VIII-Ⅱ-1. Project Research
Project 6

PR6

Analyzing Tumor Microenvironment and Exploiting its Characteristics forControlling Malignant Solid Tumors and Distant Metastatic Potential

S. Masunaga
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Human solid
tumors are thought to contain moderately large fractionsof quiescent (Q) tumor cells that are out of the cell cycle
and stop cell division, but are viable compared with established experimental animal tumor cell lines that havebeen employed for various oncology studies [1]. The
presence of Q cells is probably due, in part, to hypoxia
and the depletion of nutrition in the tumor core, which isanother consequence of poor vascular supply [1]. As aresult, Q cells are viable and clonogenic, but stop cell
division. In general, radiation and many DNA-damaging
chemotherapeutic agents kill proliferating (P) tumor cells
more efficiently than Q tumor cells, resulting in manyclonogenic Q cells remaining following radiotherapy or
chemotherapy [1]. Therefore, it is harder to control Q
tumor cells than to control P tumor cells, and many
post-radiotherapy recurrent tumors result partly from theregrowth of Q tumor cell populations that could not be
sufficiently killed by radiotherapy [1]. Further, sufficient
doses of drugs cannot be distributed within Q tumor cell
populations mainly due to heterogeneous and poor vascular distributions within solid tumors. Thus, one of the
major causes of post-chemotherapy recurrent tumors is aninsufficient dose distribution in the Q cell fractions.
With regard to boron neutron capture therapy (BNCT),
with 10B-compounds, boronophenylalanine-10B (BPA)
increased the sensitivity of the total cells to a greater extent than mercaptoundecahydrododecaborate- 10B (BSH).
However, the sensitivity of Q cells treated with BPA waslower than that in BSH-treated Q cells. The difference inthe sensitivity between the total and Q cells was greaterwith 10B-compounds, especially with BPA [2]. Q cells
showed greater potentially lethal damage repair (PLDR)
capacities than the total cells. -Ray irradiation and neutron irradiation with BPA induced larger PLDR capacities
in each cell population. In contrast, thermal neutron irradiation without the 10B-compound induced the smallest
PLDR capacity in both cell populations. The use of the10
B-compound, especially BPA, resulted in an increase in
the PLDR capacity in both cell populations, and made the
PLDR patterns of the both cell populations look likethose induced by  -ray irradiation [3]. In both the total
and Q tumor cells, the hypoxic fractions (HFs) immediately after neutron irradiation increased suddenly. Reoxygenation after each neutron irradiation occurred more
rapidly in the total cells than in the Q cells. In both cell
populations, reoxygenation appeared to be rapidly induced in the following order: neutron irradiation without
10
B-compounds > neutron irradiation following BSH administration > neutron irradiation following BPA administration >  -ray irradiation [4]. These findings concerning the difference in sensitivity, PLDR and reoxygenation
following neutron irradiation after 10B-compound administration were thought to be mainly based on the fact that
it is difficult to deliver a therapeutic amount of 10B fromcurrently used 10B-carriers throughout the target tumors,
especially into intratumor hypoxic cells with low uptake
capacities [5].

Therefore, the aim of this research project is focused on
clarifying and analyzing the characteristics of intratumor
microenvironment including hypoxia within malignant
solid tumors and optimizing cancer therapeutic modalities, especially radiation therapy including BNCT in theuse of newly-developed 10B-compound based on the revealed findings on intratumor microenvironmental characteristics.
RESEARCH SUBJECTS:
The collaborators and allotted research subjects (ARS)
were organized as follows;
ARS-1 (24P6-1): Optimization of Radiation Therapy
Including BNCT in terms of the Effect on a Specific
Cell Fraction within a Solid Tumor and the Suppressing
Effect of Distant Metastasis(S. Masunaga,Y. Sakurai, H. Tanaka, M. Takagaki, and
H. Matsumoto)
ARS-2 (24P6-2): Development of Hypoxic Microenvironment-Oriented 10B-Carriers(H. Nagasawa, S. Masunaga, K. Okuda, S. Y. Hirayama
and N. Tsurue)
ARS-3 (24P6-3): Clarification of Mechanism of Radio-Resistance in Cancer Using Optical Imaging at
Tissue Level(H. Harada, M. Hiraoka, S. Masunaga, S. Itasaka, M.
Ogura and M. Yoshimura)
ARS-4 (24P6-4): Analysis of Radiation-Induced
Cell-Killing Effect in Neutron Capture Reaction(R. Hirayama, S. Masunaga, Y. Sakurai, H. Tanaka and
Y. Matsumoto)
ARS-5 (24P6-5): Transdermal Drug Delivery Systemusing
Hyaluronan-Conjugated
Liposomes
as10
B-Carrier in Boron Neutron Capture Therapy for
Melanoma(S. Kasaoka, K. Hashimoto and S. Masunaga)
ARS-6 (24P6-6): Evaluation of Inclusion Complex ofCarborane Modified Kojic Acid and Cyclodextrin as
10
B-Carrier in Boron Neutron Capture Therapy(T. Nagasaki, S. Masunaga, M. Kirihata, H. Azuma, K.
Li, U. Shu, Y. Kagoshima, K. Hayashi, R. Kawasaki, Y.
Hattori and N. Kadono)
ARS-7 (24P6-7): Molecular design and synthesis andfunctional evaluation of anticancer and molecular targetting agents(Y. Uto, R. Tada, S, Masunaga)
ARS-8 (24P6-8): Analyzing biological effect of BNCT
from the viewpoint of the changes in oxygenation level.
(H. Yasui, S, Masunaga, O. Inanami, T. Yamamori, D.
Yamane)(Underline: Representative at each research group)
REFERENCES:[1] P. Vaupel, Semin. Radiat. Oncol. 14 (2004) 197-275.
[2] S. Masunaga et al., Int. J. Radiat. Oncol. Biol. Phys.
41 (1998) 1163-1170.
[3] S. Masunaga et al., J. Cancer Res. Clin. Oncol. 125(1999) 609-614.
[4] S. Masunaga et al., Int. J. Radiat. Oncol. Biol. Phys. 44(1999) 391-398.
[5] S. Masunaga et al., Clin. Exp. Metastasis 26 (2009)693-700.

