valproic acid, an antiepileptic drug with histone ... · valproic acid, an antiepileptic drug with...

Valproic Acid, an Antiepileptic Drug with Histone DeacetylaseInhibitory Activity, Potentiates the Cytotoxic Effectof Apo2L/TRAIL on Cultured Thoracic CancerCells through Mitochondria-DependentCaspase Activation1

M. Firdos Ziauddin2, Wen-Shuz Yeow2, Justin B. Maxhimer, Aris Baras, Alex Chua, Rishindra M. Reddy,Wilson Tsai, George W. Cole Jr., David S. Schrump and Dao M. Nguyen

Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National Cancer Institute,National Institutes of Health, Bethesda, MD, USA

Abstract

Inhibitors of histone deacetylases have been shown

to enhance the sensitivity of cancer cells to tumor

necrosis factor–related apoptosis-inducing ligand

TRAIL-mediated cytotoxicity. Valproic acid (VA), a

commonly used antiepileptic agent whose pharmaco-

kinetics and toxicity profiles are well described, is a

histone deacetylase inhibitor. This project aims to

evaluate if VA can potentiate Apo2L/TRAIL–mediated

cytotoxicity in cultured thoracic cancer cells and to elu-

cidate the underlying molecular mechanism respon-

sible for this effect. VA sensitized cultured thoracic

cancer cells to Apo2L/TRAIL, as indicated by a 4-fold

to a >20-fold reduction of Apo2L/TRAIL IC50 values

in combination-treated cells. Although VA (0.5–5 mM)

or Apo2L/TRAIL (20 ng/ml) induced less than 20% cell

death, VA + Apo2L/TRAIL combinations caused 60%

to 90% apoptosis of cancer cells. Moreover, substantial

activation of caspases 8, 9, and 3, which was observed

only in cells treated with the drug combinations, was

completely suppressed by Bcl2 overexpression or by

the caspase 9 inhibitor. Both the caspase 9 inhibitor

and Bcl2 completely abrogated the substantial cyto-

toxicity and apoptosis induced by this combination,

thus highlighting the pivotal role of the type II path-

way in this process. These findings provide a rationale

for the development of VA and Apo2L/TRAIL combina-

tion as a novel molecular therapeutic regimen for tho-

racic cancers.

Neoplasia (2006) 8, 446–457

Keywords: Histone deacetylase inhibitor, Apo2L/TRAIL, lung cancer,esophageal cancer, malignant pleural mesothelioma, mitochondria, Bcl2,caspases.

Introduction

Tumor necrosis factor–related apoptosis-inducing ligand

(TRAIL) is expressed by natural killer cells, T cells, neutro-

phils and monocytes in responses to interferons and play

physiologic roles of tumor surveillance and immune regulation

[1,2]. The extracellular domain of membrane-bound TRAIL can

be proteolytically cleaved from the cell surface to generate

soluble TRAIL. The soluble TRAIL extracellular domains homo-

trimerize through an internal zinc atom bound to the cysteine

residue at position 230 of each subunit, and this process is

crucial for trimer stability and biologic activity [3,4]. The receptor

repertoire of TRAIL is complex and consists of five members:

two functional death receptors (DR4, DR5) that contain a

conserved intracellular death domain motif; two decoy recep-

tors (DcR1, DcR2) that can bind TRAIL but lack the functional

intracellular death domain and are thus incapable of transduc-

ing a death signal; and the fifth is a soluble protein [osteopro-

tegerin (OPG)] that binds TRAIL but at a very low affinity at

physiologic condition [4]. Each homotrimerized TRAILmolecule

engages three receptors, each at the interface of its two sub-

units. Themolecular events downstreamof DR4/DR5 activation

by TRAIL are well described [4–6]. Binding of homotrimerized

TRAIL to its appropriate receptors DR4 and/or DR5 results in

receptor aggregation, recruitment of FADD, FLIP, and procas-

pases 8/10 to form the death-inducing signaling complex

(DISC) leading to caspase 8/10 activation (by autocatalytic

cleavage, the induced proximity hypothesis [7], or by homodi-

merization of the procaspases, the unified model [8]) [9]. The

DISC-activated caspase 8 then activates caspases 3, 6, and 7

by proteolytic cleavages of respective procaspases leading to

induction of apoptosis through a signaling cascade known as

the extrinsic pathway, in contrast to the mitochondria-mediated

Abbreviations: TRAIL, TNF-related apoptosis inducing ligand; NSCLC, non – small cell lung

cancer; EsC, esophageal cancer; MPM, malignant pleural mesothelioma; HDACI, histone

deacetylase inhibitor; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end

labeling; MTT, (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide; VA, valproic acid;

SMAC/DIABLO, second mitochondria-derived activator; GFP, green fluorescence protein

Address all correspondence to: DaoM.Nguyen,Clinical ResearchCenter, National Institutes of

Health, Room4W-4-3940, 10 Center Drive, Bethesda,MD 20892. E-mail: [email protected] research was supported by the Intramural Research Program of the National Cancer

Institute, National Institutes of Health, Bethesda, MD.2M. F. Ziauddin and W.-S. Yeow contributed equally to this work.

