p53 in trichostatin A induced C6 glioma cell death

https://doi.org/10.1016/j.bbagen.2011.02.006Get rights and content

Abstract

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.

Research Highlights

► Trichostatin A causes glioma cell apoptosis through activating p38MAPK–p53 cascade ► Trichostatin A increases p53 phosphorylation and acetylation ► Trichostatin A increases Bax, but decreases Bcl-2, Bcl-xl and survivin levels

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 re-expression of silenced regulatory genes [1], [3], [4], [5]. Importantly, HDACs deacetylates not only histones but also nonhistone substrates, which 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], [9], [10], [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 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], [19], [20]. Over-expressed 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], [23], [24], [25], [26], [27], [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], [23], [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], [30], [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], [37], [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], [42], [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.

Section snippets

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

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 inhibitor) affects cell viability in C6 cells. As shown in Fig. 1A, treatment of C6 cells with TSA decreased cell viability in time- and dose-dependent

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,

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-TMU-98–01 and SKH-8302-98-DR-18) from the Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.

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