Radiation-induced Reactive Oxygen Species Research Articles

Radiation oxidative stress in cancer induction and prevention

Exposure of cells to ionizing radiation causes generation of intracellular reactive oxygen species (ROS) which are implicated in the mechanism of carcinogenesis. Molecular steps involved in the transformation of normal cells to cancer cells have been enigmatic but generally believed to arise from aberration in cellular redox homeostasis. In normal cell function, a delicate balance is maintained between ROS generated in the metabolic process and level of endogenous antioxidant defense. ROS are known to regulate various cellular functions, such as cell division, signal transduction, and apoptosis. Cells experience oxidative stress when excess production of ROS occurs inside a cell upon exposure to external stressor agents. This redox imbalance affects the cellular functions due to DNA strand breaks, chromosomal aberrations, gene mutations, alteration in signal transduction, and inhibition of apoptosis leading to induction of cancer and other diseases. Radiation-induced ROS are involved in initiation and promotion of carcinogenesis. Therefore, detoxification of ROS by exogenous antioxidants including dietary polyphenols offers an important strategy for cancer prevention. Recent research results have shown that resistance of cancer stem cells to therapies is linked to low level of ROS. Interestingly, in vitro and in vivo experiments have reported that radiotherapy- and chemotherapy-induced ROS in cytosol sensitize the tumor cells to death, resulting in tumor growth retardation. This review is an attempt to delineate mechanisms of ROS in carcinogenesis and prevention by dietary compounds. Natural polyphenols and dietary antioxidants hold potential to prevent cancer. Interventions in ROS-mediated signal alteration, apoptosis activation, and modulation of epigenetic processes may offer effective cancer prevention strategy.

55 - MnTE-2-PyP and NOX4 Deletion Protects Radiation Induced Damage in Mouse Primary Prostate Fibroblasts by Inhibiting TGFβ1 Signaling Pathway

Patients with prostate cancer, receiving ionizing radiation, frequently suffer from radiation induce damage in normal tissues adjacent to the tumor. These irradiated normal cells lead to tissue fibrosis and senescence which ultimately leads to post-irradiation related chronic complexities like damage in prostate tissue, urinary urgency and frequency, erectile dysfunction, urethral stricture, and incontinence etc. Radiation-induced reactive oxygen species (ROS) was reported as the most potent causative factor for radiation damage to normal tissue. MnTE-2-PyP, a ROS scavenger, can protect the normal cell by radiation-induced damage whereas it can inhibit the growth of the cancer cell after irradiation, which is necessary for any radio protector. However, the mechanism by which MnTE-2-PyP protects from radiation-induced fibrosis is unclear. Our current study reveals the underlined molecular mechanism of radiation protection by MnTE-2-PyP in normal mouse prostate fibroblast cells. To investigate the role of MnTE-2-PyP in normal tissue protection after irradiation, primary prostate fibroblasts from C57BL/6 mice were cultured in presence or absence of MnTE-2-PyP and exposed to 2 Gy of X- rays. Results of this study indicate that, MnTE-2-PyP can protect primary prostate fibroblasts from irradiation mediated activation as measured by contraction of collagen disc and radiation-induced senescence as detected by β-galactosidase staining. We observed that MnTE-2-PyP inhibits TGFβ mediated fibroblast activation pathway by reducing the expression of the TGF beta receptor II, which in turn reduces the activation and/or expression of SMAD2, SMAD3 and SMAD4. As a result, SMAD2/3 mediated transcription of pro-fibrotic markers is reduced by MnTE-2-PyP. Due to the inhibition of the TGFβ pathway, fibroblasts treated with MnTE-2-PyP can resist radiation induced activation and senescence of the prostate fibroblast. NADPH oxidase 4 (NOX4) produces ROS and its expression is upregulated after irradiation. As was observed with MnTE-2-PyP treatment, NOX4-/- fibroblasts were protected from radiation-induced fibroblast activation and senescence. However, NOX4-/- fibroblasts had reduced levels of active TGF-β1, which resulted in decreased TGF-β signaling. Therefore, our data suggests that, reduction of ROS levels by MnTE-2-PyP or reducing NOX4 activity significantly protects normal prostate tissues from radiation induced tissue damage, but work on different components of the TGF-β signaling pathway. This study proposes a crucial insight for molecular mechanism executed by MnTE-2-PyP during its action as a radio protector. Understanding how this molecule works as a radio protector will lead to better and controlled mode of treatment during post irradiation complexities in prostate cancer patients.

