ORIGINAL PAPER
Sodium arsenite exposure inhibits AKT and Stat3 activation,
suppresses self-renewal and induces apoptotic death of embryonic
stem cells
Vladimir N. Ivanov • Gengyun Wen •
Tom K. Hei
Published online: 11 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Sodium arsenite exposure at concentration
[5 lM may induce embryotoxic and teratogenic effects in
animal models. Long-term health effects of sodium arsenite
from contaminated drinking water may result in different
forms of cancer and neurological abnormalities. As cancer
development processes seem to be originated in stem cells,
we have chosen to examine the effects of sodium arsenite
on signaling pathways and the corresponding transcription
factors that regulate cell viability and self-renewal in
mouse embryonic stem cells (ESC) and mouse neural stem/
precursor cells. We demonstrated that the crucial signaling
pathway, which was substantially suppressed by sodium
arsenite exposure (4 lM) in ESC, was the PI3K–AKT
pathway linked with numerous downstream targets that
control cell survival and apoptosis. Furthermore, the whole
core transcription factor circuitry that control self-renewal
of mouse ESC (Stat3-P-Tyr705, Oct4, Sox2 and Nanog)
was strongly down-regulated by sodium arsenite (4 lM)
exposure. This was followed by G2/M arrest and induction
of the mitochondrial apoptotic pathway that might be
suppressed by caspase-9 and caspase-3 inhibitors. In contrast
to mouse ESC with very low endogenous IL6, mouse
neural stem/precursor cells (C17.2 clone immortalized by
v-myc) with high endogenous production of IL6 exhibited a
strong resistance to cytotoxic effects of sodium arsenite
that could be decreased by inhibitory anti-IL6 antibody or
Stat3 inhibition. In summary, our data demonstrated suppression
of self-renewal and induction of apoptosis in
mouse ESC by sodium arsenite exposure, which was further
accelerated due to simultaneous inhibition of the
protective PI3K–AKT and Stat3-dependent pathways.
Keywords Embryonic stem cells Á AKT Á
Sodium arsenite Á Apoptosis
Abbreviations
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ESC Embryonic stem cells
FACS Fluorescence-activated cell sorter
FGF2 Fibroblast growth factor-2 (basic)
IETD N-Acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde)
IjB Inhibitor of NF-jB
IGF1 Insulin-like growth factor-1
IGF1R Insulin-like growth factor-1 receptor
IKK Inhibitor nuclear factor kappa B kinase
JNK C-Jun N-terminal kinase
LEHD N-Acetyl-Leu-Glu-His-Asp-CHO (aldehyde)
MAPK Mitogen-activated protein kinase
MEK MAPK/ERK kinase
NF-jB Nuclear factor kappa B
NSC Neural stem cells
PARP-1 Poly(ADP-ribose)polymerase-1
PI Propidium iodide
PPP Picropodophyllin
STAT Signal transducers and activators of
transcription
TNFa Tumor necrosis factor alpha
zVAD Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-
fluoromethylketone
V. N. Ivanov (&) Á G. Wen Á T. K. Hei
Department of Radiation Oncology, Center for Radiological
Research, College of Physicians and Surgeons, Columbia
University, 630 West 168th Street, New York, NY 10032, USA
e-mail: vni3@columbia.edu
123
Apoptosis (2013) 18:188–200
DOI 10.1007/s10495-012-0779-1
Introduction
Arsenic contamination of drinking water is a major environmental
problem that affects more than 100 millions of
people worldwide. Long-term health effects of inorganic
sodium arsenite may result in different forms of cancer and
neurological abnormalities [1, 2]. Furthermore, sodium
arsenite is a well-characterized teratogen in animal models
inducing a general embryotoxicity and causing a substantial
disruption in embryonic neurogenesis [3]. However,
the precise molecular mechanisms of these effects are
still uncertain.
Sodium arsenite at high doses ([10 lM) represents a
general cell poison acting in two directions. As a sulfhydryl
reagent, arsenite binds to the free thiol (–SH) groups of
numerous enzymes inhibiting their activities and also significantly
decreasing levels of glutathione (GSH) that further
destroys the protective cellular functions [4]. As an
inducer of production of reactive oxygen species, sodium
arsenite causes oxidative stress, mitochondrial damage and
a broad range of pathological conditions. These complementary
negative effects of sodium arsenite finally result in
the induction of cell death [5–8]. On the other hand,
chronic sodium arsenite exposure, which occurred in most
cases at low doses (1–5 lM), could notably affect signaling
pathways and substantially changing gene regulation in
cells, including probably signaling and gene regulation in
stem cells. Sodium arsenite is well-documented human
carcinogen at these doses in drinking water [2]. As cancer
development processes seem to be originated in stem cells
[9], we have chosen to examine the effects of sodium
arsenite on stem cell viability and self-renewal capacity.
The self-renewal of embryonic stem cells (ESC) and the
maintenance of their pluripotency are dependent on multifactorial
stimulation of the cell signaling pathways by certain
combinations of cytokines and growth factors that
control ‘‘stemness’’-specific gene expression. Even though
the combinations of the extrinsic factors are quite different
for mouse and human ESC, their downstream targets
include similar sets of genes, which encode transcription
factors Oct4, Sox2, Nanog, Klf4 and cMyc, the key regulators
of pluripotency of ESC [10, 11]. For mouse ESC in
culture, external signaling molecules, such as the cytokine
leukemia inhibitory factor (LIF) and several growth factors,
BMP4, IGF1/2 and Wnt, regulate their self-renewal and
pluripotency. Numerous investigations revealed a ‘‘minimal’’
combination of LIF-dependent [12, 13] and BMP-dependent
signals [14], which integrate with core transcriptional networks
and control the maintenance and proliferation of mouse
ESC together with a dynamic inhibition of the differentiation
pathways [15–17]. The critical pathways, which regulate
the self-renewal of mouse ESC and control gene expression
of the core transcription factors, are initiated by LIF:
LIF–(LIFR/gp130)–JAK2–Stat3 and LIF–(LIFR/gp130)–
PI3K–AKT, where gp130 is the common signal transduction
receptor protein of the IL6 family [13]. The relative pathways
could also be initiated by IGF1/IGFR1 or EGF/EGFR. Furthermore,
LIF–(LIFR/p130)–Ras–Raf–MEK–MAPK [13]
and EGF/EGFR–Ras–Raf–MEK–MAPK signaling pathways
are in dynamic crosstalk with Stat3- and AKT-pathways
based on mutual interference [15, 16]. In contrast, the
BMP–Smad1 pathway appears to suppress differentiation
programs of ESC [17].
