Endocrinology. First published ahead of print May 31, 2007 as doi:10.1210/en.

2006-1511

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 Gao4 Affiliations:
1

Department of Endocrinology and Metabolism, the 1st Affiliated Hospital, China

Medical University, Shenyang, China

2

Department of Molecular Biology, China Medical University, Shenyang, China Department of Geriatrics, the 1st Affiliated Hospital, China Medical University,

Shenyang, China
4

Department of Orthopedics, the 1st Municipal Hospital of Qinhuangdao, Qinhuangdao,

China
*

These authors contributed equally to this work.

Key words: GAPDH, nuclear translocation, TRAIL, thyroid cancer cell

Copyright (C) 2007 by The Endocrine Society

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:dzx_doctor@hotmail.com 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:dzx_doctor@hotmail.com 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 of GAPDH was observed in another thyroid cancer cell line KTC2, which is also sensitive to TRAIL, but not in those TRAIL insensitive cell lines: ARO, KTC1 and KTC3. These data indicate that NO-mediated S-nitrosylation of GAPDH and

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 now supporting the notion that GAPDH is a multifunctional protein (10-13). Particularly, its role as a mediator for cell death, frequently associated with oxidative stress has been highlighted (14-23). The involvement of GAPDH in apoptosis was first demonstrated in

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 this study, nuclear translocation of GAPDH was observed in TRAIL-treated FRO and KTC2 cells, which are sensitive to TRAIL-induced cell death, but not those insensitive cell lines: ARO, KTC1 and KTC3. The nuclear accumulation of GAPDH was closely

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 M. IFN was obtained from Roche Molecular biochemicals (Mannheim, Germany) and IL-1 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--actin monoclonal antibody (Chemicon, Bedford, MA) Cell culture Human thyroid cancer ARO, FRO, KTC1, KTC2 and KTC3 cells were grown in RPMI 1640 (Sigma, Saint Louis, MO) supplemented with 10% fetal bovine serum (FBS,

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 manufacturers instructions. Real time PCR analysis was performed in triplication on the ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using the SYBR Green PCR Master mix (Applied Biosystems, Warrington, UK). Primer sets specific for GAPDH (forward

5-ccaggaaatgagcttgacaaagtg-3, reverse 5-aaggtcatccctgagctgagctg-3) and -actin (forward 5-gcgagaagatgacccagatca-3, reverse 5-aaggaaggctggaagagtgc-3) was used as internal control for PCR amplification. The validity of -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 (1106 in 100 mm2 culture dishes) were lysed in a buffer containing Tris-HCl, and Triton X-100. Lysates were then incubated with RNase A and proteinase K. DNA was obtained with an equal volume of neutral phenol: chloroform: isomyl alcohol mixture (25:24:1) and then precipitated with ethanol and

sodium acetate at -20C. Equal amount (6 g) of the purified DNA were then subjected

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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 g 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 g/ml aprotinin, 1 g/ml leupeptin, and 2 g/ml pepstatin, to achieve a total reaction volume of 500 l. 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 M and incubated for 1 hour at 37C. 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 g 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, Sigma-Aldrich) according to the manufacturers instructions. After staining with annexin V-FITC and PI, samples were analyzed by fluorescence-activated cell scanner (FACScan) flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

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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 -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). Assessment of subcellular localization of GAPDH using confocal microscopy For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 15 min then permeabilized with cold acetone for 5 min. Nonspecific antibody binding was

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blocked by incubating cells with 5% normal goat serum for 1 hour. Fixed cells were incubated overnight at 4C 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 (14C)-L-citrulline, generated from (14C)-L-arginine (Amersham, Germany). The assay was performed using the NOS detect assay kit (Stratagene, Germany) according to the manufacturers instructions. Radioactivity was counted in a -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 manufacturers protocol. Briefly, culture media were filtered with 0.2-m filters. 80 l of each sample was treated with nitrate reductase and its cofactors to convert all of nitrate to nitrite before applying 100 l of the Griess reagent. Absorbance was measured at 543 nm, and nitrite concentration was determined using a standard curve of sodium