The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury.

Antioxidants are prospective radioprotectors because of their ability to scavenge radiation-induced reactive oxygen species (ROS). The hematopoietic system is widely studied in radiation research because of its high radiosensitivity. In the present study, we describe the beneficial effects of 5-methoxytryptamine-α-lipoic acid (MLA), which was synthesized from melatonin and α-lipoic acid, against radiation-induced hematopoietic injury. MLA administration significantly enhanced the survival rate of mice after 7.2 Gy total body irradiation. The results showed that MLA not only markedly increased the numbers and clonogenic potential of hematopoietic cells but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, MLA decreased the levels of ROS in hematopoietic cells by inhibiting NOX4 expression. These data demonstrate that MLA prevents radiation-induced hematopoietic syndrome by increasing the number and function of and by inhibiting DNA damage and ROS production in hematopoietic cells. These data suggest MLA is beneficial for the protection of radiation injuries.

Vitis vinifera seeds extract for the modulation of cytosolic factors BAX-α and NF-kB involved in UVB-induced oxidative stress and apoptosis of human skin cells.

Background and aimsThe depletion of the ozone layer allows overexposure of the skin to UV radiation, which is prolonged due to the increasing life expectancy, together with inappropriate life habits contribute to the increasing incidence of cutaneous malignancies. Plant extracts with antioxidant capacities are frequently employed as a means to protect skin against ultraviolet (UV) radiations, thus preventing skin cancers. In the present study we assessed a red grape seed extract (GSE) potential capacities to reduce ultraviolet B (UVB) radiation-induced reactive oxygen species (ROS) and subsequent apoptosis in a human keratinocytes cell line (HaCaT). We identified molecules and pathways modulated by the GSE through which this may exert its photoprotective effect.MethodsThe GSE was standardized according to its polyphenolic content and the most important biologically active compounds, such as epigallocatechin and epicatechin, catechin hydrate, procyanidin B and gallic acid were evidenced by high-performance liquid chromatography. According to the plant extract cytotoxicity on the HaCaT cell line, two concentrations were selected for testing from the non-toxic range: GSE1 (37.5 μgEqGA/ml) and GSE2 (75 μgEqGA/ml). The level of ROS was evaluated with CM-H2DCFDA assay, while apoptosis, Bax-α and NF-kβ p65 proteins with ELISA and confirmed by western-blot.ResultsBoth concentrations of the extract decreased the level of ROS in UVB-irradiated keratinocytes (p<0.001), whereas apoptosis and Bax-α pro-apoptotic protein were only reduced by the higher concentration (GSE2). The NF-kB p65 protein level registered increasing values in time after UVB exposure of the cells, while the tested plant extract re-established its level when its smaller concentration was used (GSE1).ConclusionThese results encourage further studies on this extract in order to identify other molecules and pathways through which this extract might exert its beneficial effects and also recommend its use as a potential photoprotective agent.

The effect of radiation-induced reactive oxygen species (ROS) on the structural and functional properties of yeast alcohol dehydrogenase (YADH)

Purpose: To determine the mechanism underlying oxidative modifications caused by radiation-induced reactive oxygen species (ROS) and elucidate their effect on the structure and function of yeast alcohol dehydrogenase (YADH), a zinc-containing protein.Materials and methods: YADH was exposed to water radiolysis products in an air atmosphere. YADH oxidation products were determined by spectrophotometric and spectrofluorimetric methods. The extent to which oxidative modifications affected enzyme activity was also studied.Results: Water radiolysis products oxidized thiol groups leading to the release of zinc ions and the destruction of tryptophan and tyrosine residues. Those processes were accompanied by alterations in protein structure such as increased surface hydrophobicity, greater tryptophan accessibility to acrylamide, and changes in the secondary structure. Structural modifications were correlated with lower enzyme activity.Conclusion: During the process of functional and structural changes in YADH exposed to reactive oxygen species, a key part is the oxidation of cysteine residues attached to zinc and the release of zinc ions from the molecule. It may be assumed that ROS induce similar changes in many other zinc-containing proteins.

