p53 in trichostatin A induced C6 glioma cell death

Biochimica et Biophysica Acta 1810 (2011) 504–513

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

p53 in trichostatin A induced C6 glioma cell death Ya-Fen Hsu a, Joen-Rong Sheu b,c, George Hsiao b,c, Chien-Huang Lin b, Tsai-Hsing Chang b, Pei-Ting Chiu c, Chun-Yu Wang c, Ming-Jen Hsu b,c,⁎ a b c

Division of General Surgery, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 7 July 2010 Received in revised form 21 January 2011 Accepted 23 February 2011 Available online 2 March 2011 Keywords: Trichostatin A Histone deacetylase p53 Survivin Apoptosis

a b s t r a c t Background: Histone deacetylase (HDAC) inhibitors were demonstrated to induce cell cycle arrest, promote cell differentiation or apoptosis, and inhibit metastasis. HDAC inhibitors have thus emerged as a new class of anti-tumor agents for various types of tumors. However, the mechanisms by which HDAC inhibition-induced cell death remain to be fully defined. Methods: In the present study, we explored the apoptotic actions of trichostatin A (TSA), a HDAC inhibitor, in C6 glioma cells. Results: TSA activated p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation and activation. P53, a proapoptotic transcription factor, in turn transactivated the expression of a proapoptotic protein, Bax. In addition, survivin, a member of inhibitor of apoptotic protein, was significantly decreased in TSA-treated C6 cells. P53 recruited to the endogenous survivin promoter region was increased and accompanied by decreasing recruitment of SP1 in response to TSA. TSA was also shown to induce IKK dephosphorylation and to suppress NF-κB reporter activity. Conclusions: TSA may cause C6 cell apoptosis through activating p38MAPK–p53 cascade resulting in Bax expression and survivin suppression. Negative regulation of IKK–NF-κB signaling may also lead to p53 activation and contribute to TSA apoptotic actions. General significance: TSA-induced p53 activation may occur through p53 modification by phosphorylation or by acetylation via IKK inactivation. The present study delineates, in part, the signaling pathways involved in TSA-induced glioma cell death. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The organization of chromatin appears to play a central role in regulating gene expression including those involved in the pathogenesis of cancer [1,2]. The function of chromatin is regulated by a variety of post-transcriptional modifications of histones including acetylation, methylation, and ubiquitination [3]. Histone modification by acetylation is maintained by the opposing activities of histone acetylases and histone deacetylases (HDACs). Excessive deacetylated level of histones has been linked to cancer pathologies by promoting the repression of tumor regulatory genes. HDAC inhibitors may cause an increase of the acetylated level of histones leading to the reexpression of silenced regulatory genes [1,3–5]. Importantly, HDACs deacetylates not only histones but also nonhistone substrates, which

⁎ Corresponding author at: Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, No. 250 Wu-hsing Street, Taipei 11031, Taiwan. Tel./fax: +886 2 27361661x3198. E-mail address: [email protected] (M.-J. Hsu). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.02.006

participate in a variety of cellular responses [6,7]. In addition, HDAC inhibitors have recently been noted for their ability to induce cell cycle arrest, differentiation, apoptosis and to attenuate metastasis in numerous cancer cell types [4,8–11]. However, the molecular mechanisms underlying HDAC inhibitors actions have not been fully delineated. Gliomas are the most common primary neoplasm in the brain. Guha et al. [12] have recently reported the molecular alterations underlying astrocytoma formation. Recent studies further demonstrated that the methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter is a specific predictive biomarker of glioblastoma and astrocytoma responsiveness to chemotherapy with alkylating agents. Thus, epigenetic therapy may be a promising and potent treatment for human neoplasia including glioblastoma and astrocytoma [13]. Therefore, we aimed to use trichostatin A (TSA), a potent HDACs inhibitor, to elucidate the apoptotic mechanisms of HDAC inhibition in C6 glioma cells. Apoptosis plays a critical role in developmental process and maintenance of tissue homeostasis [14,15]. One of the prerequisites of tumor formation and progression is the suppression of apoptosis [16]. Initiation of apoptosis is controlled by regulation of the balance between the death and survival signals perceived by a cell [17]. The

