Introduction

The three Nobel fundamental discoveries made by Roentgen (x-rays in December 1895), Becquerel (natural radioactivity in March 1896), and Curie (radium 1898) in a short time span astonishes the world scientific community, making everyone into a scientific frenzy. But the era of doom was started soon, when Edison, Tesla, and Gubbe reported radiation-induced (RI) injury in their eyes and skin on March 1896 [1]. Despite the threat, technology was rapidly spreading from bench to bedside and barely 6 months after Roentgen’s discovery, radiation was used for the first time in the treatment of gastric cancer and basal-cell carcinoma patients in France, America, and Sweden [2]. In 1902, the first case of RI cancer was reported in the hand of a radiologist [3]. Later, arm of a lab assistant named Clarence Dally was amputated due to RI blistering and subsequently, he died in 1904 [4]. Soon, Curie also reported RI skin erythema and ulceration, and later, she and her daughter Irene both died from RI leukemia. Afterward, Roentgen’s wife also joined the list of persons who died, believed to be a consequence of radiation [5]. The fundamental problem in RI damage was due to many reasons, i.e., 1. the lack of knowledge about radiation, 2. instrumentation to measure radiation dose, and 3. undefined unit in those days.

The term “radioprotection” was first used by Dale in 1942 on his articles, arguing that x-ray-induced enzymes/protein inactivity is responsible for RI cell death [6], though later in the 1960s, the concept was superseded by “DNA damage.” Subsequently, Patt et al. showed the effect of cysteine in protection against x-rays [7]. Indeed, the first breakthrough report came from Gray et al. while working at Hammersmith Hospital, London, showed that the RI damage could be minimized by 2–3 times in anoxic condition [8]. In the 1960s, many phosphorothioates compounds under the WR series was synthesized by Akerfeldt at the Walter Reed Army Institute of Research; it was another revolutionary mark in radioprotection [9]. Then from the 1970s onwards, plenty of reports came from multiple investigators dealing with radioprotectors, but due to multiple reasons, only amifostine got a nod from the FDA in 1995 to use as a co-therapy agent in RT for lowering normal tissue damage (fda.gov).

With the demise of cold war hostility in the 1990s, burgeoning incidents of cancer projected to increase by 75 % by 2030 from 2008 figure or reach to 22.2 million [10] in absolute terms as the baby boomer generation of 1970s (record population growth due to improved medical condition) become older. Indeed, the rapid increase in cancer incidence is mainly due to higher life expectancy [11] since the aged are more prone to cancer. Though in reality, the cancer incidents are declining (declined by 3.1 % year−1 in men while stable in female between 2009 and 2012 [12]) due to better hygiene, immunization, etc. [11]. The only bad side of the ongoing success in reduction of cancers may be hampered by factors like obesity, stress, and diabetes, etc. or so-called the diseases of civilization. Today, radiation is the centerpiece in cancer treatment; as of 2014, 1.1 million out of 1.67 million total cancer patients received radiotherapy (RT) in the USA alone [13]. Despite extensive uses of RT, the majority of patients only get modest benefits due to collateral damage to normal tissues. In fact, just increasing the cumulative dose by 10–20 % makes the difference between the incomplete and complete eradication of some tumors [14]. Further variables like hypoxia, radio-resistance, faulty repair system, etc. consistently escalate the radiation dose. The dictum primum non nocere or “first, do no harm” is the centerpiece in medical profession and means that physicians are duty-bound to access the benefits over the likely harm and try to overcome them later. Hence, aiming for better patient quality of life, radioprotection had evolved from obstacle kilovoltage (1900–1940) to megavoltage (1946–1996) for eliminating severe skin damage, especially when targeting deeper tumor and further adopted much safer computer-assisted (1996–ongoing) 3D conformal RT [15]. In fact, there are a couple of new promising technologies such as stereotaxic ablative body radiotherapy, fine intensity-modulated RT, volumetric modulated arc therapy, dynamic conformal arc therapy, multimodal image-guided RT, concentrating the radiation in tumor via proton particles or carbon ion therapy (Bragg’s peak), and recently introduced four-dimensional RT (where respiration is also considered) are rapidly diffusing though expensive over standard or conventional therapy [16]. But due to the ill-defined boundary and complex anatomical location, normal tissue still faced the similar fate as tumor under RT, forcing oncologists to limit the exposure dose; to achieve it, there is a need to either selectively increase the tolerance of normal tissues or radiosensitivity of tumor or both (Fig. 1). Last but not least, need of radioprotectors is also becoming apparent as a radiation countermeasure agent in space science, nuclear energy generation, medical diagnosis/imaging, nuclear fallout, and nuclear terrorism.

Fig. 1
figure 1

Present and ideal radioprotector and radiosensitizer for radiotherapy

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Radiosensitization and radioprotection

Here, we are describing promising druggable targets, which can be selectively targeted for normal tissue radioprotection or enhancing the tumor radiosensitivity or both.

Free radical scavengers

Pioneer studies of Biaglow and Bump demonstrated that the irradiation results in depletion of antioxidant pool predominantly glutathione (GSH) and increase in antioxidant enzymes including superoxide dismutase (SOD) following irradiation [17]. SOD2 or Mn-SOD is localized into mitochondrial membrane due to the presence of mitochondrial targeted peptide while SOD1 and SOD3 are localized in the cytoplasm and extracellular spaces, respectively. Delivery of SOD1 construct with mitochondrial targeting peptide or Mn-SOD (not SOD1, SOD3, or even SOD2 without mitochondrial peptide) has shown protection against the RI damage with tumor radiosensitization [17, 18]. Delivering vector construct expressing Mn-SOD is superior over protein form due to the bigger size (222 amino acids) of the latter, making it difficult to internalize by cells. Moreover, protein form is also susceptible to ONOO− mediated nitration at tyrosine-34, which makes it nonfunctional [19]. Cancer cells under extreme oxidative stress as it diminished the expression of antioxidant enzymes including glutathione peroxidase (GPx) and catalase by epigenetic modification and mutation in promoter region as a mechanism for initiation, progression, and metastasis of cancer (Fig. 2a) [20, 24]. GPx degrades H2O2; a product of SOD generated from RI superoxide anion radical (Fig. 2a). Thus, delivering Mn-SOD to tumor-bearing organism causes accumulation of cytotoxic H2O2 in the cancer cells following irradiation, while normal cells remain unaffected due to intact GPx (Fig. 2a) [17]. Moreover, cancer cells are more sensitive to Mn-SOD therapy since they are under extreme oxidative stress due to progressive upregulation of Mn-SOD as a mechanism to acquire more aggressive and invasive phenotype/enhanced malignancy (Fig. 2a) [20]. However, it does not mean that inducing ROS (by Mn-SOD) make cancer cells more malignant, as momentary ROS induction is harmful and have killing effects while under persistent ROS, cancer cells survive due to adaptive resistant by upregulating antioxidant system [25]. Delivering Mn-SOD under chemo-radiotherapeutic regime via plasmid-liposome in non-small-cell lung cancer (NSCLC) patients has been shown to reduce RI esophageal toxicity in a phase-I clinical trial [26] and now the phase-II trial is under progress (NCT00618917, clinicaltrials.gov). Therefore expression of SOD, GPx, and catalase are probably useful markers for selecting the patients eligible for co-therapy (Mn-SOD with RT).

Fig. 2
figure 2

Reactive oxygen species (ROS) in biological system. a Detoxification of ROS in normal cells; enforcement of tumorigenesis by deregulating superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in cells (adapted and modified with permission from Elsevier B.V.©2011 [20]). b Role of antioxidant in genomic instability/cancer, a proportional relation between antioxidant supplements and cancer incidents or DNA damage is mostly observed in smokers [21] while an inverse relation was observed in non or passive smokers [22, 23] so on the basis of endogenous ROS level [passive (low-level ROS) vs. non-passive smokers (high-level ROS)] we are proposing that at low level of ROS, antioxidant act as anti-cancer agents while after certain thresholds it behaves as procancer agents, however role of antioxidants at excessive level of ROS such as in radiotherapy is yet to clear

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Cerium oxide (CeO2) nanoparticles have shown to protect against the RI lung and gastrointestinal epithelium damage by scavenging free radicals (O2−, •OH) and upregulation of SOD [27]. The CeO2 nanoparticles have shown to mimetic catalase and SOD, interestingly the catalase activity lost at acidic pH, resulting in accumulation of H2O2 in the presence of superoxide radicals [28]. Cancer thrives in an acidic environment via lactic acid production; hence, using CeO2 nanoparticles could radiosensitize the tumor via accumulation of H2O2 while normal cells remain unaffected due to intact catalase activity. Additionally, modifying the charge of CeO2 nanoparticles to negative have shown to be preferentially internalized by cancer cells over normal cells [29], which may further potentiate the radiosensitization effect provided that charge modification does not influence the mimetic property.

