Radiotherapy is an effective strategy for the treatment of many kinds of cancer to kill or control the malignant tumor cells. However, its benefits have been limited due to radiation resistance. It is imperative to identify new targets and develop cytotoxic drugs to increase radiosensitivity. Radiosensitization has traditionally been performed with agents known to induce apoptosis. However, recent studies demonstrate autophagy induced by radiation also plays an important role in cancer cell fate decision, particularly in solid tumors (1). Autophagy is an evolutionarily conserved catabolic process that delivers cellular constituents, including damaged or superfluous organelles and long-lived proteins to lysosomes for degradation and recycling, thereby regulating cellular homeostasis. The key steps of autophagy include initiation, phagophore nucleation and elongation, fusion and degradation. Autophagy initiation begins with the membrane distention from either the ER or Golgi complex, followed by formation of autophagosome, a cup shaped double-membrane structure which sequesters cellular components. Subsequently, autophagosome fuses with the lysosome to form an autolysosome which is the site for degradation of the sequestered cargo by the action of the lysosomal hydrolytic enzymes (2).

More than 40 autophagy-related proteins have been identified by large-scale genetic screening in yeast and various mammalian species (3). The core pathway of mammalian autophagy comprises at least four molecular components, including: i) ULK complex (comprising ULK1, ATG13, ATG101 and FIP200), and plays a key role in the induction of autophagy. Autophagy inducers cause dephosphorylation of ULK1; the ULK1 complex then dissociates from the mTORC1 complex, which leads to increasing activity of the BECN1 complex through phosphorylation of AMBRA1 and BECN1 (Beclin 1, autophagy related); ii) core PtdIns3K complex (comprising PIK3C3, PIK3R4/Vps15 and BECN1); BECN1 is a component of core PtdIns3K complex, which participates in phagophore nucleation and elongation. Phosphorylation BECN1 recruits ATG14, AMBRA1 or UVRAG to the PtdIns3K complexes to promote autophagy (4); iii) ATG9 and VMP1, two transmembrane proteins, are critical in leading to autophagosome formation. The biogenesis of the phagophore is thought to initiate at the phagophore assembly site (PAS). Most of the ATG proteins transiently reside at the PAS, although the ultrastructure of this site and the interactions among the ATG proteins during phagophore formation are not known (5). ULK1 complex assembles at the PAS where autophagy is initiated and recruits ATG9-vesicles in order to initiate the formation of autophagosomes (6). VMP1 induced by autophagy stimulus interacts with the BECN1 BH3 domain partitioning BECN1 to the autophagic pathway. This interaction promotes the recruitment of the autophagy-specific PtdIns3K complex to the PAS and activates the PtdIns3K complex, which generates the PtdIns3p. PtdIns3p is necessary to recruit the rest of the ATG machinery, leading to autophagosome formation (7); iv) ATG12 and ATG 8/LC3, are two ubiquitin-like protein conjugation systems essential for autophagosome formation. Ubiquitin-like molecule ATG12 is activated and transferred to the E2-like conjugating enzyme ATG10 by E1-like enzyme ATG7, ultimately attached to ATG5 4, 6, 7. In addition, ATG7 and the E2-like enzyme ATG3 promote the LC3 (ATG8 in yeast) is conjugated to the lipid phosphatidylethanolamine (8). Elongation of the phagophore membrane is dependent on the ATG12 and LC3 conjugation systems (9). Most of these core autophagy pathway components are directly controlled by cellular stress signals (10,11).

Autophagy induced by radiation play bi-directional effects in cell fate decision whether cells survive or die depends on severity and duration of this phenomenon (12). When the stress is mild, autophagy can degrade and recycle damaged or unwanted cellular constituents in autophagolysosomal vesicles to provide additional energy supply during stress, which has an essential effect in quality control of organelles and cellular adaptation to stress (13). However, several reports demonstrate that various drugs in combination with radiation promote autophagic cell death rather than apoptosis significantly contributing to the antitumor effects of radiosensitizing treatment or radiotherapy in glioma (14). Hence, autophagy induced by radiation also functions as a pro-death mechanism. Besides dual activity of autophagy on tumor cell fate in vitro, recently in vivo studies demonstrate that irradiation-induced autophagy exerts a crucial activity on tumor clearance by the immune system. Autophagy induced by radiation contributes to the release of cell death-associated danger signals ATP and HMGB1 that trigger antitumor host immune responses. Furthermore, autophagy inhibition reduces radioresponses in vivo due to deficient immunogenic signaling (15,16).

