Introduction
SCLC, accounting for 15–20% of newly diagnosed lung
cancer, is an extremely aggressive malignancy with early metastasis
and poor prognosis. Despite a prompt response to chemotherapy,
relapses occur in the majority of patients with SCLC. Therefore the
development of an alternative therapy against SCLC becomes
imperative (1).
ATO has been proven to be an effective therapeutic
agent in acute promyelocytic leukemia (APL) with high complete
remission rate and prolonged survival (2,3). ATO
can induce apoptosis through PML-RARα-independent pathways in APL
or other cancer cells via p53 activation (4,5),
Bcl-2 downregulation (6,7), mitochondrial membrane depolarization
and cytochrome c release (8–10),
depletion of intracellular reduced glutathione (GSH) content and
elevation of reactive oxygen species (11,12).
More recently, the application of ATO in lung cancer treatment has
been explored in preclinical models, mainly in non-small cell lung
cancer (NSCLC). ATO induces growth inhibition and apoptosis in
NSCLC cells through G2/M cell cycle arrest (13,14),
Bcl-2 downregulation and GSH depletion (15). Recently, downregulation of
thymidylate synthase and E2F1 were observed in lung adenocarcinoma
(16,17) and mesothelioma (18). Synergistic effects have been
observed when combining ATO with cisplatin (19), buthionine sulfoximine (BSO)
(15), suberoylanilide hydroxamic
acid (20), and sulindac (21). The effect of ATO in SCLC is less
reported. In a panel of lung cancer cell lines, ATO is highly
cytotoxic to SCLC preferentially mediated through necrosis rather
than apoptosis (22).
Biologically, ATO is known to bind to proteins with
sulfhydryl (-SH) groups (23). It
targets GSH which is a thiol molecule involved in detoxification of
arsenicals (11,24). Evidently, loss of GSH and its
cytotoxic effect is not directly caused by damage of GSH-related
enzymes (glutathione peroxidase, glutathione reductase and
glutathione transferase) (25).
Nonetheless, thioredoxin (Trx) system serves as a key antioxidant
target of ATO in breast cancer cells (26). Trx system, composed of thioredoxin
reductase (TrxR), Trx and NADPH, plays an important role in
maintaining somatic redox homeostasis. Moreover, TrxR and Trx are
overexpressed in many cancers (27–29),
which take part in redox regulation of transcription factors
(30). Therefore, Trx system has
become a new target in cancer therapy (31).
In the present study, we aimed to elucidate the
mechanisms of ATO in SCLC using a cell line model, which may
provide a scientific base for future application of ATO in
treatment of SCLC.
Materials and methods
Chemicals and reagents
ATO, N-acetyl-L-cystine (NAC), buthionine
sulphoximine (BSO) and butylated hydroxyanisole (BHA) were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Apoptosis inducing factor
(AIF), Bcl-2, cleaved caspase-3, cytochrome c, PARP, SMAC,
thioredoxin and XIAP antibodies were purchased from Cell Signaling
Technology (Danvers, MA, USA).
Cell lines and culture
Five SCLC cell lines (H187, DMS79, H526, H69 and
H841) were obtained from the American Type Culture Collection
(Manassas, VA, USA). H187, DMS79, H526 and H69 cells were cultured
in RPMI-1640 medium (Gibco®, Life Technologies,
Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS,
Gibco), while H841 cells were grown in HETIS medium supplemented
with 5% FBS. Cells were incubated in 37°C with a humidified
atmosphere of 5% CO2.
Cell proliferation assay
The cytotoxicity of ATO in SCLC cell lines was
measured using
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay (ATCC) as previously performed (32).
Annexin V-phycoerythrin
(PE)/7-aminoactinomycin D (AAD) staining
Apoptosis was determined by PE-conjugated Annexin
V/7-AAD kit (BD Biosciences, CA, USA). Briefly, treated cells were
collected, washed and re-suspended in binding buffer. Cells were
stained for 15 min at room temperature with PE-Annexin V (Ex/Em =
488/578 nm)/7-AAD (Ex/Em = 546/647 nm), then read-out by Cytomics
FC 500 analyzer with FL2/FL4 channels (Beckman Coulter, CA, USA).
The cell populations in PE+/7-AAD− and
PE+/7-AAD+ quadrants were calculated.
Measurement of mitochondrial membrane
depolarization (MMD)
Briefly, cells after treatment were collected,
washed, and stained with JC-1 (5 μg/ml) at 37°C for 15 min before
reading with FL1/FL2 channels of Cytomics FC 500 analyzer.
