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Title:
Involvement of GAPDH in tumor necrosis factor (TNF)-related apoptosis-inducing
ligand (TRAIL)-mediated death of thyroid cancer cells
Short title:
GAPDH in TRAIL-mediated cell death
Authors:
Zhen-Xian Du1*
, Hua-Qin Wang2*
, Hai-Yan Zhang3, Da-Xin Gao
4
Affiliations:
1Department of Endocrinology and Metabolism, the 1
stAffiliated Hospital, China
Medical University, Shenyang, China
2Department of Molecular Biology, China Medical University, Shenyang, China
3Department of Geriatrics, the 1
stAffiliated Hospital, China Medical University,
Shenyang, China
4Department of Orthopedics, the 1
stMunicipal Hospital of Qinhuangdao, Qinhuangdao,
China
*
Endocrinology. First published ahead of print May 31, 2007 as doi:10.1210/en.2006-1511
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Corresponding author:
Zhen-Xian Du, MD, PhD, Department of Endocrinology and Metabolism, the 1st
Affiliated Hospital, China Medical University, Shenyang, 110001, China.
Phone: +86-24-81908201, FAX: +86-24-23926176 E-mail:[email protected]
Reprint requests:
Zhen-Xian Du, MD, PhD, Department of Endocrinology and Metabolism, the 1st
Affiliated Hospital, China Medical University, Shenyang, 110001, China.
Phone: +86-24-81908201, FAX: +86-24-23926176 E-mail:[email protected]
Disclosure statement:
The authors have nothing to disclose
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Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is cytotoxic to
most thyroid cancer cell lines including those originating from anaplastic carcinomas,
implying TRAIL as a promising therapeutic agent against thyroid cancers. However,
signal transduction in TRAIL-mediated apoptosis is not clearly understood. In addition
to its well known glycolytic functions, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) is a multifunctional protein including its surprising role as a mediator for cell
death. In this study, we explored the involvement of GAPDH in TRAIL-mediated
thyroid cancer cell death. In FRO cells, S-nitrosylation and nuclear translocation of
GAPDH appears to mediate TRAIL-induced cell death at least partially, as evidenced by
that pre-treatment with L-NAME, a competitive nitric oxide synthase (NOS) inhibitor
partially but significantly attenuated TRAIL-induced apoptosis through the reduction of
S-nitrosylation and nuclear translocation of GAPDH. In addition, GAPDH siRNA
partially prevented the apoptotic effect of TRAIL, although TRAIL-induced NOS
stimulation and production of NO was not attenuated. Furthermore, nuclear localization
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subsequent nuclear translocation of GAPDH might function as a mediator of
TRAIL-induced cell death in thyroid cancer cells.
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Introduction
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has gained
considerable interest in cancer therapy since it displays specific antitumoral activity
against a wide range of tumor cells and has little or no toxicity to normal cells (1, 2).
TRAIL is well recognized to induce apoptosis by interacting with two cell-surface death
receptors DR4 and DR5 (3). The signal is propagated through caspase 8 and 10, finally
leading to activation of effector caspases such as caspase 3 (4). Recently, TRAIL has
been shown to modulate the production of nitric oxide (NO), and the simultaneous
activation of both NO synthase (NOS) and effector caspases appears to be required for
induction of TRAIL-mediated antitumoral effects (5-9).
For many decades, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has
been regarded merely as a housekeeping glycolytic enzyme that exists mainly in the
cytoplasm. However, increasing evidence demonstrates that GAPDH is located in
multiple cellular compartments, including the cytosol, plasma membrane, mitochondria,
cytoskeletons, and nuclei. In addition to glycolytic function, accumulating evidence is
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primary cultures of brain neurons (18, 20, 24, 25), and this finding was soon expanded
to numerous apoptotic paradigms in diverse cell types, including neurons and
nonneuronal cells (19). Knowledge concerning the mechanisms underlying GAPDH
nuclear translocation and subsequent cell death is growing. Several lines of evidence
suggest that GAPDH may be an intracellular sensor of oxidative stress during the early
phase of the apoptotic cascade. NO stress-mediated modification of GAPDH appears to
target it to nuclear, since NO donors stimulates accumulation of nuclear GAPDH,
whereas, NOS inhibitors prevents the nuclear translocation of GAPDH (12, 13, 26-28).
An increase in nuclear GAPDH is required for its apoptotic effects, which appear to be
upstream events that mediate apoptotic signals, as evidenced by the nuclear
accumulation of GAPDH precedes chromatin condensation, nuclear fragmentation, and
a decline in mitochondrial membrane protein, as well as knockdown of GAPDH by
antisense oligonucleotides suppresses cell death (15, 19, 22, 26, 29, 30).
Based on these reports, the experiments were designed to investigate the potential
implication of GAPDH in TRAIL-induced apoptosis in human thyroid cancer cells. In
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associated with NO-mediated S-nitrosylation in FRO cells. Furthermore, both NOS
inhibitor and siGAPDH partially but significantly inhibited TRAIL-induced cell death.
Our results indicated that NO-mediated S-nitrosylation and subsequent nuclear
translocation of GAPDH might be implicated in TRAIL-mediated thyroid cancer cell
death, suggesting a general role of GAPDH as a mediator for cell death.
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Materials and Methods
Reagents and Antibodies
Human recombinant TRAIL was obtained from Calbiochem (LaJolla, CA).
