Basic characterization of 64Cu-ATSM as a radiotherapy agent.

Abstract

64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an attempt to elucidate the radiobiological basis of 64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line models. Cells were incubated with 64Cu-ATSM, and the dose-response curves were obtained by performing a clonogenic survival assay. Radiation-induced damage in DNA was evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and propidium iodide staining methods. Washout rate and subcellular distribution of 64Cu in cells were investigated to further assess the effectiveness of 64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the flow tracer analog Cu-pyruvladehyde-bis(N4-methylthiosemicarbazone). In this study, 64Cu-ATSM was shown to reduce the clonogenic survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up 64Cu-ATSM and radioactive 64Cu was highly accumulated in the cells. In the 64Cu-ATSM-treated cells, DNA damage by the radiation emitted from 64Cu was detected, and inhibition of cell proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced by radiation were observed following 64Cu-ATSM treatment. The majority of the 64Cu taken up into the cells remained in the postmitochondrial supernatant (the cellular residue after removal of the nuclei and mitochondria), which indicates that the beta- particle emitted from 64Cu may be as effective as the Auger electrons in 64Cu-ATSM therapy. These data allow us to postulate that 64Cu-ATSM will be able to attack the hypoxic tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the beta- particle decay of 64Cu.

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Basic characterization of64Cu-ATSM as a radiotherapy agent
Atsushi Obataa,b, Shingo Kasamatsuc, Jason S. Lewisd, Takako Furukawaa, Shinji Takamatsua,
Jun Toyoharae, Tatsuya Asaia, Michael J. Welchd, Susan G. Adamsd, Hideo Sajib,
Yoshiharu Yonekuraa, Yasuhisa Fujibayashia,*
aBiomedical Imaging Research Center, University of Fukui, Matsuoka, Fukui 910-1193, Japan
bGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8151, Japan
cJFE Fukui Branch, University of Fukui, Matsuoka, Fukui 910-1193, Japan
dMallinckrodt Institute of Radiology, Washington University Medical Center, St. Louis, MO 63110, USA
eResearch Center, Research and Development Division, Nihon Medi-Physics, Co. Ltd., Chiba 910-1193, Japan
Received 24 March 2004; received in revised form 29 July 2004; accepted 20 August 2004
Abstract
64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an
attempt to elucidate the radiobiological basis of64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line
models. Cells were incubated with64Cu-ATSM, and the dose–response curves were obtained by performing a clonogenic survival assay.
Radiation-induced damage in DNAwas evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and
propidium iodide staining methods. Washout rate and subcellular distribution of
effectiveness of64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the
flow tracer analog Cu-pyruvladehyde-bis(N4-methylthiosemicarbazone). In this study,64Cu-ATSM was shown to reduce the clonogenic
survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up64Cu-ATSM and radioactive64Cu was highly
accumulated in the cells. In the64Cu-ATSM-treated cells, DNA damage by the radiation emitted from64Cu was detected, and inhibition of cell
proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced
byradiationwereobservedfollowing64Cu-ATSMtreatment. Themajority ofthe64Cutakenupintothecellsremainedinthepostmitochondrial
supernatant(thecellular residueafterremoval ofthenucleiand mitochondria), whichindicates thattheh?particleemitted from64Cumaybeas
effective as the Auger electrons in64Cu-ATSM therapy. These data allow us to postulate that64Cu-ATSM will be able to attack the hypoxic
tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the h?particle decay of64Cu.
D 2005 Elsevier Inc. All rights reserved.
64Cu in cells were investigated to further assess the
Keywords:64Cu-ATSM; Tumor; Internal radiation therapy; Hypoxia; Apoptosis; PET
1. Introduction
Radionuclide therapy is one of the highly promising
approaches for treating cancer and is widely investigated in
basic research and clinical practice [1]. Radionuclide
therapies using labeled antibodies or peptides targeting
tumor-related antigens or receptors have been extensively
studied especially in the field of hematological malignan-
cies such as lymphoma and leukemia [2–4]. However,
success of these agents in solid tumors has been limited
mainly due to heterogeneous antigen expression and low
overall tumor uptake. A variety of strategies have been
employed to improve the tumor targeting and therapy effects
of such methods including combination therapy with other
modalities [5–8].