局所腫瘍制御と転移抑制を同時に目指す治療法開発のためのがん微小環境解析とその応用（京大・原子炉）増永慎一郎
採択課題番号 24P6

プロジェクトPR6-1

Combined Treatment with Continuous Administration of Tirapazamine and
Mild Temperature Hyperthermia in -Ray Irradiation in Terms ofLocal Tumor Response and Lung Metastatic Potential

S. Masunaga, Y. Sakurai, H. Tanaka, K. Tano, M. Suzuki,
N. Kondo, M. Narabayashi, A. Maruhashi and K. Ono
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Many of thecancer cells in solid tumors are non-proliferating (quiescent), and many of the features of quiescent (Q) cells arestill unknown. To improve cancer treatment, the responseof Q cells to anticancer treatment should be determined,
since often tumor cells that are quiescent in situ are stillclonogenic.
The development of bioreductive agents that are particularly toxic to hypoxic cells is considered a promising approach to solving the problem of radio-resistant tumor
hypoxia in cancer radiotherapy. Tirapazamine (TPZ), a
lead compound in the development of bioreductive hypoxic cytotoxins, in combination with radiation has beenshown to be very useful for controlling solid tumors as awhole, especially for controlling Q tumor cell populations that are rich in hypoxic region. Tumor hypoxia results from either limited oxygen diffusion (chronic hypoxia) or limited perfusion (acute hypoxia, transient hypoxia or ischemic hypoxia). Chronically hypoxic tumor
cells existing at the rim of the oxygen diffusion distancecan be killed by just a single administration of TPZ.
Acutely hypoxic tumor cells occurring sporadicallythroughout solid tumors can be killed by TPZ duringlong-term continuous administration. Namely, thelong-term continuous administration of TPZ can kill both
chronically and acutely hypoxic tumor cells.
Mild temperature hyperthermia (MTH) was reported toincrease the response of tumors to radiation by improving
oxygenation through an increase in tumor blood flow.
Further, MTH was also shown to enhance the tumor response, especially of the intratumor Q cell population, to
TPZ.
Metastasis is a leading cause of cancer deaths and involves a complex, multistep process by which tumor cells
disseminate to distant sites to establish discontinuous
secondary colonies. It was reported that acute and cyclic,
but not chronic, hypoxia significantly increased thenumber of spontaneous lung metastases in mice by a factor of about 2, and that this effect was due to the influence of the acute hypoxia treatment on the primary tumor
and not to other potential effects of the treatment such as
damage to the lung epithelium. Based on this report, werecently reported the significance of injections of anacute hypoxia-releasing agent, nicotinamide, into tumor-bearing mice as a combined treatment with highdose rate -ray irradiation in terms of reducing the num-

採択課題番号 24P6-1

ber of lung metastatic nodules.
Here, using a readily metastasizing murine melanoma
cell line, we tried to analyze the usefulness of combined
treatment with continuous long-term administration of
TPZ and MTH in radiotherapy with -rays in terms of
local tumor response and lung metastatic potential. Concerning the local tumor response, the effect not only on
the total (= proliferating (P) + Q) tumor cell population
but also on the Q cell population was evaluated using ouroriginal method for selectively detecting the response of
Q cells in solid tumors [1].
MATERIALS AND METHODS: B16-BL6 melanoma
tumor-bearing C57BL/6 mice were continuously given
5-bromo-2’-deoxyuridine (BrdU) to label all proliferating(P) cells. The tumor-bearing mice then received -ray
irradiation after a single intraperitoneal injection or 24 hcontinuous subcutaneous infusion of TPZ, either with orwithout MTH. Immediately after the irradiation, cells
from some tumors were isolated and incubated with acytokinesis blocker. The responses of the Q and total (= P+ Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumor-bearing mice, 17 days after
irradiation, macroscopic lung metastases were enumerated.
RESULTS: Continuous administration elevated the sensitivity of both the total and Q cells, especially the total
cells. MTH raised the sensitivity of Q cells more remarkably in both single and continuous administrations,probably because of more exposure to TPZ in intermediately hypoxic areas derived mainly from chronic hypoxia
through MTH. With or without irradiation, TPZ, especially administered continuously and combined with
MTH, decreased the number of lung metastases.
DISSCUSSION: The combination of continuous
long-term administration of TPZ and MTH in -ray irradiation was thought to be promising because of its potential to enhance local tumor response and repress lungmetastatic potential [2].
It was elucidated that control of the chronic hypoxia-rich
Q cell population in primary solid tumors has the potential to impact the control of local tumors as a whole,while control of the acute hypoxia-rich total tumor cell
population has the potential to impact the control of lungmetastases. Namely, in conventional radiotherapy, continuous TPZ administration combined with MTH isthought to have a great potential to control both local
solid tumors and lung metastases from the local tumors.
REFERENCES:[1] S. Masunaga et al., J. Radiat. Res. 43 (2002) 11-25.
[2] S. Masunaga, et al., Int. J Hyperthermia 28 (2012)
636-644.