Inhibitor of iNOS N-nitro-L-arginine methyl ester (L-NAME) (Calbiochem, LaJolla,
CA) was added to the culture medium at a concentration of 100 μM. IFNγ was obtained
from Roche Molecular biochemicals (Mannheim, Germany) and IL-1β was bought from
Sigma-Aldrich (Saint Louis, MO). The following antibodies were used in this study:
goat anti-lactate dehydrogenase (LDH) polyclonal antibody (abcam, Cambridge, MA),
mouse anti-GAPDH monoclonal antibody (Chemicon, Bedford, MA), mouse
anti-GAPDH monoclonal antibody (clone 6C5) (Ambion, Austin, TX), rabbit
anti-Histone H2B polyclonal antibody (Cell signaling, Danvers, MA), mouse anti-Bcl-2
monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Bax
monoclonal antibody (Sigma, Saint Louis, MO ), rabbit anti-cytokeratin 18 polyclonal
antibody (Chemicon, Bedford, MA) and mouse anti-β-actin monoclonal antibody
(Chemicon, Bedford, MA)
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Sigma, Saint Louis, MO) and the medium was changed every 3 days. Starving of the
cultures and growth to post confluence were strictly avoided. Since serum depletion per
se might induce nuclear translocation of GAPDH (31), all treatment procedures were
performed in the presence of 5% FBS. Primary normal thyroid epithelial cells were
prepared as previously described (32, 33). Six normal thyroid samples were used in
the study. Histological examination of adjacent paraffin-embedded tissue was made in
every case to confirm the normal structure of thyroid samples. The purity of thyroid cell
population was verified by staining with an antibody against cytokeratin 18 (a marker
for epithelial cells), and only cultures that contained more than 90% cytokeratin positive
cells were used for experiments. Thyroid epithelial cells were used between the second
and fourth passages.
Real time RT-PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA).
Reverse transcription was carried out using Superscript II (Invitrogen, Carlsbad, CA)
and oligo(dT)12-18 primer according to manufacturer’s instructions. Real time PCR
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5’-ccaggaaatgagcttgacaaagtg-3’, reverse 5’-aaggtcatccctgagctgagctg-3’) and β-actin
(forward 5’-gcgagaagatgacccagatca-3’, reverse 5’-aaggaaggctggaagagtgc-3’) was used
as internal control for PCR amplification. The validity of β-actin as a housekeeping
gene was confirmed by no significant change during each stress treatment.
Trypan blue analysis
Trypan blue was used to assess the percentage of cell death caused by late apoptosis
and necrosis. Cells were collected by a brief trypsin wash. Equal volume of trypan blue
dye (Sigma-Aldrich, Saint Louis, MO) was added to collected cells. Cells were counted
by hemocytometer and assessed for blue inclusion, which is suggestive of a
compromised membrane and cell death. The blue and non-blue cells were counted
blindly by two independent observers. Cell death was determined by the percentage of
blue cells in total cells. In each group, 500-1000 cells were counted per experiment.
DNA ladder assay
Following treatment, FRO cells (1×106
in 100 mm2
culture dishes) were lysed in a
buffer containing Tris-HCl, and Triton X-100. Lysates were then incubated with RNase
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to electrophoresis in a 2% agarose gel and visualized under UV light after staining with
ethidium bromide.
Caspase-3 activity assay
For caspases-3 enzymatic assays, 50 μg of whole cell extract were added to reaction
buffer containing 25 mM HEPES (pH 7.5), 4 mM CHAPS, 1 mM DTT, 1 mM PMSF, 2
μg/ml aprotinin, 1 μg/ml leupeptin, and 2 μg/ml pepstatin, to achieve a total reaction
volume of 500 μl. Ac-DEVD-AMC (Ac-Asp-Glu-Val-Asp-7-amino- 4-methylcoumarin;
Alexis Biochemicals, San Diego, CA, USA) was added to the mixture at a concentration
of 100 μM and incubated for 1 hour at 37ºC. Cleavage of the substrate was measured by
fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA) using an excitation
and emission wavelength of 360nm and 465 nm, respectively. The activities were
expressed as fluorescence increase per μg of protein.
Detection of apoptotic cell death
For cell death assays, cells were washed twice in phosphate-buffered saline and then
stained with Annexin V-FITC (Biovision, Mountainview, CA) and propidium iodide (PI,
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Nuclear Fractionation
The nuclei were obtained from the cell lysates using a sucrose gradient was
performed as described previously (19). Immunoblotting of nuclear and total fractions
was performed with GAPDH antibody. Antibodies against Histone H2B and β-actin
were used as loading controls for nuclear and total proteins, respectively. The
purification of nuclear fractions was confirmed by lack of LDH signals using an
antibody against LDH, which is exclusively localized in the cytosolic fractions.
Western blot analysis
Protein concentration was determined using a commercial protein assay kit (Pierce,
Rockford, IL). An equal amount of protein for each sample was separated by 12%
SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, Billerica,
MA). After incubation in primary antibodies, membranes were probed with appropriate
horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia, UK).
Bound antibody was visualized using an enhanced chemiluminescence reagent
(Amersham Pharmacia, UK).
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blocked by incubating cells with 5% normal goat serum for 1 hour. Fixed cells were
incubated overnight at 4ºC with a primary antibody, followed by reaction for 2 hours
with Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR), and
then counterstained with DAPI. Finally, the slides were analyzed with a LSM510
confocal laser-scanning microscope (Zeiss, Oberkochen, Germany).
Evaluation of NOS activity
The NOS enzyme activity was evaluated by determination of (14
C)-L-citrulline,
generated from (14
C)-L-arginine (Amersham, Germany). The assay was performed using
the NOS detect assay kit (Stratagene, Germany) according to the manufacturer’s
instructions. Radioactivity was counted in a β-scintillation counter (Beckmann,
Germany).
