1. Introduction
Fruits, vegetables, cereals and beverages contain more than 8000 natural compounds that are characterized as polyphenols [
1]. Depending on the number of their phenolic rings and other structural characteristics, polyphenols are classified as phenolic acids, flavonoids, stilbenes and lignans [
1]. Consumption of polyphenols and the bioavailability of their secondary metabolites has been related to the protection against a variety of pathological conditions including carcinogenesis, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases [
1].
The generation of reactive oxygen species (ROS) deregulates the endogenous antioxidant mechanisms in cells, leading to macromolecule damage, characterized in part by lipid peroxidation, DNA-protein crosslinks, base modifications, adduct formation and DNA single- and double-strand breaks [
2,
3]. These modifications initiate complex signal transduction pathways such as those involved in DNA repair, cell cycle arrest and induction of apoptosis [
4].
Protection of normal tissue from radiation-induced damage such as that occurring by accidental exposure or as side effect of conventional radiotherapy to treat malignancies [
5] is of great importance. Currently, there is an unmet need to develop a safe and effective radioprotecting pharmacological agent [
6,
7]. A large number of potential radioprotective agents have been reported, but their high cost, serious side effects and toxicity have limited their clinical usefulness [
4,
8]. On the other hand, natural compounds, especially polyphenolics, have been tested as potential radioprotectors, due to their antioxidant, anti-inflammatory, antimicrobial, immunomodulatory and anti-carcinogenic activities as well as their low toxicity profile and high availability. Polyphenolic compounds can act as free radical scavengers and inhibitors of lipid peroxidation. They upregulate pro-survival factors and cytoprotective antioxidant enzymes, as well as modulate DNA repair [
6,
9].
For the past few years, our group has been evaluating the protective effects of dietary flaxseed (FS) supplementation in preclinical murine models of oxidative lung damage such as hyperoxia, acid aspiration injury [
10], and ischemia/reperfusion injury. We determined that the protective effects of FS may be due in part to its ability to enhance antioxidant enzyme expression in lung tissues [
10,
11]. Importantly, dietary FS ameliorated the adverse effects of thoracic radiation when given both prior to exposure [
12] as well as post-exposure [
13]. In these studies, dietary flaxseed decreased radiation-induced oxidative lung tissue damage, decreased lung inflammation and prevented pulmonary fibrosis.
We have further characterized the radioprotective effects of the lignan component (FLC) of wholegrain flaxseed, enriched in the phenolic secoisolariciresinol diglucoside (SDG). The antioxidant and free radical scavenging properties of SDG are well documented [
14,
15] which is of paramount importance as the free radical scavenging ability of a compound can be directly related to its radioprotective efficacy. In an early pilot study on lung endothelial cells, SDG exhibited free radical scavenging properties when cells were exposed to gamma-irradiation [
12]. Importantly, the entire flaxseed lignan component (FLC) enriched in SDG, mediated radioprotection [
16] and radiation mitigation [
17] in mice. We recently chemically synthesized SDG (LGM2605) to allow scalable synthesis for evaluation in large scale experiments [
18]. We confirmed potent free radical scavenging and antioxidant properties of LGM2605, comparable to the commercially available SDG. We extended our evaluation of synthetic SDG and further confirm DNA radioprotective properties of the synthetic phenolic in cell free systems [
19]. However, characterization of the radioprotective properties of SDG in cells or tissues has not yet been evaluated. This study was performed in order to determine the radioprotective ability of the biphenolic SDG in three lung cell types (endothelial, epithelial and fibroblasts) against damaging gamma radiation.
In this study, we show for the first time that SDG pre-treatment protects lung cells from radiation-induced DNA damage and increases their clonogenic survival. SDG also significantly boosts the endogenous antioxidant capacity of the lung cells, increasing the gene expression and protein levels of antioxidant enzymes, such as HO-1, GSTM1 and NQO1. Our findings identify the lignan biphenolic SDG as a potential radioprotective agent for normal lung cells.
