Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells

Article abstract of DOI:10.1007/s10534-015-9852-x

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC₅₀ value was approximately 20 μM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Full text of DOI:10.1007/s10534-015-9852-x

Biometals
DOI 10.1007/s10534-015-9852-x
Anti-proliferative effect of Fe(III) complexed with
1-(2-hydroxy-3-methoxybenzaldehyde)-
4-aminosalicylhydrazone in HepG2 cells
•
Takeshi Fukushima Erina Taniguchi Hiroshi Yamada Kiyomasa Kato
•
•
•
•
•
•
•
Ayako Shimizu Yoshikazu Nishiguchi Mayu Onozato Hideaki Ichiba
Yutaro Azuma
Received: 17 February 2015 / Accepted: 25 March 2015
Ó Springer Science+Business Media New York 2015
Abstract We previously developed a chelating ligand,
1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicyl-
hydrazone (HMB–ASH), which can chelate Fe(III) to
form a complex. The HMB–ASH–Fe(III) complex
exhibits a dose-dependent anti-proliferative effect in
HepG2 cells, whereas the ligand, HMB–ASH, and
Fe(III) alone had no considerable effect. The HMB–
ASH–Fe(III) complex was composed of Fe(III):
HMB–ASH (1:2), as determined by high-performance
liquid chromatography with high-resolution mass
spectrometry. The IC50 value was approximately 20
lM, which was comparable to those of the anti-cancer
drugs oxaliplatin (OXP) and etoposide (ETP) under
the same conditions. Similar to OXP and ETP, HMB–
ASH–Fe(III) induced apoptosis in HepG2 cells, as
revealed by terminal deoxynucleotidyl transferase
fluorescein-12-dUTP nick end labeling assay.
Keywords 1-(2-Hydroxy-3-methoxybenzaldehyde)-
4-aminosalicylhydrazone Á Metal complex Á
Anti-proliferation Á Fe(III) Á HepG2 cells
Introduction
Cisplatin and oxaliplatin (OXP) are well-known
metal complex-containing cancer chemotherapeutics
that possess Pt(II) in their structure. In recent
studies, a number of metal complexes exhibited
anti-proliferative activity in cancer cells in vitro.
Recently, numerous organic ligands coordinated
with various transition and typical metal ions have
been developed, including Pt (Huang et al. 2013),
Cu (Farag et al. 2013; Genc et al. 2014), Ni (Genc
et al. 2014; Hsu et al. 2013), Fe (Hou and Wang
2005), Zn (Farag et al. 2013; Genc et al. 2014),
and Sn (Nath et al. 2014; Mahmudov et al. 2014),
which exhibit anti-proliferative activity in cancer
cells. These metal complexes could be potential
lead compounds for designing novel anticancer
drugs.
Electronic supplementary material The online version of
this article (doi:10.1007/s10534-015-9852-x) contains supple-
mentary material, which is available to authorized users.
T. Fukushima (&) Á E. Taniguchi Á H. Yamada Á
K. Kato Á A. Shimizu Á M. Onozato Á H. Ichiba
Department of Analytical Chemistry, Faculty of
Pharmaceutical Sciences, Toho University, 2-2-1
Miyama, Funabashi-shi, Chiba 274-8510, Japan
e-mail: t-fukushima@phar.toho-u.ac.jp
Y. Nishiguchi
Department of Pharmaceutical Practice, Faculty of
Pharmaceutical Sciences, Toho University, 2-2-1
Miyama, Funabashi-shi, Chiba 274-8510, Japan
Y. Azuma
The reported organic ligands have one or more
Schiff base moieties for chelating metal ions. In the
Schiff base moiety, the lone pair on the nitrogen atom
Department of Medical Biochemistry, Faculty of
Pharmaceutical Sciences, Toho University, 2-2-1
Miyama, Funabashi-shi, Chiba 274-8510, Japan
123

Biometals
in the azomethine linkage coordinates the metal ions to
form a complex. In our laboratory, three organic ligands
with Schiff base moieties, 2,4-[bis-(2,4-dihydroxyben-
zylidene)]-dihydrazinoquinazoline (DBHQ) (Kimura
et al. 2009a, b), 2,4-[bis-(2-hydroxy-3-methoxyben-
zylidene)]-dihydrazinoquinazoline (HBQZ) (Yamada
et al. 2010), and 1-(2-hydroxy-3-methoxybenzaldehy-
de)-4-aminosalicylhydrazone (HMB–ASH) (Fig. 1)
(Yamada et al. 2012), have been developed for the
fluorescence detection of Ga(III), Zn(II), and Sc(III),
respectively. Additionally, we found that the emitted
fluorescence of the HMB–ASH–Sc(III) complex was
quenched by adding exogenous Fe(III). These results
suggest that a complex of HMB–ASH–Fe(III), which
emits no fluorescence, may be formed.