腫瘍内各特定細胞集団の制御と転移制御をも目指す
癌治療(特に BNCT)の最適化

プロジェクト（京大・原子炉）増永慎一郎、櫻井良憲、田中裕基（藍野学院短期大）高垣政雄（福井大）松本英樹（済生会千里病院）山本直典（大阪医大）弘田祐己

PR6-2Development of Direct Intra-Cellular Drug Delivery System Basedon Lipidated Peptides, Pepducins

M. Tsuji, S. Masunaga1, K. Okuda, T. Hirayama andH. Nagasawa

also demonstrated before the treatment with the probe.
(a)

FRET

(b)

Flip

Laboratory of Medicinal & Pharmaceutical Chemistry,
Gifu Pharmaceutical University
1
Research Reactor Institute, Kyoto University

Dab

HS

FAM

S

Dab

SH

GSH

FAM
Pep13-FL-SS-Dab

cytosol

FRET(c)

INTRODUCTION: One of the most important issues
in the development of the boron carrier for boron neutron
capture therapy (BNCT) is the innovation of versatiledrug delivery systems across the cell membrane. We focus on the unique cell-penetrating feature of lipidated
peptides called pepducins discovered as allosteric modulators of GPCRs[1]. Unlike classical GPCR modulatorsthat act at the extracellular ligand-binding site of receptors without crossing the plasma membrane barrier,pepducins are supposed to interact with the intracellular
portion of target receptors on the inner leaflet of the lipid
bilayer. Pepducins consist of a synthetic peptide derived
from the appropriate intracellular loop domains of
GPCRs, typically 10–20 amino acids in length, and ahydrophobic moiety, e.g., palmitate. The lipid moiety
anchors in the plasma membrane and sequentially flips
across the membrane. Then, pepducins directly penetrate
cellular membrane not by endocytosis to interact with theintracellular loop domain of the GPCR. We envisage thatthe transmembrane system of pepducin could be usefulfor the development of the cell penetrating boron carrier.
We report the design and synthesis of novel bioreductively activatable FRET probes to analyze the transbilayer movement of lipidated peptides in living cells byfluorescence microscopy. We further demonstrate substantial functions of the lipidated peptide moiety to flip
across the membrane and to achieve rapid delivery offluorescein as a cargo into the cytosol using
pepducin-based probes [2].
EXPERIMENTS: To visualize the flipping movementof pepducins across the membrane in live cells, we designed two types of FRET-based imaging agents,
Pep13-FL-SS-Dab (1) and Pep13-Dab-SS-FL (2), whichwere based on the structure of the PAR1 pepducin agonist,
P1pal-13, developed by Covic et al.[1]. he probes arecomposed of pepducin and a FRET component consistingof 5(6)-FAM as fluorophore and dabcyl as quencher,connected through disulfide bond linkage (Figure 1). All
the peptide parts of the probes were obtained by Fmoc
solid-phase synthesis (SPPS). We performed live cell
imaging experiments using MCF-7 cells with probe 1 and
2 and confocal microscopy. MCF-7 cells were treatedwith the probes at the concentration of 1 µM for 15 min,and then observed with the confocal laser-scanning microscope after changing the medium and washing. Nuclear co-staining with Hoechst 33342 (10 mg/mL) was

S

FAM

S

Flip
S

GSH

Dab

FAM
HS
SH

Dab
Pep13-Dab-SS-FL

cytosol

Fig. 1 Design and synthesis of bioreductively activatable FRET based
probes.

RESULTS: Probe 1-treated cells showed a distinct
membrane- localized fluorescence pattern (Figure 2a).
While, probe 2-treated cells showed diffuse cytosolic
fluorescence (Figure2b). When the cells(a) 1
were pre-incubatedwith N-ethyl- maleimide (NEM) before the treatment(b) 1+NEM
with probe 1 in order to deplete reduced thiols both inmedium and in thecells, fluorescence(c) 112
signals were significantly low (Figure3c), indicating thatthefluorescenceenhancement is defFig 2. Confocal microscopic images
initely attributed toand bright field (BF) images of live
MCF-7 cells. (a) 1 μM of
free thiols.
Pep13-FL-SS-Dab (1) for 15 min . (b)CONCLUSION:
1 μM of Pep13-Dab-SS-FL (2) for 15
min. (c) Cells were incubated with 50
We developed novel
μM of NEM prior to treatment with 1bioreductively actiμM of 1 for 15 min.
vatableFRETprobes based on pepducins. These probes enabled for the
first time live cell imaging of the unique transbilayer
movement of pepducins, which revealed that the lipid
moiety of pepducin plays a pivotal role to flip across theplasma membrane and to tether its peptide moiety to theinner leaflet of the bilayer. Furthermore, these studies
demonstrated the great potential of applying the present
probe architect to simple and straightforward intracellular
drug delivery systems. Currently, we are optimizing the
peptide sequence and lipid structure for intracellular targeting of different cells for the development of
pepducin-based drug delivery systems.
REFERENCES:[1] L. Covic et al., Proc. Natl. Acad. Sci. USA., 99(2002) 643–648.
[2] M. Tsuji et al., Org. Biomol. Chem., 11 (2013),
3030-3037.
Green fluoro