3. Discussion
In the current study, we demonstrated for the first time that the lignan phenolic SDG can protect murine lung cells against radiation-induced oxidative damage. We observed that pre-treatment of cells with SDG decreased radiation-induced DNA strand breaks (single- and double-stranded breaks) and improved the overall cell survival as measured by clonogenic survival assay. Importantly, expression of the Nrf2-regulated enzymes, NQO1 and HO-1, was also altered by SDG treatment, indicating its important role in adaptation to oxidative stress (
Figure 6).
Figure 6.
Schematic representation of possible mechanism by which lignan phenolic SDG (LGM2605) protects lung cells against radiation exposure. SDG upregulates the phase II cytoprotective enzymes, HO-1, NQO1 and the gene levels of GSTM1; thus, when the non-malignant lung cells are exposed to ionizing radiation, less oxidative damage is induced, leading to increased clonogenic survival of the irradiated cells.
Figure 6.
Schematic representation of possible mechanism by which lignan phenolic SDG (LGM2605) protects lung cells against radiation exposure. SDG upregulates the phase II cytoprotective enzymes, HO-1, NQO1 and the gene levels of GSTM1; thus, when the non-malignant lung cells are exposed to ionizing radiation, less oxidative damage is induced, leading to increased clonogenic survival of the irradiated cells.
Our results are concordant with our previous studies in cell-free systems, in which plasmid and calf thymus DNA were exposed to gamma radiation resulting in DNA fragments of low-molecular weight, which were prevented in a dose-dependence manner by SDG [
19].
Polyphenols have the ability to protect normal tissue or cells from damaging effects of radiation by reducing ROS mediated oxidative DNA damage [
23]. In the present study, we evaluated the role of SDG against IR-induced DNA strand breaks in murine lung cells. While SSBs can more easily be repaired by the cell as compared to the DSBs, the latter are more likely to result in mutagenesis [
24]. We report here that SDG protected cellular DNA from IR-induced SSBs and importantly, from DSBs in all three cell lines that tested. Our results are concordant with other reports that show a similar protective effect from radiation-induced both DNA SSB and DDB, for other phenolics such as chrysin [
25] and epicatechin [
26], resveratrol [
27] and green tea catechin [
28].
Α key biological response to oxidative stress is the activation of the Keap1/Nrf2/ARE pathway, which regulates many antioxidant and cytoprotective genes responsible for the cellular homeostasis [
19]. Activation of this pathway prior to exposure to a genotoxic agent, such as ionizing radiation can be crucial for the fate of the cell. Here, pre-treatment of cells with the lignan phenolic SDG upregulated Keap1/Nrf2/ARE-regulated protective enzymes. Our group has previously shown that an SDG-rich diet (FLC) upregulates the antioxidant enzymes, HO-1 and NQO1, in a murine lung model of radiation. Findings of the current study support the dual role of SDG in free-radical scavenging and antioxidant defense boost [
12,
14], in protecting from radiation-induced DNA damage in normal cells.
SDG, similarly to other phenolic compounds, such as apigenin [
29], hesperidin [
30], silibinin [
31] and epigallocatechin gallate (EGCG) [
28], showed radioprotection by scavenging harmful free radicals or by increasing the cell endogenous antioxidant defense, such as trans-resveratrol [
27], EGCG [
28] and euticoside C [
32]. However, despite over 50 years of research, most agents have not proceeded past pre-clinical evaluation or have not been evaluated in large trials [
33]. Numerous natural products and their isolated purified bioactive ingredients have been shown to have antioxidant and anti-inflammatory potential [
34,
35], properties useful for a variety of pathological conditions, but most are not tolerated in humans at the concentrations required to achieve clinical efficacy [
36]. In contrast, SDG is well tolerated in humans, its clinical efficacy was demonstrated in a wide range of pathological conditions with oxidative and inflammatory components. SDG formulations (FLC) were shown in preclinical models to be radioprotective in lung with antioxidant, anti-inflammatory and antifibrotic activity, while not inhibiting tumor killing by radiation [
16]. Importantly, SDG formulations were safely administered to humans for 6–24 weeks with no reported toxicity and secondary lignan metabolites were detectable in biological fluids [
37,
38]. We have proceeded to chemically synthesize SDG (LGM2605) to enable preclinical evaluation in animal models of radiotherapy as well as subsequently in Phase I clinical trials [
18,
19].
