NFκB signaling pathway activation by a small molecule DMA confers radioprotection to intestinal epithelium in xenograft model

Free Radical Biology and Medicine 108 (2017) 564–574

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Original article

Akt1/NFκB signaling pathway activation by a small molecule DMA confers radioprotection to intestinal epithelium in xenograft model

MARK

Vinod Tiwaria, Mohammad Zahid Kamranb, Atul Ranjanc, Hemlata Nimesha, Manish Singhb, ⁎ Vibha Tandona,b, a b c

Chemical Biology Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India Special Centre for Molecular Medicine, Jawaharlal Nehru University, Delhi 110067, India Department of Cancer Biology, The University of Kansas Cancer Center, 3901 Rainbow Blvd, Kansas City, KS 66010, USA

A R T I C L E I N F O

A B S T R A C T

Keywords: Radiation Radioprotector Acute radiation syndrome Tumor Akt1/NFκB signaling

Normal tissue protection and recovery of radiation-induced damage are of paramount importance for development of radioprotector. Radioprotector which selectively protects normal tissues over cancerous tissues improves the therapeutic window of radiation therapy. In the present study, small bisbenzimidazole molecule, DMA (5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxy-phenyl)-5′-benzimidazolyl]-benzimidazole) was evaluated for in vivo radioprotective effects to selectively protect normal tissue over tumor with underlying molecular mechanism. Administration of single DMA dose prior to radiation has enhanced survival of Balb/c mice against sublethal and supralethal total body irradiation. DMA ameliorated radiation-induced damage of normal tissues such as hematopoietic (HP) and gastrointestinal tract (GI) system. Oxidative stress marker Malondialdehyde level was decreased by DMA whereas it maintained endogenous antioxidant status by increasing the level of reduced glutathione, glutathione reductase, glutathione-s-transferase, superoxide dismutase and total thiol content in hepatic tissue of irradiated mice. Mechanistic studies revealed that DMA treatment prior to radiation leads to Akt1/NFκB signaling which reduced radiation-induced genomic instability in normal cells. However, these pathways were not activated in tumor tissues when subjected to DMA treatment in similar conditions. Abrogation of Akt1 and NFκB genes resulted in no radioprotection by DMA and enhanced apoptosis against radiation. Plasma half-life of DMA was 3.5 h and 2.65 h at oral and intravenous dose respectively and 90% clearance was observed in 16 h. In conclusion, these data suggests that DMA has potential to be developed as a safe radioprotective agent for radiation countermeasures and an adjuvant in cancer therapy.

1. Introduction Radiotherapy targets cancerous cells during treatment but it has deleterious effects on surrounding normal tissues. Similarly, injury to hematopoietic (HP)/gastrointestinal tract (GI) system is major factor for acute radiation syndrome (ARS) associated death in organisms exposed to radiation [1,2]. Apart from radiotherapy, there are chances of radiation accidents such as Fukushima, Japan (2011), Tokaimura, Japan (1999), Goiânia, Brazil (1988), Chernobyl, Russia (1988), and Three Mile Island nuclear power station, United States (1979). Military and first responder which are employed to these fatal accidents areas for search-and-rescue purposes would also require radioprotectors as radiation countermeasures [3,4]. Thus, it is imperative to develop safe radioprotector for radiation countermeasures. Amifostine is only clinically approved radioprotector used for cancer patients undergoing radiotherapy [5]. It has dose limited toxicity and cause serious side ⁎

effects such as nausea, vomiting and hypotension at its maximum effective doses [6]. Recently developed CBLB502 had shown an excellent radioprotection to healthy cells over cancerous cells [7]. CBLB502 has half-life < 20 min and will require repeated dosing for desired clinical effect by i.v. route of administration [8] whereas other radioprotectors such as methylproamine, PrC-210, ON01210/Ex-RAD® [9] and 3,3′-Diselenodipropionic acid (DSePA) [10] are at various stages of development. Akt1 is known for cellular protection against ionizing radiation (IR)induced apoptosis in germ cells [11]. Prostaglandin treatment reduces apoptosis of epithelial by Akt activation [12]. In response to IR stimuli, Nuclear factor-kappa binding (NFκB), a transcription factor, activates its downstream target genes which regulates cell survival, cellular proliferation and apoptosis [13,14]. Earlier studies suggested that radioprotectors like ON01210 and CBLB502 provides radioprotection by activation of PI3K/Akt [15] and NFκB [7] signaling pathway

Corresponding author at: Special Centre for Molecular Medicine, Jawaharlal Nehru University, Delhi 110067, India. E-mail address: [email protected] (V. Tandon).

http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.029 Received 9 March 2017; Received in revised form 6 April 2017; Accepted 20 April 2017 Available online 21 April 2017 0891-5849/ © 2017 Published by Elsevier Inc.

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with optimum dose of DMA (200 mg/kg bw) prior to whole body exposure to 5, 6, 8, 9 & 10 Gy. Body weight, radiation sickness and mortality in animals were noted for 30 days. Ratio of LD50/30 mice treated with both DMA and radiation to radiation was calculated as DRF. 2.3.2. Nude mice survival 4 groups of mice containing 5 animals each were created. Group 1, sham control; Group 2, DMA treated (50 mg/kg, i.p.); Group 3, TBI 7 Gy; Group 4, treated with DMA (50 mg/kg i.p.) prior to 7 Gy TBI. Body weight, radiation sickness and mortality in animals were noted for 30 days. Survival study was carried out for a period of 30 days.