Experiments with overexpression of gene combinations,
encoding specific transcription factors, after retroviral
infection of the corresponding cDNA in ESC established
several sets of genes: Oct4, Sox2, cMyc and Klf2 [18, 19];
Oct4, Sox2, Lin28 and Nanog [20]; Oct4, Sox2, Klf4 and
Tbx [21], which could induce reprogramming fibroblasts to
pluripotency. The triumvirate of transcription factors Oct4,
Sox2 and Nanog plays the fundamental role in gene regulation,
often binding multiple closely localized sites in the
regulatory regions of the genome, creating enhanceosomes
and coordinating expression of numerous genes in ESC.
Smad1, Stat3 and the coactivator p300 appear to be additional
components of enhanceosomes [17, 22].
The main hypothesis, which had been addressed in the
present study, was that sodium arsenite might directly
target numerous signaling pathways in ESC, suppressing
self-renewal and promoting apoptosis. To prove our
hypothesis, we elucidated the effects of sodium arsenite
exposure on signaling pathways in mouse ESC with a
special attention to regulation of expression levels of key
transcription factors Oct4, Sox2 and Nanog.
During embryogenesis, sodium arsenite, which is known
as transplacental carcinogen [23], might affect survival and
proliferation of different types of stem/precursor cells,
including embryonic neural stem/precursor cells, which
can differentiate into the cells in the nervous system. We
have further suggested in the present study that sodium
arsenite exposure might target the embryonic neurogenesis
in mice via interference and interaction with cell signaling
pathways in mouse neural stem/precursor cells. We also
elucidate a possible mechanism of the resistance to apoptotic
death induced by sodium arsenite in neural stem/
precursor cells based on the IL6–Stat3 pathway.
Results
Sodium arsenite treatment modulates signaling
pathways that control self-renewal and survival
of mouse ESC
In mouse ESC exposed to graded doses of sodium arsenite
(1–6 lM, 24–48 h), there was a dramatic dose-dependent
Apoptosis (2013) 18:188–200 189
123
reduction in cell survival as shown in Fig. 1. Phase contrast
microscopy of live cell cultures demonstrated a massive
flotation of ESC (24–48 h after treatment) that was
accompanied by cell death (Fig. 1a). Annexin-V-FITC and
PI staining of control and sodium arsenite treated ES cells
revealed an increase in percentage of Annexin-V-FITCpositive
apoptotic cells (most of which were also PIpositive)
12 h after treatment with the coincident increase
in the subpopulation of the secondary necrotic (AnnexinV-FITC-negative,
PI-positive) cells (Fig. 1b). Simultaneously,
we observed significant changes in expression
levels of hallmark proteins that control cell survival and
apoptosis, such as a upregulation of the protective enzyme,
heme oxygenase-1 (HO-1), that linked with massive heme
inactivation after cytochrome-c release from mitochondria,
transcription factor FOXO3A (as a sensor of oxidative
stress), p21-WAF (as an indicator of the cell cycle
arrest) and, finally, caspase-3-mediated PARP-1 cleavage
(as an indication of irreversible apoptotic commitment)
(Fig. 1c).
FACS assays of PI-stained nuclei revealed strong dosedependent
changes in cell cycle regulation for stem cells
that resulted in G2/M arrest 24 h after arsenic treatment
followed by pronounced apoptosis 48 h after treatment
(Fig. 1d–f). As expected, total levels of cell death were
higher, than apoptotic levels after sodium arsenite exposure
of mouse ESC, due to induction of necrosis (Fig. 1d). A
relative resistance of normal cells, including embryonic
fibroblasts, to the cytotoxic effects of sodium arsenite at
low doses (\5 lM) is well-known phenomenon [24],
Apo G1 G2/M
2 42 36%
14 8 70%
17 8 60%
14 14 55%
DNA content
Cellnumber
Mouse ESC, 24 h Mouse ESC, 48 h
Apo G1 G2/M
11 48 28%
33 33 15%
41 25 14%
46 21 18%
{Total
death}
{14%}
{49%}
{68%}
{77%}
DA Mouse ESC,
no As, 24 h
no As
As
2 μμM
As
4 μM
As
6 μM
E Mouse ESC, 48 hF
0
10
20
30
40
50
60
70
80
0 0.5 1 2 4 6
%Cells
As, μM
Apoptosis
G1
G2/M
Mouse ESC, 24 h
0
10
20
30
40
50
60
0 0.5 1 2 4 6
%Apoptosis
As, μM
As+Sol
As+zVAD
As+LEHD
As (4 μM) 24 h
PARP-1
p89
0 1 2 4 As (μM)
β-Actin
p21-WAF
Mouse ESC, 24 h
p116
HO-1
FOXO3A
C
40x
2% 2%
3%
2% 6%
3%
10% 8%
2%
6% 7%
6%
Annexin V-FITC
Mouse ESC, 12 h
no As
As
2 μM
As
4 μM
As
6 μM
B
PI
Fig. 1 Sodium arsenite treatment of mouse ESC induced G2/M arrest
followed by apoptotic cell death. a Phase contrast microscopy (940
magnification) of mouse ESC (cultured as adherent cells) in the
absence and in the presence of 4 lM sodium arsenite, 24 h of
exposure. b Annexin-V-FITC and PI staining of control and arsenitetreated
ECS was followed by FACS analysis. c Western blot analysis
of expression of indicated proteins in mouse ESC 24 h after treatment
by increasing doses of sodium arsenite. b-Actin was used as a loading
control. d–f Cell cycle-apoptosis analysis of mouse ESC 24–48 h
after treatment with sodium arsenite (2–6 lM). Cells were stained by
propidium iodide and analyzed by the flow cytometry. A typical
experiment is presented in (b); pooled results of four independent
experiments are shown in (c). Error bars represent means ± SD
(p \ 0.05, Student’s t test). e Effects of a universal caspase inhibitor
zVAD-fmk (40 lM), a caspase-9 inhibitor Ac-LEHD-CHO (40 lM)
and vehicle solution (sol) that contained 0.1 % DMSO on arseniteinduced
apoptosis in mouse ESC. Error bars represent means ± SD
(p \ 0.05, Student’s t test)
190 Apoptosis (2013) 18:188–200
123
which allows us to use arsenic for treatment of sensitive
types of cancer without strong cytotoxicity for normal cells
[25, 26]. On the other hand, a higher sensitivity of ESC to
arsenic exposure described in the present study increases
our understanding of risks linked with such treatment.
