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M. Umemura et al. / Journal of Pharmacological Sciences xxx (2017) 1e8
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1. Introduction
under ambient conditions (23 C). Rabbit squamous cell carcinoma
VX2) cells were purchased from American Type Culture Collection
(
Chemotherapy is a key treatment for patients with cancer,
together with surgery and radiotherapy. However, excess admin-
istration of anti-cancer drugs is a major problem causes adverse
(ATCC) (Manassas, VA, USA). YKG (YKG-1, JCRB0746), which are
human glioblastoma malignant glioma cells, were purchased from
Japanese Collection of Research Bioresources (JCRB) Cell Bank.
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effects during treatment.
OVK18, human ovarian carcinoma cells were purchased from
Riken Bioresource Center, cell bank. Early passage cultures of each
group of cells were stored and used for these experiments. VX2 cell
0
We previously reported that an iron-salen, i.e.,
bis(salicylidene)ethylenediamine iron (Fe(Salen)) is a magnetic
compound, that has several roles in anti-cancer activity, magnetic
m
-oxo-N,N -
lines were cultured in RPMI-1640 with L-glutamine and phenol red
1
resonance imaging (MRI), and a drug delivery system (DDS).
medium containing 10% fetal bovine serum (FBS) and 1% pen-
icillinestreptomycin. YKG were cultured in D-MEM, containing 10%
FBS and 1% penicillinestreptomycin. OVK18 were cultured in MEM,
containing 10% FBS and 1% penicillinestreptomycin.
Fe(Salen) was classified as a magnetic substance that can be
made to accumulate locally using a permanent magnet, and it can
be heated up inside the tumor tissue under an alternating magnetic
field (AMF), thereby locating and targeting a tumor in situ. In our
previous study, Fe(Salen) was mostly metabolized throughout
liver-gallbladder.1 Fe(Salen) injection (50 mg/kg) did not change
serum hepatic enzyme (AST) and ALT in mice for 7 days. The excess
Fe(Salen) induced liver dysfunction and renal function.
2.2. Optical properties of Fe(Salen)eDFO mixture
The Ultraviolet Visible Absorption Spectroscopy (UVeVis)
absorbance and Fourier Transform Infrared Spectroscopy (FT-IR)
characteristics of Fe(Salen)eDFO mixture samples were deter-
mined using a UVeVis spectrophotometer (Nanodrop, Fisher-
Scientific) and a FT-IR spectrophotometer (JASCO FT/IR-4100) in
an ATR mode, respectively.
We have recently examined the use of intrinsic magnetic Fe(S-
alen) in hyper-thermic therapy, demonstrating successful targeting
of tongue cancer in a rabbit model. In the study, we examined the
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combination of systemic intravenous Fe(Salen) injection, controlled
drug delivery using a magnet, followed by hyper-thermic therapy
using AMF. We used a rabbit model of tongue cancer because the
application of magnet and AMF are relatively easy in this model. We
have also reported the anti-tumor and hyperthermia-inducing ef-
fects of Fe(Salen) in human glioblastoma (GB), both in vitro and
2.3. Sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-
[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay
A cell proliferation assay was performed using a commercially
available kit, XTT Cell Proliferation Assay Kit (ATCC) (Manassas, VA,
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in vivo. Therefore, Fe(Salen) could be a potent single-drug anti-
1
e3
cancer agent for clinical applications.
USA) according to the manufacturer's protocol.
VX2, OVK18 and
3
4
3
Conversely, excess administration of iron often induces toxic-
ities or side effect such as chronic iron overload, resulting in
increased morbidity and mortality. For example, cyclophosphamide
and ifosfamide are widely used antineoplastic agents, but their
side-effect of hemorrhagic cystitis is still encountered and it is an
important problem. Acrolein is the main molecule responsible for
this side-effect and 2-mercaptoethane sulfonate (mesna) is the
commonly used preventive agent. Mesna binds acrolein and pre-
YKG cells (5 ꢂ 10 cells/ml, 1 ꢂ 10 cells/ml and 8 ꢂ 10 cells/ml,
respectively) were seeded on 96-well plates. The inoculated plate
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was incubated at 37 C for 2 h in a 5% CO
2
humidified atmosphere.
