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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



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



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



subsequent nuclear translocation of GAPDH might function as a mediator of

TRAIL-induced cell death in thyroid cancer cells.



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



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



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.



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)



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



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



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,



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).



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



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.



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



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



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



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



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



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



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).



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,



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),



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



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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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



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



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.



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



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



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.



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 )



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 - - ++



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 )



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



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 ( % )



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



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

gapdh tnf il-1b

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