Conventionally, low linear energy transfer h-emitters
such as
radionuclide therapy [9].67Cu also has been investigated as
a candidate for radionuclide therapy.
physical and biochemical properties for radionuclide ther-
apy and67Cu-labeled monoclonal antibodies were reported
to be useful for the treatment of lymphoma and colorectal
carcinoma [9,10]. However, production of67Cu requires a
large accelerator or nuclear reactor, limiting the availability
of67Cu.
131I,
90Y and
186Re have been widely used for
67Cu has excellent
0969-8051/$ – see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2004.08.012
* Corresponding author. Tel.: +81 776 61 8430; fax: +81 776 61 8170.
E-mail address: yfuji@fmsrsa.fukui-med.ac.jp (Y. Fujibayashi).
Nuclear Medicine and Biology 32 (2005) 21–28
www.elsevier.com/locate/nucmedbio

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64Cu with a half-life of 12.7 h decays by electron capture
(41%), h?decay (0.573 MeV, 40%) and h+decay (0.656
MeV, 19%), accompanied by emission of annihilation
radiation (0.511 MeV, 38%) and g photons (1.34 MeV,
0.5%). It is a promising therapeutic radionuclide because of
its favorable h?particle emissions [11,12].64Cu is reported
to be as efficient as67Cu for tumor treatment [13,14] and
can be produced using small biomedical cyclotrons in
regular PET centers on a daily basis [15,16]. Furthermore,
because of its multiple decay modes,64Cu can be used for
real-time PET monitoring of regional drug concentration,
kinetics and dosimetry during radiation therapy if it is used
to label the therapeutic radiopharmaceuticals.
Copper-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-
ATSM; Fig. 1) has been examined extensively by our
group and others as a possible imaging agent to delineate
hypoxia within tumors [12,17,18]. Cu-ATSM is taken up
into tumor cells rapidly and efficiently and reduced by an
enzymatic system of sequential electron transport chains,
where monovalent Cu is released from the chelate to be
retained subsequently [19]. Considering these characters,
64Cu-ATSM has potential as a radiotherapy agent with an
option of real-time PET monitoring. Indeed, as a therapy
agent,
treatment of colorectal carcinoma in vivo tumor model
[20]. In the present report, we have investigated the
molecular basis of64Cu-ATSM therapy using in vitro tumor
cell models. The cell killing ability of
evaluated by colony-forming assay. Some of the typical
features of cell death derived by radiation were observed;
namely, inhibition of the cell proliferative rate and apoptotic
cell death. Subcellular localization of Cu-ATSM was
studied and compared with Cu-pyruvladehyde-bis(N4-
methylthiosemicarbazone) (Cu-PTSM), a flow tracer [21].
We also discuss the fate of64Cu in relation to cell toxicity.
This study may provide useful information for designing
effective therapy strategies and improving the radiotherapy
efficacy in cancer treatment.
64Cu-ATSM was reported to be useful for the
64Cu-ATSM was
2. Materials and methods
All chemicals were reagent grade. The64Cu at University
of Fukui was produced on a 12-MeV biomedical cyclotron
using previously reported methods [15,16]. The
Washington University was produced on a CS-15 biomed-
ical cyclotron (Cyclotron) [15]. H2ATSM was synthesized as
64Cu at
described previously [22].
mixing 200 mM glycine buffer containing
H2ATSM in dimethyl sulfoxide (20:1 by volume) [17].
Labeling efficiency was determined by radio-HPLC using
conditions described previously [19]. Radiochemical purity
and specific activity of
N99% and N56,000 GBq/mmol, respectively.
64Cu-ATSM was prepared by
64Cu and
64Cu-ATSM in all studies were
2.1. Cell culture
Mouse Lewis Lung carcinoma LL/2 cells were purchased
from Dai-Nippon Seiyaku (Japan) and were grown in a 5%
CO2-humidified atmosphere at 37 8C. Cells were routinely
maintained in Dulbecco’s modified Eagle Medium
(DMEM) (GIBCO, Grand Island, NY) supplemented with
10% fetal bovine serum.
