The Protective Roles of ROS-Mediated Mitophagy on 125I Seeds Radiation Induced Cell Death in HCT116 Cells

For many unresectable carcinomas and locally recurrent cancers (LRC), 125I seeds brachytherapy is a feasible, effective, and safe treatment. Several studies have shown that 125I seeds radiation exerts anticancer activity by triggering DNA damage. However, recent evidence shows mitochondrial quality to be another crucial determinant of cell fate, with mitophagy playing a central role in this control mechanism. Herein, we found that 125I seeds irradiation injured mitochondria, leading to significantly elevated mitochondrial and intracellular ROS (reactive oxygen species) levels in HCT116 cells. The accumulation of mitochondrial ROS increased the expression of HIF-1α and its target genes BINP3 and NIX (BINP3L), which subsequently triggered mitophagy. Importantly, 125I seeds radiation induced mitophagy promoted cells survival and protected HCT116 cells from apoptosis. These results collectively indicated that 125I seeds radiation triggered mitophagy by upregulating the level of ROS to promote cellular homeostasis and survival. The present study uncovered the critical role of mitophagy in modulating the sensitivity of tumor cells to radiation therapy and suggested that chemotherapy targeting on mitophagy might improve the efficiency of 125I seeds radiation treatment, which might be of clinical significance in tumor therapy.


Introduction
Due to its low complication rates and high efficacy-which is comparable to that of radical surgery and external beam radiation therapy-125 I seeds implantation brachytherapy has become one of the most popular treatment modalities for many unresectable carcinomas and locally recurrent cancers [1][2][3][4][5][6][7]. A series of studies have explored the molecular mechanisms through which 125 I seeds radiation exerts anticancer activity. Most studies have focused on apoptosis and cell cycle arrest resulting from DNA damage after exposure to 125 I seeds radiation [8][9][10]. However, there is growing evidence that mitochondria, which account for up to 30% of the total cell volume, may also be important extranuclear mediators of the cytotoxic effects of radiation [11,12]. Healthy mitochondria act as powerhouses, producing energy for cell function through the TCA cycle (tricarboxylic acid cycle) and oxidative phosphorylation [13]. Damage to mitochondria can lead to cell death and a variety of other problems [14].
Mitophagy, which refers to the selective removal of damaged or unwanted mitochondria, is crucial for mitochondrial quality control following stresses such as starvation, photo damage, hypoxia, and ROS production [15]. Certain physiological stresses can induce mitochondrial damage, which can cause oxidative stress and cell death triggered by the production of ROS from the mitochondrial electron transport chain (ETC). The high level of ROS can be selectively sequestered in autophagosomes and subjected to lysosomal degradation in a process termed mitophagy to promote cellular homeostasis and survival [16]. Mitophagy can thus alleviate cell injury following stress, acting as an effective antioxidant pathway and clearing increased mitochondrial or cytosolic ROS. Mitophagy has been reported to be involved in tumor resistance to therapy by maintaining healthy mitochondria [17,18].
Mitophagy is mediated by specific receptors such as NIX, BNIP3, and FUNDC1 in mammalian systems [19]. BNIP3 and NIX are two important mitochondrial stressor sensors with homology to BCL2 in the BH3 domain. Once 2 Oxidative Medicine and Cellular Longevity mitophagy is triggered, BNIP3 and NIX are selectively recruited to dysfunctional mitochondria and then bound to the conserved LC3-interacting region (LIR) of LC3-II present on autophagosome to promote removal of damaged mitochondria by the autophagosome [16,20,21]. In addition, both BNIP3 and NIX facilitate mitophagy by promoting the release of Beclin1 from the Beclin1-Bcl2/Bcl-X complex [22]. NIX and BNIP3, two hypoxia-inducible proteins that target mitochondria for autophagosomal degradation, are the transcription products of HIF-1 [23]. HIF-1 is an important predictor of tumor progression for several types of solid cancers and can regulate the transcription of a number of genes (such as BNIP3 and NIX) that are involved in mitophagy and apoptosis [24]. Several studies have shown that elevated mitochondrial ROS increases the expression of HIF-1 and its target genes BNIP3/NIX [17,25].
In the present study, we have focused on the regulatory roles of autophagy in the radiosensitivity of tumors to 125 I seeds irradiation as well as the molecular mechanisms that underlie 125 I seeds radiation induced mitophagy. We found that mitophagy significantly decreased the sensitivity of tumor cells to 125 I seeds irradiation. Thus, targeting mitophagy combined with radiotherapy may improve the therapeutic efficiency in clinical patients with tumors, which needs to be confirmed by the clinical studies.

