 1a). MTT method was used to detect cell activity of KYSE150 cells after different irradiation, and it was found that the cell activity gradually decreased with the increase of time (Fig. 1b) and dose (Fig. 1c), and the toxic effect of 125 I-CLDR on cells was more signi cant in 125 I-CLDR group (Fig. 1b-c).
Compared with SDR, more proportions of the early and late apoptosis rate were shown as well as cells at G2/M phase in 125 I-CLDR group.
To observe the death of cells after irradiation after placenta blue staining and it is found that the percentage of positive cells in 125 I -CLDR group was obviously higher than in SDR group (Fig. 2a). To detect cell apoptosis after irradiation by ow cytometry instrument and it is found that apoptosis ratio was increased signi cantly in 125 I-CLDR group (Fig. 2b), the difference was signi cant statistically (Fig. 2c) and the difference was dose dependence (Fig. 2d). Detection of the expression of apoptosisrelated proteins by Western blot and it revealed that the expression of apoptosis-precursor protein Bim and anti-apoptotic protein bcl-2 were all increased, at the same time, the expression of caspase-3, a direct indicator of apoptosis, was up-regulated in both group, and the expression of them in 125 I-CLDR group was higher than in SDR group (Fig. 2e). After irradiation, cell cycle of KYSE150 cells was detected by ow cytometer (Fig. 2f), and it was found that the ratio of G2/M phase increased, and the difference was more signi cant in 125 I-CLDR group (Fig. 2g). Furthermore, we found that the expression of CylinB1 which is to regulation of G2/M point is decreased in 125 I -CLDR group, while it is increased in the SDR group (Fig. 2h).
Cells was larger and grainy in 125 I-CLDR group.
Cell morphology in each group after irradiation were observed by phase contrast microscope. It was found that the cells were signi cantly larger and the vacuoles as well as granules increased after 125 I-CLDR irradiation (Fig. 3a). The FS and SS were detected by ow cytometry after irradiation, the results showed that the volume and granulation of cells increased signi cantly after irradiation (Fig. 3b), and the difference was statistically signi cant in 125 I-CLDR group (Fig. 3c-d).
ER stress signal was elevated, and ATP concentration was decreased in 125 I-CLDR group.
After irradiation, the cells was loaded with Mito-Tracker Green (mitochondrial Green uorescent probe) and mitochondrial uorescence intensity was observed by uorescence microscope (Fig. 4a). The intensity of mitochondrial uorescence was analyzed quantitatively by ow cytometry (Fig. 4b). The intracellular ATP concentration was reduced signi cantly after irradiation in 125 I-CLDR group (Fig. 4c).
Analysis of the expression of ER stress marker GRP78/Bip1 and downstream activation pathway molecule PERK by Real-time PCR and it was found that the expression of them were up-regulated signi cantly at 3, 6 and 12 h in 125 I-CLDR group (Fig. 4d-e).
ROS levels was elevated, and DNA damage was aggravated in 125 I-CLDR group.
KYSE150 cells was loaded with DCFH-DA probe after irradiation and the ROS level of KYSE150 cells was analyzed by ow cytometry, it is found that ROS was signi cantly increased after irradiation in both two group, but the ROS level has increased dramatically in the 125 I-CLDR group though it is declining with the increase of time (Fig. 5a), the difference was signi cant statistically (Fig. 5b). The expression levels of DNA-pks, Ku70 and Ku80, which is the components of DNA repair protein complex, were detected by Western blot and it showed the expression of DNA-pks and Ku70 was signi cantly down-regulated, while there was no signi cant difference in Ku80 in the 125 I-CLDR group (Fig. 5c).
Activation of Akt-mTOR signaling pathways was decreased, and autophagy was increased in 125 I-CLDR group.
Acridine orange (AO) staining can be used to assess the level of intracellular autophagy [17]. Detection of the red-green signal ratio by ow cytometry and it showed that the proportion was up-regulated after irradiation, and the increase was more obvious in the 125 I-CLDR (Fig. 6a). Detection of autophagy-related protein expression by Western blot and it showed that autophagy related proteins LC3-II and ATG5 were signi cantly up-regulated in the 125 I-CLDR, while Beclin1 was not changed signi cantly (Fig. 6c). Detection of the expression of Akt-mTOR pathway by Western blot and it showed that the expression of p-Akt, p-mtor and p-S6 were signi cantly down-regulated, especially at 48 h after irradiation, while the expression of total mTOR was not changed signi cantly in 125 I-CLDR group (Fig. 6b).
Discussion strictures in China [18]. Our results showed that two modes of irradiation could both inhibit the proliferation viability of KYSE150 cells, while the inhibition effects in 125 I-CLDR were signi cantly stronger compared with SDR.