Sodium arsenite (especially at doses [5 lM) could
directly target mitochondria causing mitochondrial damage,
cytochrome-c release to the cytoplasm and initiating
the caspase-9-mediated mitochondrial death pathway [27,
28]. We used a pan-caspase inhibitor zVAD-fmk (50 lM)
and specific caspase-9 inhibitor Ac-LEHD-CHO (50 lM),
to obtain further evidences of activation of the mitochondrial
apoptotic pathway in mouse ESC after arsenite
treatment. Both inhibitors, zVAD-fmk and Ac-LEHDCHO,
notably suppressed apoptotic death induced by
1–4 lM sodium arsenite, while for higher arsenic concentration
their inhibitory effects were not pronounced
(Fig. 1f) indicating on increased levels of non-apoptotic
cell death. Caspase-8 inhibitor Ac-IETD-CHO did not
exhibit substantial effects on apoptosis in ESC (data not
shown), further highlighting a role for induction of the
mitochondrial apoptotic pathway after sodium arsenite
treatment [28, 29].
In contrast to many cancer cell lines, transfer of ES cells
to the restricted media (with 1 % FBS but without LIF)
rapidly induced cell floating and high levels of cell death
by apoptosis (Fig. 2b, c). In the presence of sodium arsenite
(2 lM), already high levels of spontaneous apoptotic
death of ES cells grown in 1 % FBS were only modestly
elevated (Fig. 2b, c). On the other hand, a combination of
less restricted media (with 7.5 % FBS, without LIF) and
sodium arsenite demonstrated additive increase in apoptotic
levels of ECS (Fig. 2b). Combined treatment notably
changed the protein levels of FGFR1 and EGFR, but not
IGFR1, which could further down-regulate activating signaling
in ESC at the late time points (Fig. 2a). In summary,
these observations indicated that sodium arsenite exposure
and growth factor withdrawal had the same final destination,
induction of apoptosis, which could be mediated by
the similar signaling pathways.
Sodium arsenite exposure of ESC in the complete media
induced a transient activation of JNK and c-Jun that was
characteristic for the relatively early time point of 3–6 h
after treatment and was followed by gradual down-regulation
of JNK activity (Fig. 3a). On the other hand, the
PI3K–AKT connection, a cross-road of signaling pathways
induced by activated growth factor receptor protein kinases
(such as IGFR and EGFR) or by LIF receptor complex,
plays a central role for a general cell survival and antiapoptotic
protection [30]. Our experiments demonstrated
negative properties of sodium arsenite treatment on the
late AKT activation (16–24 h of exposure) via inhibition
of its Ser473 phosphorylation (Fig. 3a, b). As expected,
suppression of AKT after 16 h of arsenic exposure was also
accompanied by down-regulation of protein expression
levels of b-catenin (Fig. 3a) that could be protected from
degradation via AKT-mediated inactivation of GSK3b
[31]. We and others previously reported a negative regulation
of Jak2/Stat3 in normal and cancer cells by sodium
arsenite treatment [26, 32]. We further demonstrated the
negative effects of sodium arsenite treatment on Stat3
activation via Tyr705 phosphorylation in mouse ESC
(Fig. 3a). Since LIF–Jak2–Stat3 activation is crucial for
ESC proliferation and survival [12, 13], negative effect of
sodium arsenite exposure on Stat3 activation are significant
for acceleration of arsenite-induced apoptosis in ESC.
Furthermore, sodium arsenite exposure persistently upregulated
already high basal levels of MEK-dependent ERK1/
2 activity. In contrast to JNK activation, high levels of
active phospho-ERK1/2 were detected in ESC even 16 h
after treatment (Fig. 3a).
