Sphingosine kinase inhibitor, SKI-II confers protection against the ionizing radiation by maintaining redox homeostasis most likely through

Nrf2 signaling
Dhananjay Kumar Sah a, b, Yogesh Rai a, Ankit Chauhan a, Neeraj Kumari a, Madan Mohan Chaturvedi b, Anant Narayan Bhatt a, *
aInstitute of Nuclear Medicine & Allied Sciences, DRDO, Delhi, India
bDepartment of Zoology, University of Delhi, Delhi, India


Keywords: SKI-II
Nrf2 signaling Ionizing radiation Radioresistance Radioprotection Antioxidant defence DNA damage

Exposure to ionizing radiation (IR) set a series of deleterious events causing acute radiation syndrome and mortality, posing the need for a potent and safe radio-protective drug. IR induces cell death predominantly by causing oxidative stress and macromolecular damage. The pre-existing antioxidant defence machinery of the cellular system plays a crucial role in protecting the cells against oxidative stress by activation of Nrf2. The current study was undertaken to investigate the radio-protective potential of sphingosine kinase inhibitor (SKI- II), which was demonstrated to activate Nrf2 signaling. The safety and efficacy of SKI-II were evaluated with cell cytotoxicity, proliferation index, and clonogenic survival assays in different cell lines, namely Raw 264.7, INT- 407, IEC-6 and NIH/3T3 cell lines. A safe dose of SKI-II was found radio-protective in all the cell lines linked with the activated antioxidant defence system, thereby resulting in the amelioration of IR induced oxidative stress. SKI-II pretreatment also significantly reduced DNA damage, micronuclei expression, and accelerated DNA repair kinetics as compared to IR exposed cells. Reduced oxidative stress and enhanced DNA repair significantly reduced apoptosis and suppressed the pro-death signaling associated with IR exposure. Furthermore, the in-vitro observation was verified in the in-vivo model (C57 BL/6). The Intra-peritoneal (IP) administration of SKI-II, 2 h before a lethal dose of IR exposure (7.5 Gy) resulted in 75% survival. These results imply that SKI-II ameliorates IR-induced oxidative stress and cell death by inducing anti-oxidant defence system and DNA repair pathways, thus strengthening its potential to be used as radiation countermeasure.


The planned or accidental exposure of a biological system with ionizing radiation (IR) set a series of deleterious events. The high energy IR directly induces cytogenetic damage and indirectly causes the hy- drolysis of cellular aqueous medium leading to the production of reac- tive oxygen species (ROS) and reactive nitrogen species (RNS) [1,2]. These cell damaging events are countered by the cellular antioxidant defence system and DNA repair pathways. The radio-resistant cells and tissues of the body drive the homeostasis towards cell survival if the extent of damage is low; however, radio-sensitive cells die and release the oxidized macromolecules as damage-associated molecular patterns (DAMPs) [3]. The cells with a high proliferation rate or poor degree of differentiation are more sensitive at lower doses of radiation than the
less rapidly dividing or more differentiated cells [4]. The most radiation- sensitive organs include the hematopoietic, gastrointestinal (GI), skin, spermatogenic, and vascular systems, while the less sensitive is the muscles, nervous system, etc. [5]. The depletion of immature paren- chymal stem cells in specific radio-sensitive tissues becomes the primary cause of hematopoietic syndrome (HS) and gastrointestinal syndrome (GIS), together known as acute radiation syndrome (ARS). The he- matopoietic system of animals, including humans, is critically sensitive to IR, which results in the myelosuppression characterized by lympho- cytopenia, neutropenia, and thrombocytopenia. Similar events have been observed in GI, causing loss of crypts and perforations in the in- testinal tissues. Collectively, hematopoietic acute radiation syndrome (H-ARS) and gastrointestinal acute radiation syndrome (GI-ARS) in- creases the risk of infection, bleeding, and mortality [6–8].
* Corresponding author at: Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Timarpur, Delhi 110 054, India.
E-mail address: [email protected] (A.N. Bhatt).
Received 11 February 2021; Received in revised form 15 April 2021; Accepted 23 April 2021 Available online 30 April 2021
0024-3205/© 2021 Elsevier Inc. All rights reserved.

It has been well established that IR-induced ROS and RNS are the primary regulator of cell death and biological effects in radio-sensitive tissues. Excessively high ROS levels can lead to significant DNA dam- age and various cellular responses, including cell cycle arrest, senes- cence, and apoptosis [7]. Phase II detoxification enzymes usually detoxify the oxidative stress-induced cytotoxic insults and antioxidant proteins regulated by Nuclear factor erythroid-2–related factor 2 (Nrf2). Nrf2 promotes an adaptive cytoprotective response by regulating cellular redox status, thereby protects against IR-induced DNA damage and apoptosis [6]. Nrf2 is a strong trans-activator of the anti-oxidant response element (ARE); upon activation, Nrf2 dissociates from its cytosolic inhibitor KEAP1 and coordinates the up-regulation of ARE [9]. An upregulated ARE further drives the expression of electrophile detoxification and antioxidant genes to control the cellular redox state and protect the cell against oxidative damage [9]. The radio-protective role of Nrf2 is evident in a wide variety of cells, which is mediated by neutralizing ROS, reducing apoptosis, and increasing DNA repair re- sponses [7,10]. Many studies have recently shown that Nrf2 activation protects multiple tissues from carcinogens, electrophiles, inflammation and restores the radiation-induced H-ARS and GI-ARS [6,11]. Evidence demonstrates that Nrf2 activation increases macrophages’ phagocytic activity and prevents inflammation [12]. Previous studies showed that depletion of Nrf2 sensitizes the cells, whereas its overexpression confers radioresistance [13]. Therefore, it is evident that Nrf2-ARE driven detoxification and antioxidant genes play an essential role in cellular protection against oxidative stress and are therefore central to radio- protection. These findings have increased interest in identifying novel pharmacological agents, which modulate cell-specific Nrf2 activity to protect against radiation-induced systemic damage.
After CDDO-Me, a potent Nrf2 inducer, failed in a phase-III clinical trial [14], the search for a new and safe Nrf2 inducer, which can be developed as a radio-protector is on. In this study, we evaluated the radio-protective potential of a novel and alternative Nrf2 activator SKI-II ((2-(phydroxyanilino)-4-(p-chlorophenyl) thiazole). This sphingosine kinase inhibitor activated Nrf2 signaling in the nanomolar range by dimerizing and inactivating Keap1, independent of sphingosine kinase inhibition [15]. Therefore, the chances of associated toxicity of this molecule would be very less. We demonstrated that pretreatment with SKI-II stabilizes Nrf2 in cellular models, thereby activating the antioxi- dant defence system and increasing DNA repair efficiency, eventually protects from IR-induced cell growth inhibition and clonogenic cell death. Additionally, SKI-II treatment showed significant recovery from IR induced hematological alteration, thereby increasing survival effi- cacy in the in-vivo model system.

2.Material and methods
2.1.Maintenance of cell lines
All the cell lines, including mouse macrophage cells (Raw 264.7), rat intestinal epithelial cell (IEC-6), human intestinal cells (INT-407), and mouse fibroblast cells (NIH/3T3), were obtained from NCCS, Pune, India. Cell lines were maintained in their respective growth medium, high glucose DMEM and MEM supplemented with 10% fetal bovine serum (either normal or heat-inactivated as per the need of cell types). All the experiments were performed in the exponential growth phase maintained by routine passaging (every 2–3 days) in a fresh growth medium.