Cell cycle deregulation is one of the major hallmarks of cancer cells. The induction of cell cycle arrest may be an effective strategy to control the aberrant proliferation of cancer cells [19]. Our results demonstrated that 125 I radioactive seeds continuous low dose rate radiation causes the G 2 /M phase arrest of KYSE150 cells which were associated with a decrease of cyclin B1. Cyclin B1 is present in the nucleus during the G 2 /M phase, where it participates in the initiation and completion of mitosis. Lack of cyclin B1 causes lengthening of G2/M phases [20]. Thus, our results indicate that 125 I radioactive seeds continuous low dose rate radiation may modulate the expression of cell cycle-related proteins to induce G 2 /M phase arrest and inhibit the proliferation of KYSE150 cells consequently. However, decreased cyclin B1 expression is not necessarily linked to growth arrest. Thus, the sophisticated mechanism underlying the 125 I radioactive seeds continuous low dose rate radiation is still to be explored. On the other hand, autophagy defect also could lead to cell cycle arrest in G2/M phase, resulting in prolonged M phase and growth arrest [21]. Our results demonstrated that 125 I radioactive seeds continuous low dose rate radiation increases autophagy, which may also lead to cell cycle arrest in the G2/M phase and increases radiosensitivity.
Apoptosis is a cell death mechanism and can be activated by compounds targeting extrinsic and intrinsic (mitochondrial) pathways [22]. The extrinsic pathway is related to activation of cell surface death receptors such as FasL and TNF-R. Alternatively, the mitochondria-mediated intrinsic pathway may be initiated by many stress conditions. For the mitochondria-mediated intrinsic pathway, the release of cytochrome C from the mitochondrial membrane into the cytosol is fundamental to apoptosome formation and activation of the caspase family [23,25]. Members of the Bcl2 family also play a signi cant role in apoptosis by controlling mitochondrial membrane permeability and facilitating the passage of cytochrome C [26]. We found that 125 I radioactive seeds continuous low dose rate radiation induced an increase in Bcl2 levels, and further provoked sharp increase of Bim and the consequent release of cytochrome C into the cytosol. Based on our results, an imbalance of Bim and Bcl2 proteins after treatment with 125 I radioactive seeds continuous low dose rate radiation may lead to the resulting in the release of cytochrome C from the mitochondria into the cytosol to trigger caspase-3 activation, ultimately causing apoptosis in KYSE150 cells. Nevertheless, these ndings do not rule out the possibility that activation of extrinsic pathways may also be involved in 125 I radioactive seeds continuous low dose rate radiation-induced cell apoptosis, since we did not explore the effect on cell surface death receptors.
Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is another antiproliferation pathway that plays an essential role in determining cellular fate. Recently, studies demonstrated that apoptosis can induce autophagy by regulation of autophagy proteins as well as the formation of autophagosomes [12]. Our results con rm these ndings, given that 125 I radioactive seeds continuous low dose rate radiation promoted remarkable levels of autophagy activity as evidenced by the accumulation of autophagy-related proteins, such as LC3-II and ATG5. In mammalian cells, apoptosis and autophagy are regulated by several important intracellular signaling pathways, including the Akt-mTOR signaling pathway [27][28][29][30]. 125 I radioactive seeds continuous low dose rate radiation caused signi cant inhibition of Akt-mTOR and S6 phosphorylation. The Akt-mTOR signaling proteins exist in a phosphorylated form and suppress apoptosis and autophagy under normal conditions; however, when the levels of phosphorylated Akt-mTOR are downregulated, apoptosis and autophagy are triggered [31,32], indicating that downregulated of this pathway might underlie the 125 I radioactive seeds continuous low dose rate radiation induced excessive apoptosis and autophagy. In addition, 125 I-CLDR could also strong trigger caspase-3 activation, causing decreased esophageal cancer cells Akt-mTOR pathway signaling, which in turn increases autophagy. Apoptosis and autophagy are both highly regulated biological processes that may take place independently; however, in most circumstances, both processes are tightly connected by some common upstream signaling components. For example, there are common upstream signaling pathways between autophagy and apoptosis induced by ER stress, including ATF4, IRE1α, ATF6 and Ca2 + [12]. Therefore, our future studies will investigate whether both processes have substantial interconnections in inducing cell death under 125 I radioactive seeds continuous low dose rate radiation.
Compared with the X-ray single dose radiation, 125 I radioactive seeds continuous low dose rate radiation damaged the repopulation of KYSE150 cells which means there are some changes in subcellular structure of KYSE150 cells, such as endoplasmic reticulum and mitochondria. Mitochondria is the main organelles of intracellular ATP production, our results showed intracellular ATP concentration was signi cantly reduced, suggesting that 125 I radioactive seeds continuous low dose rate radiation caused damage to mitochondria of KYSE150 cells and the production of ATP was affected. Studies have demonstrated that reduction of ATP can activate AMPK, inhibiting mTOR signal and promoting the autophagy in cells [33]. Endoplasmic reticulum (ER) is a common cellular stress response that is triggered by a variety of conditions that disturb cellular homeostasis, and induces cell apoptosis [34]. Similarly, ionising radiation can activate ER stress signals [35], and radiotherapy has also been shown to induce autophagy in esophageal cancer cells through ER stress [36]. GRP78/Bip1 is an important signal molecule of ER stress, and PERK is one of the downstream signal molecules of ER stress [37]. We found that ER stress signals in esophageal cancer cell line KYSE150 were not signi cantly activated in the SDR group, while ER stress signals were signi cantly increased in the early stage after 125 I radioactive seeds continuous low dose rate radiation, suggesting that ER stress may be involved in autophagy induction of 125 I radioactive seeds continuous low dose rate radiation on KYSE150 cells. In addition, autophagy is closely related to the apoptosis induced by ER stress. ER and mitochondria are the main sources of free radicals, which play an essential role in the regulation of apoptosis [31] [31,38]. Our results showed that 125 I-CLDR could activate ER stress signal and damage mitochondrial which caused ROS levels continually to rise which led to apoptosis and autophagy increasing.