The initiation signal of autophagy induced by radiation have not been completely elucidated, although biochemical analysis performed during the last few years has identified several proteins in DNA damage repair signaling pathway or oxidative stress signaling pathway participated in modulation of autophagy. Clarifying crosstalk between autophagy and intracellular radiation response is beneficial to provide novel targets for exploiting novel radiosensitizers to enhance the effects of anticancer therapies for cancer patient.

As previously indicated ER is another target of radiation, radiation initially induces ROS production, which would activate ER membrane sensors of ER stress in IEC-6 cells and breast cancer MDA-MB-231 and MCF-7 cells (76,77). ER stress induced by radiation triggers signal transduction pathways, known as unfolded protein response (UPR) also through the induction of DNA damage (78,79). ER stress and UPR related genes are required for stress induced autophagy. The last few years have evidenced that the ER stress and autophagy processes are closely related as some of the signaling routes activated during the ER stress response are involved in stimulating autophagy.

Although the main signaling pathway of ER stress that is activated following irradiation is still a debatable one; some recent evidence suggests that PERK-eIF2a and/or IRE1a may serve as the main executing pathways of ER stress in irradiation scenarios (80–82). A link between autophagy and the ER stress has been further substantiated by the PERK-eIF2α pathway which is essential for autophagy induction after ER stress. PERK phosphorylates the eukaryotic initiation factor 2α (eIF2α) on residue serine 51, which then initiates a cascade of events that decreases the overload of misfolded proteins, thereby alleviating ER stress (83). eIF2α phosphorylation stimulates the selective translation of the ATF4 transcription factor (although general translation is shut off) and CHOP (a transcription factor induced by ATF4), which were shown to transcriptionally regulate more than a dozen ATG genes. These genes are necessary for sustained autophagy (84). PERK activated by oxidative stress can also directly inhibit mTOR or indirectly inhibits mTOR via activation of PARP1, which can induce autophagy (85). In addition, IRE1 was activated in response to a variety of cellular stressors after exposure to radiation. In mammalian cells, IRE1-JNK pathway was required for autophagy activation after ER stress (86,87). Activation of IRE1 and JNK causes Bcl-2 phosphorylation, dissociation of BECN1, activation of the PI3K complex and induces autophagy. Moreover, dysregulated autophagy may also trigger the IRE1 activity with concomitant activation of UPR, thereby dampening excessive autophagy triggered via the PERK/eIF2α pathway and pointing to a plausible feedback mechanism in the control of UPR signaling (88).

ROS is a family of highly reactive molecules which includes free oxygen radicals, like superoxide anion (O₂^•−), hydroxyl radical (OH^•), and non-radical oxygen derivatives, like the stable hydrogen peroxide (H₂O₂). ROS are unstable molecules which have the capacity to readily convert to many different viable and active forms. The superoxide radicals react to form other ROS, namely, hydrogen peroxides and hydroxyl radicals, and interconvert with RNS, which generate effects similar to ROS (89). Radiation exposure has been intimately linked to increased reactive ROS production and persistent oxidative stress in cells. Accumulating data implicate accumulation of ROS from radiolysis of water molecules, damage mitochondria and ER regulation of autophagy in oxidative stress response (66,90). Although multiple studies reported that accumulation of ROS regulated autophagy by pathways such as: i) oxidization of ATG4 leading to accumulation of autophagosomes; ii) activation of the AMPK signaling cascade inducing the initiation of autophagy through the ULK1 complex; iii) disruption of BECN1-BCL-2 interaction leading to the initiation of autophagy; or iv) alteration of mitochondria homeostasis leading to mitophagy activation (75). Furthermore, reactive oxygen/nitrogen species (ROS/RNS) generated in the context of radiation exposure are essential activators of cytoplasmic signaling cascades such as p38 MAPK, JNK, HIF-1α, which play essential roles in the regulation of autophagy (91).

p38 MAPK and JNK are two of the major MAPK subfamilies, which are serine/threonine kinases that mediate responses to various extracellular stimuli. Once activated in response to various stresses, p38 MAPK and JNK lead to phosphorylation-dependent activation of other kinases and transcription factors and play a crucial role in the modulation of autophagy (92). It has been previously reported that disruption of ROS clearance activates the p38 MAPK pathway which acts as a key regulator in ROS-mediated autophagy (93). The activation of p38 MAPK was in part responsible for the downregulation of phosphorylated mTOR and the subsequent activation of autophagy (94). p38 MAPK depletion is also found to regulate the trafficking of ATG9, a multi-spanning membrane protein that is essential for autophagy (95). Fractionated radiation activated p38 MAPK which is involved in radiation-induced stabilization of HIF-1α which play an important role in switching autophagy. Consistently, inhibition of p38 MAPK also effectively attenuated radiation-induced stabilization of HIF-1α (96).

After exposure to radiation, the expression of HIF-1α increased, then HIF-1α translocated into the nucleus. In the nucleus, HIF-1α bound to the hypoxia response element in its target promoter and promoted the transcription of its target (97). HIF-1α is a subunit of HIF1 which consists of a regulatory HIF-1α subunit and the constitutively expressed HIF-1β subunit, both are members of the basic helix-loop-helix and PER-ARNT-SIM families of transcription factors (98). Pervious opinion considered that while HIF-1β is constitutively expressed, HIF-1α is an oxygen sensitive subunit and its expression is induced under hypoxic conditions (99). Accumulating evidence has recently revealed that HIF-1α is activated in cancer cells not only under hypoxic conditions, but also in the presence of oxygen when the following conditions are satisfied. In addition to post-translational mechanisms, regulation at the level of transcription and translation initiation is also important for the activation of HIF-1α under normoxic conditions (100). Koshikawa et al reported that ROS generated in mitochondria upregulated the transcription of the HIF-1α gene via the PI3K-Akt/PKC/ HDAC pathway, leading to the accumulation and activation of HIF-1α in tumor cells (101). ROS upregulates HIF-1α transcription by activating non-hypoxic factors in a redox-sensitive manner (102). HIF-1α induces autophagy by a mechanism involving the upregulated expression of its target genes BNIP3 and its homologue NIX and the dissociation of the BECN1-BCL2 complex, leading to release of BECN1, which is capable of triggering autophagy (103,104).

Radiation activates the cell surface glycohydrolases which play a crucial role in the production of ceramide at the plasma membrane. Ceramide is localized within lipid rafts in the plasma membrane, which are specialized membrane microdomains that regulate various signaling pathways (105). Ceramide is also a central molecule of sphingolipid metabolism and involved in the regulation of autophagy at various levels (106,107). Ceramide downregulates the expression of amino acid and nutrient transporters in the context of starvation, a state that induces survival autophagy by reducing mTOR signaling pathway or activating AMPK (108,109). Further evidence indicated that ceramide increases BECN1 expression by activating JNK kinase, which in turn activates c-Jun, a known transcription factor for BECN1 expression (110). Ceramide accumulation also can induce ER stress, mTOR inhibition via TRB3 (tribbles homologue 3), which triggered lethal autophagy (111–113). Moreover, ceramide can be generated by neutral sphingomyelinase in mitochondria, which is a late event upon the cell irradiation following the early phase of ceramide accumulation at the cell plasma membrane (106). Ceramide accumulation in the mitochondria can lead to ceramide stress-induced mitochondrial fragmentation, and decrease in ATP production (114). These important mitochondrial parameter changes can induce mitophagy (115).

Cellular response to radiation-induced stress is generally mediated through the production of the second messenger, in-flux of Ca²⁺ (90,116,117). These radiation-induced changes in Ca²⁺ concentration can be elevations, oscillations or single transient changes within minutes to days after exposure to radiation. Radiation regulates intracellular Ca²⁺ concentration through several different pathways. Firstly, radiation can induce the accumulation of ceramide, which can regulate influx of extracellular Ca²⁺ at plasma membrane channel level (106). Secondly, radiation can activate PLC, the main enzyme localized at the plasma membrane and producing IP3 and consequently inducing Ca²⁺ release from ER (90,118). Several studies in mammalian cells and yeast models have demonstrated a potential role for mitochondrial parameters regulated by Ca²⁺ signals in modulating and/or triggering mitophagy (115,119). Ca²⁺ release events from the ER, the major intracellular Ca²⁺ -storage organelle that have an immediate effect on the physiological function of mitochondria and lysosomes (120). Ca²⁺ released from the ER activates members of the PKC family. In mammals, PKCθ, a member of the PKC family, is a novel factor that mediates Ca²⁺-dependent induction of autophagy in response to ER stress. Increased Ca²⁺ concentrations induce PKCθ phosporylation and its localization to cytosolic LC3 puncta (121). In addition, the CAMKK2-PRKAA-mTOR pathway is an important signaling pathway for Ca²⁺ -induced autophagy, which is also a cascade with established roles in multiple cellular processes including ribosome biogenesis and transcription in addition to cell motility and metabolism (119). In addition elevation of intracellular Ca²⁺ can activate AMPK, which plays a role in Ca²⁺ -mediated signal transduction pathways (122,123). An overview of the major intracellular autophagic triggers induced by radiation is shown in Fig. 2.

Various preclinical models revealed that autophagy is activated in irradiated tumor cells and that ATG expression patterns are upregulated following irradiation. Inhibition of autophagy genes such as ATG5 alone or a mixture of BECN1, ATG3, ATG4b, and ATG5 together leads to the strongest, and significant sensitizing effect found in radiation (124). Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of radiation therapy. Currently, multiple clinical trials have been initiated combined with autophagy inhibitors, such as hydroxy chloroquine and chloroquine, which associated with increased sensitivity to radiation (125,126). However, the role of autophagy in the resistance of cancer cells to radiation therapy remains controversial. One logical outcome of this argument is that chloroquine and hydroxychloroquine are unlikely to be appropriate drugs for this purpose, because of the fact that patients with malaria are able to endure treatment with these drugs for years, which suggests that the doses of chloroquine and hydroxychloroquine that are used effectively for malaria treatment may not actually be acting to inhibit autophagy. Another logical outcome is that various studies indicated that the sensitivity of tumor cells to radiation could be enhanced by co-treatment with rapamycin, an autophagy promoter, in various kinds of tumor cells (44,50,127).

Radiotherapy is one of the best therapeutic choices for cancer treatment (26). Radiation-induced autophagy in cancer cell lines is related to cell death mechanism and autophagy-inducing agents may act especially as radio-sensitizers or radio-resistance (128). Recent studies show that radiation can damage DNA, plasma membrane and cellular organelle such as mitochondria and ER and activate several radiation responsive pathways. During periods of cellular stress, the convergence of these pathways promotes autophagy (129). To clarify cross talk between autophagy and intracellular radiation response, we focused on the proteins and molecules that link the intracellular radiation response with autophagy. A part of these proteins of radiation responsive pathway were verified to participate in the modulation of radiation-induced autophagy. However, others have not been confirmed in the regulation of autophagy. Deep experimental research is needed to further investigate the understanding of the pathways regulating autophagy induced by radiation, which will likely offer new targets for radiotherapy treatment of certain aggressive forms of tumors. Additionally, it would indicate how to regulate the autophagic process or its related pathways that protect cancer cells from undergoing autophagy and may be beneficial to exploit radio-sensitizers. There may also be a therapeutic role for autophagy in protecting normal tissues from lethal radiation exposures. In addition, a recent study showed that autophagy may yield the opposite returns from tumor immunity perspective, irradiation-induced autophagy exerts crucial activity on tumor clearance by the immune system needing further investigation. Clearly, more work is needed to clarify crosstalk between autophagy and intracellular radiation response.