Fig. 1. Structure of DMA 3HCl.

respectively. DMA (Fig. 1), a bis-benzimidazole derivatives, has been shown as non-toxic, free radical scavenging radioprotector [16–18]. It has effective radioprotection at 1/7th dose of its maximum tolerable dose (MTD) of 2000 mg/kg [19]. DMA induces NIK mediated NFκB activation and modulates number of key regulatory pathways to overcome radiation-induced damage in vitro [18,20]. Here we have deciphered the molecular mechanism of DMA as a radioprotector in normal and tumor bearing Balb/c mice against total body irradiation (TBI). Single 200 mg/kg oral and 50 mg/kg intravenous (i.v.) DMA dose augments 80% and 100% survival respectively at 8 Gy radiation through activation of Akt/NFκB pathway, improves HP and GI conditions and maintains redox balance in vivo.

2.3.3. Tumor xenograft generation Melanoma model: 0.8×106 B16F10 cells were injected subcutaneously in the right flank of Balb/c mice (n=24). When tumor volume reached 0.5 cm3, mice were irradiated at 8 Gy as reported earlier [19]. 2.3.4. Immunohistochemistry (IHC) of cell proliferation and IL-6 in tissue Spleen cell proliferation was measured 3 days after irradiation using 5-bromo-2′-deoxyuridine (BrdU) (i.p., 100 mg/kg) as labeling agent in each mouse 2 h before euthanasia. IL-6 expression was checked in small intestine through IHC as described previously [7].

2. Materials and methods 2.1. Animals, cell lines and treatment conditions

2.3.5. Endogenous spleen colony forming assay Group 1, sham control (normal saline treated); Group 2, DMA treated (50 mg/kg bw, i.v.); Group 3, radiation control (8 Gy TBI); Group 4, treated with DMA (50 mg/kg bw, i.v.) prior to whole body exposure to 8 Gy. Mice (6 animals each) were irradiated, 2 h postadministration of DMA. The mice were sacrificed on day 10 and spleens were recovered, cleaned for blood and weighed. [spleen index=(spleen weight/body weight)×100] formula was used to calculate spleen index. Subsequently Bouin's fixative was used to fix spleens for 15 min and the number of macroscopic spleen cell colonies was counted manually [21].

Balb/c mice (25 ± 5 g) were obtained from National Institute of Nutrition, Hyderabad and experiments were conducted as per recommendation of Committee for the purpose of control and supervision of experiments on animals (CPCSEA), India and ARRIVE guidelines [19]. HEK293 cell line was obtained from National Centre for Cell Science, Pune, India whereas MRC5 cell line was kind gift from Prof. George Iliakis, University of Duisburg-Essen, Germany. Four experimental conditions were designed in all in vitro and in vivo experiments: control (untreated), DMA (50 µM for cells and indicated dose for mice), radiation (5 Gy for HEK293, 6 Gy for MRC5 and TBI in mice) and DMA+radiation (50 µM DMA+5 Gy for HEK293, 6 Gy for MRC5 and indicated DMA+radiation dose for mice). In all in vitro and in vivo experiments, DMA treatment at indicated dose was given 2 h before irradiation. Cells and mice were exposed to γirradiation using Co60 source (INMAS, Delhi, India) at 1.836 Gy/min.

2.3.6. Biochemical estimations for antioxidant enzymes and total protein in liver Mice hepatic tissues were homogenised using REMI homogenizer in phosphate buffer and centrifuged at 10,000×g for 15 min and aliquots of supernatant were separated. The supernatant was used for the biochemical estimations using standard spectrophotometric reported methods. ~100 mg of hepatic homogenate was mixed with 10 ml of 10% TCA and placed at 90 °C in a water bath for 30 min with stirring. Filtrate was dissolved with gentle warming in 0.1 mol/l NaOH. The total protein was determined by the Lowry method.

2.2. Uptake and efflux of DMA HEK293 cells (106cells/ml) were seeded and treated with complete media containing 50 µM DMA for 2 h. After 2 h incubation, cells were collected by centrifugation, resuspended in cold phosphate-buffer saline for subsequent flow cytometry analysis.

2.3.7. Total thiols estimation, lipid peroxidation Briefly, 200 µl hepatic homogenate was mixed with phosphate buffer (pH 8.0), 40 µl of 10 mM DTNB and 3.16 ml of methanol. After 10 min incubation, absorbance was measured at 412 nm and total thiol content was calculated by standard method [22]. The amount of malondialdehyde (MDA) was done by reaction with thiobarbituric acid (TBA) at 532 nm by literature method [23].

2.3. In vivo radioprotection by DMA 2.3.1. Balb/c mice survival by different mode of DMA administration against radiation Mice were grouped into 4 groups each containing 10 animals depending upon the mode of DMA administration as indicated. Group 1, sham control (saline treated); Group 2, DMA treated {200 mg/kg by oral, 50 mg/kg by i.v., i.p. & s.c.}; Group 3, radiation (saline treated followed by radiation, TBI); Group 4, treated with DMA{200 mg/kg by oral, 50 mg/kg by i.v., i.p. & s.c.} 2 h prior to radiation, TBI. Body weight, radiation sickness and mortality in animals were noted for 30 days. LD50/30 (lethal radiation dose causing 50% mortality in 30 days) for radiation and DMA+radiation treated mice were calculated from these studies. Dose reduction factor (DRF): 4 groups were created containing 10 mice each. Group 1, control; Group 2, DMA treated (200 mg/kg bw); Group 3, radiation control (5, 6, 8, 9 & 10 Gy TBI); Group 4, treated

