PTEN inhibitor bpV(HOpic) confers protection against ionizing radiation

Exposure to Ionizing radiation (IR) poses a severe threat to human health. Therefore, there is an urgent need to develop potent and safe radioprotective agents for radio-nuclear emergencies. Phosphatidylinositol-3-kinase (PI3K) mediates its cytoprotective signaling against IR by phosphorylating membrane phospholipids to phosphatidylinositol 3,4,5 triphosphate, PIP3, that serve as a docking site for AKT. Phosphatase and Tensin Homolog on chromosome 10 (PTEN) antagonizes PI3K activity by dephosphorylating PIP3, thus suppressing PI3K/AKT signaling that could prevent IR induced cytotoxicity. The current study was undertaken to investigate the radioprotective potential of PTEN inhibitor (PTENi), bpV(HOpic). The cell cytotoxicity, proliferation index, and clonogenic survival assays were performed for assessing the radioprotective potential of bpV(HOpic). A safe dose of bpV(HOpic) was shown to be radioprotective in three radiosensitive tissue origin cells. Further, bpV(HOpic) significantly reduced the IR-induced apoptosis and associated pro-death signaling. A faster and better DNA repair kinetics was also observed in bpV(HOpic) pretreated cells exposed to IR. Additionally, bpV(HOpic) decreased the IR-induced oxidative stress and significantly enhanced the antioxidant defense mechanism in cells. The radioprotective effect of bpV(HOpic) was found to be AKT dependant and primarily regulated by the enhanced glycolysis and associated signaling. Furthermore, this in-vitro observation was verified in-vivo, where administration of bpV(HOpic) in C57BL/6 mice resulted in AKT activation and conferred survival advantage against IR-induced mortality. These results imply that bpV(HOpic) ameliorates IR-induced oxidative stress and cell death by inducing AKT signaling mediated antioxidant defense system and DNA repair pathways, thus strengthening its potential to be used as a radiation countermeasure.

Cell death assays. Acridine Orange and Ethidium bromide (EtBr) staining was performed in NIH-3T3 cells to account for the apoptotic cell, as described previously 34 . Briefly, cells were cultured in 96 well plates overnight prior to treatments. At indicative timepoints, plates were centrifuged briefly and incubated in 1:1 Acridine Orange and EtBr (Sigma-Aldrich) to a final concentration of 10 μg/ml each and incubated at room temperature for 10 min. Fluorescence was visualized under an Olympus fluorescence microscope, and percent of apoptotic cells out of total cells per field were calculated. Caspase-3 and -7 activity was studied using Cell Event Caspase 3/7 activity probe (Invitrogen, Ref. C10423) following manufacturer's protocol and percent positive cells out of total cell per field represented as Caspase-3 and -7 positive. For flow cytometry-based cell death analysis, we performed AnnexinV-PI (Invitrogen) staining following the manufacturer's instructions and cells were analyzed using BD FACS ARIA.
γ-H 2 AX foci formation assay. The residual DNA-double stranded breaks were determined by the γ-H 2 AX foci formation assay. Briefly, for γ-H 2 AX immunostaining, 0.1 × 10 5 cells were cultured on coverslips, at indicative timepoints cells were washed with ice-cold PBS, and permeabilized with 0.1%v/v Triton X-100 for 10 min at room temperature and washed again with ice-cold PBS. Non-specific protein binding was blocked with 4% BSA for 60 min at room temperature. Primary antibody (γ-H 2 AX Cell Signaling Technologies, 1:800) incubation was done for 1 h at room temperature followed by washing and secondary incubation with FITC conjugated secondary antibody (Sigma-Aldrich) for 45 min at room temperature. Cells were rewashed and mounted on a clean slide with antifade mount solution with DAPI (Invitrogen, Cat #P36931). Immunofluorescence was acquired using MetaCyte γ-H 2 AX foci scan software of an automated Metafer microscope (Metasystem, Germany). Roughly 150-200 images were analyzed with and Metafer (Version) and verified by manual counts.