Measurement of NO production
The NO production was determined the level of nitrite and nitrate in the culture
media using the Griess reagent kit (Molecular Probes, Eugene, OR) following the
manufacturer’s protocol. Briefly, culture media were filtered with 0.2-μm filters. 80 μl
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actin.
Data analysis
Statistical difference were evaluated using the one-way ANOVA with Dunnett’s
post hoc test and considered significant at P<0.05.
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Results
Increased GAPDH level in FRO cells upon treatment with TRAIL
Previous studies have shown that FRO cells were sensitive to TRAIL stimulation
(35, 36). In our hands, FRO cells were very sensitive to TRAIL even in the presence of
serum, with IC50 values in the range of 10-20 ng/ml (Figure 1A). Increase in GAPDH
protein levels has been found to be associated with an increased probability of cell death
(30). We then evaluated whether TRAIL can regulate the level of endogenous GAPDH
in FRO cells using real-time RT-PCR and western blotting analyses. In FRO cells
cultured for 12 hour with various concentrations (2-50 ng/ml) of TRAIL, a significant
dose-dependent increase in GAPDH mRNA was observed. The maximum of
stimulation was reached at 20 ng/ml TRAIL (resulting in a 3 –fold increase) (Figure 1B).
The FRO cells were then treated for different period with 20 ng/ml of TRAIL prior to
the measurement of GAPDH expression. A statistically significant increase of GAPDH
mRNA was observed as early as 2 hours following TRAIL treatment and reached the
plateau at 8 hours (Figure 1C). The protein level of GAPDH significantly increased at 8
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Previous studies have suggested that nuclear translocation of GAPDH occurs in
panels of cells upon variety stressors (19-21, 28, 29). We then examined whether
TRAIL treatment redistributes GAPDH to the nucleus in FRO cells. Nuclear fractions
were purified from cells after exposure to 20 ng/ml TRAIL for different hours, and
western blot analysis was performed. GAPDH levels in a nuclear fraction, absent
initially, became apparent at 2 hours and increased substantially at 8 and 12 hours
following TRAIL treatment, on the other hand, GAPDH levels in a cytosolic fraction
demonstrated little alteration (Figure 2A). The purification of nuclear fraction without
contamination of cytosolic proteins was confirmed using antibodies against LDH
(Figure 2A). Nuclear accumulation of GAPDH was much more prominent than those in
the total cell extract, where only modest increases were observed (Figure 1C). To
ascertain whether increased expression of GAPDH in the nucleus was causally
associated with TRAIL-mediated FRO cell death, we assessed the cell viability using
trypan blue assay. Consistent with previously reported, nuclear translocation appears to
be an early event upon TRAIL treatment, as evidenced by that GAPDH began to exist in
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GAPDH immunostaining was performed. Under basal conditions, staining was
heterogeneous with 5-10% of the cells staining much more than the rest (data not
shown). GAPDH appears to be primarily localized in the cytosol of control FRO cells
(Figure 2B, upper panels), and little nuclear staining occurred (<1%, data not shown).
However, following 4 hours of TRAIL treatment, the number of cells that stained
positive for GAPDH in the nucleus significantly increased (Figure 2B, middle panels).
Most of cells revealed nuclear localization of GAPDH at 12 hours post TRAIL exposure
(Figure 2B, lower panels).
Stimulation of NOS activity upon TRAIL treatment in FRO cells
Because it has been shown that cytotoxic activity of TRAIL is mediated, at least in
part, by the production of NO in myeloid cells (7), we investigated whether TRAIL
increased NO production in FRO cells. The NOS activity was assessed in cell lysates
after treatment with TRAIL at different time points. A significant increase in NOS
activity was observed starting at 4 hours of TRAIL treatment (Figure 3A). In addition,
the supernatant of TRAIL-treated FRO cells contained increasing levels of the NO
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apoptosis in FRO cells
S-nitrosylation of GAPDH after induction of inducible NOS has been shown to
elicit its nuclear translocation, a process blocked by NOS inhibitors (28). We therefore
evaluated the presence of S-nitrosylated GAPDH in FRO cells after TRAIL treatment.
Using the biotin switch assay, we observed S-nitrosylation of GAPDH at 8 hours after
TRAIL treatment, which is prevented by the iNOS inhibitor L-NAME (Figure 4A).
Subcellular fractionation shows that GAPDH is translocated to the nucleus in response
to TRAIL, an effect that is also reversed by L-NAME (Figure 4B). In parallel
experiments, we also explored relationships between these changes in GAPDH and cell
death following TRAIL treatment. The apoptotic action of TRAIL is significantly
reduced by the iNOS inhibitor L-NAME as assessed by DNA ladder and caspase-3
activity assays (Figure 4C). The amounts of Bcl-2 and Bax proteins were unaltered at
the time periods tested (Figure 4D).
Involvement of GAPDH in TRAIL-mediated apoptosis in FRO cells
To further ascertain the importance of GAPDH for apoptotic cell death, we depleted
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Left), TRAIL significantly increased the nuclear fraction of GAPDH in control
siRNA-treated cells, whereas little nuclear localization of GAPDH was observed in
siGAPDH-treated cells (Figure 5A, Right). The apoptotic effect of TRAIL is partially
abolished by siGAPDH treatment, and no additive effect of siGAPDH and NOS
inhibitor L-NAME was observed (Figure 5B). Moreover, the influence of siGAPDH
treatment is unrelated to the formation of NO, detected by its oxidized product
nitrite/nitrate, which is similar in the presence or absence of siGAPDH (Figure 5C).