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nitrite concentrations ranging from 0 to 50 M. S-nitrosylation biotin switch assay The assay was performed as described (34). In brief, cells were lysed and reduced cysteines were blocked with 4 mM methylmethanethionsulphonate (MMTS). Subsequently, S-nitrosylated cysteines were reduced with 1 mM ascorbate and biotinylated with 1 mM Biotin-HPDP (Pierce, Rockford, IL). The biotinylated proteins were pulled down with streptavidin agarose and analyzed by western blotting. Small interfering RNA (siRNA) The following sequences were chosen for silencing the gene expression: GAPDH, CGGGAAGCUCACUGGCAUG and control, CCGUAUCGUAAGCAGUACU. The transfection of siRNA oligonucleotides was performed with oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturers recommendations. Protein Carbonyl Assay Cells were homogenized in 50 mM Tris, 150 mM NaCl, and 1% v/v Triton X-100, pH7.5, and centrifuged at 2000g for 10 min to remove tissue particles. Supernatants were assayed for protein content (Pierce, Rockford, IL) and 40 g of protein was assayed for protein carbonyls according to the manufacturers instructions (OxyBlot, Chemicon, Bedford, MA). Equivalent protein loading was confirmed by probing for

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actin. Data analysis Statistical difference were evaluated using the one-way ANOVA with Dunnetts 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 hours following TRAIL treatment (Figure 1D). Similar results were achieved using two different antibodies against GAPDH (data not shown). Effects of TRAIL on GAPDH cellular localization in FRO cells

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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 the nuclear fraction as early as 2 hours following TRAIL treatment while almost no cells were stained by trypan blue (Figure 2A). To confirm further the TRAIL-induced increased GAPDH protein in the nucleus,

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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 oxidation products nitrite and nitrate, which represent the stable end-products of NO and accumulate in the cell culture media (Figure 3B). Effect of S-nitrosylation and nuclear translocation of GAPDH on TRAIL-induced

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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 it by small interfering RNA (siRNA) with pyruvate-containing medium to compensate for any requirement of GAPDH in the glycolytic pathway (37). siGAPDH treatment significantly decreased the level of GAPDH as assessed by western blotting (Fig 5A,

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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 cells were the most resistant, almost completely insensitive after treatment for 24 hours (Figure 6A). KTC1 cells demonstrated a limit cell death, less than 20% cell death was observed (Figure 6A). FRO was most sensitive with IC50 values in the range of 10-20

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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 observed under conditions while cells were sensitized to TRAIL induced apoptosis. Consistent with these previous reports, we also observed TRAIL-induced normal thyroid epithelial cell death after exposure to IL-1 (data not shown). Upon exposure to

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TRAIL, nuclear localization of GAPDH was observed in IL-1 pretreated normal primary thyroid epithelial cells, but not in vehicle pretreated cells (Figure 7A). Similar effects were also observed in IFN 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 IFN 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-1 pretreated normal primary thyroid epithelial cells (Figure 7C), as well as in IFN 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 IFN 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, evidenced by the observation that nuclear transportation of GAPDH and accumulation of protein carbonyls (a marker of oxidative damage) was only observed in TRAIL sensitive thyroid FRO, KTC2 cells. Furthermore, nuclear localization of GAPDH was

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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), suggesting that accumulation of GAPDH might function as a upstream pathway of apoptosis. Consistent with these reports, the increase of GAPDH protein in the nucleus appeared to be an early event in the TRAIL-induced apoptotic process in FRO cells as

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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 has been demonstrated that GAPDH expression is substantially increased in human cancers of various origins(44-47). Given that GAPDH was upregulated at both mRNA and protein levels in response to TRAIL exposure in FRO cells, our results support the

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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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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 Dunnetts 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 dosedependent 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 Dunnetts 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 Dunnetts 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 -actin was used as a loading control. A representative image was presented and the ratios versus that of control (normalized by -actin) were noted at the bottom of the image (n=3). Figure 2 Translocation of GAPDH into nuclear fractions in FRO cells during

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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 -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 increase in NOS activity was observed starting at 4 hours of TRAIL treatment. (B) nitrite/nitrate in culture media was measured, and increased NO production was also observed upon TRAIL treatment. Data represent the means SD of 4 independent

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experiments performed in duplicate. * P<0.05, ** P<0.001 by one-way ANOVA with Dunnetts 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 M 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 Dunnetts post hoc test. (D) Cells were treated as A and immunoblot analysis was performed using antibodies against Bcl-2 and Bax. Figure 5 GAPDH-mediated FRO cell death upon TRAIL treatment (A) siRNA against GAPDH depletes GAPDH protein in FRO cells. 24 hours following transfected with siRNA against GAPDH or control, FRO cells were stimulated with

