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REVIEW
Development of p53-targeting drugs that increase
radioresistance in normal tissues
Shintaro Ochi, Yuichi Nishiyama, and Akinori Morita
Department of Biomedical Science and Technology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan

Abstract : Radiation damage to normal tissues is a serious concern in radiation therapy. Advances in radiotherapeutic technology have improved the dose distribution of the target volumes and risk organs, but damage to riskorgans that are located within the irradiation field still limits the allowable prescription dose. To overcome thisdose-limiting toxicity, and to further improve the efficacy of radiotherapy, the development of drugs that protect
normal tissues but not cancer tissues from the effects of radiation are expected to be developed based on molecular target-based drugs. p53 is a well-known transcription factor that is closely associated with radiation-induced
cell death. In radiation-injured tissues, p53 induces apoptosis in hematopoietic lineages, whereas it plays a radioprotective role in the gastrointestinal epithelium. These facts suggest that p53 inhibitor would be effective forradioprotection of the hematopoietic system, and that a drug that upregulates the radioprotective functions of
p53 would enhance the radioresistance of gastrointestinal tissues. In this review, we summarize recent progress
regarding the prevention of radiation injury by regulating p53 and provide new strategic insights into the development of radioprotectors in radiotherapy. J. Med. Invest. 66 : 219-223, August, 2019
Keywords : radiation, p53, apoptosis, radioresistance, molecular target-based drugs

INTRODUCTION
Recent progress high precision radiation therapy has beenremarkable, and with improvements in dose distribution haveresulted in much improved therapeutic effects. The prescription
dose has improved by about 1.3 times compared the 4-field box
technique (tolerance dose of 60 to 66 Gy) with the latest intensity-modulated radiation therapy IMRT (tolerance dose ~ 80 Gy)(1). However, because the complete segmentation of the target
volumes and risk organs is often difficult in clinical settings, riskorgans frequently suffer adverse events, especially risk organs
that are located in the vicinity of the target lesion. The result
is that the prescription dose is still limited by injury to normal
tissue. It is especially true that, when the intestinal tract, a radiosensitive tissue, is included in the irradiation field of the pelvic region, adverse events such as diarrhea, intestinal bleeding,ulceration, perforation, intestinal obstruction, and stenosis may
occur in the form of early and late effects of intestinal disorders.
In addition, because some tumors move with the movement ofprimary or surrounding organs such as by peristalsis, respiration, and swallowing, a technological innovation for minimizing
the damage of normal tissues is also necessary. Now that thephysical technology has been optimized, it now becomes imperative to further improve the tolerable dose by new technology.
We believe that biological innovations will further improve thepresent tolerable dose. Therefore, our goal is to develop a normal
tissue-selective radioprotective agent that permits a good valueof dose reduction factor (DRF; the fold change in irradiation doseto produce a given level of biological effect). We initially set thetarget value of DRF to 1.3 and more, because a value of 1.3 isReceived for publication December 11, 2018 ; accepted February 20,
2019.
Address correspondence and reprint requests to Akinori Morita, Prof.,
Ph.D., Department of Biomedical Science and Technology, Graduate
School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan and Fax : +81-88-633-9052.

excellent protection activity comparable to DRFs achieved by theradioprotective agents developed so far (2, 3).
For radiation protection, particularly important radiosensitive organs are bone marrow and the intestinal epithelium.
It is known that bone marrow death occurs with exposures of
up to 10 Gy and gastrointestinal death predominates in a dose
range from 10 to 50 Gy. These acute radiation syndromes are
attributable to radiation sensitive stem cell death in each tissue.
p53 is involved in the progression of radiation cell death, but its
functions are completely different in each tissue. In bone marrow, it acts as a mediator of radiation-induced apoptosis, but it
also acts as a resistance factor for non-apoptotic mitotic deathin the intestinal epithelium (4). Thus, it would be necessary torationally control p53 according to the radiation syndrome being
considered.