125 I Radiation Source.
The 125 I seeds used as the radiation source in this study were purchased from Ningbo Junan Pharmaceutical Technology Company (Ningbo, Zhe Jiang province, China) and were installed in an in-house model developed in our laboratory for in vitro 125 I seeds radiation. A detailed description of this model has been published earlier [26,27]. 125 I seeds have a half-life of ∼59.4 days. The experimentally applicable radiation dose rate of 125 I seeds ranged from 2.77 cGy/h to 1.385 cGy/h, which is approximate to the clinically applicable radiation dose rate used in permanent LRC brachytherapy. This model was validated by using thermoluminescent dosimetry (TLD) measurement, and the irradiation time was calculated according to the absorbed dose and initial radiation dose rate. The control cells were seeded and harvested at the same time points as the irradiated cells.

Reagents and Antibodies.
Annexin V-FITC apoptosis detection kit I was purchased from Beijing Zoman Biotechnology (Beijing, China); the ROS assay kit and mitochondrial membrane potential assay kit with JC-1 were purchased from Beyotime (Shanghai, China); the mitochondrial ROS indicator MitoSOX was purchased from Invitrogen (Carlsbad, CA, USA); N-acetylcysteine (NAC) and chloroquine (CQ) were purchased from Sigma-Aldrich; 3-methyladenine (3-MA) was purchased from Selleck Chemicals LLC (Houston, TX, USA); Ly294002 was purchased from Invitrogen; Antimycin A was purchased from Sigma-Aldrich. LipofectamineTM 2000 was purchased from Invitrogen. Diphenyleneiodonium chloride (DPI) was purchased from Gene Operation Co., Ltd. (Wuxi, China). cDNA reverse transcription kit and real-time PCR kit were purchased from Takara Biotechnology (Dalian, China); and goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, anti--actin, and anti-LC3 polyclonal antibody were obtained from Sigma-Aldrich.

Cell Culture. Human colorectal cancer cells-HCT116
cells-were kindly provided by Dr. Xiaojuan Du of Peking University Health Science Center, Beijing, China. The HCT116 cells were grown in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin, and 100 g/mL streptomycin, in a humidified atmosphere containing 5% CO 2 at 37 ∘ C.

Clonogenic Survival
Assay. HCT116 cells in appropriate numbers were seeded in 35 mm dishes to ensure the formation of about 100 colonies per dish. After 24 hours, the cells were exposed to 125 I seeds irradiation in the model. At the desired time points, the cells were removed from the radiation source and cultured for 10-12 days [28]. They were then fixed with absolute ethanol and glacial acetic acid in a 1 : 1 ratio at 4 ∘ C for 30 minutes and stained with methylene blue for 2 hours. Finally, the samples were washed with cold phosphatebuffered saline (PBS) and dried at room temperature. The number of colonies per dish was counted and the survival fraction (SF) was calculated as PE (tested group)/PE (0-Gy group), where PE was (colony number/number of inoculating cells) × 100%. The experiment was repeated two times in duplicate. The survival fraction values were presented as the mean ± SD for the respective radiation doses.

Western Blot Analysis.
Cells were lysed in a radio immunoprecipitation assay (RIPA) buffer (50 mM of pH-7.4 Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, and 1 mM of pH-7.4 EDTA) with protease and phosphatase inhibitor cocktails for 15 minutes on ice and then centrifuged for 30 minutes at 12,000 and 4 ∘ C. Bicinchoninic acid (BCA) protein assay kits (Beyotime Biotech, Shanghai, China) were used to measure the protein concentrations of protein supernatant. Equal amounts of the protein samples were denatured at 95 ∘ C for 5 minutes and then separated on dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond-P; Amersham, Buckinghamshire, UK) for 2 hours at 100 V on ice. Membranes were blocked with 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 1 hour at room temperature and then incubated overnight at 4 ∘ C with the indicated primary antibody. After washing thrice with TBST, the membranes were blotted with the respective secondary antibody for 1 hour at room temperature. The membranes were incubated with enhanced chemiluminescence (ECL) detection kits (Pierce Biotechnology, Rockford, IL, USA) to visualize proteins. Expression of human -actin was detected as a loading control. labeled Annexin V and propidium iodide (PI) for 15 minutes in binding buffer according to the manufacturer's instructions to detect cell apoptosis. Then, fluorescence was detected by flow cytometry (Beckman Coulter, Miami, FL, USA) to assay the percentage of cell apoptosis.