53BP-1 foci formation assay. 53BP-1 foci formation was carried out in HEK cells stably transfected with a 53BP1-GFP plasmid (a gift from Dr. Deepak Saini's Laboratory, Indian Institute of Sciences, Bangaluru, India) using Lipofectamine 2000 (Invitrogen). For imaging, roughly 0.075 × 10 6 cells were cultured on sterile coverslips in a 35 mm PD with 2 ml growth medium, 24 h before treatment. 48 h post-treatment GFP fluorescence was observed, and images were captured using a fluorescence microscope (Olympus IX51, Japan) with 20X (objective) × 10X (eyepiece) magnification. Average foci formed per cell were counted.
Micronuclei assay. Approximately 1 × 10 5 cells were grown in a PD 35 mm for micronuclei assay, as described earlier 33 . Cells were washed and fixed at indicated timepoints in Carnoy's fixative (3:1 v/v, Methanol: Acetic Acid) at 4 °C overnight. Fixed cells were dropped on pre-chilled glass slides, air-dried, and stained with 10 μg/ml of DNA specific fluorochrome, diamidino-2-phenylindole dihydrochloride, DAPI, prepared in phosphate buffer (0.05 M Na 2 HPO 4 ·2H 2 O, 0.05%, Tween-20, 0.01 M citric acid, pH 7.4) in dark, at room temperature for 15 min. Stained slides were rinsed in PBS, mounted in an aqueous mounting medium (PBS-Glycerol, 1:1 v/v). Micronuclei fraction was scored using the criteria suggested by Countryman and Heddle 35 , from ~ 1000 cells per group from triplicate slides under a fluorescence microscope using a UV excitation filter. Micronuclei fraction (MF), i.e., the percentage of cells with micronuclei, was then calculated as MF(%) = N m /N t × 100, where N m is the number of cells with micronuclei, N t is the total number of cells analyzed.
Measurement of reactive oxygen species. Intracellular  In-vivo experiments. Adult, 6-8 weeks old C57BL/6 mice, with an average weight of 22-26 g, were issued from the Experimental Animal Facility of INMAS, DRDO, Delhi, India. They were distributed randomly in groups of 6 per cage and kept at 22 ± 2 °C and 12-12 h/light-dark cycle and were given a standard laboratory rodent diet (Golden Feeds, Delhi, India) and water ad libitum. The power analysis to compute the sample size was done using the Power analysis tool available at the National Centre for the Replacement Refinement and Reduction of Animals in Research, London, UK (https ://eda.nc3rs .org.uk). The acclimatization of animals was done one week prior to the experiments. The animals were administered bpV(HOpic) (Intraperitoneal route) 4 h before irradiation, and all animals were exposed to whole-body irradiation of 7.5 Gy using a 60 [39][40][41] . The radiation-induced cytotoxicity and proliferation index was assessed by SRB and growth kinetics assays respectively at indicative timepoints to evaluate the radioprotective potential of PTENi. The bpV(HOpic) was shown to be cytoprotective against IR up to 5 μmol/L in NIH-3T3, Raw 264.7, and INT-407 cell lines. The drug-induced toxicity was not apparent up to 5 μmol/L in the cell lines we tested; however, the drug alone showed toxicity at 10 μmol/L in all the three cell lines (Fig. 1A). The proliferation index of irradiated cells was compared with cells treated with two best cytoprotective concentrations of bpV(HOpic) (100 nmol/L and 500 nmol/L) one hour before irradiation. A time-dependant increase in proliferation index was observed in all three cell lines we tested when treated with bpV(HOpic), 1 h before irradiation as compared to their respective controls (Fig. 1B). In clonogenic assays also, increased clonogenicity was evident in all three irradiated cells treated with bpV(HOpic) in similar experimental conditions (Fig. 1C). Taken together, these results demonstrate that bpV(HOpic) induces radioresistance in cells. www.nature.com/scientificreports/ blot analysis of key apoptotic proteins. For this and subsequent mechanistic studies, we selected the NIH-3T3 cell model as bpV(HOpic) treatment showed good cellular protection against radiation injury in this cell line. The appearance of condensed or fragmented orange chromatin upon EtBr uptake, a characteristic of apoptotic cells that have lost their membrane integrity, was pronounced in γ-irradiated cells at 48 h post-exposure (40.23% ± 3.9 vs. 1.1% ± 0.2 in control cells; p < 0.01; Fig. 2A), which was diminished by the pretreatment of bpV(HOpic) (14.1% ± 3.79; p < 0.01; Fig. 2A). A similar trend was observed with Annexin V/PI flow cytometric analysis, where γ-irradiation lead to increased apoptosis at 48 h post-irradiation. In contrast, bpV(HOpic) conferred protection