Protein levels of transcription factors Oct4, Sox2 and
Nanog, which together with Stat3 and c-Myc were
assembled in the core transcription factor circuitry of ESC
[12], started to decrease in dose-dependent manner 6 h
after sodium arsenite treatment; this decrease was well
pronounced 16 h after treatment (Fig. 3a). It reflected a
new balance between expression of genes, encoding Oct4,
Sox2 and Nanog, and the corresponding protein degradation
that was established in conditions of sodium arsenite
exposure. Quite surprisingly, total p53 levels were relatively
stable after sodium arsenite exposure (Fig. 3a)
without a notable effect on its Ser15/Ser20 phosphorylation
(data not shown). We also did not observe any substantial
effects of low doses (2–4 lM) of sodium arsenite on IKK
activation via suppression of its phosphorylation, as well as
on the subsequent IjBa phosphorylation mediated by
IKKb in ESC. Even though BMS345541 (10 lM), a specific
inhibitor of the IKK–NF-jB pathway, demonstrated
strong negative effects in ESC and finally down-regulated
levels of the nuclear NF-jB p65-(phospho-Ser536) subunit
(Fig. 3c).
An additional usage of specific inhibitors of tyrosine
kinase activity of growth factor-receptors, which control
proliferation and survival of ESC, demonstrated strong
negative effects of PPP, an inhibitor of IGF1R kinase
Fig. 2 Effects of low serum media and sodium arsenite on cell cycle
and apoptosis of mouse ESC. a Western blot analysis of expression
levels of growth factor receptors in mouse ESC 24 h after treatment
with 2 lM sodium arsenite in the media with 15 % or with 1 % FBS.
b Cell cycle-apoptosis analysis of mouse ESC 24 h after treatment
with sodium arsenite (1–2 lM) in three types of media: with 15 %
FBS, 7.5 % FBS and 1 % FBS. Cells were stained by propidium
iodide and analyzed by the flow cytometry. c Phase contrast
microscopy (980 magnification) of mouse ESC (cultured as adherent
cells) in media with 15 % FBS or 1 % FBS in the absence or in the
presence of 2 lM sodium arsenite, 24 h of exposure
c
Apoptosis (2013) 18:188–200 191
123
Mouse ESC, 24 h
0 2 0 2 As (μμM)
IGF1R (p95)
FGFR1 (p145)
EGFR (p170)
FGFR1 (p120)
FGFR1 (p175)
4 41 31%
Apo G1 G2/M
Control
(15% FBS)
As 2 μM
(15% FBS)
14 15 57%
(1% FBS) 36 29 18%
41 16 31%
DNA content
Cellnumber
BA
15 15 1 1 FBS(%)
Mouse ESC, 24 h
C Mouse ESC, 24 h
80x
(1% FBS)
Control
(15% FBS)
As 2 μM
(15% FBS)
As 2 μM
(1% FBS)
As 2 μM
(1% FBS)
As 1 μM
(1% FBS)
As 1 μM
(15% FBS)
13 20 48%
34 27 22%
As 2 μM
(7.5% FBS)
35 23 24%
(7.5% FBS) 16 31 33%
As 1 μM
(7.5% FBS)
29 20 32%
192 Apoptosis (2013) 18:188–200
123
activity, on activation of PI3K–AKT. PD152035, an
inhibitor of EGFR kinase activity, as well as LY294002, an
inhibitor of PI3K, exhibited similar effects that finally
coincided with effects of sodium arsenite 24 h after treatment
(Fig. 3b). As expected, MEK-ERK activation was
also dependent from activation of both IGF1R- and especially
EGFR-dependent signaling pathways, as was shown
by negative effects of specific upstream inhibitors, PPP and
PD152035, on these signaling pathways. Small molecule
inhibitor of MEK, U0126 (10 lM), blocked MEK-ERK
activation that was opposite to arsenite action (Fig. 3b, the
bottom panel).
To further evaluate effects of sodium arsenite on cell
signaling, we compared its action with effects of specific
molecule inhibitors on regulation of the cell cycle and
apoptosis in ESC. The rapid response with pronounced
induction of apoptosis in ESC was observed for LY294002
(40 lM), a PI3K–AKT inhibitor, and BMS345541
(10 lM), an IKK–NF-jB inhibitor (Fig. 4a, b). Furthermore,
PPP and PD153035, inhibitors of IGF-1R and EGFR
kinase activity, respectively, and upstream suppressors of
AKT activation, induced strong changes in the cell cycle
distribution and high levels of apoptosis (Fig. 4a, b).
Inhibition of Stat3 with Stat3-inhibitor-6 (50 lM) also
resulted in notably increased levels of apoptosis (Fig. 4b).
In contrast, suppression of MEK-ERK by U0126 increased
G1 arrest and induced a modest apoptosis. SP600125, a
JNK inhibitor, and SB203580, a MAPK p38 inhibitor, did
not increase basal levels of apoptosis (Fig. 4b). Caspase-
3-dependent cleavage of PARP-1 provided a further proof
of apoptotic commitment in ESC induced by LY294002
and BMS345541 (Fig. 4c). Taken together, these data
indicated that suppression of PI3K–AKT and Stat3-mediated
signaling by sodium arsenite treatment of ESC could
be critical for deregulation of the cell cycle and the
subsequent apoptosis.
Inhibition of IKK–NF-jB also substantially decreased
survival of ESC (Fig. 4a, b). A general protective function
of NF-jB in embryogenesis, especially in neurogenesis, is
well known [33]. However, sodium arsenite treatment at
Fig. 3 Effects of sodium
arsenite treatment on signaling
pathways and corresponding
transcription factors in mouse
ESC. a–c Western blot analysis
of indicated proteins was
performed 3, 6, 16 and 24 h
after treatment with increased
doses of sodium arsenite. Panels
(b) and (c) demonstrate effects
of small molecule inhibitors of
cell signaling pathways: PPP
(0.5 lM), an inhibitor of IGF1R
protein kinase activity;
PD153035 (20 lM), an
inhibitor of EGFR protein
kinase activity; LY294002
(40 lM), an inhibitor of PI3K–
AKT; U0126 (10 lM), an
inhibitor of MEK-ERK;
BMS345541 (10 lM), an
inhibitor of IKK–NF-jB. All
inhibitors were dissolved in
0.1 % DMSO; control cultures
were treated with 0.1 % DMSO
Apoptosis (2013) 18:188–200 193
123
low doses did not notably affect the NF-jB pathway and
NF-jB-dependent transcription in ESC (see Fig. 3c). In
contrast to numerous cancer lines, the NF-jB pathway in
ESC probably does not critically involve regulation of the
resistance to sodium arsenite. Interestingly, the summary
effects of sodium arsenite treatment on regulation of the
ESC cell cycle-apoptosis were quite similar to an upstream
inhibition of IGR1/IGF1R-mediated signaling by PPP.