2.2. Uptake of64Cu-ATSM into cells
Previous reports show that the uptake of
was dramatically increased under hypoxic conditions [23];
therefore, we followed the reported methods during the
uptake phase. Briefly, cells were trypsinized and collected in
a polypropylene tube (Falcon, Becton Dickinson, Lincoln
Park, NJ). Cell numbers were counted with a hemocytom-
eter and the cells were resuspended in serum-free DMEM to
a concentration of 1?106cells/ml. Ten million cells were
transferred to a three-necked flask and hypoxic gas (95%
N2, 5% CO2) was passed over the cells at 37 8C for 1.5 h.
64Cu-ATSM was then added to the flask and incubated for
1 h. After incubation, aliquots of the cell suspension were
removed and the cells were pelleted from the reaction media
to calculate the percentage uptake of
tional aliquots of cells were transferred to a polypropylene
tube and resuspended with fresh DMEM containing 10%
fetal bovine serum for further experiments.
64Cu-ATSM
64Cu-ATSM. Addi-
2.3. Clonogenic survival assay
The radiotherapy effect of64Cu-ATSM was measured by
performing a clonogenic survival assay under normoxic
condition [24,25] because continuous hypoxia treatment
itself affected cell viability. The64Cu-ATSM-treated cells,
resuspended in fresh medium, were counted using a
hemocytometer and diluted properly. Cells were seeded
into 60-mm dishes (Falcon, Becton Dickinson, Lincoln
Park, NJ) and cultured in a humidified 5% CO2/95% air
atmosphere at 37 8C. After 5 days of incubation, the cells
were stained with 3% Giemsa solution, and colonies
containing more than 50 cells were counted as survivors.
The absolute plating efficiencies of control cells were
34.1F9.7%. The surviving fraction was determined as the
ratio of live colonies in the
populations relative to the glycine-treated control.
64Cu-ATSM-treated cell
2.4. Cell growth assay
After the treatment, cells were seeded in six-well plates
(Falcon, Becton Dickinson, Lincoln Park, NJ) and cultured
Fig. 1. Structure of Cu-ATSM.
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under normoxic condition. Cells were trypsinized and
counted using the trypan blue dye exclusion test at the
specified time points.
2.5. Comet assay (single cell gel electrophoresis)
To detect the damage to DNA induced by
comet assay was performed after
[26]. Glycine-treated control cells and64Cu-ATSM-treated
cells were seeded in 100-mm dishes (Falcon, Becton
Dickinson) and cultured under normoxic condition. Six
hours after the treatment, cells were trypsinized and counted
using the trypan blue dye exclusion test. Viability of cells
was N90%. The cells were sedimented by centrifugation
and adjusted to the concentration of 1?105cells/ml with
phosphate-buffered saline. The cells were treated with a
64Cu, the
64Cu-ATSM treatment
CometAssay Kit (Trevigen, Gaithersvurg, MD) according to
the instruction manual from the manufacturer. After
electrophoresis, assay slides were treated with CometAssay
Silver Staining Kit (Trevigen) to visualize the assay results.
Data analysis was performed using Komet v. 4.0.2 software
(Kinetic Imaging, Wirral, UK).
2.6. Measurement of apoptotic cell death
Early stages of apoptosis were identified using an
Annexin V-FITC Apoptosis Detection Kit I (Becton Dick-
inson, San Jose, CA) [27]. After the uptake period, cells
were seeded in six-well plates and cultured under normoxic
condition. At the specified time points, cells were collected
and stained with Annexin V-FITC/propidium iodide (PI)
according to the manufacturer’s protocol. Level of apoptosis
was quantified using a Becton Dickinson FACSCalibur
system and analyzed using CellQuest v. 3.1 software
(Becton Dickinson, San Jose, CA). Cells that were Annexin
Fig. 2. Clonogenic survival of LL/2 cells treated with64Cu-ATSM. LL/2
cells were treated with different concentrations of64Cu-ATSM for 1 h under
hypoxic conditions to trap
treatment, surplus64Cu-ATSM was removed and cells were resuspended
in fresh culture medium and the radioactivity taken up by the cells was
measured. Cells were seeded in 6-cm dishes and cultured for 5 days, and the
colonies containing more than 50 cells were counted. The surviving
fractions determined as described in Materials and methods was plotted
against the amount of64Cu taken up by the cells. Values are the mean and
S.D. (n=6).