Nuclear translocation of GAPDH upon exposure to TRAIL in a panel of thyroid
cancer cell lines
To clarify whether nuclear translocation is a FRO cells-specific or a general
phenomenon in response to TRAIL treatment, we further investigated GAPDH
translocation upon TRAIL treatment in a panel of undifferentiated thyroid cancer cell
lines: ARO, KTC1, KTC2 and KTC3. We first evaluated the responsiveness of various
cell lines by treatment with increasing concentrations of TRAIL for 24 hours. These
thyroid cancer cell lines had different levels of sensitivity to TRAIL. ARO and KTC3
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ng/ml (Figure 1A), KTC2 cell lines had intermediate levels of sensitivity with IC50 in
the range of 500-1000 ng/ml (Figure 6A). GAPDH was observed in the nuclear fraction
in TRAIL sensitive FRO and KTC2 cells, but not ARO, KTC1 and KTC3 cells (Figure
6B). Since GAPDH is implicated in oxidative stress-mediated cell death, as well as
reactive oxygen species have been shown to be involved in TRAIL-mediated
cytotoxicity (6, 38), we then evaluated the degree of oxidative damage upon TRAIL
treatment. TRAIL treatment caused dramatic accumulation of protein carbonyls, a
well-known marker of oxidative damage in FRO and KTC2 cells. In contrast, no or
little alterations in protein carbonyls were observed in ARO, KTC1 and KTC3 cells
(Figure 6C). Our data thus indicated a close relation among nuclear translocation of
GAPDH, sensitivity to TRAIL and degree of oxidative damage in thyroid cancer cells in
vitro.
Previous studies have shown that cytokines could sensitize primary thyroid
epithelial cell or otherwise resistant thyroid carcinoma cell lines including ARO cells
(33, 39, 40), we then investigated whether nuclear transportation of GAPDH could be
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TRAIL, nuclear localization of GAPDH was observed in IL-1β pretreated normal
primary thyroid epithelial cells, but not in vehicle pretreated cells (Figure 7A). Similar
effects were also observed in IFNγ pretreated ARO cells, while TRAIL alone had no
effect on ARO viability (data not shown), nor nuclear localization of GAPDH was
observed (Figure 7B); pretreatment with IFNγ significantly sensitized ARO cells to
cytotoxicity induced by TRAIL (data not shown) and at the same time, nuclear
localization of GAPDH was detectable under this condition (Figure 7B).
Immunostaining confirmed the nuclear localization of GAPDH in IL-1β pretreated
normal primary thyroid epithelial cells (Figure 7C), as well as in IFNγ pretreated ARO
cells (Figure 7D). To further confirm the potential role of GAPDH in TRAIL-induced
cell death, siRNA against GAPDH was used to knock down GAPDH in ARO cells
(Figure 7E). Downregulation of GAPDH significantly inhibited the sensitizing effect of
IFNγ on TRAIL-induced ARO cell death (Figure 7F).
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Discussion
We have here demonstrated for the first time that GAPDH is implicated in the
anti-tumor activity of TRAIL in thyroid cancer cells in vitro. GAPDH is a well known
example of a multifunctional enzyme, with involvement in apoptosis as one of its
intriguing functions. A wide range of apoptotic stimuli activate NO formation, which
S-nitrosylates GAPDH. The S-nitrosylation therefore confers upon nuclear translocation
of GAPDH, enabling it to affect apoptosis (27). Previous studies have shown that
TRAIL activates iNOS and induces the generation of NO in lymphoblastic and
myeloma cell lines (7). Likewise, in our hands, TRAIL promotes the generation of NO
in FRO cells. Consistently, we found the occurrence of S-nitrosylated GAPDH and
redistribution of GAPDH to the nucleus following TRAIL treatment. Furthermore, NOS
inhibitor L-NAME significantly decreased the abundance of this modified form and
nuclear localization of GAPDH, as well as inhibited cell death mediated by TRAIL
treatment. Nuclear transportation of GAPDH appeared to correlate with the degree of
oxidative damage and the sensitivity to TRAIL in thyroid cancer cells in vitro,
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also observed in cytokines pretreated primary normal thyroid epithelial cells as well as
otherwise resistant ARO cells. Previous studies have shown that ROS is involved in
TRAIL-mediated cytotoxicity (6, 38), overexpression of antioxidant molecules is thus
possibly implicated in conferring resistance to TRAIL. Nuclear accumulation of
GAPDH becomes prominent after treatment with the genotoxic agents (19, 41, 42) or
other types of stress (22) and is accompanied by apoptotic cell death (15). Increased
expression of GAPDH is essential for induction of apoptosis of cerebellar granule cells
(23, 43), and the level of nuclear GAPDH has been linked to the sensitivity of human
leukemia cells to thiopurine treatment (42). Coupled with these findings, our study
contributes in indicating GAPDH might function as a general mediator of apoptosis
upon treatment with a broader spectrum of cytotoxic agents.
Other groups have stated the importance of nuclear translocation of GAPDH in
apoptosis induced by a variety of death stimuli, such as serum withdrawal and
ischemia-reperfusion, high glucose (19, 22, 24, 29). However, survival signals may be
able to reverse GAPDH nuclear translocation, therefore allowing cells to recover (31),
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evidenced by that nuclear accumulation of GAPDH already occurred when almost
100% of cells were still viable by trypan blue exclusion assay.