against radiation-induced apoptosis and reduced the percentage of the late apoptotic population (37% to 22%). However, the total apoptotic population (both late and early) was reduced to 32% in bpV(HOpic) treated cells as compared to 57% in radiation alone treatment (Fig. 2B). We also assessed the caspase-3 and -7 activity as well as the protein levels of cleaved caspase-3 in response to radiation. We observed that significantly low percentage (nearly 4.9% ± 1.01 at 12 h; 9.7% ± 2.6 at 24 h; 12.9% ± 0.4 at 48 h; p < 0.01; Fig. 2C) of bpVHOpic pretreated cells showed caspase-3 and -7activity as compared to radiation alone treated cells, in which a high number of cells showed caspase-3 and -7 activity at all of the timepoints we assayed (14.14% ± 1.64 vs. 2.1% ± 1.03 of control at 12 h; 19% ± 3.6 vs. 3.4% ± 0.99 at 24 h; 30.5% ± 4.7 vs. 2.4% ± 0.7 of control at 48 h; p < 0.01; Fig. 2C). A nearly threefold difference in caspase-3 cleavage was also observed between irradiated cells and cells pretreated with bpV(HOpic) before irradiation (4.5 fold ± 0.5 vs. 1.6 ± 0.13 at 24 h; p < 0.01; Fig. 2D). Besides, bpVHOpic pretreated cells showed a reduced level of the radiation-induced pro-apoptotic protein Bax and augmented level of pro-survival protein Bcl-xL. These results suggest that bpVHOpic pretreatment protects cells from radiationinduced apoptosis.

bpV(HOpic) reduces IR-induced DNA damage.
Besides apoptosis, radiation-induced DNA damage mediated mitotic catastrophe is another cause of death in radiation exposed cells. DSBs are the most lethal form of DNA damage; failure to resolve DSBs leads to cell death. The IR-induced DSBs are the main contributing factors for the loss of clonogenicity and increased cell death observed in cells exposed to IR 33 . Hence, the effect of PTEN inhibition on IR-induced DNA damage was assessed. The histone H2AX that undergoes phosphorylation (γH2AX) at the sites of DNA DSBs serves as a hallmark for DSBs detection. A time-dependent change in γH2AX foci, indicative of residual DNA-DSBs, was observed in NIH-3T3 cells after irradiation, detectable as early as 30 min post IR exposure (30.7 ± 1.8 vs. 4.5 ± 0.7 foci/cell in control; p < 0.01; Fig. 3A). However, bpV(HOpic) pretreatment efficiently reduced the number of detectable residual γH2AX foci per cell after IR exposure at 30 min (17.3 ± 1.9 foci/cell vs. 30.7 ± 1.8 in the irradiated group; p < 0.01; Fig. 3A). Further, the number of foci was found to be significantly lower in bpV(HOpic) pretreated cells at all the time points than radiation alone (Fig. 3A). To further strengthen the results of γH2AX foci formation assay, HEK cells stably expressing 53BP1-GFP plasmid were used as a reporter assay for IR-induced DNA double-stranded breaks. Using these reporter HEK cell line, we determined the effect of PTENi on 53-BP1 activation, a critical component of the NHEJ pathway. Cells showed increased amount of 53-BP1 foci 48-h post-irradiation (17.0 ± 1.6 vs. 5.1 ± 0.95 foci/cell in control; p < 0.01) that was significantly decreased by bpV(HOpic) in irradiated cells (9.6 ± 1.6 foci per cell; p < 0.01) (Fig. 3B). Therefore, the effect of bpV(HOpic) on DNA repair kinetics is rather a generalized observation and not specific to any species or cell line. Failure to repair DSBs leads to chromosomal aberrations that are manifested as micronuclei in daughter cells after mitosis, indicating cytogenetic damage. The kinetics of micronuclei formation was followed in NIH-3T3 (until 96 h) post-irradiation. We noted a significantly reduced number of micronuclei positive cells in the bpV(HOpic) pretreated group (Fig. 3C). Moreover, the protein levels of key sensors and regulators of DNA DSBs repair pathways were also analyzed 1-h post-radiation exposure by western blot. bpV(HOpic) pre-treatment led to a significant increase in all three components of MRN complex; interestingly, only MRE-11 (1.6 fold ± 0.2 vs. IR p < 0.01) and NBS-1 (1.9 ± 0.3 fold vs. IR p < 0.01) levels were increased in irradiated NIH-3T3 cells pre-treated with bpV(HOpic). A similar trend was observed with the protein levels of key components of the HR and NHEJ DNA repair pathways ( Fig. 3D and S3). These results suggest that PTEN inhibition through bpVHOpic significantly reduces radiation-induced DNA-DSBs and cytogenetic damage through enhanced DNA repair. Reduced cytogenetic damage results in enhanced clonogenicity and cell proliferation; therefore, this observation is in line with the enhanced clonogenic potential of bpV(HOpic) treated cells after radiation exposure.