Nitroxides and its derivatives are recycling free radical scavengers and were shown to reduce the RI damage in different model system by ROS scavenging [30]. Selective targeting of GS-nitroxide (JP4-039) to mitochondria has shown to protect oral mucosa from RI damage in Fancd2−/− mice without affecting tumor radiosensitivity [31]. Tempol is another molecule that has been shown to protect mammalian system against RI cytotoxicity in in vitro and in vivo (without tumor protection) [32]. Lack of tumor protection by Tempol is due to conversion of Tempol from radioprotective form to non-radioprotective form Tempol-H by bioreduction in hypoxic area. Topical application of Tempol in the scalp has shown to reduce the RT-induced alopecia in brain metastatic cancer patients in a phase I trial [32]. Erker et al. had demonstrated the chemopreventive activity of Tempol in tumor-bearing mice; however, from the study, more questions emerged than answers, like was the effect was due to Tempol or Tempol-H, what was the role of hypoxia, etc. [33]. But still, nitroxide derivatives including Tempol are promising in RT as radiosensitizers, besides radioprotectors, due to its SOD mimetic property which further amplified at acidic pH [20].

Melatonin, a hormone secreted by the pineal gland has shown to protect against RI damage in different biological systems by free radical scavenging and enhancing SOD and GPx activity [34, 35]. However, its efficacy in RT remains disputable. Administration of melatonin (20 mg day−1) in glioblastomas patients undergoing RT has shown a survival enhancement by 1 year in 6 (14) patients in comparison to only 1 (14) in RT arm [36]. However, in a subsequent phase II clinical trial involving brain metastases patients, melatonin (20 mg day−1) failed to increase the patient survival [37]. Indeed, the clinical trials are ongoing to test the efficacy of melatonin on protection against RT-induced oral mucositis/xerostomia and fatigue in cancer patients (NCT02332928, NCT02430298).

Genistein [38] and fullerenes (hydroxylated) [39] have been shown to reduce the RI lethality in irradiated mice. Pretreatment of mice with genistein (200 mg kg body weight) has been shown to reduce the RI GI damage in mice along with enhancing the radiosensitivity of implanted tumor [38]. Silibinin, a mixture of flavonolignans present in milk thistle (Silybum marianum) seed is another promising candidate and can be used as a co-therapy agent. Silibinin has mitigated the RI lung injury and improved the survival of tumor-bearing mice without interfering with tumor radiosensitivity [40]. However, despite enough preclinical evidence, using antioxidants in RT always had a concern over the possibility of protecting tumor from cytotoxic therapeutic-free radicals, and further, may promote the mutagenesis by protecting the extensively damaged cells from apoptosis directly or indirectly by apoptosis inhibition since ROS also act as secondary messengers in apoptosis signaling. The suspicion was not unfounded as in a large (29133 subject) epidemiological study, the incidence of lung cancer was reported to increase dramatically in heavy smokers after vitamin E and β-carotene consumption [21]. In two separate investigations using transgenic mice prone to spontaneous brain cancer and mammary tumor, the fast clearing of the tumor by apoptosis when kept under antioxidant-free diets were shown [41, 42]. In another study supplementing N-acetylcysteine and vitamin E was shown to markedly increase tumor progression and consequently reduce survival in B-RAF and K-RAS-induced lung cancer mouse model [43]. Therefore, it seems that antioxidants may be useful in the reduction of cancer initiation at lower levels of oxidative stress and once the threshold is crossed, they may start behaving as procancer agents (Fig. 2b) [22]. The supplementation of β-carotene was shown to increase DNA damage in smokers while decreased in nonsmokers [23]; therefore, patient lifestyle could be taken into consideration before recommending any antioxidant supplementation, and it is also true for the general public who are taking plenty of antioxidants. In scrutiny of antioxidants one must differentiate between high and low dose of antioxidant supplementation (vital for maintenance of basic function i.e., essential vitamins and antioxidant present in food since numerous epidemiological studies have demonstrated the anti-cancer effects of diet rich in antioxidant substance). In preclinical studies, moderate/intermediate dose of antioxidants were shown to reduce the efficacy of radiation and induce the proliferation of cancer cell [44, 45]. In a clinical trial combining high dose of nutrients i.e., β-carotene, vitamin C, niacin, selenium, coenzyme Q10, and/or zinc with standard therapies (e.g., surgery, chemotherapy, radiation therapy, and hormonal therapy) in nonmetastatic breast cancer patients had been shown to shorten disease-free survival and breast cancer-specific survival in the nutrient-supplemented group than non-supplemented group [46]. In contrast to that, investigation from two separate groups in randomized trial using pentoxifylline and α-tocopherol in patients with NSCLC and melatonin in patients with brain glioblastomas has shown an improvement in survival following RT [36, 46–48]. In a subsequent study, the observed radiosensitization (brain metastasis) or enhanced patient survival with melatonin was not observed in a randomized phase II trial [37]. Moreover interpretation of the clinical results is also quite difficult since doses which limit or induce the cancer growth vary among species and tumor type [44]. Hence, clinical trials are still ongoing to evaluate the efficacy of low and high antioxidant diets in the prevention of RI toxicity (NCT02195960 and NCT00486304). Importantly, β-carotene and α-tocopherol may be given a special consideration in RT with modulating the partial pressures of oxygen (pO2) since β-carotene changes its behavior from antioxidant to prooxidant with increasing pO2 while α-tocopherol works as a strong antioxidant at higher pO2; hence, oxygen pressure-dependent behavior may be exploited in enhancing the radiotherapeutic index [49]. Nevertheless, the post-RT administration of antioxidant such as EGCG, vitamins E and C, β-carotene, etc. has been shown to help in the reduction of RI toxicity in multiple normal organs in different clinical trials [50–52].

Administration of amifostine before RT was shown to protect against the RI mucositis, acute/late xerostomia and dysphagia, and other RI symptoms in normal tissues in NSCLC, diffuse intrahepatic tumor, and bladder cancer patients without compromising the patient’s survival [53–56]. Indeed, despite the considerable body of evidence; it disappointed in a few other clinical trials [57, 58]. In fact some serious adverse effects was also noted in some preclinical and clinical studies involving RT [59, 60]. In fact, Amifostine treatment to tumor-bearing mice had shown to reduce the tumor radiosensitivity by inducing the antioxidant enzymes both in normal and tumor tissues [61]. However, it constitutively induces the antioxidant enzymes; therefore, it could be useful in minimizing the effect of occupational and high background radiation exposure [61]. The differential effects of amifostine stem from its metabolism, which allow it to concentrate rapidly in normal tissues instead in tumor, due to the requirement of being dephosphorylation (WR-1065) by alkaline phosphatase, an enzyme highly expressed in the normal cell before being incorporated. Despite being selective, it is not widely used in clinical practice due to the report of adverse side effects such as hypotension, fatigue, etc. [59, 60]; hence, investigations are still ongoing to evaluate its safety/efficacy. Thomas et al. designed a novel glutathione-based pro-drug PB-42 which works on similar mechanism as amifostine shown to increase GSH pool selectively in normal cells and abrogated cisplatin-induced nephrotoxicity, and the effect was remained unaffected by GSH synthesis inhibitor buthionine sulfoximine (BSO) [62]. Data from previous studies demonstrated that GSH depletion by BSO enhance the tumor radiosensitivity, but severely affects the normal tissues [63]. Therefore, PB-42 may have major clinical significance in RT.