2.2.Experimental animals
C57BL/6 adult female mice (6-8 week old) with an average weight of 22–25 g were randomly distributed in a group of 6 per cage and kept at room temperature in 12–12 hour light-dark cycle, with a standard lab- oratory rodent diet (Golden Feeds, Delhi, India) and water ad libitum. Acclimatization of the animal was done one week before the
experiments. The study protocols were reviewed and approved by the Institute’s Animals Ethics Committee (Institutional Ethical Committee Number: INM/IAEC/2018/21).

2.3.Drug treatment and irradiation
According to experimental settings, cells were grown in either well plates (6, 12, 24, and 96) or 35 and 60 mm Petri dishes overnight at 37 ◦ C with 5% CO2 in the cell culture incubator. The next day, cells in their log phase were treated with SKI-II (100 nmol/l to 5000 nmol/l), 2 h before exposure to gamma (γ)-radiation. SKI-II was dissolved in DMSO (1 mg/ml; w/v), further dilution was made in culture medium and added to the culture dishes/plates to obtain the desired concentration (01 to 5000 nmol/l) before exposure to IR. Cells were incubated following IR exposure at 37 ◦ C with 5% CO2 in the cell culture incubator till further processing. For different cell lines, the radiation dose is mentioned in the respective figure legends. Whereas most of the in-vitro study was carried out in Raw 264.7 cells using 2 Gy radiation dose. In mice, SKI-II was administered by intraperitoneal injection 2 h before being exposed to a single dose of radiation (2 or 7.5 Gy) followed by multiple SKI-II treatment doses up to 72 h (at the interval of 24 h each). Un-anaesthetized mice (not more than four at a time) were irradiated with a single dose of 7.5 Gy. After irradiation, mice of all the treatment groups were housed in the institute’s central animal facility and were fed on a standard diet and water throughout the study period. All radiation treatments were carried out at the institutional 60Cobalt-Teletherapy Unit (Bhabhatron II, Panacea Medical Technologies Pvt. Ltd., India) under adjusted field size/SSD to obtain a fixed-dose rate of 1 Gy/min.

2.4.Analysis of cell viability by sulphorhodamine-B staining
Cells were seeded in 96-well plates at a density of 3000 cells per well in 200 μl of the growth medium, incubated overnight. The next day, cells were treated with SKI-II (100 nmol/l-5000 nmol/l) 2 h before irradia- tion and kept for incubation. At 48 h post-irradiation growth medium was removed, and cells were fixed in 10% (w/v) trichloroacetic acid for 1 h at 4 ◦ C. Cells were washed twice with double deionized water to remove excess fixative and air-dried followed by staining with 0.4% (w/
v) SRB solution (prepared in 1% (v/v) acetic acid) and incubated at 37 ◦ C for 30 min. Excess stain was removed with 1% acetic acid solution, and plates left to air dry. The protein-bound dye was dissolved in 200 μl of 10 mM Tris base solution (pH 10). The coloured solution’s optical density was read at 510 nm wavelength on a microplate reader (Biotek Instruments, USA) [16].

2.5.Cell proliferation/growth kinetics assay
Cells were seeded at a density of 0.075 × 106 or 0.1 × 106 in 35 or 60 mm culture dishes according to cells doubling time. After all drug and radiation treatments, cells were harvested by trypsinization/scraping and counted on Neubauerschamber (Paul Marienfeld, Germany) at indicative time-points under 10× objective and 10× eyepiece magnifi- cation using a compound light microscope (Olympus CH30, Japan).
2.6.Clonogenic cell survival assay
Clonogenic cell survival of SKI-II treated cells before irradiation was analyzed by macrocolony assay as described previously [17]. Briefly, cells were seeded in triplicates at very low density (100 to 3200 cells per plate) in 60 mm Petri dishes. After treatment with SKI-II 2 h before irradiation (0–8 Gy), cells were grown undisturbed to form colonies at 37 ◦ C in a CO2 incubator. When microcolonies were visible at 7–10 days, cultures were terminated, fixed in 10% Methanol, and stained with 1% crystal violet solution. Stained colonies were scored (at least 50 cells/
colony), and plating efficiency (PE) was calculated as given formula PE = (No. of colonies counted / No. of cells plated) × 100. The surviving

fraction (SF) was calculated as SF = PE of the Treated group / PE of control. The survival curve for all the cell lines has been fitted with a linear-quadratic model [18], alpha and beta values were analyzed using GraphPad Prism (Version 8.0a for Window OS).

2.7.Acridine orange-ethidium bromide staining

Acridine orange-ethidium bromide (AO-EtBr) staining was per- formed in Raw 264.7 according to the protocol described by Deborah Ribble with minimal modifications [19]. Briefly, cells were cultured in 96 well plates overnight before treatments as described earlier. At indicated time point (24 h), plates were centrifuged briefly and incu- bated in 1:1 acridine orange and EtBr (Sigma-Aldrich) solution to a final concentration of 100 μg/ml each and incubated at room temperature for 5–10 min. Images were captured under a fluorescence microscope (Olympus IX51 Fluorescence Microscope, Japan) at 10 × 10× magnifi- cations. The frequency of apoptotic cells was calculated for each group by counting the dead and live cells per field from 10 images for each group.

2.8.Caspase-3/7 activity assay
Activation of caspases was assessed by cell event Caspase 3/7 activity probe (Invitrogen) following manufacturer’s protocol, briefly cells grown in 96 well plates followed by treatments and irradiation (as mentioned above). Further culture medium was replaced with a fluo- rescent probe and incubated for 30 min. After washing with PBS, fluo- rescence cells were captured under a fluorescence microscope at 10
× 10× magnifications. The stained cells showing green dots represented active caspase-3/7. These dots/foci were counted from every ten images
for each group.

2.9.Cell death analysis by Annexin V-propidium iodide staining

Exponentially growing cells were treated with SKI-II followed by irradiation. At 24 post-treatment, cells were washed twice with PBS and harvested by gentle scraping. Cells were suspended in 1× binding buffer (provided in the kit) at a density of 1.0 106cells/ml. In 100 μl of
binding buffer, 5 μl of Annexin V-Alexa flour and 10 μl of PI (10 μg/ml) was added to flow tube and vortexes gently. The cells were incubated in the dark at room temperature for 15 min, followed by the acquisition using FACS Aria cell sorter (Becton Dickinson, USA) and data analysis using the FACS Diva software (Becton and Dickinson, USA). The per- centages of Annexin-V +ve/-ve and PI +ve/-ve cells were estimated by applying appropriate gates.

