Neurochem Res (2014) 39:2334–2342 DOI 10.1007/s11064-014-1432-x

ORIGINAL PAPER

Indirect Effects of Radiation Induce Apoptosis and Neuroinflammation in Neuronal SH-SY5Y Cells Yasmeen Saeed • Bingjie Xie • Jin Xu • Hailong Wang • Murtaza Hassan • Rui Wang Ma Hong • Qing Hong • Yulin Deng



Received: 14 June 2014 / Revised: 24 August 2014 / Accepted: 4 September 2014 / Published online: 17 September 2014 Ó Springer Science+Business Media New York 2014

Abstract Recent studies have evaluated the role of direct radiation exposure in neurodegenerative disorders; however, association among indirect effects of radiation and neurodegenerative diseases remains rarely discussed. The objective of this study was to estimate the relative risk of neurodegeneration due to direct and indirect effects of radiation. 60Co gamma ray was used as source of direct radiation whereas irradiated cell conditioned medium (ICCM) was used to mimic the indirect effect of radiation. To determine the potency of ICCM to inhibit neuronal cells survival colony forming assay was performed. The role of ICCM to induce apoptosis in neuronal SH-SY5Y cells was estimated by TUNEL assay and Annexin V/PI assay. Level of oxidative stress and the concentration of inflammatory cytokines after exposing to direct radiation and ICCM were evaluated by ELISA method. Expression of key apoptotic protein following direct and indirect radiation exposure was investigated by western blot technique. Experimental data manifest that ICCM account loss of cell survival and increase apoptotic induction in neuronal SH-SY5Y cells that was dependent on time and dose. Moreover, ICCM stimulate significant release of inflammatory cytokines i.e., tumor necrosis factor TNF-alpha (P \ 0.01), Interleukin-1 (IL-1, P \ 0.001), and Interleukin-6 (IL-6, P \ 0.001) in neuronal SH-SY5Y cells and elevate the level of oxidative stress (MDA, P \ 0.01). Up-regulation of key apoptotic protein expression i.e., Bax, Bid, cytochrome C, caspase-8 and caspase-3 confirms the toxicity of ICCM to neuronal cells. This study provides the evidence that indirect effect

Y. Saeed  B. Xie  J. Xu  H. Wang  M. Hassan  R. Wang  M. Hong  Q. Hong  Y. Deng (&) School of Life Sciences, Beijing Institute of Technology, Beijing 100081, People’s Republic of China e-mail: [email protected]

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of radiation can be as much damaging to neuronal cells as direct radiation exposure can be. Hence, more focused research on estimation risks of indirect effect of radiation to CNS at molecular level may help to reduce the uncertainty about cure and cause of several neurodegenerative disorders. Keywords Indirect effect of radiation  Apoptosis  Inflammatory cytokines  Radiation  Neurodegeneration  Neuroinflammation Abbreviations ICCM Irradiated cell conditioned medium TUNEL Terminal deoxynucleotidyl transferaseassay mediated dUTP-biotin nick end-labeling assay hrs Hours MDA Malondialdehyde

Introduction Accumulating evidence describes the occupational and accidental exposure to radiation and its damaging effect on human health such as cancer, acute radiation sickness, central nervous system damage and degenerative diseases [1, 2]. Nevertheless damage to the central nervous system is among the major challenge for radiobiology. However, frequent use of radiation in medical diagnosis and nuclear industry may contribute to the etiology of neurodegenerative disease [3]. At the moment, it is very well known that CNS is a radiosensitive organ and the tissues in central nervous system have no or very limited regenerative