HPLC-grade methanol (MeOH), and acetonitrile
(CH3CN) were obtained from Wako Pure Chemicals
(Osaka, Japan). Water was used after purification
using a WR600G instrument (Nihon Millipore K.K.,
Tokyo, Japan). Special-grade ammonium acetate
([97.0 %) was procured from Kanto Chemical Co.
Inc. (Tokyo, Japan).
Synthesis of HMB–ASH
HMB–ASH was synthesized according to our previ-
ous paper (Yamada et al. 2012). Briefly, methyl
4-aminosalicylate (4.0 g, 23 mmol) was added to
80 % hydrazine monohydrate (12 mL), and the mix-
ture was refluxed for 90 min. After evaporation, the
residue was added to distilled H2O (20 mL) to obtain a
crude crystal. The crude crystal was recrystallized in
EtOH to obtain 4-aminosalicylic hydrazide (yield
78.4 %). Subsequently, 4-aminosalicylic hydrazide
(0.5 g, 3.0 mmol) in EtOH (100 mL) was reacted with
o-vanillin (0.45 g, 3.0 mmol) for 2 h in a water bath at
100 °C. After cooling to room temperature, the
precipitate was collected by filtration and was recrys-
tallized from MeOH to obtain the final product, HMB–
ASH (yield 82.5 %).
FAB-MS: m/z 302 (M ? H)?; Anal. Calcd. for
C15H15O4N3: C, 59.80; H, 5.02; N, 13.95. Found: C,
60.02; H, 4.89; N, 13.77. 1H NMR (DMSO-d6) (ppm):
12.27 (s, 1H), 11.67 (s, 1H), 11.02 (s, 1H), 8.55 (s, 1H),
7.59 (d, 1H), 7.06 (d, 1H), 6.96 (d, 1H), 6.80 (t, 1H),
6.14 (m, 2H), 6.03 (d, 1H), 5.88 (s, 1H), 3.81 (s, 3H).
13C NMR (DMSO-d6) (ppm): 166.31, 162.33, 163.33,
155.25, 148.42, 148.02, 147.63, 129.44, 121.42, 119.46,
114.31, 106.41, 101.88, 99.86, 56.34. FT-IR (solid
phase) (cm-1): 3358 (vNH), 3236, 1352 (vOH), 1599
(vC=N), 1250 (vO-CH3). Melting point: 252–254 °C.
Among the ligand–metal ion combinations we
previously developed, the HMB–ASH–Fe(III) complex
was chosen for the present study because of its ease
synthesis (Yamada et al. 2012) and sufficient stability.
We evaluated the anti-proliferative action of HMB–
ASH–Fe(III) in HepG2, K562, and MCF7 cells using
the WST-1 assay. Consequently, we found that the
HMB–ASH–Fe(III) complex had an intense anti-
proliferative effect in HepG2 cells. We further
analyzed the chemical structure of the HMB–ASH–
Fe(III) complex by ultraviolet (UV) spectroscopy and
high-performance liquid chromatography with mass
spectrometry (LC–MS). To elucidate whether the
HMB–ASH–Fe(III) complex induced apoptosis, DNA
fragmentation was examined by TdT-mediated dUDP
nick end labeling (TUNEL) assay.