All studies so far have demonstrated an increase in GAPDH in the nucleus,
however, changes in cytosolic GAPDH protein levels during apoptosis varies,
depending on the stimuli and cell types (20, 24, 30). In this study, we did not see a
marked change in cytosolic GAPDH in FRO cells exposed to TRAIL. It is also still
speculated whether the nuclear GAPDH results from translocation of pre-existing
protein in the cytosol or from newly synthesized protein. Our results suggest both,
considering increased nuclear GAPDH as early as 2 hours following TRAIL treatment,
when total GAPDH had little increase.
GAPDH has been commonly considered as a constitutive housekeeping gene and
widely used as a control molecule. However, there is overwhelming evidence
suggesting that its use as an internal standard is inappropriate. Several lines of evidence
indicate that GAPDH is involved in various biological processes such as endocytosis,
control of gene expression, DNA replication and repair and apoptosis(41). Moreover, it
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idea that it should be caution to use GAPDH as an internal control.
In summary, given the interest of TRAIL as a promising candidate reagent for
cancer therapy and the importance to understand mechanisms underlying
TRAIL-mediated anti-tumor effects, we have investigated the role of GAPDH in
TRAIL-induced apoptosis in thyroid cancer cells. This is the first study to show that
GAPDH pathway is involved in TRAIL-mediated apoptosis, indicating a general role of
this classical glycolytic protein in apoptosis.
Acknowledgements
We thank Dr Junichi Kurebayashi (Kawasaki Medical University, Japan) for generously
providing KTC1, KTC2 and KTC3 cell lines.
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References
1. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W,
Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT,
Schuh JC, Lynch DH 1999 Tumoricidal activity of tumor necrosis
factor-related apoptosis-inducing ligand in vivo. Nat Med 5:157-163
2. Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B,
Hillan K, Totpal K, DeForge L, Schow P, Hooley J, Sherwood S, Pai R,
Leung S, Khan L, Gliniak B, Bussiere J, Smith CA, Strom SS, Kelley S, Fox
JA, Thomas D, Ashkenazi A 2001 Differential hepatocyte toxicity of
recombinant Apo2L/TRAIL versions. Nat Med 7:383-385
3. Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki A, Akiba H, Okumura
K, Yagita H 1999 Involvement of TNF-related apoptosis-inducing ligand in
human CD4+ T cell-mediated cytotoxicity. J Immunol 162:2639-2647
4. Wang X 2001 The expanding role of mitochondria in apoptosis. Genes Dev
15:2922-2933
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 28/49
(eNOS) activity and its localization within the human vein endothelial cells
(HUVEC) in culture. J Cell Biochem 97:782-794
6. Lee MW, Park SC, Kim JH, Kim IK, Han KS, Kim KY, Lee WB, Jung YK,
Kim SS 2002 The involvement of oxidative stress in tumor necrosis factor
(TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa
cells. Cancer Lett 182:75-82
7. Secchiero P, Gonelli A, Celeghini C, Mirandola P, Guidotti L, Visani G,
Capitani S, Zauli G 2001 Activation of the nitric oxide synthase pathway
represents a key component of tumor necrosis factor-related apoptosis-inducing
ligand-mediated cytotoxicity on hematologic malignancies. Blood 98:2220-2228
8. Huerta-Yepez S, Vega M, Jazirehi A, Garban H, Hongo F, Cheng G,
Bonavida B 2004 Nitric oxide sensitizes prostate carcinoma cell lines to
TRAIL-mediated apoptosis via inactivation of NF-kappa B and inhibition of
Bcl-xl expression. Oncogene 23:4993-5003
9. Hussain SP, Trivers GE, Hofseth LJ, He P, Shaikh I, Mechanic LE, Doja S,
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 29/49
10. Sirover MA 1999 New insights into an old protein: the functional diversity of
mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta
1432:159-184
11. Chuang DM, Hough C, Senatorov VV 2005 Glyceraldehyde-3-phosphate
dehydrogenase, apoptosis, and neurodegenerative diseases. Annu Rev
Pharmacol Toxicol 45:269-290
12. Hara MR, Cascio MB, Sawa A 2006 GAPDH as a sensor of NO stress.
Biochim Biophys Acta 1762:502-509
13. Hara MR, Snyder SH 2006 Nitric Oxide-GAPDH-Siah: A Novel Cell Death
Cascade. Cell Mol Neurobiol 26:525-536
14. Kragten E, Lalande I, Zimmermann K, Roggo S, Schindler P, Muller D, van
Oostrum J, Waldmeier P, Furst P 1998 Glyceraldehyde-3-phosphate
dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466
and R-(-)-deprenyl. J Biol Chem 273:5821-5828
15. Carlile GW, Chalmers-Redman RM, Tatton NA, Pong A, Borden KE,
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 30/49
16. Saunders PA, Chalecka-Franaszek E, Chuang DM 1997 Subcellular
distribution of glyceraldehyde-3-phosphate dehydrogenase in cerebellar granule
cells undergoing cytosine arabinoside-induced apoptosis. J Neurochem
69:1820-1828
17. Chen RW, Saunders PA, Wei H, Li Z, Seth P, Chuang DM 1999 Involvement
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53 in neuronal
apoptosis: evidence that GAPDH is upregulated by p53. J Neurosci
19:9654-9662
18. Ishitani R, Sunaga K, Hirano A, Saunders P, Katsube N, Chuang DM 1996
Evidence that glyceraldehyde-3-phosphate dehydrogenase is involved in
age-induced apoptosis in mature cerebellar neurons in culture. J Neurochem
66:928-935
19. Sawa A, Khan AA, Hester LD, Snyder SH 1997 Glyceraldehyde-3-phosphate
dehydrogenase: nuclear translocation participates in neuronal and nonneuronal
cell death. Proc Natl Acad Sci U S A 94:11669-11674
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 31/49
21. Saunders PA, Chen RW, Chuang DM 1999 Nuclear translocation of
glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis.
