Targeted and Off-Target (Bystander and Abscopal) Effects of Radiation Therapy: Redox Mechanisms and Risk/Benefit Analysis

Abstract

Significance: Radiation therapy (from external beams to unsealed and sealed radionuclide sources) takes advantage of the detrimental effects of the clustered production of radicals and reactive oxygen species (ROS). Research has mainly focused on the interaction of radiation with water, which is the major constituent of living beings, and with nuclear DNA, which contains the genetic information. This led to the so-called target theory according to which cells have to be hit by ionizing particles to elicit an important biological response, including cell death. In cancer therapy, the Poisson law and linear quadratic mathematical models have been used to describe the probability of hits per cell as a function of the radiation dose. Recent Advances: However, in the last 20 years, many studies have shown that radiation generates "danger" signals that propagate from irradiated to nonirradiated cells, leading to bystander and other off-target effects.

Critical issues: Like for targeted effects, redox mechanisms play a key role also in off-target effects through transmission of ROS and reactive nitrogen species (RNS), and also of cytokines, ATP, and extracellular DNA. Particularly, nuclear factor kappa B is essential for triggering self-sustained production of ROS and RNS, thus making the bystander response similar to inflammation. In some therapeutic cases, this phenomenon is associated with recruitment of immune cells that are involved in distant irradiation effects (called "away-from-target" i.e., abscopal effects).

Future directions: Determining the contribution of targeted and off-target effects in the clinic is still challenging. This has important consequences not only in radiotherapy but also possibly in diagnostic procedures and in radiation protection.

Keywords: abscopal effects; bystander effects; nontargeted effects; radionuclide therapy; radiotherapy; targeted effects.

FIG. 1.

Kinetic description of the ROS produced by water radiolysis. Ionizing radiation induces excitation and ionization of water molecules in a very short time. Excited H₂O* molecules can then dissociate to generate H^• and highly reactive HO^• that can be produced also by transfer of one proton from ionized water molecules H₂O^+•. Ejected electrons can be thermalized to produce hydrated electrons e⁻_aq, or react with H⁺ or O₂ to produce H^• and O₂^−• respectively. Radical recombination reactions also can occur, mostly after irradiation with high LET particles, leading to the production, for example, of H₂O₂ or H₂ through recombination of two HO^• or H^• radicals, respectively. H₂O₂, hydrogen peroxide; LET, linear energy transfer; O₂^−•, superoxide anion; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Mechanisms of 8-oxodGuo and FapydGuo formation through direct or indirect effects of irradiation. The direct effect produces a guanine radical cation that, following dehydration, produces a neutral radical 1 that can also be formed by addition of HO^• (produced through the indirect effect) onto the guanine moiety. Oxidation of 1 gives rise to 8-oxodGuo, whereas reduction of 1 leads to FapydGuo production. 8-oxodGuo, 8-oxo-7′8-dihydro-2′-deoxyguanosine; dGuo, 2′-deoxyguanosine. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Radiation-induced DNA damage. Different types of DNA lesions that could be produced by ionizing radiations, including (A) base (B) modifications, abasic sites, SSB or DSB, intrastrand crosslink, tandem DNA lesions involving two adjacent modifications. (B) Types of DSB and non-DSB clustered DNA lesions involving the combination of all possible DNA lesions in one or two DNA helix turns (82). DSB, double-strand break; SSB, single-strand break. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Mechanisms of formation of complex DNA lesions (including tandem 8-oxodGuo-dF lesions and G[8-5]C intrastrand crosslinks) at dG-dC sequences mediated by a single oxidation event. After HO^• reaction on the cytosine base, in the absence of oxygen, the produced radical can react with an adjacent guanine base, thus producing a G[8-6]C intrastrand crosslink. On the contrary, in the presence of oxygen, the cytosine radical is trapped by molecular oxygen, thus producing a peroxyl radical. This peroxyl radical can react with an adjacent purine base, thus generating an unstable endoperoxide that, on decomposition, gives rise to tandem lesions constituted of two adjacent oxidative DNA lesions. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Radiation-induced lipid peroxidation and sphingolipids. (A) Lipid peroxidation and formation of PUFA decomposition products. (B) Phospholipids contain two hydrophobic long-chain fatty acids linked to an alcohol (usually glycerol) and a hydrophilic group made of a phosphate group. Similarly, sphingolipids contain a long-chain sphingoid base (such as sphingosine) linked via an amide to long-chain fatty acids, and to one polar head group that makes them amphipathic molecules. Head groups differentiate sphingolipids from ceramides (phosphorylcholine constituting SM and hydroxyl group, respectively). (C) Irradiation induces rapid formation of ceramide through the hydrolysis of SM by ASMase. 4-HNE, 4-hydroxy-2-nonenal; ASMase, acid sphingomyelinase; MDA, malondialdehyde; PUFA, polyunsaturated fatty acids; SM, sphingomyelin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