2.3.8. Estimation of reduced glutathione Glutathione was measured according to the Ellman's method [24]. 2.3.9. Superoxide dismutase (SOD), Glutathione-S-transferase (GST) and Glutathione reductase (GR) activity Superoxide dismutase (SOD) activity was assayed according to the Marklund and Marklund method [25]. CDNB was used as substrate to determine GST activity. The reaction mixture contained 1 mM of CDNB, 1 mM GSH in 0.1 M phosphate buffer (pH 6.5). GSH-CDNB conjugate formation was estimated at 340 nm and the activity was calculated by using ε=9.6 mM−1 cm−1 [26]. NADPH oxidation rate by GSSH was 565

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(Santa Cruz Biotechnology Inc.) according to the manufacturer's instructions for knockdown of Akt gene. Annexin-V staining and clonogenicity of normal and Akt depleted HEK293 cells at four treatment conditions with 50 µM dose of DMA for 2 h were performed as reported previously [18,20]. Δp65-HEK293 cells generation is included in Supplementary data. Controlscram and Δp65-HEK293 cells were treated with 50 μM DMA for 2 h and clonogenicity was performed as described earlier [17,18].

used as a standard measure of enzymatic activity. The oxidation of 1 pmol of NADPH/min under these conditions is used as a unit of glutathione reductase activity [27]. 2.3.10. Quantitative real-time PCR Group 1, control; Group 2, DMA treated (50 mg/kg, i.v.); Group 3, TBI 8 Gy; Group 4, treated with DMA (50 mg/kg i.v.) prior to TBI 7 Gy. 2 µg of total RNA was used for the synthesis of cDNA by reverse transcription. The cDNA was amplified using 1 µl of the reaction products in 10 µl with respective primers for 45 cycles. Each cycle consisted of 10 s of denaturation at 95 °C, 60 s of annealing at 60 °C, and 60 s of extension at 72 ⁰C. The primers used for cDNA amplification (forward and reverse) are mentioned below Akt FP 5′-GTAGCCAATGAAGGTGCCAT-3′;Akt RP 5′-ATGAGCGACGTGGCTATTG-3′ p21 FP 5′-ATGTCCAATCCTGGTGATGT-3′, p21 RP 5′-TGCAGCAGGGCAGAGGAAGT-3′,GAPDH FP5′- AATGTGTCCGTCGTGGATCTGA-3′, GAPDH RP 5′-GATGCCTGCTTCACCACCTTCT-3′; Bcl2 FP 5′AGGATAACGGAGGCTGGGTA-3′, Bcl2 RP 5′-GCAGCAAGCTACTCAGACGA-3′; GADD45α FP 5′-TGGTGACGAACC-CACATTCAT-3′, GADD45α RP5′- ACCCACTGATCCATGTAGCGAC-3′; NFκB p65 FP 5′CTTCCTCAGCCATGGTACCTCT-3′;RP 5′-CAAGTCTTCATCAGCATCAAACTG-3′.

2.3.15. Pharmacokinetics of DMA Pharmacokinetics of DMA from mice blood was performed at 50 and 100 mg/kg dose of DMA given intravenously and orally respectively. Bioanalysis and pharmacokinetics analysis of samples were performed as reported previously [19]. 2.3.16. Statistical analysis Error bars are ± SD (in vivo) and ± SEM (in vitro) from 3 independent experiments. Statistical significance was determined using the Student's t-test and the one-way ANOVA followed by Tukey's Multiple Comparison as posthoc test for in vivo results using Graph Pad Prism software (version 5.0) software. P < 0.05 was considered significant data. Significance was calculated by Log-rank test in animal survival study.

2.3.11. Extraction of cytosolic and nuclear proteins 2.3.11.1. Cytosolic protein extraction. Cells after treatment were collected by trypsinization and washed twice with ice-cold PBS. Cell pellet was resuspended in five volumes of cytoplasmic extraction buffer (CEB) [10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.3% NP-40 and 1X protease inhibitor cocktail] to the size of cell pellet for 5 min with intermediate mixing. Protein solution was centrifuged at 3000 rpm for 5 min at 4 °C and supernatant was harvested as cytoplasmic extract. Protein content was estimated by Bradford reagent and equal total protein was loaded for desired protein detection by western blot.

3. Results 3.1. In vivo radioprotection by DMA DMA showed rapid uptake reaching its highest intensity in 2 h with gradual decrease in its intensity in HEK293 cells (Fig. S1). Therefore, in the subsequent experiments we used DMA concentration 2 h prior to irradiation. Mice treated with DMA at 200 mg/kg through oral route 2 h prior to irradiation showed 100%, 100%, 82%, 18% and 12% survival at 5, 6, 8, 9 and 10 Gy respectively (Fig. 2A) with a DRF of 1.28. Intravenous (i.v.) administration of DMA at 50 mg/kg 2 h prior to 8 Gy and 9 Gy irradiation exhibited 100% and 50% mice survival respectively whereas radiation only treated mice survival were found to be 40% and 0% for 8 and 9 Gy respectively (Fig. 2B). 50 mg/kg intraperitoneal (i.p.) DMA administration 2 h prior to 8 Gy irradiation showed 55% mice survival whereas, all irradiated mice died after 8 days (Fig. 2C). Similarly 20% mice survived when 50 mg/kg DMA was administered subcutaneously (s.c.) 2 h prior to 8 Gy irradiation (Fig. 2D). Next, we investigated the survival of nude mice by DMA pretreatment against lethal irradiation dose to access the translational implication of DMA. Our data revealed that all nude mice died on 12th day at 7 Gy radiation whereas, 40% nude mice survived by intraperitoneal injection of 50 mg/kg DMA 2 h prior to irradiation (Fig. 2E). Gradual weight loss was observed in irradiated mice whereas mice pretreated with DMA showed considerable regain of weight after initial 9 days of irradiation with delayed appearance of radiation sickness (Fig. S2).

