Introduction
Nasopharyngeal carcinoma (NPC), which has the highest incidence in Southeast Asia, remains one of the leading causes of cancer-related mortality in the Cantonese region of Southern China. Radiotherapy is the major treatment for NPC (1). However, radioresistance remains a serious obstacle to successful treatment in many cases (2,3). Thus, an effective way to improve radiation sensitization of nasopharyngeal carcinoma is by identifying the mechanisms involved in NPC radiation resistance. Autophagy is a catabolic process involved in cell growth, development and homeostasis, maintaining a balance between the synthesis, degradation and subsequent recycling of cellular products. The process starts with the formation of the autophagosome or autophagic vacuole. The vacuole membrane then fuses with the lysosomal compartment to deliver the contents into the organelle lumen, where they are degraded and the resulting macromolecules are recycled (4,5). Recent studies have revealed the importance of autophagy in the immune response, inflammatory response, cardiovascular disease, cancer and neurodegenerative disease (6–9). However, the major role that autophagy plays in cancer is controversial (10). Genetic knockout of autophagy-related genes enhances the development of spontaneous malignancies whereas mice deficient in autophagy-related genes exhibit sensitivity to radiotherapy, chemotherapy or immunotherapy (10–13).
Poly(ADP-ribose) polymerase-1 (PARP-1), activated by DNA strand breaks, participates in the DNA repair process physiologically (14). PARP-1 has been implicated in the G2/M cell cycle checkpoint and has been shown to play an important role in the recovery of DNA damage in in vivo studies. Specifically, PARP-1 has been demonstrated to be an important mediator of DNA base excision repair, which is important in the repair of single-stranded breaks. Moreover, PARP-1 is also known to bind the more lethal double-stranded DNA breaks (15–17). IR induces DNA strand breaks, which mediate its cytotoxic effects. Excessive activation of PARP-1 mediates ionizing radiation (IR)-induced cell death under the status of oxidative stress and DNA damage (18,19). However, it remains elusive whether and how PARP-1 activation is involved in autophagy and the exact function of PARP-1-mediated autophagy under oxidative stress and DNA damage in CNE-2 cells. Recently, a study demonstrated that IR induces autophagy through a novel autophagy signaling mechanism linking PARP-1 activation to the mammalian target of rapamycin (mTOR) pathway in prostate cancer cell (20).
Questions concerning the role that autophagy plays in response to IR in CNE-2 cells and whether it promotes cell survival or induces cell death and the methods to promote radiosensitivity by regulating autophagy are yet unanswered. Thus, it is not surprising that the bulk of research has focused on the regulation of autophagy in a variety of models (8,11,20–26). Our data illustrate the importance of IR-induced autophagy, and we demonstrated that autophagic inhibition may be used as a new method to promote radiosensitivity. PARP-1-mediated autophagy plays a cytoprotective role in IR-induced CNE-2 cell death as suppression of autophagy greatly sensitizes IR-induced cell death.
Materials and methods
Cell culture and irradiation conditions
CNE-2, a human NPC cell lines, was purchased from the Cancer Hospital of Shanghai Fudan University, and was cultured in RPMI-1640 medium (HyClone, USA) supplemented with 10% fetal calf serum (Gibco, USA), penicillin (100 U/ml), streptomycin (100 U/ml) and maintained at 37°C in a humidified incubator with 5% CO2. All irradiations were delivered using 6-MV X-rays with a linear accelerator (Elekta, Sweden) with a dose rate of 220 cGy/min; SSD, 100 cm.
Reagents
Chloroquine diphosphate (CDP), an inhibitor of autophagy, was purchased from MP Biomedicals (Santa Ana, CA, USA). Rapamycin (RAPA), an inducer of cellular autophagy, was purchased from LC Laboratories (Woburn, MA, USA). PARP-1 inhibitor, 3-amino benzamide (3AB), was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and rabbit anti-human microtubule-associated protein 1 light chain 3 (MAP1LC3B) was purchased from Sigma. Rabbit anti-PARP-1 primary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and the rabbit anti-PAR primary antibody was supplied by BD Biosciences (San Jose, CA, USA). The GAPDH primary antibody was purchased from Boster Co. (Wuhan, China), and the goat anti-mouse/rabbit IgG secondary antibody was purchased from the KPL Co.; Annexin V-FITC apoptosis necrosis detection kit was purchased from Nanjing Kaiji Co. (Nanjing, China).