J Neurochem 72:925-932
22. Dastoor Z, Dreyer JL 2001 Potential role of nuclear translocation of
glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress. J
Cell Sci 114:1643-1653
23. Ishitani R, Chuang DM 1996 Glyceraldehyde-3-phosphate dehydrogenase
antisense oligodeoxynucleotides protect against cytosine
arabinonucleoside-induced apoptosis in cultured cerebellar neurons. Proc Natl
Acad Sci U S A 93:9937-9941
24. Sunaga K, Takahashi H, Chuang DM, Ishitani R 1995
Glyceraldehyde-3-phosphate dehydrogenase is over-expressed during apoptotic
death of neuronal cultures and is recognized by a monoclonal antibody against
amyloid plaques from Alzheimer's brain. Neurosci Lett 200:133-136
25. Ishitani R, Sunaga K, Tanaka M, Aishita H, Chuang DM 1997
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 32/49
26. Hara MR, Thomas B, Cascio MB, Bae BI, Hester LD, Dawson VL, Dawson
TM, Sawa A, Snyder SH 2006 Neuroprotection by pharmacologic blockade of
the GAPDH death cascade. Proc Natl Acad Sci U S A 103:3887-3889
27. Hara MR, Snyder SH 2006 Nitric Oxide-GAPDH-Siah: A Novel Cell Death
Cascade. Cell Mol Neurobiol 26:525-536
28. Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y,
Takahashi M, Cheah JH, Tankou SK, Hester LD, Ferris CD, Hayward SD,
Snyder SH, Sawa A 2005 S-nitrosylated GAPDH initiates apoptotic cell death
by nuclear translocation following Siah1 binding. Nat Cell Biol 7:665-674
29. Kusner LL, Sarthy VP, Mohr S 2004 Nuclear translocation of
glyceraldehyde-3-phosphate dehydrogenase: a role in high glucose-induced
apoptosis in retinal Muller cells. Invest Ophthalmol Vis Sci 45:1553-1561
30. Senatorov VV, Charles V, Reddy PH, Tagle DA, Chuang DM 2003
Overexpression and nuclear accumulation of glyceraldehyde-3-phosphate
dehydrogenase in a transgenic mouse model of Huntington's disease. Mol Cell
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 33/49
Biol 80:419-427
32. Arscott PL, Knapp J, Rymaszewski M, Bartron JL, Bretz JD, Thompson
NW, Baker JR, Jr. 1997 Fas (APO-1, CD95)-mediated apoptosis in thyroid
cells is regulated by a labile protein inhibitor. Endocrinology 138:5019-5027
33. Bretz JD, Mezosi E, Giordano TJ, Gauger PG, Thompson NW, Baker JR, Jr.
2002 Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid
epithelial cells. Cell Death Differ 9:274-286
34. Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH 2001
Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell
Biol 3:193-197
35. Petrella A, Festa M, Ercolino SF, Zerilli M, Stassi G, Solito E, Parente L
2005 Induction of annexin-1 during TRAIL-induced apoptosis in thyroid
carcinoma cells. Cell Death Differ 12:1358-1360
36. Ahmad M, Shi Y 2000 TRAIL-induced apoptosis of thyroid cancer cells:
potential for therapeutic intervention. Oncogene 19:3363-3371
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 34/49
38. Izeradjene K, Douglas L, Tillman DM, Delaney AB, Houghton JA 2005
Reactive oxygen species regulate caspase activation in tumor necrosis
factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell
lines. Cancer Res 65:7436-7445
39. Wang SH, Mezosi E, Wolf JM, Cao Z, Utsugi S, Gauger PG, Doherty GM,
Baker JR, Jr. 2004 IFNgamma sensitization to TRAIL-induced apoptosis in
human thyroid carcinoma cells by upregulating Bak expression. Oncogene
23:928-935
40. Mezosi E, Wang SH, Utsugi S, Bajnok L, Bretz JD, Gauger PG, Thompson
NW, Baker JR, Jr. 2004 Interleukin-1beta and tumor necrosis factor
(TNF)-alpha sensitize human thyroid epithelial cells to TNF-related
apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7
and bid, and the down-regulation of p44/42 mitogen-activated protein kinase
activity. J Clin Endocrinol Metab 89:250-257
41. Sirover MA 1997 Role of the glycolytic protein, glyceraldehyde-3-phosphate
8/3/2019 GAPDH TNF IL-1B
http://slidepdf.com/reader/full/gapdh-tnf-il-1b 35/49
novel protein complex distinct from mismatch repair binds thioguanylated DNA.