2.3.11.2. Nuclear protein extraction. The cell pellet obtained after extraction of cytoplasmic proteins was resuspended again in CEB without NP-40. It was centrifuged at 3000 rpm for 5 min at 4 °C and supernatant was discarded. This step was repeated twice to avoid cytoplasmic proteins contamination in nuclear proteins. Equal volume of nuclear extraction buffer (NEB) [20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 25% Glycerol, and 1×protease inhibitor cocktail] was added to nuclear pellet and incubated on ice for 10 min with intermediate mixing. Nuclear pellet solution was centrifuged at 14,000 rpm for 5 min at 4 °C and supernatant was harvested as nuclear protein extract. Protein content was estimated by Bradford reagent and equal total protein was loaded for desired protein detection by western blot. 2.3.12. Chromatin immunoprecipitation (ChIP)-PCR assay ChIP-PCR assay was carried out as described previously [28] for genes targeted by NFκB such as IκBα, IL8 and Naf1 in HEK293 cells treated with 50 µM of DMA for 2 h.

3.2. DMA does not protect tumor against radiation 2.3.13. Immunoblotting analysis We used following antibodies: Akt, pAkt (Ser-473), GSK3β, pGSK3β, Bad, PTEN, IKKα/β, pIKKα/β, IκBα, NFκB (p65) and GAPDH. HRPconjugated mouse or rabbit secondary antibody (Abcam) were used using standard protocol [18]. Equal amount of MRC5 and Balb/c mice intestine protein from each condition was used to determine Akt kinase activity using Kinase Kit (CST, #9840) following the manufacturer's instructions.

As DMA showed significant radioprotection in vivo, we evaluated whether DMA specifically protects normal tissue or it also protect tumor against radiation. It was observed that when B16F10 melanoma tumor bearing mice (TBM) subjected to 50 mg/kg i.v. DMA administration 2 h before 8 Gy irradiation the average tumor volume at day 14th was found to be 2873.9, 2269.5, 1605.5 and 1804.125 mm3 for control, DMA, irradiated and DMA+radiation conditions respectively (Fig. 2F). There was 50% survival of TBM when DMA was administered prior to radiation as compared to radiation only treated mice, 0% on 14th day (Fig. 2G). This data suggested that DMA treatment prior to radiation provided no radioprotection to tumor.

2.3.14. Annexin-V staining and clonogenicity assay with Akt depleted and NFκB p65 knockdown (Δp65-) cells HEK293 cells were transfected with Akt-siRNA and control-siRNA 566

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Fig. 2. In vivo radioprotection by DMA. Percentage survival of (A) Balb/c mice at 5, 6, 8, 9 and 10 Gy TBI, with or without oral 200 mg/kg DMA pretreatment (n=10/group), (B) 8 and 9 Gy TBI, with or without 50 mg/kg, i.v. DMA pretreatment (n=10/group), (C) 8 Gy TBI, with or without 50 mg/kg, i.p. DMA pretreatment (n=10/group), (D) 8 Gy TBI, with or without 50 mg/kg, s.c. DMA pretreatment (n=10/group) (E) Nude mice (n=5/group) at 7 Gy TBI with or without 50 mg/kg, i.p. DMA pretreatment. (F) Effect of 50 mg/kg i.v. DMA on tumor growth in TBM treated at 8 Gy TBI (n=6/group). (G) Percentage survival of TBM after 8 Gy TBI, with or without 50 mg/kg, i.v. DMA pretreatment (n=6/group).*p value < 0.05 as compared to control.

Similarly, in TBM, DMA pretreatment to irradiation restored mice intestine and spleen almost equivalent to control group (Fig. 3B & S3). Through immunohistochemistry, it was observed that DMA pretreatment to irradiation increased BrdU-positive cells as compared to irradiated control in spleen tissue (Fig. 3C). Higher expression of IL6 was observed in DMA pretreatment to irradiation condition as compared to irradiated control in small intestine tissue (Fig. 3C).

3.3. DMA ameliorates radiation-induced damage in HP/GI tissues Disruption of villi and crypts were observed in 8 Gy TBI animals' intestines (Fig. 3A). DMA (50 mg/kg, i.v.) pretreatment to irradiation restores the crypts and villi height almost equivalent to control group (Fig. 3A). There was loss of spleen white pulp in irradiated mice whereas, it appeared normal in control and DMA groups. DMA pretreatment to radiation restored the spleen white pulp (Fig. 3A). Liver of TBI mice indicated fragmentation of hepatocytes and showed pyknotic and inflammatory kupffer cells whereas these cells number were reduced in DMA pretreatment to irradiation group (Fig. 3A).