Materials and methods
Chemicals
Atomic spectrophotometry-grade FeCl3 (1000 ppm in
0.1 N HCl), special grade dimethylsulfoxide (DMSO),
Preparation of HMB–ASH–Fe(III) complex
Forty microliters of 24 mM HMB–ASH in DMSO was
mixed with 16 mM FeCl3 (60 lL) in H2O and
100 mM NH3–NH4Cl buffer (1013 lL; pH 7.4). The
mixed solution was incubated for 10 min at room
temperature after mixing with vortex. Subsequently,
0.1 NaOH (87 lL) was added to prepare 800 lM
HMB–ASH–Fe(III) complex. The solution was then
diluted with culture medium, as described below, to
prepare 12.5, 25, 50, 100, and 200 lM HMB–ASH–
Fe(III) complex solution. For the blanks without
Fig. 1 Chemical structure of HMB–ASH
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Biometals
HMB–ASH or Fe(III), DMSO (40 lL) or H2O (60 lL)
was added to prepare the solution.
subjected to UV spectrometry by JASCO V-650
(Jasco, Tokyo, Japan).
LC-atmospheric pressure chemical ionization
(APCI)-MS
Cell culture and anti-proliferation assay
Human hepatocellular carcinoma HepG2 cells and
human breast cancer MCF7 cells (JCRB Cell Bank,
National Institute of Biomedical Innovation, Osaka,
Japan) were cultured in Dulbecco’ s modified Eagle’s
medium [Nissui Pharmaceutical Co., Ltd. (Tokyo,
Japan)] supplemented with 10 % fetal bovine serum
(FBS), and human leukemia K562 cells (JCRB Cell
Bank) were cultured in RPMI-1640 medium [Nissui
Pharmaceutical Co., Ltd. (Tokyo, Japan)] supplement-
ed with 10 % FBS [Nissui Pharmaceutical Co., Ltd.
(Tokyo, Japan)]. The anti-proliferative effect of the
complexes was assayed in 96-well microplates using
WST-1 reagent (Dojindo Laboratories Co., Ltd.,
Kumamoto, Japan). Briefly, HepG2 and MCF7 cells
(1.0 9 105 cells/mL) were suspended and seeded into
96-well plates at a density of 5.0 9 103 cells/well/50
lL. K562 cells were also suspended (2.0 9 105 cells/
mL) and seeded at a density of 1.0 9 104 cells/well/50
lL. The cells were pre-incubated at 37 °C for 24 h in a
humidified atmosphere containing 5 % CO2. Subse-
quently, 50 lL of the complex solution (12.5, 25, 50,
100, or 200 lM) was added to each well and incubated
for 48 h (n = 3).
HMB–ASH (100 lM, 200 lL) was mixed with 200
lM Fe(III) solution (100 lL) and 10 % CH3CN-
100 mM ammonia-buffered solution (pH 8.0, 700 lL),
and the solution was mixed vigorously for 15 s. The
solution (50 lL) was injected through an autosampler
onto an LC–time-of-flight (TOF)-MS system, com-
prising an Agilent 1200 series HPLC system (Agilent
Technologies, Santa Clara, CA, USA) and a TOF-MS
(JMS-T100 LP AccuTOF LC-Plus) equipped with an
atmospheric pressure chemical ionization (APCI)
source (JEOL Co. Ltd., Tokyo, Japan). The separation
column was
a CAPCELL PAK C18 MG-II
(100 mm 9 4.6 mm; i.d.: 3 lm; Shiseido Co., Ltd.,
Tokyo, Japan), and the mobile phase was 5.0 mM
CH3CO2NH4 in H2O:CH3CN (90:10) (A) and CH3CN
(B). The mobile phase was eluted by a linear gradient
program; 0 min A/B = 70/30, 12.0 min A/B = 42/
58, 12.1 min A/B = 42/58, 22.0 min A/B = 42/58,
22.1 min A/B = 70/30, and 32 min A/B = 70/30.