Flow Cytometry Analysis of Intracellular ROS and
Mitochondrial ROS. The ROS assay kit was used to assess intracellular ROS levels. After exposure to 2 Gy of radiation adherent HCT116 cells were washed with serum-free RPMI 1640 medium and stained with 5 M of DCFH-DA at 37 ∘ C for 30 minutes. Then, labeled HCT116 cells were trypsinized and analyzed by flow cytometry.
MitoSOX6 Red mitochondrial superoxide indicator selectively targets mitochondria to detect mitochondrial ROS. Postirradiation adherent HCT116 cells were washed with serum-free RPMI 1640 medium and incubated with 10 M of MitoSOX Red mitochondrial superoxide indicator at 37 ∘ C for 30 minutes. After the treatment, HCT116 cells were trypsinized and analyzed by flow cytometry.

Flow Cytometry Analysis of Mitochondrial Membrane
Potential. One hundred thousand HCT116 cells were collected and washed once with PBS after irradiation. Mitochondrial membrane potential was detected by flow cytometry using a mitochondrial membrane potential assay kit with JC-1. Under normal conditions, JC-1 accumulates in the mitochondrial matrix in the form of J-aggregates, which emit a red fluorescence; however, when the mitochondrial membrane potential collapses, JC-1 exists as a monomer that cannot enter mitochondria and emits a green fluorescence. The ratio of the JC-1 red signal (J-aggregates) to the JC-1 green signal (monomer) was used as a measure of the mitochondrial membrane potential. Data were analyzed by FCS Express V3 software (De Novo Software, Glendale, CA, USA).

Immunofluorescence
Analysis. Cells were cultured on coverslips in 35 mm dishes. After the indicated dose of 125 I seeds irradiation, the cells were fixed with 4% formaldehyde in PBS for 15 minutes at 37 ∘ C. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 15 minutes on ice and subsequently blocked by goat serum. After incubation with primary antibodies overnight at 4 ∘ C, LC3 and TIM23 were detected with the polyclonal anti-LC3 antibody and the monoclonal antibody against TIM23, respectively. LC3 were detected with FITC-(Fluorescein Isothiocyanate-) conjugated goat anti-rabbit IgG. TIM23 were labeled with TRITC-(Tetramethylrhodamine Isothiocyanate-) conjugated goat anti-mouse IgG. Nuclei were stained with 4 ,6-diamidino-2phenylindole (Beyotime, China). Fluorescence images were captured with an LSM 510 Zeiss confocal microscope (Carl Zeiss Jena, Germany).

Detection of Cellular ATP Levels.
Firefly luciferase-based ATP assay kit (Beyotime, China) was used to measure cellular ATP level. After exposure to the 2 Gy of radiation, HCT116 cells were lysed and centrifuged at 12,000 for 5 minutes. Cell lysates (100 L) were mixed with 100 L of ATP detection working dilution in a 96-well plate, and the cellular ATP levels were measured using the BioTek Synergy 2 Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). A BCA assay kit was used to determine the protein concentration of cell lysates (1 L) of each group. The ratio of cellular ATP level to the protein concentration was used to evaluate total ATP level.

Statistical Analysis.
Experiments were performed at least three times in duplicate. Statistical analyses were performed using the statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The two-tailed -test was applied to compare group means. Statistical significance was set at ≤ 0.05.