Hoechst 33342 + Bright field

採択課題番号 24P6-2 低酸素微小環境を標的とするボロンキャリアの開発
プロジェクト（岐阜薬大・薬化学）辻美恵子、鶴江範彦、奥田健介、平山 祐、永澤秀子（京大・原子炉）増永慎一郎

PR6-3

Upstream Activators of HIF-1 as Targets for Radiosensitization

H. Harada, S. Masunaga1 and M. Hiraoka2
Group of Radiation and Tumor Biology, Career-Path
Promotion Unit for Young Life Scientists, Kyoto University.
1
Research Reactor Institute, Kyoto University
2
Department of Radiation Oncology and Image-applied
Therapy, Graduate School of Medicine, Kyoto University.

INTRODUCTION: Effect of conventional therapies
for cancer patients, such as chemotherapy, radiation
therapy, and surgery, is known to be affected by various
intracellular and extracellular factors in malignant tumors.
One of the most influential factors is activity of a transcriptional factor, hypoxia-inducible factor 1 (HIF-1).
HIF-1 is a heterodimer composed of an -subunit(HIF-1) and a -subunit (HIF-1) and its activity ismainly dependent on stability and transactivating activityof HIF-1, which are strictly regulated by
-KG-dependent
enzymes,
prolyl-4-hydroxylases(PHDs) and factor-inhibiting HIF-1 (FIH-1), respectively.
PHDs hydroxylate proline residues, P402 and P564, inthe oxygen-dependent degradation domain (ODD domain) of HIF-1 in both oxygen- and -KG-dependent
manner. The hydroxylations trigger polyubiquitilationand subsequent proteasomal degradation of HIF-1.
FIH-1 hydroylates asparagine residue, N803, in the
C-terminal transactivation domain (C-TAD) of HIF-1,resulting in the suppression of its transactivating activity
under normoxic conditions. On the contrary, HIF-1 becomes active under hypoxic conditions because of theinactivation of these hydroxylases, and then, interacts
with its binding partner, HIF-1. Resultant heterodimer,
HIF-1, binds to its cognate enhancer sequence, the hypoxia-responsive element (HRE), and induces transcriptions of various genes related to the improvement of oxygen-availability (angiogenesis), adaptation of cellular
metabolism to hypoxia (metabolic reprogramming), andescape from hypoxia (invasion and metastasis of cancer
cells). Indeed, clinical as well as basic researches haveshowed that HIF-1 expression level correlates with apoor prognosis and incidences of both tumor recurrence
and distant tumor metastasis, justifying targeting HIF-1
and its upstream activators for cancer therapy. However,molecular mechanisms behind the activation of HIF-1
have not been fully elucidated yet, which makes it difficult to develop strategies for eradication of cancers.
EXPERIMENTS & RESULTS: In order to explore
novel genes which are responsible for the activation of
HIF-1, we established a new screening method (Fig. 1).
Using an artificial promoter composed of five repeats of

採択課題番号 24P6-3

hypoxia-response elements (HRE) and human CMV
minimal promoter (5HRE promoter: 5HREp), we newly
constructed a plasmid expressing blasticidin S resistantgene (bsd) in a HIF-1-dependent manner (5HREp-bsd
herein). NIH3T3 cells stably transfected with the
5HREp-bsd (NIH3T3/5HREp-bsd) were sensitive to
blasticidin S under normoxic conditions because of insufficient expressions of both HIF-1 and BSD. After introducing
human
cDNA
library
intothe
NIH3T3/5HREp-bsd cells, we cultured them in blasticidin S-containing medium under normoxic conditions
with an expectation that some cDNAs encode upstream
activators of HIF-1 and consequently induce BSD expression.

Fig. 1. Schematic Diagram of the Gene Screening Method
for Novel Activators of HIF-1.

Through such a gene screening experiment, we could
actually acquire surviving colonies which showed blasticidin S-resistance compared to the parental cell line. We
rescued cDNA from one of the colonies by PCR and
identified it as the gene encoding wild type isocitrate dehydrogenase 3alpha (IDH3), a subunit of IDH3 composed of two , one , and one  subunits. In order toexamine whether IDH3 induces HIF-1 activity, we performed a luciferase assay using the 5HREp-luc reporter
gene, which expresses Luciferase under the control of
5HREp. Overexpression of IDH3 resulted in
up-regulation of HIF-1 activity in various cancer celllines under normoxic and hypoxic conditions. We plan to
examine whether targeting IDH3 enhances therapeutic
effect of radiation in next experiments [1-3].
REFERENCES (papers published from our lab):[1] Zhu Y et al., Oncogene, 32 (2013) 2058-2068.
[2] Yoshimura M et al., Biomed Res Intl., (2013) Article
ID 685308.
[3] Yeom CJ et al., Int J Mol Sci. 13 (2012)
13949-13965..