PTEN inhibitor alleviates IR-induced oxidative stress.
Since oxidative stress is the major contributing factor to the IR-induced macromolecular damage, we evaluated the role of PTENi in regulating IR-induced ROS and resulting oxidative stress. The total cellular ROS was measured at 4 and 24 h post-irradiation. Radiation exposure resulted in a significant increase in total cellular ROS at 4 h (1.5 ± 0.10 fold; p < 0.01; Fig. 4A), which was further augmented at 24 h (2 ± 0.27-fold; p < 0.01; Fig. 4A). A significant (~ twofold at 4 h and 2.6-fold at 24 h) decrease in radiation-induced total cellular ROS was observed at both early and late timepoint in cells pretreated with bpV(HOpic). Moreover, we observed nearly three-fold increased radiation-induced mitochondrial ROS in NIH-3T3 cells at 4 and 24-h post-irradiation. However, bpVHOpic pretreatment led to a marginal yet significant reduction in mitochondrial ROS at 4 h (1.2-fold). At later timepoint, a reduction in IR-induced mito-ROS levels was better (twofold vs. IR) in bpV(HOpic) pretreated cells (Fig. 4B). We also measured the level of reduced glutathione (GSH) under similar experimental conditions. PTENi alone resulted in a modest (nonsignificant) increase in the levels of GSH initially that were significantly reduced in irradiated cells (3.04 ± 0.14 vs. 4.24 ± 0.32 μg/mg protein in control at 4 h; p < 0.01; Fig. 4C). However, cells pretreated with bpV(HOpic) before irradiation showed enhanced GSH levels (5.74 ± 0.54 μg/mg protein p < 0.01; Fig. 4C) at 4 h and later time point also. We also examined the effect of PTENi on the protein levels of critical free radical metabolizing enzymes in irradiated NIH-3T3 cells. IR induced reduction in the levels of catalase, MnSOD, and glutathione   www.nature.com/scientificreports/ reductase (GR) were found to be significantly replenished in co-treated cells (Fig. 4D). Consistent with this, PTENi pretreated cells also showed enhanced enzymatic activity of both catalase and total SOD enzymes, thus strengthening the radiation-induced oxidative stress modulating potential of PTENi bpV(HOpic) (Fig. 4E,F). We further assayed the IR induced oxidative damage to protein and lipids. For protein, we followed the total cellular carbonyl content up to 4 h of post-IR exposure. We found a nearly tenfold increase in total carbonyl content in IR-exposed NIH-3T3 cells at 4 h (39.59 ± 7.16 vs. 4.11 ± 1.44 nmol/mg protein in control; p < 0.01; Fig. 4G). By contrast, bpV(HOpic) pretreatment leads to a 3.6-fold decrease in total carbonyl content with respect to radiation alone, 4 h post-irradiation (11.13 ± 1.21 nmole/mg protein; p < 0.01; Fig. 4G). At late timepoints (24 h post IR-exposure), IR elevated the levels of lipid peroxidation-end product MDA to nearly two-fold (1.89 ± 0.16 vs. 0.88 ± 0.07 nmoles/mg protein in control; p < 0.01), that was reduced by the bpV(HOpic) pretreatment significantly (1.29 ± 0.08 nmoles/mg protein; p < 0.01; Fig. 4H). These data indicated that bpV(HOpic) suppresses IR-induced oxidative stress in cells by strengthening the antioxidant defense mechanism. bpV(HOpic) confers radio-protection by activating AKT signaling. PTEN inhibitors are known to activate AKT signaling, and studies have shown that activated AKT signaling modulates the radiation response of malignant tissues. However, most of the studies reported the implication of AKT signaling in malignancies, and it is not clear if activated AKT signaling could modulate the radiation response of healthy tissue to