The lignan phenolic SDG can be considered as a potential protective agent against radiation-induced oxidative damage to non-malignant lung cells. The protective effects of SDG from radiation damage in cells are mediated in part by free-radical scavenging and the boost of antioxidant defenses. Further studies are needed to validate SDG as an effective radioprotective agent with clinical usefulness.
4. Materials and Methods
4.1. Cell Lines
Fibroblasts and endothelial cells were isolated from C57/Bl6 mouse as described previously [
39]. For fibroblast isolation, mouse lungs were harvested, minced, and incubated with dispase (2 mg/mL) for 45 min. Cell suspension was plated out and fibroblasts were cultured as described previously. Cells were used while in passages 3–10. Pulmonary microvascular endothelial cells (PMVEC) were isolated from murine lungs as described previously [
40]. Briefly, freshly harvested mouse lungs were treated with collagenase followed by isolation of cells by adherence to magnetic beads coated with mAb to platelet endothelial cell adhesion molecule (PECAM). Epithelial cells (C10) cells were originally derived from a normal BALB/c mouse lung explant and are non-tumorigenic [
41], contact-inhibited, and have alveolar type 2 cell features at early passage (kindly provided by Dr. Alvin Malkinson, University of Colorado, Denver, CO, USA).
4.2. Reagents
Secoisolariciresinol Diglucoside (SDG) is commercially available (ChromaDex, Inc., Irvine, CA, USA) or was chemically synthesized based on a published synthetic pathway [
18] and is designated as LGM2605. Initial experiments were performed with the commercially available SDG, which is costly and prohibitively expensive to permit animal dosing in long-term studies. Additionally, extraction methods to obtain large amounts of SDG are complex [
42,
43,
44], require special resources and expertise and result in variable yields. A synthetic path to obtain SDG was thus pursued [
18] to achieve the needed quality control, lot-to-lot uniformity, and dose and cost parameters consistent with pharmaceutical development. The chemically synthesized LGM2605 has remarkably similar antioxidant properties as the parent, natural compound [
19]. Please note that since we have shown in several studies that commercially available SDG and synthetic SDG (LGM2605) behave identically with respect to their antioxidant and DNA radioprotective properties, we use SDG and LGM2605 interchangeably in the text.
Comet assay kit was purchased from Trevigen, Inc., (Gaithersburg, MD, USA). P-Histone H2AX (rabbit mAb) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Phosphate buffered saline (PBS), Bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine, glucose 1 g/L, without sodium bicarbonate, HEPES buffer, trypsin, bovine serum albumin (BSA), ethylenediamine tetra acetic acid (EDTA), 4,6 diamidino 2-phenyl indole (DAPI), Fetal bovine serum (FBS), Collagenase, Triton-X 100 and dispase were purchased from Sigma-Aldrich, St. Louis, MO, USA.
4.3. COMET Analysis
Exponentially growing cells were cultured and treated with SDG (50 μM) at different time intervals prior to irradiation (2 Gy). Cells were processed for comet assay as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, cells (1 × 105 cells/mL in PBS) were mixed with LMAgarose® (1:10, v/v) and immediately pipetted onto CometSlide™. Cells were then lysed (4 °C, 30 min) and kept in dark for unwinding (RT). Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (200 Amp) for 25 min. Slides were washed twice with distilled water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen). At least 150 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet Assay IV, Perceptive Instruments Ltd., Haverhill, MA, USA). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera with a 40× objective lens.