One of the major challenges in RT is how to deplete glutathione selectively from tumor to increase tumor vulnerability without affecting the normal cell radiosensitivity. GSH is synthesized from l-γ-glutamyl-l-cysteine and glycine by glutathione synthetase [64]. l-γ-glutamyl-l-cysteine forms from glutamine and cysteine by γ-glutamylcysteine synthetase (target of GSH synthesis inhibitor BSO), a rate-limiting enzyme [64]. Cancer cells frequently overexpress the glycine decarboxylase (GLDC) as a mechanism to divert the glycine towards DNA synthesis, while normal cells have plenty of glycine [65–67]. Hence, we are hypothesizing that supplementing l-γ-glutamyl-l-cysteine with BSO during RT could protect the normal tissue by increasing GSH pool due to excess glycine while at the same time BSO inhibits the endogenous GSH production in both normal and cancer cells resulting in radiosensitization of cancer.

Targeting hypoxic condition

Historically, hypoxia is the softest target recognized by clinicians due to the physiological difference between hypoxic cancer and normoxic normal cells. Systemic induction of hypoxia by cardiovascular alterations, hemoglobin function manipulating (RSR-13), over-oxygen consumption, reducing the respiration rate or by overexpression of the oxygen-labile subunit of hypoxia-inducible factors (HIF) complex (HIF1/2/3α), result in the induction of pro-angiogenic cytokines, i.e., VEGF and bFGF, known to help in radioprotection. HIF is known to promote neovascularization regulated by prolyl hydroxylase domain (PHD), and it has been observed that inhibition of PHD shown to help in reducing the radiation-induced (RI) lethality by improving the epithelial integrity of the GI tract [68]. However, in RT, deoxygenation may work only in exceptional circumstances where oxygen does not modulate the radiosensitivity [69]. HIF modulation in RT is zero sum game as HIF inhibition has shown to radiosensitize the tumor [70, 71]; hence, the above-mentioned approach is not feasible in RT.

Hypoxia-activated prodrugs (HAPs) are cyclic non-toxic prodrugs which are converted into free radical species by addition of electron (one or two electrons) by reductive enzymes under hypoxic condition causing DNA damage and DNA adducts formation, while under aerobic condition, it again oxidized or converted back to parent molecule after reacting with oxygen or by SOD (an enzyme highly expressed in normal cells). Misonidazole was the first tested drug in RT despite being known to cause neuropathy and used in successive large randomized clinical trial [72]. As anticipated, these trials failed to demonstrate any major clinical outcome in RT but provided proof of principle [73]. Another breakthrough work was done by Zeman et al., using tirapazamine (SR-4233), originally screened as an herbicide in 1972, has shown to induce the selective RI cytotoxicity to hypoxic cells (near ×200), and the rest was history [74]. Tirapazamine acted as a backbone in the development of many improved HAP. Five different chemical moiety, i.e., quinones, aromatic and aliphatic N-oxides, transition metal, and nitro(hetero)cyclic group has shown to change under anaerobic condition [75]. HAPs are classified on the basis of oxygen threshold required for their activation. Class I HAPs (SN-30000, SR-4233, benzotriazine) require a higher threshold for action than class II HAPs (PR-104A, TH-302). Tirapazamine (SR-4233) has shown promising results in preclinical and early clinical studies; however, it was disappointed in phase III clinical trial in combination with RT; hence, currently, it is not in use [76]. Then, focus was shifted towards nimorazole, a less toxic version of misonidazole shown to reduce the locoregional reoccurrence rate without any toxicity to normal tissues following RT in randomized DAHANCA 5 phase III clinical trial; however, it failed to improve survival [73]. The low clinical utility of nimorazole was due to the absence of suitable biomarkers to access the tumor hypoxic condition for screening patients required for trial. In a recent retrospective study classifying patients on the basis of expression of 15 hypoxic signature genes, a statistically better locoregional tumor control and disease-specific survival in patients classified as more hypoxic, than less hypoxic patients was shown [77]. TH-302 (Threshold Pharma) is a novel prodrug of alkylating agent “bromo-isophosphoramide mustard” that showed promising results in preclinical and clinical studies. In two separate investigations, combining TH-302 and radiation with either VEGF-A, or mTOR inhibitor (to inhabit the pro-tumor HIF-mediated angiogenic response under hypoxic condition) has shown to reduce the growth of implanted human sarcomas [78] and renal cell carcinoma (RCC) [79], respectively, in mice model. In fact TH-302, with VEGF-A and radiation combination, the tumor remained dormant for 3 months after cessation of therapy [78]. In another co-treatment study, using TH-302 with radiation showed hypoxic tumor radiosensitization without causing cytotoxicity to normal tissue in rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals [80]. In a phase II clinical trial combining TH-302 with doxorubicin, a promising result was shown [81]. Hence, in the future, combination of TH-302 with RT is promising; however, the study must consider utilizing hypoxic cell markers for restricting therapy to only those who have significant numbers of hypoxic cells either it would get similar fate as nimorazole.

Furthermore, reducing pO2 via limiting the blood supply using vasoconstrictor (i.e., norepinephrine, phenylephrine, epinephrine, etc.) [82] under co-therapy (RT and HAP) regimen certainly reduced the RI toxicity in normal tissues; while tumor therapeutic response could be enhanced or remains affected, depending on tumor vascularization. Tumors under severe hypoxia have very poor vascularization; hence, further reducing oxygen in the body may not significantly alter the tumor local hypoxic environment hence oxygen enhancement ratio (OER) would remain unaffected while even if its reduced, the reduced OER in tumor further compensated by enhanced HAPs cytotoxicity. However, use of vasoconstrictor must be taken with care since association between tumor blood supply and hypoxia is yet to clear and currently guided by two conflating hypothesis steal and anti-steal effects [83]. Steal effects assumed the fully dilated tumor vessel so vasodilator caused relocating of blood from the tumor to the rest of the body while anti-steal suspected the fully constricted tumor vessel so vasodilator cause relaxation of tumor vessel and reverse the blood flow; so, the vasoconstrictor is only useful under the anti-steal mechanism. Furthermore, currently, no study has been done showing the sensitivity of normal and tumor tissues blood vessels towards vasoconstrictor or vasodilator. Further, we hypothesized that tumor vessels may lose the sensitivity towards vasoconstrictor as an evolutionary feature since they need to maximize the blood supply.

Tumor hypoxia can be considered as oxygen demand surpassing supply. Hence alternatively reducing the oxygen demand as effective as increasing the oxygen to get the same end point (hypoxia elimination) [84]. Metformin is the drug used to treat type II diabetes shown as tumor radiosensitization in multiple preclinical studies [85]. It had been shown to reduce the risk of cancer occurrence when used as a monotherapy agent [86]. However, action mechanism of metformin is not clear and multiple possible explanations are under the current discourse. One explanation is that the metformin reduces the circulating insulin which has mitogenic and prosurvival effects on cells expressing insulin receptors [85]. Another explanation is that the metformin activates the AMPK results in the suppression of mTOR, the main regulator of AKT [86]. The last explanation is the metformin protects the CD8+ tumor-infiltrating lymphocytes from apoptosis [86]. Recently, one fascinating mechanism has been proposed that metformin inhibits the complex-I activity of mitochondrial electron transport chain, and inhibition was anticipated to reduce the oxygen consumption [85]. As expected, metformin has shown to diminish the oxygen consumption in in vitro and reduces the hypoxia in tumor results in tumor growth inhibition and improved survival [85]. Hence, combining metformin with RT seems promising as many cancer patients already received metformin treatment without report of any adverse effects. Recently, few clinical trials have been started to study the synergy of metformin with chemoradiotherapy in NSCLC patients (NCT02186847, NCT02115464).

