Autophagy induced by suberoylanilide hydroxamic acid in Hela S3 cells involves inhibition of protein kinase B and up-regulation of Beclin 1

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Abstract

Histone deacetylase inhibitors are promising chemotherapeutic agents and some are in clinical trials. Several molecular mechanisms have been invoked to describe their effects on cancer cells in vivo and in vitro. Autophagy has been observed in response to several anticancer reagents and has been demonstrated to be responsible for cell death. However, the exact mechanism of this phenomenon is still not clear. Here we demonstrated that suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, induces nonapoptotic cell death with several specific features characteristic of autophagy in Hela S3 cells. Suberoylanilide hydroxamic acid inhibits the activity of the mammalian target of rapamycin, a negative regulator of macroautophagy which induces the formation of autophagosomes in a Beclin 1- and autophagy-related 7-dependent manner. This process is mediated by Akt and tuberous sclerosis 2 as is demonstrated by inhibition by continuous active Akt plasmid transfection and RNA interference of tuberous sclerosis 2. Our data provide the first evidence that suberoylanilide hydroxamic acid induces autophagy in Hela S3 cells through interference with the mammalian target of rapamycin signaling pathway. These findings suggest that suberoylanilide hydroxamic acid may induce autophagic cancer cell death via its specific pathway, and invite further investigation into the detailed mechanism of this pathway to explore this compound's full potential as a chemotherapeutic agent.

Introduction

Autophagy is a process characterized by the appearance of double- or multi-membrane vesicles which engulf cytoplasm and organelles such as mitochondria or endoplasmic reticulum, with subsequent degradation by fusion with the cell's own lysosomal system (Gozuacik & Kimchi, 2004). It serves as a cell survival mechanism in starving cells through the degradation of intracytoplasmic cellular components (Baehrecke, 2005). Autophagy is also implicated in certain human diseases, such as cancer (Liang et al., 1999), neurodegenerative disease (Yuan, Lipinski, & Degterev, 2003) and myopathies (Nishino et al., 2000). Clarke suggested that autophagic (or type 2) programmed cell death (PCD) is a cell death pathway distinct from apoptotic (type 1) PCD (Clarke, 1990).

Although the presence of abundant autophagic vacuoles in dying cells of multiple organisms suggests that autophagy plays a causative role in cell death, the role of autophagy in cancer is controversial (Hippert, O’Toole, & Thorburn, 2006; Takeuchi et al., 2005). It is unclear whether autophagy directly brings about cell death. Recent works have proven that, at least in vitro, autophagy may cause cell death (Shimizu et al., 2004, Yu et al., 2004). With the discovery of the homology of yeast ATG in mammalians, recent progress in the characterization of the molecular mechanism controlling autophagy has brought a renewed interest in this process.

Histone deacetylase (HDAC) inhibitors specifically affect tumor cells while leaving untransformed cells relatively unscathed, and these inhibitors show promise as chemotherapeutic agents (Johnstone, 2002; Marks, Richon, Miller, & Kelly, 2004). Some of them are under clinical trial phase 1-2. HDAC inhibitors have been categorized into several groups by their chemical structures including: (i) short-chain fatty acids such as butyrates; (ii) hydroxamic acids such as trichostatin A (TSA), SAHA and oxamflatin; (iii) cyclic tetrapeptides containing a 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) moiety such as trapoxin A; (iv) cyclic peptides not containing the AOE moiety, such as apicidin; and (v) benzamides (Marks, Richon, & Rifkind, 2000). The cytotoxic effects of HDAC inhibitors are associated with activation of differentiation programs, inhibition of cell cycle, induction of apoptosis and/or production of reactive oxygen species (ROS) (Johnstone, 2002, Li et al., 2005; Munro, Barr, Ireland, Morrison, & Parkinson, 2004; Place, Noonan, & Giardina, 2005; Rebbaa, Zheng, Chu, & Mirkin, 2006; Ruefli et al., 2001). In addition to these mechanisms, a recent study showed that SAHA, a HDAC inhibitor, could induce caspase-independent cell death in Hela cells and cell death could not be prevented by Bcl-xl overexpression or Apaf-1 knock out. Therefore, it was suggested that autophagic cell death was responsible for this event (Shao, Gao, Marks, & Jiang, 2004).