ROS are critical signaling molecules that regulate many signal transduction pathways, and excessive generation of ROS may interfere with cellular signaling pathways and activate subsequent apoptosis and autophagy processes. Therefore, ROS are considered one of the earliest hallmarks of these cell death processes compared to other markers [31]. For instance, ROS have been shown to lead to the induction of the intrinsic pathway of apoptosis by regulating the activity of the Bcl2 family protein via two main mechanisms: direct oxidation of Bcl2 leading to abrogation of its antiapoptotic activity and increasing the ubiquitination of Bcl2 [39]. ROS generation has also been shown to regulate Akt-mTOR signaling pathway activity and is subsequently associated with the classical markers of autophagy such as induction of LC3 and conversion of LC3-I to LC3-II [40]. Given that exposure to 125 I radioactive seeds continuous low dose rate radiation signi cantly enhanced ROS production in KYSE150 cells, it is possible that the 125 I radioactive seeds continuous low dose rate radiation triggered apoptosis, autophagy, and downregulated of the Akt-mTOR signaling pathway are related to excessive ROS generation. In addition, ROS can also induce DNA damage [41]. Recent studies have provided mechanistic insight into how ROS can also in uence the cellular response to DNA damage. DNA lesions is rstly important to identify the speci c lesions responsible for initiating apoptosis before the apoptotic executing pathways can be elucidated [42]. γ-H2AX represents the damage of DNA [43], our previous data proved that γ-H2AX increased signi cantly after 125 I radioactive seeds continuous low dose rate radiation [44]. DNA-PK Ku70 and Ku80 is a component of the DNA double strand breaks repair apparatus, and cells de cient them are hypersensitive to ionising radiation [45][46][47]. It suggests that DNA-PK may also have roles in apoptosis [48,49]. In sum, it is agree with our results that 125 I-CLDR caused ROS levels continually to rise, as a result, more severe DNA damage and impaired the repair ability to KYSE150 cells, which led to apoptosis increasing.
Taken together, our ndings con rmed that 125 I-CLDR could strongly inhibit KYSE150 cancer cells. Furthermore, we revealed that autophagy might be an important fate which Akt-mTOR pathway might involve in: 125 I-CLDR could activate ER stress signal and damage mitochondrial which caused ROS levels continually to rise which led to apoptosis with strong trigger caspase-3 activation, causing decreased esophageal cancer cells Akt-mTOR pathway signaling, which in turn increases autophagy, leading to cell cycle arrest in the G2/M phase and increases radiosensitivity. These ndings will be helpful to support the application of esophageal stent loaded with 125 I seeds in clinic.

Conclusion
In summary, our study provides a bene cial exploration of biological effects and the potential cellular  Figure 1 KYSE150 cells were more radiosensitive to 125I-CLDR than SDR. a. Cell dose survival curves after two types of irradiation. b. Cell activity in each group at different time points after 4Gy irradiation. c. Cell activity in each irradiation group after 72h of different irradiation doses. * P < 0.05; * * P < 0.01; * * * P < 0.001. irradiation. * P < 0.05; * * P < 0.01; * * * P < 0.001.

Figure 3
Cells became bigger and grainy in 125I-CLDR group. a. Cell morphology of each group at 72h after 4Gy irradiation by inverted phase contrast microscope (20×). b. Cytometry detection of FS and SS in each group at 72h after 4Gy irradiation. c. Cell size after irradiation. d. Cell granularity after irradiation. * * * P < 0.001.

Figure 4
ER stress signal was elevated and ATP concentration was decreased. a. Mitochondrial uorescence intensity in each experimental group at 24h after 4Gy irradiation. b. MFI of mitochondria in each group at 24h after 4Gy irradiation. c. intracellular ATP concentration at 24h after 4Gy irradiation. d. The mRNA level of GRP78/Bip1 after 4Gy irradiation. e. The mRNA level of PERK after 4Gy irradiation.* * P < 0.01; * * * P < 0.001. Figure 5 ROS levels was elevated and DNA damage was aggravated. a. ROS levels in each group at 72h after 4Gy irradiation. b.ROS levels in each group at different time points after 4Gy irradiation. c. Expression of DNApks, Ku70 and Ku80 in protein level after irradiation. * P < 0.05; * * P < 0.01; * * * P < 0.001.