Nitric oxide (NO) is a widely studied molecule in inducing radiosensitization by modulating the blood flow in poorly vascularized tumor; however, only few reports are available dealing with radioprotection. Increasing NO level by NO donor DEA/NO or blocking the interaction between CD47 and TSP-1, has been shown to reduce the RI mortality in mice model [83, 87]. The role NO was anticipated in CD47, and TSP-1 interaction as both inhibit the NO/cGMP signaling. However, an enhancement in autophagy was found to be the reason behind radioprotection; interestingly, blocking interaction also radiosensitize the implanted melanoma or squamous lung tumors in mice [88, 89]. RRx-001 is a multifaceted anti-cancer agent, mediates biological action by epigenetic modification through ROS, RNS, etc. [83]. RRx-001 is a NO-donating compounds release NO locally in a biphasic manner due to different metabolism over classical NO agents/donors. Evidence has demonstrated that RRx-001 binds selectively with hemoglobin (at NO binding sites beta-cysteine 93) and glutathione in a rapid and irreversible manner [83]. The glutathione-RRx-001 adduct could increase the oxidative stress, but it is rapidly excreted; while RRx-001-Hemoglobin adduct remains in circulation till the destruction of RBCs. The initial burst of NO following RRx-001 administration is due to replacement of NO from hemoglobin resulting in rapid and transient local vasodilatation [83]. The local vasodilatation allows the flow of oxygenated blood resulting in enhancement of tumor radiosensitivity. The deoxygenated hemoglobin further enhances the NO content by converting the nitrite present in serum at the local site due to its nitrite reductase activity of hemoglobin under hypoxic condition; hence, compensating the missing nitrite synthetase activity of tumor under hypoxic condition [90]. Additionally, the RRx-001 binding with hemoglobin further potentiates the nitrite reductase activity of later enhancing the NO production resulting in better oxygenation and radiotherapeutic efficacy [90]. In an in vitro study, RRx-001 has been shown to augment the DMF by 1.9 times in radio-resistant hypoxic cells [83]. In the extended in vivo study, administration of 5 or 6 mg RRx-001 kg−1 body weight shown to synergistically enhance the radiosensitivity of SCCVII syngeneic tumor in mouse after local or whole body irradiation (WBI). Amazingly, RRx-001 also protected the intestinal stem cells or GI tract from lethal (10–15 Gy) WBI [83]. Hence, in view of the promising result, RRx-001 warranted further study in clinical model system.

Targeting DNA damage repair and cell cycle

Poly ADP ribose polymerase (PARP) is an attractive target which can be exploited in RT. PARP inhibitors have shown to radiosensitize the BRCA1 (breast cancer susceptibility 1) and BRCA2 mutated breast and ovarian cancers [91]. BRCA is known to play a crucial role in the repair of double-strand DNA by homologous recombination (HR) while PARP involves in the repair of single-strand DNA damage via base excision repair [92]. Hence, blocking the base excision repair pathway by PARP inhibitor results in synthetic lethality in BRCA-deficient or HR-defunct cells, due to accumulation of damage DNA [91]. In normal cell, RI DNA damage could be repaired by HR and non-homologous end joining after PARP inhibition. Interestingly, PARP inhibitor 3-aminobenzamide has also been shown to inhibit the RI irreversible loss of salivary gland fluid secretion by inhibiting the activation of the transient potential melastatin-like 2 resulting in the reduction of RI xerostomia in C57BL/6 mice [93]. Although BRCA1/2 mutation is only limited to breast and ovarian cancers but the mutation in other component of HR pathway such as HR RAD51B (in prostate cancer, acute myeloid leukemia, lipoma, colorectal cancer, non-small-cell lung cancer, pancreatic, and uterine leiomyoma [94, 95]); PTEN (in glioblastoma multiforme, prostate and endometrioid endometrial cancer [96]); RECQL4 (in osteosarcoma, prostate tumor, and basal/squamous cell skin carcinomas [95, 97]); MRE11 (in endometrial carcinomas [98]), EXO-1 (in HNPCC and prostate cancer [99]) and RAD54 and CtIP (in non-Hodgkin’s lymphoma and colon cancer [100]); Nbs1 (in melanoma, neck and head cancer, and colorectal cancer [99]); and FANCF (in leukemia and cervical cancer [96]) also makes HR dysfunctional, hence targeting PARP is a promising approach in anti-cancer therapy. Although these mutations may be rare, but if considering >200 proteins critical for the onset and regulation of HR [100] make it attractive to investigate the efficacy of PARP inhibitors in RT. PARP inhibitor has shown to sensitize the endometrial carcinomas expressing defunct MRE11 provided a proof of principle concept [98]. Importantly, defect in HR pathway can be easily detected by DNA methylation-specific microarrays and homologous recombination deficiency test. PARP is also known to act as cofactor for NF-kB; hence, blocking off major survival pathway could further add some therapeutic value [101].

Loss or perturbations of p53 function is the most common feature of human cancers resulting in loss of G1 checkpoint hence cells solely rely on G2 checkpoint regulated by Wee1 following DNA damage [102]. Evidence has shown that inhibition of Wee1 in p53-deficient tumors causes tumor radiosensitization [103]. Additionally, inhibition of Wee1 does not contribute the RI cytotoxicity into normal cells due to the presence of intact G1 blockage [103, 104]. Under chemotherapeutics regimen, Wee1 inhibitors such as PD-166285 and MK-1775 has demonstrated the potential usefulness without causing toxicity to normal cells [104, 105]. Therefore, inhibition of Wee1 is quite attractive in RT. Recently, few clinical trials have been initiated to study the synergy of MK-1775 or AZD1775 under chemoradiotherapy (RT and temozolomide or cisplatin) in glioblastoma multiforme and cervical cancer patients (NCT01849146, NCT01958658).

The key regulator of DDR is ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein kinases, have overlapping properties [106]. The ATM monitors all phases of cell cycle in response to double-strand breaks (DSBs) while ATR is responsible for the S and G2 phases of the cell cycle [106, 107]. Normal cells have functional G1 and G2 checkpoints while majority of the cancers are deficient in p53/ATM resulting in loss of G1 checkpoint and therefore slowly dependent on ATR/Chk1 led G2 checkpoint. Hence, it has been hypothesized that inhibiting ATR could result in accumulation of damaged DNA resulting in mitotic catastrophe while normal cells remain protected due to the functional G1 checkpoint; however, initial research mired over the concern of ATR essentiality. ATR disruption has shown to result in chromosomal fragmentation, tumor genesis, and early embryonic lethality [108]. ATR code for vital function; hence, patient with ATR mutation or in rarely occurred Seckel syndrome are naturally hypomorphic (partial loss of protein function). A recent study in ATR mouse model of Seckel syndrome has shown loss of ATR can be reverted by functional p53 [109]. The deletion of p53−/− in ATR-mosaic knockout mice reported to exacerbated tissue degeneration and induced synthetic lethality [110]. Subsequently, these two breakthrough reports worked as guiding path in ATR therapy. VE-821, a novel ATR inhibitor has shown to synergistically enhance the effect of cisplatin in ATM or p53-defective (H23) cell lines, while p53 wild-type normal fibroblast cell line (HFL1) remained unaffected [111]. HeLa cells were radiosensitized by VE-821 having suppressed p53 while normal cells expressing wild-type p53 was unaffected [112]. VE-822 (VX-970, Vertex), an analog of VE-821 having strong ATR inhibition property demonstrated a strong radiosensitizing and chemosensitizing effect in cancer cells both in in vitro and in vivo studies by suppressing the HR without affecting the normal cells [113]. Another ATR inhibitor AZ20 (AstraZeneca), under monotherapy regimen, has shown to reduces the growth of LoVo tumor xenografts in mice [114]. AZD6738 (AstraZeneca) is another improved version of AZ20 induce synthetic lethality in p53 or ATM-deficient cells or tumor in response to chemotherapy and ibrutinib hence could be useful in RT [115]. Caffeine is another ATR inhibitor radiosensitize the cells but it also blocks the ATM; thus it may cause toxicity to normal cells [116]. Additionally, due to extreme replicative stress oncogenic cell becomes more sensitive to ATR inhibition than normal cells, therefore targeting ATR pathway is a promising strategy in RT. Now, phase I clinical trial is underway, investigating the side effects and best dose of VX-970 under RT in brain metastases (NSCLC) and HPV-negative head and neck carcinoma patients (NCT02567422, NCT02589522). ATR inhibition has also shown to induce the synthetic lethality in cell-deficient XRCC1 a protein-involved repair of SSB [117]. Indeed, due to unavailability of the crystal structure of ATR, pose a major challenge in developing selective ATR inhibitor. Additionally, the large size (2644 amino acids, UniProt: Q13535) of ATR makes it difficult to be cloned, expressed, and isolate protein in purified form required for screening inhibitors in kinase-based assay. The long sequence of ATM also poses a challenge in routine clinical diagnosis, which is further aggravated by missense variants (mostly) spread across the coding sequence as gene does not have any mutational hotspot (NCBI Gene ID: 472). The frequent missense mutation results in the loss or reduction of ATM expression; hence, immunohistochemistry may be a viable tool to inspect the ATM status in cancer patients [118].