DNA damage-mediated activation of ATM signaling pathways. (A) The DNA damage repair mechanism includes sensors, transducers, and effectors. Sensors, such as the MRN complex, recognize the DNA structure modifications induced by DNA DSBs. Transducers are ATM (and ATR involved in HRR) PI3K involved in phosphorylation of many effector proteins that control cell cycle progression (CHK1 and CHK2), DNA repair (via, for instance, NHEJ mechanisms), and apoptosis. The intrinsic apoptotic signaling pathway relies on p53 stabilization, followed by PUMA transcription and BAX activation. MDM2 ubiquitin ligase activity plays a central role in keeping a low p53 concentration in non-ATM-activated cells. On ATM activation, MDM2 ubiquitin ligase activity is inhibited and p53 stabilized. PUMA frees the proapoptotic BAX/BAK proteins (members of the BCL2-family) that can transfer to mitochondria where they promote cytochrome C, AIF, and SMAC release and further activation of caspases. In the extrinsic apoptotic signaling pathway, death receptor (TNF-R, TRAIL-R, and FAS) activation leads to BID cleavage by caspase 8 and further release of cytochrome C. ATM-mediated phosphorylation of H2AX triggers recruitment of MDC1, which in turn recruits more MRN complexes and activated ATM to the damaged chromatin. This promotes H2AX phosphorylation and spreads ATM and H2AX phosphorylation over a large chromatin domain. (B) ATM structure. ATM is a 350 kDa protein (3056 amino acids) and a member of the PIKK family. It includes HEAT repeats, FAT, PI3K, and FATC domains. The HEAT repeats allow NBS1 binding. Post-translational modifications include autophosphorylation (at Ser 1981, Ser 367, Ser 1893, Ser 2996) and TIP 60 acetylation at Lys 3016 (170, 185, 231, 282). 53BP1, p53-binding protein 1; AIF, apoptosis-inducing factor; ATM, ataxia-telangiectasia mutated; ATR, ATM- and Rad3-related; BID, BH3 interacting-domain death agonist; BRCA1, breast cancer type 1; CHK1, checkpoint kinase 1; CHK2, checkpoint kinase 2; FAT, FRAP-ATM-TRRAP; FATC, FAT C-terminal; HEAT, Huntington-elongation factor 3-protein phosphatase 2A-TOR1; HRR, homologous recombination repair; MDC1, mediator of DNA damage checkpoint protein 1; MDM2, mouse double minute 2 homologue; MRN, MRE11–RAD50–NBS1; NHEJ, nonhomologous end joining; PI3K, phosphatidylinositol-3-kinase; PIKK, phosphatidylinositol 3-kinase-related kinase; PUMA, p53-upregulated modulator of apoptosis; SMAC, second mitochondria-derived activator of caspases; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Schematic representation of the bystander and abscopal responses. Targeted effects are observed only in irradiated cells, while nontargeted effects are observed in nonirradiated cells. Nontargeted effects include bystander and abscopal responses. (A) Radiation-induced targeted effects concern (i) nuclear DNA and extranuclear targets, (ii) mtDNA, Ca²⁺-mediated mitochondrial production of ROS and RNS, (iii) ER, as a Ca²⁺ storage place, and (iv) cell