Open access peer-reviewed chapter - ONLINE FIRST
Abstract
Radiation therapy is a cornerstone of cancer treatment, targeting tumor cells through DNA damage and subsequent induction of various forms of cell death. This chapter explores the multifaceted biological effects of Radiation therapy (RT), highlighting its ability to trigger different lethal and non-lethal death mechanisms. The mechanisms underlying these responses involve complex interactions between radiation-induced DNA damage, reactive oxygen species production, and disruption of cellular homeostasis. RT therapeutic efficacy is influenced by factors such as tumor type, microenvironment, and the balance between cell death and survival pathways. Advances in understanding how RT impacts cell death mechanisms, including the modulation of ferroptosis and pyroptosis, have unveiled new opportunities to enhance radiosensitivity and overcome tumor resistance. Furthermore, non-lethal processes, such as senescence and mitotic catastrophe, underscore the potential of RT to suppress tumor progression through mechanisms beyond direct cytotoxicity. This chapter emphasizes the need for integrating molecular insights with clinical applications to optimize the efficacy of RT while minimizing damage to healthy tissues. By examining emerging strategies, such as the exploitation of immune responses and targeting tumor microenvironmental factors, this work provides a comprehensive foundation for advancing radiotherapy in oncology.
Keywords
- radiotherapy
- cell death
- pyroptosis
- ferroptosis
- senescence
- mitotic catastrophe
- irradiation
1. Introduction
Cancer is the second greatest cause of death after heart disease and has long been a global health concern [1]. Radiation therapy (RT) is one of the main treatment modalities. It is used both as curative and palliative for almost all solid tumors. RT is frequently used in conjunction with immunotherapy, cytotoxic chemotherapy, and surgery as the first line for treating cancer [2]. Radiation kills cells in a way that is not specific to tumor cells, and the best way to deliver RT is to strike a balance between maximizing the dosage to the tumor and minimizing the damage to healthy tissue [3]. DNA is RT’s main intracellular target; it is harmed by ionizing radiation (IR) either directly or indirectly via reactive oxygen species, which sets off a series of events that may result in cell death. DNA double-strand breaks (DSBs) are caused by radiation and can be repaired via non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ functions throughout the cell cycle, whereas HR is limited to the S and G2 phases. Furthermore, a third mechanism called alternative NHEJ was just identified: it repairs damaged DNA by utilizing tiny areas of microhomology within the break sites [4]. Therefore, HR and NHEJ pathways detect and effectively repair damaged DSBs, and NHEJ is the major mechanism for repairing RT-induced DNA damage [5]. A high radiation-associated deletion burden may indicate the sensitivity of recurring malignancy following RT and has been linked to poor survival. On the other hand, inadequate or ineffective DNA repair processes cause cell death [6]. Factors such as the rate of mitosis, the degree of differentiation, and the total and fractional radiation doses significantly affect the extent of cell death or resistance to treatment [7, 8]. Cell death is the cytological effect of radiation on the human body. In this context, it is known as proliferative death and interphase, which are further classified based on the molecular process [9, 10]. According to cell death classification, it is possible to distinguish between regulated cell death, generally known as programmed cell death (PCD), including: apoptosis, oncosis, necroptosis, eryptosis, ferroptosis, pyroptosis, parapoptosis, parthanatosis, mitoptosis, alkaliptosis, methuosis, oxeiptosis, sarmoptosis, Wallerian degeneration, transneuronal degeneration, NETosis, entosis, emperipolesis, anoikis, cornification, immunological cell death, mitotic cell death, autophagy-dependent cell death, autosis, and lysosome-dependent cell death, while the accidental cell death refers to the necrosis [11]. IR affects multiple biological targets and activates different pathways, resulting in diverse types of cell death depending on the radiation dose and environmental factors [9]. Due to the diversity of cell phenotypes, cell cycle phases, doses of radiation, and even cell subregions, different cell death types can occur. Furthermore, it is more difficult to differentiate and categorize the different types of cell death due to the presence of signaling and initial activation molecules shared by all of them, as well as due to complex crossovers in cellular molecules. This chapter serves as a critical foundation for understanding the biological basis of RT by exploring its essential role in oncology and examining its mechanisms, biological effects, and potential to drive advances in therapeutic approaches. An in-depth discussion is devoted to the fundamental impact of radiation at the cellular level, highlighting how various types of radiation induce damage to key cellular components such as DNA, proteins, and membranes. This chapter offers a comprehensive analysis of some cell deaths triggered by radiation exposure, including apoptosis, necroptosis, pyroptosis, and ferroptosis. Additionally, it explores non-lethal processes such as senescence and mitotic catastrophe. Furthermore, the chapter examines the diverse mechanisms through which radiation exerts its effects. It will also provide a thorough understanding of the processes that underline the therapeutic and cytotoxic actions of radiation and highlight novel strategies intended to increase its effectiveness in the treatment of cancer. This comprehensive approach ensures a deeper understanding of the biological complexities of RT and paves the way for innovative strategies to overcome limitations, such as tumor radioresistance, thus refining its application in clinical oncology.