2.10.Measurement of reactive oxygen species and mitochondrial membrane potential
Intracellular reactive oxygen species (ROS) were assessed using the oxidant-sensitive fluorescent probes CM-H2DCFDA for total cellular ROS and MitoSOx Red for Mitochondrial ROS. Both probes obtained from Molecular Probes (Eugene, U.S.A.). According to the manufacturer protocol, cells were harvested and washed with PBS and incubated in probe buffer (1 mM CaCl2, 1 mM MgCl2, 5 mM Glucose) with either probe (20 μM CM-H2DCFDA or 5 μM MitoSox Red) at 37 ◦ C for 30 min. The excess stain was washed off using cold PBS. Cell suspended in PBS was analyzed on BD FACS ARIA III flow cytometer in their respective fluorescence channels. Mitochondrial membrane potential was deter- mined at respective time point following incubation of treated cells with TMRM (5 nmol/l/ml; 30 min; 37 ◦ C), in incubation buffer as mentioned above; cells were washed with PBS to remove excess stain followed by analysis on BD FACS ARIA III flow cytometer in their respective fluo- rescence channels.
2.11.Measurement of lipid peroxidation
Levels of lipid peroxidation were assayed by measuring the malon- dialdehyde (MDA) levels in the thiobarbituric acid reactive substances (TBARS) assay [20]. At indicated time points, cells were harvested and homogenized in ice-cold Tris-KCl buffer (10 mM Tris-HCl, 150 mM KCl, at pH 7.4). One volume of clear homogenate was heated for 45 min in a boiling water bath with two volumes of 0.37% w/v thiobarbituric acid and 15% w/v trichloroacetic acid. After cooling the solution and removing the precipitate, the clear supernatant’s absorbance was read at 532 nM on a 96 well-plate spectrophotometer. Finally, lipid peroxida- tion levels were calculated using a molar absorption coefficient of 155 mM–1 cm-1 and normalized with respective group protein content. Data presented as nanomoles of MDA formed per milligram of protein.

2.12.Analysis of superoxide dismutase enzyme activity

According to the manufacturer, the enzymatic activity of anti- oxidant enzymes superoxide dismutase was determined using Oxise- lect Superoxide Dismutase Activity assay kit (Cell Biolabs Inc.) instruc- tion. Briefly, 1–5 × 106cells were lysed in 1 mL of cold 1× lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.1 mM EDTA, 0.5% Triton-X 100) and incubated on ice for 10 min. Cell supernatant was collected after centrifugation at 12,000g for 10 min. Samples were prepared in 96 well plates having all the reaction components and incubated for 1 h at room temperature. Absorbance was read at 490 nm on a microplate reader. The results were represented as a relative fold change in the SOD activity of each group with respect to control.
2.13.Estimation of reduced glutathione (GSH) levels
The reduced glutathione (a non-enzymatic anti-oxidant) level was measured by its reaction with 5, 5′ -dithiobis-(2-nitrobenzoic acid) (DTNB or Ellman’s reagent) as described earlier [21]. Briefly, 0.05 ml of 25% TCA was added to 0.1 ml of cell homogenate to precipitate the protein. After cooling on ice for 5 min, the tubes were centrifuged at 10,000 rpm for 5 min at 4 ◦ C. From this, 0.1 ml of the supernatant was mixed with 0.9 ml of 0.2 M phosphate buffer (pH 8.0) and 2.0 ml of freshly prepared Ellman’s reagent (0.6 mM in 0.2 M phosphate buffer pH 8.0). The absorbance of the yellow-coloured product formed [5-thio- 2-nitrobenzoic acid (TNB)] was read after a 10-minute incubation at 412 nm in a spectrophotometer against the reagent blank (containing 5% TCA instead of homogenate). A standard curve of GSH was prepared using concentrations ranging from 2 to 50 μg of GSH in 5% TCA, and the amount of glutathione was expressed as μg/mg protein.
2.14.Protein expression analysis by western blotting
The protein expression of Nrf2, Keap1, SOD, catalase, HO-1, Bcl2, Bcl-xl, Bax, P53, PARP, and loading controls beta-actin and lamin were evaluated by immunoblotting. Cells harvested at indicated time points were lysed in RIPA lysis buffer (45 mM HEPES, 50 mM KCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 0.1% Triton-X100, 1 mM PMSF, 1 mM bezamidine, 1× protease (Pierce Biotechnology) and phosphatase (Roche) inhibitors cocktail) and total protein concentration in lysate was quantified using BCA-Assay kit (Sigma Aldrich). Equal amounts of pro- teins (40–50 μg) per sample were resolved on 10–15% SDS-PAGE as per respective molecular weight and transferred to a 0.2 μm PVDF mem- brane (MDI Membrane Technologies, India). All primary antibodies were procured from Cell Signaling Technologies Inc., USA or Sigma- Aldrich and were used at 1:1000 dilutions. After overnight incubation (14 h) of primary antibody at 4 ◦ C, membranes were washed 4 times (each for 10 min) with Tris-buffered saline with 0.1% Tween-20 Detergent TBST followed by incubation with the appropriate HRP con- jugated secondary antibody (1:3000) procured from Santacruz Biotechnology, Inc., USA. Following 2 h incubation at room


























Fig. 1. SKI-II treatment is safe and induces Nrf2 levels in cells at subtoxic doses.
A & B. SRB assay was performed to examine the effect of SKI-II induced cytotoxicity in Raw 264.7 (A) and INT407 cells (B) for 48 and 72 hours (h) respectively at the indicated concentrations. Data presented as percentage (%) cell survival with respective control. C & D. Immunoblotting was performed to assess the effect of indicated SK I-II concentrations on the basal level expression of Nrf2 at 2 h post treatment in Raw 264.7 (C) and INT 407 (D) cells. Blot images of both the cell lines are presented with densitometric evaluation (values mentioned below to each respective protein) as fold change with respect to control. E & F. Raw 264.7 (E) and INT 407 cells (F) were further subjected to time-dependent immunoblotting to analyze the effect of SKI-II (100 nmol/L) induced change in the Nrf2 expression level at the indicated time points. Statistical significance was determined and data are expressed as the mean ± SD (n = 3) *p < 0.05 vs. control.

temperature, membrane again washed 4 times with TBST and image developed using Forte Western HRP substrate (Merck-Millipore, USA) on ImageQuant LAS500 chemiluminescence CCD camera (GE Health- care, USA). Densitometry analysis was done using ImageJ 1.52K (NIH, USA).

2.15. γ-H2AX foci detection assay

Assessment of radiation-induced DNA damage was done by micro- scopic evaluation of γ-H2AX [22]. Briefly, for γ-H2AX immunostaining, 0.1 105 cells were grown in 35 mm petri dish on coverslips before any
treatments. After appropriate treatments, cells were gently washed with ice-cold PBS, followed by fixation and permeabilization with Acetone: Methanol (1:1) v/v for 10 min at -20 ◦ C and washed twice with TBST, blocked with 5% goat serum (in TBST) for 30 min at room temperature. After that, cells were incubated with monoclonal anti-γ-H2AX antibody diluted as 1:1000 in 1% BSA in TBST for 1 h at room temperature. Further washing of primary antibody followed by incubation with FITC conjugated IgG (dilution 1:1500 in 1% BSA in TBST) in the dark for 1 h at room temperature. Finally, cells were washed, and coverslip mounted over slide using SlowFade™ Gold AntifadeMountant (Thermo Fisher Scientific, USA) containing DAPI as the DNA-specific counterstain. Each


































(caption on next page)