membrane, as the site of ion channels, NADPH oxidase, growth factor and death receptor localization, lipid peroxidation leading to 4-HNE or MDA production, and production of ceramide that acts as a second messenger or is involved in ceramide-enriched large platforms (lipid rafts). Bystander effects are observed in neighboring cells (in contact or not with the irradiated cells) that have not been crossed by ionizing particles. Intercellular cross talk is mediated by gap junctions (GJIC) or through the release of soluble factors, including cytokines, ROS, and RNS. Exosomes containing mRNA, microRNA, and DNA can also be released. (B) Abscopal effects are observed at long distance from the irradiation site (e.g., localized breast irradiation). Consequently, the biological effects must be investigated at the whole-body scale. Abscopal effects may involve the immune system through the release of DAMPs that are recognized by antigen-presenting cells (e.g., dendritic cells that will present antigenic peptides to CD4 and CD8 T lymphocytes for immune response activation). The cell response to targeted and nontargeted effects can result in cell death, cell transformation, or cell survival and these possible outcomes have to be taken into account in the field of cancer therapy and radiation protection. 4-HNE, 4-hydroxy-2-nonenal; DAMPs, damage-associated-molecular-patterns; EBRT, external beam radiotherapy; ER, endoplasmic reticulum; GJIC, gap junction intercellular communication; mtDNA, mitochondrial DNA; NAD(P)H, nicotine adenine dinucleotide phosphate; NOX, NAD(P)H oxidase; RNS, reactive nitrogen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Endogenous sources of ROS and enzymatic antioxidant defenses. (A) O₂^−•, hydroxyl radical (HO^•), and H₂O₂ are produced by endogenous sources that reduce O₂. The main sources are mitochondria through ATP production and oxidative (oxygenase, dehydrogenase, oxidase) enzymes, such as NAD(P)H, and xanthine oxidase, lipoxygenase, myeloperoxidase. Oxygenase enzymes (e.g., lipoxygenase) oxidize substrates by transferring one electron, while oxidizing a cofactor [e.g., NAD(P)H] in the presence of oxygen. Dehydrogenases use organic substrates as an electron acceptor (e.g., quinones, NAD⁺). Oxidases just use O₂ as an electron acceptor. The yield of radiation-induced DNA lesions (/Gy/cell) is rather low compared with that produced by endogenous stress [averaged values from Goodhead (102), Pouget et al. (241, 245), Sage and Shikazono (262), and Ward (329)]. (B) Superoxide can be dismuted into H₂O₂ by the action of superoxide dismutase enzymes that possess a metal transition ion (Mn³⁺, Cu²⁺, Fe³⁺, or Ni³⁺) to catalyze the reaction. In the presence of M⁽ⁿ⁾⁺ metal ions, the resulting H₂O₂ can be broken down into HO^•+OH⁻ and M⁽ⁿ⁺¹⁾, according to the Fenton reaction. The latter reaction can also be mediated by catalase and GPx (22, 76, 80). GPx, glutathione peroxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 9.