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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 Dunnetts 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 represent the mean SD (n=3). * P<0.05, ** P<0.001 by one-way ANOVA with Dunnetts post hoc test. (B) Cells were treated with 1000 ng/ml (FRO cells, 20 ng/ml) of TRAIL for 8 hours. Nuclear proteins were extracted and Western blot analysis was

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performed. (C) Cells were treated as B and protein carbonyls were evaluated using Oxyblot according to manufacturers instructions. An antibody against -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-1 (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 IFN (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-1 (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 with or without IFN (100 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. (E) siRNA against GAPDH depletes GAPDH protein

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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 IFN (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 IFN-pretreated ARO cells. 24 hours following transfected with siRNA against GAPDH, ARO cells were pretreated for 24 hours with or without IFN (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 Dunnetts post hoc test.

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Figure 1 A
100 Apoptotic cell (%) 80 60 40 20 2 5 10 20 50 (ng/ml)

**

**

** *

0 TRAIL 0

B
GAPDH mRNA level (ratio versus control)

5 4 3 2 1 2 5 10 20

** **

**

0 TRAIL 0

50 (ng/ml)

C
5
GAPDH mRNA level (ratio versus control)

4 3 2 1 0 0 1 2 4

** **

** *

12

24 (h)

D
0 2 4 8 12 24 (h) GAPDH
Mean SD 1.00 0.93 1.37 1.86 2.12 1.68 0.11 0.07 0.13 0.17 0.09

Actin

Figure 2

A Nuclear fraction

12

(h) GAPDH H2B LDH

Cell Viability (%) 100 100 91 69 Cytosolic fraction

38 GAPDH LDH Actin H2B

GAPDH

GAPDH/DAPI

GAPDH/DAPI (m)

0h

4h

12h

Figure 3

200 NOS activity (% of 0 h treatment) 160 120 80 40 0 0 2 4 8

** * *

12

24 (h)

B Production of nitrite/nitrate

(M) 140 120 100 80 60 40 20 0 0 2 4 8 12 24 (h)

**

** **

Figure 4

L-NAME TRAIL

- + - - +

+ +
SNO GAPDH Total GAPDH Actin

L-NAME TRAIL

- + - - +

+ +
GAPDH Histone H2B LDH

L-NAME TRAIL

- + - - +

+ +

Cell Viability (%) 100 100 45 72

(fluorescence increase/g protein)

1500

Caspase-3 activity

1000

500

L-NAME TRAIL

+ +

+ +

L-NAME TRAIL

- + - - +

Bcl-2 Bax Actin

Figure 5

A
GAPDH siRNA control siRNA

GAPDH Actin

+
GAPDH H2B

B
GAPDH siRNA control siRNA L-NAME TRAIL
(fluorescence increase/g protein)

Caspase-3 activity

- - + + - + - - - - + - + + +

*
1500

* NS

1000

500

GAPDH siRNA control siRNA L-NAME Cell Viability (%) 100 37 60 67 TRAIL

+ +

C
Production of nitrite/nitrate

(M) 120 100 80 60 40 20 0

NS

GAPDH siRNA control siRNA L-NAME TRAIL

+ +

Figure 6

A
100 Apoptotic cell (%) 80 60

** **

**
ARO KTC1 KTC2

40

*
20 0 TRAIL 0

KTC3

125 250 500 1000 2000 (ng/ml)

AR

O T FR K

C1 K

2 C3 TC KT
GAPDH H2B

TARIL
KTC1 KTC1 KTC2 KTC3 ARO FRO KTC2 ARO KTC3 FRO

Protein carbonyls

Actin

Figure 7

A TRAIL Nuclear fraction 0 2

IL-1 4 8 12 24

vehicle 4 8 12 24 (h) GAPDH H2B

Cytosolic fraction

GAPDH actin

B TRAIL Nuclear fraction 0 2

IFN 4 8 12 24

vehicle 4 8 12 24 (h) GAPDH H2B

Cytosolic fraction

GAPDH actin

Figure 7

GAPDH

GAPDH/DAPI

GAPDH

GAPDH/DAPI

vehicle/TRAIL

IL-1/TRAIL

GAPDH siRNA control siRNA

F +

40 35 30 25 20 15 10 5 0

GAPDH Actin

control siRNA GAPDH siRNA

Apoptotic cell (%)

Nuclear fraction

GAPDH H2B

IFN

TRAIL

IFN/TRAIL

IFN/TRAIL

vehicle/TRAIL