2. Principles of radiation therapy
RT is a cornerstone of cancer treatment in different solid tumors, using ionizing radiation (IR) as a physical agent to target and kill cancer cells [12, 13, 14]. IR generates electrically charged particles, ions, by transferring energy to the cells in the tissues it penetrates, thus directly destroying tumor cells or leading to genetic damage that ultimately leads to their elimination [15].
2.1 Types of radiation used in cancer treatment
The most common kind of radiation utilized in cancer treatment is photons, which include gamma and X-rays. Photon beams have a very low charge and mass. X-rays and γ-rays are both classified as low linear energy transfer (low-LET) and consist of massless energetic particles. Cathode ray tubes and linear accelerators, for example, emit X-rays when they excite electrons. γ- rays, on the other hand, come from radioactive compounds decaying. Photon radiation is routinely used for its versatility and ability to effectively penetrate tissue, making it suitable for treating various types of cancer. Particle radiation, on the other hand, involves the use of charged or neutral particles, which have unique physical and biological properties compared to photons [16]. Electron beams are commonly used in routine RT; these are particularly effective for treating tumors near the body surface because their penetration into deeper tissue is limited. Proton beams are a highly precise form of particle radiation [15]. Proton therapy exploits the Bragg peak phenomenon; this provides the maximum energy accumulation at the tumor location while sparing surrounding healthy tissue. Proton therapy is, therefore, particularly useful for pediatric cancers and for adult tumors located close to vital structures, including tumors of the skull base and spinal cord [17]. Neutron beams are produced by neutron generators, which direct proton beams onto a target. These show high-LET and induce more extensive DNA damage than photons (Figure 1). Despite their greater biological efficacy, their application is limited due to the technical complexity and cost of generating neutron particles and building treatment facilities. Synchrocyclotrons and synchrotrons create heavy ion beams such as helium, carbon, nitrogen, argon, and neon. These particles have a high-LET and are particularly effective against radioresistant tumors such as sarcomas, renal cell carcinomas, melanomas, and glioblastomas. Particle radiation has a higher LET than photons, offering greater biological efficacy, especially for tumors resistant to conventional photon therapy [18].
Figure 1.
Schematic representation of DNA damage caused by different types of ionizing radiation. High Linear Energy Transfer (LET) radiation, such as alpha particles (α) and protons (p), induces complex DNA lesions. Low-LET radiation, including electrons (e−, β−, and β+) and electromagnetic radiation (γ-rays and X-rays), primarily causes single-strand breaks (SSBs) and double-strand breaks (DSBs).
2.2 Types of cell damage induced by radiation
RT hit all the cellular components; indeed, proteins, lipids, the nucleus, mitochondrial DNA, and other constituents can all be impacted [19, 20]. However, the nuclear genome is the main target of radiation. IR destroys molecular bonds and induces numerous lesions caused through direct energy deposition and/or free radicals, commonly known as reactive oxygen species (ROS), including O2 and OH generated during water hydrolysis [21, 22, 23, 24]. The amount and severity of damage is determined by the radiation’s quality, intensity, and absorbed dose [25]. In general, low-LET particle radiation, like photons, X-rays, and γ-rays, is less detrimental compared to high-LET particle radiation, which comes from protons, α-particles, and heavy ions. High-LET particles produce dense ionization by storing energy in the medium, exhibiting greater biological effects than low-LET radiation, which exhibits a uniform and sparse spatial distribution of ionization in cells [17, 26]. Single-strand breaks (SSBs), DSBs, oxidative base damage, and some clustered DNA lesions are caused by low-LET radiation, while complex clustered DNA lesions are induced by high-LET radiation [19]. Upon IR, SSBs and DSBs represent radiation-induced DNA damage, while clustered DNA damage, base damage, and cross-linking can also be distinguished, thus leading to DNA damage responses (DDRs). IR can also induce non-targeted effects, including those induced by bystander signals that occur in non-irradiated cells after receiving signals from nearby irradiated cells, whose trigger signals are very diverse, including ROS, reactive nitrogen species (RNS), and cytokines with proinflammatory action [27, 28, 29]. Signaling molecules can then cross gap junctions between cells, diffuse over long distances in the extracellular environment or bloodstream, and be transported by mediators like exosomes or another carrier [30]. IR can also trigger non-targeted effects by affecting some cellular organelles, such as mitochondria [31]. Regardless of the direct or indirect effects caused by RT, its main objective is to disable the proliferative ability of cancer cells and ultimately eliminate them: once the DNA is irreparably damaged, cells cease dividing and undergo death [15]. Following IR, cell death can be classified as interphase or proliferative death. Indeed, IR can cause a variety of cellular fates, including lethal and non-lethal processes (Figure 2) [3, 32].