Mol Pharmacol 59:367-374
43. Berry MD, Boulton AA 2000 Glyceraldehyde-3-phosphate dehydrogenase and
apoptosis. J Neurosci Res 60:150-154
44. Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M, Sawada K,
Sakiyama S 1987 Enhanced expression of a glyceraldehyde-3-phosphate
dehydrogenase gene in human lung cancers. Cancer Res 47:5616-5619
45. Schek N, Hall BL, Finn OJ 1988 Increased glyceraldehyde-3-phosphate
dehydrogenase gene expression in human pancreatic adenocarcinoma. Cancer
Res 48:6354-6359
46. Kim JW, Kim SJ, Han SM, Paik SY, Hur SY, Kim YW, Lee JM, Namkoong
SE 1998 Increased glyceraldehyde-3-phosphate dehydrogenase gene expression
in human cervical cancers. Gynecol Oncol 71:266-269
47. Revillion F, Pawlowski V, Hornez L, Peyrat JP 2000
Glyceraldehyde-3-phosphate dehydrogenase gene expression in human breast
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Figure legends
Figure 1 Upregulation of GAPDH upon TRAIL treatment
(A) Dose-response curves of FRO thyroid cancer cells treated with TRAIL. FRO cells
were treated with different concentrations of TRAIL for 24 hours in the presence of 5%
FBS and subjected to Annexin V-FITC and PI staining. Data represent the mean ± SD
(n=3). * P<0.05, ** P<0.001 by one-way ANOVA with Dunnett’s post hoc test. (B)
Dose course of induction of GAPDH upon TRAIL treatment. FRO cells were treated
with different concentrations of TRAIL for 12 hours. Real-time RT-PCR demonstrated
dose–dependent increase of GAPDH mRNA upon TRAIL treatment. Data represent the
mean ± SD (n=3). * P<0.05, ** P<0.001 by one-way ANOVA with Dunnett’s post hoc
test. (C) Time course of induction of GAPDH upon TRAIL treatment. FRO cells were
treated with 20 ng/ml of TRAIL for different period. Data represent the mean ± SD
(n=3). * P<0.05, ** P<0.001 by one-way ANOVA with Dunnett’s post hoc test. (D)
FRO cells were treated with 20 ng/ml of TRAIL for different hours. Total proteins were
extracted and western blot analysis was performed. An antibody against β-actin was
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TRAIL treatment
(A) Western blot analysis of nuclear or cytosolic extracts of cells. FRO cells were
treated with 20 ng/ml of TRAIL for different hours and western blot analysis was
performed on both nuclear and cytosolic fractions. Antibodies against histone H2B and
β-actin were used as loading controls for nuclear and cytosolic fractions, respectively.
Purification of nuclear fraction was confirmed using an antibody against LDH. The
western blot is representative of three independent experiments. Simultaneously, cell
viability was assessed and noted at the bottom of the image.
(B) Cells were processed for GAPDH staining. Prior to TRAIL treatment, GAPDH
staining was almost excluded from the nucleus in FRO cells (upper). The number of
GAPDH nuclear positive cells was increased after 4 hours (middle) upon TRAIL
treatment. At 12 hours post exposure to TRAIL, a large population of cells revealed
nuclear localization of GAPDH (lower). GAPDH/DAPI(m) indicates magnified images.
Figure 3 Stimulation of NOS activity in FRO cells upon TRAIL treatment
(A) FRO cells were treated with TRAIL (20 ng/ml) for the indicated times, significant
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experiments performed in duplicate. * P<0.05, ** P<0.001 by one-way ANOVA with
Dunnett’s post hoc test.
Figure 4 S-nitrosylation and nuclear translocation of GAPDH in TRAIL-treated
FRO cells
(A) NO generated from NOS causes S-nitrosylation of GAPDH. FRO cells were treated
with vehicle, 100 μM L-NAME, 20 ng/ml TRAIL for 8 hours, or pre-treated with
L-NAME for 1 hour then stimulated with TRAIL for 8 hours. Cell lysates were
subjected to the biotin switch assay. (B) GAPDH translocates to the nucleus upon
TRAIL treatment. FRO cells were treated as in A. Nuclear fractions were analyzed by
western blotting. (C) NOS inhibitor significantly attenuates TRAIL-mediated cell death.
FRO cells were treated as in A, nuclear DNA fragmentation (left) and caspase-3 activity
(right) was then analyzed. Cell death evaluated by trypan blue staining was noted at the
bottom. * P<0.05, by one-way ANOVA with Dunnett’s post hoc test. (D) Cells were
treated as A and immunoblot analysis was performed using antibodies against Bcl-2 and
Bax.
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TRAIL for another 8 hours. Total cell lysates (Left) and nuclear fractions (Right) were
subjected to western blotting. (B) siRNA against GAPDH partially inhibits cell death in
TRAIL-treated FRO cells. 24 hours following transfected with siRNA against GAPDH,
FRO cells were stimulated with TRAIL or pretreatment with L-NAME then stimulated
with TRAIL for another 8 hours. Nuclear DNA fragmentation (left) and caspase-3
activity (right) was then analyzed. Cell death evaluated by trypan blue staining was
noted at the bottom. * P<0.05, NS, no significant difference, by one-way ANOVA with
Dunnett’s post hoc test. (C) siGAPDH has no effect on the NO generation. FRO cells
were treated as B, nitrite/nitrate concentration in the media was measured by the Griess
reagent (n=3). NS, no significant difference.
Figure 6 Close relations among the sensitivity to TRAIL, nuclear transportation of
GAPDH and degree of oxidative stress in thyroid cancer cells in vitro
(A) Dose-response curves of a panel of thyroid cancer cells treated with TRAIL.
Thyroid cancer cells were treated with different concentrations of TRAIL for 24 hours
in the presence of 5% FBS and subjected to Annexin V-FITC and PI staining. Data
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performed. (C) Cells were treated as B and protein carbonyls were evaluated using
Oxyblot according to manufacturer’s instructions. An antibody against β-actin was used
as a loading control.