Fig. 2. SKI-II confers radioresistance in various cells.
A, C, E, G. Radioprotective effect of SKI-II pretreatment (100 nmol/L) was examined by cell growth assay. Cells were irradiated as A. Raw 264.7 (2 Gy), C. INT 407 cells (6 Gy), E. IEC-6 (6 Gy) and G. NIH-3T3 (4 Gy), further cell number enumerated at 24, 48, and 72 h post-IR with respective control. Graph plotted as Nt/N0, whereas column graph chart represents the Nt/N0 of the last time point for each of the cell line. B, D, F, H. Radiation dose-response curve is indicated in B. Raw 264.7, D. INT 407, F. IEC-6, and H. NIH-3T3 cells. The D10 (10% survival) value of SKI-II + IR and IR alone was estimated by linear quadratic equation and presented in the inset graph of each respective cell line. I. The correlation between the radio-protective role of SKI-II pre-treatment and Nrf2 was validated by using NrF2 inhibitor Brusatol. Cells were pre-incubated with SKI-II, followed by immediate treatment of 50 nmol/l of Brusatol and irradiated (2 Gy). Further growth kinetics was performed in the mentioned treatment groups at indicated time points, and data presented as Nt/N0. For the better appreciation of the difference in cell number among the indicated treatment groups column graph is presented adjacent to the line graph, with the obtained value of Nt/N0 at 48 h post-irradiation. Data are expressed as the mean ± SD (n = 3) *p < 0.05 vs. control, #p ≤ 0.05, with respect to IR treatment alone and @≤0.05 SKI-II + IR vs. SKI-II + Brusatol + IR.
slide was pre-scanned under 10× objective followed by γ-H2AX foci counting at 63× magnification in an automated Metafer microscope (MetaSystems, Germany) using the MetaCyte γ- H2AX foci scan soft- ware. About 500 nuclei were scanned per slide to calculate the fre- quency of γ- H2AX foci as well as the number of foci per cell [23].
2.16.Assessment of cytogenetic damage by micronuclei formation
About 0.1 million cells were grown in 35 mm culture dishes over- night for micronuclei assay. After individual treatments, cells were allowed to grow for varying intervals. Cells were harvested at every 12- hour interval, washed with PBS, and fixed by Carnoy’s fixative (3:1 V/V, methanol:GAA). Fixed cells were dropped over clean, chilled glass slides, air-dried overnight, and stained with DNA specific dye, Hoechst (1 μg/ml) in phosphate buffer (0.45 M Na2HPO4⋅2H2O and 0.01 M citric acid in the ratio of 9:1, final pH: 7.4) containing 0.05%, Tween-20 for 30 min in the dark at room temperature. After washing the excess stain with distilled water followed by PBS, the slides were mounted in PBS-glycerol (1:1) and observed under a fluorescence microscope (Olympus IX51, Japan) using a UV excitation filter. A total of 1000 cells were analyzed for counting the number of cells containing micronuclei. The frequency of cells with micronuclei called the M-fraction (MF) was calculated as MF (%) = Nm / Nt × 100 where Nm is the number of cells with micronuclei, and Nt is the total number of cells analyzed [24].
2.17.Determination of animal survival against radiation
C57BL/6 mice were administered with the intraperitoneal injection of SKI-II (0.1 mg/kg body weight) with multiple doses (one dose at 2 h pre and three doses post-irradiation (7.5 Gy) at the interval of 24 h each) and monitored for survival till 30 days. The animals’ body weight was also recorded every alternate day for analyzing the effect of SKI-II on the radiation-induced decline in body weight. The survival curves were drawn by the Kaplan- Meier method [25].

2.18.Analysis of peripheral blood

Mice were administered SKI-II by intra-peritoneal injection, 2 h before being exposed to whole-body irradiation of 2 Gy followed by multiple SKI-II doses up to 72 h. Blood was collected from the retro- orbital plexus of mice into EDTA coated vacutainers at 01, 03, 07, 14, 21, and 28 days post-irradiation. Blood was allowed to mix thoroughly on a rotary shaker, and complete blood cell counts were obtained within 30 min of blood collection using automated hematology analyzer Euro count Plus (Medsourcebiomedicals, India). The analyzed parameters included total white blood cells (WBCs), lymphocyte number, gran- ulocyte number, RBC, and platelets.
Results were considered significant at p < 0.05.

3.1.SKI-II stabilizes Nrf2 on low and safe doses
The growth-inhibitory and cytotoxic activity patterns of SKI-II were screened in the Raw 264.7 and INT-407 cell lines using cell proliferation assay (SRB). The percentage of cell survival against increasing concen- tration range (100–5000 nmol/l) of SKI-II was evaluated at 48 h (continuous exposure) post-treatment in Raw 264.7 (murine macro- phage) and INT-407 (human intestine epithelial) cells (Fig. 1A & B). SKI- II did not show any toxicity up to 500 nmol/L in both the cell lines (Fig. 1A & B). Further, an increase in the concentration range (700 nmol/L–5000 nmol/l) showed cytotoxic activity in a concentration- dependent manner only in Raw 264.7 cells (Fig. 1A). Interestingly, in INT-407 cells, all SKI-II tested concentration ranges were found to be safe (Fig. 1B). Next, we performed immunoblotting to access the Nrf2 expression in a concentration (0–1000 nmol/l) and time (0–24 h) dependent manner in both the cell lines. Relative fold change analysis with respect to control observed with significant upregulation of Nrf2 at all the tested concentration with respect to control in both the cell lines (Fig. 1C & D). Nevertheless, maximum Nrf2 expression noted at 100 nmol/L (~1.8fold) and 200 nM (~2.8 fold) of SKI-II in Raw 264.7 and INT-407 cells, respectively. Moreover, compared to control SKI-II (100 nmol/L) treatment induced a significant change in the activation of Nrf2 protein, initially detected at 2 h in Raw 264.7 and 1 h in INT-407 cells (Fig. 1E & F). Although the degree of Nrf2 expression varies with time in both the cell lines; however, significant change remained persistent up to the last tested time point (24 h).
Taken together these finding suggests that SKI-II (<100 nmol/l) is nontoxic and ensure the upregulation of Nrf2 protein in both the cell lines. Therefore, we chose SKI-II (100 nmol/l) for further experiments.

3.2.SKI-II confers radioresistance

We next examined if SKI-II induced basal level increase in Nrf2 expression gives radio-protective efficacy in Raw 264.7, INT-407, IEC-6 and NIH/3T3 cells following exposure to IR. We performed cell growth assay and colony formation (clonogenic) assay in all these cell lines. Evaluation of growth kinetics data showed that pretreatment of SKI-II (100 nmol/l) followed by IR results in significant cell growth recovery

Table 1
The table represents the summary of the Linear Quadratic Analysis of the different cell line.
Groups α (Gy-1) β (Gy-2) α/β (Gy) D10 (Gy)

2.19. Statistical analysis

Data were analyzed using GraphPad Prism (Version 8.0a for Window OS), Microcal Origin 5.0 (Origin labs, for Window OS), and the experi- mental results were expressed as mean ± SD. All experiments were carried out twice and in triplicate every time. The student’s t-test was performed to determine the statistical significance between the groups.
Raw264.7 cells IR 0.195 0.092
SKI-II + IR 0.073 0.074
IEC-6 cells IR 0.235 0.024
SKI-II + IR 0.028 0.043
INT-407 cells IR 0.223 0.001
SKI-II + IR 0.001 0.017
NIH3T3 cells IR 0.049 0.054
SKI-II + IR -0.028 0.048
02.672 05.93
01.245 07.13
09.883 07.35
00.654 10.37
36.360 09.06
05.413 13.57
-00.059 08.19
00.908 11.18


