Related results with final induction of apoptosis were also
obtained after EGF/EGFR inhibition (Fig. 4a, b). Quite
surprisingly, that in spite of common downstream targets,
suppression of either IGFR1 or EGFR resulted in pronounced
apoptosis in ESC, indicating incomplete interchangeability
of the signaling pathways initiated by these
receptor protein kinases.
Next, we evaluated effects of a relatively low dose of
sodium arsenite (2 lM) in combination with inhibitors of
signaling pathways on self-renewal and apoptosis in mouse
ESC, as well as on protein expression levels of key transcription
factors, Oct4, Sox2 and Nanog. Treatment of ESC
with sodium arsenite (2 lM) in combination either with
LY294002, or with BMS345541 additively increased
Fig. 4 Effects of small molecule inhibitors of signaling pathways on
cell cycle and apoptosis of mouse ESC. Inhibitors of main signaling
pathways used are: PPP (0.5 lM), an inhibitor of IGF-1R protein
kinase activity; PD153035 (20 lM), an inhibitor of EGFR protein
kinase activity; LY294002 (40 lM), an inhibitor of PI3K–AKT;
BMS345541 (10 lM), an inhibitor of IKK–NF-jB; U0126 (10 lM),
an inhibitor of MEK-ERK; SP600125 (20 lM), an inhibitor of JNK;
SB203580 (40 lM), an inhibitor of MAPK p38; and STAT3
inhibitor-6 (20 lM). All inhibitors were dissolved in 0.1 % DMSO;
control cultures were treated with 0.1 % DMSO. a, b Cell cycleapoptosis
analysis of mouse ESC 24–48 h after treatment with a
specific inhibitor of signaling pathway. Treated cells were stained by
propidium iodide and analyzed by flow cytometry. A typical
experiment (one from four) is presented in (a); pooled results of
four independent experiments are presented in (b). Error bars
represent means ± SD (p \ 0.05, Student’s t test). Total death levels
were determined by cell counting using trypan blue staining and light
microscopy. c Western blot analysis of caspase-3-dependent PARP-1
cleavage was performed 24 h after exposure to indicated inhibitors.
b-Actin was used as a loading control. d Cell cycle-apoptosis analysis
of mouse ESC 48 h after treatment with specific molecule inhibitors
of cell signaling in the presence or in the absence of sodium arsenite
(2 lM); control cultures were treated with 0.1 % DMSO alone or in
combination with sodium arsenite. Error bars represent means ± SD
(p \ 0.05, Student’s t test). e, f Western blot analysis of protein
expression levels 24 h after treatment of mouse ESC with sodium
arsenite (2 lM), LY294002 (40 lM), BMS345541 (10 lM) alone or
in combination
194 Apoptosis (2013) 18:188–200
123
apoptotic levels, achieving or surpassing levels induced by
6 lM sodium arsenite alone (Fig. 4d). Some enhancing
effects on arsenite-induced apoptosis were observed with
U0126 and Stat3-inhibitor-6, while SP600125 and
SB203580 did not exhibit notable effects (Fig. 4d).
LY294002 alone or in combination with sodium arsenite
(2 lM) strongly decreased protein levels of Nanog
(Fig. 4e). Positive regulation of Nanog expression by
PI3K–AKT signaling via inhibition of GSK3 was previously
described [34]. In contrast, LY294002 and sodium
arsenite (2 lM) only modestly decreased Oct4 and Sox2
levels, acting alone or in combination. On the other hand,
additive negative effects of LY294002 and sodium arsenite
were observed for b-catenin protein levels, highlighting
again a role of PI3K–AKT–GSK3b in stabilization of
b-catenin protein levels and a role of sodium arsenite as an
inhibitor of this pathway (Fig. 4e).
BMS345541 alone or especially in combination with
sodium arsenite (2 lM) strongly decreased Oct4 protein
expression, revealing a possible role of IKK–NF-jB (in
concert with other transcription factors) for control of Oct4
expression. Both agents also decreased Sox2 levels, but
without additive effects in combination (Fig. 4f). On the
other hand, upregulation of transcription factor FOXO3A
protein levels induced by sodium arsenite was not affected
by simultaneous BMS345541 treatment (Fig. 4f). Results
obtained exhibited relatively mild effects of lower doses of
sodium arsenite (1–2 lM) on key transcription factors in
ESC; however, an increase in concentration of sodium
arsenite (up to 4–6 lM) resulted in a significant downregulation
of the levels of key transcription factors, Oct4,
Sox2 and Nanog (see Fig. 3). So, protein levels of
b-catenin, Nanog and Oct4 were under control of the
PI3K–AKT pathway; furthermore, Oct4 and Sox2 levels
were also dependent from IKK–NF-jB in mouse ESC.
Combinations of sodium arsenite (2 lM) with either
LY294002 or BMS345541 demonstrated additive suppression
of b-catenin and Oct4 levels, respectively.