Figure 7 Nuclear localization of GAPDH in cytokine-sensitizing normal thyroid
epithelial cells or otherwise resistant ARO cells
(A) Normal primary thyroid epithelial cells were pretreated for 4 days with or without
IL-1β (50 U/ml) then stimulated with TRAIL (1000 ng/ml) for the indicated times in the
presence of 5% FBS. Western blot analysis was performed on both nuclear and
cytosolic fractions. (B) ARO cells were pretreated for 24 hours with or without IFNγ
(100 U/ml) then stimulated with TRAIL (1000 ng/ml) for the indicated times in the
presence of 5% FBS. Western blot analysis was performed on both nuclear and
cytosolic fractions. (C) Normal primary thyroid epithelial cells were pretreated for 4
days with or without IL-1β (50 U/ml) then stimulated with TRAIL (1000 ng/ml) for 8
hours in the presence of 5% FBS and subjected to GAPDH staining. Arrow head
indicates nuclear localization of GAPDH. (D) ARO cells were pretreated for 24 hours
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in ARO cells. 24 hours following transfected with siRNA against GAPDH or control,
ARO cells were pretreated for 24 hours with IFNγ (100 U/ml) then stimulated with
TRAIL (1000 ng/ml) for 8 hours in the presence of 5% FBS. Total cell lysates (Upper)
and nuclear proteins (Lower) were subjected to Western blotting analysis. (F) siRNA
against GAPDH significantly inhibits TRAIL-induced cell death in IFNγ-pretreated
ARO cells. 24 hours following transfected with siRNA against GAPDH, ARO cells
were pretreated for 24 hours with or without IFNγ (100 U/ml) then stimulated with
TRAIL (1000 ng/ml) for 24 hours in the presence of 5% FBS and subjected to Annexin
V-FITC and PI staining. Data represent the mean ± SD (n=3). * P<0.05 by one-way
ANOVA with Dunnett’s post hoc test.
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0
20
40
60
80
100
0 2 5 10 20 50
Figure 1
(ng/ml)
C
B
TRAIL
*
**
****
A
A p o p t o t i c c e l l ( % )
****
**
0
1
2
3
4
5
0 2 5 10 20 50TRAIL (ng/ml)
G A P D H m R N A
l e v e l
( r a t i o v e r s u s c o
n t r o l )
0
1
2
3
4
5
0 1 2 4 8 12 24 (h)
**
****
*
G A P D H m R N A l e v e l
( r a t i o v e r s u s c o n t r o l )
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Figure 3
B
0
40
80
120
160
200
0 2 4 8 12 24
0
20
40
60
80
100
120
140
0 2 4 8 12 24
*
***
*
*
****
**
(h)
(h)
A
N O S a c t i v i t y
( % o
f 0
h t r e a t m e n t )
P r o d u c t i o n o f n i t r i t e / n i t r a t e
(μM)
Figure 4
AL NAME - - ++
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B
A
SNO GAPDH
Total GAPDH
Actin
L-NAME
TRAIL
-+- -
-+
++
L-NAME
TRAIL
-+- -
-+
++
GAPDH
Histone H2B
LDH
C L-NAME
TRAIL
-+- -
-+
++
0
500
1000
1500
L-NAME - - ++
Cell Viability (%) 100 100 45 72
*
C a s p a s e - 3 a c t i v i t y
( f l u o r e s c e n c e
i n c r e a s e / μ g p r o t e i n )
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Figure 5
B
GAPDH
Actin
Cell Viability (%) 100 37 60 67
L-NAME
TRAIL
-+-
- -+
+
+
60
80
100
120
C
GAPDH siRNA
control siRNA
--
--+ +
+ -
L-NAME
TRAIL-
+- - -+
+
+
GAPDH siRNA
control siRNA
--
--+ +
+ -
GAPDH
H2B
AGAPDH siRNA
control siRNA
--+
+
--+
+
*
*
NS
NS
0
500
1000
1500
C a s p a s e - 3 a c t i v i t y
( f l u o r e s c e n c e i n c r e a s e / μ g p r o t e i n )
o f n i t r i t e / n i t r a t e
(μM)
Figure 6
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B
GAPDH
A R O
0
20
40
60
80
100
0 125 250 500 1000 2000
ARO
KTC1
KTC2
KTC3
TRAIL (ng/ml)
H2B
C
K T C 3
K T C 2
K T C 1
F R O
A R O
K T C 3
K T C 2
K T C 1
F R O
TARIL
Protein
b l
A R O
K T C
3
K T C 2
K T C 1
F R O
A
*
**
****
* *
A p o p t o t i c c e l l ( % )
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Figure 7
H2B
GAPDH
actin
GAPDH
AIL-1β vehicle
0 2 4 8 12 24 0 2 4 8 12 24 (h)
Cytosolic fraction
Nuclear fraction
B vehicle
0 2 4 8 12 24 0 2 4 8 12 24 (h)
Nuclear fraction
IFNγ
H2B
GAPDH
actin
GAPDHCytosolic fraction
TRAIL
TRAIL
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C GAPDH GAPDH/DAPIDGAPDH GAPDH/DAPI
v e h i c l e / T R A I L
I L - 1 β / T R A I L
v e h i c l e / T R A I L
I F N γ / T R A I L
GAPDH siRNA
control siRNA
--+
+
GAPDH
Actin
E control siRNA
GAPDH siRNA
A p o p t o t i c c e l l
( % )
IFNγ/TRAILTRAILIFNγ0
5
10
15
2025
30
35
40F
*
H2B
GAPDHNuclear fraction
Figure 7