Blank control wells contained medium without Fe(Salen). Cells
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were incubated for 24 h, at 37 C, and 5% CO
2
and Fe(Salen) was
ml of the activated-
added with or without DFO. After incubation, 50
XTT solution was added to each well. The plate was returned to the
incubator for 3 h. The wells containing the cells and blank controls
were measured using a micro plate reader.
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vents it from direct contact with uroepithelium.
Suitable antidotes for Fe(Salen) are also required in clinical use.
Because Fe(Salen) is an organic compound that includes iron (Fe), we
focused on deferoxamine mesylate (DFO). DFO is derived from
Streptomyces pilus,5 and it is a hexadentate siderophore molecule
that has high affinity for Fe, resulting in formation of a stable 1:1
complex.5 Over the past 30 years, DFO has been a gold standard for
controlling body iron which can prolong survival and prevent po-
tential organ dysfunction, and it is generally considered safe and
efficacious.7 Currently, DFO is widely used to treat Fe overload-
2.4. Measurement of reactive oxygen species
Measurement of reactive oxygen species (ROS) was performed
1,2
as previously reported. The cells were plated in 96-well culture
,6
5
plates (1 ꢂ 10 cells/ml) overnight. The cells were then treated with
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7.5 or 15
mM Fe(Salen) in the presence or absence of DFO at 37 C for
24 h. The intracellular ROS level was then measured using the
0
0
fluorescent dye 2 ,7 -dichlorofluorescein diacetate (DCFH-DA)
(SigmaeAldrich, Tokyo) as previously described. In the presence of
5
,8
9,10
associated disease such as thalassemia
allergic or anaphylactic reaction to DFO are rare.
using intra-lysosomal ferritin and ferrioxamine, allows the chelated
and tumors,
and
1
1,12
0
0
DFO-Fe chelation
oxidant, DCFH is converted into the highly fluorescent 2 ,7 -
dichlorofluorescein. The cells were first washed with PBS, and
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complex to be excreted by the kidney and in the feces via the bile.
serum-free MEM containing 10 mM DCFH-DA was added to each
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In this study, our aim is to develop a DFO-assisted antidote
method in Fe(Salen) administration, where the iron-chelating
agent binds free iron in Fe(Salen), thereby boosting the efficacy of
Fe(Salen) in cancer treatments and also reducing possible side
effects.
well, and then incubated at 37 C for 40 min. ROS production was
measured using a microplate reader equipped with a spectrofluo-
rometer (PerkinElmer ARVO MX, Yokohama) at an emission
wavelength of 538 nm and excitation wavelength of 485 nm.
2.5. Acute side effect of Fe(Salen) and survival experiment in mice
2
. Materials and methods
Animal experiments were performed according to the Yoko-
2.1. Reagents and cell culture
hama City University guidelines for experimental animals. Male ICR
mice (n ¼ 50) weighting 30.0e38.5 g were purchased from Nihon
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DFO (ꢀ92.5%) salt was purchased from Sigma (St. Louis, MO,
SLC. Fe(Salen) and DFO were mixed, incubated at 37 C for 10 min,
USA). Fe(Salen) (>95.0%) was purchased from Tokyo Chemical In-
and sonicated before injection. Avertin (12.5 g/L 2,2,2-
tribromoethanol, 25 ml/L 2-methyl-2-butanol) was injected intra-
peritoneally for general anesthesia.
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dustry (Japan). Fe(Salen) was sonicated for 6 h at 4 C, and was used
in normal saline suspension. Sample preparation was conducted
Please cite this article in press as: Umemura M, et al., The iron chelating agent, deferoxamine detoxifies Fe(Salen)-induced cytotoxicity, Journal
Umemura, Masanari
