Nrf2 Expression and Activation After Radiation Injury

Introduction

The potential and unwanted  disruption and destruction of normal tissue are the major concerns that limit the use of radiation therapy [1] The initial cellular damage in response to radiation exposure occurs in less than 1 picosecond and the ROS generated are mostly quenched within 5 minutes   [2, 3]. However, even after the dispersion or quenching of ROS, ionizing radiation induces longer lasting increases in oxidative stress. This is due to decreases in cellular antioxidants and detoxification molecules, and damage induced mitochondrial dysfunction which causes secondary production of ROS.[4, 5]. Thus, while radiation therapy is a first line treatment for brain and spinal cord tumors and metastases there is concern over the long term cognitive impairments typically associated with its use [6, 7].  The duration of cellular responses to radiation can vary greatly from restoration of normal expression occurring within 24h to continued dysfunction and dysregulation continuing out to 2 months [8, 9]. This variation is likely due to differing cell type types of radiation, dose rates of radiation, and total dose.

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Homeostasis of the nervous system relies heavily on a careful redox balance, with reactive oxygen species having impacts on cell fate, neurogenesis, cell survival, and apoptosis and senescence [10]. Thus, the brain and other neurological tissues may experience profound and long lasting effects of radiation exposure, and the effectiveness of protective cellular responses are critical for mitigating potential injury and promoting tissue repair.

The antioxidant response element (ARE) is responsible for the transcriptional activation of antioxidant genes under basal and oxidative stress conditions [11]. Nuclear factor E2-related factor 2 (Nrf2), a potent transcription factor activated under conditions of redox stress, is credited with binding and activation of ARE to produce one of the cell’s most potent and immediate antioxidant defenses in response to ROS exposure [12]. Activation of ARE produces an increase in phase II antioxidant proteins including glutathione S-transferase (GST), manganese superoxide dismutase (MnSOD), catalase, and thioredoxin [13, 14]. Under basal normoxic conditions Nrf2 is post transcriptionally regulated by Kelch-like erythroid cell-derived protein (Keap1) which binds Nrf2, preventing its entry into the nucleus and aids in ubiquitylation and degradation of Nrf2 [15]. Oxidative stress causes Nrf2 release from Keap1, allowing it to translocate to the nucleus and induce expression of the phase II antioxidant genes to improve cell survival in both normal and tumorigenic cells [16-19]. Knockdown of Nrf2 induces increased cellular sensitivity to oxidative stressors in murine models and in vitro tumor and normal tissue [17, 20, 21].

In contrast, knockdown of Keap1, disruption of Keap1-Nrf2 binding or pharmacological amplification of Nrf2 produces cytoprotective responses compared to controls when subjected to oxidative stress both in murine models and in vitro in NCM460 colonic cells, primary neonatal rat astrocytes  [22-24]. Survival after radiation injury, therefore, may be attributed to at least two mechanisms: quenching of ROS, and effective DNA repair [25]. Central to these mechanisms is upregulation of Nrf2 to activate early responses to control redox stress, and thereby promote cell survival. Specific antioxidant enzymes downstream of Nrf2 activation have also been demonstrated to play a role in cellular survival in vitro and in survival in murine models of redox stress. These endogenous antioxidant enzymes are: manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx), thioredoxin (THX), and catalase [26, 27]

Research into cancer cells has indicated that higher expression and activity of antioxidant enzymes and their products is key to survival after oxidative stress. Radiation resistant glioma subtypes demonstrate  increased activity of MnSOD, GPx, and catalase compared to their more radiation sensitive parent strain both before and after radiation injury [28]. Similarly exogenous overexpression of GPx, MnSOD or THX reduces apoptosis and improves survival after oxidative stressors from ionizing radiation or staurosporine in murine models as well as tumor cells and normal cells [29-35].