ATM is another protein shown to cause synthetic lethality with some specific mutations. ATM kinase inhibitors [119] have shown to radiosensitize the cells carrying Fanconi anemia gene mutations, a common mutated or lost gene in cancer [120]. ATM inhibition resulted in the radiosensitization of bladder cancer cells with DAB2IP gene defects [121], ovarian, endometrial, cervical cancer [122], glioma [119], and glioblastoma stem-like cells [123]. However, despite the fact that currently, no ATM inhibitor is under clinical development over the concern of normal tissue cytotoxicity. Inhibition of ATM by KU-55933 or KU-60019 resulted in persistence DNA damage following irradiation due to defective repair in normal cells [124, 125]. Subsequent study has shown that cells possessing kinase dead (kd) ATM protein is more harmful to the cell than complete loss of ATM gene and, in fact, ATM knockout mice were viable in comparison to embryonic lethality in kd ATM mice [119, 126]. It has been hypothesized that binding of kd ATM to DNA DSBs site causing hindrance in the binding of other protein destined to start alternative repair makes little use of ATM inhibitors in clinical practice. However, brief administration of ATM inhibitor KU59403 shown to chemo-sensitize the human colon cancer xenografts (SW620 and HCT116) in mice without showing any cytotoxicity to normal tissue, further raise the possibility of ATM inhibitor development for RT [127]. The outcome of the study was that ATM inhibitor must be administrated briefly and only with the topoisomerase I (camptothecin) and II inhibitor (etoposide and doxorubicin). However, future investigation must target the ATM either by inhibiting at translation level or by disrupting the interaction between HEAT repeats of ATM and carboxy-terminal FXF/Y motif of Nbs1 [128] with the novel drug instead of targeting kinase activity of ATM.

Inhibitors of cyclin-dependent kinases (CDKs) are the emerging class of the radioprotective agents given the cells is least radiosensitive at mitosis especially during late S (due to HR) and quiescence (G0) phase [129]. Cell enters into cell cycle by D-type/CDK4/6 complex mediated hyperphosphorylation of Rb and subsequent release of E2F to start transcription of gene required for cell division. The entry in cell cycle is inhibited by p16INK4A, an inhibitor of CDK4/6 [130]. Cancer cell often deregulates the cell cycle checkpoints as a mechanism to promote the uninterrupted cellular proliferation even in presence of damaged DNA by amplification of CDK4/6 and D-type cyclins, or loss of p16 and correlated with resistance to therapies [130]. Administration of PD0332991 and 2BrIC, an inhibitor of CDK4/6, 4 h prior or 20 h after the irradiation has shown to induce the G1 arrest reversibly in Rb-positive cells resulted in the protection of the bone marrow progenitors cells and all peripheral blood lineages: platelets, erythrocytes, myeloid cells, and peripheral blood lymphocytes [131]. Further, PD0332991 does not protect the Rb-deficient tumors as they develop independently to CDK4/6 [131]. However, it does not mean that it is only effective in patients having Rb-deficient tumors, as CDK inhibitor induces the irreversible blockage causing induction of senescence and apoptosis in cancer cells having functional p53 and mTOR and former act an inhibitor of later, while in normal cells, it induces reversible quiescence, a radioresistant state [14, 132]. Indeed, subsequent studies were not highly conclusive. PD0332991 has shown to be protected against carboplatin-induced hematopoietic injury in Rb-deficient breast cancer mice, but failed to show similar effects in Rb-positive MMTV–HER2 mice [132]. While in another study, combined treatment of PD-0332991 and RT have shown to increase the survival benefit compared with either therapy alone in Rb1-positive glioblastoma intracranial xenografts implanted mice [133]. Treatment with PD-0332991 or 2BrIC have shown to increase the radioresistance of Rb1-positive melanoma cells and immortalized fibroblasts [133]. Hence, with cautions, a clinical trial has been initiated to evaluate the G1T28 (CDK4/6 inhibitor) in reducing the carboplatin-induced hematopoietic injury in SCLC (a common Rb-negative tumor) (NCT02499770). Sorafenib or Nexavar, an FDA-approved drug for liver and kidney cancer, is also a promising candidate; it synergistically reduces the tumor size with radiation without causing normal tissue injury in mice model via acting on at G1-S cell cycle checkpoint and currently under different phase I–II clinical trials involving RT [134].

Inhibiting cell death

The inhibition of RI cell death could be another strategy in radioprotection as p53-deficient mice are shown to be less sensitive to radiation compare to wild type [135]. In subsequent studies, inhibition of p53 by sodium orthovanadate or AS2 had shown to enhance the survival of lethally irradiated mice [136]. Inhibition of the p53 by antisense was also found to be helpful in the radioprotection of mice following TBI (15 Gy) [137]. Therefore, blocking off p53 by its inhibitors nutlin-3 sounds good in radioprotection but given its role in tumor suppression may discourage its uses; however, it has been observed that the temporary and reversible suppression of p53 by genetically or pharmacologically agents found to be helpful in rescuing the large numbers of cell from the clench of apoptosis without increasing the chance of secondary cancer [14]. Many tumors undergo RI apoptosis by the p53-independent pathway, hence blocking p53 in that scenario does not affect tumor radiosensitivity [138, 139]. Differential phosphorylation of p53 also decides the action of p53 between rescuer (DNA repair) and destroyer (apoptosis) [140]. So, selectively targeting the p53 sites, responsible for apoptosis using novel drug could be another strategy to reduce the RI damage. However, clinical utility of p53 inhibitors are very limited due to p53-independent apoptosis as seen in multiple organs including hematopoietic progenitor cells [138, 141], GI track [142], and endothelial cells (follow ceramide-dependent apoptotic pathway instead of p53-dependent). In fact, p53-deficient mice are more prone to RI GI damage than wild-type [142] and subsequently, targeting ceramide by anti-ceramide antibody, showed to protect against RI GI syndrome [143]. Therefore, it is needed to explore beyond p53.

Peroxisome proliferator-activated receptor (PPAR) is another druggable target that could be exploited in RT. Fenofibrate (Abbott), a PPAR agonist, has shown to reduce RI damage in the peripheral cortex but failed to restore hippocampal-dependent cognitive functions [144], while in another study, it induced the hippocampal neurogenesis by protecting the newborn cells and inhibition of microglial activation following irradiation (10 Gy) in mice [145]. The difference in result seems to be due to species and exposure multiplicity. In the other study, it has been shown to enhance the radiosensitivity of human esophageal carcinoma cell lines (Eca-109 and TE1) [100]. The differential effect of Fenofibrate seems to mediate via halting the cell at G2/M transition following RI DNA damage resulting in mitotic catastrophe in cancer cell due to defunct DNA repair machinery while normal cell successfully repairs the damaged DNA. Rosiglitazone, another promising PPAR agonist, has shown to radiosensitize the implanted A549 lung tumor in CD1 mice by decreasing the expression of NF-kB and TGF-β, without affecting the normal pulmonary tissue [146]. Catalase inhibition has shown to radiosensitize the cancer and normal cells due to the accumulation of cytotoxic H2O2 as a byproduct of RI ROS (Fig. 2a) [147, 148]. Exposing the rat primary astrocytes and their cancer counterpart (C6 glioma cells) to different PPAR agonist, i.e., PPAR-α (GW3276 and WY-14643) and PPAR-γ (CP086 or darglitazone, troglitazone, rosiglitazone, CP096, 9cRA) have shown to selectively enhance the catalase expression in normal cells [149]. The maximum difference in expression was observed with CP096 where it was reduced by 38 % in cancer cell while 137 % enhancement in normal cells was observed after 48 h of treatment [149]. Interestingly, the catalase expression was blocked by PPARγ-dominant negative plasmid, indicated altered expression is not due to enhancement of messenger RNA (mRNA) stability instead due to de novo expression [149]. Hence, in view of differential effects of PPAR agonist warranted further studies to elucidate its full potential in RT.