Inhibition of Autophagy Enhanced the Sensitivity of Tumor
Cells to 125 I Seeds Radiation. To investigate how autophagy affects the radiosensitivity of tumor cells, clonogenic survival assay was performed to assess the response to 125 I seeds radiation with and without pretreatment with autophagy inhibitors. HCT116 cells were pretreated with the autophagy inhibitors 3-MA (0.5 mM) or Ly294002 (10 M) 2 hours prior to being exposed to 2 Gy and 4 Gy 125 I seeds radiation. The ratios of surviving fraction between 3MA group and control group were 0.81, 0.48 for total doses of 2 and 4 Gy, respectively ( Figure 1(a)). What is more, the ratios of surviving fraction between Ly294002 and control group were 0.83 and 0.59 for total doses of 2 and 4 Gy, respectively (Figure 1(b)). We found a statistically significant decrease in the clonogenic survival fraction in HCT116 cells pretreated with 3-MA or Ly294002 at 2 Gy and 4 Gy ( ≤ 0.05), suggesting that these autophagy inhibitors have a radiosensitizing effect on HCT116 cells. Neither 3-MA nor Ly294002 was specific autophagy inhibitor.
We further investigated whether knockdown of LC3 expression, a conserved gene required for mammalian autophagy, affected the radiosensitivity of HCT116 cells. Clonogenic survival assay was used to assess the response to 125 I seeds Oxidative Medicine and Cellular Longevity      radiation after transfection with LC3 siRNA or nonrelated control siRNA duplex. The expression of LC3 in HCT116 cells transfected with LC3 siRNA duplex significantly decreased compared to nonrelated control siRNA group (Figure 1(c)). Following irradiation the ratios of surviving fraction between LC3 siRNA group and nonrelated control siRNA were 0.66, 0.51 for total doses of 2 and 4 Gy, respectively. We found that knockdown of LC3 expression sensitized HCT116 cells to 125 I seeds radiation at 2 Gy and 4 Gy ( ≤ 0.05, Figure 1(d)), suggesting that inhibition of autophagy had a radiosensitizing effect on HCT116 cells.

Inhibition of Autophagy Promoted Apoptosis Induced by 125 I Seeds Radiation
. In previous studies we had shown that 125 I seeds radiation induces apoptosis in colorectal cancer cells. To examine whether the enhanced radiosensitivity of autophagy inhibitors was due to increased apoptosis, the percentage of apoptotic cells was measured by double staining with Annexin V and PI and assayed by flow cytometry. We found that the proportion of PI − /Annexin V + and PI + / Annexin V + cell population in HCT116 cells increased from 13.9% to 19.5% following treatment with 125 I seeds radiation plus the autophagy inhibitor 3-MA ( ≤ 0.05, Figures 2(a) and 2(b)). To confirm this phenomenon, the same experiments were duplicated with autophagy inhibitor Ly294002 instead of 3MA (Figure 2(c)). The percentage of apoptosis increased from 19.1% to 30.1% in cells treated with 125 I seeds radiation plus Ly294002 ( ≤ 0.05, Figure 2 ( ≤ 0.05, Figures 2(e) and 2(f)). These results suggested that inhibition of autophagy increased the sensitivity to 125 I seeds radiation by enhancing apoptosis.