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In contrast depletion or knockdown of these antioxidants together or separately can produce sensitization to oxidative stressors in both murine models and tissue culture samples. Most obvious, complete knockdown of MnSOD/SOD2 produces a neonatal lethal mutation in mice in which offspring are produced but die weeks after birth [36]. Downregulations of catalase expression are blamed for the autosomal disorder Granular Corneal Dystrophy Type II which promotes oxidative stress and apoptosis in corneal fibroblasts even in the absence of injury [37]. Prolonged depletion of glutathione or thioredoxin significantly decreases survival in murine models and tumor and normal tissue culture [38-42]. There is abundant evidence in the literature that these antioxidants are necessary for protection against oxidative stress. Moreover, their absence induces cell death in a variety of cell types, indicating their necessity.

Based on this evidence it seemed reasonable to believe that antioxidant response would be necessary for survival and proliferation after radiation injury. The hypothesis of this project was that NHAs would utilize upregulate multiple antioxidant pathways to effectively handle the production of ROS which occurs after radiation injury. This ability to handle radiation injury was set to be measured in the cytoplasm by DCF and in the mitochondria by MitoSOX. Further, long term damage from ROS would be measured by survival of the cells and ability to produce ATP, as this is a mechanism for measuring mitochondrial function. I hypothesized that the radiation resistant normal human astrocytes would display a robust antioxidant response compared to mesenchymal stem cells (MSC)s and cells and this would be the cause of their ability to survive and proliferate after high dose of radiation

Methods

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Nrf2 expression and activation after radiation injury

Cells were irradiated then cultured for 30min, 2h and 6h. Cells were washed (3 times with phosphate buffered saline [PBS]), then treated for 10 min with 4% paraformaldehyde (room temperature) and washed again (3 times with PBS). Cells were permeabilized with 70% ethanol (10 min, room temperature), blocked with 5% normal donkey serum in PBS, 1 h, 37^oC, and incubated for overnight at 4^oC with Nrf2 antibody (Novus Biologicus) diluted 1:400 in 5% normal donkey serum in PBS. Cells were washed 3 times with PBS and then incubated for 1 h in Donkey anti-rabbit IgG (Thermo Fischer, A21206) in PBS at room temperature. Cells were washed (3 times, PBS), incubated with DAPI (4′,6-diamidino-2-phenylindole) (5 min, room temperature, in dark) diluted 1:1000 in PBS, then mounted and visualized with an Olympus BX61 fluorescence microscope (Olympus, Center Valley, PA) using 10× magnification at 488nm. Nrf2 positive nuclei were be counted. Approximately 100 DAPI-positive cells per plate were be counted.

Thioredoxin

Protocol was similar to methods for Nrf2. Antibody was obtained from xx (catxx).

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Reactive Oxygen Species Assay

MSCs and NHAs were irradiated or sham irradiated at 70% confluence in 96 well assay black bottom plates (Corning, Corning NY, cat#3603) then treated with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Thermofisher, Waltham MA, cat#D399) or Mitosox (Thermofisher, cat#m36008). Briefly, cells were washed twice in pre-warmed PBS then incubated with 5uM mitosox for 20 minutes at 37^oC or with 10um H₂DCFDA for 20min at 37^oC. Cells were then read on a microplate reader at excitation and emission wavelengths as recommended by the company.

Western Blotting

Cells were grown to 70% confluence then irradiated and collected 15minutes, 2h, 6h, 12h, 24h, 48h, 72h post radiation injury. These cells were then treated with RIPA buffer and Holt protease and phosphatase cocktail. Cells were sonicated for 10 seconds in a cold room before being centrifuged at 15000xg for 15 minutes at 5^oC. B-actin (), Nrf2, MnSOD, catalase, thioredoxin

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ATP measurements

Cells were washed with warm PBS then trypsinized once they reached 1.5×10⁶ or 4.5×106. They were then centrifuged at 1000rpm for 5min at 4^oC. Cells were then washed twice with cold PBS and centrifuged at 10,000rpm for 30 seconds. Supernatant was discarded and pellets were treated with 0.1% triton x-100 for 1 hour on a shaker in the cold room. Cells were then centrifuged at 14,000 rpm for 10 minutes at 4^oC. The supernatant was moved to a new tube then flash frozen in liquid nitrogen.