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Fig. 3. SKI-II pretreatment ameliorates IR induced cell death.
A. Photomicrograph showing AO/EtBr stained Raw 264.7 cells in the indicated treatment groups at 24 h post irradiation (10× objective; scale bar 20 μM). B. Quantitative assessment indicating the average of scored %dead cells (derived information from A) obtained from 10 different microscopic fields of view. C. Representative image of flow cytometry scatterplots depicting the Annexin-V (Alexa Fluor 488)/Propidium Iodide staining of Raw 264.7 cells at 24 h post-irradiation (2 Gy) in the mentioned treatment groups. Different apoptotic phase events (%) represents by Quadrants (Q1-Q4). The percentages of cells indicated in each quadrant as the means ± SD (n = 4) from two independent experiments. D & E. Representative photomicrographs Caspase-3/7 activity in the indicated treatment groups of Raw 264.7 cells (10× objective; scale bar 20 μM) (D) and the column graph (E) presented as the % of Caspase-3/7 positive cells per field (derived information from Fig. D) at 24 h post irradiation (2 Gy). F. Raw 264.7 cells were pretreated with SKI-II, 2 h before irradiation and whole cell lysates were subjected to immunoblotting for the analysis of key pro-apoptotic and pro-survival proteins at 24 h post irradiation with respective control. Densitometric evaluation was carried out (as values mentioned below to each respective protein) and data presented as relative fold change in the indicated protein expression with respect to untreated control. Error bars are mean ± SD (n = 4) from two independent experiments; **p < 0.01 compared to control; and #p < 0.05 with respect to IR treatment alone.

in all the cell lines, compared to IR treatment alone (Fig. 2A, C, E & G). Cumulative cell proliferation index of INT-407, IEC-6 and NIH/3T3 cells quantified at 72 h, and observed with a significant increase of ~39%
5.56) with respect to IR treatment alone (Fig. 2C, E & G). However, in (+
Raw 264.7 cell proliferation index quantified at 48 h (72 h time point omitted due to plateau phase) and showed a 43% increase, compared to
IR treated cells (Fig. 2A). Additionally, SKI-II pretreatment appeared to prevent the reproductive cell death and thus substantially retain the clonogenic potential of all the cell lines as compared to IR treatment alone (Fig. 2B, D, F & H). Interestingly, the magnitude of an increased surviving fraction in the IEC-6 cells showed significant difference in a dose-dependent (at 2, 4, 6 & 8 Gy) manner with respect to IR treated cells (Fig. 2F). Moreover, a significant increase in the surviving fraction was observed even at a higher dose (8 Gy) in INT-407 and NIH/3T3 cells, compared to IR treatment alone (Fig. 2D & H). Further results of sur- viving fraction from all the four cell lines were analyzed using a linear- quadratic model to confirm the SKI-II mediated radio-resistance (Table 1). The D10 value (10% Survival dose) of all the four cell lines were showed a considerable increase in the SKI-II + IR treatment group, compared to IR alone. The high α/β ratio in IR alone treated group in- dicates that the overall damage was considerably increased as compared to SKI-II and IR treated cells. The low α/β ratio and high D10 dose for the SKI-II pretreated cells indicates enhanced SKI-II mediated radio- resistance. To check if increased radioresistance in these cell lines is attributed to the SKI-II mediated NRf2-upregulation, we inhibited the Nrf2 signaling using Brusatol, which reverted the SKI-II mediated radi- oresistance in SKI-II pre-treated cell following irradiation. These results are also advocating the role of Nrf2 in SKI-II induced radioresistance.

3.3.SKI-II impedes IR induced cell death
We next questioned the mechanistic aspect of SKI-II mediated radi- oresistance by exploring cell death pathways in Raw 264.7 cells. Qual- itative image of Et/AO staining viewed and analyzed in terms of live cells (green; Et-/AO+), early apoptotic with more accumulation of AO stain (bright green) due to nuclear condensation (Et-/AO++), and late apoptotic cells showed orange in colour because of dye merging (Et+/
AO+). Microscopic image and derived quantitative information revealed that early and late apoptotic cell populations were observed most frequently and significantly high in the IR treatment alone, compared to SKI-II pretreated cells followed by IR (Fig. 3A & B). Moreover, early and late apoptotic phase events were further confirmed by using Annexin-V/
PI assay. SKI-II pretreatment conferred protection against IR-induced apoptosis and reduced the percentage of both late (Annexin-V+/PI+) and early (Annexin-V+/PI-) apoptotic population collectively from 53.3% to 24.7% in Raw 264.7 cells (Fig. 3C). These observations were further validated by caspase3/7, the critical mediator of the mitochondrial-dependent intrinsic apoptosis pathway [26]. SKI-II pre- treated cells following IR treatment showed a significant decrease in caspase 3/7 activity compared to IR treatment alone (Fig. 3D & E). Additionally, increased caspase3/7 activity is further linked with the upregulated expression of cleaved PARP in IR-treated cells (Fig. 3F). Moreover, the substantial increase in the expression of p53 following IR exposure was notified as ~4 fold in IR treated cells whereas confined to
1.46 fold in cells pretreated with SKI-II with respect to untreated control cells (Fig. 3F) The p53 protein is known to regulate radiation-induced apoptosis [27]; therefore, as compared to IR treatment alone the sub- stantially reduced level of p53 in SKI-II pretreated cells might be linked with the reduced cell death following irradiation. Apart from that reduced levels of pro-apoptotic protein Bax and enhanced level of anti- apoptotic proteins Bcl-2 and Bcl-xL observed in SKI-II pretreated cells following IR as compared to IR treatment alone resulted in the protec- tion of cells from radiation-induced cell death (Fig. 3D).
These results suggest that SKI-II pre-treatment confers radio- resistance by regulating the induction of key apoptotic determinants eventually resulting in reduced cell death following irradiation.

3.4.SKI-II treatment attenuates IR-induced oxidative stress

The major cause of radiation-induced cell death is IR induced oxidative stress and macromolecular damage [28]. Because post IR exposure oxidative stress is the major contributing factor for macro- molecular damage, and as we observed a significant reduction in cell death in SKI-II treated cells; hence, SKI-II’s role was further examined for the regulation of IR induced ROS and oxidative stress. A follow-up analysis of total cellular ROS kinetics revealed a nearly 2 fold increase in the untreated group as early as 4 h while reaching its peak of 4.3 fold at 36 h post-IR exposure. In SKI-II treated groups, a significant decrease in the IR-induced total ROS was evident at both early and late time points (viz. 4, 8, 14, 18, and 36 h) as compared to untreated control (Fig. 4A). We observed no significant change in IR-induced mitochon- drial ROS at earlier time points, though a small difference was observed between the control and SKI-II + IR group which may helpful in Nrf2 nuclear translocation, a four-fold increase in IR-induced mitochondrial ROS was seen at 24 h post-irradiation that was significantly diminished with SKI-II pretreatment (Fig. 4B). Hyperpolarized mitochondria acti- vate the apoptotic pathways and enhance the cell kill. Therefore, we analyzed mitochondrial membrane potential, an indicator of mito- chondrial health, under a similar experimental setup. At the early time points, no significant differences were observed in any group; however, at 24 h post-IR exposure, cells showed nearly 3-fold high MFI of TMRM, which indicated membrane hyperpolarization that was dampened nearly 2-fold by SKI-II pretreatment (Fig. 4C). A significant reduction in the protein levels of anti-oxidant enzymes like HO-1, SOD, and catalase was also observed in radiation alone group cells but replenished by SKI- II pretreatment (Fig. 4D). An increase in SOD expression was signifi- cantly correlated with the upregulated enzymatic activity in SKI-II pre- treated cells followed by irradiation compared to IR treatment (Fig. 4E). A similar trend was observed in the level of reduced glutathione (GSH) which observed with a significant increase in the SKI-II treated groups compared with the untreated IR exposed group at all the tested time points post-IR-exposure (Fig. 4F). In a similar experimental condition these results were further correlated with the IR-induced oxidative damage to macromolecules was further assessed from lipid peroxidation by-product- MDA levels which was increased significantly in IR alone as compared to SKI-II + IR treated cells (Fig. 4G). The Nrf2 is a vital regulator of the cellular antioxidant response mechanism where its cellular localization, activity, and expression are tightly regulated at



