Detection of glutathione (GSH) and
reactive oxygen species (ROS)
Cellular GSH level was measured by using
5-chloro-methylfluorescein diacetate (CMFDA, Ex/Em = 522/595 nm,
Invitrogen, CA, USA) fluorescence as described previously (33). After treatment, cells were washed
and incubated for 30 min at 37°C with 5 μM CMFDA in FBS-free
medium, followed by incubation for 40 min at 37°C with complete
medium. CMF fluorescence intensity was determined using a Cytomics
FC500 flow cytometer. Hydrogen peroxide
(H2O2) and superoxide were measured by
2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA,
Ex/Em = 500/520 nm) and dihydroethidium (DHE, Ex/Em = 518/605 nm)
(Invitrogen) staining, respectively. Treated cells were washed and
incubated for 20 min at 37°C with 1 μM of H2DCFDA or 1
μM DHE in FBS-free medium following flow cytometry.
Immunofluorescence
Oxidative DNA damage was assessed using
8-hydroxy-2-deoxyguanosine (8-OHdG) immunofluorescence. Cells were
seeded on slides before treatment. Slides were washed and blocked
for 60 min with 10% normal goat serum in PBS, followed by
incubation at 4°C overnight with monoclonal anti-8-OHdG antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The slides
were then incubated for 2 h with FITC-conjugated goat anti-mouse
IgG (Santa Cruz Biotechnology, Ex/Em = 500/520 nm), cover-slipped
with UltraCruz™ Mounting Medium (Santa Cruz Biotechnology) and
observed with Eclipse E-800 (Nikon, Tokyo, Japan). DAPI (Santa Cruz
Biotechnology) staining was used to visualize the nucleus.
Western blotting
Total protein was extracted with RIPA buffer (PBS,
1% Nonidet-P-40, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate)
containing protease inhibitors. Nuclear protein and mitochondrial
protein were collected with NE-PER Nuclear and Cytoplasmic
Extraction kit and Mitochondria Isolation kit respectively (Pierce
Biotech, Rockford, IL, USA) as described by the manufacturer.
Proteins (40 μg) were separated in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred
to nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK),
immunoblotted with corresponding primary and secondary antibodies,
and detected using ECL detection kit (GE Healthcare). Image
analysis was carried out with ImageJ (Research Services Branch,
National Institute of Mental Health, Bethesda, MD, USA).
DNA fragmentation assay
Cellular DNA was extracted with standard DNA
phenol/chloroform extraction method (34). DNA (0.1 μg/sample) was separated in
1.5% agarose gel and bands were visualized on a UV transilluminator
at 302 nm.
Redox western blot analysis
Redox forms of Trx1 were separated as previously
described (26,35). Trx1 was carboxymethylated in
guanidine-Tris solution (6 M guanidine-HCl, 50 mM Tris, 1 mM EDTA,
30 mM iodoacetic acid) at pH 7.5. After incubation at 37°C for 30
min, proteins were desalted and concentrated using centrifugal
filter device (Millipore, Billerica, MA, USA). Proteins were
separated with native gel and western blotting was performed. Redox
potential was calculated with Nernst equation: Eh, Trx1 = 240 mV +
30 × ln (ratio of Trx1Ox and Trx1Red)
(36).
Statistical analysis
Data were presented as mean ± standard deviation
(SD), with between-group differences analyzed by two-tailed
Student’s t-test. A p-value <0.05 was considered statistically
significant. Linear regression was used to calculate
IC50 values. All statistical analyses were performed
using Prism 5 (GraphPad Software, La Jolla, CA, USA).
Results
Cell growth inhibition and apoptosis
induced by ATO
Five SCLC cell lines were incubated for 48 h with
different concentrations of ATO. Cell proliferation was suppressed
in a dose-dependent manner with IC50 values in
clinically reachable levels (Fig.
1A). H841 cell line was chosen due to its adherent property for
apoptotic assay. ATO induced dose-dependent apoptosis (Fig. 1B) in H841 cells accompanied by
increased cleaved caspase-3 and PARP (Fig. 1C).
ATO-induced MMD, GSH depletion and ROS
elevation
ATO prompted a dose-dependent shift from red to
green fluorescence that indicated MMD in H841 cells (Fig. 2A). This was associated with
downregulation of Bcl-2 and XIAP as well as release of AIF and SMAC
to cytosol (Fig. 2B). Low
concentration of ATO (2.5 μM) reduced 60% of GSH content (Fig. 2C) and increased
H2O2 by >1.5-fold compared with control
H841 cells (30500±6150 vs 13522±3146) (Fig. 2D). Nonetheless, superoxide content
remained unchanged except at the highest concentration (10 μM) of
ATO treatment (Fig. 2D).
ATO induced oxidative DNA damage and
fragmentation
Treatment with ATO (5 μM) resulted in stronger FITC
fluorescence (green) than in the control group, in keeping with
increased 8-OHdG content in H841 cells upon exposure to ATO
(Fig. 3A). This is associated with
appearance of lower molecular weight DNA fragments with ATO
treatment (Fig. 3B).