3.4. DMA exhibits protection of HP system in Balb/c mice A significant reduction in spleen index was observed in TBI mice 567

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Fig. 3. DMA ameliorates radiation-induced damage in normal tissues. H & E staining of tissue sections were performed 3 days postirradiation (8 Gy) with and without DMA treatment (50 mg/kg, i.v.) (A) Intestine (20× & 40×magnification), spleen, and liver (40× magnification). Arrow represents white pulp (WP) and red pulp (RP) in spleen. (B) H & E staining of intestine section from TBM at 20× & 40× magnification. (C) Immunohistochemical analysis of BrdU uptake in spleen and IL6 protein expression in intestine tissues at 40× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

pared to irradiated group (Fig. 4D). 8 Gy TBI induced significant decrease (2.02 ± 0.20) in GST level while treatment with DMA (50 mg/kg, i.v.) resulted in insignificant decrease (2.76 ± 0.32) in GST level compared to the control group (3.20 ± 0.30). Administration of DMA prior to radiation induced significant increase (2.78 ± 0.25) in GST content compared to irradiated group (Fig. 4E). DMA did not show any significant change (16.96 ± 1.35) in SOD activity whereas 8 Gy TBI induced significant decrease (10.68 ± 2.4) compared to control group (20.49 ± 0.83) (Fig. 4F). Increased levels of SOD with DMA pretreatment to radiation (15.50 ± 0.60) showed protection of the mice from the acute radiation effects (Fig. 4F). 8 Gy TBI showed a significant decrease in total thiol contents (0.47 ± 0.02) in mice liver homogenates as compared to control tissues (0.81 ± 0.05) (Fig. 4G) which was restored by DMA pretreatment (0.91 ± 0.08) indicates maintenance of desired level of SH groups.

(0.21 ± 0.06) compared to sham control (0.40 ± 0.09). Pretreatment with DMA (50 mg/kg, i.v.), improved the spleen index (0.33 ± 0.02) compared to the radiation control. Whereas single dose DMA treatment alone showed spleen index of 0.41 ± 0.05 (Fig. 4A). Significant increase in endogenous colony forming units on 10th day post irradiation was also observed in DMA pretreated animals (11.25 ± 1.7) compared to radiation control (5.25 ± 1.5). DMA treatment alone exhibited no significant change in comparison to sham control (0.25 ± 0.5) (Fig. 4A). In this experiment, the enhanced spleen cell colony counts in irradiated mice pretreated with DMA suggest that DMA is a protective agent for hematopoietic cells. Therefore, it can be stated that DMA gave significant protection to the hematopoietic system, resulting in increased survival rates even after 30 days of whole-body irradiation.

3.5. DMA regulates radiation induce redox balance in mice 3.6. DMA regulates cell proliferation and apoptosis related genes in intestine of Balb/c mice

Intravenous DMA (50 mg/kg) administration resulted in insignificant change (0.19 ± 0.06) in MDA level compared to control group (0.22 ± 0.03) in hepatic tissues. 8 Gy TBI induced a significant increase in MDA level (0.76 ± 0.29) compared to control group at 24 h postirradiation. DMA pretreatment significantly decreased the MDA level (0.20 ± 0.04) as compared to radiation (Fig. 4B) thus restoring the damage caused by lipid peroxidation. Insignificant change in reduced glutathione (GSH) levels was observed in mice liver pretreated with DMA (50 mg/kg, i.v.) (5.07 ± 0.09) as compared to control animals (5.69 ± 0.26) (Fig. 4C). 8 Gy TBI resulted in significant decrease in GSH levels (2.16 ± 0.34) whereas pretreatment with DMA significantly restored the GSH levels in mice liver tissues (3.73 ± 0.69) as compared to radiation control (Fig. 4C). 8 Gy TBI induced decrease in the activity of glutathione reductase compared to control group (Fig. 4D). Treatment with DMA 2 h prior to irradiation resulted in a significant increase in enzyme activity com-

Based on our earlier microarray studies [20], we estimated expression of Akt1, NFκB, Gadd45α, p21 and Bcl-2 genes at transcription level in TBI mice intestine tissue (Fig. 5A). It was observed that DMA (50 mg/ kg, i.v.)+radiation (8 Gy) condition increased the expression of Akt1, NFκB and Gadd45α genes with respect to control (Fig. 5A). p21 expression was downregulated in DMA (50 mg/kg, i.v.)+radiation (8 Gy) condition with respect to radiation (Fig. 5A). Antiapoptotic gene Bcl-2 showed no significant response either in radiation or DMA+radiation conditions (Fig. 5A). 3.7. DMA activates Akt1/NFκB pathway in vitro as well as in vivo Since DMA pretreatment increased Akt and NFkB expression at transcription level, we evaluated their expression at translational level 568

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Fig. 4. DMA regulates radiation induced redox balance in murine. (A) Effect of DMA (50 mg/kg, i.v.) on spleen excised 10 days after 8 Gy irradiation in Balb/c mice (n=6). Bar graphs show comparison of spleen index among different groups; and comparison of number of spleen colonies among different groups. *P value < 0.05 as compared to saline control; **P value < 0.05 as compared to radiation control. Effect of DMA (50 mg/kg bw i.v.), radiation (8 Gy) and their combination on the level of (B) malondialdehyde (MDA) (C) reduced GSH. (D) GR Activity (E) GST Activity (F) SOD and (G) total thiol content in Balb/c mice liver tissue. Error bars are standard deviation (SD) for n=6. *P value < 0.05 as compared to saline control; **P value < 0.05 as compared to radiation control.