(caption on next page)

Fig. 4. SKI-II reduces the IR-induced oxidative stress in cells by activating the antioxidant defence mechanism.
A. Total ROS production in Raw 264.7 cells was estimated as the indicated function of time following irradiation (2 Gy) in the mentioned treatment groups. At the indicated time points cells were harvested and subjected to spectrophotometric analysis with H2-DCFDA fluorescent probe the presented ROS kinetics is the relative fold change in mean fluorescent intensity with respect to untreated control. B. Mitochondrial ROS production in Raw 264.7 cells was estimated at 4 and 24 h post- irradiation (2 Gy) using MitoSox staining in the mentioned treatment groups. Data presented as the change in mean fluorescent intensity with respect to untreated control. C. Effects of SKI-II pre-treatment on the mitochondrial membrane potential (MMP) in irradiated Raw 264.7 cells (2 Gy) presented at indicated time points and treatment groups. Data are presented as change in mean fluorescence intensity (MFI) with respective control. D. SOD activity was determined in Raw 264.7 cells as early as 4 h post irradiation in the indicated treatment groups and presented as fold change with respect to control. E. Immunoblotting was carried out in Raw 264.7 cells 4 h post-irradiation in the indicated treatment groups and densitometric evaluation is presented below to each respective proteins. F. Lipid peroxidation assay was performed using the TBARS method at indicated time points in Raw 264.7 cells. Graph shows the malondialdehyde (MDA) content as (nmol/mg protein) in the mentioned treatment groups. G. The cellular GSH content was estimated in Raw 264.7 cells at indicated time points and the data presented as (μg/mg protein). H. Effect of SKI-II on the expression of Nrf2 and Keap1 was examined by immunoblotting in the indicated treatment groups of Raw 264.7 cells at 4 h post-irradiation. I, Immunoblotting was performed to assess the nuclear localization of Nrf2 following SKI-II treatment in Raw 264.7 cells at 2 h post-irradiation in the indicated treatment groups. Data are expressed as the mean ± SD (n = 3) *p < 0.05 vs. control and #p ≤ 0.05, ##p ≤ 0.01 with respect to IR treatment alone.
several levels. We observed a reduction in the levels of Keap-1, an Nrf2 repressor protein, in cells treated with SKI-II, while an increase in the Nrf2 protein levels was noted. In contrast, a significant increase in the Keap-1 and a dip in NRf2 protein levels were observed in IR- exposed cells (Fig. 4H). The nuclear localization of the stabilized Nrf2 is a critical determinant for activating the critical targets of the antioxidant response mechanism. SKI-II pretreatment led to an increased ratio of nuclear Nrf2 in cells exposed to IR (Fig. 4I). These results suggest that SKI-II regulates IR-induced oxidative stress and associated macromo- lecular damage by strengthening the antioxidant defence mechanism most likely by Nrf2 upregulation and its nuclear localization.

3.5.SKI-II reduces IR-induced DNA damage

The DNA DSBs are the major contributing factors to IR-induced cell death and loss of clonogenicity. Thus, we investigated the effect of SKI-II on IR-induced DNA damage, where we observed an increased γH2AX foci per cell [22], indicative of DNA-DSBs, at 60 and 120 min in cells exposed to radiation. In contrast, SKI-II pretreatment significantly reduced the γH2AX frequency in cells exposed to radiation at all the time-points we assessed (Fig. 5A & B), suggesting reduced DNA DSBs. Micronuclei formation, a chromosomal aberration/cytogenetic damage arising out of unresolved DNA DSBs, is another cause of loss of clono- genicity by inducing mitotic catastrophe in cells following IR exposure. We followed the IR-induced micronuclei kinetics (M-fraction) and Cell growth kinetics (Nt/No) in Raw 264.7 cells until 72 h and observed a significant reduction in the micronuclei positive cells in SKI-II pretreated groups exposed to radiation (Fig. 5C). The cells pretreated with SKI-II has a significantly low M-fraction value with a high rate of cell prolif- eration (Nt/No) while IR alone group found a high M-fraction value and low rate of cell proliferation. These data suggest that SKI-II reduces DNA DSBs and cytogenetic damage, possibly through better DNA repair.

3.6.SKI-II confers protection against IR in animals
Further to check if SKI-II induced radio-protection observed in cellular models can also translate to the animal model, we checked the radio-protective potential of SKI-II in C57BL/6 mice by performing a 30- day survival analysis. The animals were administered with SKI-II 0.1 mg/kg body weight of mice (average weight, 23 g), intraperitoneal in- jection (i.p.), 2 h before being exposed to a single lethal dose of 7.5 Gy followed by 3 doses post-irradiation each at 24-h interval. Animals were observed for signs of radiation sickness and mortality for 30 days post- irradiation treatment. Animal survival was then analyzed using the Kaplan Meier survival curve (Fig. 6A). At this lethal radiation dose, the percent survival of vehicle group animals was 17%, accompanied by radiation sickness symptoms such as loss of weight and dehydration. However, it was interesting to observe 75% animal survival with maintained health at the end of the study in the SKI-II treated animals (p
< 0.01).
3.7.SKI-II protects from radiation-induced cytopenia
As we discussed above, IR-induced hematopoietic syndrome is one of the major cause behind IR induced mortality. Therefore, we analyzed the hematopoietic parameters of IR exposed animals. The peripheral blood cell indices are a reliable and straightforward assay for assessing hematopoietic injury and its amelioration by a countermeasure agent [29]. Since the low dose radiation is sufficient to cause myelosup- pression, the hematopoietic recovery was studied in C57BL/6mice exposed to 2 Gy radiation and compared with the effect of SKI-II at day 01 to day 28 post-treatment. Radiation-induced severe depletion of white blood cell counts were observed at 24 h following whole body irradiation in both SKI-II pretreated and radiation alone mice compared to the un-irradiated/sham irradiated animals (p < 0.01). However, SKI- II treatment significantly inhibited the initial dip in both WBCs and lymphocytes and showed accelerated recovery, which is quite delayed in irradiated animals (Fig. 6B). The total leukocyte count in the irradiated animal at 24 h post-irradiation was significantly low with respect to SKI- II pretreated animals (3.145 ± 1.180 in SKI-II + IR group vs. 1.24
± 0.257 in IR group). Moreover, the total leukocyte count did not show complete recovery; however, SKI-II treatment initiated recovery from day 14 onwards and almost recovered to baseline till day 28 (p < 0.05). The lymphocyte and granulocyte number also reached their nadir values just after 24 h post-irradiation and showed a similar pattern of recovery in the presence of SKI-II. Like total WBCs, the initial dip in lymphocyte counts were significantly less in SKI-II treated animals and recovered entirely at day 21 than radiation alone, which did not show recovery till day 28 (Fig. 6C). For granulocytes, the decline was similar to that of lymphocytes until day 7 after irradiation. The counts were very low till day 14, which was indicative of severe neutropenia in the irradiated cohort (Fig. 6D). SKI-II instigated recovery from day 7 onwards (p < 0.05). However, SKI-II treatment most effectively mediated the recovery from radiation-induced lymphocytopenia. Platelet counts were the lowest between days 7 and 14 in radiation-treated animals; however, there was a slight incline in SKI-II treated group on the 7th day of ra- diation (Fig. 6E). Further, there was no change in RBC following 2 Gy irradiation (Fig. 6F).