Received 12 December 2005; Revised 28 March 2006; Accepted 29 March 2006.

Copyright D 2006 Neoplasia Press, Inc. All rights reserved 1522-8002/06/$25.00

DOI 10.1593/neo.05823

Neoplasia . Vol. 8, No. 6, June 2006, pp. 446 – 457 446

www.neoplasia.com

RESEARCH ARTICLE

intrinsic death signaling pathway activated following cyto-

toxic stresses [10,11]. Moreover, proteolytically active cas-

pase 8 can also process Bcl2-interacting domain (BID) to

yield truncated BID (tBID), which translocates to the mito-

chondria and, through interaction with Bak and Bax [12–14]

on the mitochondria outer membrane, mediates the release

of multiple proapoptotic proteins including cytochrome c,

apoptosis-inducing factor, and second mitochondria-derived

activator (SMAC/DIABLO), thus linking the extrinsic to the

intrinsic death signaling pathways [10,11,15,16]. Cyto-

chrome c, together with cytosolic apoptotic protease activat-

ing factor 1, procaspase 9, and ATP, forms the apoptosome

that activates caspase 9. Activated caspase 9 processes

caspases 3/7, which proteolytically cleave and activate cas-

pase 8, thus forming a positive amplification feedback loop

to further activate the apical caspase 8 [1]. Death ligand–

mediated induction of apoptosis is further classified into type I

or type II depending on the involvement of the intrinsic

(mitochondria mediated) death signaling cascade in the

effective execution of apoptosis. Type I cells undergo death

receptor–mediated apoptosis independent of mitochondria

(and thus not sensitive to Bcl2/BclXL), whereas type II cells

rely on the intrinsic pathway for efficient apoptosis (and

apoptosis is abrogated by Bcl2, BclXL or by the selective

caspase 9 inhibitor Z-LEHD-fmk) [17].

Recombinant human TRAIL, in its Zn2+-containing homo-

trimer form known as Apo2L/TRAIL (Genentech, Inc., South

San Francisco, CA), in contrast to other forms of recombinant

TRAIL, has been shown to be tumor selective in that it rapidly

and profoundly induces apoptosis of susceptible cancer cells

while sparing normal cells especially primary human hepato-

cytes [3,18,19]. Systemic administrations in nonhuman pri-

mates were well tolerated with no apparent toxicity to the

liver or the bone marrow [19,20]. Malignant tumors of di-

verse histology (cultured cell lines and tumor specimen)

amply express DR4 and/or DR5, yet significant proportions

of receptor-positive cultured cancer cells exhibit resistance

to the cytotoxic effect of Apo2L/TRAIL in vitro [21]. The

molecular basis of this intrinsic or acquired resistance to

TRAIL-induced cytotoxicity in various cancer cell lines is

complex and multifactorial [22]. Fortunately, this limitation

can be overcome by combining recombinant TRAIL receptor

ligand with cancer chemotherapeutic agents (standard cyto-

toxic drugs like cisplatin [23–26], CTP-11 [19], or others

[24,26–29] as well as experimental anticancer drugs [30])

[31,32]. Whereas the underlying mechanisms responsible

for the synergistic interactions between chemotherapeutics

and TRAIL receptor agonists to mediate profound induction

of apoptosis are incompletely understood, it appears that

recruitment/activation of the intrinsic death pathway (mito-

chondria mediated) in combination-treated cells plays the

crucial role [28,33,34].

Histone deacetylase inhibitors (HDACIs) are structurally

diverse chemical compounds that share common biologic

properties of inducing core histone hyperacetylation leading

to gene expression and of mediating potent antitumor effects

[35–37]. Some HDACIs are either naturally occurring com-

pound like sodium butyrate (a fatty acid metabolite found

in high concentration in the lumen of the large intestine) or

a pharmacologic compound such as valproic acid (VA, a

commonly prescribed antiepileptic drug), whereas others are

complex chemicals isolated from culture broths of micro-

organisms (depsipeptide, apicidin, or trichostatin A) or syn-

thetic derivatives (MS-275, CI-994). HDACIs are subdivided

into four fundamental groups: short-chain fatty acids (sodium

butyrate, phenylbutyrate, VA), synthetic benzanides deriva-

tives (MS-275, CI-994), cyclic tetrapeptides (depsipeptide,

trapoxin, apicidin), and hydroxamic acids [trichostatin A,

suberoylanilide hydroxamic acid (SAHA), LAQ8240] [35].