64Cu metabolically into cells. After the
Fig. 3. The effect of carrier-free64Cu and64Cu-ATSM on the survival rate
of LL/2 cells. A clonogenic survival assay was performed with a control
group (treated with glycine solution), a64Cu-glycine solution-treated group
(938.3F41.65 ACi added to 107cells) and a
(896.7F35.84 ACi added to 107cells). Viability compared with control
group was 91.43F24.64% and 0.1247F0.2874%, respectively. *Pb.0001,
significant degrease compared with control (Student’s t test, n=3).
64Cu-ATSM-treated group
Table 1
64Cu uptake ratio and radioactivity in tumor cells after 1 h of treatment
% UptakeRadioactivity in
tumor cells (Bq/cell)
64Cu-glycine
64Cu-ATSM
2.47F0.46
61.4F17.7
0.0857F0.0038
2.04F0.08
Data are expressed as the meanFS.D. (n=3).
Fig. 4. The typical microscopic images following the comet assay at 6
h after treatment. Images of a normal cell (a) and DNA-damaged cell (b) are
shown. In the64Cu-ATSM-treated groups, over 90% of total cells suffered
from DNA damage and displayed damaged pattern as shown in b.
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V-FITC-positive and PI-negative were identified as apopto-
tic cells as described previously [28,29].
2.7. Retention and intracellular distribution of64Cu-ATSM
and64Cu-PTSM in LL/2 cells
Cells were incubated in hypoxic gas (95% N2, 5% CO2)
for 1.5 h at which point either64Cu-ATSM or64Cu-PTSM
was added to the media. Following a 1-h incubation, the
media was removed and the cells in fresh medium were
seeded in 100-mm dishes, cultured under normoxic
condition and recollected at 0, 1, 3, 6, 12 and 24 h after
the treatment. Cells were washed twice with an ice-cold
isolation medium (0.25 M sucrose buffered to pH 7.4 with
10 mM HEPES). Aliquots of cells were separated and the
radioactivity in the cells was counted using a g-counter
(ARC-2000, ALOKA, Japan). Another aliquot of the cells
was resuspended in ice-cold lysis buffer (0.005% SDS
buffered to pH 7.4 with 10 mM HEPES) and homoge-
nized. After homogenization, the P1 fraction (crude nuclear
fraction) was obtained by centrifugation at 1000?g for
5 min at 4 8C. The supernatant was centrifuged at
8000?g for 10 min at 4 8C to yield the P2 (crude
mitochondrial) and S2 (the postmitochondrial supernatant
which contains the cellular residue after removal of the
nuclei and mitochondria) fraction. Radioactivity in each
fraction was measured.
3. Results
Fig. 2 shows the radiation dose–response curve for the
LL/2 cells treated with
was decreased in a dose-dependent manner.
uptake of 1.50 Bq/cell produced 99% killing of the cells.
Mock treatment with H2ATSM or nonradioactive Cu-ATSM
did not affect the survival ratio of LL/2 cells (data not
shown), which also indicates that hypoxia treatment during
uptake phase did not affect the cell viability by itself.
The effect of bfreeQ64Cu ion and64Cu-ATSM on tumor
cells was also compared using the clonogenic survival assay.