Exposure to ionizing radiation activates several macromolecular damaging cascades and triggering several cellular response signaling pathways. Several molecules reducing the extent of IR-induced macro- molecular damages by modulating the antioxidant defence system of the cells are currently envisaged in radioprotection. Nrf2/ARE axis plays a vital role in protecting cells from IR induced oxidative stress and cellular death. Nrf2 regulates DNA repair, cell cycle checkpoints, apoptosis, and senescence, determining the fate of cellular response towards radiation exposure [30]. Nrf2 signaling also regulates hematopoietic stem cell proliferation, crucial for protecting the exposed individual from radiation-induced cytopenia. Based on this, we hypothesized that the
































Fig. 5. Effects of SKI-II treatment on radiation induced DNA-DSBs and cytogenetic damage in Raw 264.7 cells.
A. Representative photomicrographs of γ-H2AX foci formation for each treatment group are shown in irradiated Raw 264.7 cells at indicated time points. Cellular images displayed here as DAPI (in the left panel) γ-H2AX foci (middle panel) and a composite image of γ-H2AX and DAPI (right panel) in the indicated treatment groups and time points (63× objective; scale bar 5 μM). B. For the better appreciation of radiation-induced γ-H2AX foci formation minimum of 100 cells from each group replicates were scored and plotted at different time intervals (X-axis) in the indicated treatment groups. C. The line diagram is plotted between Micronuclei fraction (M-fraction) and Nt/N0 to correlate the cytogenetic damage with cell proliferation. Details are given in the method section. Error bars are mean ± SD (n = 4) from two independent experiments. #p < 0.05 vs. SKI-II + IR with respect to IR treatment alone.





























Fig. 6. SKI-II confers protection against the lethal effect of radiation in vivo.
A. C57 BL/6 female mice 6–8 weeks of age were taken for survival study. The number of mice kept as control and IR (n = 6), for SKI-II treatment group (n = 12). The mice were intraperitoneally (IP) administered with SKI-II (0.1 mg/kg body weight) 2 h before irradiation (7.5 Gy). Mice were under observation for the signs of premorbid state during the 30 days analysis period. Kaplan–Meier survival plot indicating the effect of SKI-II on the survival of mice compared with IR alone. B–F. Hematological analysis including WBC (B), lymphocytes (C), granulocytes (D), platelets (E) and RBC (F) was carried out by taking 50 μl blood at the indicated time points from Retro-orbital bleeding performed using Eurocount hematology analyzer, represented as count per litre. Statistical significance is presented as #p < 0.05 vs. SKI-II + IR with respect to IR treatment alone.