GSH, GSSG assay

Cells were irradiated or sham irradiated and collected at  24, 48, and 72 hours post injury. This assay was performed as previously described [43]. Briefly cells were trypsinized, collected, then 500ul was taken for BCA assay. The remaining solution was spun at 200xg, 5 min, 4c , then washed 2x with 1x PBS on ice.1% perchloric acid was used to resuspend the cells before they were sonicated 10 sec in a cold room. Cell lysates were centrifuged 14,000xg for 10 min at 4^oc.   Samples were stored in -20C until used. Frozen cell lysates were thawed on ice then brought to pH 6.5-7.0 by potassium hydroxide-3-(N-morpholino)propanesulfonic acid (MOPS). Then samples were sonicated and centrifuged 14,000xg at 4oc for 10 min. Oxidized glutathione of varying concentrations between 1-10nmol were used as reference. The Assay buffer solution (0.1M sodium phosphate  (mono and dibasic were mixed evenly) with 5 mM EDTA, pH 7.4) was used to dilute standards and samples. 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB), NADPH and glutathione reductase were utilized as described to produce reaction mixtures which were added to samples and standards [44]. The reaction was measured at 410nm absorbance for 3 minutes at 1 minute intervals on  Synergy H1 BioTek spectrophotometer. Oxidized glutathione, glutathione reductase, DTNB, and NADPH were all purchased from Sigma Aldrich.

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Superoxide Dismutase Activity assay

The activity levels of SOD was measured via an SOD determination kit (Fluka/Honeywell, Morris Plains NJ, cat19160). Cells were grown to 70% then irradiated and extracted at indicated time points using a modified buffer (0.5% sodium deoxycholate, 1% triton X, Holt Protease and Phosphatase inhibitor) in 1xPBS. Enzyme working solution and WST working solutions were added and blanks were used according to manufacturer’s protocol. Samples were incubated in a 96 well plate for 20min at 37^oC then read at 450nm to calculate SOD activity.

Results

ROS expression and impact on cells

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Radiation injury can produce long lasting alteration in ROS expression in cells [45]. Increased and uncontrolled ROS are linked to cell dysfunction and death . To measure this potential change in the cell DCF and Mitosox were used to measure ROS in cytoplasm and mitochondria, respectively. The expression of ROS after radiation injury via DCF or mitosox varied depending on run though general trends could be observed. In both NHAs and MSCs the DCF assay indicated short term (1-12 h) and long term (24-72 h) alterations in ROS expression in the cytoplasm (Fig. 1). In MSCs and NHAs the mitosox assay indicated excess superoxide production observed shortly after radiation injury (1-6 h) in both cell types; NHAs did not display long term excess superoxide production long term in most assays while MSCs did (Fig. 2). Changes in time points of peaks for ROS production was potentially effected by passage number of cells and an inability to serum starve and control cell cycle.

ATP

To understand if increases in ROS production were relevant for cell death and  mitochondrial dysfunction we next looked at ATP production. A trend towards significant decrease in ATP production was observed in MSCs, but did not reach significance. ATP production was not significantly decreased in either cell type (Fig. 3).

Nrf2

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Prior studies in A549 lung cancer cells, immortalized T cell Jurkats, and immortalized human corneal endothelial cells have shown that ionizing radiation is capable of upregulating Nrf2 expression within 6h post-radiation [42, 46, 47]. It was thus anticipated that Nrf2 expression and nuclear localization would occur within this time period for NHAs and MSCs. However, Nrf2 expression was below detection in MSCs at all time points (Fig. 5). In contrast, NHAs displayed a basal level of both Nrf2 expression and its localization to the nucleus (Fig. 4). Unexpectedly, there was no significant increase of Nrf2 entry into the nucleus at any time point post radiation injury in NHAs.