Changes in MAPKs expression during ATO
treatment
Elevation of p-Erk1/2 was detected after 30 min of
ATO treatment and it was sustained for ≥48 h (Fig. 4A) while p-P38 and p-JNK were
unaltered (data not shown). Nonetheless, application of specific
p-Erk1/2 inhibitor (PD98059) (20 μM) decreased p-Erk1/2 expression
induced by ATO (Fig. 4B) but did
not reverse ATO-induced cell death and MMD (Fig. 4C).
Effects of antioxidants on cell death
induced by ATO
NAC, a cysteine donor in GSH synthesis, was used to
determine the role of GSH and ROS in ATO-induced cell death in H841
cells. NAC (10 mM) was shown to effectively restore GSH content and
reduce H2O2 after ATO exposure (Fig. 5A). This was associated with
reversal of apoptotic cell death (39.6±5.3 vs. 5.3±1.2% for ATO vs
ATO + NAC) and MMD (34.5±4.5 vs. 5.6±0.4% for ATO vs. ATO + NAC)
(Fig. 5B). In addition, NAC
reversed the alterations in apoptosis-related proteins induced by
ATO (Bcl-2, XIAP, cleaved caspase-3 and cleaved PARP) (Fig. 5C). On the other hand, a different
antioxidant BHA suppressed H2O2 induced by
ATO but failed to restore GSH content. Consequently, BHA only
partially reversed apoptosis and MMD induced by ATO (Fig. 5D).
BSO potentiated cytotoxic effect of ATO
by further disturbing the redox status
BSO, an inhibitor of γ-glutamylcysteine synthetase,
was used as a GSH inhibitor. BSO potentiated the cytotoxic effect
of ATO in H841 cells, as shown by increased apoptotic death
(PE+ by flow cytometry in 17 vs. 44%: ATO vs. ATO +
BSO), enhanced MMD (17 vs. 42%: ATO vs. ATO + BSO), increased
cleavage of caspase-3 and downregulated XIAP (Fig. 6A). Nonetheless, administration of
10 mM NAC only partially rescued the H841 cells from apoptosis and
MMD (Fig. 6A). Interestingly, BSO
caused more accumulation of H2O2 than further
depletion of GSH when combined with ATO (Fig. 6B), resulting in increased oxidative
DNA damage (8-OHdG-FITC signal) in BSO/ATO group compared with
either ATO or BSO alone (Fig.
6C).
Trx1 is one of the important components of Trx redox
system responsible for maintaining redox equilibrium in mammalian
cells. ATO reduced total Trx1 protein expression in H841 cells,
which could be further downregulated by BSO but was reversed by NAC
(Fig. 6D). Based on Trx redox
western blot analysis, both oxidized and reduced forms of Trx1 were
downregulated by ATO, nevertheless, redox potential (Eh) remained
the same in different treatment groups (Fig. 6E).
Discussion
In the past decade, ATO has been used as a highly
effective anticancer therapy in leukemia (particularly APL) with or
without all-trans retinoic acid and achieving high rate of complete
remission (2,3). Nonetheless, it has been increasingly
reported to exert cytotoxic effect in other forms of haematological
malignancies and solid cancers such as breast (37), ovarian (38), cervical (39), and lung (20) cancers. The mechanisms and pathways
involved are highly diverse and cell type-specific, though
knowledge is scarce in SCLC.
In this study, a panel of five SCLC cell lines was
tested for the effect of ATO on cellular proliferation, with
IC50 values in clinical achievable concentrations
(3).
Caspase-dependent apoptosis is classified into
intrinsic (mitochondria-mediated) and extrinsic (death
receptor-mediated) pathways (40).
Both mechanisms converge to activate the apoptotic executioner
caspase-3 and -7, which consequently cleave polyADP-ribose
polymerase (PARP) that impairs DNA repair. Classically, cleaved
caspase-3 and cleaved PARP are widely adopted apoptotic markers. In
this study, both markers were elevated after ATO treatment in H841
cells, indicating cell death via the caspase-dependent pathway
(41).
AIF is one of the main mediators in cell death,
which is liberated from mitochondria and translocated to nucleus
resulting in DNA damage. It has been suggested that nuclear
translocation of AIF accompanied by activation of receptor
interacting protein 1 (RIP1) are markers of necroptosis (42). Nonetheless, the binding of AIF with
DNA provokes caspase-independent chromatin condensation and DNA
fragmentation (43–46). In our H841 cell line model, apart
from triggering MMD accompanied by AIF release and nuclear
translocation, ATO treatment also increased RIP1 expression (data
not shown). Taken together, cell death initiated by ATO in H841
cells could be mediated through both caspase-dependent apoptosis
and AIF-induced apoptosis or necroptosis.