Western blotting
CNE-2 cells were washed with ice-cold PBS twice and lysed at 4°C. The lysates were centrifuged with 12,000 rpm at 4°C for 30 min, at a centrifugal acceleration of 18,500 × g. Protein content in the supernatants was determined using the BCA Protein Assay kit (Beyotime, Nanjing, China). Equal amounts of protein (25 μg) were submitted to a 15% sodium dodecyl sulfate-polyacrylamide gel and then electrotransferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking for 2 h, the membranes were incubated with appropriate antibodies overnight: anti-LC3B (1:3,000), anti-PARP-1 (1:1,000), anti-PAR (1:1,500) and anti-GAPDH (1:800). After washing and incubating with fluorescently labeled goat anti-mouse/rabbit IgG secondary antibody (1:5,000), the fluorescence intensity was detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Autophagosome detection
Transmission electron microscopy (TEM) (H-600 IV; Hitachi, Tokyo, Japan) was utilized for analyzing the ultrastructural images of autophagosomes and autolysosomes. CNE-2 cells were harvested by trypsinization, washed twice with PBS, and fixed with ice-cold glutaraldehyde (3% in 0.1 M cacodylate buffer, pH 7.4) for 24 h. The cells were post-fixed in OsO4 and dehydrated in a graded series of 70 to 100% acetone and then embedded in Epon 812. One micrometer thin sections were cut, double stained by uranium tetraacetate and lead citrate trihydrate, and viewed using TEM with a scanning attachment.
Flow cytometry
The samples were washed with phosphate-buffered saline (PBS) twice and centrifuged at 1,500 rpm for 5 min, at a centrifugal acceleration of 1,800 × g. The cells were suspended in 500 μl of binding buffer [Annexin V-fluorescein isothiocyanate (FITC) kit; Kaiji, Nanjing, China], containing 5 μl of Annexin V-FITC and 5 μl of PI for determination of phosphatidylserine exposure on the outer plasma membrane. After incubation for 5–15 min at room temperature in a light-protected area, the samples were quantified by flow cytometry (BD FACSCalibur, San Jose, CA, USA).
Assessment of cell viability using MTT assay
Cells were plated into 96-well plates (1×103 cells/well, 200 μl cell suspension/well) and cultured overnight to allow for cell attachment. After irradiation (0, 24, 48 and 72 h) with 6 Gy X-rays, 20 μl of MTT (5 g/l) was added into each well. The cells were then incubated at 37°C for 4 h, the supernatant was removed and 200 μl DMSO was added. When the blue crystals were dissolved, a 96-well multiscanner autoreader (Bio-Rad M550; Bio-Rad, Hercules, CA, USA) was used to measure the absorbance value at 490 nm for each well. The survival rate was calculated as follows: (OD values of the experimental samples/OD values of the control) × 100%.
Clonogenic survival assay
CNE-2 cells were enzymatically dissociated with trypsin and seeded at 200, 200, 400, 600, 1,000, 5,000 and 10,000 cells/well. The cells were then irradiated (2 ml of the cell suspension/well) at room temperature with 6 MV X-rays with an Elekta linear accelerator with a dose rate of 220 cGy/min, accordingly, with the exposure dose corresponding to 0, 1, 2, 4, 6, 8 and 10 Gy. Fourteen days later, cells were fixed with 75% ethanol and stained with Giemsa, and colonies containing >50 cells were counted. The surviving fraction was calculated as: Survival fraction (SF) = experimental group colony forming efficiency/control group colony forming efficiency; Colony formation efficiency (PE) = the number of colonies/plant cell number. Experiments were conducted in triplicate. Survival curves were fitted using the linear-quadratic model (y=exp(-(α*x+β*x2))) using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).