MnSOD expression and activity

Manganese superoxide dismutase (MnSOD), an inducible enzyme located in the mitochondrial matrix, catalyzes the conversion of superoxide to hydrogen peroxide and oxygen [48]. The hydrogen peroxide is then hydrolyzed to water and oxygen by either catalase or the glutathione system. MnSOD knockout produces embryonic lethal mice, indicating its expression and activity are essential for life [49]. While it was anticipated that expression would increase after radiation injury, to respond to oxidative stress, no increase was observed in NHAs, while a statistically significant increase was observed in MSCs at 12h (Fig. 6). Since activity of an enzyme is not always correlated with expression levels we then sought to measure the activity of MnSOD at both short (15m and 2h) and longer time points (24, 48, 72h) post 10 Gy radiation. But there was no statistical difference in activity between control and irradiated or between NHA and MSC, at any time point (Fig. 7).

Catalase

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Catalase expression has previously been shown to protect cells from radiation damage. Knockdown of catalase in a glioma cells produced sensitization to radiation and hydrogen peroxide induced oxidative stress [50]. It was anticipated that catalase expression would increase as a mechanism to handle radiation induced oxidative stress. However, again no significant change in expression was observed after radiation injury in either cell type, at any time point (Fig. 8). Additionally, no difference in expression of MnSOD or Catalase was observed between control levels of MSCs and NHAs (Fig. 9).

Thioredoxin

Thioredoxins act as electron donors and ROS scavengers through their disulfide active sites, allowing them to protect cells from oxidative stress [51]. Thioredoxin requires thioredoxin reductase (TrxR) for the reduction of its active site; TrxR expression directly impacts the reduced or oxidized state of thioredoxins within the cell [52]. Thioredoxin is also capable of translocating from the cytoplasm to the nucleus within an hour after 400 J/m2 UVB or 10Gy x ray radiation stimulation in HeLa, MCF-7 and immortalized human keratinocytes (HSC-1)s [53, 54]. Once inside the nucleus, thioredoxin aids in modulation of redox factor 1 (Rel-1) and activator protein 1 (AP-1) factors. Thioredoxin entry into the nucleus improves cell proliferation and DNA repair through this cascade involved in DNA repair and cell proliferation [55].To measure thioredoxin expression and localization immunofluorescence was performed on NHAs and MSCs (Fig. 10, 11respectively).  . Thioredoxin was expressed in the nucleus in control conditions and did not rise to significance after radiation injury in either cell type, at any time.

GSH

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Previously it was shown in U373-MG and HCT116 cells that increased glutathione biosynthesis via activation of the (PKR)-like endoplasmic reticulum kinase (PERK) pathway was associated with increased radiation resistance and enhanced survival [56]. Similar increases in B16M melanoma tumor cell and HeLa cell survival, metastatic capacity and radiation resistance have been linked to high GSH content [57, 58]. Conversely, depletion of GSH in these B16M and modified radiation resistant HeLa cells (HeLaR) caused increased sensitivity to ROS, radiation sensitivity, and cytotoxicity [58, 59].  It was thus anticipated that higher levels of glutathione would be associated with radiation resistance in the normal human astrocytes. While NHAs did display an increase in glutathione expression at 24h post-exposure, which trended towards significance, MSC expression of glutathione was almost double that of NHAs at all time points observed (Fig. 12). MSC expression was statistically higher than NHAs via student T test at 48 h and 72 h post radiation injury.

Discussion

Previous research in normal and cancerous cell cultures have indicated that ROS may persist after radiation injury [60]. Uncontrolled ROS may lead to senescence, mutagenesis or cell death. Our previous research had indicated NHAs proliferate after radiation injury while MSCs senesce. We had anticipated that the NHAs would be more efficient than MSCs in handling ROS production and would not display spikes in ROS at late time points (24-72h) after radiation injury. In both DCF and mitosox assays we saw ROS spikes. These spikes were not consistent between experiments. This may be due to alterations of endogenous ROS expression among the cells as they reached different points in the cell cycle.