HDACIs induce differentiation, cell cycle arrest, and/or apop-

tosis of cancer cells in culture and in in vivo animal models

[35–37]. Multiple HDACIs (SAHA, depsipeptide, MS275)

have been shown to have anticancer properties in phase I

and II clinical trials [35,38–40]. The antitumor activity of

HDACIs has been attributed to both their ability to inhibit de-

acetylases (leading to accumulation of hyperacetylated his-

tones and alteration of gene transcription) and their ability to

suppress mitogenic signal transduction pathways through

downregulation of oncoprotein expression [41] as well as

their effect on the phenotypic expression of Bax, Bak, Bcl2,

and BclXL leading to a net increase in the ratio of pro- versus

antiapoptotic proteins of the Bcl2 superfamily and the apop-

togenicity of the mitochondria [42–46]. It is the latter property

of HDACIs that we wished to exploit to potentiate the cyto-

toxic effect of Apo2L/TRAIL in cultured thoracic cancer cells

(cancer cell lines derived from tumors of the lung, the eso-

phagus, or the pleura). VA, a commonly prescribed anti-

epileptic drug of which the pharmacokinetics and toxicity

profiles are well documented [47,48], has recently been

shown to be an HDACI [49–51] and to exhibit anticancer

activity in in vitro and in vivo animal models [52–54]. The aim

of this study was to critically evaluate the cytotoxic effect of

the combination of VA and Apo2L/TRAIL in a panel of cul-

tured thoracic cancer cells. We observed that VA + Apo2L/

TRAIL combination synergistically induced profound cyto-

toxicity and apoptosis of cultured thoracic cancer cells

through the mitochondria-dependent (type II) pathway.

Materials and Methods

Cell Lines and Reagents

Cultured non–small cell lung cancer (NSCLC) cells H460

and H322; esophageal cancer (EsC) cells TE2 and TE12;

and malignant pleural mesothelioma (MPM) cells H211 and

H513 were maintained in RPMI 1640 culture medium sup-

plemented with fetal calf serum (10% vol/vol), L-glutamine

(1 mM), and antibiotics [streptomycin (100 mg/ml)/penicillin

(100 U/ml)]. Normal human primary fibroblast and human

umbilical vein endothelial cells (HUVEC) were purchased

from Cambrex Bio Science (Walkerville, MD) and grown in

their special culture media as per instructions of the vendor.

Apo2L/TRAIL was obtained from Genentech Inc. through an

institutional M-CRADA. VA was purchased from Alexis (San

Diego, CA). Selective caspase 8 or caspase 9 inhibitors were

purchased from R&D Systems (Minneapolis, MN). Bcl2-

Valproic Acid Potentiates Apo2L/TRAIL Cytotoxicity Ziauddin et al. 447

Neoplasia . Vol. 8, No. 6, 2006

overexpressing stable transductants of TE2 (TE2Bcl2) and

H513 (H513Bcl2) cells were created by retrovirally mediated

gene transfer using Bcl2-expressing viral vector containing

green fluorescence protein (GFP) as a selectable marker

(generously provided by P. Robbins, National Cancer Insti-

tute) and previously published transduction techniques [55].

Vector control stable transductants of similar cancer cells

were created using GFP-expressing retrovirus. The magni-

tude of GFP fluorescence in Bcl2 stable transfectants closely

correlated with the level of Bcl2 expression [55]. Cells with

the highest level of GFP were selected by cell sorting for

further culture and expansion. Bcl2 was detected using intra-

cellular staining with phycoerythrin (PE)-conjugated anti-

Bcl2 monoclonal antibody (clone Bcl-2/100, Alexis) after cell

fixation and permeabilization using the Cytofix/Cytoperm kit

from BD Biosciences (San Jose, CA). Flow cytometry con-

firmed 100% of cells expressing high levels of Bcl2. Western

blot analysis demonstrated very high levels of Bcl2 in all

Bcl2-expressing stable transductants.

Flow Cytometric Analysis of TRAIL Receptors

Cancer cells (controls or treatedwith VAat 1.0 or 5.0mM�24 hours) were washed with PBS with 5% BSA and incu-

bated with biotinylated mouse antihuman antibodies for

DR4, DR5, DcR1, and DcR2 (1 mg/500,000 cells; R&D

Systems) for 30 minutes at room temperature. Excess un-

bound antibodies were removed by one wash with PBS and

5% BSA; cells were further incubated with streptavidin-PE

(30 mL/500,000 cells; R&D Systems) for 30 minutes and

washed once before being submitted for flow cytometry.