4.4. Immunostaining
For immunostaining of γ-H2AX, cells were plated on glass coverslips (5000 cells/coverslip), pre-treated (6 h) with 50 μM SDG and irradiated (2 Gy). At desired time interval, cells were fixed (4% para-formaldehyde), washed and blocked with PBST (PBS + 0.1% TritonX-100 containing 5% goat serum, 1% BSA). Cells were incubated with γ-H2AX antibody (1:200) overnight at 4 °C followed by washing with PBST (3 × 5 min) and incubation with secondary antibody (Alexa fluor® 488, Invitrogen, CA, USA) for 1 h at RT. Nuclei were counterstained with DAPI and visualized under fluorescence microscope with a 20× objective lens.. Total cells (blue) γ-H2AX-positive cells (green) were counted per field and percentage of γ-H2AX positive cells were calculated. A minimum of 500 cells was counted for each treatment and the experiment was repeated twice.
4.5. Flow Cytometry for γ-H2AX
For FACS analysis, cells were trypsinized and washed with PBS. Cells were then fixed (Fix/Perm buffer, eBioscience, San Diego, CA, USA), for 45 min and washed thereafter using permeabilization wash buffer (BioLegend, San Diago, CA, USA). Cells were resuspended in 200 μL rabbit monoclonal phospho-histone γ-H2AX (Ser139) antibody conjugated to Alexa fluor® 488 (1:100 v/v, Cell Signaling Technology, Danvers, MA, USA) and incubated for 30 min at 4 °C. Cells were washed again with wash buffer and analyzed. The CyAn ADP (Advanced Digital Processing) flow cytometer (Dako, Glostrup, Denmark) was used to measure γ-Η2AX and positive cells were quantified using Summit Software (Dako, Glostrup, Denmark).
4.6. Clonogenic Survival
Exponentially growing cells were plated as single cells and incubated overnight. Cells were treated with various doses of the lignan SDG (10–50 μM) 6 h prior to irradiation (2, 4, 6 and 8 Gy). Lignan dose was selected based on animal studies to be within the physiological levels reached in the blood circulation when 10% Flaxseed is ingested [
10,
13]. Cells were irradiated with a Mark 1 cesium (Cs-137) irradiator (J.L. Shepherd, San Fernando, CA, USA) at a dose rate of 1.7 Gy/min. Colonies were stained and counted 10 to 15 days after irradiation and surviving fraction was calculated.
4.7. Quantitative Real Time PCR (qPCR)
Quantitative polymerase chain reaction (qPCR) was performed as previously described [
22]. Briefly, TaqMan
® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA) were used. To evaluate the effect of SDG treatment on the mRNA expression of antioxidant genes, individual TaqMan
® gene expression assays were selected for antioxidant enzymes (heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), and glutathione
S-transferase mu 1 (GSTM1)).
Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti
® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔC
T method as shown previously [
22].
4.8. Western Blotting
Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Complete™, Mini, EDTA-free, Sigma-Aldrich). Total protein content was determined by BCA Protein Assay (Thermo Scientific). Total protein content was determined by BCA Protein Assay (Thermo Scientific). Samples were loaded on 8%–12% NuPAGE gel (Invitrogen, Carlsbad, CA, USA). Electrophoresis was performed at 200 V for 1 h. Transfer of proteins to PolyScreen PV transfer membrane (PerkinElmer Life Sciences, Boston, MA, USA) was performed for 2 h, at 25 volts. Membrane was blocked overnight in 5% non-fat dry milk in phosphate buffered saline. Protein levels of heme oxygenase 1 (HO-1) and NQO1 were detected using rabbit monoclonal anti-mouse antibodies, following manufacturer recommended dilutions (Abcam, Cambridge, MA, USA). Peroxidase-conjugated Donkey anti-rabbit IgG was used as a secondary antibody. (Jackson Laboratories, West Grove, PA, USA). Membranes were developed using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA) and bands were visualized on standard X-ray film (HyBlot CL, Denville Scientific Inc., Metuchen, NJ, USA). Densitometric analysis of the bands was performed using ImageJ (NIH) software.
4.9. Statistics
Results are expressed as mean ± SEM. Survival curve for clonogenic assay was prepared using GraphPad Prism 6 software. Statistical differences among groups were determined using one-way analysis of variance (ANOVA). When statistically significant differences were found (p < 0.05), individual comparisons were made using the Bonferroni/Dunn test (Statview 4.0).