125 I Seeds Radiation Induced Mitophagy in Tumor Cells.
To investigate whether 125 I seeds radiation could induce autophagy in HCT116 cells, LC3-I and LC3-II were used as autophagy markers to estimate the induction of autophagy and the overall autophagic flux to permit correct interpretation of the results. Immunofluorescence staining with anti-LC3 antibody was performed with HCT116 cells following exposure to 0, 0.5, 1, and 2 Gy of 125 I seeds radiation (Figure 3(a)). As shown in Figure 3(b), the number of LC3 punctuations per cell increased gradually after exposure to 0, 0.5, 1, and 2 Gy of 125 I seeds radiation. Western blot analysis, performed to examine the transformation of soluble LC3 (LC3-I) to the LC3-II form, showed that the ratio of LC3-II to LC3-I increased gradually after treatment with indicated doses of 125 I seeds radiation (Figure 3(c)). This provided further evidence that 125 I seeds radiation induced autophagy in a dosedependent manner in HCT116 cells.
Autophagy involves two key processes: the formation of autophagosomes and the formation of autolysosomes. In the initial stage, membrane distant from either the Golgi or ER sequesters cellular components to form autophagosome. At a relatively late stage of autophagy, autophagosome fuses with the lysosome to form an autolysosome, where LC3-II and sequestered cellular components were degraded by lysosomal hydrolytic enzymes [31][32][33]. There are two causes for the accumulation of LC3-II: one is increase of autophagosome formation and the other is inhibition of the formation of autolysosomes which is the site for degradation of autophagosomes [34]. To identify which of these two mechanisms was responsible for the autophagy caused by 125 I seeds radiation, we then investigated whether elevated LC3-II levels were due to the upregulation of autophagosome formation or the escape of autophagosome-lysosome fusion. Chloroquine (CQ), which acts as a late-stage autophagy inhibitor by blocking autophagosome-lysosome fusion, was used to treat the irradiated HCT116 cells for 6 hours before harvest, and the cells were examined under a confocal microscope (Figure 4(a)). As expected, treatment with CQ could make further increase of the number of LC3-II punctuations per cell induced by 125 I seeds radiation ( ≤ 0.05, Figure 4(b)). Moreover, whole cell lysates from treated cells were analyzed by Western blotting in parallel at the same time. The results showed that CQ could make further increase of the ratio of LC3-II to LC3-I induced by 125 I seeds radiation (Figure 4(c)). These suggested that 125 I seeds radiation induced LC3-II punctuation accumulation was due to the increase of autophagosome formation rather than inhibition of lysosome-autophagosome fusion.
We also used electron microscopy, the gold standard for detecting autophagy, to investigate whether 125 I seeds radiation induced autophagy in HCT116 cells. Transmission electron microscope (TEM) analysis revealed that a large number (c) HCT116 cells were exposed to 125I seeds radiation (2 Gy) with or without chloroquine for 6 hours, respectively. The ratio of LC3II/I was measured by Western blotting analysis. * indicates significant difference ( ≤ 0.05) as compared to the control group.
of mitochondria in autophagosome-like vacuoles appeared in the cytoplasm after exposure to 2 Gy of 125 I seeds radiation ( Figure 5(a)). This phenomenon was consistent with the result of immunofluorescence analysis. Following exposure to 2 Gy of 125 I seeds radiation, the cells were double indirect immunofluorescence staining with an anti-TIM23 antibody (red) and an anti-LC3 antibody (green) to detect the mitochondrial marker protein TIM23 and autophagosome marker protein LC3, respectively. The confocal image showed LC3 punctuation colocalized with mitochondria after 125 I seeds radiation. Besides, this colocalization induced by 125 I seeds radiation increased significantly after treatment with CQ, which could block autophagosome-lysosome fusion to amplify autophagy ( Figure 5(b)). These results further supported that 125 I seeds radiation induced robust mitophagy by promoting autophagosome formation.

125 I Seeds Radiation Impaired Mitochondria and Induced
Oxidative Stress. To study whether 125 I seeds radiation could induce mitochondrial damage, we examined a series of indices that can reflect mitochondrial dysfunction. Immunofluorescence staining was performed with anti-TIM23 antibody to detect the mitochondrial marker protein TIM23. The fluorescence confocal image showed that 125 I seeds radiation induced changes in mitochondrial morphology, altering it from the elongated linear network form to a floccular and dot form (Figure 6(a)). In addition, mitochondrial membrane potential was analyzed with flow cytometry (Figure 6(b)); we found that 125 I seeds radiation impaired mitochondrial function and induced decrease in mitochondrial membrane potential ( ≤ 0.05, Figure 6(c)). It has been reported that mitochondrial dysfunction induced by radiation results in reduction of ATP synthesis owing to disruption of the proton gradient across the mitochondrial membrane [35,36]. We next investigated whether 125 I seeds radiation induced mitochondrial dysfunction led to alterations in ATP production. Cellular ATP level was evaluated by firefly luciferase-based ATP assay kit; the results showed the level of total ATP decreased significantly after Oxidative Medicine and Cellular Longevity HCT116 cells were exposed to 125 I seeds radiation alone or in combination with CQ (40 mol) for 6 hours. Following fixation, the cells were immunostained with an anti-TIM23 antibody (red) and an anti-LC3 antibody (green) and visualized by confocal microscopy. The representative confocal microscopy images are shown. Small squares denoted the area zoomed in region of the images and white arrows denoted mitophagy punctuation induced by 125 I seeds radiation.
125 I seeds radiation ( ≤ 0.05, Figure 6(d)). After exposure to 2 Gy of 125 I seeds radiation, cells were stained with the mitochondrial ROS and subjected to flow cytometry analysis ( Figure 6(e)). Results showed that 125 I seeds radiation elevated levels of mitochondrial ROS in HCT116 cells ( ≤ 0.05, Figure 6(f)). Similarly, postirradiation cells were stained with the intracellular ROS probes and analyzed by flow cytometry (Figure 6(g)). Results showed that 125 I seeds radiation elevated levels of intracellular ROS in HCT116 cells ( ≤ 0.05, Figure 6(h)). Together, these data suggested that 125 I seeds radiation induced oxidative damage in mitochondria.