Figure 2.
Schematic representation of some biological effects induced by RT. Lethal events (apoptosis, ferroptosis, necroptosis, and pyroptosis) are shown in shades of red, while non-lethal effects (senescence and mitotic catastrophe) are highlighted in yellow.
3. RT inducing PCD
3.1 Apoptosis
Apoptosis is a vital homeostatic process involved in morphogenesis during early development that can also be activated in pathological conditions. It is one of the main mechanisms of cell death following anticancer therapy, particularly active upon RT. Morphologically, it presents with cellular shrinkage and the formation of apoptotic bodies [15]. In addition, the formation of vesicles on the membrane, compacting chromatin with nuclear edge localization, and fragmented DNA are frequently observed. Mitochondria are the cellular organelles primarily involved in this type of cell death [33]. In apoptosis, two different mechanisms can be distinguished: intrinsic or extrinsic mediated pathway; both pathways converge in the activation of caspase. These proteins are enzymes with cysteine protease activity considered essential in both inflammation and apoptotic pathways; they are classified as initiators: caspases-2, −8, −9, and, −10, and effectors: caspases-3, −6, and − 7, and oversee the primary catalytic events during the intrinsic and extrinsic pathways of apoptosis [33, 34]. The extrinsic apoptotic pathway is modulated by external signals via the activation of death receptors, including Fas receptors, DR4/DR5, tumor necrosis factor receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-R) present on the surface of several cells [35, 36]. TNF receptors are composed of extracellular cysteine-rich domains and a cytoplasmic domain, the death domain, implicated in the transmission of the death signal from the outside to the inside of the cell [37]. On the cell surface, the death receptor interacts with its specific ligands, recruiting adaptor proteins including Fas-associated protein with death domain (FADD), tumor necrosis factor receptor-associated death domain protein 1 (TRADD), and subsequently downstream factors such as caspase-8 [38, 39]. The death receptor, once it binds to specific ligands, recruits adaptor proteins such as Fas-associated protein with death domain such as FADD, TRADD, and then downstream factors such as caspase-8 [38, 39]. Crucially, ligand binding recruits and dimerizes death receptors, exposing the death domains and activating caspases-8 and 10 [40]. Finally, active caspase-8 and -10 trigger apoptosis by cleaving and activating caspases 3, 6, and 7 [41, 42]. DNA damage and p53 activation start the intrinsic apoptosis process, also referred to as the mitochondrial apoptosis pathway, which sets off a series of events that culminate in mitochondrial outer membrane permeabilization. The Bcl-2 protein family includes both pro- and anti-apoptotic proteins and is in charge of controlling the intrinsic pathway. Cell fate is ultimately determined by the heterodimeric connections between Bcl-2 proteins via the Bcl-2 homology domains: BH1, −2, −3, and − 4 [43]. Pro-apoptotic proteins such as Bax and Bak can initiate alterations in mitochondrial membrane potential after the death signal has been activated because BH3-only domain proteins can neutralize or activate the anti-apoptotic protein Bcl-2. Once cytochrome c, an intermembrane protein of mitochondria, is released into the cytosol, it assembles with APAF-1 and pro-caspase 9 to form the apoptosome. Within the apoptosome, activated caspase-9 cleaves and activates the apoptosis effector proteins caspase-3, −6, and − 7 [44, 45]. Cellular irradiation can affect mitochondrial membrane permeability via an intrinsic caspase-dependent pathway, resulting in greater release of pro-apoptotic proteins into the cytoplasm, therefore initiating a series of apoptotic cascades [3]. Indeed, several studies have indeed demonstrated that RT triggers the intrinsic pathway; in particular, the p53 protein has an important function in modulating the redox state in response to oxidative stress caused by IR. Following IR, cellular DNA is damaged, and in response, there is the activation of the proteins ATM and ATR. In turn, these proteins rapidly lead to the activation of the tumor suppressor protein, p53, in detail after IR, ATM protein, phosphorylates p53. The activation of p53 causes Bcl-2 proteins to release Bax, thus promoting apoptosis [12, 46, 47]. p53 regulates the transcriptional activation of apoptosis mediating proteins, particularly Bcl-2, Bax, Puma, and Noxa [48, 49]. Furthermore, p53 can induce the production of SOD2 and GPX1 by binding to their promoters, resulting in an antioxidant response [50, 51]. p53 plays a crucial role in modulating the cellular redox state in response to oxidative stress induced by IR. Crucially, alterations in apoptotic mediators can significantly impact the cellular response to RT. Mutations in p53, frequently observed in radioresistant tumors, impair the activation of the intrinsic