Statistical analysis
Statistical data are presented as means ± standard deviation (SD). All data were subjected to analysis of variance (ANOVA) with significant differences among means identified by LSD multiple range tests using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). The criterion for statistical significance was taken as P<0.05. At least three independent experiments were performed.
Results
Irradiation-induced accumulation of autophagosomes
Intracellular autophagosomes were observed by TEM in CNE-2 cells after IR. CDP is widely used as an inhibitor of autophagy and RAPA is widely used as an inductor of autophagy. We, therefore, examined whether CDP or RAPA had effects on the autophagy in CNE-2 cells. As shown in Fig. 1, subcellular structure analysis revealed typical morphological features of autophagy in CNE-2 cells 24 h after treated with IR. Autophagosomes decreased in the CNE-2 cells treated with 10-Gy irradiation combined with 40 μM CDP, but increased in cells treated with 10-Gy irradiation combined with 20 nM RAPA. However, in the untreated cells or cells treated with CDP or RAPA alone for 24 h, normal nuclei surrounded by cytoplasm with normal appearing mitochondria were presented, and only occasional autophagosomes were observed.
LC3-II expression level in CNE-2 cells treated with CDP or RAPA combined with IR
To further determine the effect of IR on autophagy, the conversion of cytosolic LC3-I to LC3-II was examined in the CNE-2 cells. LC3-II protein level demonstrated a slight dose- and time-dependent increase induced by IR in the CNE-2 cells (Fig. 2A and B). LC3-II levels were correlated with the extent of autophagosome formation. Western blot assays showed that the levels of LC3-II were strongly elevated in the 10 Gy group and at 48 h following IR; a similar phenomenon was observed in CNE-2 cells. Significant differences in the expression level of LC3-II were found between the different experimental groups (F=231.68, P<0.01). In the RAPA group, the LC3-II level was increased, and in the CDP group the LC3-II level was also increased, indicating that RAPA-induced autophagy occurred. CDP inhibits the phenomenon of autophagy. CDP destroys the structure and function of the lysosomal to inhibit autophagy, resulting in autophagic lysosomal aggregation. LC3-II was not effectively degraded, thus the LC3 expression level was upregulated in the CDP-treated group, confirming that CDP inhibits autophagy. Compared with the untreated group, the level of LC3-II was significantly higher in the RAPA + 10 Gy group (P<0.01) and CDP group (P<0.01) (Fig. 2C).
PARP-1 activation contributes to irradiation-induced cell autophagy
PARP-1 is readily activated in response to DNA damage and is well associated with cell death. As shown in Fig. 3A and B, increased formation of the PAR polymer, a direct result of PARP-1 activation, was detected as early as 10 min after IR exposure. To verify whether PARP-1 activation contributes to the cell autophagy induced by IR, cells were treated with 10 Gy X-rays in the presence or absence of 3-amino benzamide (3AB), a specific PARP-1 inhibitor. Pretreatment with 3AB significantly inhibited IR-induced PAR formation. PAR and LC3-II both showed a certain degree of dose-dependence. Changes in PAR content were noted obviously earlier than LC3-B changes, and PAR was soon degraded. This suggests that PAR may be upstream of the signaling pathways. In order to further verify if the PARP-1 signaling pathway is upstream, we used the PARP-1 chemical inhibitor 3AB, autophagy inhibitor CDP and autophagy inducer RAPA to pretreat CNE-2 cells, and 2 h after 10 Gy IR, cells were collected and the protein was extracted, and changes in the levels of LC3-I and -II and PAR were determined. (Fig. 3C and D). The results clearly suggested that PARP-1 activation contributed to IR-induced cell autophagy. TEM examination further confirmed this finding (Fig. 4).
Autophagy or PARP-1 inhibition increases the apoptosis rate in CNE-2 cells following irradiation
IR combined with autophagy inhibitor or inducer caused CNE-2 cell apoptosis; 48 h after IR, the apoptosis rates of CNE-2 cells in the untreated group, CDP group, RAPA group, 3AB group, IR group, IR + CDP group, IR + RAPA group and IR + 3AB group, were 7.71±0.90, 8.29±1.60, 9.20±1.59, 9.91±0.75, 26.63±3.11, 31.28±1.58, 34.19±1.15 and 30.80±3.33%, respectively. Significant differences in the apoptosis rates were observed between the groups (F=109.69, P<0.01). The apoptosis rate was significantly higher in the CDP + IR, RAPA + IR and 3AB + IR group than that in the IR alone group (P<0.01) (Fig. 5A). At 24 or 48 h after IR, a similar phenomenon was observed, but at 72 h after IR, RAPA or 3AB combined with IR promoted cell survival rather than enhanced CNE-2 cell apoptosis (Fig. 5B).
Autophagy or PARP-1 inhibitor contributes to inhibition of CNE-2 cell proliferation and survival rate after irradiation
MTT assay was used to further elucidate whether the sensitivity of CNE-2 cells to IR occurred through autophagy or PARP-1 inhibition. The absorbance value indicates the proliferation and survival of cells; the higher the absorbance value, the higher the number of surviving cells. The proliferation rate of the CDP, RAPA or 3AB combined with 6 Gy IR groups was lower than that of the IR alone group (Fig. 6A). There were significant differences in the number of surviving cells and the survival rate between the different treatment groups 72 h after IR (F=50.40, P<0.01) (Fig. 6A). The survival rates of the CDP, RAPA or 3AB combined with 6 Gy IR group were significantly lower than that of the IR alone group, at each time point (P<0.01) (Fig. 6B).
Autophagy or PARP-1 inhibitor contributes to the radiation sensitization of CNE-2 cells
As expected, the effect of CDP, RAPA and 3AB on the lethality of IR was ascertained by clonogenic survival assay. GraphPad Prism 5.0 Software using the linear-quadratic model was used to calculate the radiobiology parameters and fitting dose survival curve (Table I and Fig. 7). Compared with IR alone, the IR + CDP group caused increased radiosensitivity of CNE-2 cells; RAPA or 3AB combined with IR showed a similar role and suggests that autophagy inhibition may be an approach to enhance the lethality of radiation.
Discussion
Apoptosis is a process of programmed cell death, autophagy is a process of programmed cell survival (27). Yet, too much or too little autophagy can damage cells. In some cases, autophagy can cause cell death. Several reports in the early literature also called autophagy ‘type II programmed cell death’, but now it is a misnomer (28,29). At present, concerning the relationship between autophagy and radiation sensitization of tumor cells, several scholars believe that inhibition of autophagy of tumor cells can improve the effect of radiotherapy (11,21,23). Chen et al(21) found that autophagy inhibitor 3-methyladenine (3-MA) combined with radiation increased the apoptosis of esophageal squamous carcinoma cells. Restraining autophagy increased the cytotoxicity of radiotherapy in esophageal carcinoma cells and arrested the cell cycle in the G2/M phase, increasing the sensitivity of radiotherapy. Ito et al(23) found that autophagy inhibitors, 3-MA and bafilomycin A1, increased the sensitivity of malignant glioma U373-MG cells. Meanwhile, radiation caused increased DNA double chain ruptures after inhibition of autophagy. Thus, autophagy inhibitors may become a type of new radiotherapy sensitization agents for malignant glioma. Several researchers also suggest that inducing autophagy improves the effect of radiotherapy on tumors (20,22). Cao et al(20) found that mTOR inhibitors, rapamycin and RAD001, inhibit mTOR function and induce autophagy, consquently improving the radiotherapy effect in breast cancer cells.
The present study demonstrated that radiation causes autophagy, and LC3-II protein levels were increased in a dose- and time-dependent manner induced by irradiation in CNE-2 cells. LC3-II is currently the only protein identified which is present in autophagic bodies and autophagy-lysosome membrane (30). Our research also detected autophagy body formation and the quantity in nasopharyngeal carcinoma cells by TEM which is the gold standard of autophagy detection, which further corroborated the test results of the western blot analysis.