a similar extent. Therefore, to assess the role of AKT, we followed the kinetics of AKT activation upon bpV(HOpic) treatment. As evident by the phospho-protein/total protein expression ratio, PTEN inhibition led to a time-dependent increase in AKT phosphorylation in NIH-3T3 as well as Raw 267.4 macrophages (Fig. 5A). Exposure to IR also results in activation of AKT as a cell survival mechanism, and the same was evident in our results where we observed a nearly fourfold fold increase in AKT phosphorylation, 1-h post-IR exposure. However, the extent of AKT activation with bpV(Hopic) pretreatment was already ~ sixfold at the time of irradiation and maintained even after 1 h post-IR exposure.
Moreover, the bpV(Hopic) and IR induced AKT activation could be reversed by pretreatment with AKT inhibitor MK-2206 (Fig. 5B). To further explore the role of AKT signaling in PTENi conferred protection against IR, we performed growth kinetics analysis after inhibiting Akt activation in irradiated NIH-3T3 cells. The pharmacological and the genetic blockage of the AKT pathway through its allosteric inhibitor MK-2206 and AKT1/2 siRNA, respectively, resulted in the reversal of the radioprotection conferred by the bpV(HOpic) in NIH-3T3 cells (Fig. 5C,D). This data indicates that bpV(HOpic) confers protection against IR by activating AKT signaling.

bpV(HOpic) confers radioresistance by elevating glycolysis.
AKT exerts its radio-modulating effect by increasing the expressions of key metabolic proteins, thereby increasing the glycolysis 42 and enhanced glycolysis is also known to induce radioresistance in cells 33 . In agreement with the previous reports, increase in the levels of the key regulators of glycolysis was evident in cells treated with bpV(HOpic) where up to 1.5 fold increase in Glut 1 was observed at 4 and 24 h time intervals; and up to 1.6 fold increase in HKII was found at 4 h and 24 h as compared to their respective untreated controls; p < 0.01. Further, bpV(HOpic) significantly prevented the IR-induced reduction in levels of Glut 1 and HKII proteins (Glut 1 = 1.76 fold vs. 0.8 in IR at 24 h; p < 0.01; HKII = 1.8 fold vs. 0.7 in IR at 24 h; p < 0.01) (Fig. 6A). Mitochondrial translocation of HKII, the first enzyme of the glycolysis pathway, is known to increase the glycolytic flux, cell proliferation, and is critical for the regulation of mitochondria-dependant apoptosis. We estimated the levels of HKII in mitochondria by western blot analysis, where a significant decrease in mitochondrial HKII was observed in NIH-3T3 cells exposed to ionizing radiation. The reduced levels of mt-HKII were not only replenished but increased by bpV(HOpic) treatment of NIH-3T3 cells. The level of glucose uptake was followed in bpV(HOpic) treated NIH-3T3 cells postirradiation. At the time of irradiation (one-hour post-PTENi treatment), there was a significant increase in the mean fluorescence intensity of glucose fluorescent analog 2-NBDG in PTENi treated NIH-3T3 cells (1.2-fold; p < 0.01). A modest yet statistically significant increase in glucose uptake was also observed at 2 h (p < 0.01) and 4 h (p < 0.05) time-intervals after irradiation in cells treated with bpV(HOpic) (Fig. 6C). Further, we also noted a net gain in total ATP content per cell in irradiated cells that received bpV(Hopic) pretreatment (1668.69 ± 31.97 vs. 801.82 ± 35.03 pmol/cell in IR; p < 0.01; Fig. 6D). These results indicate that bpV(HOpic) protects against the lethal effect of IR through AKT-induced enhanced glycolysis.