We also evaluated a role for mTOR in response of ESC
to arsenic exposure. Activation of mTOR, a key regulator
of protein synthesis in the cell, is a well-known downstream
effect of the PI3K–AKT pathway [35]. Our data
further demonstrated negative effects of sodium arsenite
treatment (24–48 h) on the late AKT activation through
inhibition of Ser473 phosphorylation and the subsequent
down-regulation of its downstream target, mTOR activation,
in ESC 24 h after treatment (Fig. 5a, b). We also
expected that sodium arsenite-mediated suppression of
mTOR activity was critically involved in the regulation of
arsenite-induced apoptosis. Surprisingly, rapamycin
(1–5 nM), a specific inhibitor mTOR activity, induced G1
arrest of the ESC proliferation, rather than apoptosis
(Fig. 5b). It probably indicated a significance of other
downstream targets of AKT in regulation of anti-apoptotic
response.
In summary, our data demonstrated profound, but differential
effects of sodium arsenite exposure on cell signaling
pathways regulating the cell cycle, general cell
survival and apoptosis in mouse ESC, balancing between
cell cycle arrest, apoptosis and differentiation.
Effects of sodium arsenite exposure on immortalized
mouse neural stem/progenitor cells (clone C17.2)
We used in our study surrogate mouse neural stem/progenitor
cells (C17.2), which were isolated from neonatal
mouse cerebellum. Propagation and stem-like behavior of
this cell line was augmented via v-myc transduced by
MMLV retrovirus vector [36]. Interestingly, the DNA
profile of these cells, which were stained with PI, indicated
a high level of aneuploidy (Fig. 6a). It was correlated with
results of the former study that detected aneuploidy in
0 24 48 24 48 24 48 h
A 0 0 0 2 2 4 4 As (μμM)
Mouse ESC
AKT-P(S473)
AKT
β-Actin
mTOR-P
(S2448)
mTOR
0
10
20
30
40
50
60
70
80
90
0 1 2 5
%Cells
Apoptosis G1 G2/M
B
Mouse ESC, 48 h
Rapamycin (nM)
Fig. 5 Sodium arsenite exposure suppresses mTOR activation in
mouse ESC. a Western blot analysis of indicated proteins was
performed 24 and 48 h after treatment with increased doses of sodium
arsenite (0, 2 and 4 lM). b Cell cycle-apoptosis analysis of mouse
ESC 48 h after treatment with rapamycin (1–5 nM), a mTOR
inhibitor. Cells were stained by propidium iodide and analyzed by
the flow cytometry. Pooled results of four independent experiments
are shown. Error bars represent means ± SD (p \ 0.05, Student’s
t test)
Apoptosis (2013) 18:188–200 195
123
C17.2 cells using chromosome staining [37]. Unexpectedly,
our data demonstrated a high resistance of C17.2 cells
to sodium arsenite-induced apoptosis in culture conditions:
modest apoptotic levels were detected only 48 h after
arsenite treatment at dose 8 lM (Figs. 6c, 7a). A recent
study also proven anti-apoptotic resistance of C17.2 cells,
demonstrating pronounced apoptosis at extremely high
doses of sodium arsenite, such as 40 lM [38]. In contrast,
LU294002, a PI3K–AKT inhibitor, and two upstream
inhibitors of PI3K–AKT signaling, PPP and PD153035,
were able to induce pronounced apoptosis in C17.2 cells
48 h after treatment (Fig. 6c). As expected, total cell death
levels were higher than apoptotic levels in treated C17.2
cells (Fig. 6c). Western blot assay revealed very high basal
levels of active ERK1/2, which were further upregulated by
arsenite, but modest levels of active AKT-phospho-Ser473
that were downregulated by sodium arsenite exposure. It
was accompanied by a partial suppression of GSK3b
phosphorylation and relatively modest down-regulation of
b-Catenin protein levels (Fig. 5b). Inducible expression of
HO-1, a hallmark protective enzyme, served as an internal
control of initiation of arsenite-dependent intracellular
activities (Fig. 6b). What was a reason for substantial
resistance of C17.2 cells to sodium arsenite treatment?
Previously performed profiling of gene expression in C17.2
cells demonstrated massive changes in gene expression,
including increased production of IL6 in this cell line,
compared to the primary mouse neural stem cells [37].
Mouse ESC, which are critically dependent on the exogenous
LIF, an IL6-related cytokine, also produce very low
levels of the endogenous IL6 [39]. We further confirmed
these data detecting the high basal and TNFa-induced
levels of IL6 secretions in the culture media of C17.2 cells,
compared to MEF or ESC (Fig. 6b). Addition of anti-IL6
Mouse NSC (C17.2) , 24 h
Apo G1 G2/M
1 53 31%Control
(no As)
1 54 29%As
2 μμM
6 39 39%PPP
0.5 μM
16 59 16%PD153035
10 μM
DNA content
22 56 13%
Apo G1 G2/M
2 64 21%
4 66 21%
45 42 8%LY294002
40 μM
Cellnumber
{Total
Death}
{5%}
{12%}
{68%}
24 49 12% {56%}
23 61 9% {54%}
48hC
0 1 2 4 As (μM) 6 h
C17.2
AKT-P(S473)
AKT
HO-1 (p32)
ERK1/2
ERK1/2-P(T202,Y204)
B
1 48 36%As
8 μM
15 43 24% {24%}
A (2N 4N)
Mouse
melanocytes
Mouse NSC
(C17.2)
DNA content
Cellnumber
Mouse ESC
GSK3β-P(S9)
β-Catenin
GSK3β
β-Actin
16 h
Fig. 6 Effects of sodium arsenite exposure on mouse neural stem/
precursor cells (C17.2). a Comparison of DNA content of mouse cell
lines using PI staining and FACS analysis. b Western blot analysis of
indicated proteins after arsenic treatment of C17.2 cells. c Cell cycleapoptosis
analysis of C17.2 cells 24–48 h after indicated treatment.