Elevated ROS Is Critical for Both Mitophagy and Apoptosis Induced by 125 I Seeds Radiation.
There is increasing evidence that oxidative stress is responsible for autophagy [25]. To confirm the role of ROS in mitophagy and apoptosis induced by 125 I seeds radiation, we treated HCT116 cells with N-acetyl-L-cysteine (NAC; a ROS scavenger), 125 I seeds radiation, or both. Mitochondrial ROS and intracellular ROS generation were then analyzed using the corresponding probe for flow cytometry analysis. As shown in Figure 7(a), NAC decreased 125 I seeds radiation induced accumulation of mitochondrial ROS. Statistical results from three independent experiments in duplicate are shown in Figure 7(b) ( ≤ 0.05). Besides, NAC decreased accumulation of intracellular ROS induced by 125 I seeds radiation (Figure 7(c)). Experiments were duplicated three times and data are shown in Figure 7(d) ( ≤ 0.05). Protein levels of LC3-II and LC3-1 were determined by Western blotting in parallel. As shown in Figure 7(e), the rise in ratio of LC3-II to LC3-1 that was induced by 125 I seeds radiation lowered when the cells were treated with NAC. Moreover, immunofluorescence staining showed that NAC could decrease the LC3 punctuation accumulation induced by 125 I seeds radiation (Figure 7(h)). We next utilized mitochondrial respiratory chain inhibitor to investigate the role of mitochondrial ROS on mitophagy induced by 125 I seeds radiation. DPI (50 uM) and Antimycin A (50 uM), which act as mitochondrial respiratory chain inhibitor by blocking electron flow at mitochondrial respiratory chain complexes I and III, respectively, were used to treat the irradiated HCT116 cells for 18 hours before harvest. As shown in Figures 6(f)-6(h), the treatment of DPI and Antimycin A could promote the accumulation of mitochondrial ROS and further increase mitophagy induced by 125 I seeds radiation.
We also used NAC, the ROS scavenger, to examine whether apoptosis correlated with the level of ROS induced by 125 I seeds radiation. Cells were doubled stained with the probes Annexin V and PI to evaluate the percentage of apoptosis by flow cytometry analysis (Figure 8(a)). Flow cytometry analysis showed that NAC decreased the proportion of apoptosis induced by 125 I seeds radiation ( ≤ 0.05, Figure 8(b)), indicating that ROS play an important role in mitophagy and apoptosis in HCT116 cells.

The Accumulation of Mitochondrial ROS Induced by 125 I Seeds Irradiation
Is Critical for mRNA Expression of HIF-1 and BNIP3/NIX. We investigated whether 125 I seeds radiation influenced the expression of HIF-1 and its target genes. Real-time PCR was performed to detect the expression of HIF-1 and its target genes BNIP3 and NIX. As shown in    Figure 9(a)), BNIP3 ( ≤ 0.05, Figure 9(b)), and NIX ( ≤ 0.05, Figure 9(c)).
To determine whether ROS induced by 125 I seeds radiation can raise the expression of BNIP3 and NIX, which is a crucial signal transduction pathway to regulate mitophagy, we examined the expression of HIF-1 , BNIP3, and NIX at the mRNA level by using real-time PCR. As shown in Figure 9, NAC decreased the expression of HIF-1 ( ≤ 0.05, Figure 9(d)), BNIP3 ( ≤ 0.05, Figure 9(e)), and NIX ( ≤ 0.05, Figure 9(f)). And the treatment of Antimycin A could further increase the mRNA expression of HIF-1 ( ≤ 0.05, Figure 9(g)), BNIP3 ( ≤ 0.05, Figure 9(h)), and NIX ( ≤ 0.05, Figure 9(i)) induced by 125 I seeds radiation, indicating that mitochondrial ROS induced by 125 I seeds irradiation might be critical for mitophagy and apoptosis through HIF-1 -BNP3/NIX pathways.