induction of Nrf2 would facilitate the neutralization of IR-induced ROS, activation of antioxidant pathways, DNA repair, and cell proliferation, thereby protecting the cells from cytotoxicity induced by exposure to IR [7].
With a very high proliferation rate, the hematopoietic and gastro- intestinal tissues are more susceptible to IR insult as compared to other organs with low cell proliferation rate [11]. The damage caused by oxidative stress to these organs is of concern for an effective radio- protector’s efficacy. Upregulation of Nrf2 with SKI-II 2 h before IR exposure was correlated with enhanced survival of cells originated from hematopoietic, intestinal, and fibroblast tissue (Fig. 2A, C, E, G). Exposure to IR results in the loss of clonogenicity as cells cannot divide and produce progeny due to loss of their protein and DNA synthesis, these type of cells are considered dead [7]. SKI-II treated cells showed an increase in clonogenic cell survival and cell proliferation compared to untreated cells following irradiation. SKI-II retreatment showed a sig- nificant increase in the radioresistance, tested in all the cell lines (Fig. 2B, D, F, H).
Exposure to IR induced macromolecular damages, which activates numerous cell death pathways such as apoptosis, necrosis, autophagy, senescence, and mitotic catastrophe. Apoptosis is the major mode of death observable post-exposure to irradiation, manifested through a series of events. Post irradiation apoptosis is easily identified by its hallmarks such as condensation of chromatin (pyknosis), cell shrinkage, loss of structural and functional integrity of plasma membrane, intra nucleosomal chromatin breakage [28,31]. In our results, we found a significant increase in the pyknosis in untreated control cells, while it was reduced in the cells treated with SKI-II before IR exposure (Fig. 3A &
B). The early apoptosis is marked by the loss in the membrane asym- metry, while a loss of integrity marks the late apoptotic event. Our experimental data suggest a remarkable decrease in the total population (early apoptotic) in the SKI-II pretreated cells when compared with the untreated cells subjected to irradiation (Fig. 3C). For the execution of apoptosis, the activation of caspase through its proteolytic cleavage is critical. Activated caspase regulates apoptosis through several key reg- ulators through their proteolytic cleavage [32]. Apoptosis induced by irradiation is known to follow the intrinsic pathways (mitochondrial), and canonical extrinsic (mediated through the death receptors) path- ways and caspase activation may occur irrespective of the apoptotic mode [26]. In line with this fact, in our results also a high amount of effector caspase-3 and -7 activity was observed in IR exposed cells, which was found to be reduced with SKI-II pretreatment before IR (Fig. 3D & E). The intrinsic apoptotic pathways involve the per- meabilization of the outer mitochondrial membrane, thus disrupting mitochondria’s structure and function regulated by pro-and anti- apoptotic Bcl-2 family proteins [26]. The reduced cell death and cas- pase3/7 activation in combination (SKI-II and IR) correlated with a significant reduction in pro-apoptotic protein (Bax) and an increase in anti-apoptotic protein (Bcl-xL) levels in SKI-II pretreatment before irradiation (Fig. 3F). Besides routine DNA repair function, PARP plays a crucial role in regulating pathological and physiological functions. PARP-1 is one of several known cellular substrates of caspases. Cleavage of PARP-1 by caspases is considered to be a hallmark of apoptosis [33]. The result showed that pretreatment with SKI-II reduces the cleavage of PARP-1 protein when compared with untreated control subjected to irradiation. This finding also correlates with enhanced DNA repair observed in SKI-II treated cells before IR exposure (Fig. 5). Further, p53 regulates major apoptotic signaling pathways and its increased levels for a longer duration after IR exposure indicates residual macromolecular damage and induction of apoptosis in cells [34]. We found that pre- treatment with SKI-II, which induces the Nrf2 level in cells, reduces the p53 protein level post IR exposure, while it is high in the IR alone (Fig. 3F). This result is in line with earlier observations by other groups, where they have demonstrated that the regulation of mitochondrial redox post cytoplasmic irradiation, reduces the p53 level and promotes cell survival. Additionally, these observations also found in correlation
with our finding of increased mitochondrial ROS at 4 h post irradiation in combined treatment. Possibly this enhanced mitochondrial ROS promoted the Nrf2 translocation to the nucleus contributing to sup- pressing the p53 level and associated cell death [35,36].
It indicated that SKI-II plays a protective role possibly by inducing the Nrf2 levels and reducing the p53 regulated apoptosis induced by IR exposure. In IR exposed cells, high levels of ROS upregulates the guardian of genome integrity, p53 and provokes a pro-oxidant response to further increase the oxidative stress for inducing the p53-dependent apoptotic processes to eliminate the cells with mutated and damaged genome from the system, in order to suppress tumorigenesis. However, p53 regulates the oxidative stress in a biphasic manner, at a low level of oxidative stress and damage, it upregulates the Nrf2 to protect the cells from oxidative stress induced cell death and when oxidative stress crosses the level, it induces cell death [37]. Similarly in our results, we also observed that SKI-II pretreatment controlled the IR induced oxidative stress and DNA damage repair efficiently (Figs. 4 & 5) and therefore p53 levels were found nearly similar to control (Fig. 3F) as compared to IR alone. Taken together, these observations suggest that on the contrary of IR alone exposed cells, where p53 induced apoptosis was observed, SKI-II pretreatment regulated IR induced oxidative stress and DNA damage repair leading to protective phasic regulation of p53.
The deleterious effect of IR exposure is primarily manifested through oxidative stress, water radiolysis leads to potent oxidants such as reac- tive oxygen species (ROS), reactive nitrogen species (RNS). Failure of cellular machinery in these stressors’ clearance results in oxidative damage to the macromolecules like lipids, proteins, and DNA [38]. IR exposure leads to the disturbance of redox homeostasis that was evident by an increase in the level of reactive oxygen species, reduction in the level of reduced glutathione, activity and levels of key antioxidants enzymes, oxidized lipids, and protein contents of the cells, the second and subsequent waves of ROS observed builds steadily and forms bigger waves leading to more cell death (Fig. 4A–G). Accumulated oxidative stressors are the major factors responsible for the macromolecular damages that result in cell death/apoptosis. Oxidation of membrane lipids results in MDA formation, which is a key marker for stress-induced tissue damage [39]. The increased MDA level observed in the IR exposed cells further contributes to the impairment of the membrane’s structural and functional integrity (Fig. 4F). IR exposure also results in the per- turbations of the electron transport chain which results in the over- production of mitochondrial ROS, further contributing towards oxidative stress. Pretreatment with SKI-II reduced the primary wave of IR induced ROS and subsequent delayed oxidative stress burst also by reducing the level of total ROS, and the mitochondrial ROS also main- tains the mitochondrial membrane potential indicator of mitochondrial health (Fig. 4C). SKI-II pretreatment further replenishes the enzymatic (HO-1, catalase, SOD) and non-enzymatic (GSH) antioxidant defence and enhances the activity of the SOD enzyme (Fig. 4D). Nrf2 is a key regulator of ARE which in turns activate the antioxidant defence in the cellular system [30]. Our results also showed that the pretreatment with SKI-II increase the level of Nrf2 and reduces the level of Keap1, which negatively regulates the Nrf2 (Fig. 4H). Therefore, it appeared that the maintained cellular redox status and reduced cell death in irradiated cells by SKI-II pre-treatment is possibly led by Nrf2 upregulation (Fig. 4I), while their accumulated level results in macromolecular damage and cell death. The high level of total cellular ROS and mito- chondrial superoxide result in accumulating stressors in the cells causing DNA damage. IR induces the double-strand breaks if it remains unre- paired and could be lethal and leads to affected cell death. Cells sensitive to radiation also lacks the efficient DNA repair mechanism, those cells escape apoptosis even after the accumulated damaged DNA die through mitotic catastrophe linked cell death [7]. Therefore, efficient DNA repair is crucial for regulating radiation induced cell death. In the cells pre- treated with SKI-II, the observed reduction in cell death could be due to the faster and efficient DNA repair process. Several pre-existing pieces of evidence show the role of Nrf2 in DNA repair processes. Activation of
Nrf2 provides radioresistance to cells by activating fast and efficient DNA repair [7]. Our results also correlated with these studies and pro- vided considerable evidence that the radio-protective role of SKI-II is might be attributed to the Nrf2 upregulation contributing to the efficient and fast clearance of IR induced DNA lesions (Fig. 5A & B). The faster and efficient DNA repair kinetics were observed in the SKI-II pretreated cells, resulting in reduced mitotic catastrophe (micronuclei)/apoptosis probably contributing to enhanced clonogenicity as observed in this study.
IR induced oxidative stress, DNA damage and p53 mediated apoptotic death is the major cause of death in fast proliferating leuko- cytes leading to radiation-induced hematopoietic syndrome [40]. In hematological studies, we demonstrated that pharmacological inter- vention to increase Nrf2 expression by SKI-II treatment before radiation exposure not only reduces the IR induced cytopenia in WBCs but also helps in early recovery from hematopoietic syndrome (Fig. 6B–F). This observation gained support from an animal survival study, where SKI-II conferred 75% survival against IR alone. The results obtained in this study suggest that SKI-II confers radioprotection by regulating IR induced ROS through enhanced antioxidant defence signaling and faster DNA repair, which in turn reduce the DNA damage and associated mitotic death. Further, we have observed that SKI-II mediated increase in the basal level of Nrf2 expression is transient (Fig. 1E & F), which may reduce the adverse impact on normal cell physiology when translated as a drug.
In conclusion, our findings suggest that SKI-II confers radioresistance most likely through the induction of Nrf2/ARE and eventually alleviate the IR induced cytotoxic insult. Probably, this is the first report of sphingosine kinase inhibitor, SKI-II in contracting IR induced cell death and animal mortality. Further, understanding the mechanism of SKI-II induced Nrf2 activation and its potential role in radioprotection in vivo will pave the way for utilizing it as a possible radiation counter- measure drug during nuclear accidents.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We acknowledge the members of the Metabolic Cell Signaling Research Laboratory, staff members of the INMAS institutional experi- mental facility, staff members of the Department of Zoology, University of Delhi for their constant support and coordination. We are thankful to Dr. Nabo K. Chaudhury and Dr. I Prem Kumar, INMAS for extending instrument support for some experiments. We extend our sincere grati- tude to Prof. Umesh Rai (Head of the Department) Department of Zoology, University of Delhi for his continuous moral and administrative support. This study was funded by buildup grant from Defence Research and Development Organisation (DRDO), Government of India. DKS was a recipient of a fellowship from the Department of Biotechnology (DBT), Government of India. While YR, NK and AC received fellowship support from Indian Council of Medical Research (ICMR), Government of India.


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