プロジェクト低酸素誘導性因子 1（HIF-1）を活性化する新規遺伝子の探索と機能解析、および局所腫瘍制御への展開（京大・キャリアパス）原田浩（京大・原子炉）増永慎一郎（京大院・医）平岡真寛

BPA Concentration-Dependency of HSG Cell Inactivation by
Neutron Irradiation

R. Hirayama, Y. Matsumoto, S. Masunaga1, Y. Sakurai1,
H. Tanaka1 and Y. Furusawa

100 colonies per dish. After 14 days of incubation, thecolonies were fixed with 10 % formalin solution andstained with 1 % methylene blue in water. Colonies
consisting of more than 50 surviving cells were scored.
The survival curves were fitted by the single-hit model:
SF = exp (-α∙D). SF and D are the surviving fraction
and the dose (Gy-1), respectively.

Research Center for Charged Particle Therapy, National
Institute of Radiological Sciences
1
Research Reactor Institute, Kyoto University

RESULTS: The survival curves for neutrons are shownin Fig. 1. The D10 value of neutron with and without
BPA were 0.2 and 2.8, respectively. The D10 value of
X-rays was 4.7 Gy. The RBE values for with and without BPA were 19.9 and 1.7, respectively. The survival
curves of the coefficient of determination (R2) wereabove 0.8. We will retest and add the dose points and
BPA concentrations. These results suggest that he large
RBE was induced by low energy heavy particles (α andLi) from boron neutron capture reaction.

0.1

0.01

0.001

EXPERIMENTS: Human salivary gland tumor (HSG)
cells were grown in Eagle’s minimum essential medium
supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin)
under humidified air with 5% CO2 at 37˚C. The HSG
cells were incubated with 25 µg/ml BPA for 3 h before
irradiation. The cells were suspended at a density of
about 7 × 105 cells/ml containing 25 µg/ml BPA. The
cells in Polypropylene tubes (NUNC) were irradiated at
the remodeled heavy water facility at the KURRI.
Total fluencies of thermal neutron, epithermal neutronand fast neutron were measured by means of gold foil
activation analysis. The gamma ray dose including
secondary gamma rays was measured with a thermo luminescence dosimeter. Boron concentrations in thecells were taken to be equivalent to those in the medium
as reported previously [4].
After irradiation, cells were seeded in triplicate onto 60
mm (Φ) culture dishes at densities to give approximately

1

1Surviving Fraction

INTRODUCTION:
Excellent dose distribution ofneutron capture reaction of boron atom induces high relative biological effectiveness (RBE) and the low oxygen
enhancement ratio (OER). These phenomena are commonly assumed to be an interaction between cells andlow energy heavy particles (α and Li) resulting from theboron atom fissions in the cells. However, there has
been little study done concerning the action of the particles on living cells. We have investigated contributionsof indirect actions of radiation in cell killing by heavy
ions with radical scavenger that selectively reduces theindirect action [1-3].
Therefore, it is important that how these mechanisms
can be made to clear through a thorough basic research inboron neutron capture therapy is urgently discussed.
The main object of this year is to make clear an influenceof boron neutron capture reaction at high concentration of
10
B-para-boronophenylalanine (BPA) on HSG cell inactivation.

0

Surviving Fraction

PR6-4

n
n + 25ppm BPA
200kV X-raysnn + 25ppm BPA
200kV X-rays

0.1

0.01
2
4
6
8
Physical
0.001 Dose (Gy)

0

2
4
6
8
Physical Dose (Gy)Fig. 1. Survival curves for HSG cells exposed to neutrons in the absence (circle) and presence (square) of 25µg/ml BPA. The survival parameters were calculated
from the data by a curve fitting using: SF = exp (-α∙D).

REFERENCES:[1] A. Ito et al., Radiat. Res., 165 (2006) 703-712.
[2] R. Hirayama et al., Radiat. Res., 171 (2009) 212-218.
[3] R. Hirayama et al., Radiat. Phys. Chem., 78 (2009)1175-1178.
[4] Y. Sakurai and T. kobayashi, Nucl. Instrum. Methods
Phys. Res. Sect. A., 453 (2000) 569-596.