Mitochondrial electron transport chain and NAD(P)H oxidase as endogenous source of ROS. (A) NADH and succinate produced during the citric acid cycle are used as electron donors for ATP synthesis by the ETC. ETC consists of complex I (NADH coenzyme Q reductase), complex II (succinate dehydrogenase coenzyme Q), complex III (coenzyme Q cytochrome C reductase), and complex IV (cytochrome C oxidase). The final acceptor molecule O₂ is reduced to H₂O. However, a small percentage of electrons can leak at complex I and complex III and can reduce O₂ into O₂^−•. (B) The second major source of ROS is NOX. NOX contains membrane proteins (gp91^phox or NOX-2 and p22^phox that constitute the flavocytochrome b558, and the small G Rap1A protein). During NOX activation (for instance, in neutrophils, or by Ca²⁺ or radiation), cytosolic proteins (p40^phox, p47^phox, p67^phox and G Rac2) are recruited to the membrane and NADPH binds to NOX and transfer electrons to FAD and further across the membrane to O₂ (108, 208). ETC, electron transport chain; FAD, flavin adenine dinucleotide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 10.

Lipid rafts: ceramide-enriched large platforms. (A) The cell membrane consists of a lipid bilayer that includes proteins and cholesterol. According to the fluid-mosaic model, lipids can rotate laterally and between bilayers. The lipid distribution in the cell membrane is associated with specific cellular functions. Specifically, the cell membrane can include glycolipids, phospholipids (e.g., phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine), and sphingolipids, among which SM is predominant. Their resistance to disruption allowed the identification of membrane domains enriched in ceramide. Ceramide is produced via ASMase-mediated hydrolysis of SM following RNS and ROS activation and ASMase translocation to the outer layer of the cell membrane. Interaction between the resulting lipids and proteins leads to the coalescence of microdomains (lipid rafts) into ceramide-enriched large platforms. (B) These platforms can promote clustering of receptors and activation of signaling pathways. Besides its role in lipid rafts, ceramide is also a second messenger (58, 167, 346). CER, ceramide; GPL, glycophospholipids; TM, transmembrane. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 11.

Interplay between Ca²⁺ and radiation-induced oxidative stress [extensively reviewed in Decrock et al. (68)]. Irradiation increases the intracellular Ca²⁺ level (oscillations or single transient changes occurring within minutes to days after irradiation). Radiation-induced ATP release by irradiated cells can activate ATP-gated P2X receptor cation channels (P2X receptors) present on the cell membrane, thus allowing Ca²⁺ entry into the cell. It can also activate P2Y receptors that have been identified as phospholipase C activators. Ca²⁺ can also be released from the ER through calcium-induced calcium release mechanisms that involve IP₃Rs or RyRs. IP₃ is produced (with diacylglycerol) during hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C. Phospholipase C is activated by Ca²⁺, GPCRs, ROS, RNS, receptor and nonreceptor tyrosine kinase (e.g., HER1 and 3). Ca²⁺ can activate ion channels and binds to calmodulin before activation of the serine/threonine protein phosphatase calcineurin. Ca²⁺ can also activate protein kinase C that in turn activates, by phosphorylation, the MAPK pathway and phospholipase A2, at the origin of COX-2 activity modulation. It can activate transcription factors (NF-κB, AP1) that promote various downstream pathways (iNOS, COX-2). Released Ca²⁺ can also be taken up by mitochondria via VDAC and MCU and modulation by the ER-mitochondria-tethering proteins GRP75 (mitochondrial heat shot protein HSP70) and MFN 1 and 2 (involved in mitochondrial fusion). The increase in mitochondrial Ca²⁺ level is accompanied by ROS, an RNS increase, mtDNA damage, altered ATP synthesis, mitochondrial depolarization, and release of cytochrome C and caspase 3 that will amplify IP₃R activity. COX-2, cyclooxygenase-2; GPCRs, G-protein-coupled receptors; iNOS, inducible nitric oxide synthase; IP₃, inositol trisphosphate; IP₃Rs, IP₃ receptors; MAPK, mitogen-activated protein kinase; MCU, mitochondrial Ca²⁺ uniporter; MFN, mitofusin; NF-κB, nuclear factor kappa B; RyRs, ryanodine receptors; VDAC, voltage-dependent anion channel. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 12.

The MAPK and PI3K signaling pathways. The four major MAPK signaling pathways (ERK1/2, ERK5, JNK1/2, and p38) regulate cell survival (ERK1/2 and ERK5) and apoptosis (JNK1/2 and p38) via the expression of transcription factors. The PI3K signaling pathway is involved in cell growth via AKT1/2 proteins. Death (Fas/CD95, TNF-R, DR3-5) and growth (HER family and IGF-1 receptors) receptors are located in lipid rafts and can be activated during coalescence of ceramide-enriched raft platform. They facilitate the cross talk between death or growth signaling from the membrane environment and intracellular signaling cascades. FAK and PYK-2 serve as scaffold proteins to facilitate the functional integration of focal adhesion proteins, such as paxillin, involved in Ca²⁺ homeostasis and can phosphorylate PI3K. ERK, extracellular signal-related kinase; FAK, focal adhesion kinase; IGF-1, insulin-like growth factor 1; JNK, c-JUN N-terminal kinase; PTEN, prime time entertainment network. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 13.

NF-κB activation mechanisms. The antiapoptotic NF-κB consists of five heterodimers, among which the most common form involves p65 and p50. It is kept in the cytoplasm by its interaction with IκB. On activation (e.g., by ATM, TNFα, IL1), the IKK complex, which is made of IKK-α (IKK1), IKK-β (IKK2), and the regulatory subunit IKK-γ/NEMO, phosphorylates and targets IκB for ubiquitination and degradation by the proteasome, while NF-κB can enter the nucleus and activate its target genes. IKK, IκB kinase; NEMO, NF-κB essential modulator. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 14.