apoptotic pathway, thus limiting the efficacy of RT. Alterations in p53 have been shown to be associated with liver cancer recurrence and radioresistance in patients. Mutations in the TP53 gene are also associated with the downregulation of BCL2 family proteins such as Bcl-xs or Bax proteins, leading to acquired resistance and poor efficacy of RT against this tumor [52]. In addition, under hypoxic conditions, the efficacy of RT is reduced due to the downregulation of pro-apoptotic members of the Bcl-2 family and the upregulation of anti-apoptotic ones, promoting radioresistance. However, targeting Bcl-2 with the inhibitor ABT-263 potentiates the RT effect, overcoming resistance both in vitro and in vivo [53]. Caspase modulation may also impact the outcome of RT. Indeed, it has been shown that activated caspase-3 promotes the release of several growth factors from irradiated tumor cells that stimulate the proliferation of adjacent cells, promoting post-irradiation angiogenesis. For this reason, the combination of RT with caspase-3 inhibitors could be a new and promising therapeutic strategy to substantially reduce tumor recurrence due to post-irradiation angiogenesis [54, 55]. In contrast, tumor cells become more sensitive to RT due to the high production of pro-apoptotic proteins. Indeed, a crucial role of the Bax protein, which mediates the mitochondrial apoptotic pathway in prostate cancer cells within minutes after irradiation, has been reported [56].
3.2 Pyroptosis
Pyroptosis is a type of PCD, considered as an immunogenic cell death (ICD). It is modulated by both inflammatory caspases and the gasdermin superfamily proteins (GSDM). The GSDM proteins are inactivated because the C-terminal repressor domain masks their N-terminal pore-forming regions [57]. Once triggered, the caspase cleaves the GSD protein, releasing the N-terminal domain. Then, this domain binds to membrane lipids and perforates the membrane, causing membrane perforation and resulting in changes in osmotic pressure and swelling until the membrane ruptures [58]. Three pathways lead to pyroptosis: the first pathway is triggered by the caspase-1 pathway, which cleaves GSDMD into N- and C-terminal domains when the inflammasome forms and attaches to pro-caspase-1 via the adaptor protein the apoptosis-associated speck-like protein containing a CARD (ASC). As a result, the cytokines IL-1β and IL-18 are activated. After binding to the cell membrane, N-GSDMD leads to pore formation on the membrane, favoring the release of cellular contents. In the second pathway, bacterial LPS triggers the activation of caspase-4/5/11, which cleaves GSDMD to cause pyroptosis and simultaneously triggers the activation of caspase-1. In the Gasdermin C (GSDMC) caspase-8 cleavage pathway, apoptosis is converted to pyroptosis by PD-L1. Other pathways leading to pyroptosis involve distinct mechanisms, including the GSDMD caspase-3/8 cleavage pathway, the GSDME caspase-3/8/9 cleavage process, the GSDMB GZMA cleavage pathway, and the GSDME GZMB cleavage pathway [59]. Pyroptosis has attracted increasing interest in cancer RT due to its ability to both directly trigger cancer cell death and encourage the immune response to eliminate any remaining tumor cells after RT, possibly enhancing therapeutic effectiveness [60, 61]. The pyroptosis mechanism is more frequently activated following treatments with higher radiation doses [58, 59]. Indeed, in the literature, it has been reported that high-LET radiation can induce pyroptosis more than low-LET radiation [62, 63, 64]. The NLRP3 inflammasome has been shown to be activated after RT, causing pyroptosis in liver cells and bone marrow-derived cells [65, 66]. Furthermore, it has been demonstrated that upon IR, tumor cells generate damage-associated molecular patterns (DAMPs) triggering pyroptosis [59, 67]. IR activates pyroptosis and subsequently stimulates an antitumor immune response. Interestingly, the release of pyroptosis-specific signals could trigger the activation of immune cells that can target metastases far from the irradiated primary tumor [59]. An important player in the modulation of pyroptosis after RT treatment is undoubtedly the immune system: as already mentioned, this type of cell death is deeply linked to the immune system and closely related to its activation. Interestingly, NLRP3 has been demonstrated to greatly enhance antigen presentation, innate immune function, and T cell activation following RT [68]. Another crucial element in the induction of pyroptosis after RT is hypoxia. It is well known that high levels of hypoxia can interfere with RT, conferring radioresistant properties to the tumor [69]. IR can produce ROS in the TME due to water radiolysis. Then, ROS activates caspase-8 and GDSM with pyroptosis as a result [70]. Furthermore, free radicals can cause caspase 9/3-dependent GDSME activation; thus, low oxygen levels may limit ROS production in the tumor after RT by reducing the activation of pyroptosis [61]. RT can activate pyroptosis, exploiting