採択課題番号 24P6-4
中性子捕捉反応における細胞致死機構の放射線作用解析
プロジェクト（放医研・重粒子医科学センター）平山亮一、松本孔貴、古澤佳也（京大・原子炉）増永慎一郎、
櫻井良憲、田中浩基

PR6-5

Electroendocytosis of Boron-Conjugated Hyaluronic Acid NanoParticles
for Tumor Targeting in Boron Neutron Capture Therapy

S. Kasaoka, K. Hashimoto, S. Masunaga1, Y. Sakurai1,
H. Tanaka1 and K. Ono1
Department of Pharmaceutical Science, Hiroshima International University
1
Research Reactor Institute, Kyoto University

INTRODUCTION: Hyaluronic acid (HA) is a candidate for active targeting ligand to melanoma, many ofwhich overexpress the hyaluronic acid receptors CD44
and RHAMM [1]. Electroendosytosis, low electric fields(LEF) induced endocytic-like process, is novel concept as
a complementary method to uptake of macromoleculesby cells [2]. Such uptake is induced by the production ofhydrogen ions with electrolysis [3]. The elevated concentration of extracellular hydrogen ions which bind to theanionic charged sites on the external membrane leaflet
would result in local reduction of charge density on theexternal membrane surface. In this study, we have developed a novel boron delivery system using the combination of the electroendosytosis and HA conjugated withborocaptate (BSH) for boron neutron capture therapy(BNCT). This BSH-bearing HA nanoparticles(B-HA-NPs) are expected to deliver boron atoms intomelanoma cells by electoroendosytosis and receptor-mediated endocytosis for antitumor application in
BNCT.
EXPERIMENTS: BSH was dissolved in acetone andadded to N-Succinimidyl 6-Maleimidobenzoate with triethylamine catalysis. The mixture was incubated at room
temperature for 2 h under argon. Sodium hyaluronate(M.W. = 4,200, 78,000, 960,000) was dissolved in deionized water. To this solution, the highly acidic ion exchange resin, Dowex-100, was added, and the slurry wasstirred for eight hours. The acidic solution was neutralized with 0.2M tetrabutylammonium hydroxide, forming
a quaternary ammonium salt of hyaluronate and the tetrabutylammonium group (HA-TBA). The solution wasfrozen and lyophilized to yield the dry product. The
HA-TBA was dissolved in DMSO and added to BSH
derivative was of conjugation with diethylamine catalysis.
The mixture was incubated at room temperature for 48 hunder argon. The solution was dialyzed and lyophilized

採択課題番号 24P6-5

to yield the dry product.
B16F10 murine melanoma cells were pre-incubated with
25 ppm of B-HA-NPs at 37°C for 2 hours before neutron
irradiation. The exposure of cells to pulsed LEF was performed in 24 well plate between parallel stainless steel
electrodes (20 V/cm). The cells were rinsed twice in PBS
and suspended in fresh medium. After neutron irradiation
the cells were plated into plastic Petri dishes 60 mm indiameter at 200 cells per dish. They were incubated foran additional 7 days to allow colony formation.
RESULTS: B-HA-NPs had high stability (89-95%) in
the retention of 10B during storage at 4-37°C for 2 weeks.
All borocaptate-loaded formulations had no cytotoxic
effects. B-HA-NPs (M.W.=78,000, 960,000) were readilybound to melanoma cells, and were internalized by electroendocytosis and receptor-mediated endocytosis. As
shown in Fig. 1, B-HA-NPs with exposure to pulsed LEF
showed higher suppression of growth of melanoma cells
than without exposure to pulsed LEF. This result suggested novel boron delivery system using the combination of the electroendocytosis and receptor-mediated endocytosis of B-HA-NPs is useful in BNCT.

Fig. 1. Suppression of the colony formation of B16F10
cells after in vitro BNCT.

REFERENCES:[1] D. Peer and R. Magalit, Neoplasia, 6 (2004) 343-353.
[2] Y. Antov et al., Biophy J., 88 (2005) 2206-2223.
[3] N. Ben-Dov et al., PLoS One, 7 (2012) 50299-50306.

新規皮膚透過性メラノーマ標的化ボロン送達システムを用いた中性子捕捉療法の開発（広国大・薬）笠岡 敏、橋本佳奈（京大・原子炉）増永慎一郎

プロジェクトPR6-6

Kojic Acid-Appended Carorane/Hydroxypropyl--Cyclodextrin Complex
for BNCT of Murine Melanoma

R. Kawasaki, T. Nagasaki, J. Li, H. Azuma, S. Masunaga1,
Y. Sakurai1, M. Kirihata2 and Y. Hattori2
Graduate School of Engineering, Osaka City University
1
Research Reactor Institute, Kyoto University
2
Graduate School of Life and Environmental Sciences,
Osaka Prefecture University

INTRODUCTION: Boron neutron capture therapy(BNCT) has been attracted great deal of attention as apotentially useful modality for malignant melanoma [1].
The selective and effective delivery of 10B compounds
into melanoma cells is a key process in order to improvethe efficacy of melanoma BNCT. Kojic acid is wellknown to work as an excellent whitening agent for melanocytes by a strong tyrosinase inhibition [2]. This factsuggests that kojic acid possess a specific affinity formelanocytes. Previously, we evaluated kojic acid appended o-carborane (CKA) as BNCT boron agent formelanoma. Kojic moiety functionalized not only to accumulate selectively into melanoma cells, but also to localize nuclei. As these results indicated that CKA waspromising to be a boron agent for melanoma BNCT,
herein, we evaluated CKA as BNCT agent in vivo.
EXPERIMENTS: Water-soluble complex of CKA
with hydroxypropyl--cyclodextrin (CKA/HP--CD, Fig.
1) was prepared with high-speed vibration milling technique [3]. B16BL6 murine melanoma cells were grown in
RPMI mediums containing 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin at 37°C with CO2 atmosphere. C57/BL6J mice were transplanted with 2 x 105
B16BL6 cells into the left thigh. After 10 days of transplantation, intraperitoneal injections of boron compounds(1500 ppm or 4500 ppm of 10B concentration; 200 l)were carried out for the evaluation of tumor accumulation
and BNCT effect. Boron concentration was measured by
ICP-AES (Vista-MPX, Seiko instruments Inc.).