The molecular pathways through which autophagy is regulated in cancer cells are not very clear. How SAHA may initiate this process is unknown. In mammalian cells distinct classes of phosphoinositide 3-kinase (PI3K) control the macroautophagic pathway with opposite effects (Petiot, Ogier-Denis, Blommaart, Meijer, & Codogno, 2000). The class III PI3K complex, including Beclin 1, which is homologous with yeast Atg6, plays a stimulatory role in autophagy (Baehrecke, 2005). As shown in Fig. 1, autophagy is also regulated by a kinase cascade consisting of class I PI3K, PI3K-dependent kinases (PDK) 1, protein kinase B (Akt/PKB), tuberous sclerosis (TSC) 1/TSC2 and mTOR, the mammalian target of rapamycin (Shintani & Klionsky, 2004). The class I PI3K, activated by insulin receptor and insulin receptor substrate protein, converts PtdIns(4,5)P2 to PtdIns(3,4,5)P3, which then activate Akt by PDK1. Disruption of the PI3K/Akt signaling pathway, culminating in inhibition of Akt, which is a central regulator of cell survival, has been found to be associated with autophagy induced by a variety of agents in cancer cells. Activated Akt has been linked to signaling pathways involved in aberrant cell growth and differentiation regulation characteristic of carcinogenesis, as well as to resistance to cancer chemotherapy making it an attractive target in drug discovery strategies. Thus, understanding the role of SAHA in modulating this key pathway of cell survival is important.

One proposed downstream effector of Akt involved in the regulation of autophagy is mTOR kinase, which inhibits autophagy by phosphorylating downstream substrate possibly analogous to Atg1 or other ATG gene products (as demonstrated in yeast) (Shintani & Klionsky, 2004). Akt contributes to the positive regulation of mTOR activity through an inhibitory effect of TSC-2, a GTPase activating protein that reduces the activity of Rheb, an activator of mTOR (Fig. 1).

The purpose of this study was to examine in Hela S3 cells whether SAHA induces autophagy, and if so, to identify the signaling pathway involved. Our data show that SAHA indeed caused autophagy in Hela S3 cells through class III PI3K/Beclin 1 and the PI3K-Akt-mTOR signaling pathway. This autophagy was inhibited by RNAi of Beclin 1, TSC2 and ATG7, and ATG7 has been identified as a key autophagy gene required for autophagic vacuoles formation in yeast and mammals (Kim, Dalton, Eggerton, Scott, & Klionsky, 1999; Komatsu et al., 2005). Together, these data unveil activation of autophagy induced by SAHA in Hela S3 cells and the underlying autophagy-regulating signaling pathway.

Section snippets

Reagents and antibodies

SAHA and Z-VAD-FMK were purchased from ALEXIS, Inc. (Lausen, Switzerland). Z-IETD-FMK and Z-LEHD-FMK were from BioVision, Inc. (CA, USA). Acridine orange was from Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were from Gibico BRL (Grand Island, NY, USA). Anti-Beclin 1, anti-tuberin (C-20) and anti-actin antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-p70 S6K, anti-phospho p70 S6K (Thr-389), anti-mTOR, anti-phospho

SAHA induces caspase-independent cell death in Hela S3 cells

Hela S3 cells were treated with 5 μM SAHA, an effective concentration for HDAC inhibition, for different lengths of time. As shown in Fig. 2A, SAHA induced time-dependent cell death.

Apoptosis is an important PCD pathway and it is mediated by caspases. To investigate whether SAHA-induced cell death was caspase-dependent, we treated Hela S3 cells with 5 μM SAHA for 72 h n the presence or absence of several caspase inhibitors, including Z-Val-Ala-DL-Asp-fluoromethyl ketone (Z-VAD-FMK), a potent

Discussion

HDAC inhibitors are promising chemotherapeutic agents, because of their significant anticancer activity and few side effects (Marks et al., 2004). SAHA is a hydroxamic acid HDAC inhibitor with high potency that is currently under evaluation in clinical trials. Previous studies showed that HDAC inhibitor-induced cell death was accompanied by activation of caspases, which are mediators of apoptosis (Pyo et al., 2005; Weidle & Grossmann, 2000). However, both a previous report (Shao et al., 2004)

Acknowledgements

We are very grateful to Dr. Tamotsu Yoshimori (National Institute of Genetics, Mishima, Japan) for the GFP-LC3 plasmid and LC3 antibody, Dr. R. Freeman (University of Rochester, Rochester, NY, USA) for the cAkt plasmid and Dr. William Dunn (College of Medicine, University of Florida, FL, USA) for the Atg7 antibody. This study was supported partially by The LiFu Educational Foundation.

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