in vitro as well as in vivo. Our kinase assay data revealed that DMA+radiation condition increased Akt kinase activity in mice intestine and MRC5 cells with respect to control (Fig. 5B, C & S4 A, B). In line

with this, we observed higher expression of phosphorylated Akt1 (pAkt1, Ser-473) protein in mice intestine as well as in vitro (HEK293 and MRC5 cells) in DMA+radiation condition (Fig. 5D, E & S4C, S5A, 569

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Fig. 5. DMA activates Akt1/NFκB pathway. (A) Effect of DMA(50 mg/kg, i.v.) on the mRNA expression of Akt, Bcl-2, Gadd45, p21 and NFκB genes from Balb/c mice intestine tissue (n=3).*p < 0.05 compared to the control, #p < 0.05 compared to irradiated (8 Gy TBI) group. Western blot analysis of Akt kinase assay from (B) mice intestine tissue (n=3/group) and (C) MRC5 cells. (D) Western blot analysis of indicated proteins from mice intestine tissue (n=3/group) (E) Western blot analysis of proteins from whole cell protein from MRC5 and HEK293 cells. Western blot analysis of indicated proteins from (F) cytosolic and nuclear extract of HEK293 cells. C denotes Control, D-50 mg/kg i.v. and 50 µM for cells, R-5 Gy for HEK293 cells, 6 Gy for MRC5 cells and 8 Gy TBI in Balb/c mice, DR-DMA+radiation as mentioned respectively. (G) ChIP-PCR analysis showing DMA activates NFκB signaling using promoter specific primer for IκBα, IL8 and Naf1. 10% total input DNA was used as loading control. Isotype-matched IgG was used as negative control.

3.8. DMA doesn’t modulate Akt1/NFκB in tumor tissue

B) as suggested by western blotting. Similarly, we found increased NFκB expression in mice intestine as well as in vitro in DMA+radiation condition (Fig. 5D, E & S4C, S5A, B). Accordingly, we observed higher expression of NFκB p65 in cytoplasmic and nuclear extract in DMA+radiation condition as compared to control (Fig. 5F & S5C, D). All these data suggest that DMA+radiation condition increases pAkt1 and NFkB expression in vitro as well as in vivo. Further, to confirm these observations, we evaluated the expression of various other genes related to Akt1 and NFkB signaling. Our data revealed that DMA+radiation condition increased the expression of phosphorylated GSK3β (pGSK3β) in vitro as well as in vivo (Fig. 5D, E & S4C, S5A, B). However total GSK3β remains unchanged. We did not observe any change in expression level of PTEN protein in vitro (Fig. 5E & S5A, B) however; there was significant reduction in the expression of PTEN protein in mice intestine (Fig. 5D & S4C). In line with increased NFκB expression in DMA+radiation condition, we observed decrease in IκBα level in vitro as well as in vivo (Fig. 5D, E & S4C, S5A, B). In addition, we found higher expression of pIKKα/β in DMA+radiation condition as compare to control in mice intestine (Fig. 5D & S4C). Furthermore, we performed ChIP-PCR assay and showed that 2 h incubation with 50 µM DMA induced the binding of NFκB to its promoter of genes such as IκBα, IL8 and Naf1 (Fig. 5G). Thus DMA activates Akt1/NFκB pathway to regulate cell survival.

As DMA pretreatment to radiation activates Akt1 and NFκB signaling in vitro as well as in mice intestine, we evaluated whether DMA (50 mg/kg, i.v) modulates these signaling in tumor tissue or not. Surprisingly, our data revealed that DMA+radiation condition did not increase the expression of NFκB and pAkt1 in tumor tissue (Fig. 6). Similarly, IKKα/β and pIKKα/β remained unaffected in DMA+radiation condition in tumor (Fig. 6). IκBα, Akt1 and GSK3β proteins level were constant in DMA, radiation and DMA+radiation condition as compare to control tissue (Fig. 6). PTEN was overexpressed in both radiation and DMA+radiation condition as compare control tissue (Fig. 6). To the best of our knowledge, we reported first time where modulated signaling pathway was evaluated in tumor tissues under similar treatment condition in TBM. 3.9. Inhibition of Akt1 and NFκB limits radioprotection abilities of DMA Our data suggested that DMA provides radioprotection by targeting Akt1 and NFκB signaling. We confirmed these observations by abrogating Akt1 and NFκB gene. First, we knockdown Akt1 gene in HEK293 cells using siRNA approach (Fig. S6) and measured apoptotic cells. We observed increase number of apoptotic cells (23% at 3 h, 21% at 6 h, and 22% at 24 h) in DMA+radiation condition in Akt1 knockdown 570