Cells were stained by propidium iodide and analyzed by the flow
cytometry. Total cell death levels were determined using Trypan blue
staining
196 Apoptosis (2013) 18:188–200
123
inhibitory mAb (5 lg/ml) into the culture media substantially
increased levels of sodium arsenite-induced apoptosis
in C17.2 cells (Fig. 7c), highlighting a protective effect of
the endogenous production of IL6. Since IL6 is a powerful
inducer of Stat3 activation, we further treated C17.2 cells
with Stat3-inhibitor-6 (20 lM) alone or in combination
with sodium arsenite, significantly increasing the basal and
arsenite-induced levels of apoptosis (Fig. 7d). It also
demonstrates a protective role of IL6-induced Stat3 activation
against arsenite-induced apoptosis. Taken together,
our results and published data allow us to consider C17.2
cells as model line with a strong resistance to sodium
arsenite exposure in vitro, due to upregulation of IL6-Stat3dependent
signaling.
Discussion
A positive role of the PI3K–AKT pathway in regulation of
embryonic stem cell and neural stem cell self-renewal has
been well established [34, 40–42]. Two specific targets of
this pathway are the ‘‘stemness’’ factors Nanog and Oct4 in
mouse ESC [34]. Furthermore, the PI3K–AKT pathway is
involved in regulation of numerous cell functions, including
activation of mTOR that regulates a general protein
synthesis in the cell [35]. A significance of PI3K–AKT/
b-catenin and Wnt/b-catenin signaling in the maintenance
of self-renewal of ESC and NSC cells was also elucidated
in detail [43, 44]. Suppression of GSK3b activity by AKT
that resulted in stabilization of protein levels of b-catenin
and cyclin D1 was one of the critical events in regulation of
proliferation and survival of neural stem cells [45]. There is
no surprise that cancer cells do use the same strategy for
increasing proliferation and survival based on activation of
the PI3K–AKT pathway [46].
The results of the present study also indicated that
one of the main and common targets for negative regulation
by sodium arsenite was the PI3K–AKT pathway in
both mouse ESC and NSC. Consequently, the current data
and previous studies demonstrated that efficient protection
against sodium arsenite action in ESC, NSC and
in numerous cancer cell lines might be achieved by
Fig. 7 Upregulation of
apoptosis in mouse neural stem
cells through inhibition of IL6.
a Nestin (red) immunostaining
and DAPI (blue) staining of
mouse NSC (C17.2) 48 h after
sodium arsenite (8 lM)
exposure. Apoptotic nuclei are
shown by the white arrows.
b Levels of IL6 (ng/ml) in the
culture media of non-treated or
TNFa-treated MEF, ESC and
C17.2 (500,000 cells/well) were
determined by ELISA 24 h after
treatment. c Dose-dependent
levels of apoptosis (determined
by PI staining with the
subsequent FACS assay) in
mouse C17.2 cells 48 h after
sodium arsenite exposure. AntiIL6
inhibitory antibody or
control IgG (5 lg/ml) were
added to C17.2 cells before
sodium arsenite treatment.
d Effect of Stat3-inhibitor-6
(20 lM) on sodium-arsenite
induced apoptosis in C17.2 cells
48 h after treatment. Error bars
in the (b)–(d) represent
means ± SD (p \ 0.05,
Student’s t test) (Color figure
online)
Apoptosis (2013) 18:188–200 197
123
over-activation of the PI3K–AKT pathway [41, 47]. This
pathway could be permanently induced in cells by variety
of growth factors and cytokines, such as LIF, FGF2, EGF
and IGF1/2, which could be present in the complete media
in vitro, by these growth factors produced by helper cells of
microenvironment or by endogenously produced growth
factors via cell paracrine and autocrine mechanisms
in vivo. In contrast, a general inhibition of the PI3K–AKT
pathway by LY294002 or by sodium arsenite (but not
mTOR inhibition) induced pronounced apoptosis that was
additively increased by combined treatment using both
agents. Arsenite-induced mitochondrial damage, the subsequent
cytochrome-c release and caspase-9/caspase-3
mediated apoptosis could substantially decrease a survival
of mouse ESC after sodium arsenite exposure.
Effects of sodium arsenite on its downstream targets in
ESC were further investigated in the present study. Besides
negative regulation of targets of the PI3K–AKT pathway,
such as Oct4 and Nanog, sodium arsenite exposure further
down-regulated the Jak2–Stat3 pathway and its downstream
targets, including Stat3/Oct4-dependent expression of Sox2
in ESC. It finally suppressed the whole core transcription
factor circuitry that control self-renewal of mouse ESC,
including Stat3, Oct4, Sox2 and Nanog [12, 22].
Negative regulation of IKK–NF-jB activities by sodium
arsenite (5–10 lM) is a well-established phenomenon for
many normal and cancer cell lines [48, 49]. However, for
ESC investigated in the present study, sodium arsenite
(2–4 lM) actually did not affect IKK–NF-jB activation,
due to probably increased compensatory production of
endogenous cytokines, including TNFa [50] that by itself
was a powerful inducer of the NF-jB activation pathway.
This problem, however, needs a more precise analysis,
since numerous protein–protein interactions of key transcription
factors, such as Stat3–NF-jB p65 [51] or
Stat3–Nanog [52] could substantially complicate general
regulation of NF-jB-dependent and Stat3-dependent gene
expression in stem cells.