PTEN inhibition confers protection against radiation in animals.
We further wanted to check whether the bpV(HOpic) induced and AKT mediated cytoprotection in vitro model can translate in vivo. For this, we administered the bpV(HOpic) (1 mg/kg body weight) intraperitoneally in C57BL/6 mice, and induction of AKT signaling in animals treated with bpV(HOpic) was investigated by immunoblotting. A significant induction of AKT signaling was evident from pAKT/AKT levels in gastrointestinal and hematopoietic tissues of bpV(HOpic) treated mice (Fig. 7A). After ensuring the bpV(HOpic) induced AKT activation in radiosensitive tissues in animals, we conducted an animal survival experiment following whole-body radiation exposure. We observed ~ 16% survival in mice exposed to a dose of 7.5 Gy whole-body irradiation, whereas bpV(HOpic) pretreatment provided survival advantage in radiation-exposed animals that was found to be significant in logrank tests (58%; p < 0.001; n = 12 per group), however, the result was also found very close to significance in trend analysis by same method (p = 0.06) (Fig. 7B). Taken together, these findings demonstrated an in-vitro and in-vivo radioprotective potential of PTENi, bpV(HOpic).

Discussion
Exposure to IR causes cell macromolecular damages, thereby triggering a number of cellular responses and signal transduction pathways 1,43 . Several signaling modifier molecules reducing the extent of IR-induced macromolecular damage are currently envisaged in radioprotection. PI3K-AKT axis is a vital signaling pathway that regulates DNA repair, cell cycle checkpoints, apoptosis, and senescence to determine the fate of cells following exposure to IR 13,16,17,44 . The activity of AKT is negatively regulated primarily via PTEN phosphatases by reversing the effects of PI3K action (converting PIP3 back to PIP2, through dephosphorylation of PIP3) 20,21 . Therefore, the critical balance between PI3K and PTEN activities has a significant influence on AKT signaling and cell survival following stress exposures. Several PTEN inhibitors have been employed in oxidative stressinduced conditions where they are shown to protect the tissue from oxidative damage [22][23][24][25] . Based on this, we hypothesized that inhibition of PTEN would facilitate the DNA repair and ROS clearance, thereby protecting the cells from IR-induced cytotoxicity. The hematopoietic and gastrointestinal systems are the most sensitive organ system to IR-induced cellular damage where exposure to doses 2-5 Gy leads to the manifestation of H-ARS and doses exceeding 5 Gy results in GI-ARS 2, 38 . Hence, oxidative damage to these organ systems is of concern for the efficacy of an effective radioprotector. Inhibition of PTEN by bpV(HOpic) one hour before IR-exposure shown to protect the cells of hematopoietic, intestinal, and fibroblast tissue origin against the radiation-induced cytotoxicity (Fig. 1A). Upon IR-exposure, the loss of clonogenicity occurs as cells are unable to divide and produce progenies owing to their lost ability to synthesize proteins and DNA; such cells are considered dead 33 . Compared with untreated cells, a gain in cell proliferation and clonogenic cell survival was evident in all three cell lines pretreated with bpV(HOpic) before IR exposure, indicating that PTENi treated cell lines showed significantly increased radioresistance (Fig. 1B,C).
IR induced macromolecular damage activates numerous cell death pathways. Where apoptosis is the majorly observed mechanism of cell death, executed through a series of molecular events. The well-characterized hallmarks of radiation-induced apoptosis 45,46 , like pyknosis (irreversible chromatin condensation) and loss of plasma membrane asymmetry and integrity, were markedly evident in our results that were abrogated by bpV(HOpic) treatment ( Fig. 2A). Our data also indicated a notable reduction in total apoptotic cells (early and late) in bpV(HOpic) pretreated cells when compared to radiation alone treated cells (Fig. 2B). Activation of caspases through their proteolytic cleavage is critical for the execution of apoptosis. This correlated with our results of a high amount of effector caspases-3 and -7 activity and cleaved caspase 3 protein levels in cells exposed to IR. The bpV(HOpic) treatment shown to reduce the caspases activity as well as protein levels of activated caspases (Fig. 2C,D). A significant reduction of pro-apoptotic proteins (Bax) and an increase in anti-apoptotic protein (Bcl-xL) levels in drug-treated groups indicated that bpV(HOpic) plays a protective role by reducing the radiation-induced apoptosis (Fig. 2D).