the signal cascade of the canonical mechanism, the inflammasome-mediated pathway regulated by the activation of caspase-1 and inflammatory response. The canonical pyroptosis pathway is activated upon IR at different levels of the signal cascade and exploits different events that trigger the assembly of the inflammasome, direct activation of caspase-1, and ROS production. It has been shown that IR leads to the formation of inflammasomes containing NOD-like receptors, in particular NLRP1 and NLRP3 [71]. The formation of these cytosolic complexes triggers the activation of caspase-1, catalyzing the activation of proinflammatory cytokines. Subsequently, caspase-1 causes the cleavage of GSDMD, the final effector of pyroptosis. In addition, it has been shown that IR is able to trigger pyroptotic processes, directly activating the caspase-1 and downstream processes, in an inflammasome-independent manner [72]. ROS generated in the tumor microenvironment (TME) and within the cell are essential in the setting of pyroptosis upon RT. Indeed, studies have shown that RT can activate NLRP3 and the pyroptosis pathway once ROS related to glycolysis or those related to mitochondrial activity (mitoROS) accumulate [59, 73]. Also, the immune system is crucial for pyroptosis activation after RT: following irradiation, macrophages present in the TME can activate the p38 pathway MAPK-NLRC4-caspase-1 and trigger this process [74]. RT is also able to activate pyroptosis via a pathway that is not dependent on inflammasome formation or on caspase-1 activation. γ-radiation has been shown to cause cleavage of GSDM via the caspase-9/caspase-3 pathway in several solid tumors [72]. Following RT, antitumor immunity can also be activated by inducing GSDME-mediated pyroptosis, which transforms the tumor from “cold” to “hot,” making it more susceptible to the immune response [61, 75]. On the other hand, pyroptotic tumor cells may secrete inflammatory modulators that encourage tumor cell repopulation and development [76]. In addition, pyroptotic cells can, in turn, release multiple inflammasomes to damage normal tissues [77]. It was reported that different pharmacological agents can modulate pyroptosis; for instance, the caspase 1 inhibitor Vx-765 used in combination with RT has shown promise in preventing pyroptosis and reducing radiation-induced damage [78]. In addition, key regulators of immune checkpoints, such as PD-1 and its ligand, PD-L1, are connected to pyroptosis. Under hypoxic conditions, PD-L1 undergoes nuclear translocation, which facilitates GSDMC transcriptional activation. Clinical studies indicate that individuals treated with radiation and PD-L1 inhibitors can undergo pyroptosis, which destroys tumor cells; these patients have a higher survival rate than those treated with PD-L1 inhibitors alone [79]. In another study, GSDME-high-expressing tumor cell lines, such as lung, liver, breast, and glioma, have been demonstrated to cleave GSDME in a dose- and time-dependent way following irradiation, and different forms of irradiation may cause pyroptosis. Moreover, pyroptosis was considerably activated when irradiation and DNA-damaging chemotherapeutic drugs like cisplatin or etoposide were combined [61].
3.3 Necroptosis
Necroptosis is triggered by the activation of a death receptor on the plasma membrane, the receptor-interacting protein kinase-1 (RIPK1). The TNF-α-TNFR complex recruits proteins such as RIPK1, TRADD, cIAP1, and TRAF2 to form the pro-survival complex I [80]. This complex can be deubiquitinated, resulting in subsequent complexes, such as complex II. These, in turn, can induce both apoptosis and necrosis; in particular, complex IIa, which consists of TRADD, FADD, and caspase-8, promotes apoptosis via caspase activation [81]. Apoptosis is promoted by caspase-8’s interaction with RIPK1, RIPK3, and FADD to create complex IIb, which cleaves RIPK1 and RIPK3 to render them inactive. Complex IIc, commonly referred to as the “necrosome,” is generated when caspase-8 activity is blocked [82]. This complex is composed of RIPK1, RIPK3, and FADD. The necrosome develops after RIPK1 activates RIPK3, then RIPK3 recruits MLKL and promotes its phosphorylation. Once MLKL becomes phosphorylated, it creates oligomers that migrate to the cell membrane, altering its permeability and causing necrosis [3, 83]. RT can activate necroptosis mechanisms by stimulating different pathways [84]. In irradiated cancer cells, there is an abnormal accumulation of cytoplasmic DNA cells [85]. In addition, a correlation has been observed between the increase of cytosolic DNA and the activation of the ZBP1-MLKL pathway. Indeed, following RT, ZBP1-MLKL necroptotic signaling connects tumor cell damage to anticancer immune responses [86]. The cGAS-STING signaling, activated autonomously, can communicate with the ZBP1-MLKL pathway, thus generating a positive feedback effect between these two signaling pathways. Following this, irradiated cancer cells constantly maintain two useful pathways to drive inflammation processes, so the necroptotic pathway mediated by ZBP1-MLKL can increase antitumor immunity through communication