Inhibition of glutamine metabolism counteracts pancreatic cancer stem cell features and sensitizes cells to radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) cells utilize a novel non-canonical pathway of glutamine metabolism that is essential for tumor growth and redox balance. Inhibition of this metabolic pathway in PDAC can potentially synergize with therapies that increase intracellular reactive oxygen species (ROS) such as radiation. Here, we evaluated the dependence of pancreatic cancer stem cells (PCSCs) on this non-canonical glutamine metabolism pathway and researched whether inhibiting this pathway can enhance radiosensitivity of PCSCs. We showed that glutamine deprivation significantly inhibited self-renewal, decreased expression of stemness-related genes, increased intracellular ROS, and induced apoptosis in PCSCs. These effects were countered by oxaloacetate, but not α-ketoglutarate. Knockdown of glutamic-oxaloacetic transaminase dramatically impaired PCSCs properties, while glutamate dehydrogenase knockdown had a limited effect, suggesting a dependence of PCSCs on non-canonical glutamine metabolism. Additionally, glutamine deprivation significantly increased radiation-induced ROS and sensitized PCSCs to fractionated radiation. Moreover, transaminase inhibitors effectively enhanced ROS generation, promoted radiation sensitivity, and attenuated tumor growth in nude mice following radiation exposure. Our findings reveal that inhibiting the non-canonical pathway of glutamine metabolism enhances the PCSC radiosensitivity and may be an effective adjunct in cancer radiotherapy.

Abstract 3310: Targeting MUC1-mediated metabolism sensitizes pancreatic cancer cells to radiation therapy

Abstract Overexpression of MUC1 promotes growth promoting metabolic alterations in pancreatic cancer cells. We investigated the role played by MUC1-mediated metabolic alterations in radiation response of pancreatic cancer cells. Our findings indicate that cell survival and clonogenicity of pancreatic cancer cells upon radiation treatment correlate with MUC1 expression. MUC1 expression reduced radiation-induced reactive oxygen species (ROS) production and DNA damage, whereas, increased glucose uptake and lactate secretion upon irradiation. Metabolomic analysis through liquid chromatography-coupled tandem mass spectrometry approach revealed that pancreatic cancer cells exhibit spontaneous metabolic alterations upon irradiation. MUC1 expression further altered radiation-induced carbon flux especially in glycolysis and pentose phosphate pathway. We further investigated radiation-sensitization of MUC1-expressing cells by targeting glycolysis or pentose phosphate pathway using metabolic inhibitors, and observed that pre-treatment with 3-bromopyruvate (BrPA) effectively abrogates MUC1-mediated radiation-resistance by elevating ROS and DNA damage. BrPA treatment also reduced MUC1-induced carbon flux into glycolysis and pentose phosphate pathway, upon irradiation. Hence, our findings demonstrate that MUC1-mediated radiation-resistance in pancreatic cancer cells can be effectively targeted through inhibition of glycolysis. Citation Format: Venugopal Gunda, Jaime Abrego, Surendra K. Shukla, Nina V. Chaika, Fang Yu, Tadayoshi Bessho, Chi Lin, Pankaj K. Singh. Targeting MUC1-mediated metabolism sensitizes pancreatic cancer cells to radiation therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2015-3310

Enhancement of radiotherapy by ceria nanoparticles modified with neogambogic acid in breast cancer cells.

Radiotherapy is one of the main strategies for cancer treatment but has significant challenges, such as cancer cell resistance and radiation damage to normal tissue. Radiosensitizers that selectively increase the susceptibility of cancer cells to radiation can enhance the effectiveness of radiotherapy. We report here the development of a novel radiosensitizer consisting of monodispersed ceria nanoparticles (CNPs) covered with the anticancer drug neogambogic acid (NGA-CNPs). These were used in conjunction with radiation in MCF-7 breast cancer cells, and the efficacy and mechanisms of action of this combined treatment approach were evaluated. NGA-CNPs potentiated the toxic effects of radiation, leading to a higher rate of cell death than either treatment used alone and inducing the activation of autophagy and cell cycle arrest at the G2/M phase, while pretreatment with NGA or CNPs did not improve the rate of radiation-induced cancer cells death. However, NGA-CNPs decreased both endogenous and radiation-induced reactive oxygen species formation, unlike other nanomaterials. These results suggest that the adjunctive use of NGA-CNPs can increase the effectiveness of radiotherapy in breast cancer treatment by lowering the radiation doses required to kill cancer cells and thereby minimizing collateral damage to healthy adjacent tissue.

Ginsenoside Rb2 Attenuates UV-B Radiation-Induced Reactive Oxygen Species and Matrix Metalloproteinase-2 through Upregulation of Antioxidant Components in Human Dermal Fibroblasts

Aims: The antiphotoaging activities of ginsenoside Rb2 on the skin, one of the predominant protopanaxadiol-type ginsenosides, were evaluated in cultured human dermal fibroblasts. Methods: The antiphotoaging activity was examined by analyzing the levels of reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity as well as cell viability for fibroblasts under UV-B irradiation. Results: When cultured fibroblasts were exposed to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced ROS elevation and MMP-2 on both activity and protein levels, while it exhibited an enhancing activity on total GSH level and SOD activity diminished by UV-B irradiation. However, Rb2 could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Conclusion: Ginsenoside Rb2 plays a photoprotective role against UV-B-induced oxidative stress in human dermal fibroblasts, which implies its skin antiphotoaging potential.

HIF-1 Alpha Regulates the Response of Primary Sarcomas to Radiation Therapy through a Cell Autonomous Mechanism.

Hypoxia is a major cause of radiation resistance, which may predispose to local recurrence after radiation therapy. While hypoxia increases tumor cell survival after radiation exposure because there is less oxygen to oxidize damaged DNA, it remains unclear whether signaling pathways triggered by hypoxia contribute to radiation resistance. For example, intratumoral hypoxia can increase hypoxia inducible factor 1 alpha (HIF-1α), which may regulate pathways that contribute to radiation sensitization or radiation resistance. To clarify the role of HIF-1α in regulating tumor response to radiation, we generated a novel genetically engineered mouse model of soft tissue sarcoma with an intact or deleted HIF-1α. Deletion of HIF-1α sensitized primary sarcomas to radiation exposure in vivo. Moreover, cell lines derived from primary sarcomas lacking HIF-1α, or in which HIF-1α was knocked down, had decreased clonogenic survival in vitro, demonstrating that HIF-1α can promote radiation resistance in a cell autonomous manner. In HIF-1α-intact and -deleted sarcoma cells, radiation-induced reactive oxygen species, DNA damage repair and activation of autophagy were similar. However, sarcoma cells lacking HIF-1α had impaired mitochondrial biogenesis and metabolic response after irradiation, which might contribute to radiation resistance. These results show that HIF-1α promotes radiation resistance in a cell autonomous manner.

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O2(-)) and hydrogen peroxide (H2O2), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H2O2), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O2(-) formation.

Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 against ionizing radiation

Radiation-induced skin injury is a serious concern during radiotherapy. However, the molecular mechanism underlying the pathogenesis of radiation-induced skin injury has not been extensively reported. Most biological functions are performed and regulated by proteins and noncoding RNAs, including microRNAs (miRNAs). The interplay between mRNA and miRNA has been implicated in disease initiation and progression. Technical advances in genomics and proteomics have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathogenesis of radiation-induced skin injury. In this study, we compared the protein and miRNA expression in rat skin irradiated with a 45-Gy electron beam with expression from adjacent normal tissues. We found 24 preferentially expressed proteins and 12 dysregulated miRNAs in irradiated skin. By analyzing the protein and miRNA profiles using bioinformatics tools, we identified a possible interaction between miR-214 and peroxiredoxin-6 (PRDX-6). Next, we investigated the expression of PRDX-6 and the consequences of its dysregulation. PRDX-6 is suppressed by radiation-inducible miR-214 and is involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred radioresistance on cells, decreased cell apoptosis, and preserved mitochondrial integrity after radiation exposure. In addition, in vivo transfection with PRDX-6 reduced radiation-induced reactive oxygen species and the malondialdehyde concentration and ameliorated radiation-induced skin damage in rats. Our present findings illustrate the molecular changes during radiation-induced skin injury and the important role of PRDX-6 in ameliorating this damage in rats.

Amelioration of Radiation-induced Skin Injury by HIV-TAT-Mediated Protein Transduction of RP-1 from Rana pleurade

Radiation-induced reactive oxygen species (ROS) can damage DNA and most other biological macromolecules in skin and radiation-induced skin injury is a serious concern for radiation therapy. Skin possesses an extremely efficient antioxidant system, which is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. Amphibian skin is a multifunctional organ, which protects against dangers of various oxidative stresses. Recently, a small peptide called RP-1 was isolated from the skin secretions of Rana pleurade, which shows strong antioxidant activity. However, this RP-1 peptide is limited because its inability to across the cell membrane. Protein transduction domains (PTDs) have demonstrated high efficiency for facilitating the internalization of both homologous and heterogeneous proteins into cells. This study aims to elucidate the protective effects of a HIV-TAT (TAT) PTD-coupled RP-1 fusion protein (TAT-RP1) on radiation-induced skin injury in vitro and in vivo. The synthesized fusion TAT-RP1 peptide can be incorporated into human keratinocyte HaCaT cells in a dose- and time-dependent manner without cytotoxicity. We then evaluated the protective role of TAT-RP1 against ionizing radiation. TAT-RP1 supplementation increased anti-superoxide anion ability of HaCaT cells and decreased HaCaT cell radiosensitivity to irradiation. Moreover, TAT-RP1 was able to penetrate the skin of rats, entering epidermis as well as the dermis of the subcutaneous layer in skin tissue. Topical spread of TAT-RP1 promoted the amelioration of radiation-induced skin damage in rats. These results suggest that TAT-RP1 has potential as a protein therapy for radiation-induced skin injury.

Short term UV-B radiation-mediated transcriptional responses and altered secondary metabolism of in vitro propagated plantlets of Artemisia annua L.

In vitro propagated Artemisia annua plantlets were exposed to low dose (2.8 W m−2) of UV-B (280–315 nm) radiation for different short-term (1, 2, 3 and 4 h) durations that resulted in 103 and 100 % enhanced artemisinin and flavonoid yield respectively after 3 h of UV-B irradiation. UV-B irradiation resulted in reduced chlorophyll a, b and carotenoids while the concentrations of UV-B-absorbing anthocyanins and phenolics were induced. Under UV-B radiation, superoxide radical formation increased in a time dependent manner and was maximum at 4 h. This lead to differential responses of enzymatic antioxidants (superoxide dismutase, catalase, peroxidases and glutathione reductase) to withstand the UV-B radiation-induced reactive oxygen species burst. The transcript analysis through RT-PCR revealed the significant up-regulation of HMGR, DXR, IPPi, FPS, ADS, CYP71AV1 and RED1 gene transcripts leading to higher artemisinin accumulation. Differential expression of stress regulated genes (AOX1a, NDB2, UPOX, PAL, LOX) helped the plant towards better adaptability at least up to 3 h UV-B irradiation period. Simultaneous clustering of genes and UV-B treatments using complete linkage and a euclidean distance matrix revealed that 3 h irradiation period is optimum for high artemisinin yield with better tolerance. Histological studies revealed the larger trichome size in UV-B treated plants as compared to untreated plants. The present study conclude that short term UV-B treatment to in vitro raised A. annua may be a safe approach for continuous supply of high artemisinin producing plantlets with simultaneous action of stress- regulated genes to keep the plant healthy.