CONVENTIONAL METHODS FOR PREVENTINGRADIATION INJURIES USING ANTIOXIDANTS
Ionizing radiation generates reactive oxygen species (ROS)
such as •OH and H2O2 through water-splitting reactions in theliving body. ROS are highly reactive with respect to cell membranes and DNA, leading to gene mutation and cell death. The
use of antioxidants to scavenge ROS is a useful way for preventing radiation injury. In 1948, a few years after the atomic bombs
were dropped in Japan, Patt et al. demonstrated that the aminoacid cysteine, which contains a thiol group, showed radioprotective effect in animal experiments (5). This finding attracted
international attention especially from countries involved in theCold War. Several thiol-containing radical scavengers were subsequently developed as radioprotectors. Among them, amifostine(WR-2721, S-2 [3-aminopropylamino]-ethylphosphorothioic acid;
Ethyol) is the most representative one (6).
Antioxidative agents should be administered prior to exposure to radiation, since ROS production occurs at the momentof exposure. Although this type of agent is unsuitable for use in

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S. Ochi, et al. Radioprotective p53-targeting drugs

unanticipated exposure situations such as the Fukushima andChernobyl nuclear disasters, it has a great utility when used ina timely manner. The US Food and Drug Administration (FDA)
currently approves amifostine as the only radioprotector aimed
at protecting salivary glands in radiation therapy to head andneck cancers, but the dosage is strictly limited due to a variety
of side effects (e.g. nausea, vomiting, and hypotension) (3, 6). In
addition, most antioxidantive agents, including amifostine, also
have a disadvantage, in that they increase radioresistance incancer tissue due to their non-specific protective effects.

EFFICACY OF p53 REGULATION IN CANCER TREATMENT
In recent years, some protective agents have been developed that enhance radioresistance by controlling the molecularmechanism of cell death without relying on antioxidant activity.
The p53 regulatory agent selectively protects normal tissue
cells from radiation damage, which have, as a matter of course,
normal p53 function. In contrast, the p53 regulatory agent does
not protect many cancer cells from radiation injury, because
nearly half of all types of cancer have lost their p53 function by
oncogenic suppression or mutation. Therefore, the p53 regulatory agent is expected not only to function as a radioprotector in
nuclear disasters but also as a reducing agent for alleviating theside-effects associated with cancer treatment by overcoming thedose-limiting toxicities of radiation therapy and chemotherapy(Figure 1). Although the risk of promoting carcinogenesis as adisadvantage of p53 suppression is often pointed out, several
studies have shown that this is not a concern by using genetically modified mice. These studies have reported that the genetic
impairment of the apoptosis-inducing activity of p53 does not increase carcinogenic risk, and its function as a tumor suppresser
is independent from that of inducing apoptosis, cell cycle arrest,and senescence (7-9). Hence, transient pharmacological p53
regulation is an ideal strategy for protecting normal tissues incancer therapy.

Figure 1 Specific protection of normal tissues during cancer treat-

ment. A mutation in the TP53 gene that encodes p53 is found in nearly
half of all human cancers. This is a major cause of differences in the
biological response to various stimuli including radiation between
normal and cancer tissues. The p53 regulator permits normal tissues
having wild p53 to be specifically protected against DNA damage-induced cytotoxicity, and is expected to function as a protective agent
for the normal tissues during radiotherapy and chemotherapy.