3.4 Ferroptosis
Ferroptosis is a cell death type triggered by iron accumulation and subsequent peroxidation of phospholipids. Indeed, it is considered an iron-dependent cell death, distinguished by an increase of lipid-derived ROS [94]. Ferroptotic cells exhibit physically tiny dysmorphic mitochondria with decreased cristae and condensed membranes. The identity of the executor proteins in ferroptosis is yet unclear, in contrast to classical Regulated cell death (RCD), which entails the involvement of proteins that carry out cell death, for instance, gasdermin D for pyroptosis, caspase for apoptosis, and the protein MLKL for necrosis [95]. Ferroptosis is mainly activated by the breakdown of cellular antioxidant defenses, particularly the glutathione (GSH)-dependent glutathione peroxidase 4 (GPX4) pathway, which protects against lipid peroxidation [96, 97]. Iron-dependent lipid peroxidation is a hallmark of this type of PCD and is caused by redox imbalance. Under physiological conditions, the redox balance of lipids is necessary for cell viability. Lipids are essential because they act as buffers for ROS, thus preserving the dynamic equilibrium between reduced and oxidized states. The oxidation of polyunsaturated fatty acids (PUFA) present in the phospholipids of cell membranes, mediated by lipoxygenases, triggers ferroptosis. These enzymes catalyze the reaction between PUFA, susceptible to lipid peroxidation because of double bonds in their hydrocarbon chains, and ROS, converting them into chemically active toxic lipid peroxides. Furthermore, lipoxygenase activity and lipid peroxide accumulation also depend on the availability of intracellular iron, which acts as an essential cofactor for these reactions [98, 99]. Ferroptosis is regulated by several metabolic and molecular pathways: maintenance of redox homeostasis, concentration and accumulation of iron in cells and tissues, mitochondrial activity, and finally, metabolism of amino acids, lipids, and sugars. A crucial factor in the regulation of intracellular iron is dependent on phosphorylase kinase gamma 2 (PHKG2), which plays a role in modulating iron metabolism, influencing its availability in the cytoplasm. Once the intracellular iron pool increases, an environment favorable to lipid peroxidation is generated, pushing the cell toward ferroptosis. Ferroptosis is strongly related to tumor biology and has recently been identified as a target to prevent cancer development. On one side, ferroptosis seems to be an innate mechanism of tumor suppression [100, 101, 102]. Ferroptosis induction not only inhibits tumor development but can also boost immunotherapy responses and overcome resistance to current cancer treatments [102]. The damage induced by IR can be indirect, caused by water radiolysis and ROS accumulation. Therefore, there is a strong link between RT and ferroptosis; understanding the link between these two elements could open new avenues in the field of radiobiology and radiation oncology [103]. Ferroptosis can act in synergy with radiation to increase ROS production, compromising the antioxidant system and inhibiting tumor growth. ROS accumulation contributes to the decrease of GSH and activates genes related to DNA damage in tumor cells. Lipid peroxidation, a defining feature of ferroptosis, is triggered by RT via at least two parallel pathways. The generation of ROS by radiation encourages lipid peroxidation, which can strip PUFAs of their electrons to create fatty acid radicals. These are unstable carbon-centered radicals that react quickly with molecular oxygen to produce lipid peroxide radicals (PUFA-OO•). These radicals can then extract H• from other molecules through Fenton reactions, resulting in the formation of lipid hydroperoxides (PUFA-OOH). Furthermore, ACSL4, which is essential for mediating the manufacture of PUFA-PL, a class of lipids that are especially vulnerable to peroxidation, is expressed more when exposed to radiation [98]. In addition, ferroptosis can increase radiation sensitivity through iron overload, disruption of the antioxidant system, and lipid peroxidation [104]. After radiation exposure, iron increases in organs due to heme degradation in local tissues. Iron then accumulates and induces nonenzymatic phospholipid peroxidation through three different pathways:
It causes water radiolysis and the generation of products such as hydroxyl radicals, hydrogen peroxide, and hydrated electrons. Subsequently, hydroxyl radicals can generate lipoxide radicals and phospholipid hydroperoxides.
Ionizing radiation causes hemorrhage-dependent iron accumulation and an increase in phospholipid hydroperoxides [105].
Radiation-induced iron-dependent Fenton chain reaction with hydrogen peroxide, which further increases the level of hydroxyl radicals. All these processes generate nonenzymatic phospholipid peroxidation products that trigger ferroptosis [106].