Abstract B130: Radiodermatitis prevention by topical norepinephrine in post-surgical breast cancer patients

Abstract Purpose: This study was a feasibility and exploratory study to evaluate the safety and efficacy of topical norepinephrine in preventing radiodermatitis in patients receiving whole breast external beam radiation therapy after breast conserving surgery. Materials &amp; Methods: In preclinical experiments to test whether co-application of a topical vasoconstrictor would limit the systemic uptake of a topically applied, radioprotective, oxygen free radical scavenger, it was observed that the vasoconstrictor alone was radioprotective, preventing radiation dermatitis when administered alone as a control. This radioprotection is consistent with a mechanism based on local, transient vasoconstriction within the skin, which reduces local tissue oxygenation, limits the formation of radiation-induced reactive oxygen species, and protects skin stem cells from radiation damage. Topical norepinephrine was well tolerated in two Phase I safety studies conducted prior to this study, one in normal volunteers and one in cancer patients receiving palliative radiation therapy for bone metastases. Tested doses elicited transient skin blanch, which is indicative of reduced blood flow secondary to local vasoconstriction, but did not induce blood pressure elevation, local skin necrosis, or any other significant adverse event. Most of the adverse events that were possibly, probably, or definitely related to study medication were mild skin reactions, typically including pruritis, that occurred at or near the application site. There were no serious adverse events. The present study was a nonrandomized, open-label safety and exploratory study in post-surgical breast cancer patients treated with 45 to 50 Gray to the whole breast. A 10-16 Gray boost to the lumpectomy region was permitted. Eligible patients were women, age 18 years or older, who underwent breast conserving surgery for Stage Ia (T1, N0, M0), Stage Ib (T0 or 1, N1mic, M0) or Stage IIa (T&amp;lt;3cm, N0, M0) infiltrating ductal or lobular carcinoma of the breast or ductal carcinoma in situ (DCIS). The radioprotectant dose was a topical application of 3.0 mL of norepinephrine HCl (82.3 mg/mL in 70% ethanol) applied to the same 50-cm2 treatment site within the lateral aspect of the whole breast radiation field (i.e. in or near the axilla) about 20 minutes prior to each daily radiotherapy fraction. Results: 10 patients completed the protocol. No new safety concerns were identified. There were no serious adverse events, and no occurrences of norepinephrine-associated damage to irradiated skin. Assessments of radiodermatitis severity at the treatment site provided evidence for local reductions in radiodermatitis severity, compared to surrounding (untreated) control areas, in a majority of the treated patients. Conclusions: Topical norepinephrine demonstrated promising preliminary results in reducing radiation dermatitis in women undergoing whole breast irradiation, with minimal toxicity. Dose escalation, an expansion of the dose volume to permit the treatment of the entire skin area at risk for radiodermatitis, will precede a randomized, placebo-controlled evaluation of safety and efficacy. NCT01263366. Citation Format: James F. Cleary, Bethany M. Anderson, Jens C. Eickhoff, Deepak Khuntia, William E. Fahl, Allen L. Thunberg. Radiodermatitis prevention by topical norepinephrine in post-surgical breast cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B130.

Selenoprotein P Inhibits Radiation-Induced Late Reactive Oxygen Species Accumulation and Normal Cell Injury

Radiation is a common mode of cancer therapy whose outcome is often limited because of normal tissue toxicity. We have shown previously that the accumulation of radiation-induced late reactive oxygen species (ROS) precedes cell death, suggesting that metabolic oxidative stress could regulate cellular radiation response. The purpose of this study was to investigate whether selenoprotein P (SEPP1), a major supplier of selenium to tissues and an antioxidant, regulates late ROS accumulation and toxicity in irradiated normal human fibroblasts (NHFs). Flow cytometry analysis of cell viability, cell cycle phase distribution, and dihydroethidium oxidation, along with clonogenic assays, were used to measure oxidative stress and toxicity. Human antioxidant mechanisms array and quantitative real-time polymerase chain reaction assays were used to measure gene expression during late ROS accumulation in irradiated NHFs. Sodium selenite addition and SEPP1 overexpression were used to determine the causality of SEPP1 regulating late ROS accumulation and toxicity in irradiated NHFs. Irradiated NHFs showed late ROS accumulation (4.5-fold increase from control; P<.05) that occurs after activation of the cell cycle checkpoint pathways and precedes cell death. The mRNA levels of CuZn- and Mn-superoxide dismutase, catalase, peroxiredoxin 3, and thioredoxin reductase 1 increased approximately 2- to 3-fold, whereas mRNA levels of cold shock domain containing E1 and SEPP1 increased more than 6-fold (P<.05). The addition of sodium selenite before the radiation treatment suppressed toxicity (45%; P<.05). SEPP1 overexpression suppressed radiation-induced late ROS accumulation (35%; P<.05) and protected NHFs from radiation-induced toxicity (58%; P<.05). SEPP1 mitigates radiation-induced late ROS accumulation and normal cell injury.

Radioprotective effect of epicatechin in cultured human fibroblasts and zebrafish

Radiation-induced normal cell damage limits the delivery of high-dose radiation to targeted cancer. This study investigated the effect of epicatechin (EC), a minor component of green tea extracts, on radiation-induced cellular damage in vitro in primary cultured human fibroblasts and in vivo in a zebrafish model. Cell viability, proliferation and wound-healing efficacy, mitochondrial membrane potential, and reactive oxygen species (ROS) generation as well as changes in the signaling pathway related to apoptosis were investigated in fibroblasts. The therapeutic effects of EC were explored in a zebrafish model. EC increased clonogenic survival and restored the migration ability of the fibroblasts after irradiation. EC inhibited radiation-induced ROS generation, mitochondrial dysfunction and cell death. EC significantly reduced the expression of p-JNK, p-38, and cleaved caspase-3 compared with their significant increase after radiation treatment. EC attenuated the radiation-induced embryotoxicity in a zebrafish model. These results suggest that EC represents an effective means of reducing cellular damage and facilitating wound healing after radiation exposure.

Effect of N-acetyl-l-cysteine on Saccharomyces cerevisiae irradiated with gamma-rays

Ionizing radiation (IR) induces DNA strand breaks (DSBs), base damage, inhibition of protein activity, apoptosis by reactive oxygen species (ROS). Detoxification or removal of generated ROS can reduce oxidative damage. Antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase are immediately triggered for ROS scavenging. N-acetyl-l-cysteine (NAC) having a thiol, a precursor for reduced glutathione (GSH), is known as one of the antioxidants. In this study, the effect of NAC as an antioxidant and a radioprotector was investigated on survival rate, transcriptional level of antioxidant enzymes gene, and protein level including SOD activity and intracellular GSH in yeast Saccharomyces cerevisiae W303-1A strain mutated YBP1 gene irradiated with gamma-rays. NAC did not protect the gamma-ray-induced cell death. The gene expression of antioxidant enzymes including SOD1, SOD2, GPX1, and GPX2 was induced by gamma-rays. In contrast, the pretreatment of NAC reduced the expression of these genes. NAC reduced SOD activity and intracellular GSH level in yeast. These data suggest that NAC is able to reduce radiation-induced ROS levels in vivo but does not protect yeast cells against radiation-induced death.

Responses of Mn2+speciation inDeinococcus radioduransandEscherichia colito γ-radiation by advanced paramagnetic resonance methods

The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) "antioxidant" Mn(2+)-metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)-scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn(2+) speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn(2+) of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn(2+) of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn(2+) speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn(2+) complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn(2+) speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants.

Reactive oxygen species activation of MAPK pathway results in VEGF upregulation as an undesired irradiation response

Radioresistance limits the effectiveness of radiotherapy in head and neck squamous cell carcinoma. We previously demonstrated post-radiogenic mitogen-activated protein kinase (MAPK) pathway activation and vascular endothelial growth factor (VEGF) release resulting in reduced tumor cell response. Here, we examined the association of this mechanism with the induction of reactive oxygen species (ROS) under irradiation (IR). Intracellular ROS after IR were measured. We modeled radiation-induced ROS by exposure of two SCC lines to H2 O2 and evaluated the impact of irradiation and ROS on ERK phosphorylation by Western blot, immunohistochemistry, and ELISA. We found elevated pERK levels after treatment with IR and H2 O2 , which could be distinctly suppressed by U0126. Immunohistochemistry and ELISA revealed increased intracellular VEGF levels after H2 O2 application. Our data show that irradiation-induced ROS activate the MAPK pathway and release of VEGF. As VEGF is known to be released after cellular distress resulting in cytoprotection, the described mechanism is potentially of importance for therapy success.