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4. Discussion DMA controls radiation-induced DNA damage through free radical quenching with significant reduction in IR-induced foci such as γH2AX, 53BP1 and Rad51 [20]. Based on our earlier in vitro studies [18,20], present in vivo studies clearly establishes DMA as safe and effective radioprotective agent. Single dose of DMA injected either through oral, i.v., i.p. or s.c. routes provides protection against TBI in mice. DRF is a measure of effectiveness of a radioprotector. DMA has DRF of 1.28 when administered orally. Recilisib/ON01210 has US FDA investigational new drug (IND) status with DRF of 1.16 at 500 mg/kg (s.c)[30]. Genestein, another radioprotector, has DRF of 1.16 when injected s.c at 200 mg/kg [33]. Also, DMA is better than other radioprotectors such as tempol [31] and cystamine and comparable to Rajgira extract [32]. Amifostine, an FDA approved radioprotector, showed rapid clearance from the plasma with half-life of < 1 min and more than 90% clearance occurred within 6 min [33]. However, DMA by most common modes of administration has sufficient half-life (3.5 h for oral and 2.65 h for i.v.) with significant radioprotection (Figs. 2 & 8A, B). Exposure to ionizing radiation causes adverse side effect and animal mortality due to radiation induced GI and HP syndrome. DMA pretreatment to radiation improves survival of mice by recovering intestinal crypts and villi. Similarly, increased survival of TBM indicates protection of normal tissue (intestinal crypts and villi) against radiation (Fig. 3). IL6 helps in recovery of radiation-induced ARS [34]. DMA pretreatment increases IL6 expression in mice intestine (Fig. 3). Endogenous spleen colony formation after exposure to radiation in animals indicates proliferation, stimulation and survival of cells (stem cells) [35]. DMA pretreatment in irradiated mice showed less hematopoietic injury by increasing the proliferation of spleen cells with increase in spleen index (Fig. 4A). These results clearly indicate DMA ameliorates radiation induced damage of GI and HP system. Further TBI alters antioxidant defense systems in the body that lead to acute and long term tissue damage [36]. DMA pretreatment to radiation protects liver cells against radiation damage by suppressing cellular MDA level and increasing reduced glutathione, glutathione reductase, glutathiones-transferase, superoxide dismutase and total thiol content (Fig. 4B, C, D, E, F & G). Radioprotectors activate Akt1 and NFκB signaling pathway in normal tissue to protects against radiation damage [9]. Recently, Rosen and coworkers showed that DIM (3,3′-diindolylmethane) provides protection against TBI by activating NFκB signaling [37]. Our data revealed that DMA treatment protects mice intestine by increasing pAkt1 expression with concomitant increase in pGSK3β, a direct target of Akt1 (Fig. 5D & E). PTEN, a negative modulator of Akt1 signaling, is downregulated by DMA treatment in vivo. Similarly, our finding suggests that DMA treatment increases NFκB expression with decrease in expression of NFκB Inhibitory protein IκBα in vitro as well as in mice intestine (Fig. 5D & E). In addition, DMA treatment promotes nuclear movement of NFκB and induces the binding of NFκB transcription factor to its promoter suggesting a probable mechanism by which DMA activates NFκB signaling (Fig. 5F & G). In line with these observations, DMA does not provide radioprotection in Akt1 and NFκB abrogated cells (Fig. 7). One of the mechanism by which radiation induces Akt and NFκB system is by reactive oxygen species (ROS) intermediate signaling in cells [38,39]. However, we has proposed earlier that DMA is a free radical scavenger and minor groove binding ligand [16]. DMA treatment prior to radiation scavenges ROS and rescued the cells from ROS induced damage [16]. It is important to note that minor groove binder is known to modulate gene transcription by binding at DNA binding region of genes [20,40]. Thus, Akt1/NFκB activation by DMA is through gene transcription modulation. It is well reported that activation of Akt1/NFκB pathway promotes the transcription of wide range of antiapoptotic and cell prosurvival pathway such as Bcl-2, Bad etc. Akt activation by DMA following radiation inhibited GSK3β which is known to activate glycolysis and glucose transport and suppresses apoptosis

Fig. 6. DMA does not modulate Akt1/NFκB pathway in tumor tissues. Western blot analysis of indicated proteins from melanoma tissue (n=3/group) where C denotes Control, D-50 mg/kg i.v., R- 8 Gy TBI, DR-DMA+radiation as mentioned respectively.

cells (Fig. 7B). However, number of apoptotic cells were 12% at 3 h, 9% at 6 h and 7% at 24 h in DMA+radiation condition in control SiRNA treated cells (Fig. 7A). Importantly, by using clonogenic survival assay we observed that DMA provides minimal radioprotection in Akt1 knockdown cells in DMA+radiation condition (Fig. 7C). There was 40.6% and 7.5% radioprotection in control siRNA and Akt1 siRNA treated cells respectively at 5 Gy (Fig. 7C). Next, we knockdown NFκB gene in HEK293 cells using shRNA approach and efficacy of DMA in NFκB p65knockdown (NFκB Δp65-) cell line was studied (Fig. 7D). There was no radioprotection observed in NFκBΔp65- cells by DMA with 1.02 DMF in NFκB Δp65-HEK293 and 1.32 DMF in scrambled control cells (Fig. 7E). An increased expression of pAkt1 and pGSK3β was observed in controlscrm and NFκB Δp65-HEK293 cells but basal level of above protein was constant (Fig. 7F). NFκB p65 expression was very low in NFκB Δp65-HEK293 cells whereas, it has high expression in DMA+radiation condition in controlscrm cells. Subsequently the expression of IκBα was lower in DMA+radiation condition and pIKKα/β were elevated in DMA+radiation condition in both the cell lines. PTEN was almost constant in all conditions in both cell lines (Fig. 7F). Similarly, we observed minimal radioprotection by DMA in presence of Akt and NFκB inhibitors (LY294002 and PS1145 respectively) in HEK293 cells (Fig. S7). In control cells DMA provided 40%, 35% and 16% radioprotection at 24, 48 and 72 h respectively (Fig. S7A). LY294002 treated cells showed 15%, 10%, and 4% radioprotection at 24, 48 and 72 h respectively (Fig. S7B). PS1145 treated cells showed was reduced to 4%, 3.5%, and 2.4% radioprotection by DMA at 24, 48 and 72 h respectively (Fig. S7C). When HEK293 cells were treated with both LY294002 and PS1145 simultaneously radioprotection observed by DMA was 3%, 2.2%, and 2% at 24, 48 and 72 h respectively (Fig. S7D).