On the other hand, MMD is associated with
mitochondrial release of another apoptosis mediator SMAC, in which
cytosolic SMAC can inhibit apoptosis inhibitor protein (XIAP)
leading to subsequent release of caspases that initiate apoptosis
(40). Our finding of SMAC release
and XIAP downregulation during treatment with ATO in H841 cells
suggests the possibility of caspase-3 activation via SMAC
release/XIAP inhibition rather than cytochrome c
release/caspase-9 activation. This is in keeping with previously
reported mechanism of caspase-dependent apoptosis via
SMAC/XIAP/caspase activation in other cancer cell line models with
ATO treatment (47,48).
GSH depletion and H2O2
accumulation with ATO treatment in H841 cells detected in this
study are in keeping with previous reports (11,49–52).
Nonetheless, the type and amount of ROS needed to initiate cell
death by ATO are still controversial, which can vary with different
cell lines and dose of ATO (53).
Indeed, the role of ROS in causing cell death induced by ATO was
uncertain in other studies (54,55).
Based on our findings in H841 SCLC model, oxidative stress with ATO
treatment was due to both H2O2 production and
GSH depletion, in which NAC (a GSH precursor) (56) could reverse these alterations
resulting in restoration of mitochondrial membrane potential. In
leukemia cell line U973, intracellular GSH depletion could trigger
Bax translocation to mitochondria and provoke membrane
permeabilization resulting in apoptosis (57). In H841 cell line, we demonstrated
downregulation of Bcl-2 upon ATO treatment, abrogating the
protection on mitochondria. The mechanism of ATO-induced Bcl-2
downregulation has not been clearly elucidated. Nonetheless Bcl-2
has manifested an antioxidant-like effect in response to either
oxidative stress or GSH depletion (58,59).
Reversal of ATO-induced Bcl-2 downregulation with NAC in our
experiments has suggested the mechanistic role of oxidative stress
and GSH depletion.
BHA is an effective scavenger of free radicals
widely used in food industry. It has been shown to be effective in
scavenging H2O2 to rescue cells from the DNA
damage (60–63). In our study, adding BHA with ATO in
H841 cells was capable of reducing H2O2
without effective restoration of GSH content compared with ATO
alone, resulting in partial reversal of cell inhibition and
apoptosis induced by ATO. It is possible that depletion of GSH with
ATO treatment is not only due to consumption by
H2O2 mediated via glutathione peroxidase
(GPx) (64) but also direct
consumption by ATO (65). In
addition, oxidative DNA damage was more pronounced in ATO treatment
with BSO compared with ATO alone, associated with significant GSH
depletion due to increased H2O2 as previously
reported (66,67). Taken together, GSH depletion plays
a more important role in ATO-induced cell death in SCLC, similar to
previous findings in NSCLC (66).
Thioredoxin system was reported to be an important
target of ATO in some cancers (26,68).
In H841 cell line model, ATO downregulated both reduced and
oxidative form of Trx1 without affecting redox status. This is
discrepant from previous report, which indicated that ATO could
inhibit thioredoxin reductase and consequently decrease the reduced
form of Trx1 (68). Although the
activity or expression of thioredoxin reductase has not been
measured, our findings suggest that ATO targets the total Trx1
rather than just the reduced form in H841 cells.
MAPKs are potential ROS response factors,
nonetheless, p-JNK and p-p38 were not involved in ATO-induced cell
death in our H841 cell line model (data not shown), which is
different from other cancer models (69–71).
Although upregulated p-Erk1/2 was detected after ATO treatment in
H841 cells, specific Erk inhibitor PD98059 failed to revert the
effects induced by ATO on MMD and cell death. It suggests that
p-Erk1/2 is not an effective mediator leading to cell death induced
by ATO in SCLC, which may serve in other biological actions
(72,73).
In conclusion, the mechanisms of cell death induced
by ATO in SCLC are mainly dependent on altered redox homeostasis
that comprises GSH depletion, H2O2
accumulation and mitochondrial depolarization. Both
caspase-dependent pathway mediated via Bcl-2/SMAC/XIAP/Caspase-3
and caspase-independent pathway mediated via AIF would take part in
causing apoptosis and necroptosis in SCLC.
Abbreviations:
|
ATO
|
arsenic trioxide
|
|
BHA
|
butylated hydroxyanisole
|
|
GSH
|
glutathione
|
|
NAC
|
N-acetyl-L-cysteine
|
|
SMAC/DIABLO
|
second mitochondria-derived activator
of caspases
|
|
Trx1
|
thioredoxin 1
|
|
XIAP
|
X-linked inhibitor of apoptosis
protein
|
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