Autophagy inhibitor CDP damages the structure and function of lysosomes to inhibit autophagy. It causes autophagy-lysosome to gather together and the degradation of LC3-II is reduced. RAPA is an autophagy inducer and causes Atg13 dephosphorylated and Atg1 activation by inhibiting mTOR activity to induce autophagy (24). In this study, CDP inhibited CNE-2 cell autophagy and RAPA induced CNE-2 cell autophagy caused by irradiation (31). It is important to note that CDP, by destroying the lysosome structure and function, inhibits autophagy, causes autophagy-lysosome together, and reduces LC3-II degradation. LC3-II failed to undergo effective decomposition, thus in the CDP-treated group, LC3-II expression level was increased, which confirmed that CDP inhibits autophagy; the characteristic of CDP is different from the autophagic inhibitor 3-MA (21).
Flow cytometry detected the apoptosis rate of the different groups at 24, 48 and 72 h after radiation. Our study showed that the apoptosis rate of the CDP + IR group significantly increased at the three time points compared with the IR alone group, which indicated that autophagy inhibitors can improve the sensitivity of the early response of nasopharyngeal carcinoma cells to IR. Although the autophagy inducer RAPA combined with IR significantly decreased the apoptosis rate at 72 h after radiation compared with the IR alone group, it significantly increased rather than decreased the apoptosis rate at 24 or 48 h after radiation. The results concerning the effect of RAPA were contradictory, and the reasons may be as follows: i) the characteristics of the RAPA drug itself, which was used widely as an antifungal drug; ii) physiological autophagy is a dynamic balance; a dysregulated balance may cause damage; 10 Gy IR combined with RAPA have synergy, and excessive autophagy may be also a type of cell death consequently leading to an increase in the apoptosis rate. Autophagy is a double-edged sword. Autophagy caused by IR inhibits the cell death process and plays a protective role. Thus, inhibiting autophagy can increase the radiation sensitization of CNE-2 cells. However, excessive autophagy may also cause cell death (6,7,12,13). According to the radiation biology, the surviving cells after radiation require concern. Only those cells that lose their proliferation capability and are unable to form effective clone cells are unable to cause cancer recurrence. We used a quadratic linear model to construct dose survival curves and calculate the radiation biology parameters. The results suggest that autophagy inhibitors significantly increased radiation sensitization, autophagy inducers showed a similar effect. The proliferation of the cells treated with an autophagy inhibitor combined with IR decreased.
In the present study, we identifed a novel function for PARP-1 in mediating IR-induced autophagy and such autophagy plays a pro-survival function in IR-induced cell death. Although at the 72-h time point, 3AB promoted CNE-2 cell survival rather than enhanced cell apoptosis, a clonogenic survival assay was performed to evaluate long-term cell survival. The result showed that the PARP-1 inhibitor contributed to radiation sensitization of CNE-2 cells (16). It appears that PARP-1 is able to elicit dual pathways with opposite functions in response to oxidative stress (31), as illustrated in Fig. 5B. The decision of cell life or death is dependent on the balance between apoptosis and autophagy mediated by these two distinct pathways. Oxidative DNA damage includes modifications to bases and the sugar phosphates, as well as single- or double-strand DNA breaks. Such damage leads to PARP-1 activation, suppression of ATP production and finally cell death. Furthermore, overactivation of PARP-1 produces large quantities of PAR polymers, leading to autophagy, and finally cell survival.
In summary, we demonstrated a novel function of PARP-1 in the regulation of IR-induced autophagy through the mTOR signaling pathway, and such autophagy serves as a cell survival mechanism against IR-mediated cell death. Autophagy inhibitors and PARP-1 inhibitors contributed to the radiation sensitization of CNE-2 cells. This suggests that CDP or 3AB may be used as adjuvant treatment for nasopharyngeal carcinoma. Further animal experiments and clinical tests are warranted to verify our findings.