Fig. 1. Water-soluble CKA/HP-β-CD complex.

採択課題番号 24P6-6

Fig. 2. Tumor-accumulation of boron in tumor-bearing
mice using CKA/HPβ-CD (A). The tumor to normal tissue
ratio (T/N) (B).

Fig. 3. Survival curves of tumor-bearing mice after
neutron irradiation (5.0 x 1012 fluence cm-2) in KUR
atomic reactor.
RESULTS AND DISCUSSION:
In vivo tumor-accumulation studies were performed withCKA/HP--CD complex (Fig. 2). After 1 hour of administration, highest concentration (ca. 8 ppm of 10B) was
observed. These phenomena are consistent with in vitro
melanoma selectivity. The antitumor effect of BNCT was
evaluated on basis of the survival of mice (Fig. 3). All the
non-treated mice did not survive after 22 days of neutron
irradiation, and their average survival rate was 14 days.
When 1500 ppm of 10B solutions were used, average survival rate were 19 and 22 days with L-BPA/fructose and
CKA/HP--CD complex, respectively. Moreover, longsurvival rates (25 daays) were observed with 4500 ppm
of 10B solutions of CKA/HP--CD complex. Kojic acid-appended carborane (CKA) is promising to be an efficient boron agent toward melanoma BNCT.
REFERENCES:[1] H. Fukuda et al., Radiat. Res., 138 (1994) 435-442.
[2] Y. Mishima, Pigment Cell Res., 14 (2001) 47-70.
[3] K. Komatsu et al., J. Chem. Soc., Perkin Trans. 1,(1999) 2963-2966.

メラノーマ中性子捕捉療法への適応を目指した薬剤送達システムに関する研究

プロジェクト

（阪市大・工）長﨑 健、東 秀紀、李 家暐、鹿子嶋祐太、林高一郎、河崎 陸、櫻本昌士、湯川寛子（阪府大・生命環境科）切畑光統、服部能英、門野尚之（京大・原子炉）増永慎一郎

PR6-7

Design, Synthesis and Phrmacokinetic Analysis of Boron Tracedrugs UTX-51

Y. Uto, R. Tada, H. Hori and S. Masunaga1
Institute of Technology and Science, Graduate School
The University of Tokushima
1
Research Reactor Institute, Kyoto University

INTRODUCTION: The evaluation of pharmacokinetic
properties and ADME-tox of drug candidates, being under development, and drugs, available on the market, hasrecently become an increasingly important factor of drug
discovery and development, because of increased needsof targeted drugs with less adverse effects. This needs
accelerate medicinal chemists to develop drugs withhigher traceability, even in their whole lifetime. Radiolabeled compounds have been still available with someinherent problems such as their half-life and the specificregulation of experimental facilities. For the purpose ofovercoming these problems and creating traceable drugs
without RI forever, we are developing boron tracedrugs
with their “on demand” traceability and their physicalforce for neutron dynamic therapy (NDT). We previously
developed boron tracedrugs UTX-42, UTX-43, and
UTX-44, which possess antioxidant potency [1]. Among
boron tracedrugs tested previously, we choose the boron
tracedrug, UTX-51, for our present NDT study to explore
their dynamic, beyond chemical, effects when acquiredby weak thermal neutron irradiation of human serum Gc
protein, which is related with serum protein-quality control, treated with the boron tracedrug UTX-51.
EXPERIMENTS: The curcuminoid boron tracedrugs
UTX-51 (it contains boron isotopes, B-10 and B-11 with
their natural abundance ratio) was used for neutron dynamic therapy. Thermal neutron irradiation was performed using a reactor neutron beam with a cadmium(Cd) ratio of 9.4. The neutron fluence was measured fromthe radioactivation of gold foils at the front of the sample
tubes, and the average neutron fluence determined fromthe values measured was used. Contaminating γ-ray doses, including secondary γ-rays, were measured withthermoluminescence dosimeter powder at the front of thesample tubes. The absorbed dose was calculated using theflux-to-dose conversion factor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed to detect the decomposition by thermal
neutron irradiation of human serum Gc protein treatedwith the boron tracedrug UTX-51. The evaluation of thepharmacokinetic property of UTX-51 is performed using
chick embryo model. After UTX-51 was intravenously
injected into blood vessel in the chorioallantoic membrane, each internal organs were isolated and the boron
concentration was measured by neutron capture prompt
gamma activation analysis (PGAA).

採択課題番号 24P6-7

RESULTS: The combination of 0.5 nmol Gc protein (54KD, 27 µg) with three different B-10 concentration of 10,
25. and 50 nmol of the boron tracedrug UTX-51 (MW
416.12 g/mol; 50 nmol or 20.8 µg, 125 nmol or 52.0 µg,
and 250 nmol or 104.0 µg, for 10 nmol B-10, 25 nmol
B-10, and 50 nmol B-10, respectively) showed a decreasein band intensity after neutron irradiation (Fig. 1). The
pharmacokinetic property of UTX-51 by chick embryomodel was shown in Fig. 2. Although the concentrationof UTX-51 in the heart and a brain decreased with time
progress, while it’s rose 12 hours after injection in bloodand decreased after that.
In conclusion, all doses of the boron tracedrug UTX-51
caused destructive dynamic damage against Gc protein
during thermal neutron irradiation.