Immunotherapy

Radiotherapy induces a tumor-specific response represented by upregulation of MHCs, pro-inflammatory molecules and their receptors, cell adhesion molecules, dendritic cell maturation, and their cross-presentation ability; hence, radiation can be considered as a perfect in situ vaccine. The role of immune system in tumor response was first suggested in 1979, demonstrating the reduced radiotherapeutic efficacy in mice lacking T cell repository [150]. Following irradiation the upregulation of ligands such as CXCL9, CXCL10, CXCL16, and retinoic acid early inducible (RAE-1) in the tumor takes place [151]. The CXCL9, CXCL10, and CXCL16 enhanced the recruitment of CXCR6+ effector CD8 T and CD4 TH1 cells in the tumor, while RAE-1 interacts with NKG2D present in effector T cells resulting in the generation of effective T cell response against tumor [152]. However, inhibitory checkpoint receptor CTLA-4, PD-1, PD-L1, BTLA, TIM-3, CD160, and LAG-3 cause obstruction in the response [153]. Inhibiting the CTLA-4 in RT has shown to induce the successful T cell-mediated anti-tumor response, while only anti-CTLA-4 treatment was failed to show any anti-tumorigenic activity [152, 154]. Since effect was only seen in the single exposure; it is further required to evaluate with fractionation radiation for wider use. Some phase I clinical trial are ongoing to check the efficacy of anti-CTLA-4 in RT. Blocking of PD1 with pembrolizumab in ipilimumab-refractory advanced melanoma patient shown exceptional efficacy under monotherapy resulted in FDA approval in 2014 [84, 155, 156]. Furthermore, targeting CTLA-4 and PD1 has shown a significantly longer progression-free survival in comparison to individual modality in metastatic melanoma patients [157]. In glioblastoma multiforme tumors mice model, combining PD-1 blockade with localized radiation therapy has shown a synergistic effect in survival [158]. The identical therapy in B16-OVA melanoma or 4T1-HA breast carcinoma tumor model has shown a batter tumor control [159]. Combining anti-PD-1 antibody with single (10 Gy) or fractionated (2 Gyx5) RT has shown to promote the anti-tumor T cell response, reducing the local accumulation of myeloid-derived suppressor cells and reduced the IFN-γ mediated induction of PD-1 [160, 161]. The underlying mechanism behind the effects of PD-1 is more or less similar to anti-CTLA-4 therapy and since anti-PD-1 antibodies are milder toxic than anti-CTLA-4 antibodies [84, 156, 162]; therefore, PD-1 seems promising in clinical RT. Further understanding of PD-1 and CTLA-4 pathway is also promising in autoimmunity treatment and tissue transplantation including β cell for treating type 1 diabetes. Moreover, BTLA TIM-3, CD160, and LAG-3 are other promising inhibitory molecules that could be targeted for generating a successful anti-tumor response. Indeed, RT always does not promote anti-tumor response. It also induces a protumor response represented by enhanced immunosuppressive Treg-cell representation, influx of CD11b+ myeloid cells and M2 macrophage infiltration (Fig. 3) [163–165]. The enhancement in Treg is probably mediated by hypoxia raised from RT-induced disrupted vasculature. The RT-induced influx of myeloid cells into tumors can be inhibited by blocking of HIF1α/CXCL12/CXCR4 [156, 161] or CSF1/CSF1R [156, 162] axes for better radiosensitization of the tumor.

Fig. 3
figure 3

Radiation and immune response. Radiation generates an oxidative, apoptotic, and inflammatory response that promotes the enhanced antigen presentation by antigen presenting cells (APCs) including dendritic cells (DC) results in cytotoxic T cells (CTL) activation and tumor destruction (left side). However, to resist oxidative response, tumor tissues generates a proangiogenic, anti-inflammatory and antioxidative response represented by increase in number of T regulatory (Treg), myeloid-derived suppressor cells (MDSC), Nrf2, M2 macrophages, TH1 cells, and its cytokines, i.e., TGF-β, IL-10, IL-4 (right side)

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Radioprotection by growth factors inducers

Hematopoietic growth factors have been used from long to rescue the hematopoietic and progenitor cells following irradiation. Now, it is advanced towards non-hematopoietic factors, i.e., keratinocyte growth factor (KGF). Preclinical studies demonstrated the radioprotective effects of KGF against RI intestinal damage and pneumonia [166, 167]. Interestingly, no tumor cell proliferation was seen, indicates the functional difference in signaling between normal and cancer cells. Palifermin (rKGF-2) reduced the chemoradiation-induced oral mucositis in randomize trial involving head and neck cancer patients and now recommended by FDA for reducing severe mucositis in patient undergoing myeloablative hematopoietic stem cell transplants with TBI [168–170]. However, it has disappointed in other clinical settings hence currently not recommended for wider clinical uses [171, 172]. GM-CSF administration in pre and post-RT has shown to reduce the RI severe oral mucositis in multiple randomized, head and neck cancer patients RTOG trial [173–175]. Interestingly, administration of HSV GM-CSF in squamous cell cancer of the head and neck (SCCHN) patients undergoing chemoradiation therapy has shown to lessen the locoregional recurrence and enhances the survival of two thirds of patients in phase I/II clinical trial [176]. The differential effect of GM-CSF was mediated by tumor oncolysis and immunomodulation and hence due to promising nature, further trial expected to start soon. Erythropoietin has shown to reduce the radiation and tumor-induced anemia without affecting the radiosensitivity of tumor in preclinical studies; however, results from successive clinical studies were not highly encouraging [177–179]. Failure of erythropoietin seems due to inappropriate patients under suitable modality since erythropoietin causes loss of tumor radioresistance due to neovascularization and moreover, the later effect also increases the chemotherapeutic drug diffusibility in tumor result in better tumor control in addition to offering protection to normal tissues from chemoradiation-induced toxicity. Therefore, to avoid failure in future trials, hypoxia must be taken into consideration and only used under chemoradiation settings. In a recent co-treatment study combining erythropoietin with carboplatin has shown to limit the growth of A549 and H838 NSCLC-xenografts in mice [180]. Thrombopoietin is another promising molecule in RT [181, 182]. Velafermin (rhFGF-20) was shown to protect against RI cheek pouch mucositis induced by fractionated radiation [183].

Role of inflammation

Radiation has shown to polarize the monocytes and residential macrophages into M1 and M2 macrophages by classical (TH1-based programs) and alternative (TH2-based programs) pathway, respectively, which subsequently participate in inflammation (M1) and tissue reconstruction (M2). Radiation polarizes the macrophages into M1 which subsequently play an important role in generation of anti-tumor immunity operated from NF-kB/TH1 cytokines axis (Fig. 3). Macrophages (M2) polarize in response to RI M1 response or to counteract RI oxidative response (ROS, NF-kB) for homeostasis by upregulating antioxidative (Nrf-2) and anti-inflammatory pathway. M2 macrophage has shown to suppress the DC function and Th1 type adaptive immunity resulting in tumor growth promotion by Nrf-2/TH2 cytokines axis. Furthermore, Nrf-2 induction has demonstrated to shift the metabolic flux towards NADPH regeneration and purine biosynthesis by pentose phosphate pathway for tumor growth [156, 184]. Irradiation potentiates the macrophage to upregulate the IL-1, TNF-α, NO, and a variety of growth factors (PDGF, IGF-1) [185]. Following irradiation tumor becomes hypoxic due to the destruction of the vasculature, result in HIF-1 and HIF-2 mediated infiltration of macrophages [186]. Irradiation to MT1A2 tumor-bearing mice, results in accumulation of MMP9 expressing CD11b+ myelomonocytes at tumor site essential for vascular restoration and tumor growth and selective depletion of them has shown to cause tumor growth inhibitions in pre-irradiated tissue [187, 188]. Other studies also demonstrated a better tumor control following depletion of macrophages (M2) from irradiated tumor-bearing animal [189]. Hence targeting macrophage is promising in RT.