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capacity. Thus, radiation can potentially provoke peculiar inflammatory response that might play vital role in pathogenesis of neurodegenerative disorders [4]. Moreover, highly rated morbidity and mortality are associated with acute or chronic neurodegenerative disease. Unfortunately; to date no effective treatment is available for complete cure of neurodegenerative disorders. Therefore, it is of utmost importance to limit the damages that cause neuronal death and to define every possible source of stress that has potency to induce acute or chronic neurodegenerative damages [5–8]. Particularly, in chronic neurodegenration, environmental factors have also been indicted among the major sources [9]. Though, the health risks associated with direct radiation exposure have been extensively studied however, indirect and delayed effects of radiation risk specifically to CNS are still under consideration and require more evidence for comprehensive knowledge [10]. For instance, brain damage to the embryo induced by prenatal exposure to ionizing radiation is a major concern for radioprotection [11–13] but it also points towards involvement of indirect effects of radiation in neurodegenration. Radiation has wide range biological effects that are not only limited to its direct exposure but number of indirect and delayed effects are also involved [14–20]. Hence, radiation exposure can trigger several signaling factors particularly inflammatory cytokines and reactive oxidative species that further cause bystander damage to neurons that never been exposed to radiation by triggering apoptotic cascade and cause detrimental conditions for neurogenesis that ultimately leads towards chronic neurodegeneration [21]. These evidence appear to focus on indirect and delayed effects of ionizing radiation that may enhance the occurrence of chronic neurodegenerative disorders. The aim of this study was to figure out the damaging effect of indirect radiation exposure to neuronal cells. This study was conducted on basis of various end points i.e., cell survival, apoptotic and pro-inflammatory responses and expression of key proteins involved in apoptotic cascade. Our data reported the toxicity of ICCM to human neuroblastoma (NB) cells via activation of inflammatory cytokines and oxidative stress that lead towards up-regulation of apoptotic protein expression.

Materials and Methods Cell Line and Culture Conditions Neuroblastoma SH-SY5Y cell line was cultured by using Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12, Invitrogen, Carlsbad,CA) supplemented with 10 % fetal bovine serum (FBS) and solution penicillin/streptomycin

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(100 IU/ml; 100 lg/ml) (both from invitrogen), and incubated at 37 °C in a humidified atmosphere containing 5 % CO2/95 % air. Irradiation and Irradiated Cell-Conditioned Medium (ICCM) Neuroblastoma SH-SY5Y cells were exposed to ionizing radiation source Gamma cell 60Co irradiator (dose rate, 0.95 Gy/min). 60Co gamma ray radiation service was provided by the China Institute of Atomic Energy (Beijing, China). To evaluate the potency of indirect effect of radiation to induce damage in non-irradiated neuronal SH-SY5Y cells, the medium transfer experiment was adopted in this study. Cells growing exponentially at 85 % confluence were irradiated (0–20 Gy) at room temperature at a dose-rate of 0.95 Gy/min using a 60C0 irradiator. Control flasks were sham irradiated (0 Gy) and handled under the same conditions as the irradiated cells. The medium removed from irradiated and sham irradiated (0 Gy) cell cultures were centrifuged at 3,000 rpm at room temperature and filtered using 0.22 um filter. ICCM was used for further experiments. Colony-Forming Assay The ability of ICCM to induce cell death in non-irradiated cells was assessed by Clonogenic survival assay. Neuronal SH-SY5Y cells were exposed to (ICCM) at various doses ranging from 0 to 20 Gy. For Clonogenic survival assay neuroblastoma SH-SY5Y cell cultures were trypsinized, counted with a Coulter counter, and aliquots of the cells were re-plated into 100-mm-diameter dishes. Then exposed to ICCM (generated from exposure to direct irradiated cells at 0–20 Gy dose) and incubated for 12 days with one medium renewal at day 6. After 12 days of incubation cells were fixed with formaldehyde and stained with Giemsa. Surviving fractions were calculated as described elsewhere [22]. Cells in fresh medium served as controls. Subsequent studies were conducted with ICCM from 10 Gy dose of radiation. Annexin V Assay Detection of apoptosis and membrane permeability was tested with the Annexin V-FITC/PI (KTK101-100) Apoptosis Detection Kit for Flow Cytometer. This experiment was designed to estimate effective time duration of medium (during which it remains in contact with irradiated cells) that further induce neuronal cell death. For this purpose cell cultures were exposed to 24, 48 and 72 hrs old ICCM at 10 Gy dose. While 24 and 72 hrs old medium from control cells served as control, fluorescent signals were analyzed by flow cytometer.

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TUNEL Assay For further confirmation and quantification of apoptosis in ICCM treated, directly irradiated, and control groups of neuronal cells, terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end-labeling assay (TUNEL assay) was used. Experiment was performed with Millipore apopTag Plus Peroxidase in situ detection kit (S7101) by following kit instructions. Mean and SD were calculated from three independent experiments. Enzyme-Linked Immunosorbent Assay (ELISA) Inflammatory cytokines i.e., IL-6, IL-1, TNF-alpha and MDA level in the media and in the cell lysate from control cells, directly irradiated cells and ICCM treated cell were measured by using Enzyme Linked Immunosorbent Assay (ELISA) according to kit instructions (R&D Systems, MN, USA). Protein levels were calibrated on a micro plate reader at 450 nm. All standards, controls and samples were run in triplicate.

Fig. 1 Determination of clonogenic survival in neuronal (SH-SY5Y) cells. SH-SY5Y cells were exposed to ICCM of graded doses of gamma-particles i.e., 0, 3, 5, 7, 10, 15 and 20 Gy. Data were pooled from three independent experiments. Values represent ‘‘mean ± SD’’

quantitatively determined using density analysis software (MetaMorph Imaging System, Meta Imaging Series 4.5) and the density ratio represented the relative intensity of each band against those of controls in each experiment.

Western Blot Analysis Statistics Analysis Expression of key apoptotic proteins i.e., caspase-3, caspase-8, cytochrome-C, Bcl-2, Bid and Bax was detected by western blot assay. Cells were washed twice in cold PBS, and lysed in ice-cold lysis buffer containing 150 mM NaCl, 1.0 % Nonidet-P40 and 50 mM Tris–Hcl (pH 8.0). For determination of apoptotic proteins expression whole cells lysate was used except for cytochrome C. Expression of cytochrome c was determined in cytosol. For isolation of cytosol cells were centrifuge at 800 g for 15 min and the supernatant was used for isolation of cytosol and mitochondria. Then the supernatant was again centrifuged at 6,000 g for 15 min to pellet the mitochondria, supernatant was preserved as crude cytosol while pellet contain mitochondria. Crude cytosol was centrifuged for 1 hrs at 100,000 g in a swing-bucket ultracentrifuge. Decant the supernatant and save as pure cytosol to determine the concentration of cytochrome C by western blot technique [23]. One Hundred micrograms of protein were electrophoresed by SDS-PAGE and then blotted to an Immobilon-P 0.22 mm membrane (Millipore, Billerica, MA, USA), followed by blocked with 5 % nonfat milk in 1 % TBST and incubated with primary antibodies overnight. The secondary antibody was conjugated to horseradish peroxidase (CWBIC, Beijing, China), and antigen–antibody complexes were detected by the chemiluminescence (Millipore). Both the primary and secondary antibodies were diluted in 5 % nonfat milk in TBST. The primary antibodies were caspase-3 (1:800) caspase-8 (1:1,000), cytochrome-C (1:800), Bcl-2 (1:800), Bid (1:1,000) and Bax (1:800) (Santa Cruz, USA). Intensity of each band was

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Data is expressed as mean ± SD from at least three independent experiments. Statistical differences between mean values in two groups were evaluated using T test analysis. The differences among more than two groups were analyzed by one-way ANOVA and Tukey’s post hoc test. The differences were considered statistically significant at a P value \0.05 (*), \0.01 (**) or (*** P \ 0.001).

Results ICCM Induces Cell Death in Neuronal SH-SY5Y Dependent on Time and Dose of Radiation Neuronal SH-SY5Y cells were exposed to irradiated cell conditioned medium (ICCM) at various doses of radiation ranging from 3 to 20 Gy and cell survival was estimated by colony forming assay. However, the cells that received medium from sham irradiated (0 Gy) cells served as control. It was observed that ICCM cause loss of cell survival that was dependent on dose of radiation. According to Fig. 1 the percentage of clonogenic survival by ICCM at different doses of radiation i.e., 0, 3, 5, 7, 10, 15 and 20 Gy were 99.7 ± 0.41, 80.9 ± 0.47, 62.1 ± 0.64, 49.9 ± 0.56, 28.33 ± 0.36, 14.9 ± 0.68 and 8 ± 0.17 %, respectively. ICCM from 10 Gy doses of irradiation causes prominent Clonogenic inhibition that was 28.33.0 ± 0.36. In view of this data ICCM from 10 Gy dose of irradiated cells was selected for further study.

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Fig. 2 Assessment of apoptosis by AnnexinV/PI in Neuronal SHSY5Y cells after treatment with ICCM at various time points ranging from, 24 hrs (2C), 48 hrs (2D) and 72 hrs (2E) while 24 and 72 hrs old medium from sham irradiated (0 Gy) cells served as control (Fig. 2a, b). Cells were considered to be in early apoptosis, that were stained with annexin V but not with PI (annexin V?/PI-), and cells that were stained with both (annexin V?/PI?) were considered to be necrotic or late apoptotic. Cells that were stained with PI alone or (annexin V-/PI?) were necrotic. At least 10,000 cells for each

sample were analyzed in each experiment. Figure 2f represents statistical analysis of apoptotic cells number and that of necrotic cells. Data are shown as mean ± SD from at least three separate experiments. Comparison among control group and ICCM treated groups represented as * P \ 0.05, ** P \ 0.01, *** P \ 0.001; difference between 72 hrs control and 72 hrs ICCM treated cells denotes by arrow line (** P \ 0.01, *** P \ 0.001). Comparison between 48 hrs old ICCM and 72 hrs old ICCM treated groups; (## P \ 0.01, ### P \ 0.001)

To determine the time point, at which indirect effect of radiation can induce significant cell death by apoptosis, we next treated neuronal SH-SY5Y cells with ICCM of different time duration that ranges from 24 to 72 hrs while medium from control cells served as control. Cell death was analyzed by AnnexinV/PI assay. Figure 2f indicates that early apoptosis was significantly higher in SH-SY5Y cells (*** P \ 0.001) after treatment with 48 and 72 hrs old ICCM as compare to the cells exposed to control medium. However, significantly higher late apoptosis was observed when SH-SY5Y cells were exposed to 48 hrs (*** P \ 0.001) and 72 hrs (** P \ 0.01) old ICCM, as compare to medium from control cells. Significant increase

in necrosis (** P \ 0.01) was observed in the cells that were treated with 72 hrs old ICCM as compare to 72 hrs old control medium. Hence, after exposure to 48 hrs old ICCM the most preeminent response was apoptosis while treatment with 72 hrs old ICCM caused necrosis in neuronal SH-SY5Y cells. Based on these results, further experiments were designed with 48 hrs old 10-Gy ICCM at 10 Gy dose. ICCM Induces Apoptosis in Neuronal SH-SY5Y Cell The quantification of apoptosis in neuronal SH-SY5Y cells was estimated by TUNEL (Peroxidase) staining method; in

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Fig. 3 Quantification of radiation induced apoptosis by Terminal Deoxynucleotidyl Transferase-mediated dUTPbiotin nick end labeling assay (TUNEL assay) in neuronal SHSY5Y cells after exposure to direct radiation and ICCM. Arrows are indicating the TUNEL positive cells that were characterized by yellowish brown color (Color figure online)

that TUNEL positive cells were characterized by yellowish brown color. Results were calculated by scoring approximately 1,000 cells in total for TUNEL-positive cells in each experiment. According to scored data the number of TUNEL positive cells in neuronal SH-SY5Y cells that received ICCM (Fig. 3d), was significantly higher (** P \ 0.01) (Fig. 3c) as compare to directly irradiated cells (Fig. 3b). ICCM Elevates Cytokines Level and Oxidative Stress in Neuronal SH-SY5Y Cells It has been reported recently that pro-inflammatory cytokine activation may directly contribute to neurodegeneration [24]. The potency of ICCM to provoke inflammatory cytokines and oxidative stress in neuronal SH-SY5Y cells was investigated by ELISA. Our data infer that ICCM can potentially elevates the level of TNF-alpha, IL-1, IL-6 and MDA level in neuronal SH-SY5Y cells as direct radiation does. Figure 4b is the demonstration of significant increase of TNF-alpha level in the medium from directly irradiated (** P \ 0.01) and ICCM treated (** P \ 0.01) SH-SY5Y cells as compare to medium from control cells. However,

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statistical analysis indicates that TNF-alpha concentration was higher in medium from directly irradiated cells (# P \ 0.05) as compare to medium received from ICCM treated cells (Fig. 4b). Figure 4f represent the TNF-alpha level in cell lysate after direct radiation (** P \ 0.01) exposure and ICCM treatment (** P \ 0.01) as compare to control cells, while no significant difference in TNF-Alpha level was observed between directly irradiated and ICCM treated SH-SY5Y cells (Fig. 4f). Figure 4a and e indicates the significant increase in concentration of IL-1 in cell lysate (** P \ 0.001) (Fig. 4e) and in medium (** P \ 0.01) (Fig. 4a), respectively, obtain from SHSY5Y cells after exposing to direct radiation and ICCM compare to control. However, no significant difference was observed between directly irradiated and ICCM treated groups. Figure 4c and g illustrates that IL-6 level in cell lysate (*** P \ 0.001) (Fig. 4g) and in the medium (*** P \ 0.001) (Fig. 4c) from directly irradiated and ICCM treated neuronal SH-SY5Y cells was significantly increased as compared to control cells. While statistical analysis demonstrate significant difference between directly irradiated and ICCM treated groups both in the medium (## P \ 0.01) and in the cell lysate (# P \ 0.05) of

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Fig. 4 Direct irradiation and ICCM evoked inflammatory cytokines and oxidative stress in Neuroblastoma SH-SY5Y cells (Fig. 4a, e) IL-1 level was determined in medium and cell lysate (respectively) from SH-SY5Y cells in all groups (i.e., control, Directly Irradiated and ICCM treated). (Fig. 4b, f) TNF-alpha level was detected in medium and cell lysate (respectively) from all groups (i.e., control, Directly Irradiated and ICCM treated) (Fig. 4c, g) IL-6 level was measured in medium and cell lysate (respectively) from all groups (i.e., control, Directly Irradiated and ICCM treated). (Fig. 4d, h) MDA level was

determined in medium and cell lysate (respectively) from all groups (i.e., control, Directly Irradiated and ICCM treated). Inflammatory cytokines and MDA level in the medium and in cell lysate was measured by using enzyme linked Immunosorbent assay (ELISA) according to kit instructions (both groups i.e., directly Irradiated and ICCM treated; compared with Control, * P \ 0.05, ** P \ 0.01, *** P \ 0.001; directly irradiated versus ICCM treated, # P \ 0.05, ## P \ 0.01; ns, non significant)

neuronal SH-SY5Y cells (Fig. 4c, g). Figure 4d demonstrate significant increase in malondialdehyde (MDA) level in medium from directly irradiated (** P \ 0.01) and ICCM (* P \ 0.05) treated cells as compare to control whereas MDA level was significantly higher (# P \ 0.05) in directly irradiated cells as compare to ICCM treated cells. Figure 4h is demonstration of MDA level in cell lysate, according to results direct irradiation exposure and ICCM treatment significantly elevate the MDA level (** P \ 0.01) in neuronal SH-SY5Y cells as compare to control cells; however, the difference in MDA level between directly irradiated and ICCM was not significant (Fig. 4h).

ICCM in initiating apoptotic cascade in neuronal SH-SY5Y cells. The expression of key apoptotic protein involved in mitochondrial apoptotic pathway i.e., caspase3, caspase-8, Bid, Bax, Bcl-2 and cytosolic cytochrome c were analyzed by western blot assay. Results shown in (Fig. 5c, d) revealed significant expression of effector caspase-3 (* P \ 0.05) and caspase8 (* P \ 0.05) following treatment with ICCM similar in way as direct radiation induced (Fig. 5c, d). Bid, a substrate of caspase-8 also activated after treatment with ICCM (* P \ 0.05) as compared to control neuronal SHSY5Y cells (Fig. 5f). Significant reduction in Bcl-2 expression (** P \ 0.01) in directly irradiated and ICCM treated (* P \ 0.05) was observed (Fig. 5g). While upregulation of Bax expression (* P \ 0.05) was observed in directly irradiated as well as ICCM exposed (* P \ 0.05) neuronal SH-SY5Y cells (Fig. 5e). Figure 5b demonstrates that ICCM can induce significant release of cytochrome C (** P \ 0.01) in cytosol. However, no significant difference was observed between directly radiated and ICCM

ICCM Triggers Up-Regulation of Key Apoptotic Protein Expression in Neuronal SH-SY5Y Cells Direct radiation exposure is among the prominent factors that initiate apoptotic cascade in neuronal cells. The main objective for this part of study was to evaluate the role of

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Fig. 5 Expression levels of indicated proteins from the control, ICCM treated and directly irradiated human neuroblastoma (SHSY5Y) cells was determined by Western blot analysis. Quantification of cytochrome C (Fig. 5b), cleaved caspase-3 (Fig. 5c), caspase-8 (Fig. 5d), Bid (Fig. 5f), Bax (Fig. 5e), Bcl-2 (Fig. 5g) was determined in control, directly irradiated and ICCM tread group of SH-SY5Y cells by Western blotting analysis. Protein expression was analyzed

by densitometry analysis as described in ‘‘Materials and Methods’’. Relative density was calculated as the ratio of cytochrome-c, caspase3, caspase-8, Bid, Bax and Bcl-2 expression to beta-actin expression (both groups i.e., directly Irradiated and ICCM treated; compared with control, * P \ 0.05, ** P \ 0.01, *** P \ 0.001; Directly Irradiated versus ICCM treated, # P \ 0.05, ## P \ 0.01; ns nonsignificant)

treated cells in expression of above described apoptotic protein expression. Hence, this data infer that ICCM can be as toxic as direct radiation exposure in provoking the factors like up-regulation of Bax expression and decreased in Bcl-2 expression that enhance the release of cytochrome-c into cytosol that ultimately lead towards increased activity of caspase-3 and result in apoptosis of neuronal SH-SY5Y cells (Fig. 5). These findings proposed that indirect effect of ionizing radiation cause damage to neuronal cell that might follow mitochondrial apoptotic pathway.

increase in apoptosis was observed in SH-SY5Y cells after exposure to 48 hrs old ICCM however, after exposure to 72 hrs old ICCM increase in necrosis was observed. Nevertheless, this data also depicts that when SH-SY5Y cells were treated with 72 hrs old medium from control group of cells, the dominating response was necrosis (*** P \ 0.001). It might depicts that after 72 hrs (even without any external stress such as radiation) cells strive through nutrient starvation and reduced oxygen supply that result in the depletion of ATP [25] that might changes the composition of nutrient in the medium of cultured SHSY5Y cells that finally lead towards necrosis. Hence, these results demonstrate that indirect effect of radiation is dose and time dependent phenomenon. Quantification of apoptosis by TUNEL assay manifest higher number of TUNEL positive cells after exposure to ICCM as compare to cell that were exposed to direct radiation (Fig. 3c). This data may infer that direct radiation exposure caused severe injury to neuronal SH-SY5Y cells that promptly results in cell death in a way that necrosis dominate over apoptosis [26]. However, ICCM dominantly induce apoptosis in neuronal SH-SY5Y cells (Fig. 3c). It has been documented previously that neurological disorders are caused either by necrosis or apoptosis in neuronal cells [27–30] depending on the magnitude of stimulus for instance strong stimulus like direct radiation exposure may cause acute illness by necrosis mode of cells death.

Discussion Effect of ionizing radiation on neurodegeneration has been widely discussed previously. However, it has been documented that indirect effects of radiation are detrimental for neurogenesis as well [21]. The scope of this study was to evaluate the role of indirect effect of radiation in neurodegeneration. This study infer that ICCM exposure dominantly reduce the cell survival and increases apoptosis in neuroblastoma SH-SY5Y. Figure 1 demonstrates that as the dose of irradiation exposed to ICCM was increased neuronal SH-SY5Y cells loose the cell survival (Fig. 1). Furthermore, as the time duration, in which ICCM remain intact with irradiated cells was increased; the numbers of apoptotic cells was also increased (Fig. 2). Preeminent

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Whereas milder or indirect stress conditions like delayed or indirect radiation exposure may results into chronic disorders specifically via apoptotic cell death [9]. Figure 4 illustrates that direct radiation exposure and ICCM can potentially provoke inflammatory cytokines and oxidative stress biomarker MDA in neuronal SH-SY5Y cells. This study also suggests that inflammatory cytokines and oxidative stress play a role in the transmission of radiation-induced bystander damage to neurons that never been exposed to radiation [21]. Congeneric to preceding evidences these results may speculate that combined actions of inflammatory cytokines particularly IL-1 and TNF-alpha may enhance neurodegeneration in CNS [31, 32]. Nevertheless increase in IL-6 level halts the neurogenesis process that lead towards chronic neurodegenerative disorders [33]. Furthermore, inflammatory cytokines are known to initiate apoptotic cascade in neurons and glial cells [24]. While apoptosis is predominant feature of acute and chronic neurological disorders [34–36]. It has been determined in preceding work that inflammatory cytokines such as TNF-alpha induces and regulates the activation of apoptotic protein especially upstream caspase [37, 38]. Hence, in chronic neurodegenerative diseases apoptosis is consider as leading form of cell death [39, 40]. Western blot results exhibit that ICCM up-regulate the expression of key apoptotic protein involved mitochondrial apoptotic cascade i.e., (pro-apoptotic Bax, pro-apoptotic Bid, cytochrome-C, caspase 8 and effector caspase 3 and decrease the expression of anti-apoptotic Bcl-2). These findings may speculate that indirect effect of radiation trigger apoptosis by mitochondrial pathway (Fig. 5). As considerable preceding research work have demonstrated that Bax and Bcl-2 are antagonistic to one another, in that pro-apoptotic Bax promotes the release of cytochrome c and other apoptotic factors whereas anti-apoptotic Bcl-2 blocks the release of these factors [41]. However, Bid protein, (a Bcl-2 family member), is crucial for caspase-8mediated mitochondrial damage and cell death [42]. Upregulation in expression of these apoptotic proteins may provide evidence that indirect effect of radiation can effectively cause apoptosis in neuronal cells that might have roots in mitochondrial induced apoptosis. However, for complete understanding of underlying signaling mechanism more focused research is required. In conclusion, the data presented here revealed various aspects of damage to neuronal SH-SY5Y cells by indirect radiation exposure. A schematic drawing of the proposed mechanisms of ICCM induced apoptosis in neuronal SHSY5Y cells is shown in (Fig. 6). This study also proposed that ICCM induced apoptosis in neuroblastoma SH-SY5Y cells via activation of caspase that might involve interaction among inflammatory cytokines and modulation of the

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Fig. 6 A schematic drawing of the proposed mechanisms of ICCM induced apoptosis (see text for details)

apoptotic protein expression particularly Bid, Bax and Bcl-2 (Fig. 6). However, the exact nature of the precise molecular mechanisms of action remained to be determined in future studies. This data may contribute to understand the risks of neurodegeneration associated with exposure to indirect or delayed effect of radiation. Acknowledgments The authors thank the support from the National Basic Research Program of China (973 Program, 2012YQ040140), and partially supported by Foundation of China (No. 211110205 5311077). The authors also acknowledge the support provided by Beijing Institute of Technology and all colleagues in the laboratory.

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Indirect effects of radiation induce apoptosis and neuroinflammation in neuronal SH-SY5Y cells.

Recent studies have evaluated the role of direct radiation exposure in neurodegenerative disorders; however, association among indirect effects of rad...
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