Fig. 1. NDT of Gc protein using a boron tracedrug
UTX-51.

Fig. 2. Pharmacokinetic property of UTX-51 by chickembryo model.
REFERENCES:[1] E. Nakata et al., Adv. Exp. Med. Biol., 737 (2012)
301-306.

ホウ素を有する抗癌剤・分子標的薬剤の分子設計・合成
プロジェクトと機能評価（徳島大・ソシオテクノサイエンス）宇都義浩、多田 竜、堀 均（京大・原子炉）増永慎一郎

PR6-8The Effect of Boron Neutron Capture Reaction on the Cell Survival ofTumor Cells Preconditioned with Intermittent Hypoxia

H. Yasui, M. Nagane, S. Masunaga1, T. Yamamori and
O. Inanami
Laboratory of Radiation Biology, Graduate School of
Veterinary Medicine, Hokkaido University
1
Research Reactor Institute, Kyoto University
INTRODUCTION: Hypoxia has been known to be afeature associated with tumor radioresistance. Recently,
in addition to chronic hypoxia due to the limitation of theoxygen diffusion, intermittent hypoxia whose frequency
can range between a few cycles per minutes to hours is
receiving increased attention [1]. It is because this type ofhypoxia has been reported to have an influence on tumor
malignancy through the up-regulation of pro-survival
pathways [2]. Our previous study revealed that the preconditioning of murine squamous cell carcinoma(SCCVII) cells with intermittent hypoxia decreased their
sensitivity to X-irradiation compared to persistent hypoxia or normoxia. However, it has not been investigatedwhether intermittent hypoxia induces cellular radioresistance to the high-linear energy transfer (LET) radiation
as well as the low-LET X-irradiation. It is well known
that low energy heavy particles (α and Li), produced fromboron neutron capture reaction, induces high relative biological effectiveness (RBE). In this study, we aimed toreveal the effect of the preconditioning of intermittent
hypoxia on the reproductive cell death induced by boron
neutron capture reaction in SCCVII cells.
EXPERIMENTS: SCCVII cells were grown in minimum essential medium alpha supplemented with 10%
fetal bovine serum under humidified air with 5% CO 2 at
37°C. Two million cells attached to a 60-mm plastic dishwere pretreated with persistent hypoxia for 4 hours orintermittent hypoxia with 4 cycles of 1-hour hypoxia interrupted by 30-minute reoxygenation, according to theprotocol illustrated in Fig. 1. Hypoxic induction was performed by placing dishes in the commercial hypoxic
chamber (MIC-101; Billups-Rothenberg Inc.) and continuously passing a gas mixture of 95% N2 and 5% CO2.
Concomitant with hypoxic treatment,
10 µg/ml
10
B-para-boronophenylalanine (BPA) was incorporated tocells for 4 hours prior to irradiations. Cell suspensions inpolypropylene tubes (2×106 cells/tube) were irradiatedunder normoxia by neutrons from the 1 MW Research
Reactor at the KURRI. The total neutron fluencies were
measured by gold foil activation analysis. The gamma-ray dose including secondary gamma rays was measured with a Mg2SiO4 (Tb) thermo luminescence dosimeter. The calculation of total absorbed dose was performed
according to the previous report [3]. Irradiated cells were

採択課題番号 24P6-8

rinsed, counted, and then diluted to plate onto triplicate a
60-mm plastic dish. After 7 days’ incubation, colonies
were rinsed with PBS, fixed by 10% formalin solution
and stained by 1% methylene blue. The number of colonies consisting of more than 50 cells was counted. The
surviving fraction at each dose was calculated with respect to the plating efficiency of the non-irradiated control and dose-response curves were plotted.

Fig. 1. Schematic experimental protocols.
RESULTS: We show the preliminary result of clonogenic assay in Fig. 2. It is unclear whether the survival curve
has an initial linear slope derived from contaminatedgamma-ray in each group. The enhancement rate by
BNCT obtained from the D10 values (1.75 Gy and 4.00
Gy for BPA[+] and BPA[-], respectively) was about 2.29.
There was no difference in the radiosensitivities among
the three groups pretreated with normoxia, persistent hypoxia, or intermittent hypoxia. We need to make moredetailed investigations to compare with the case of photon beam.

Fig.2. Dose-response curves for SCCVII cells.
REFERENCES:[1] H. Yasui et al., Cancer Res., 70 (2010) 6427-6436.
[2] M.W. Dewhirst et al., Radiat. Res., 172 (2009)653-665.
[3] Y. Kinashi et al., Radiat. Oncol., 6 (2011) 106-113.

酸素分圧変動に着目した中性子捕捉療法のプロジェクト
生物学的効果に関する基礎的検討（北大・獣医）安井博宣、永根大幹、山盛 徹、稲波 修（京都大・原子炉）増永慎一郎