IR induces double-strand DNA breaks, which are potentially lethal and leads to cell death. Radiosensitive cells also lack efficient DNA repair capacity, those cells which escape apoptosis even after accumulating radiation-induced DNA damage die through mitotic catastrophe linked cell death 33,47,48 . Therefore, DNA repair plays a crucial role in regulating radiation-induced cell death. The observed reduction in cell death could be due to the better and faster DNA repair process in groups pretreated with PTEN inhibitors. Several lines of evidence exist where the direct and indirect role of the PI3K-AKT pathway has been shown to have implication in DNA repair processes 7,18,[49][50][51] . The activation of the PI3K-AKT pathway due to the inactivation of PTEN has been correlated with radioresistance 52, 53 . Our results corroborated with these studies where we observed a faster and better clearance of IR induced residual DNA lesions (Fig. 3A,B). The components of DNA repair pathways are shown to be under the regulation of PI3K-AKT axis [54][55][56] . The bpV(HOpic) pretreatment led to an increased induction and activation in the key components of the DNA DSB repair pathways (Fig. 3D and S3), which could be one possible mechanism for the faster and better clearance of DNA lesions in irradiated cells. Aberrant mitosis generates abnormal chromosome segregation, which on cell division leads to the formation of nuclear anomalies like micronuclei 47 . Time kinetics of micronuclei expression in irradiated cells clearly revealed that bpV(HOpic) pretreatment significantly reduced the number of micronuclei (Fig. 3C). Further, increased proliferation index, clonogenicity, and reduced cell death observed with bpV(HOpic) pretreatment (Figs. 1, 2) could be attributed to this.  www.nature.com/scientificreports/ The detrimental biological effects of IR are primarily orchestrated through oxidative stress, where exposure of cellular water to IR generates potent oxidants viz. reactive oxygen species (ROS) and reactive nitrogen species (RNS). Failure of cellular antioxidant defense mechanisms to metabolize these oxidants results in the oxidation of biomolecules like lipids, proteins, and DNA 1 . Exposure to IR resulted in a significant amount of oxidative stress that was evident in the form of increased total and mitochondrial ROS, reduced glutathione www.nature.com/scientificreports/ levels, key antioxidant enzymes protein levels, and their activity; oxidized total cellular protein and lipid content ( Fig. 4A-H). The accumulating oxidative stress, due to increased amounts of free radicals generated by IR exposure, is the major contributing factor for the macromolecular damage, culminating in cell death/apoptosis. The increased levels of oxidized biomolecules viz. lipids and proteins observed in IR exposed cells could be due to the free radical-mediated damage to the cell membranes and proteins, that further contributes to reduced enzymatic antioxidant defense systems (Fig. 4). These observations have direct implications in the high levels of residual DNA damage and cell death in IR exposed cells. Exposure to IR also results in the perturbations of the electron transport chain leading to mitochondrial ROS overproduction, further contributing to mitochondrial ROS-mediated oxidative stress. Pretreatment of bpV(HOpic) reduced the IR-induced oxidative stress and also replenished the enzymatic (Catalase and SOD) and non-enzymatic (GSH) antioxidant defense systems in cells.