Due to the conflicting nature of this data we sought a second method of measuring cellular health: production of ATP. An inability to produce ATP due to damage to the mitochondria would lead to cell dysfunction or cell death. Since we saw no significant cell death in either cell type after radiation no major changes in ATP production were anticipated. While their was a trend towards decreased ATP production in MSCs neither cell type displayed a significant drop in ATP production.

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If the cells were able to maintain ATP production and were not dying they had to be utilizing some mechanisms to control ROS. We therefore focused first on MnSOD, due to its location in the mitochondria. However, MnSOD expression and activity did not increase in NHAs.

Next, since Nrf2 is the central molecule involved in activation of the antioxidant response its expression and location in the cell was investigated. No significant increases in Nrf2 entry into the nucleus is observed in NHAs between control and times after irradiation. Interestingly, no Nrf2 is visible in MSC samples. Indicating that NHAs may have a higher total expression of Nrf2 which continuously acts to maintain low ROS levels. However, this could not be corroborated via western blotting where no bands were observable.

Pathways downstream of Nrf2 including: thioredoxin and catalase similarly failed to show any significant differences between NHAs and MSCs after radiation injury. Interestingly, it seems there is a statistically significant amount of glutathione in MSC controls compared to NHA controls. This difference disappears at 24h and becomes significant once more at 48h and 72h.  Suggesting that NHAs do upregulate GSH in response to radiation injury whereas MSCs maintain a consistent amount.

I had expected a more robust antioxidant response after radiation injury. While there were some trends towards significance in the NHA data, there is no clear cut upregulation of activity or expression after injury. However, Nrf2 levels appeared significantly higher in NHAs at all time points compared to MSCs. There is emerging evidence of Nrf2 involvement in DNA repair [21]. Perhaps then the major method for astrocyte radiation survival is through its DNA repair response. While knockdown of the antioxidant response would likely be catastrophic for cell survival it seems to not be the main driver for cell proliferation and repair.

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Figure 1 Effect of 10Gy x-ray radiation injury on normal human astrocyte and mesenchymal stem cell production of reactive oxygen species in the cytoplasm. Normal human astrocytes (NHA) and normal human adult mesenchymal stem cells (MSC) were plated at approximately 70% confluence in 96 well plates and sham irradiated or exposed to 10Gy x-ray. DCF was applied as previously described and read via spectrophotometer at the indicated time points. Graphs show cells plated in triplicate ± SEM. Negative controls were subtracted from irradiated and control samples, then irradiated samples were divided by controls. A value of 1 indicates readings for irradiated cells were equivalent to readings for control cells

Figure 2 Effect of 10Gy x-ray radiation injury on NHA and MSC production of reactive oxygen species in the mitochondria. NHAs and MSCs were plated at approximately 70% confluence in 96 well plates and sham irradiated or exposed to 10Gy x-ray. MitoSox was applied as previously described and read via spectrophotometer at the indicated time points. Graphs show cells plated in triplicate ± SEM. Negative controls were subtracted from irradiated and control samples, then irradiated samples were divided by controls. A value of 1 indicates readings for irradiated cells were equivalent to readings for control cells

Figure 3 Neither MSCs nor NHAs display significantly decreased ATP levels compared to control. NHAs or MSCs were grown to about 1.5 x 10^6 or 4.5 x 10^6 cells then prepared at indicated time points. Representative data are shown from n=3 independent experiments. Graphs show means ± SE of three independent experiments;* indicates p≤0.05

Mesenchymal stem cells, display increased expression of MnSOD but not catalase, Normal Human Astrocytes display no increase in expression of either protein. Normal human astrocytes (NHA) or normal human adult mesenchymal stem cells (MSC) were grown to 70% confluence and sham irradiated or exposed to 10 Gy X-ray. Cell lysates were prepared at the indicated time points, and western blots were performed for MnSOD (A,B) or catalase (c,d) or for β-actin (A-D) as a loading control. Representative data are shown from n=3 independent experiments. Graphs show means ± SE of three independent experiments;* indicates p≤0.05.