The NF-κB and cytokine receptor pathways in the bystander response. DNA DSBs in irradiated cells activate ATM and NEMO that in turn assemble IKK before IκB ubiquitination and proteosomal degradation. Released NF-κB enters the nucleus and induces the transcription of target genes, such as those encoding cytokines. Secreted cytokines can in turn bind to receptors on bystander cells. On binding, receptors will activate NF-κB-responsive element-containing molecules (e.g., cytokines, COX-2, and iNOS), thus contributing to ROS and RNS production and transmission of bystander signals in a self-sustained process. The MAPK and JAK2-STAT3 pathways are also activated by IL8 and IL6, respectively. The TGFβ receptor also can activate NOS. β-Cat, β-catenin; JAK2, Janus kinase 2; PGE2, prostaglandin E2; STAT3, signal transducer and activator of transcription 3; TGF, transforming growth factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 15.

Complex DNA damage leads to immune signaling. (A) Repair of a clustered damaged DNA site: a challenging task. On induction by ionizing radiation of complex DNA damage, such as a DSB and two oxidative DNA lesions (a damaged base, red star, and an apurinic/apyrimidinic, AP, site), at least two DNA repair pathways and several DNA repair proteins will be activated. For the base damage, BER is the main repair pathway, while for the DSB, only NHEJ will be considered here for simplicity. In all cases, the most basic proteins and enzymes are described. For short-patch BER, a DNA glycosylase will recognize and remove the damaged base and the repair should be completed by the concerted activity of AP endonuclease 1 (APE1), a DNA polymerase, and ligase III to seal the broken ends. In the nearby DSB area (a few bp apart), the Ku heterodimer (Ku70/80) initiates NHEJ by binding to the free DNA ends and engaging other NHEJ factors, such as DNA-PK, XRCC4, and DNA ligase IV, to the DSB site. DNA-PK becomes activated on DNA binding and then phosphorylates a number of substrates, including p53, Ku, and the DNA ligase IV cofactor XRCC4. Phosphorylation of these factors is believed to further facilitate DSB processing. For ligation, the ends must be partially processed by the nucleases Artemis, MRE11/RAD50/NBS1 complex, and FEN-1. Moreover, as shown by advanced fluorescence microscopy, the formation of each DSB is rapidly accompanied by phosphorylation of thousands of histone H2AX molecules (γH2AX). The MRN complex functions as a sensor of DNA ends and activates the ATM kinase that phosphorylates CHK2, p53, and H2AX in flanking chromosomal regions. (B). Systemic effects. Processing of clustered DNA damage can lead to unrepaired and persistent DNA damage that can cause cell senescence or cell death (i.e., apoptosis). This can trigger the extracellular release of different “danger” signals or damage-associated molecular patterns (DAMPs: ATP, short DNAs/RNAs, ROS, heat shock proteins [HSPs], high-mobility group box [HMGB]-1, S100 proteins, and others). DAMPs activate different PRRs, such as TLRs and the formation of inflammasomes, a process that leads usually to inflammation and immune-related pathologies. Interestingly, recent evidence (see section IV.B.1) suggests a direct interaction between different PRRs and DNA repair proteins. Cell damage or death can also lead to the release of several cytokines and chemokines that can regulate immune responses. PRR activation usually results in NF-κB-mediated release of various proinflammatory cytokines, such as IFNs, IL-1, IL-6, IL-8, VEGF, EGFR, and TNFα. The activation of APCs, for instance, dendritic cells and macrophages, will induce primarily the innate immune response (activation of T cells) and most rarely the adaptive immune response (mediated by B cells). In all cases, the constant triggering of the immune system might generate many detrimental systemic effects for the organism. Positive immunomodulation is usually mediated by the action of regulatory (suppressor) T cells (i.e., Treg cells), suppressor macrophages, and immunosuppressive cytokines to maintain overall tissue homeostasis. APCs, antigen-presenting cells; BER, base excision repair; DNA-PK, DNA-dependent protein kinase; EGFR, epidermal growth factor receptor; PRRs, pattern recognition receptors; Treg cells, regulatory T cells; TLRs, Toll-like receptors. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars