Cadmium-induced malignant transformation of rat liver cells:
Potential key role and regulatory mechanism of altered apolipoprotein
E expression in enhanced invasiveness
Masayo Suzukia
, Shuso Takedaa
, Noriko Teraoka-Nishitania
, Akane Yamagataa
Takahiro Tanakaa
, Marika Sasakia
, Natsuki Yasudaa
, Makiko Odaa
, Tatsuji Okanoa
Kazuhiro Yamahiraa
, Yuta Nakamuraa
, Takanobu Kobayashib
, Katsuhito Kinob
Hiroshi Miyazawab
, Michael P. Waalkesc
, Masufumi Takiguchia,
a Laboratory of Xenobiotic Metabolism and Environmental Toxicology, Faculty of Pharmaceutical Sciences, Hiroshima International University (HIU), 5-1-1
Hiro-koshingai, Kure, Hiroshima 737-0112, Japan bKagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1, Shido, Sanuki, Kagawa 769-2193, Japan
Raleigh, NC 27709, USA
A R T I C L E I N F O
Article history:
Received 26 January 2017
Received in revised form 13 March 2017
Accepted 13 March 2017
Available online 16 March 2017
Keywords:
Cadmium
Malignant transformation
Apolipoprotein E
DNA hypermethylation
Cell invasion
TRL 1215 cells
A B S T R A C T
Cadmium is a transition metal that is classified as human carcinogen by the International Agency for
Research on Cancer (IARC) with multiple target sites. Many studies using various model systems provide
evidence of cadmium-induced malignancy formation in vivo or malignant cell transformation in vitro.
Nonetheless, further studies are needed to completely understand the mechanisms of cadmium
carcinogenicity. Our prior studies have utilized a rat liver epithelial cell line (TRL 1215) as a model for
cadmium-induced malignant transformation. In the present study, we focused on the molecular
mechanisms of this malignant transformation, especially with regard to hyper-invasiveness stimulated
by cadmium transformation. By performing a series of biochemical analyses on cadmium transformed
cells, it was determined that cadmium had significantly down-regulated the expression of apolipoprotein
E (ApoE). ApoE was recently established as a suppressor of cell invasion. A key factor in the suppression of
ApoE by cadmium appeared to be that the metal evoked a 5-aza-20
-deoxycytidine-sensitive
hypermethylation of the regulatory region of ApoE, coupled with interference of the action of liver X
receptor a (LXRa), a transcriptional regulator for ApoE. Furthermore, the expression of LXRa itself was
suppressed by cadmium-mediated epigenetic modification. Re-expression of ApoE clearly abrogated the
cell invasion stimulated by cadmium-induced malignant transformation. Together, the current results
suggest that the cadmium-mediated enhanced cell invasion is linked to down-regulation of ApoE during
malignant transformation these liver cells.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
Cadmium is a highly toxic transition metal, and is classified as a
human carcinogen by the International Agency for Research on
Cancer (IARC) (IARC, 2012). Many studies have shown the linkage
between cadmium exposure and human cancer or cancer in rodent
models, and in vitro cadmium has been associated with malignant
cell transformation. There is evidence for relationship between
cadmium exposure to human and cancer of the liver (Waalkes,
2000). Previous reports showed that cadmium induce tumors of
the liver in rodents (Waalkes and Rehm, 1994; Waalkes, 2000).
However, the precise mechanism or mechanisms of cadmium
carcinogenesis are not well defined and further studies are
required to completely understand its mechanisms. Since binding
of cadmium to DNA is weak, cadmium is thought to be a heavy
metal exhibiting an indirect genotoxic/mutagenic profile (Waalkes
and Poirier, 1984; Waalkes, 2000). Epigenetic changes have been
Abbreviations: IARC, International Agency for Research on Cancer; ApoE,
Apolipoprotein E; LXR, liver X receptor; LXRE, liver X receptor response element; 5-
aza-dC, 5-aza-20
-deoxycytidine; ABCA1, ATP-binding cassette transporter; SREBP-
1c, sterol regulatory element-binding protein-1c.