Discussion
Presently available evidence is in favor of autophagy as a novel cancer therapy target [37]. Autophagy is a dynamic process characterized by lysosomal degradation of cytoplasmic components such as cytoplasmic proteins and intracellular organelles [38]. The process requires the formation of the double-membrane autophagosome, which sequesters and transports the cytoplasmic cargo to the lysosome for degradation [39]. Recent studies show that autophagy plays an important role in cancer cell fate decisions following stress condition such as starvation, hypoxia, and chemotherapy [40]. Several studies have shown that elevated level of autophagy is associated with radio-resistance of various cancers [41,42]. Multiple clinical trials have been performed to study the effects of conventional anticancer radiotherapy combined with autophagy inhibitors [37]. A number of autophagy inhibitors, such as chloroquine (CQ), hydroxychloroquine (HCQ), and 3-methyladenine (3-MA), have been applied in clinical trials. The combination of autophagy inhibitors and radiotherapy has been shown to provide survival benefits and increased lifespan in patients with cancer [43][44][45]; however, the specific role of autophagy induced by 125 I seeds radiation has not yet been determined.
In the present study, we first showed that both autophagy inhibitors 3-MA and Ly294002 could decrease the survival fraction of HCT116 cells after 125 I seeds radiation (Figures 1(a)  and 1(b)). Moreover, knockdown of LC3 expression sensitizes HCT116 cells to 125 I seeds radiation, indication that inhibition of autophagy has a radiosensitizing effect on HCT116 cells (Figure 1(d)). Next, we explored how autophagy inhibitors modified the response of HCT116 cells to 125 I seeds radiation. We found that inhibition of autophagy increased the proportion of apoptosis induced by 125 I seeds radiation (Figures 2(a)-2(f)). Autophagy induced by multiple forms of cellular stress-such as hypoxia, ROS accumulation, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens-interact with apoptosis to control cell survival and death [46]. We therefore consider it possible that 125 I seeds radiation triggers autophagy to protect colorectal cancer cells from apoptotic cell death. LC3 is an important marker for detection of autophagy. It exists in two forms: LC3-I and LC3-II. LC3-I, a soluble cytosolic protein, is initially synthesized in an unprocessed form, proLC3. Once autophagy is triggered, LC3-I is modified to LC3-II which is recruited to autophagosomal membranes and showed punctuation-like distribution. The amount of LC3-II punctuation is reliably correlated with the extent of autophagosome formation [47]. LC3 lipidation can be evaluated by observing the ratio of LC3-II to LC3-1 on immunoblots. The accumulation of autophagosome can be measured by TEM image analysis or immunofluorescence analysis of LC3 punctuation [34]. In the present study, we identified 125 I seeds radiation induced autophagy (Figures 3(a)-3(c)) and autophagic flux (Figures 4(a)-4(c)) by immunofluorescence and Western blotting for the lipidation of LC3, respectively. Furthermore, the TEM images revealed the presence of autophagic vacuoles with mitochondria inside (Figure 5(a)), which was consistent with the results of immunofluorescence analysis ( Figure 5(b)). On the basis of these findings we concluded that 125 I seeds radiation robustly induced mitophagy.
Mitophagy is a cargo-specific autophagy that selectively removes damaged mitochondria [48]. As a cell death executor, in addition to its more traditional roles in bioenergetics and metabolism, mitochondria are now recognized to play an important role in radiation induced cellular responses [11]. Mitochondrial damage could induce mitophagy to control mitochondrial number and quality to match metabolic demands [49]. Mitochondria are crucial energy organelles where large amounts of ATP are produced using the electrochemical gradient generated across mitochondrial inner membrane by the ETC [50]. Mitochondrial membrane potential supplies proton-motive force to promote translocation of proton from mitochondrial matrix to the intermembrane space, which is necessary for ATP synthesis. In addition to being the power center of the cell, mitochondria also generates reactive oxygen species (ROS), which originate from the electron leak from ETC. It has been well known that damage of mitochondria presented typical alterations   in mitochondrial morphology accompanied by biochemical changes, such as decreased ATP, decreased mitochondrial membrane potential, and increased mitochondrial ROS [51]. We therefore measured mitochondrial parameters after exposure to 2 Gy of 125 I seeds radiation. We demonstrated that 125 I seeds radiation had a significant impact on mitochondrial function, causing cell-specific disruption of mitochondrial membrane potential, reduction in the production of ATP, and accumulation