TGF-β is a cytokine strongly induced following irradiation and play a role in tumor survival. Knocking out the Smad3 (S3KO), a downstream signaling intermediate in the TGF- β pathway, in mice has been shown to protect the skin from RI damage as demonstrated by decreased epithelial acanthosis and dermal fibrosis [190]. Inhibition of TGF-β following radiation exposure has shown to help in priming tumor antigens specific T cells, results in better tumor control [191] in addition to radioprotection [192]. TGF-β has also shown to inhibit collagen production in fibroblasts, resulting in fibrosis reduction following RT [193]. However, in the clinical setting, care must be taken since TGF-β also was shown to help in the maintenance of genomic integrity [156, 184, 194].

Protecting the genomic integrity of cells against radiation

The role of radiotherapeutic radiation in the occurrence of secondary cancer (SC) remains controversial and untenable as most of the arguments rely on data extrapolated from World War II atomic bombings life span survivor, where a proportional relation was observed between cancer onset and radiation dose (0.1–2.5 Gy). However, if the frequency of SC calculated using the theoretical model developed from the above-mentioned studies, the chance of SC would be much higher than reported in clinical settings, it means that the risk of cancer caused by radiation is highly extravagated [16]. Interestingly, this linear nonthreshold (LNT) model still widely used by different regulatory agencies [195]. Even the relative risk of SC declined after a certain dose as with increasing dose probability of cell killing is increased or the risk of SC decreased since once cell died it cannot undergo transformation. Therefore, due to uncertainty, radiation is categorized as a weak carcinogen [16]. Although many papers are claiming a relation between RT and SC but in a recent long-term follow-up study involving 12,247 Hodgkin patients does not show any increase in secondary malignancy in comparison to chemotherapy-treated patients [194]. A meta-analysis comprising 762,468 breast cancer patients do not show any link between radiotherapy and second thyroid cancer [196]. Furthermore, in two large cohort studies, no link between RT dose and SC was observed in childhood cancer survivor and atomic bomb survivor or prostate cancer patients and sugary-treated patients [197, 198]. In a recent large pooled cohort of pelvic cancers patients treated with surgery or radiotherapy did not show any significant difference in developing SC [199]. Moreover, even if any relation between both, the variables like tumor microenvironment, genetic predisposition, altered nutrition, immunodeficiency, personal lifestyle, age, and social condition, etc. pose a challenge to draw a conclusive relationship. Furthermore, long-term exposure to low-dose radiation induces cancer via carcinogenesis while direct chromosomal damage and abrupt failure of signaling events in addition to carcinogenesis is responsible for RT-induced SC so both are incomparable. However, it is not a denying fact on the involvement of RT radiation in SC development, though it develops independently to RT dose. Hence, to understand the possibility of developing SC in RT, focus is needed at the individual level rather then on finding a relation between SC and exposure dose as genetic polymorphism is known to play a major role in radiation sensitivity. Recently, Radiogenomics Consortium (supported by NCINIH, USA) has established to work together, share data/samples, perform meta-analyses, identify SNPs, and biomarkers responsible for adverse effects including SC following RT. Response to lymphocytes have shown to act as an interpreter of RI toxicity in patients undergoing RT and could be used for selecting patients for RT [200]. Telomere length is another reliable marker that can be used to predict the RT-induced acute toxicity and SC before RT [201]. This observation has confirmed in a recent large sample clinical study involving childhood cancer survivors where a relation between less telomere content and treatment-related thyroid cancer was observed [202]. In a large cohort study of patients who has undergone RT, a significant relation between telomere shortening and development of cardiovascular disease [203] was shown. In another cohort study, appearance of RT-induced SC in patients was shown to have a cutoff value for telomere at 6.6 kb in comparison to 9.7 kb where no complication was observed [204]. These studies could serve as a tool for selecting treatment type for better prognosis in cancer patients as prostatectomy could be a better option for those having shorter telomere while brachytherapy or RT for prostate patients having longer telomere length.

Modulation of the cell signaling

Cancer cells require huge quantity of iron for survival, angiogenesis, and metastasis [205]. It is also required for maintenance of the ribonucleotide reductase (RR) activity involved in the conversion of ribonucleotides to deoxyribonucleotides (dNTPs) required for DNA synthesis [206]. Iron chelation has shown to cause hypophosphorylation of the retinoblastoma protein resulting in the reduction of cyclins expression including A, B, and D. Iron chelation by Triapine (3-aminopyridine-carboxaldehyde thiosemicarbazone) (Vion Pharma Ltd.) have shown to radiosensitize the cancer cells [207]. Studies performed over the years using normal cells or animal have demonstrated the reduction in RI damages in the presence of iron chelators since iron is known to participate in hydroxyl radicals generation via Fenton reaction following irradiation [208, 209]. Additionally, due to the difference in iron requirement for normal metabolism, makes cancer cells more sensitive towards iron deprivation than normal cells. Therefore, iron chelating is a quite promising strategy in RT and requires a robust investigation. Triapine is one iron chelator which is undergoing phase I/II clinical trial for evaluation of its synergy with RT and cisplatin combination in cervical or vulvar cancer patients (NCT02595879, NCT02466971, NCT01835171).

Data from earlier studies has demonstrated that radiation induces the expression of NF-kB as a mechanism to reduce the RI lethality via activating prosurvival pathway [210]. LPS has shown to improve the survival following WBI in mice [211]. Flagellin, (an NF-kB inducer from bacterial origin act via TLR-5) has shown to reduce the RI lethality in tumor-bearing mice [212]. However, in contrast to that, subsequent studies have demonstrated that inhibition of NF-kB results in radioprotection. NF-kB inhibition by ethyl pyruvate (EP) or CDDO-TFEA (RTA401) and RTA 408 (Reata Pharma. USA) were found to reduce the RI lethality in zebrafish embryo [213] and GI damage in mice [214], respectively. The EP or CDDO-TFEA (RTA401) was abrogated by proteasomal inhibitor PS-341 (Bortezomib/VELCADE) in zebrafish [213]. The role of RI NF-kB currently guided by two alternative hypothesis revolving around inflammation. Data from burn studies has demonstrated the pro-hematopoietic effects of low to moderate level of inflammation while a high level of inflammation has shown to cause oxidative stress and apoptosis. Therefore, it seems like suppression of RI NF-kB-led inflammation may be the reason behind the later effect while the lower level of inflammation may act as hematopoietic simulative in former studies. Elucidation of the role of RI NF-kB is also quite difficult due to associated embryonic lethality [215]. NF-kB constitutively activated in many cancers as a mechanism to help in the initiation, progression, and invasiveness resulting in poor treatment outcome in preclinical and clinical studies [216]. Inhibition of NF-kB via Icariin or DMAPT has shown to radiosensitize the colorectal cancer and NSCLC, respectively [217]. Furthermore, RTA-408 also showed to radiosensitize the CWR22Rv1, LNCaP/C4-2B, PC3, and DU145 xenografts in mice model in addition to protection of GI tract from RI damage [214]. Therefore, targeting one of major prosurvival pathway in cancer treatment may helpful for better RT outcome.