* Corresponding author.
E-mail address: [email protected] (M. Takiguchi).

http://dx.doi.org/10.1016/j.tox.2017.03.014

0300-483X/© 2017 Elsevier B.V. All rights reserved.
Toxicology 382 (2017) 16–23
Contents lists available at ScienceDirect
Toxicology
journal homepage: www.elsevier.com/locate/toxicol
shown to be involved in the cadmium-induced carcinogenesis; for
example, both the degree of genomic DNA methylation and the
enzyme activity responsible for DNA methylation (DNA methyl￾transferases) are increased by long-term cadmium exposure
(Takiguchi et al., 2003; Benbrahim-Tallaa et al., 2007; Jiang
et al., 2008). Moreover, expression of tumor suppressor genes or
genes involved in apoptosis was known to be down-regulated
through their DNA hypermethylation in cadmium-exposed cells
(Benbrahim-Tallaa et al., 2007; Wang et al., 2012; Yuan et al., 2013).
Therefore, in order to elucidate the mechanisms underlying
cadmium-induced carcinogenesis, it is important to investigate
the cadmium-induced carcinogenesis by focusing on the epige￾netic changes.
Apolipoprotein E (ApoE) is a key gene for the regulation of lipid
metabolism and cholesterol homeostasis (Mahley, 1988). ApoE has
a liver X receptor (LXR) response element (LXRE) in its promoter
region (Laffitte et al., 2001; Lu et al., 2009; Yue and Mazzone,
2009), and ApoE expression is directly regulated by LXRa (Ulven
et al., 2004; Lu et al., 2009). Recently, it has been demonstrated that
ApoE molecule is a suppressor for cell invasion as a novel
physiological function (Bhattacharjee et al., 2011; Pencheva et al.,
2012). In general, cell proliferation, invasion, and migration are
increased as cells undergo malignant transformation. This is true
for cells malignantly transformed by cadmium (Takiguchi et al.,
2003). Thus, if ApoE plays an important role in control of cell
invasion it is possible that ApoE activity could be altered during
cadmium-induced hyper-invasiveness associated with malignant
transformation. Any such altered ApoE expression could be from
epigenetic modification of the ApoE gene in cadmium-exposed
cells.
The rat liver epithelial cell line TRL 1215 (TRL 1215 cells) has
been widely used as an in vitro model for metal-induced
malignant transformation (Zhao et al., 1997). We have previously
shown that malignant transformation in TRL 1215 cells is induced
by exposure to 2.5mM cadmium for 10 weeks (Takiguchi et al.,
2003). In addition, these cadmium-induced malignant trans￾formants show persistent genomic DNA hypermethylation despite
placing cells in medium freed from cadmium for up to an
additional 4 weeks (Takiguchi et al., 2003), implicating cadmium￾associated epigenetic changes in concert with transformation.
These cells also show the hallmarks of acquired malignancy
with cadmium treatment, including greatly increased invasion
(Takiguchi et al., 2003). Given this aspect of increased invasion
together with the persistent epigenetic changes in seen cadmium
transformed TRL 1215 cells we suspected genes involved with cell
invasion and susceptible to epigenetic control could be a factor,
Thus, in the present study, we focused on the ApoE in the
malignant transformation induced by cadmium and investigated
whether ApoE expression is epigenetically regulated by cadmium
in TRL 1215 cells, to reveal any relationship between epigenetic
changes on ApoE expression and stimulated cell invasion during
cadmium-induced malignant transformation.
2. Materials and methods
2.1. Reagents
5-Aza-20
-deoxycytidine (5-aza-dC) and LXR agonist GW3965
were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Santa
Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-ApoE
antibody and anti-actin antibody were purchased from Santa Cruz
Biotechnology and Sigma-Aldrich, respectively. Anti-goat IgG
antibody and anti-rabbit antibody were purchased from Sigma￾Aldrich and Vector Laboratories (Burlingame, CA, USA), respec￾tively.
2.2. Cell cultures and treatments
The TRL1215 cells were originally derived from neonatal rat
liver that was finely minced, untreated and cultured as described (
Idoine et al., 1976). The cell line arose spontaneously and can be
subjected to long-term culture while maintaining hepatocyte
features but without acquiring a malignant phenotype (spontane￾ous immortalization). The cells were cultured in RPMI 1640
(Sigma-Aldrich) supplemented with heat inactivated 10% fetal
bovine serum (FBS) (Equitech-Bio, Kerrville, TX, USA), under an
atmosphere of 5% CO2 at 37 C. TRL 1215 cells were exposed to
2.5mM cadmium chloride for 10 weeks, and then the cells were
cultured in cadmium-free medium for 4 weeks (Fig. 1A).
2.3. Cell viability analysis (MTS assay)
In the cell viability study (Fig. 1C), cells were seeded into 96-
well plates at a density of 5000 cells/well. After 48 h of
Fig. 1. Evidence of transformation of liver TRL 1215 cells by chronic cadmium
exposure. (A) Method for treatment with cadmium. TRL 1215 cells chronically
exposed to 2.5mM cadmium for 10 weeks, followed by placing cadmium-free
medium for an additional 4 weeks. (B) Effects of chronic exposure to cadmium on
the morphology of TRL 1215 cells. (C) Effects of chronic exposure to cadmium on cell
proliferation. Data are expressed as the percent of the passage-matched control
(control), and represent the mean  S.E. (n = 3). *Significantly different (P < 0.05)
from the passage-matched control.
M. Suzuki et al. / Toxicology 382 (2017) 16–23 17
incubation, cell viability was analyzed using the CellTiter 96
Aqueous One Solution Cell Proliferation Assay (MTS reagent;
Promega, Madison, WI, USA), according to the reported protocols
(Takeda et al., 2016). In the trials in Fig. 8B, cells were seeded into
96-well plates at a density of 5000 cells/well, and 1mM 5-aza-dC,
1mM GW3965 or 1mM 5-aza-dC/1mM GW3965 were added. After
48 h of incubation, MTS assay was performed. Test chemicals were
prepared in appropriate organic solvents including DMSO. Control
incubations contained equivalent additions of solvents with no
measurable influence of vehicle observed on cell viability at the
final concentrations used.
2.4. Cell invasion assay
Cell invasiveness was assayed in Corning BioCoat Matrigel
Invasion Chamber 24-well Plate 8.0 mm (Corning, Corning, NY,
USA). The chamber was inserted to 24-well plate filled with RPMI
1640 medium with 10% FBS as the chemoattractant. For the
invasion assay, the control or cadmium exposed TRL 1215 cells
were suspended in serum-free RPMI 1640 medium and added to
the inserted chamber. In addition, the control cells were treated
with and without(Control) an inhibitory antibody specific for ApoE
(50mg/mL) (Fig. 4). The cells were incubated for 24 h at 37 C under
5% CO2. The cells that invaded through the Matrigel and onto the
lower surface of the membrane were fixed with 70% ethanol,
stained with 0.5% crystal violet, and counted under a microscope.
2.5. DNA microarray analysis
Total RNA was isolated from 2.5mM cadmium-exposed TRL
1215 cells or passage-matched (non-cadmium treated) control
cells using RNeasy Mini Kit (Qiagen, Inc. Hilden, Germany). The
specific gene expression pattern in cadmium-exposed TRL 1215
cells was examined by DNA microarray analysis in comparison
with non-cadmium treated control cells. cRNA labeling were
conducted using a Quick Amp Labeling Kit (Agilent, Palo Alto, CA,
USA). Labeled cRNA (Cy5) was hybridized to rat 12 K oligo DNA
microarray (CustomArray Inc., Bothell, WA, USA). Specific hybrid￾ization was analyzed using a GenePix 4000 B array scanner (Axon
Instrument, Union City, CA), and evaluated as a scatter-plot graph
for gene expression. Ecogenomics Inc. (Fukuoka, Japan) provided
assistance with the experiments.
Fig. 2. Effect of chronic exposure to cadmium on invasiveness of TRL 1215 cells. (A)
Scheme for cell invasion assay. (B) Effects of exposure to cadmium for 10 weeks on
cell invasion. Data are given as the percent of the passage-matched control, and
represent the mean  S.E. (n = 3). *Significantly different (P < 0.05) from the
passage-matched control. (C) Total number of invaded cells (see Methods) after
the treatment with 2.5mM cadmium (+) or without cadmium (–) for 10 weeks and
more 4 weeks in cadmium-free medium. Insert figure, representative results of cell
invasion assay. Representative data from two independent experiments with
duplicate are shown.
Fig. 3. Effects of chronic exposure to cadmium on ApoE expression. (A) DNA
microarray analysis. (B) Real-time RT-PCR analysis. Data in A and B are expressed as
a fold induction with the passage-matched control set as 1 and are given as the
mean  S.E. (n = 3). *Significantly different (P < 0.05) from the passage-matched
control. (C) Western blot analysis. b-actin was used as a loading control. Results
shown for typical assessment.
Fig. 4. Effect of an inhibitory antibody specific for ApoE on invasiveness of TRL 1215
cells. Upper panel: Representative results of cell invasion assay. Lower panel: Cell
invasion tended to be stimulated by an inhibitory antibody specific for ApoE (ApoE
Ab) compared to non-cadmium treated control cells (Control). Assay for Cd￾stimulated cell invasion was also performed. Data are expressed as a fold-change
with the passage-matched control set as 1 and are given as the mean  S.E. (n = 3).
*Significantly different (P < 0.05) from the passage-matched control.
18 M. Suzuki et al. / Toxicology 382 (2017) 16–23
2.6. Analysis of real-time reverse transcription-polymerase chain
reaction (real-time RT-PCR)
Total RNA was isolated by TRIzol RNA Isolation Reagent
(Thermo Fisher Scientific, Waltham, MA, USA) following the
manufacturer’s instructions. The mRNA (125 ng) was then
reversetranscribed into cDNA using High-Capacity cDNA Reverse
Transcription Kit (Thermo Fisher Scientific). Real-time quantitative
RT-PCR assays were performed with Fast SYBR Green Master Mix
(Thermo Fisher Scientific) and Applied Biosystems StepOne Real￾Time PCR System (Thermo Fisher Scientific). The primers used
were b-actin (sense) 50
-AAG TCC CTC ACC CTC CCA AAA G-30 and
b-actin (antisense) 50
-AAG CAA TGC TGT CAC CTT CCC-30
. The
primer used were ApoE (sense) 50
-TCT GTG GCT ACC AAC TCC ATT
G-30 and ApoE (antisense) 50
-GGC GTA GGT GAG GGA TGA TC-30
The primers used were LXRa (sense) 50
-AGT GTC GCC TTC GCA AAT
G-30 and LXRa (antisense) 50
-TCC TCT TCT TGA CGC TTC AGT TTC-30
The primers used were LXRb (sense) 50
-AAG CTG GTG AGC CTG
CGC-30 and LXRb (antisense) 50
-CGG CAG CTT CTT GTC CTG-30
. The
primers used were the ATP-binding cassette transporter (ABCA1)
(sense) 50
-GTG GAT GGT TTG GCG CTA AA-30 and ABCA1 (antisense)
50
-GGG AAA CAA CCC AGT CAG TAT TG-30
. The primers used were
the sterol regulatory element-binding protein-1c (SREBP-1c)
(sense) 50
-GAC AAG ATT GTG GAG CTC AAG GA-30 and SREBP-1c
(antisense) 50
-TGT GCT GTA AGA AGC GGA TGT AG-30
. ApoE, LXRa,
LXRb, ABCA1 and SREBP-1c mRNA levels were normalized to
b-actin mRNA levels. Relative quantification was performed using
the DDCt method.
2.7. Western blot analysis
Proteins were separated by sodium dodecyl sulfate (SDS)-
polyacrylamide gel electrophoresis (PAGE) (12% gels), and trans￾ferred to a polyvinylidene difluoride (PVDF) membrane (Millipore,
Billerica, MA). Antibodies for ApoE (1:1000 dilution) and b-actin
(1:10,000 dilution) were used as primary antibodies to detect ApoE
and b-actin, respectively. Anti-goat IgG antibody (1:5000 dilution)
and anti-rabbit IgG antibody (1:5000 dilution) conjugated
peroxidase were used as secondary antibodies, and ECL Prime
Western Blotting Detection Reagent (GE Healthcare Japan Corpo￾ration, Tokyo, Japan) were used for detection.
2.8. Data analysis
Differences were considered significant when the P value was
calculated as less than 0.05. Statistical differences between two
groups were calculated by the Student’s t test. Other statistical
analyses were performed using Scheffe’s F test, a post hoc test for
analyzing results of ANOVA testing. These calculations were
performed using Statview 5.0 J software (SAS Institute Inc., Cary,
NC, USA).
3. Results and discussion
3.1. Malignant transformation of TRL 1215 cells by chronic cadmium
exposure
TRL 1215 cells were cultured continuously for 10 weeks in the
presence of 2.5mM cadmium, followed by an additional 4 weeks in
cadmium free media (total 14 weeks) (Fig. 1A). After 14 weeks, the
cells exhibited a spindle-like morphology with pronounced
elongation compared to cells cultured in cadmium-free media
which show a cuboidal, epithelial like configuration (Fig. 1B). We
counted the cell numbers as an indication of proliferative capacity
using the MTS assay. As shown in Fig. 1C, cell proliferation, which is
typically enhanced in malignant cell populations, of the chronic
cadmium-exposed cells was much higher than the passage￾matched control, indicating that cadmium stimulated this
hallmark of cancerous conversion. This is consistent with our
previous results where TRL 1215 cells treated with cadmium for 10
weeks showed enhanced proliferation (Takiguchi et al., 2003). In
addition, in cells exposed to cadmium in the present study for 10
weeks there was an increased invasiveness compared to the
passage-matched control (1.5-fold) (Fig. 2B). After an additional 4
weeks of culture in medium freed from cadmium, the chronic
cadmium-exposed cells showed an even more markedly enhanced
invasiveness compared to the passage-matched cells (7.4-fold
increase) (Fig. 2C, see also inset). This suggests that once TRL 1215
cells are transformed by cadmium, the malignant process is
“imprinted” and continues to higher aggression levels even in the
absence of further metal exposure.
3.2. Chronic cadmium exposure induces down-regulation of ApoE
expression in a DNA methylation-dependent manner
We performed DNA microarray analysis to investigate genes
impacted by malignant transformation via chronic exposure to
cadmium in TRL 1215 cells treated for 10 weeks in the presence of
cadmium, followed by an additional 4 weeks without cadmium
(see Fig. 1A); among 3779 genes investigated the expression of 76
genes was significantly altered compared to passage-matched
control (P < 0.05). Consistent with the hyper-proliferative nature of
the cadmium exposed cells, the expression of CDC2, a cell cycle
control gene, was highly up-regulated (5.8-fold). We also sought to
investigate potentially key molecular events responsible for
Fig. 5. Effects of chronic exposure to cadmium on ApoE expression as a function of
time. (A) Time course analyses (0, 10, or 14 w) of ApoE expression levels in TRL 1215
cells were performed after the treatment with 2.5mM cadmium (+) or without
cadmium (–) (see also Fig. 1A). Data are expressed as a fold induction based on 0 w
(indicated as 1), as the mean  S.E. (n = 3). (B) The suppressed level of ApoE
expression was restored by 1mM 5-aza-dC. ApoE expression levels were analyzed
by real-time RT-PCR. Data are expressed as a fold induction based on cells treated
with cadmium for 14 w, as the mean  S.E. (n = 3). *Significantly different (P < 0.05)
from the Cd 14 w cells. (C) Cadmium-stimulated cell invasion was abrogated by the
presence of 1mM 5-aza-dC. Data expressed as the mean of two independent results.
M. Suzuki et al. / Toxicology 382 (2017) 16–23 19
malignant transformation, especially with regard to cell invasion
by cadmium. A recent gene of focus in the literature involved in cell
invasion has been ApoE, and chronic cadmium exposure caused
the expression of ApoE to be markedly decreased (0.26-fold)
(Fig. 3A). Recently, it has been demonstrated that constitutive
expression of ApoE will abrogate cell invasion (Bhattacharjee et al.,
2011; Pencheva et al., 2012). As shown in Fig. 3B, ApoE expression
was decreased to 0.11-fold of control (14 weeks cultured) using
real-time RT-PCR analysis (qRT-PCR) (Fig. 3B) confirming array
results. In addition, cadmium-induced ApoE down-regulation was
demonstrated at the protein level (0.26-fold of control) by Western
blot analysis (Fig. 3C). We also found non-cadmium treated control
cell invasion tended to be stimulated by an inhibitory antibody
specific for ApoE (ApoE Ab) (Fig. 4). As expected, in this experiment
Fig. 7. Effect of 5-aza-dC on reduced LXRa expression induced by chronic cadmium
exposure. TRL 1215 cells that had been treated with cadmium for 10 w, followed by
additional culture time (4 w) in the absence of cadmium (indicated as Cd 14 w) were
studied. Real-time RT-PCR analysis of ApoE was performed using the cells in the
presence or absence of 1mM 5-aza-dC for 48 h. Data are expressed as a fold
induction based on the passage-matched control set to 1 and data are given as the
mean  S.E. (n = 3). *Significantly different (P < 0.05) from the passage-matched
control. **Significantly different (P < 0.05) from the cadmium-treated group (Cd
14 w).
Fig. 6. Effect of chronic exposure to cadmium on LXRa and LXRb expression. (A)
LXRa and (B) LXRb expression levels were analyzed by real-time RT-PCR. Data are
expressed as a fold induction based on the passage-matched control set to 1 and
data are given as the mean  S.E. (n = 3). *Significantly different (P < 0.05) from the
passage-matched control.
Fig. 8. Effects of 5-aza-dC, GW3965, or 5-aza-dC plus GW3965 on ApoE expression.
(A) TRL 1215 cells were treated with cadmium for 10 w, followed by additional
culture time (4 w) in the absence of cadmium were compared to the vehicle-treated
controls. Real-time RT-PCR analysis of ApoE was performed using the cells after 48 h
of exposure to 1mM 5-aza-dC, 1mM GW3965, or 1mM 5-aza-dC plus 1mM
GW3965. Data are expressed as a fold induction based on the vehicle-treated
control set to 1 and data are given as the mean  S.E. (n = 3). *Significantly different
(P < 0.05) from the vehicle-treated control. **Significantly different (P < 0.05)
between 5-aza-dC-treated group and 5-aza-dC plus GW3965-treated group. (B)
Cells were treated with the same regimen as above in (A) and cell viability was
determined by the MTS assay. Data are expressed as the percent of the vehicle￾treated control, and data are given as the mean  S.E. (n = 6).
20 M. Suzuki et al. / Toxicology 382 (2017) 16–23
Cd evoked significant stimulation of cell invasion. Together these
results suggested that cadmium suppresses the expression of ApoE
and thereby stimulates TRL 1215 cell invasion during cancerous
conversion.
To obtain mechanistic insight into the alteration of ApoE
expression by cadmium, we prepared additional total RNA samples
from TRL 1215 cells at 0 (indicated as Control = 1) and 10 or 14
weeks after treatment with (+) or without () cadmium (Fig. 5A).
The expression level of ApoE was decreased (0.42- or 0.22-fold) by
culture for 10 or 14 weeks, even in the absence of exposure to
cadmium. ApoE was additionally down-regulated 0.19-fold by
cadmium exposure for 10 weeks (Cd 10 w, +). Moreover, ApoE
expression was further down-regulated in the cells cultured for 14
weeks (i.e., an additional 4 weeks after the 10 weeks exposure to
cadmium; Cd-free). These results indicated that cadmium-induced
transformation accelerated the down-regulation of ApoE expres￾sion. As reported previously, DNA methyltransferase activity and
its expression were increased by chronic exposure to cadmium
(Benbrahim-Tallaa et al., 2007; Yuan et al., 2013), and some gene
expression, such as p16, RASSF1A, and caspase-8, were down￾regulated in the cadmium-exposed cells (Benbrahim-Tallaa et al.,
2007; Wang et al., 2012; Yuan et al., 2013). In addition, we reported
that the degree of DNA methylation was significantly increased in
cells cultured for 4 additional weeks even in the absence of
cadmium (Takiguchi et al., 2003). Thus, we suspected that ApoE
expression maybe modulated by DNA hypermethylation. In
support of this concept, when chronic cadmium-exposed TRL
1215 cells (14W) were treated with 5-aza-dC, a DNA-demethylat￾ing agent, ApoE expression was indeed increased 10-fold (Fig. 5B),
which means that 5-aza-dC can completely recover from the
cadmium down-regulation of ApoE (0.016-fold) to the control
levels (0.22-fold) (see Fig. 5A). Given that ApoE suppression by
cadmium appears epigenetically controlled and alterable by
altering DNA methylation, 5-aza-dC treatment should mitigate
the invasiveness of cadmium-transformed TRL 1215 cells that
occurs via up-regulation of ApoE (Fig. 5B). Indeed, as seen in
Fig. 5C, when compared to control cadmium alone cells, cell
invasion was suppressed to 0.16-fold after treatment with the
epigenetic modifier (+5-aza-dC). These results strongly suggest
that ApoE expression is down-regulated by DNA hypermethylation
in malignant transformed TRL 1215 cells by cadmium.
Fig. 9. Effects of chronic exposure to cadmium on ABCA1 and SREBP-1c expression. (A and B) TRL 1215 cells treated with cadmium for 10 w, followed by additional culture
time (4 w) in the absence of cadmium were compared to the passage-matched control. Real-time RT-PCR analysis of ABCA1 and SREBP-1c was performed. Data are expressed
as a fold induction based on the passage-matched control set to 1 and data are given as the mean  S.E. (n = 3). *Significantly different (P < 0.05) from the passage-matched
control.(C and D) Real-time RT-PCR analysis of ABCA1 and SREBP-1c was performed using the cells in the presence or absence of 1mM GW3965 for 48 h. Data are expressed as
a fold induction based on the vehicle-treated control set to 1 and data are given as the mean  S.E. (n = 3). *Significantly different (P < 0.05) from the vehicle-treated control.
M. Suzuki et al. / Toxicology 382 (2017) 16–23 21
3.3. Involvement of LXRa in the cadmium-mediated down-regulation
of ApoE expression
There is one LXRE in the promoter region of ApoE (Laffitte
et al., 2001; Lu et al., 2009; Yue and Mazzone, 2009), and its
expression appears to be directly regulated by the transcription
factor LXRa (Ulven et al., 2004; Lu et al., 2009). Thus, it is unclear
whether ApoE expression is regulated either by local DNA
hypermethylation, or by LXRa, or by some combination of both.
Therefore, we next investigated levels of LXRa, an isoform with
restricted expression primarily to liver, together with LXRb, an
isoform expressed in almost all tissues (Song et al., 1995). The
results indicated LXRa expression was significantly down￾regulated (0.36-fold of control) in the chronic cadmium-exposed
cells (Fig. 6A), whereas LXRb expression was not altered by
cadmium treatment (Fig. 6B). This suggests specific involvement
of LXRa in cadmium-mediated ApoE expression in transformed
TRL 1215 cells. As shown in Fig. 7, the suppression of LXRa
expression induced by chronic cadmium (Cd 14 w) was clearly
restored to control levels by 5-aza-dC treatment.
It appears that ApoE expression can be increased by the
treatment with LXR agonist GW3965 in 3T3-L1 fibroblasts (Yue
and Mazzone, 2009). If LXR agonists act similarly in TRL 1215 cells,
GW3965 itself might be “effective” in stimulation of ApoE even
though LXRa expression is reduced. In TRL 1215 cells chronically
exposed to cadmium, GW3965 had no significant effect on the
expression of ApoE alone while 5-aza-dC treatment was very
effective (Fig. 8A). The question becomes how did 5-aza-dC alone
up-regulate the expression of ApoE in the absence of an exogenous
agonist like GW3965? D-Glucose has been shown to be an
endogenous ligand for LXRa (Mitro et al., 2007). Since our media
supplies D-glucose as a nutrient at high levels (i.e., 11.1 mM), ApoE
might be able to be maximally activated by D-glucose-liganded
LXRa after being freed from DNA hypermethylation by 5-aza-dC.
Importantly, ApoE expression was further increased by the co￾treatment of 5-aza-dC and GW3965 compared to 5-aza-dC alone
(Fig. 8A). These various individual or combination treatments did
not affect cell viability (Fig. 8B).
3.4. ABCA1 and SREBP-1c are up-regulated by GW3965, a LXRa
agonist
It remains unclear whether among LXRa-regulated genes if
cadmium selectively suppressed the expression of ApoE through
DNA hypermethylation or if this was a generalized occurrance.
ABCA1 and SREBP-1c are direct target genes of LXRa in addition to
ApoE (Costet et al., 2000; Repa et al., 2000). The expression of
ABCA1 and SREBP-1c was decreased 0.54-fold and 0.63-fold,
respectively, by cadmium (Fig. 9A and B), but the magnitude was
much less than ApoE (see Fig. 3B). In addition, in contrast to the
case with ApoE (see Fig. 8A), ABCA1/SREBP-1c expression was
significantly increased by GW3965 treatment, 6.4-fold and 1.6-
fold, respectively (Fig. 9C and D). Taken together with evidence
that 5-aza-dC treatment had no significant effect on the expression
of the ABCA1 and SREBP-1c genes (data not shown), it appears that
ApoE expression is specific for with cadmium transformation in
this cell line and modulated by DNA hypermethylation status of
this particular gene (Fig. 10).
4. Conclusions
In this study, we showed that cadmium-stimulated cell
invasion is inhibited by DNA demethylation, which is associated
with increased expression of ApoE in TRL 1215 cells. Further works
are needed to clarify how cadmium specifically induces DNA
hypermethylation in the regulatory region of ApoE. These results
strongly indicate that cadmium-induced down-regulation of ApoE
requires mechanistic events associated with LXRa expression and
DNA hypermethylation (Fig. 10).
Conflicts of interest
We have no actual or potential conflicts of interest.
Acknowledgement
This study was supported in part by a Grant-in-Aid for Scientific
Research [15790081 (to M.T.)] from the Japan Society for the
Promotion of Science (JSPS) KAKENHI.
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