of both cellular and mitochondrial ROS (Figure 6). These results indicated that oxidative stress induced by 125 I seeds radiation increased the concentration of reactive oxygen species and led to mitochondrial dysfunction by inhibiting mitochondrial respiration. The sources of mitochondrial ROS overproduction are the electron leak from mitochondrial ETC. Complexes I and III of ETC are regarded as major sites of mitochondrial ROS producers [52]. Specific inhibitors can inhibit complexes I and III of ETC and induce the leak of electrons, which results in a higher rate of mitochondrial ROS [53]. We used ROS inhibitor or mitochondrial ROS enhancer to influence the level of mitochondrial ROS and investigate the role of mitochondrial ROS in mitophagy induced by 125 I seeds radiation. As a ROS scavenger NAC can block intracellular and mitochondrial ROS induced by 125 I seeds radiation. After treatment with NAC, LC3-II accumulation induced by 125 I seeds radiation decreased (Figures 7(a)-7(e)). Antimycin A, which blocks electron flow at complex III, is well known for increasing mitochondrial ROS and further increases mitophagy induced by 125 I seeds radiation [54]. Diphenyleneiodonium (DPI) acting as a cellular superoxide production inhibitor by inhibition of mitochondrial respiratory chain complex I NADH reductase resulted in the potential to increase the production of mitochondrial superoxide [55]. As shown in Figures 7(f)-7(h), the treatment of DPI and Antimycin A raised mitochondrial ROS and further raised mitophagy induced by 125 I seeds radiation. These results suggested that the accumulation of mitochondrial ROS induced by 125 I seeds radiation is an important intracellular factor that contributes to trigger mitophagy.
ROS can damage cellular components and activate numerous signaling pathways to induce mitophagy. HIF-1 activated by ROS is an important signaling protein that regulates mitophagy by transcriptional regulation of its target genes BNIP3 and its homologue NIX [17,56]. ROS upregulates HIF-1 transcription by activating nonhypoxic factors in a redox-sensitive manner [57]. Therefore, we investigated whether the accumulation of ROS induced by 125 I seeds radiation activated HIF-1 and its target genes BNIP3 and NIX and thus triggered mitophagy. We demonstrated that 125 I seeds radiation elevated mitochondrial ROS and the expression of HIF-1 and its target genes BNIP3 and NIX (Figures  9(a)-9(c)). It was noteworthy that inhibition of ROS by NAC not only reduced the ROS level but also the expression of HIF-1 and its target genes BNIP3 and NIX (Figures 9(d)-9(f)). Furthermore, mitochondrial electron transport chain inhibitors (DPI and Antimycin A) raised the accumulation  Figure 9: Mitochondrial ROS induced by 125 I seeds irradiation is critical for the expression of HIF-1 and its target gene. (a-c) HCT116 cells were exposed to 2 Gy of 125 I seeds radiation. Real-time PCR was performed to detect the expression of HIF-1 (a), BNIP3 (b), and NIX (c).
(d-f) HCT116 cells were pretreated with and without 5 mM of NAC for 4 hours and then either mock exposed or exposed to 2 Gy of 125 I seeds radiation. The level of mRNA of HIF-1 (d), BNIP3 (e), and NIX (f) was detected by real-time PCR after treatment. (g-h) The irradiated HCT116 cells were treated with or without 50 uM of Antimycin A for 18 hours before harvest. The level of mRNA of HIF-1 (g), BNIP3 (h), and NIX (i) was detected by real-time PCR after treatment. * indicates a significant difference ( ≤ 0.05) between NAC treated and untreated cells.
of mitochondrial ROS level and the expression of HIF-1 and its target genes BNIP3 and NIX (Figures 9(g)-9(i)). These results suggested that mitochondrial ROS induced by 125 I seeds radiation upregulated the expression of HIF-1 and its target genes BNIP3 and NIX to trigger mitophagy. BNIP3 and NIX can interact with BCL2 and BCL-XL to play a proapoptotic role. Deregulation of BNIP3 or NIX expression is associated with apoptosis [58]. We therefore hypothesized that ROS accumulation induced by 125 I seeds radiation might play a critical role in cell fate determination. To test this theory, we used NAC to explore how ROS accumulation affects cell fate. Strikingly, NAC inhibited the percentage of apoptosis induced by 125 I seeds radiation (Figures  8(a) and 8(b)), suggesting that mitochondrial ROS accumulation induced by 125 I seeds radiation not only triggered mitophagy through upregulation of the expression of HIF-1 and its target genes but was also involved in the cellular decision of apoptosis.

Conclusions
Our results show that the high level of mitochondrial ROS generated by 125 I seeds radiation induced mitochondrial damage upregulates the expression of HIF-1 and its target genes BNIP3 and NIX and triggers mitophagy. Mitophagy serves to reduce oxidative damage and ROS levels through the removal of damaged mitochondria, thus protecting HCT116 cells from apoptosis.