The flow rate was constant at 0.8 mL min-1, and the
column temperature was maintained at 35 °C. The
APCI-MS conditions were as follows: negative ion
mode; needle voltage set at -4501 V; and the ring lens
and orifice 1 and 2 voltages set at -20, -80, and
-15 V, respectively. Nitrogen was used as the
nebulizing and desolvation gas, and the pressure was
maintained constant at 0.608 MPa. The desolvation
chamber and orifice 1 temperatures were set to 420 and
80 °C, respectively. Simultaneously, UV detection
was carried out at 335 nm. Data were obtained using
Mass Center software, MS-56010MP (JEOL).
After incubation for 48 h, 10 lL of WST-1 reagent
was added and incubated for 2 h. The absorbance at
450 nm was measured and analyzed using a Beckman
Coulter DTX 800 Multimode Detector plate reader
(Beckman Coulter, Brea, CA, USA). The obtained
data were expressed as mean standard error (S.E.)
(n = 3).
DNA fragmentation
UV spectra of HMB–ASH–Fe(III) complex
DNA fragmentation was assessed using the Dead-
EndTM Fluorometric TUNEL System (Promega KK,
Tokyo, Japan). Briefly, HepG2 cells were seeded
(1.0 9 105 cells/well) in 4-well glass plates, and were
pre-incubated for 24 h. Subsequently, 100 lM HMB–
ASH (50 lL) in the presence or absence of Fe(III) or
100 lM OXP or etoposide (ETP) was added and
incubated for 24 h. After the incubation, the cells were
washed with 200 lL of PBS, and then treated with 4 %
Five milliliters of HMB–ASH in DMSO (1.0 mM)
was diluted with methanol in a volumetric flask
(50 mL) to prepare a 100 lM solution. The HMB–
ASH solution (1.0 mL) was mixed with 0.90–90 lM
Fe(III) solution (0.1–2.0 mL) and 100 mM ammonia-
buffered solution (1.0 mL, pH 8.0), and incubated for
10 min at room temperature. The mixed solution was
diluted to 5.0 mL with methanol by volumetric flask.
The combined solution was manually mixed, and was
123

Biometals
Results
formaldehyde solution (200 lL). The treated cells
were placed on ice for 25 min. Subsequently, the cells
were washed twice with 200 lL of PBS and treated
with 0.1 % TritonÒ X-100 (100 lL) for 5 min at room
temperature. After washing with 200 lL of PBS, the
cells were incubated for 5 min, and mixed with 50 lL
of equilibration buffer containing the nucleotide mix
(fluorescein-12-dUTP) and recombinant terminal
deoxynucleotidyl transferase (rTdT). The cells were
incubated at 37 °C for 60 min. Subsequently, 500 lL
of saline sodium citrate buffer (SSC, 209) diluted
tenfold with H2O was added, and samples were
incubated for 15 min to stop the enzyme reaction.
After washing twice with 200 lL of PBS, apoptotic
cells were examined by confocal fluorescence mi-
croscopy. The stained cells were scanned using the
confocal laser scanning microscope, LSM 510 Meta
(Carl Zeiss, Oberkochen, Germany, ex. 488 nm,
40 9 water-immersion objective lens).
Anti-proliferative action of the HMB–ASH–
Fe(III) complex
We first examined the anti-proliferative action of the
HMB–ASH–Fe(III) complexes on HepG2 cells, as
compared to HMB–ASH or Fe(III) alone using a WST-
1 assay. As shown in Fig. 2, HMB–ASH or Fe(III)
alone had no effect on the HepG2 cells (Fig. 2a, b). In
contrast, HMB–ASH–Fe(III) exhibited a dose-depen-
dent anti-proliferative effect in HepG2 cells (Fig. 2c).
Interestingly, the anti-proliferative effect of the HMB–
ASH–Fe(III) complexes was remarkable high in
HepG2 cells, whereas slight inhibition was observed
in MCF7 and K562 cells (Fig. 3a). These results
suggest that HMB–ASH–Fe(III) complexes are re-
markably potent growth inhibitors in HepG2 cells. The
IC50 value was approximately 20 lM. The anti-
proliferative effect of the HMB–ASH–Fe(III) com-
plexes was more potent than the traditional chemother-
apy, and was similar to OXP under the present
experimental conditions (Fig. 3b). Thus, HMB–
ASH–Fe(III) complexes may have similar anti-prolif-
erative potency to ETP and OXP in HepG2 cells.
Flow cytometry
As described above, HMB–ASH–Fe(III) complex,
OXP, and ETP were added to HepG2 cells and
incubated for 24 h. After incubation, cells were washed
with 2 mL of PBS, followed by incubation for 5 min at
room temperature. Subsequently, the cells were de-
tached by adding 0.02 % EDTA in PBS (100 lL),
followed by incubation at 37 °C for 15 min. The
collected cells were centrifuged at 1000 g for 3 min at
4 °C, and the supernatant was removed. The pelleted
cells were treated with 1 % formaldehyde solution (100
lL) and were incubated for 20 min on ice. After
centrifugation at 1000 g for 3 min at 4 °C, the super-
natantwasremoved.Thepelletedcellsweretreatedwith
0.1 % TritonÒ X-100 (50 lL), and were incubated for
5 min on ice. The cells were suspended in 1 mL of PBS
and centrifuged at 1000 g for 3 min at 4 °C. After
removing the supernatant, equilibration buffer (100 lL)
was added and incubated for 60 min at 37 °C. EDTA
(20 mM, 1 mL) was added and vortexed to stop the
reaction. After centrifugation, the pelleted cells were
resuspended in 100 lL of PBS. The resultant cells were
mixed with 1 mL of BD FACSFlow Sheath Fluid (BD
Biosciences, San Jose, CA, USA) and analyzed by a
flowcytometer. Two-colorflowcytometricanalysiswas
performed using a FACS Calibur flow cytometer (BD
Biosciences). Analysis was done using FlowJo software
(Tree Star, Ashland, OR, USA).
UV spectra of HMB–ASH–Fe(III) complex
Although our previous work suggested HMB–ASH
binds Fe(III) (Yamada et al. 2012), the chemical
structure of the HMB–ASH–Fe(III) complex was
unknown. To better understand the chemical structure
of the HMB–ASH–Fe(III) complex, the binding ratio
of HMB–ASH to Fe(III) was evaluated by UV
titration. Figure 4a shows the changes in the HMB–
ASH UV spectra as a function of Fe(III) concentration.
As the Fe(III) concentration increased, the UV spectra
changed, with an isosbestic point at 364 nm, suggest-
ing that HMB–ASH bound Fe(III) to form a complex.
Below 364 nm, the absorbance decreased with an
increase in Fe(III) concentration. In contrast, the
absorbance was increased above 364 nm. Using the
absorbance at 460 nm, a molar ratio plot (Fig. 4b) was
drawn, because it prevented the absorbance of Fe(III)
itself. The molar ratio plot indicated that HMB–ASH
can bind to Fe(III) at a molar ratio of 2:1. In addition,
the apparent binding constant K, calculated by Benesi-
Hildebrand plot (Roy et al. 2007), was 3.06 9 107,
based on the absorbance changes at 460 nm.
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Biometals
Fig. 2 Anti-proliferative effects of HMB–ASH (a), Fe(III) (b), and the HMB–ASH–Fe(III) complexes (c) in HepG2 cells (closed
circle with solid line). [mean S.E. (n = 3)]
Fig. 3 (a) Anti-proliferative effects of the HMB–ASH–Fe(III)
complexes on HepG2 (closed circle with solid line), MCF7
(closed square with dotted line), and K562 cells (closed triangle
with dashed line). [mean S.E. (n = 3)], and (b) Anti-
proliferative effects of the HMB–ASH–Fe(III) complexes
(closed circle with solid line), oxaliplatin (closed square with
dashed line), and etoposide (closed triangle with dotted line) in
HepG2 cells. [mean S.E. (n = 3)]
Fig. 4 UV-Vis spectrum of
HMB–ASH with Fe(III)
(a) and molar ratio plot at
460 nm (b). Dashed arrow
in (a) indicates an increase
in Fe(III) concentration
(100, 200, 400, 600, 800,
and 1000 lM)
Chromatographic and accurate mass analysis
by LC–TOF-MS
at approximately 8.6 min may be the HMB–ASH–
Fe(III) complex, because it may have an increased
hydrophobicity as compared to HMB–ASH. As
determined by LC–TOF-MS analysis, the peak at
8.6 min was a peak of HMB–ASH–Fe(III) complex,
of which the m/z was 654.11620 (Fig. 6a), indicating
that the elemental composition of HMB–ASH–Fe(III)
was C30H26O8N6Fe (error -1.17 ppm). From these
accurate mass data, the presumed structure of the
HMB–ASH–Fe(III) complex was two molecules of
Figure 5 shows representative chromatograms for
HMB–ASH (Fig. 5a) and HMB–ASH mixed with
Fe(III) (Fig. 5b), obtained by reversed-phase HPLC
with UV detection at 335 nm. HMB–ASH itself eluted
at approximately 4 min. However, after adding
Fe(III), HMB–ASH peak at 4 min completely disap-
peared. Rather, a new peak eluted at 8.6 min. The peak
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Biometals
Fig. 5 Chromatograms of
HMB–ASH (a) and the
HMB–ASH–Fe(III)
complexes (b), obtained by
reversed-phase HPLC with
UV detection (335 nm).
Experimental details are
described in the text
Fig. 6 Mass spectrum of
the HMB–ASH–Fe(III)
complexes obtained by LC–
TOF-MS (a) and the
presumed chemical
structure of HMB–ASH–
Fe(III) complexes (b)
Discussion
HMB–ASH bound to Fe(III), with deletion of four
protons in 2 HMB–ASH ([2HMB–ASH ? Fe(III)-
4H]-) (Fig. 6b).
The purpose of this study was to elucidate the anti-
proliferative effect of metal complexes comprised of
a Schiff-base in cell lines in vitro using HMB–ASH, a
previously developed compound. The anti-prolif-
erative effect of the HMB–ASH–Fe(III) complex
was relatively weak in a human breast cancer cell
lines (MCF-7 cells) and chronic myelogenous
leukemia cell line (K562 cells); however, the human
liver carcinoma cell line, HepG2, was significantly
inhibited by HMB–ASH–Fe(III) complexes. There-
fore, HMB–ASH–Fe(III) complexes may have selec-
tive and pronounced activity against HepG2 cells. To
date, several metal complexes containing Co(II),
Ni(II), Zn(II), Pt(II), Ru(II), Sn(II), or Cu(II) have
shown anti-proliferative effects in HepG2 cells
(Alexandrova et al. 2012; Chen et al. 2013; Dyakova
et al. 2014; Farag et al. 2013; Mahmudov et al. 2014;
Rubino et al. 2014; Tan et al. 2009; Thati et al. 2007).
This is the first report of HepG2 cell growth
inhibition using complexes chelating the transition
metal, Fe(III). Generally, iron is one of the most
important elements for cellular proliferation. Thus,
complex formation between iron-depleting agents
DNA fragmentation and flow cytometry
Using confocal fluorescence microscopy, fluorescein-
12-dUTP fluorescence spots (TUNEL-positive cells)
were clearly observed in cells treated with HMB–
ASH–Fe(III) complexes, as well as those treated with
OXP and ETP (Fig. 7c, d, e). In contrast, few
fluorescence spots were observed in HepG2 cells
treated with HMB–ASH without Fe(III) (Fig. 7b).
These results were in agreement with the data in
Fig. 2, in which HMB–ASH–Fe(III) complex, but not
HMB–ASH alone, exhibited anti-proliferative effects.
To further confirm apoptosis in HepG2 cells, a flow
cytometric analysis was carried out. As shown in
Fig. 8a, HMB–ASH–Fe(III) complex-induced DNA
fragmentation may occur because the HMB–ASH–
Fe(III) complex exhibits similar shifts to OXP, as
compared with the control. The area of fluorescence in
the TUNEL-positive cells after HMB–ASH–Fe(III)
complex treatment was nearly similar to that of OXP
(Fig. 8b).
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Biometals
Fig. 7 Apoptosis detection
using the TUNEL assay in
HepG2 cells treated with
control (a), HMB–ASH (b),
HMB–ASH–Fe(III)
complexes (c), oxaliplatin
(d), and etoposide (e) after
24 h, as viewed under laser
scanning confocal
microscopy. Magnification
at 60 9 10
Fig. 8 Flow cytometric analysis of HepG2 cells treated with control (medium, black), HMB–ASH–Fe(III) complex (blue), and
oxaliplatin (orange) (a), and the integrated fluorescence intensity (b). (Color figure online)
and Fe(III) in cells can induce apoptosis (Yu et al.
2012). To date, several iron chelators, such as
desferrioxamine (DesferalÒ), 5-hydroxypyridine-2-
carboxaldehyde thiosemicarbazone, and pyridoxal
isonicotinoyl hydrazone, could serve as anti-cancer
drugs for refractory leukemia, gastrointestinal tumor,
and children with neuroblastoma etc. (Kovacevic
et al. 2011; Yu et al. 2012). These compounds have
chelation sites in their structures to capture Fe(II) or
Fe(III) in cancer cells. HMB–ASH can also chelate
Fe(III) with a binding constant of 107. Thus, it is
hypothesized that HMB–ASH may act as an iron
chelator in HepG2 cells. However, HMB–ASH itself
has no anti-proliferative effect, suggesting that the
complex of HMB–ASH and Fe(III) is crucial for the
anti-proliferative effect on HepG2 cells. When
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Biometals
Mr. T. Abe, T. Sakamoto, and Miss M. Hanaoka for their
technical assistance.
prepared using the batch method, the HMB–ASH–
Fe(III) complex is relatively stable, due to the binding
constant that is 107 order of HMB–ASH to Fe(III) in
weak basic pH. In addition, a clear single peak for
HMB–ASH–Fe(III) was observed using reversed-
phase chromatography with a mobile phase of
5.0 mM ammonium acetate solution (pH 7.0). Given
the high stability under neutral to weak basic pH
conditions, it seems unlikely that the HMB–ASH–
Fe(III) complex dissociated completely in cells.
Therefore, it is likely that the anti-proliferative effect
of the HMB–ASH–Fe(III) complex differs from the
mechanism of other iron-deprived chelators.
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The anti-proliferative effect of the HMB–ASH–
Fe(III) complexes is likely related to DNA fragmen-
tation, as was clearlyobserved usingthe TUNEL assay.
This was similar to other anticancer drugs, including
OXP and ETP. Based on these results, the HMB–ASH–
Fe(III) complex induces apoptosis in HepG2 cells.
In the present study, we failed to elucidate the three-
dimensional structure of the HMB–ASH–Fe(III) com-
plex by X-ray crystallography. We tried a preliminary
extraction of the HMB–ASH–Fe(III) complex using
liquid–liquid extraction with CH2Cl2 to obtain a single
crystal; however, the extract was a black powder, and
not a single crystal. As shown in Fig. S1, the powder
also exhibited anti-proliferative activity in HepG2
cells, indicating that the powder contains the HMB–
ASH–Fe(III) complex. The powder had an identical
UV and mass spectra to those in Figs. 4a and 6a;
however, a single crystal of the HMB–ASH–Fe(III)
complex could not be obtained by recrystallization.
Therefore, structural X-ray analysis was not per-
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was assumed, as shown in Fig. 6 b, by LC–TOF-MS
analysis and UV absorption spectrometry, three-
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We showed that HMB–ASH–Fe(III) complexes
have an anti-proliferative effect specifically in HepG2
cells via the induction of apoptosis, and that this effect
was comparable to that of ETP and OXP in HepG2
cells. In order to develop HMB–ASH–Fe(III) com-
plexes as a novel anti-proliferative compound, further
work is necessary to clarify the detailed mechanism of
action elucidate the structure by X-ray crystallography.
Acknowledgments The authors thank Drs. Iwasa and Iizuka,
Toho University for their valuable discussions on this study, and
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