The elevated expression of specific markers for lipid peroxidation, including MDA, 4-HNE, and prostaglandin-endoperoxide synthase 2, indicates the lipid peroxidation observed in tumor cell lines subjected to IR. This confirms the inference between radiotherapy and ferroptosis. Furthermore, mitochondria typical of ferroptosis show morphological changes in irradiated cells; these morphological features are strongly influenced by the dose of radiation administered [107]. To maintain redox balance and prevent ferroptosis, cells use the x_c system, which consists of two main subunits: a catalytic subunit, SLC7A11, and a regulatory subunit, SLC3A2, connected by disulfide bonds. These subunits work synergistically to ensure the import of extracellular cystine and the export of intracellular glutamate [108]. After importing cystine, it is converted to cysteine, an essential precursor to produce GSH. GSH is used by cells to prevent ferroptosis, as it has an antioxidant power that neutralizes free radicals and acts as a cofactor for the GPX4 enzyme, which is essential for reducing the peroxidation of phospholipids [109]. According to several studies, it was demonstrated that ionizing radiation reduces the levels of SLC7A11, GSH, and GPX4 in cells, compromising the cellular antioxidant protection system and promoting the lipid peroxides accumulation, thus inducing ferroptosis [106, 107]. Moreover, ROS generated after irradiation triggers ATM expression, which inhibits SLC7A11 and ACSL4, increasing fatal lipid peroxidation and then inducing ferroptosis. It has also been demonstrated that ACSL4 abrogation prevents ionizing radiation-induced ferroptosis, hence increasing radioresistance [98]. RT causes a significant increase in iron levels within target tissues [110]. Since iron serves as a catalyst for phospholipid peroxidation and the start of ferroptosis, IR significantly raises iron levels in target tissues. As a result, iron metabolism promotes ferroptosis and is essential to the body’s reaction to IR. Transmembrane protein FPN1 is responsible for moving iron from the cell to the external environment. To preserve the tissue redox equilibrium, it has been demonstrated that IR damage can result in a localized upregulation of FPN1 expression in response to iron buildup [111]. Specifically, rats treated with IR showed a substantial rise in FPN1 in their livers following a 25 Gy X-ray treatment [112]. Another study reported that 4 Gy of radiation increases FPN1 expression in the bone marrow of irradiated mice [106]. Ionizing radiation has been shown to have the effect of increasing serum iron. Indeed, gamma radiation has been shown to cause an increase in serum iron levels in irradiated mice [113]. The increase of iron could be related to the oxidation of ferrous ions present in the blood by the radiolysis products of water caused by ionizing radiation [114]. Furthermore, upon IR, an increase of regulatory proteins was observed, including transferrin, which transports iron, transferrin receptor, and ferritin. In addition, by silencing transferrin, decreased radiation-induced cell death [115, 116].
4. RT-induced non-lethal processes: Mitotic catastrophe and cell senescence
4.1 Mitotic catastrophe
Mitotic catastrophe (MC) is an innate tumor suppressive mechanism that blocks cells from surviving. It is considered as incomplete mitosis, DNA damage, and checkpoint errors [117]. MC morphological aspects include multiple centrosomes, misaligned chromosomes, abnormal mitotic spindles, micronuclei, and irregular nuclei [118]. This mechanism induces the formation of polyploid and aneuploid cells, which experience a variety of cellular stressors, such as elevated levels of ROS, and show increased sensitivity to treatments [119]. This mechanism is induced by genome damage and composed of mitotic death, interphase death, aberrant cell division, and genomic instability; it occurs as a mitotic arrest and non-immunogenic apoptosis dependent on Bax and Bak proteins [120]; instead of being a distinct type of PCD, mitotic catastrophe is a phase that comes before other forms of cell death. MC can block cell proliferation and induce cell death [118]. More appropriately, MC is caused by chemical or physical stressors that result in aberrant chromosome segregation. Both endogenous and external factors can result in mitotic abnormalities. Specifically, it is considered one of the key mechanisms for RT, chemotherapy drugs like doxorubicin, camptothecin, and paclitaxel, as well as other treatments with antitumor effects [119]. High levels of replicative stress and mitotic stress leading to abnormal ploidy are examples of endogenous sources. Cells that survive mitosis with chromosomal segregation abnormalities may initiate an inflammatory response by recognizing cytosolic DNA or RNA via cGAS or mitochondrial antiviral signaling [120]. Mechanistically, tumor cells are particularly vulnerable to mitotic aberrations because of their genetic instability, so they are vulnerable to the induction of MC. RT-induced DNA damage causes tumor cells, leading to MC. The G2/M checkpoint is inactivated, and consequently, the CDK1-cyclin A complex is not blocked, thus promoting mitosis. Furthermore, RT leads to hyper-amplification of the centrosome resulting from the inactivation of p53 and consequently also of p21, which activates the CDK2-cyclin E/A complex [3]. Bipolar mitotic spindles during mitosis can result from hyper-amplification of the centrosome. The formation of bipolar mitotic spindles leads to abnormal chromosome segregation, with the formation of giant cells with abnormal nuclear morphology and multiple nuclei ensuring MC. It has been shown that following treatment with BI2536, a compound with mitosis-regulating activity that induces MC, making oral cancer cells