When approximately 34.8 MBq of
was inoculated, the survival rate of cells was not affected,
whereas the equivalent amount of
produced nearly complete killing of LL/2 cells (Fig. 3). The
64Cu uptake ratio in each treatment was 2.47F0.459% and
61.4F17.7%, respectively, which means the radioactivity
64Cu-ATSM. Clonogenic survival
64Cu-ATSM
64Cu-glycine solution
64Cu-ATSM treatment
Fig. 5. Tail moment ratio in the comet assay. Forty cells were selected ad
libitum and then analyzed for the tail moment using the Comet v. 4.0.2
software. Data are presented as a ratio to the nontreated control groups (bar,
1 S.E.). **Pb.01, significant increase (Student’s t test).

Fig. 6. Growth curve of LL/2 cells after
Proliferation of the cells was inhibited 24 h after the treatment. Data are
presented as normalized cell numberFS.D. relative to time 0 (n=3).
64Cu-ATSM treatment.
Fig. 7. Apoptosis of LL/2 cells after64Cu-ATSM treatment. Floating and
adherent cells were collected, stained with Annexin V-FITC and PI, then
analyzed by FACS using CellQuest software. A significant increase in
apoptosis was observed with64Cu-ATSM-treated cells from 36 h after the
treatment. Data points are the average percentage of apoptotic cells and
S.D. (n=3). *Pb.05, significant increase (Student’s t test).
Fig. 8. Washout rate of64Cu from LL/2 cells; 1?107cells were treated with
64Cu-ATSM and radioactivity in the cell fractions was measured at each
time point. Data are shown as ratio to the initial radioactivity after the decay
correction. Each data point is the averageFS.D. (n=3).
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taken up into tumor cells was 0.0857F0.0038 and
2.04F0.08 Bq/cell, respectively. The result suggests that
intracellular uptake is the major factor in the efficient tumor
cell killing by64Cu-ATSM (Table 1).
To elucidate the mechanism of cell killing by
ATSM, the comet assay was performed 6 h after the64Cu-
ATSM treatment. Fig. 4 shows a typical microscopic image
of the results. In the64Cu-ATSM-treated groups, over 90%
of the total cells showed comet tails. The tail moment ratio
was significantly increased in the
groups compared with64Cu-glycine and nontreated control
groups (Fig. 5) (nontreated group, P=.0085;64Cu-glycine-
treated group, P=.0085). These results indicated that the
radiation emitted from the intracellular64Cu caused DNA
strand breaks, with the maximum recoil energy from the
transmutation of64Cu to its highly charged daughter nucleus
potentially increasing the cell killing ability. This damage in
the DNA is thought to be one of the major triggers of cell
death pathways. Fig. 6 shows the proliferation rate of LL/2
cells after the treatment. In the64Cu-ATSM-treated group,
cell proliferation was completely inhibited after 24 h and a
significant apoptotic fraction was seen 36 h after the
treatment (Pb.05) (Fig. 7).
Both the washout rate and subcellular distribution of
64Cu were investigated in this study. Also, a direct
comparison of the subcellular localization was made
between64Cu-ATSM and64Cu-PTSM. With64Cu-ATSM,
defining the amount of radioactivity associated with the
cells at the time of resuspension as 100%, 38.0% of the
radioactivity remained at 3 h posttreatment and 31.2% at 6
h posttreatment (Fig. 8). Considering the half-life of64Cu,
the effect of radiation emitted from64Cu up to 12 h after
treatment dominates in this study: about 14,200 decays
occurred in one cell up to 12 h after treatment (Fig. 9). In
the subcellular fractionation comparison, the distribution of
64Cu from both
similar (Fig 10). For both64Cu-ATSM and64Cu-PTSM, the
majority of the radioactivity remained in the S2 fraction
over the 24 h. A transition of the radioactivity from the S2
64Cu-
64Cu-ATSM-treated
64Cu-ATSM and
64Cu-PTSM was very
and mitochondria fraction to the nuclear fraction was noted
over the 24-h period (Fig. 10).
4. Discussion
64Cu-ATSM has distinct accumulation mechanisms that
are based on hypoxic tumor-selective metabolism by some
enzymes [19]. Equipped with the radionuclidic character-
istics of
tumor cells,
radiotherapy agent that provides a new strategy for the
treatment of tumors [20]. In this study, we investigated the
cytotoxic effect of64Cu-ATSM using clonogenic survival
assays in an in vitro cell culture model.
decreased the clonogenic survival of LL/2 cells in a dose-
dependent manner where 99% cell killing occurred when
1.50 Bq/cell of64Cu was taken up into the cells.
The nonligand binding form of64Cu (Cu-glycine) did not
affect the survival rate of LL/2 cells, whereas the equivalent
amount of radioactivity of
duced the survival rate to about 0.1% (Pb.0001). In this
experiment, the incubation medium containing
exchanged into fresh culture medium after the treatment,
64Cu and the high and specific accumulation in
64Cu-ATSM is making a new type of
64Cu-ATSM
64Cu-ATSM significantly re-
64Cu was
Fig. 9. Total decay frequency of intracellular64Cu. Based on the washout
result shown in Fig. 8, the total decay frequency was calculated for a cell
taking up a 99% cell killing dose of 1.50 Bq/cell. Radioactivity at each
time point was corrected for a half-life of 12.7 h and the area under the
curve was calculated.
Fig. 10. Subcellular distribution of64Cu following64Cu-ATSM or64Cu-
PTSM treatment. After64Cu-ATSM and64Cu-PTSM were metabolized in
the cells, the majority of the64Cu was retained in the microsomal/cytosol
fraction. A transition of radioactivity from the mitochondria and S2
fractions to the nuclear fractions was noted. Data are expressed as the mean
percentage of total cellular radioactivity (n=4).
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meaning that the64Cu taken up into tumor cells prior to the
exchange was the only source of the cell killing. The64Cu
taken up into the cells was dramatically different between
the64Cu-glycine-treated groups and the64Cu-ATSM-treated
groups, 0.0857F0.0038 and 2.04F0.08 Bq/cell, respective-
ly. This may be the major cause for the differences in the
subsequent survival rates. Furthermore, considering that
there was no difference in the survival rate between the
nontreated control and64Cu-glycine groups, the radioactive
emissions from outside of the cells during the 1-h treatment
in the flask did not affect the tumor cell survival. To be
brief, the most effective character of
accumulate radioactive
produces the ability to kill tumor cells.
Radiation causes direct and/or indirect effects on
irradiated cells. One of the well-known direct effects is
damage to DNA. DNA damage by ionizing radiation
predominantly consists of single-strand breaks, but also
includes double-strand breaks, alkali-label sites, and oxi-
dized purines and pyrimidines [30–32]. We confirmed with
the alkali comet assay that a significant increase in DNA
damage was observed with64Cu-ATSM-treated groups at 6
h after treatment. Comet assay allows us to detect the DNA
fragmentation in apoptotic and/or necrotic cells [33].
However, we could not detect significant increases in the
number of apoptotic cells as well as necrotic and/or dead
cells using the Annexin V-FITC/PI staining method at the
same time point. For this reason, the DNA damage observed
after the treatment was considered to be caused directly by
the radiation from intracellular64Cu and not by apoptosis.
Radiation-induced DNA damage elicits a variety of
cellular responses including apoptosis as one of the major
mechanisms of cell death in radiation therapy [34–36]. In
this study, apoptosis marker-positive cells significantly
increased in number (Pb.05) 36 h after the treatment when
detected by the Annexin V-FITC/PI staining method,
preceded by the inhibition of cellular proliferation 24 h after
the treatment. In an X-irradiation study, high-dose irradia-
tion was reported to induce a rapid and strong apoptotic
response, whereas low-dose irradiation induced a slow and
mild apoptosis; each type of apoptosis was defined as
premitotic apoptosis and postmitotic apoptosis, respectively
[37]. In the postmitotic apoptosis, cell death occurs after one
or a few cell divisions, not immediately after the radiation,
and cell cycle arrest is observed at the G2/M phase. In our
preliminary study, we also observed increase of G2/M phase
cells, namely, G2/M arrest, in the
groups at 12–24 h after the treatment. These findings
indicated that the typical cell death after
treatments is through postmitotic apoptosis.
It is conceivable that64Cu-ATSM would be able to exert
stronger cytotoxicity via premitotic apoptosis if a higher
radiation dose of
However, considering radiotherapy in clinical setting, doses
should be reduced to avoid adverse effects on nontarget
tissues. In this regard, the ability of64Cu-ATSM to induce
64Cu-ATSM is to
64Cu into the cells and as such
64Cu-ATSM-treated
64Cu-ATSM
64Cu-ATSM was given to the cells.
mild but steady cytotoxicity and postmitotic apoptosis
confirmed in this study is desirable as a radiotherapy agent.
Recently, it has been reported that the damage to DNA
by
about 40% of
Auger electrons with high linear electron transfer [20].
Auger electrons are expected to bring very high toxicity if
they are in close proximity to DNA. The tissue penetration
range of Auger electrons emitted from64Cu is about 5 Am
[20] and is shorter than the diameter of most tumor cells, so
it is well recognized that the radiotoxicity of Auger electron
emitters depends strongly on their distribution within the
cell. The most severe effects have been observed when the
Auger emitter is localized in the nucleus [38]. Therefore,
also in the case of64Cu, it is necessary for the radiocopper to
localize in the nucleus and around the DNA to exert
cytotoxicity through Auger electrons. In this study, we
investigated the subcellular distribution of64Cu after uptake
into tumor cells and showed that the radioactivity existed
mainly in the S2 fraction with the radioactivity slowly
migrating into the nucleus over time. Furthermore, nonra-
dioactive Cu-ATSM did not affect the survival rate, which
indicated the Cu ions themselves were not cytotoxic at the
concentrations used in this study. Based on these findings,
h?particles emitted from intracellular64Cu as well as the
Auger emissions of
considered to be the major cytotoxic sources in64Cu-ATSM
therapies. Moreover, the maximum recoil energy from the
transmutation of64Cu to its highly charged daughter nucleus
may also increase the cell killing ability. The multiplicity of
decay mode of64Cu seems to be working in favor of the
antitumor effect of64Cu-ATSM. Previous reports showed a
significant portion of
thiosemicarbazone), an analog of the series of Cu(II)-
bis(thiosemicarbazones) compounds, is delivered to the cell
nucleus [21] and our current studies have demonstrated
similar uptake patterns.
The tissue penetration range of h?particles emitted from
64Cu is up to several hundred of micrometers. For this
reason, the cytotoxic effect of the h?particle of64Cu can
range not only to the single cell that take up64Cu but also to
the surrounding cells that do not take up64Cu, which could
increase the antitumor effect in vivo64Cu-ATSM therapy.
This feature becomes important when treating in vivo tumor
masses because aggregation of the cells with heterogeneous
characters is expected in vivo tumors.
The washout rate of64Cu from tumor cells is relatively
high in our experimental model. At 3 h after the treatment,
radioactivity in the cells was reduced to 38.0% of the total
uptake at the end of the treatment. However, in the
biodistribution study using tumor-bearing animal models,
accumulation of64Cu-ATSM into tumor cells was reported
to be highest at 4 h after injection [20]. High washout rate
observed in our assay system might be limited only to in
vitro model and actual accumulation of64Cu in the tumor
might continue for much longer periods. If so, the
64Cu might be derived from Auger electrons because
64Cu decays by electron capture emitting
64Cu in the nuclear fraction are
67Cu-pyruvaldehyde-bis(N4-methyl-
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therapeutic effectiveness of64Cu-ATSM might be higher in
the in vivo tumors.
Based on the washout rate and half-life of
calculated the total number of atomic disintegrations of64Cu
occurring in the tumor cells when 1.50 Bq/cell of64Cu, a
99% cell killing dose, was taken up into cells (Fig. 9). Under
these conditions, approximately 10,000 atomic disintegra-
tions occurred within 6 h after the treatment, and about
14,000 within 12 h. That is, 10,000–14,000 atomic
disintegrations per cell might be enough for killing tumor
cells. D37 value (37% cell death) [39] was ca. 4500 decay
per cell when biological washout from the cells was taken
into account for the calculation, which was one fourth of the
value previously reported (16,000 decay per cell) [40]. The
actual area under time–radioactivity curve in the present
study (Fig. 8) was about one fourth of the estimated value
from nonwashout model, so that the present result is
considered to be more accurate as actual retention pattern
was included into the calculation.
In this study, the cells were kept under hypoxic condition
during uptake phase of64Cu-ATSM to realize high retention
of64Cu for 1 h, then cultured for 5 days at the maximum in
normoxic condition to evaluate solely the effect of radiation,
excluding the effect of hypoxia on cell viability. As hypoxia
is considered to lessen the radiation toxicity in general, the
cell killing ability determined in this study in normoxic
tumor cells might be higher than that expected in
continuously hypoxic tumor cells. On the other hand, the
present results can be used for the estimation of cell toxicity
in normoxic nontumor cells. Hypoxia selectivity of64Cu-
ATSM is critical for realizing tumor-selective treatment. In
our human studies using62Cu-ATSM, the highest accumu-
lation was found in lung tumor (tumor/blood=3.00F1.50),
followed by liver (liver/blood=2.45F1.03), whereas other
normal tissues showed relatively low accumulation (heart/
blood=1.84F0.35, lung/blood=0.43F0.09) [41]. High ab-
dominal accumulation observed in mice [23] might be a
result of hepatobiliary secretion of64Cu after metabolism.
From these findings, cytotoxicity in the liver and, to a lesser
extent, in the intestine arise as a possible problem in64Cu-
ATSM treatment. Roughly 75% of the blood entering the
liver is from portal vein and 25% from hepatic artery. Thus,
considerable part of the liver seems to be hypoxic and it
might be responsible for the rather high uptake of
ATSM in the liver. If so, radiation effect of64Cu in the liver
could be estimated in a similar manner as in hypoxic tumor
cells. Physiological excretion route of Cu is from liver to
duodenum. Radioactive64Cu injected as Cu-ATSM is also
expected to follow this route after released from the chelate.
As shown in the present study, extracellular64Cu did not
show significant cell toxicity, so that radioactivity in the
duodenum is less likely to become a source of adverse effect
in the treatment.
In vitro cell uptake study reported previously, Cu-ATSM
showed some uptake also in normoxic tumor cells [23].
However, in our PET studies in normal human, selective Cu
64Cu, we
64Cu-
accumulation was found only in the liver but no other
tissues (unpublished data). Thus, the uptake in the normoxic
tumor cells might be from a characteristic of tumor cells. In
fact, tumor cells expressed microsomal electron transport
enzymes and they played a major role in reductive retention
of Cu-ATSM in tumor cells, especially under hypoxic
conditions [19]. Thus, Cu-ATSM can be evaluated mainly
as a marker of hypoxia and, in some part, as a marker of
tumor-selective gene expression by means of microsomal
enzyme expression, in clinical practice.
5. Conclusion
64Cu-ATSM has been shown to be effective in the
treatment of tumors.
radioactive64Cu into hypoxic tumor cells and the emitted
h?particles caused lethal damage to DNA leading to
postmitotic apoptosis and mild but steady cell death. Since
Cu-ATSM showed low but considerable uptake in normoxic
cells in vitro and may be taken up in normoxic cells in vivo,
the therapeutic index need to be carefully considered. In
order to ensure the safety and effectiveness of64Cu-ATSM
treatment, contribution of hypoxia on both64Cu accumula-
tion and radiation sensitivity of target and/or nontarget cells
and possible adverse effects to the excretion route should
be clarified.
64Cu-ATSM efficiently delivered
Acknowledgments
Part of this study is supported by a Grant-in-Aid for
Scientific Research (Kakenhi No. 1430274) from JSPS,
Research and Development Project Aimed at Economic
Revitalization from the Ministery of Education, Culture,
Science, Sports and Technology Japan, and the 21st Century
COE program bBiomedical Imaging Technology Integration
ProgramQ from JSPS.
The production of Cu-64 at Washington University is
supported by NCI R24 CA86307. Additional financial
support for the studies at Washington University came
from the United States Department of Energy grant
(DE-FG02-87ER60212).
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