Figure 4 Nrf2 expression is present in NHAs but increase in nuceli after radiation does not occur. NHA were sham irradiated or exposed to 10 Gy X-ray irradiation. Immunohistochemistry was performed at the indicated time points. Panels show representative images of Nrf2 or DAPI staining. Nrf2 expression within nuclei, as measured by overlap between DAPI and Nrf2 were counted in random fields to reach a minimum of 100 cells total to determine average numbers of Nrf2 presence in the nuclei. This was done for n = 3 independent experiments. Bar graphs indicate Nrf2 in nuclei of all cells counted ± SEM. * indicates p < 0.05 from control.

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Figure 5 MSCs display no basal level of Nrf2 nor do they display upregulation after radiation injury. MSC were grown to 70% confluence and sham irradiated or exposed to 10 Gy X-ray irradiation. Immunohistochemistry was performed at the indicated time points. Panels show representative images of Nrf2 or DAPI staining. The presence of Nrf2 was supposed to be found in the nucleus. However Nrf2 expression was as low as background. No statistics were done.

Figure 6. MSCs  but not NHAs display a statistically significant increase in MnSOD at 12 h after radiation injury MnSOD. Normal human astrocytes (NHA) or normal human adult mesenchymal stem cells (MSC) were grown to 70% confluence and sham irradiated or exposed to 10 Gy X-ray. Cell lysates were prepared at the indicated time points, and western blots were performed for MnSOD andfor β-actin (A) as a loading control. Representative data are shown from n=3 independent experiments. Graphs show means ± SE of three independent experiments;* indicates p≤0.05.

Figure 7 Mesenchymal stem cells and Normal human astrocytes display similar levels of  activity for MnSOD at control levels and at select time points after radiation injury. NHA and MSCs were grown to 70% confliuence then sham irradiated or exposed to 10Gy x-ray irradiation. Lysates were collected in modified RIPA buffer and activity assays were performed according to manufacturer’s protocol at selected time points.

Figure 8 Neither MSCs nor NHAs display increased catalase expression after radiation injury. NHAs and MSCs were grown to 70% confluence and irradiated at 10 Gy or sham irradiated and harvested at indicated time points. Westerns were performed for catalase and -actin.

Figure 9. NHAs and MSCs show equivalent expression of MnSOD and catalase in control cells. NHAs and MSCs were grown to 70% confluence and irradiated at 10 Gy or sham irradiated and harvested at indicated time points. Westerns were performed for catalase, MnSOD and -actin.

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Figure 10 Thioredoxin is present in nuclei of NHAs at basal levels, and this does not rise to a statistically significant degree after radiation injury. NHAs were sham irradiated or irradiated with 10 Gy x ray at indicated time points. Panels show representative images of THX or DAPI staining THX expression within the nucleus, as measured be overlap between DAPI and THX were counted in random fields to reach approximately 100 cells. This was done for n = 3 independent experiments. Bar graphs indicate THX in nuclei of all cells counted ± SEM. * indicates p < 0.05 from control.

Figure 11 Thioredoxin is present in nuclei of MSCs at basal levels, and this does not rise to a statistically significant degree after radiation injury. MSCs were sham irradiated or irradiated with 10 Gy x ray at indicated time points. Panels show representative images of THX or DAPI staining THX expression within the nucleus, as measured be overlap between DAPI and THX were counted in random fields to reach approximately 100 cells. This was done for n = 3 independent experiments. Bar graphs indicate THX in nuclei of all cells counted ± SEM. * indicates p < 0.05 from control.

Figure 12 MSCs maintain higher glutathione levels up to 72h after radiation compared to NHAs. NHAs and MSCs were grown to 70% confluence then prepared for glutathione assay or BCA assay to establish protein concentrations. Cell lysates were prepared at the indicated time points then glutathione concentration was determined as previously described. Representative data are shown from n=3 independent experiments. Graphs show means ± SE of three independent experiments;* indicates p≤0.05.

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