SEARCHING FOR CHEMICAL COMPOUNDS THATREGULATE RADIATION-INDUCED APOPTOSIS
In our initial attempts to identify chemical compounds that
regulate radiation-induced apoptosis, we found that sodium
orthovanadate (vanadate) acts on both the p53-mediated transcription-dependent and -independent pathways, and protects
mice from total-body 12 Gy-irradiation, which is a superlethal
dose that kills bone marrow stem cells, and also produces severe,
but not lethal effects itself, including damage to the intestines,
a process that is generally referred to as gastrointestinal death(10). Vanadate was originally identified as an inhibitor of theinduction of p41, which was detected in irradiated human T-cellleukemia MOLT-4 cells using a two-dimensional electrophoretic
analysis (11). In subsequent experiments we found that p41 is a
caspase-mediated cleavage product of a nuclear protein, called
SETβ (p42) (12). However, vanadate had no direct inhibitory
effect on caspase activity. Further analyses of the apoptotic
pathway upstream of caspase activation revealed that vanadate
inhibits p53. It is known that p53 acts as a transcription factor inan early response to radiation. In addition, it was also found that
vanadate induced a conformational change in p53 to an inactive
form without affecting the phosphorylation and accumulationof p53 after DNA damage, resulting in the loss of DNA binding
activity and the transcriptional activity of p53 target genes. Furthermore, the use of cell lines having different TP53 status and ap53 knockdown cell line revealed that the anti-apoptotic actionof vanadate was required for p53, leading us to conclude thatthe target molecule of vanadate is p53 (13). Moreover, vanadate
was superior to other known p53 inhibitors, such as pifithrin
α (PFTα) (14) and PFTμ (15) in suppressing p53-dependent
apoptosis, presumably because of its wide spectrum of anti-p53
activities that inhibited not only the transcription-dependent
pathway but the transcription-independent pathway as well, in
which p53 directly acts on mitochondrial Bcl-2 family molecules.
An excellent radioprotective activity was also demonstrated by invivo radioprotection assays where vanadate rescued 60% of micethat had been treated with total-body 12 Gy-irradiation, a procedure that killed all of the control mice that had been treated withvehicle or a representative p53 inhibitor, cyclic PFTα, within 12
days. This result clearly indicated that vanadate was the firstp53 inhibitor capable of rescuing gastrointestinal death induced
by total-body irradiation (TBI) (10). In addition, a follow-up
study revealed that vanadate functions, not only as a protective
agent, but also as a mitigative agent when administered “after”
the mice were exposed to radiation (16). These results suggestthat the blockade of both p53 pathways is an excellent strategy
for maximizing radioprotective effects by inhibiting the actionof p53.
However, according to reports by other investigators, the inhibition of apoptosis is effective for the protection of TBI-inducedbone marrow death, but it is not effective for protection against
gastrointestinal death. In addition, it was also reported that
increasing p53 is effective for protection against gastrointestinal
death (4). These conclusions were arrived at based on a series ofan abdominal irradiation experiments called sub-total-body irradiation (SBI), designed to avoid bone marrow death by shielding
the bone marrow of the mouse fore leg with lead. Indeed, an
innate immune agonist CBLB502 (17), which activates the p53
antagonist NF-κB, and also vanadate showed limited efficacy
against gastrointestinal death by around 12 Gy. These results
suggest that the efficacy of p53-suppressing radioprotectors may
be limited to exposure below levels of about 12 Gy, in which thehematopoietic syndrome and gastrointestinal syndrome are
combined. Therefore, in the case of a dose to the abdominal region that exceeds 13 Gy, enhancing the anti-cell death function

The Journal of Medical Investigation Vol. 66 August 2019of p53 seems to be appropriate. It has also been shown that p21(Cdkn1 a)-deficient mice are susceptible to gastrointestinal deathby abdominal irradiation. Moreover, super p53 mice, which haveone more copy of Trp53, show resistance to intestinal death by abdominal irradiation when compared with wild type mice. Taken
together, the p53-p21 pathway is quite important for radioresistance of the intestinal epithelium (4, 18). Clinically, severe bonemarrow suppression is not assumed in radiation therapy, except
for bone marrow transplantation, which is performed by TBI
with a chemotherapeutic agent. Therefore, we decided to search
for a new radioprotective agent, with the goal of protecting riskorgans in the abdominal and pelvic region against radiotherapy.

REGULATION OF p53 FUNCTION BY ZINC CHELATION
We focused on the degenerative action of vanadate on p53
that showed the highest radioprotective effect among the p53
inhibitors tested (10). It is known that this denaturation actionof p53 is caused by the dissociation of zinc ions (19) or substituting zinc with other metal ions (20) in zinc ion binding sites thatare located between the DNA-binding domain of p53 and target
DNA (Figure 2). Thus, a zinc (II)-chelating agent was consideredas the first candidate for use as a new radioprotective agent.
However, there are no reports showing that zinc (II) chelators
inhibited p53-dependent apoptosis. In addition, there wereconcerns regarding the influence of the wide range of chelating
toxicity. Therefore, we first attempted to search for zinc chelators(tridentate to hexadentate ligands) that inhibited p53-dependent
apoptosis, in an attempt to show the p53 denaturation effectwith low cytotoxicity. As a result, we found that Bispicen, atetradentate ligand, had excellent p53 inhibitory effects andapoptosis inhibiting effects (21). Although Bispicen was able toinhibit p53-dependent apoptosis in cultured cells, it did not show
protective effect against total-body irradiated mice, presumably
due to its chelating toxicity.