In contrast to ESC, immortalized mouse neural stem/
precursor cells C17.2 demonstrated a high resistance to
sodium arsenite exposure (see Fig. 6c). However, this situation
is based on massive changes in gene regulation for
immortalized mouse NSC (C17.2), compared to the primary
neural stem cells, including strongly increased
endogenous production of IL6 [37]. This reminds a correlation
between N-Myc amplification/overexpression and a
strong IL6 production by human neuroblastomas [24, 53].
Indeed, using anti-IL6 antibody, we increased a sensitivity
of C17.2 cells to sodium arsenite-mediated apoptosis.
Suppression of the main downstream signaling target of
IL6, Stat3, by specific small molecule inhibitor also substantially
upregulated arsenite-induced apoptosis in C17.2
cells (Fig. 7c, d).
Sodium arsenite was previously described as a twosided
sword, which is a well-known human carcinogen and
an effective therapeutic agent for treatment of some types
of cancer, especially for treatment of acute promyelocytic
leukemia [25]. Better understanding of mechanisms of
cytotoxic action of sodium arsenite, for example, in the
nervous system and narrowing the window of its specific
effects for cancer, such as neuroblastoma [54], is a priority
for the development the anti-cancer therapy.
Materials and methods
Materials
Sodium arsenite, fibronectin, laminin and polyornithine
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
PI3K inhibitor LY294002, IKK inhibitor BMS-345541,
STAT3 inhibitor-6 S3I-201 (also known as NSC 74859),
IGF-1R kinase inhibitor picropodophyllin (PPP), PI3K
inhibitor LY294002, MEK inhibitor U0126, MAPK p38
inhibitor SB203580 and caspase inhibitors zVAD-fmk,
LEHD and IETD were purchased from Calbiochem (La
Jolla, CA, USA). Annexin-V-FITC was obtained from BD
Bioscience (San Jose, CA, USA).
Maintenance of mouse ESC
We used in the present study ESC line W4 from
129/SvEvTac mice. Mouse ESC were grown in DMEM
with 15 % FBS, 1 % glutamine, 1 % nonessential amino
acids, 0.1 mM b-mercaptoethanol, and 105
U/ml LIF
(ESGRO) on fibronectin coated dishes.
Maintenance of mouse neural stem cells (C17.2),
mouse embryonic fibroblasts and mouse melanocytes
Mouse surrogate neural stem cells (C17.2 clone) and
mouse embryonic fibroblasts (MEF) were grown in DMEM
with 15 % FBS, 1 % glutamine, and 1 % penicillin/streptomycin.
The neural stem cells (C17.2 clone) were kindly
provided by Dr. E.Y. Snyder (Sanford-Burnham Research
Institute, San Diego, CA). Mouse melanocytes were grown
in TICVA medium.
Immunocytochemistry analysis
Cells were fixed with 4 % paraformaldehyde in PBS for
60 min. Immunochemical staining was performed using
standard protocols. Cells were stained for the undifferentiated
NSC marker, nestin (using mAb from Millipore,
Temecula, CA, USA) and for localization of the nuclei
using DAPI.
198 Apoptosis (2013) 18:188–200
123
Apoptosis studies
For induction of apoptosis, cells were exposed to sodium
arsenite (1–10 lM) alone or in the presence of small
molecule inhibitors of cell signaling pathways. Apoptosis
was then assessed by PI staining and quantifying the percentage
of hypodiploid nuclei (pre-G1) or by quantifying
the percentage of AnnexinV-FITC-positive cells, both
PI-negative and PI-positive. Flow cytometric analysis was
performed on a FACS Calibur flow cytometer (Becton–
Dickinson) using the CellQuest program. Determination of
total cell death levels was performed by Trypan blue
staining.
Western blot analysis
Total cell lysates (50 lg protein) were resolved on SDSPAGE,
and processed according to standard protocols. The
monoclonal antibodies used for western blotting included:
anti-b-actin (Sigma, St. Louis, MO, USA); anti-caspase-8,
anti-caspase-9, anti-caspase-3 (Cell Signaling, Danvers,
MA, USA); The polyclonal antibodies used included antiphospho-p44/p42
MAP kinase (T202/Y204) and anti-p44/
p42 MAP kinase; anti-phospho-JNK and anti-JNK1-3;
anti-phospho-cJun (S73) and anti-cJun; anti-phospho-AKT
(S473) and anti-AKT; anti-phospho-p65 (S536) NF-jB
and anti-p65 NF-jB, anti-phospho-STAT3 (Y705) and
anti-STAT3; anti-phospho-FOXO3A (S253) and antiFOXO3A;
anti-b-Catenin, anti-Oct4, anti-Sox2, antiNanog
and anti-PARP-1 (Cell Signaling, Danvers, MA,
USA); anti-Ars2 (Santa Cruz Biotechnology, CA, USA);
anti-HO-1 (Enzo Life Sciences, Plymouth Meeting, PA,
USA). The secondary antibodies were conjugated to
horseradish peroxidase; signals were detected using the
ECL system (Thermo Scientific, Rockford, IL, USA).
ELISA
Antibody pair used in sandwich ELISA for this study is
commercially available. Kit to detect IL6 was from Life
Technologies/Invitrogen (Carlsbad, CA, USA).
Statistical analysis
Data from 3 to 4 independent experiments were calculated
as means and standard deviations. Comparisons of results
between treated and control groups were made by the
Students’ t-tests. A p value of 0.05 or less between groups
was considered significant.
Acknowledgments We would like to thank Drs. Adayabalam Balajee,
Mercy Davidson, Peter Grabham, Bo Zhang and Howard Lieberman
for advice, critical reading of manuscript and discussion. We
would like to thank Dr. E.Y. Snyder (Sanford-Burnham Research
Institute, San Diego, CA) for mouse neural stem cells (clone C17.2).
This work was supported by Environmental Center Grant P30
ES009089 and NIH Grant P01 CA049062.
Conflict of interest None.
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