EGFR is the growth factor responsible for radioresistance in many cancers. Targeting EGFR in tumor with cetuximab, gefitinib, and erlotinib in monotherapy had shown promising results. Indeed, in some studies, it has failed due to heterogeneity in EGCR expression in tumor. In phase III clinical trial administering cetuximab in head and neck cancer patients undergoing RT has shown to increase the post-therapy survival to 49 months in comparison to 29 months in RT arm [218, 219]. Despite showing synergy with cisplatin and radiation in sensitization of tumor cells in preclinical studies [220], cetuximab highly disappointed in subsequent clinical trials [221]. Administrating cetuximab in locally advanced rectal cancer patients under chemoradiation regimen does not show any benefits [222]. The failure of study seems to be due to blocking of tumor cell proliferation by cetuximab since proliferating cells are more sensitive to chemoradiotherapy so blocking the target of chemoradiotherapy could be reason behind poor prognosis. Concurrent administration of cetuximab, and cisplatin in stage III/IV head and neck cancer patients undergoing RT in a large (N = 891) randomized RTOG trial, failed to demonstrate any disease-free or overall survival [223]. The complete failure of therapy in the above-mentioned case yet to clear, previous studies have suggested the oropharyngeal tumors that are mostly HPV positive likely to benefit from the combination and hence, the study (RTOG 10-16) was carried out. In fact, patients reported acute toxicity, notably mucositis in cetuximab arms. However, despite the outcome and waste of resources, many prospective clinical trials of cetuximab in RT are still undergoing in different treatment settings (NCT02123381, NCT00956007, NCT01614938).

Exploiting the cellular autofluorescence property

Schaue et al. had noticed an increase in cellular autofluorescence at 450 nm following irradiation in different human and murine cell lines and concluded as general phenomena [224]. The conclusions were drawn from the study that autofluorescence is radiation dose-dependent phenomena, and proportional to the level of cell radiosensitivity, i.e., hematopoietic cells have a high level of autofluorescence. The autofluorescence is high in normal cells in comparison to cancer cells. Today, photodynamic therapy is emerging as promising treatment agents in various diseases including cancer [225]. Therefore, we are hypothesizing that using photo-reactivated drug or photodynamic therapy could immensely help in protecting normal tissue by exploiting cellular autofluorescence following irradiation. Today, many photosensitizer drugs such Temoporfin, Porfimer sodium, Methyl aminolevulinate, Hexvix, Talaporfin, or aspartyl chlorine have been used in the treatment of different form of cancer; in fact, a few of them have been shown to improve the RT efficacy [226]. The currently used photosensitizer actively untaken by cancer cells require much longer wavelength (700–850 nm) for excitation then emitted by cell autofluorescence (450 nm). Hence, due to multiple advantages, it is quite promising to develop photo protectors that exclusively reactivated at lower wavelength once the cells are irradiated.

ACE inhibitors

Renin-angiotensin system was discovered in the maintenance of fluid balance and blood pressure. However, subsequent studies have shown that targeting it could be helpful in radioprotection, especially against renal toxicity. Several protease inhibitors such as captopril, enalapril, penicillamine, pentoxifylline, L-158, 809, etc. acting via rennin-angiotensin system has shown to be helpful in the reduction of RI prophylaxis in renal and lung tissues. The use of ACE inhibitors and angiotensin-II receptor blockers have demonstrated a new avenue in the radioprotection especially where the high dose of exposure is imminent; however, in one study, it does not confer protection to the intestinal injury; therefore, it seems that it is offering tissue-specific protection [227]. Inhibition of angiotensin system shown to help in improving the outcome of RT in clinical trials by reducing the radiation pneumonitis [228].

Early growth response 1 (Egr1), known to induce apoptosis by activating the apoptotic genes is another promising transcription factor in radioprotection. Inhibiting EGR 1 with MMA (mithramycin A) in in vivo or knocking out Egr1 in mice (Egr1−/−) have shown to protect the RI GI damage by decreasing the ratio of Bax/Bcl-2 [229]. Egr1 is also known to require for the survival of various cancer including prostate, gastric, and kidney cancers. Earlier studies have shown that targeting Egr1 is helpful in radiosensitization of different cancer cells. Currently, MMA is being investigated for its clinical efficacy (phase II) in the lung, esophageal, neoplasms, and breast cancer patients (NCT01624090).

Radiation mitigation

Radiation mitigators are the agents used in radiation countermeasure following exposure from different sources, i.e., nuclear detonation, terrorist events or accidental exposure that sometimes even happens in clinics during RT or due to mishandling of isotopes [230]. FDA recommended the potassium iodide in 1982 as an emergency radiation countermeasure to protect thyroid gland against radioiodine. However, after 9/11, three more drugs radiogardase (against Cesium-137 and thallium-201), calcium and zinc salt of DTPA (against several transuranic ions, i.e., plutonium, americium, curium, etc.) were added to the list (2003–2004).

Bone marrow is the most radiosensitive and important organ required for survival. In radio-mitigation, the best option is the transplantation or inducing the proliferation of surviving stem and progenitor cells. The 9/11 terrorist attacks dramatically change the trajectory of security initiatives in the USA. Strategic National Stockpile Radiation Working Group in a consensus document and Center for Disease Control and Prevention (CDC) has recommended the bone marrow transplantation [231]. However, salivary gland is the only organ so far which functionality restoration has been demonstrated following irradiation. In an earlier study, many cytokines and hormones had shown to help in radio-mitigation; however, most of them work if they administrated before 24-h post-exposure since most of the cell lost in first 24 h after irradiation from the peak at 4 h [232]. Nevertheless, the US government is keen on mitigator for national stockpiles which can even effectively work after 24 h after exposure. Therefore, mechanism for radioprotection must not lie in apoptosis inhibition. Instead, it must revolve around the hematopoietic cells such as mesenchymal stem cells (MSCs) and bone marrow. Targeting of thrombomodulin-protein C pathway or mobilizing the progenitor’s cells after 24 h of irradiation has shown to mitigate the RI lethality in mice [233, 234]. MSCs are other cells that can be used in radio-mitigation. In fact, due to radioresistance over bone marrow and other radioresistant cell lines; currently, MSCs are under intense investigation. Indeed, in few studies, it was also shown to act as a potential source of tumorigenesis in the long term due to the acquisition of some genetic modification like telomerase shortening [235, 236].

Statins are HMG-CoA reductase inhibitors originally developed as lipid control agent has shown to mitigates the RI damages. Simvastatin has been shown to protect the cardiac system from infections in the rat when administered 9 days after irradiation. Therefore, statins could be exploited in both RT and radio-mitigation for RI cardiac problems [237]. Anticoagulants activated protein C is another promising candidate in radioprotection shown to induce the hematopoiesis, resulting in improved survival after administrating as late as 24 h after lethal exposure [233].

Future perspective

Today, RT represents the major anti-cancer modality. However, it failed to demonstrate a significant outcome despite carrying out multiple targeted clinical trials. These studies failed due to various reasons such as drug toxicity, wrong patient selection, lack of information about suitable cancer biomarkers, and heterogeneous disease process. With emergence of molecular biology, cancer biology has made tremendous progress in sorting out the physiological differences between cancer and normal cells. This provides a bedrock for designing novel drugs to selectively enhance the radiosensitivity of tumor in addition to protecting normal tissues. However, every new drug must go through rigorous preclinical studies to understand the basic mechanism and avoid failure in subsequent clinical trial as iniparib, shown to be a PARP inhibitor failed to demonstrate any clinical benefits in phase III trial [238] and further study showed that it is not only a poor inhibitor of PARP but also quite structurally distinct from other PARP inhibitors [239]. Hence, rigorous preclinical studies can avoid resources wastage [223] and discarding of potentially useful targets [73, 77]. The other challenge lies in mice model system as the majority of study for human tumor xenotransplantation is conducted in immunosuppressed mice, and we know that immune system proactively interacts with tumor; therefore, to impart the role of human immune system, humanized mice could serve as a better alternative.

Targeting tumor with monotherapy may give a selective advantage to clones harboring resistant mutant, resulting in the tumor reemergence/relapse [240]. Hence, targeting local environment may be advantageous as it equally affects all cells irrespective of clonal variation. As targeting hypoxia automatically reflected in all cells and make them radiosensitive irrespective of oxygen consumption by individual cells. The tumor is heterogeneous, evolutionary, and plastic in nature which consistently evolves and adopts new conditions until colonized in distant location hence in successful cancer elimination multitargeted therapies acting in synergy are quite promising. In last reduction of tobacco/alcohol/drug consumption, better hygienic-/pollution-free environment, obesity control, immunization, and healthy lifestyle certainly help in winning the war against cancer and may reduce 30–40 % death by cancer. In conclusion, future outcome of RT is entirely dependent on information about continued cancer biomarker identification, cancer type categorization and sorting out the fundamental difference between cancer and normal cells.