Figure 2 Structure of the p53/DNA complex created from the Pro-

tein Data Bank (PDB code : 1 TUP) (24) using a molecular viewer,
Molmil (25). The p53 molecule contains a zinc ion (red sphere) locating at the binding site to target DNA. Three cysteines (yellow) residues and one histidine (blue) residue constitute a zinc-finger structure(represented by stick bond model) in the DNA binding domain of the
p53 molecule (represented by ribbon model).

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8-HQ DERIVATIVES REGULATE p53 FUNCTIONSAND SUPPRESS RADIATION INJURIES
Therefore, we next searched for 8-hydroxyquinoline (8-HQ)based radioprotective agents. The characteristic feature of 8-HQ
is that the ligands are bidentately coordinated to the zinc (II) ion(Figure 3A). These bidentate chelating agents generally havea low chelating activity, and would be expected to show a low
toxicity to living cells and organisms. They were also expected
to affect the DNA-binding activity of p53 by virtue of being
accommodated into the niche between p53 and the target DNA
without depriving p53 of a zinc ion. We investigated whether thesynthesized 8-HQ derivative had an inhibitory effect against
p53-dependent radiation-induced apoptosis, and found thatseveral compounds showed anti-apoptotic effects (22). Amongthem, AS-2 (5,7-bis(N-methylaminosulfonyl)-8-hydroxyquinaldine) protected mice from an exposure of 8 Gy, which inducesbone marrow death (Figure 3 B, C) (23). However, most of the
8-HQ derivatives, including AS-2, that we have examined sofar, except for 5-chloro-8-quinolinol (5CHQ), showed radioprotective efficacies against bone marrow death, but had no effecton gastrointestinal death. The limited radioprotective efficacies
appeared to be the limit of radioprotection by p53 inhibition, as
predicted by results reported by Kirsch, et al. (4). However, wefound that 5CHQ had a unique radioprotective activity during
our evaluation of a series of 8-HQ derivatives. 5CHQ enhances
the p53-mediated induction of antiapoptotic p21 and suppresses
the induction of proapoptotic PUMA, which consequently leads
to the prevention of p53-dependent cell death (2). The protective
effect of 5CHQ on the mice bone marrow death after 7.5 Gy-TBI
had a limited effect, with half of the mice being rescued. However, its pharmacological effect of enhancing p21 induction wasconsidered to be effective for protecting against gastrointestinal
damage. Therefore, we performed SBI to avoid bone marrow
death, and examined the radioprotective effect on the acutegastrointestinal syndrome. As expected, 5CHQ showed a significant protective effect on the mice gastrointestinal death after
SBI, and its DRF was 1.3, which has reached the original target
value for radioprotection (Figure 4) (2).

Figure 3 Radioprotective effects of AS-2, a derivative of 8-HQ, onradiation-induced hematopoietic injury. A, 8-HQ and zinc-ion complex formation. B, Structural formula of AS-2. C, Thirty-day survival
rates in mice after total-body X-irradiation. Female ICR mice (8 wks)
received a single intraperitoneal injection of AS-2 (80 mg/kg) 30 min
before 8 Gy of total-body X-irradiation. AS-2 rescued 100% of the micefrom bone marrow death caused by the irradiation. (Modified form
ref. 23)

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Figure 4 5CHQ protects mice from gastrointestinal death induced by a high dose of abdominal γ-irradiation. Female ICR mice (8 wks) received a single intraperitoneal 5CHQ injection (60 mg/kg) 30 min before the irradiation.
Mice treated with 5CHQ showed a significantly higher survival rate at day-30 after the irradiation compared withmice that had been treated with DMSO. (Modified form ref. 2)

CONCLUSION
In this review, we summarize the efficacy of regulating p53
for preventing radiation injuries. Among the p53 regulators examined by the authors, 5CHQ was found to have a quite uniqueproperty in that it increased p53 functions that are associatedwith radioresistance without inhibiting the activity of p53. This
modulator may become a seed compound for developing newagents that assist cancer treatment in radiotherapy and chemotharpy. Further studies will clearly be needed to clarify detailed
pharmacological mechanism of 5CHQ and to assess potential
clinical applications by using animal tumor models.

ACKNOWLEDGEMENTS
This work was supported, in part, by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of
Science (grant number 16K10396).

CONFLICTS OF INTEREST
All authors declare no conflicts of interest.

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