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members of inhibitor of apoptosis proteins (IAP) family, particularly, survivin (BIRC5), were reported to play a crucial role in regulating apoptosis and contributed to tumor progression [18–20]. Overexpressed survivin gene is observed in tumor cells in most cancer diseases [21]. The regulation of survivin gene expression largely occurs at the transcription level [22]. The promoter region of the survivin gene contains many transcription factor binding sites. These transcription factors include Sp1, HIF-1α, c-myc, Stat3 and tumor suppressors Rb and p53 [20,22–28]. In particular, activation of SP1 leads to the induction of survivin whereas p53 may counteract the binding of SP1, thereby suppressing survivin expression [22–24]. However, the role of SP1 and p53 in regulating survivin expression following HDAC inhibition is still unknown. The characterization of survival signaling pathways stimulated by various growth factors has revealed the causal role of nuclear transcription factor-κB (NF-κB) in the suppression of apoptosis [29–31]. NF-κB interacts with a specific inhibitor named IκBα in the cytoplasma [32]. A variety of stimuli which activate the IκBα kinase (IKK) may cause IκBα phosphorylation and subsequent degradation leading to NF-κB nuclear translocation [33,34]. Nuclear NF-κB induces the expression of a number of genes whose products can promote cell survival and protects cells from apoptosis. Elevated levels of NF-κB are frequently detected in cancers. Blocking the IKK–NF-κB pathway has thus been a promising strategy in cancer therapies. The Bcl-2 family comprises proteins with antiapoptotic and proapoptotic function, which regulated the mitochondrial apoptotic pathway [35]. Bax, a proapoptotic member of Bcl-2 family, plays an important role in promoting apoptosis through oligomerization, mitochondria membrane disruption and subsequent release of cytochrome c. In contrast to playing the role in survivin suppression, tumor suppressor p53 has been shown to induce apoptosis by causing mitochondrial dysfunction via transactivation of Bax expression [36–38]. Activation of p53 entails phosphorylation of its serine residues, primarily Ser15 [39]. We have previously demonstrated that p38 mitogen-activated protein kinase (p38MAPK) mediated p53 Ser15 phosphorylation and subsequent Bax expression in the apoptotic paradigm of cerebral endothelial cells [40]. In another hand, p53-dependent apoptosis is also regulated by the opposing activities of histone acetyltransferases (p300/CBP) and HDACs [41–43]. Recent studies further demonstrated that IKK signaling may also participate in the regulation of p53 acetylation and activation [44,45]. We aimed to determine whether the transcription factor p53 contributes to HDAC inhibition-induced cell death. Results from the present study provide experimental evidence to support the contention that activation of the p38MAPK–p53–Bax pathway contributes to TSA-induced C6 cell apoptosis. Negative regulation of IKK–NF-κB signaling and survivin downregulation may also contribute to TSA apoptotic actions. 2. Materials and methods 2.1. Reagents DMEM, optiMEM, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA); antibody specific for α-tubulin was purchased from Novus Biologicals (Littleton, CO); anti-mouse and anti-rabbit IgG conjugated alkaline phosphatase antibodies, normal rabbit IgG (control IgG) and rabbit polyclonal antibodies specific for p53, survivin and p21 were from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Bax, Bcl-2 or Bcl-xL were from GeneTex Inc (Irvine, CA); antibodies against p53 acetylated at Lys 379, IKK phosphorylated at Ser180/Ser181 or acetylated-lysine were from Cell Signaling (St. Louis, MO); trichostatin A (TSA) and sirtinol were from Calbiochem (San Diego, CA); Taq DNA polymerase was purchased from Takara (Otsu, Japan); a chromatin immunoprecipitation (ChIP) assay kit, and TurbofectTM in vitro transfection reagent were purchased from Upstate Biotechnol-

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ogy (Lake Placid, NY); construct of PG13-luc with p53 binding sites (Addgene plasmid 16642) as described previously[46] was kindly provided by Dr. Bert Vogelstein. The reporter plasmid, NFκB-Luc, Renilla-luc and Dual-Glo luciferase assay system were purchased from Promega (Madison, WI). All materials for immunoblotting were purchased from Bio-Rad (Hercules, CA). All other chemicals were obtained from Sigma (St. Louis, MO). 2.2. Cell culture C6 glioma cell line was obtained from the American Type Culture Collection (Livingstone, MT), and cells were maintained in DMEM containing 10% FCS, 100 U/ml of penicillin G, and 100 μg/ml streptomycin in a humidified 37 °C incubator. 2.3. Immunoblot analysis Immunoblot analyses were performed as described previously[40]. Briefly, cells were lysed in extraction buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, 2 mM PMSF, 5 mM DTT, 0.5% NP-40, 0.05 mM pepstatin A, and 0.2 mM leupeptin. Samples of equal amounts of protein were subjected to SDS-PAGE and transferred onto a NC membrane which was then incubated in TBST buffer (150 mM NaCl, 20 mM Tris–HCl, and 0.02% Tween 20; pH 7.4) containing 5% non-fat milk. Proteins were visualized by specific primary antibodies and then incubated with alkaline phosphatase-conjugated secondary antibodies. Immunoreactivity was detected using NBT/BCIP following the manufacturer's instructions. Quantitative data were obtained using a computing densitometer with a scientific imaging system (Kodak, Rochester, NY). 2.4. Cell viability assay Cell viability was measured by the colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [47]. 2.5. Flow cytometric analysis Flow cytometric analyses were performed as described previously [40]. Briefly, cells were cultured in 6 cm dishes. After reaching confluence, cells were treated with vehicle, TSA or sirtinol for 24 or 48 h. Cells were then washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) and resuspended in ice-cold 70% ethanol at 0 °C overnight. Cells were washed for 5 min with 0.4 ml phosphate-citric acid buffer, pH 7.8, containing 50 mM Na2HPO4, 25 mM citric acid, and 0.1% Triton X-100 and subsequently stained with 0.5 ml of propidium iodide (PI) staining buffer containing 0.1% Triton X-100, 10 mM PIPES, 100 mM NaCl, 2 mM MgCl2, 100 μg/ml RNase A, and 50 μg/ml PI for 30 min in the dark before flow cytometric analysis. Cells were filtered on a nylon mesh filter. The samples were analyzed by the FACScantoII and FACSDiva program (BD Biosciences, San Jose, CA). Each experiment was repeated at least three times. Apoptotic cells were also detected by annexin V labeling. The labeling was performed at 37 °C by treating cells with annexin V (2 μg/ml) for 15 min. The staining was then immediately analyzed by the FACScantoII and FACSDiva program. 2.6. DNA fragmentation ELISA Cellular DNA fragmentation ELISA assay kit (Roche Diagnostics) was applied to measure apoptotic cell death by detection of 5′-Bromo2′-deoxy-uridine (BrdU)-labeled DNA fragments in cytoplasm of cell lysates, according to manufacturer's instructions.

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2.7. Transfection and luciferase reporter assays Cells were transfected with PG13-luc (Addgene plasmid 16642) or NF-κB-luc plus renilla-luc using Turbofect reagent (Upstate). Cells with or without treatments were then harvested, and the luciferase activity was determined using a Dual-Glo luciferase assay system kit (Promega), and was normalized on the basis of renilla luciferase activity. 2.8. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed following the instructions of Upstate Biotechnology. Briefly, cells were cross-linked with 1% formaldehyde at 37 °C for 10 min and then rinsed with ice-cold PBS twice. Cells were then harvested in 0.2 ml SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1, 1 mM PMSF, 0.05 mM pepstatin A, and 0.2 mM leupeptin) and sonicated five times for 15 s each, followed by centrifugation for 10 min. Supernatants were collected and diluted in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris–HCl, pH 8.1) followed by immunoclearing with 80 μl of protein A-agarose slurry for 1 h at 4 °C with gentle rotation, and an aliquot of each sample was used as “input” in the polymerase chain reaction (PCR) analysis. The remainder of the soluble chromatin was incubated at 4 °C overnight with SP1 and p53 antibodies (SantaCruz CA) or control IgG (SantaCruz). Immune complexes were collected by incubation with 60 μl of protein A-agarose slurry (Millipore) for 2 h at 4 °C with gentle rotation. The complexes were washed sequentially in the following three washing buffers for 5 min each: a low-salt immune complex washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl), a high-salt immune complex washing buffer (0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl), and a LiCl immune complex washing buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.1). Precipitates were washed two times with Tris-EDTA buffer. The complexes were eluted twice with two 100-μl aliquots of elution buffer (1% SDS, 0.1 M NaHCO3). The cross-linked chromatin complex was reversed in the presence of 0.2 M NaCl and heating at 65 °C for 4 h. DNA was purified using GFX™ DNA purification spin columns (GE Healthcare). PCR was carried out using Taq DNA polymerase for hot start (Takara), according to the manufacturer's protocol. Ten percentage of the total purified DNA were used for the PCR in 50 μl of reaction mixture. The 257-bp of survivin promoter fragment between −265 and − 9 was amplified by using a primer pair; sense: 5′-cac gcc cag cta att ttt gt-3′ and anti-sense: 5′-tca aat ctg gcg gtt aat gg-3′ in 35 cycles of PCR under the following condition; at 95 °C for 30 sec, at 56 °C for 30 sec and at 72 °C for 60 sec. The PCR products were analyzed by 1.5% agarose gel electrophoresis.

inhibitor) affects cell viability in C6 cells. As shown in Fig. 1A, treatment of C6 cells with TSA decreased cell viability in time- and dosedependent manners. TSA at 30, 100 or 300 nM significantly decreased cell viability by 7.4 ± 2.1%, 19.0 ± 6.9% and 30.3 ± 4.3% in cells exposure to TSA for 24 h, respectively (n = 4). TSA treatment for 48 h further decreased cell viability by 16.1 ± 2.6%, 45.2 ± 5.4% and 66.0 ± 2.5% at concentrations of 30, 100 or 300 nM (n = 4) (Fig. 1A). Sirtinol, a class III HDAC inhibitor, did not significantly affect cell viability after 24 h treatment. However, sirtinol at 1, 3 or 10 μM decreased cell numbers by 3.1 ± 5.1%, 19.2 ± 9.9% and 25.8 ± 6.9% in cells exposed to sirtinol for 48 h, respectively (n = 4) (Fig. 1B). 3.2. TSA, but not sirtinol, induced C6 cell apoptosis To elucidate whether TSA or sirtinol induces apoptosis, flowcytometric analysis was then used. As shown in Fig. 2A, the percentage of PI-stained cells in the apoptotic region (Apo, sub-G0/G1 peak) was significantly increased after TSA treatment for 24 h (Fig. 2Ab–d) or 48 h (Fig. 2Af–h) compared with the vehicle-treated group (Fig. 2Aa and Fig. 2Ae). The compiled data are shown in the bottom in Fig. 2A. TSA at concentrations of 30, 100 or 300 nM significantly induced cell apoptosis by 23.3 ± 4.6%, 25.1 ± 3.5%, and 22.6 ± 3.5% after TSA exposure for 24 h, respectively (n = 4) (Fig. 2Ab–d). Treatment of TSA for 48 h further increased cell apoptosis by 35.7 ± 5.7%, 43.1 ± 7.3%, and 51.6 ± 3.7% at the concentrations of 30, 100 or 300 nM (n = 3) (Fig. 2Af–h). It appears that cells treated with vehicle for 24 or 48 h show no difference (Fig. 2Aa: 7.7 ± 1.1% and Fig. 2Ae: 9.4 ± 1.3%). However, sirtinol did not significantly increase the percentage of PIstained cells in the apoptotic region after sirtinol exposure for even

2.9. Statistical analysis Results are presented as the mean± S.E. from at least three independent experiments. One-way analysis of variance (ANOVA) followed by, when appropriate, the Newman–Keuls test was used to determine the statistical significance of the difference between means. A p value of b 0.05 was considered statistically significant. 3. Results 3.1. TSA decreased cell viability Eighteen mammalian HDACs have been identified and are grouped into four classes [48]. The class II HDACs can be further subdivided into class IIa and class IIb, based on the presence in class IIa members of extended C terminal tails that are essential in regulating their function [49]. We first determined whether TSA (a class I and II HDAC

Fig. 1. Effects of TSA and sirtinol on cell viability in C6 cells. A, Cells were treated with vehicle or TSA at indicated concentrations for 24 or 48 h, and cell viability was then determined by the MTT assay. Each column represents the mean ± S.E.M. of four independent experiments performed in triplicate. *p b 0.05, compared with the control group. B, Cells were treated with vehicle or sirtinol at indicated concentrations for 24 or 48 h, and cell viability was then determined by the MTT assay. Each column represents the mean ± S.E.M. of at least three independent experiments performed in triplicate. *p b 0.05, compared with the control group.

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Fig. 2. TSA-induced C6 cell apoptosis. A, Cells were treated with vehicle or TSA at indicated concentrations for 24 or 48 h. After treatment, the percentage of apoptotic cells was then analyzed by flow cytometric analysis of PI-stained cells as described in Materials and methods. Compiled results are shown at the bottom. Each column represents the mean ± SEM of at least three independent experiments. *p b 0.05, compared with the control group. B, Cells were treated with vehicle or sirtinol at indicated concentrations for 24 or 48 h. After treatment, the percentage of apoptotic cells was then analyzed by flow cytometric analysis of PI-stained cells as described in Materials and methods. Each column represents the mean ± S.E.M. of six independent experiments. C, Cells were treated with vehicle or TSA at indicated concentrations for 48 h. DNA fragmentation was then determined using Cellular DNA fragmentation ELISA assay kit (Roche Diagnostics) as described in Materials and methods. Each column represents the mean ± SEM of three independent experiments. *p b 0.05, compared with the control group. D, Cells were treated with vehicle or TSA at indicated concentrations for 48 h. The percentage of annexin V-stained cells was then analyzed by flow cytometric analysis as described in Materials and methods. Results shown are representative of four independent experiments.

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48 h, respectively (n = 6) (Fig. 2B). To investigate the apoptotic mechanisms elicited by HDAC inhibition, we thus used TSA in the following experiments. A hallmark of apoptosis is the degradation of DNA into a specific fragmentation pattern. BrdU was thus used to label DNA fragments in elucidating whether TSA induces DNA fragmentation. As shown in Fig. 2C, BrdU-labeled DNA fragments were significantly increased in samples from cells exposure to TSA (30–300 nM) while vehicle was without effect. As compared to the vehicle-treated group, treatment of TSA for 48 h significantly increased DNA fragmentation by 44.2 ± 9.5%, 103.8 ± 5.9%, and 142.9 ± 17.8% at the concentrations of 30, 100 or 300 nM, respectively (n = 3) (Fig. 2C). Loss of membrane asymmetry is another hallmark of apoptosis. In healthy cells, the distribution of the phosphatidylserine groups in the plasma membrane is

asymmetrical such that the groups are directed toward the inside of the cell. During apoptosis, the phosphatidylserine groups are exposed to the exterior of the cell membrane. Annexin V was thus used to detect phosphatidylserine residues, which are exposed on the cell surface of apoptotic cells. As shown in Fig. 2D, annexin V-labeled cells were significantly increased after exposure to TSA (30–300 nM) while vehicle was without effect. These results suggested that TSA induced cell death by apoptosis in C6 cells. 3.3. p53 activation in TSA-induced cell death p53 is a transcription factor that plays a key role in the regulation of cell apoptosis. We then explored the role of p53 in TSA-induced death signaling. p53 modified by acetylation on its lysine residues has been shown to contribute to HDAC inhibition-induced p53 activation [41,42,50]. We examined the levels of acetylated proteins elicited by TSA in C6 cells. The level of the lysine acetylated protein with molecular weights about 55 kDa was increased from 10 min to 120 min after TSA (100 nM) treatment and lasted for at least 120 min when probed with anti-acetylated lysine antibody (Cell Signaling) (Fig. 3A). To confirm whether TSA induced p53 acetylation, anti-acetylated p53 antibody was used. As shown in Fig. 3B, TSA significantly induced p53 acetylation in a time-dependent manner. In addition, phosphorylation of serine residues, primarily Ser15, also leads to p53 activation [39,40]. We thus examined the effect of TSA on p53 phosphorylation at Ser15. TSA caused an increase in p53 phosphorylation at Ser15 in a timedependent manner. Phosphorylation began at 10 min, peaked at 1 h and lasted for at least 2 h after TSA treatment (Fig. 3C). To determine whether p53 transactivity is increased in cells exposed to TSA, a reporter construct containing a p53 DNA-binding site upstream of a basal promoter linked to a luciferase reporter gene (PGl3-Luc) [46] was used. As shown in Fig. 3D, cells treated with TSA (100 nM) for 24 h caused a significant increase in PG13-luciferase activity by 2.4 ± 0.6 folds. These results suggested that TSA may activate p53 in C6 cells. 3.4. Bax upregulation and survivin downregulation in TSA-induced C6 cell apoptosis p53 has been shown to induce apoptosis by causing mitochondrial dysfunction via transactivation of Bax [37,38]. We, therefore, examined whether TSA is capable of inducing Bax expression in C6 cells. As shown in Fig. 4A, TSA elevated cellular level of Bax in a dose-dependent manner. Treatment of TSA for 48 h significantly induced Bax expression by 1.4 ± 0.1 folds, 2.0 ± 0.3 folds, and 2.6 ± 0.6 folds at the concentrations of 30, 100 or 300 nM, respectively (n = 5) (Fig. 4A). In addition, the protein levels of anti-apoptotic Bcl-2 family members, Bcl-2 and Bcl-xl, were decreased in cells exposed to TSA. Treatment of TSA for 48 h decreased Bcl-2 level by 36.7 ± 4.0%, 25.4 ± 7.9%, and 37.1 ± 11.4% at the Fig. 3. TSA-induced p53 activation in C6 cells. A, Cells were treated with vehicle or 100 nM TSA for various time intervals as indicated. Cell lysates were then prepared and subjected to immunoblotting with anti-acetyl-lysine antibody. Typical bands representative of four separate experiments with similar results are shown. B, Cells were treated with vehicle or 100 nM TSA for various time intervals as indicated. Cell lysates were then prepared and subjected to immunoblotting with anti-acetyl-p53 antibody. Equal loading in each lane is reflected by similar intensities of p53 at the bottom. Compiled results are shown at the bottom. Each column represents the mean ± S.E.M. of five independent experiments. *p b 0.05 compared with the control group. C, Cells were treated with vehicle or 100 nM TSA for various time intervals as indicated. Cell lysates were then prepared and subjected to immunoblotting with anti-pSer15–p53 antibody. Equal loading in each lane is reflected by similar intensities of p53 at the bottom. Compiled results are shown at the bottom. Each column represents the mean ± S.E.M. of five independent experiments. *p b 0.05 compared with the control group. D, Cells were transiently transfected with PG13-luc with p53 binding sites (p53-luc, Addgene plasmid 16642) and renilla-luc for 24 h and treated with 100 nM TSA for another 24 h. PG13luciferase assay was then determined as described in Materials and methods. Data represent the mean ± S.E.M. of five independent experiments performed in duplicate. *p b 0.05 as compared with the control group.

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Fig. 4. Effects of TSA on Bax and survivin levels in C6 cells. Cells were treated with vehicle or TSA at indicated concentrations for 48 h. Protein levels of Bax (A), Bcl-2 (B), Bcl-xl (C) or survivin (D) were then determined by immunoblotting. Equal loading in each lane is reflected by similar intensities of α-tubulin at the bottom. Compiled results are shown at the bottom. Each column represents the mean ± S.E.M. of at least three independent experiments. *p b 0.05 compared with the control group. E, Cells were incubated with 100 nM TSA for 2 h and ChIP assay was performed as described in Materials and methods. Typical traces representative of four independent experiments with similar results are shown.

concentrations of 30, 100 or 300 nM, respectively (n = 4) (Fig. 4B). Bclxl level was decreased by 18.3 ± 5.4%, 35.4 ± 8.2%, and 38.3 ± 13.4% in cells exposed to 30, 100 or 300 nM TSA, respectively (n = 5) (Fig. 4C). We also examined whether TSA affects survivin expression, since p53 was also shown to suppress the expression of survivin, which was reported to play a crucial role in regulating apoptosis [23,24]. Results from Fig.4D demonstrated that TSA dose-dependently suppressed survivin expression. Treatment of TSA for 48 h significantly suppressed survivin expression by 51.5 ± 14.2%, 71.4 ± 15.3%, and 86.5 ± 4.6% at the concentrations of 30, 100 or 300 nM, respectively (n = 3). Furthermore, several lines of evidence demonstrated that activation of SP1 may leads to the induction of survivin whereas p53 may counteract the binding of SP1, thereby suppressing survivin expression [22–24]. To determine whether SP1 or p53 is recruited to the endogenous survivin promoter region in response to TSA signaling, we performed ChIP experiments in C6 cells stimulated with TSA. We used primers encompassing the

survivin promoter region between −265 and −9, which contains putative SP1 and p53 binding sites. As shown in Fig. 4E, p53 binding to the survivin promoter region (−265/−9) was readily increased after TSA exposure. On the other hand, SP1 binding to the survivin promoter region (−265/−9) was decreased after TSA exposure. In addition, the survivin promoter region (−265/−9) was detected in the cross-linked chromatin sample before immunoprecipitation (Fig. 4E, Input, positive control). These results therefore established that TSA induces the recruitment of p53 and p53 may counteract the binding of SP1 to the promoter region of the endogenous survivin gene leading to survivin downregulation. 3.5. TSA induced p38MAPK and AMP-activated protein kinase (AMPK) activation in C6 cells We next explored the signaling molecules that may contribute to TSA-induced p53 activation. We have previously demonstrated that

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Fig. 5. Effects of TSA on p38MAPK, AMPK and IKK phosphorylation in C6 cells. Cells were treated with 100 nM TSA for various time periods as indicated. The phosphorylation status of p38MAPK (A), AMPK (B) and IKK (C) were then determined by immunoblotting with anti-pThr180/Tyr182–p38MAPK, anti-pThr172–AMPK or anti-pSer180/Ser181–IKK antibody. Equal loading in each lane is reflected by similar intensities of α-tubulin at the bottom. Compiled results are shown at the bottom. Each column represents the mean ± S.E.M. of at least five independent experiments. *p b 0.05 compared with the control group. D, Cells were transiently transfected with κB-luc and renilla-luc for 24 h and treated with 100 nM TSA for another 24 h. κB-luciferase assay was then determined as described in Materials and methods. Data represent the mean ± S.E.M. of three independent experiments performed in duplicate. *p b 0.05 as compared with the control group.

p38MAPK signaling was responsible for p53 Ser15 phosphorylation in cerebral endothelial cells [40]. We thus examined whether the extent of p38MAPK phosphorylation is altered in C6 cells after TSA exposure. As shown in Fig. 5A, TSA caused an increase in p38MAPK phosphorylation in a time-dependent manner. Phosphorylation began at 20 min, and lasted at least 60 min after TSA treatment (Fig. 5A). AMP-activated protein kinase (AMPK) was recently shown to be causally related p38MAPK activation and subsequent apoptosis [51,52]. In addition, Maclaine et al. [53] demonstrated that AMPK was one of the upstream protein kinases that phosphorylate p53. We next explored whether TSA activated AMPK in C6 cells. The time-course of TSA-induced AMPK phosphorylation is shown in Fig. 5B. TSA increased AMPK phosphorylation in a time-dependent manner. Dephosphorylation of IKKs including IKKα and IKKβ has been shown to switch CBP binding preference from NF-κB to p53, resulting in an increase of p53 acetylation [44,45]. We thus determined the effect of TSA on IKK signaling. TSA caused IKKα/β dephosphorylation in a time-dependent manner (Fig. 5C). This TSA action was also accompanied by the decrease of NF-κB reporter activity, which plays a critical role downstream of IKK. Treatment of 100 nM TSA for 24 h significantly suppressed NF-κB reporter activity by 68.9± 2.9%, respectively (n = 3) (Fig. 5D). 3.6. p38MAPK inhibition attenuated TSA-induced p53 activation, Bax, Bcl-2 and Bcl-xl modulation and cell death in C6 cells We next determined whether p38MAPK or AMPK contributes to TSA-induced C6 cell death. As shown in Fig. 6A, the specific p38MAPK inhibitor, p38 inhibitor III (0.3–3 μM), significantly restored TSA-

decreased cell viability. However, the specific AMPK inhibitor, compound C, did not alter cell viability in cells exposed to TSA (Fig. 6A). Pretreatment of cells with p38 inhibitor III (0.3–3 μM) also inhibited TSA-induced Bax expression (Fig. 6B) and restored TSAdecreased Bcl-2 (Fig. 6C) and Bcl-xl levels in C6 cells (Fig.6D). In addition, the protein levels of Bax, Bcl-2 and Bcl-xl were not altered by the presence of compound C (Fig. 6B–D). To ascertain the linkage between p38MAPK signaling and p53 activation downstream of TSA, PG13-luc report construct was employed. As shown in Fig. 6E, the TSA-induced increase in PG13-luciferase activity was markedly attenuated in cells pretreated for 30 min with 3 μM p38 inhibitor III by 22.3 ± 5.5%, respectively (n = 5). Based on these results, we suggest that TSA activation of p38MAPK occurs upstream of p53 activation, Bax, Bcl-2 and Bcl-xl modulation and subsequent cell death. 4. Discussion Gliomas are the most common primary brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. In the development of strategies for cancer therapies, significant advances have been achieved in clinical trials including the use of HDAC inhibitors in cancer therapy. However, the molecular mechanism underlying HDAC inhibition-induced cell death is not yet well understood. Results from the present study, similar to those reported recently [54,55], show that HDAC inhibition by TSA induced cell death in glioma cells. We also demonstrated that the activation of the p38MAPK signaling cascades followed by p53 activation and subsequent Bax expression and survivin

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Fig. 6. Effects of compound C and p38 inhibitor III on TSA actions on cell viability, Bax, Bcl-2 and Bcl-xl modulation, and p53 activation in C6 cells. A, Cells were pretreated for 30 min with vehicle, compound C (1–3 μM) or p38 inhibitor III (1–3 μM), followed by treatment with 100 nM TSA for another 48 h. Cell viability was then determined by an MTT assay. Each column represents the mean ± S.E.M. of six independent experiments performed in triplicate. *p b 0.05, compared with the control group. #p b 0.05, compared with the group treated with TSA alone. Cells were pretreated for 30 min with vehicle, compound C (1–3 μM) or p38 inhibitor III (1–3 μM), followed by treatment with 300 nM TSA for another 48 h. Protein levels of Bax (B), Bcl-2 (C), Bcl-xl (D) were then determined by immunoblotting. Typical bands representative of three separate experiments with similar results are shown. E, Cells were transiently transfected with PG13-luc and renilla-luc for 24 h. Cells were then treated for 30 min with vehicle or p38 inhibitor III (1–3 μM), followed by treatment with 100 nM TSA for another 24 h. PG13-luciferase assay was then determined as described in Materials and methods. Data represent the mean ± S.E.M. of five independent experiments performed in duplicate. *p b 0.05, compared with the control group.

downregulation contributes to TSA-induced C6 glioma cell death. Negative regulation of IKK pathway by TSA may also participate in TSA apoptotic action. HDAC inhibition-induced apoptosis may involve alteration of members of Bcl-2 protein family, mitochondrial dysfunction and caspases activation. A number of studies have indicated that p53 plays an important role in promoting cell apoptosis by regulating the transcription of apoptosis-related genes. The expression of Bax, a proapoptotic Bcl-2 member, is induced by p53 [56] while survivin is suppressed [22,28] in response to selected stress signals. We noted in the present study that TSA elevated Bax expression and suppressed survivin expression in C6 cells. The results from CHIP assay indicated that p53 may counteract the binding of SP1 to the promoter region of the endogenous survivin gene in cells exposure to TSA. These observations suggested that p53 may be causally related to TSA-induced alterations of Bax and survivin expression, which led to C6 cell apoptosis. In the present study, we also found that TSA caused a decrease in the protein levels of Bcl-2 and Bcl-xl, two anti-apoptotic Bcl-2 members. The precise mechanism involved in TSA-decreased Bcl-2 and Bcl-xl levels in C6 cells remains to be established. Activation of p38MAPK may contribute, at least in part, to TSA actions, since p38MAPK signaling blockade by p38 inhibitor III restored TSA-decreased Bcl-2 and Bcl-xl levels. The control of survivin protein expression may also occur at several levels in addition to transcription. Hu et al. [57] recently demonstrated that survivin protein degradation may occur through post-translational substrate-dependent process. In addition, several studies suggested that HDAC inhibitors including TSA may regulate apoptosis by

decreasing the half-life of several oncogenic proteins through activating the proteasomal degradation pathway [58,59]. It raises the possibility that TSA may activate certain signaling cascades to decrease survivin protein expression by not only transcriptional, but also post-transcriptional or post-translational mechanisms. Furthermore, TSA was shown recently to induce cell cycle arrest by transcriptional modulation of survivin in HeLa cells [60]. In addition to survivin, TSA was also shown to modulate the expressions of p21Cip1/Waf1 and cyclin D1, two key regulator genes of the cell cycle in c6 cells (our unpublished data). These observations suggested that TSA may not only induced apoptosis, but also cell cycle arrest in C6 cells. Accumulating evidence has indicated that post-translational modifications of p53, including sumoylation, acetylation and phosphorylation could modulate p53 activity [42]. p53 acetylation is regulated by the opposing activities of histone acetyltransferases (p300/CBP) and HDACs [41–43]. Recent studies have indicated that IKK-mediated phosphorylation may switch the protein binding preference of CBP from p53 to NFκB resulting in concurrent upregulation of NF-κB-mediated genes and downregulation of p53-mediated genes [44,45]. We noted that TSA caused an increase in p53 acetylation in C6 cells. IKK dephosphorylation and subsequent NF-κB downregulation was also shown in TSA-treated cells. Together, these observations suggest that TSA may induce p53 acetylation by inhibiting HDACs directly or by inactivating IKK signaling indirectly. Whether TSA altered the status of p53 phosphorylation has not been previously demonstrated. We show in the present study that treatment of C6 cells with TSA caused p53 phosphorylation at Ser 15. p53 phosphorylation by p38MAPK contributed to several apoptotic

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via IKK inactivation (Fig. 7). The present study delineates, in part, the signaling pathways involved in TSA-induced cell death. Conflict of interest statement None. Acknowledgments We would like to thank Dr. Bert Vogelstein for the kind gift of the PG13-luc construct (Addgene plasmid 16442). This work was supported by grant (NSC 98-2320-B-038-007-MY3) from the National Science Council of Taiwan and grants (SKH-TMU98–01 and SKH-8302-98-DR-18) from the Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. References

Fig. 7. Schematic summary of the signaling pathway involved in TSA-induced C6 glioma cell apoptosis.

signaling pathways [40,61]. In agreement with these observations, we noted that TSA significantly induced p38MAPK phosphorylation and that P38 inhibitor III significantly inhibited TSA-increased PG13luciferase activity and subsequent cell death. Thus, it is plausible that TSA may activate the p38MAPK cascade to cause p53 activation and subsequent cell death in C6 cells. The differential mechanisms of TSA actions in driving these two separate signaling pathways leading to p53 acetylation and phosphorylation remain to be elucidated. It is likely that two different pathways culminating in p53 activation, respectively, are in cooperation. Additional works are needed to characterize the interrelationship between p38MAPK and IKK signaling cascade in TSAinduced C6 cell death. Moreover, we show in the present study that TSA caused AMPK activation whereas AMPK inhibition by compound C did not altered TSA actions on Bcl-2 family members and subsequent cell death. Several lines of evidence demonstrated that AMPK plays a critical role in cell autophagic death [62]. Whether TSA activation of AMPK leads to autophagy and what the precise mechanism is that causes TSAinduced AMPK activation in C6 cells need further investigation. In conclusion, the present study shows that TSA may activate the p38MAPK–p53 signaling pathway to cause Bax expression and survivin repression in C6 glioma cells. TSA-induced p53 activation may occur through p53 modification by phosphorylation or by acetylation

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