4.2 Cellular senescence
Cellular senescence is considered an irreversible arrest of the cell cycle [123]. Various stressors, such as DNA damage, oncogene activation or mutations linked to cancer, alterations in mitochondria, reactive metabolites, hyperoxia or hypoxia, proteotoxic stress, and extracellular signals, infections, leading to cell deformation, can trigger cell cycle arrest [124]. This process is characterized by increased senescence-associated β-galactosidase activity and senescence-associated secretory phenotype (SASP), and activation of cell cycle inhibitory pathways like p53/p21CIP, p16INK4A/pRB pathways, as well as the DNA damage response signaling [125]. This irreversible cell cycle arrest is typically triggered by oncogene activation or therapeutic interventions such as chemotherapy and RT. Proinflammatory cytokines, chemokines, growth factors, and extracellular matrix components or metalloproteases are among the soluble elements that make up the full SASP profile [126]. Senolytic drugs can be employed as adjuvant therapy; indeed, it has been observed that dasatinib with quercetin or ABT263 in conjunction with RT can considerably increase the
5. Conclusions
In summary, the intricate interplay between RT and cellular mechanisms highlights the complexity of its biological effects and therapeutic potential. RT remains a cornerstone of oncology, targeting DNA and other cellular components to induce different types of cell death. In this chapter, some types of RT-induced PCD were discussed, including apoptosis, necroptosis, pyroptosis, and ferroptosis, as well as non-lethal processes such as senescence and mitotic catastrophe. Furthermore, it has been reported that some of these mechanisms, although distinct, often converge on shared molecular pathways, underscoring the need for deeper understanding to improve treatment outcomes. RT-induced processes such as mitotic catastrophe and cellular senescence further underscore its role in halting tumor progression. Despite its efficacy, challenges such as radioresistance and collateral damage to healthy tissues persist. Advances in targeting the tumor microenvironment, harnessing immune responses, and modulating non-lethal processes are paving the way for improved strategies. New insights into ferroptosis and pyroptosis as radiosensitization pathways show promise in overcoming limitations. Ultimately, this chapter highlights the importance of integrating molecular insights with clinical practice, fostering innovation to optimize the therapeutic impact of RT while minimizing adverse effects, thus advancing the frontiers of cancer treatment.
Acknowledgments
The authors thank the National Plan for NRRP Complementary Investments (PNC, established with the decree-law 6 May 2021, n. 59, converted by law n. 101 of 2021) in the call for the funding of research initiatives for technologies and innovative trajectories in the health and care sectors (Directorial Decree n. 931 of 06-06-2022) - project n. PNC0000003 - AdvaNced Technologies for Human-centrEd Medicine (project acronym: ANTHEM). In addition, this work thanks MUR-PRIN/PNRR2022:P2022F3YRF; PNRR-MAD-2022-12376723; MNESYS “A multiscale integrated approach to the study of the nervous system in health and disease”—NextGenerationEU—CUP: B83C22004960002. The authors thank NABUCCO - NUovi fArmaci e Biomarkers di risposta e resistenza farmaCologica nel Cancro del colon rettO Prog n. F/260018/03/X51 - CUP: B29J24001270005. Epi- MET - Funzionalizzazione delle aberrazioni (epi)genomiche nei tumori metastatici Fondo Crescita Sostenibile - Accordi per l’Innovazione D.M. 31.12.2021, D.D. 18.03.2022 n. 34; n. project F/310034/03/X56 (VANVITELLI) CUP: B29J24000550005.
Abbreviations and acronyms
radiotherapy | |
ionizing radiation | |
double-strand breaks | |
non-homologous end joining | |
homologous recombination | |
linear energy transfer | |
reactive oxygen species | |
reactive nitrogen species | |
damage-associated molecular patterns | |
tumor microenvironment | |
programmed cell death | |
immunogenic cell death | |
gasdermin | |
fas-associated death domain | |
tumor necrosis factor receptor-associated death domain | |
tumor necrosis factor | |
TNF-related apoptosis-inducing ligand receptor | |
gasdermin D | |
mixed lineage kinase domain-like protein | |
Z-DNA binding protein 1 | |
cyclic GMP-AMP synthase | |
stimulator of interferon genes | |
nuclear factor Kappa-light-chain-enhancer of activated B cells | |
ataxia-telangiectasia mutated | |
glutathione peroxidase 4 | |
glutathione | |
polyunsaturated fatty acids | |
acyl-CoA synthetase long-chain family member 4 |
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