3.10. Pharmacokinetic of DMA Oral DMA (100 mg/kg) administration lead to maximum plasma concentration (445.8 ng/ml) in 1.5 h (tmax) and declined to basal level in 16 h (Fig. 8A). Half-life of DMA was found to be 3.5 h (Fig. 8A). Area under curve (AUC) calculated was 668.7 ng/ml (Fig. 8A). When DMA was injected at 50 mg/kg through intravenous route, maximum plasma concentration was 4291.95 ng/ml with half-life of 2.65 h (Fig. 8B), suggesting rapid absorption by tissue or faster elimination. The volume of distribution (Vss) of DMA was larger than total blood volume of mouse (Fig. 8B) indicating DMA distribution in extravascular system [29]. However, systemic clearance of DMA was higher (11.7 L/h/kg) than the hepatic blood flow (5.4 L/h/kg) indicating an extrahepatic elimination (Fig. 8B). 571

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Fig. 7. DMA offer no radioprotection in Akt1 depleted and NFκB Δp65- cell-lines. Percentage Annexin-V+ and Annexin-V+PI+ cells in (A) control siRNA and (B) Akt1 siRNA transfected HEK293 cells at 3 h, 6 h and 24 h with 50 µM DMA, radiation (5 Gy) and DMA(50 µM)+Radiation(5 Gy) (C) Clonogenic survival assay of control siRNA and Akt1 siRNA transfected HEK293 cells with 2 h 50 µM DMA treatment different radiation doses. (D) Western blot analysis to confirm NFκB Δp65- in HEK293 cell-line. (E) Clonogenic survival of scramble HEK293 (Controlscrm) and NFκB Δp65- HEK293 with 2 h 50 µM DMA treatment followed by indicated radiation doses. (F) Western blot analysis of indicated proteins from Controlscrm and NFκB Δp65- HEK293 cells where C denotes Control, D-50 µM DMA, R-5 Gy,DR-DMA+Radiation as mentioned respectively. *P value < 0.05 as compared to control was considered significant.

complex and hypoxic environment. It has been reported that PTEN level is reversibly inactivated by ROS level [44]. Further, we observed increased PTEN protein level in radiation and DMA+radiation condition in tumor tissue (Fig. 6). Compelling evidences suggest that upregulation of PTEN negatively regulates Akt signaling [45] and NFκB expression [46]. Thus, suggesting, due to increased PTEN expression, Akt and NFkB systems were not activated in tumor tissues. Our earlier studies showed that DMA accumulate in a low concentration in the tumor than normal tissue [19] and thus could not exhibit its transcriptional modulatory effect on Akt1 and NFκB system. Taking together, we suggest that DMA does not provide radioprotection to tumor because it fails to activate Akt1 and NFκB signaling with low accumulation in tumor. All these results indicate that DMA modulates Akt1/NFκB pathway and proteins involved in cell cycle, DNA repair, apoptosis and cell survival pathway to render radioprotection in Balb/c mice (Fig. 8C).

induction [41]. Further activated Akt is directly involved in DNA repair to promote cell survival [42]. It also phosphorylates IκBα, promoting degradation of IκBα, thereby increases activity of cell survival factor NFκB. In addition to cellular proliferation and inhibition of apoptosis, NFκB signaling induces expression of various inflammatory factors like IL-6 that have radioprotective properties. In recent years, development of radioprotective agents gained considerable importance in U.S and worldwide [43]. As a result, various radioprotective agents have been developed and many of them have promising preclinical efficacy. However, successful translation of these agents from bench to patient bedside has been hindered due to the lack of specificity. Interestingly, we found that DMA specifically protects mice intestine without protecting tumor tissues in tumor bearing mice (Fig. 2F & G). Further, mechanistic studies revealed that DMA does not activate Akt1 and NFκB signaling in tumor (Fig. 6). Generally tumor has 572

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Fig. 8. Pharmacokinetic of DMA. DMA concentration in Balb/c mice plasma following (A) 100 mg/kg oral (B) 50 mg/kg, i.v. mode of administration (n=3/timepoint). (C) Schematic representation showing minor groove binder DMA binds genomic DNA and provides radioprotection by activating Akt1/NFκB signaling.

Competing interests

In conclusion, DMA has the potential to develop as a safe radioprotective agent. It can be administered safely with most of the route of administration with considerable plasma half-life. DMA specifically protects normal tissue over tumor by targeting Akt1/NFκB signaling.

There is no conflict of interest of any authors. Acknowledgments

Funding

Authors are thankful to INMAS for radiation and nude mice facility, NII for radiation facility.

VTi and AR are thankful to CSIR, MZK to UGC- DSKPDF (Grant number BSR/BL-14/0256) and HN to ICMR (Grant number 45/26/ 2009-PHA/BMS) for fellowship. ICMR and UGC-UPE, India for project funding.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.04. 029.

Author contributions References VTi, MZK, AR, HN, MS performed experiments. VTi, AR, HN, VT analyzed data, designed and supervised the study.

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