Since oxidative stress is a major driving factor of IR-induced cytotoxicity, our observations suggest that the bpV(HOpic) ability to reduce IR-induced oxidative stress may be the primarily responsible factor for the reduction in the DNA damage and subsequently, cell death. Several lines of evidence exist where the PI3K-AKT axis has been shown to regulate cellular responses to IR, and transient activation of AKT signaling was a major driving factor to our hypothesis in this study to confer protection against the deleterious effects of IR. To explore the role of AKT signaling in bpV(HOpic) conferred radioprotection against IR, we examined the induction of AKT signaling post-bpV(HOpic) administration in cells and animals. Significant induction of AKT signaling could be seen as early as one-hour post bpV(HOpic) administration, both in-vitro and in-vivo. IR is also known to induce AKT signaling, and radiation-induced AKT phosphorylation (Fig. 5B) can be seen as a cellular stress response. However, by the time AKT activation takes www.nature.com/scientificreports/ place after radiation exposure, the pro-death pathway may dominate and overcome the AKT induced pro-survival pathways like antioxidant defense mechanisms and DNA repair pathways. On the other hand, preactivation of AKT signaling in bpV(HOpic) pretreated cells can prepare the cells to cope up with the radiation-induced oxidative and DNA damaging stress. Further inhibition of AKT phosphorylation using MK2206 reverts the bpV(HOpic) induced radioresistance in NIH-3T3 cells, suggesting the role of AKT in PTENi induced radioresistance (Fig. 5). Cells undergoing DNA repair process require a continuous supply of energy as DNA repair is indeed a completely ATP dependant process. In our previous study 33 , we have shown that cells with stimulated glycolysis have faster kinetics of IR-induced DNA lesions clearance. The potential role of the PI3K/AKT signaling pathway in stimulating glycolysis is well documented, where AKT regulates multiple steps in glycolysis that include inducing glucose transporters (GLUT) gene expression and enhancing hexokinase activity by translocating it to mitochondrial outer membrane 17, 57-60 . AKT induced hexokinase activity has a key role in regulating glucose uptake by converting glucose to glucose-6-phosphate 61 . In agreement with this, our results showed significant induction in the protein levels of Glut-1 and HKII, as well as glycolysis with the bpV(HOpic) treatment, which results in a net increase in cellular ATP (Fig. 6A-D). In addition to that, HKII is also known to play a role in regulating the apoptosis by binding to the mitochondria and inhibiting the Bax-induced cytochrome c release 62 . In our results also a significant increase in mitochondrial bound HKII was observed (Fig. 6B), which might be responsible for reduced IR-induced cell death in bpV(HOpic) treated cells. The faster DNA repair kinetics and diminished cell death observed in bpV(HOpic) pretreated cells could be attributed to the enhanced glycolysis (Fig. 6C), resulting in a net reduction in mitotic catastrophe/apoptosis and enhanced clonogenicity as evident in our study.

GI PBMCs
Actin Bone Marrow C S1 S2 S3 C S1 S2 S3 C S1 S2 S3 and Spleen] were harvested 4 h post 1 mg/Kg body weight bpV(HOpic) administration (n = 3 per group), intraperitoneally and 10% tissue homogenate was lysed and subjected to immunoblotting for pAKT and AKT levels. Lane C is a sample from control animal; Lanes S1-S3 are samples from animals 1-3. (B) Effect of bpV(HOpic) pretreatment (1 mg/Kg body weight; IP) on the survival of whole-body irradiated C57BL/6 mice. Statistical significance was measured using log-rank test. n = 12 per group, ***p < 0.001. www.nature.com/scientificreports/ With regard to this, we demonstrated here that pharmacological inhibition of PTEN protects against the lethal effect of radiation through AKT activation. Our study shows that bpV(HOpic) confers radioprotection by enhanced ROS clearance through better antioxidant signaling and faster DNA repair, thereby reducing the residual DNA damage and mitotic death. Furthermore, a single dose pretreatment of PTENi resulted in a declined cell death in an animal cell model and conferred a survival advantage to animals. However, inducing oncogene AKT by inhibition of PTEN could be the limitation for using this molecule as a radio-protector. Still, transient inhibition of PTEN has been investigated previously, where up to F2 progenies were found to be free from any tumors or other forms of chronic illness 32 . In line with this, our findings gained further support from the animal survival studies where bpV(HOpic) showed a very good trend in radio-protection, conferring 58% survival against IR. However, further investigation on large sample size, dose, route, and time of administration before IR exposure needs further exploration to derive any conclusions and further enhance the radio-protective effect of bpV(HOpic). Taken together, these findings clearly suggested that PTEN inhibition has the potential of alleviating IR induced cell cytotoxicity in an AKT dependent manner. To our knowledge, this is the first report of PTENi usage in counteracting the radiation-induced cellular damage. Further understanding of the mechanism of bpV(HOpic) conferred radioprotection in vivo will pave the way for utilizing PTEN inhibition as a possible target for the development of radiation countermeasure drugs.