The levels of receptor expression were quantified by the PE

mean fluorescence intensity index (MFII, the ratio of PEmean

fluorescence intensity of cells incubated with antireceptor

antibody and the backgroundPEmean fluorescence intensity

in cells incubated with IgG isotype control) and the percent-

ages of cells gated positive for receptor expression.

Cytotoxicity and Apoptosis Assays

Cells were seeded in 96-well microtiter plates at prede-

termined plating densities appropriate for each cell line [(1.0–

1.5) � 104 cells/well]. After an overnight incubation, cells

were treated with either VA (0.5, 1, or 5 mM) concurrently

with Apo2L/TRAIL (5 to 100 ng/ml; VA + Apo2L/TRAIL) or

12 hours pretreatment of VA followed by addition of Apo2L/

TRAIL in the presence of VA (VA/Apo2L/TRAIL). Cell viability

was quantified by (4,5-dimethylthiazo-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) at 36 hours after the onset of

Apo2L/TRAIL exposure. Cell viability after Apo2L/TRAIL

treatment or combinations of VA and Apo2L/TRAIL treat-

ments were calculated as percentages of untreated controls

or VA-treated controls (to normalize for the very mild growth-

inhibitory effect of VA, which was less than 15% at the highest

concentration of VA at 5mM), respectively. TheApo2L/TRAIL

IC50 values (indices of cellular sensitivity to Apo2L/TRAIL)

were estimated from the respective dose-response curves.

Apoptosis after treatments with Apo2L/TRAIL (10 or 20ng/ml)

alone or in combination with VA (0.5 to 5.0 mM) was deter-

mined by the terminal deoxynucleotidyltransferase-mediated

dUTP nick end labeling (TUNEL)–based Apo-bromodeoxy-

uridine (BrdU) assay (BD Pharmingen, San Jose, CA) for late

apoptosis (48 hours after Apo2L/TRAIL exposure).

Caspase Activity Assay

Specific enzymatic activity of caspases 8, 9, and 3 at

intervals after the onset of treatment with Apo2L/TRAIL (10

or 20 ng/ml) with or without VA (0.5 mM) combination was

measured by a fluorometric kit (R&D Systems). Cells were

treated with VA alone (0.5 mM), Apo2L/TRAIL alone (20 ng/

ml), or VA +Apo2L/TRAIL combination and serially harvested

at 2-hour intervals and assayed for caspase activity. The

specific caspase activity, normalized for total proteins of cell

lysates, was then expressed as fold of the baseline caspase

activity of untreated control cells.

Western Blots

Control or VA-treated cultured thoracic cancer cells were

harvested after 12 hours of VA treatment in Laemmli lysis

buffer for Western blot analysis for the expression of FADD,

pro-caspase 8 (BD Biosciences, 1:250 and 1:500 dilution,

respectively), TRADD (Santa Cruz Biotechnology, Santa

Cruz, CA, 1:200 dilution), FLIP (Alexis, 1:200 dilution), Bax,

Bak, Bcl2 and BclXL, BID (all purchased from Cell Signaling

Technology, Beverly, MA at 1:500 dilution), acetylated his-

tone 3 and histone 4 (Upstate Biotechnology, Waltham, MA

at 1:1000 dilution.). For detection of acetylated histones,

acidic cell lysates were prepared as described in the Web

site of the Chromatin and Gene Expression Group headed

by Prof. Bryan Turner (University of Birmingham, UK; http:

//medweb. bham.ac.uk/research/chromatin/protocol/

extraction.html). Blots were also probed for b-actin (Oncogene

Research Products, Cambridge, MA, 1:5000 dilution) to verify

equal protein loadings. The primary antibodies were probed

with HRP-conjugated antimouse or antirabbit secondary anti-

bodies and detected with West Dura chemiluminescence

(Pierce Biotechnology, Rockford, IL).

Statistical Analysis

Data are presented as means ± SEM of three indepen-

dent experiments each performed in duplicates. Two-tailed

Student’s t test was used for statistical analysis and P values

less than .05 were considered statistically significant.

Results

Mild Growth Inhibitory Effect of VA on Thoracic Cancer

Cells In Vitro

Valproic acid induced significant accumulation of hyper-

acetylated histone protein H3 and H4 in H322, H513, or

TE12 cells (Figure 1A). Continuous exposure of cultured

thoracic cancer cells to VA for 96 hours led to a mild dose-

dependent reduction of cell proliferation (IC50 values ranging

from 3.2 to 5.0 mM; Figure 1A) that was mainly attributable

to induction of cell cycle arrest at G1/S checkpoint (data

not shown) and a weak induction of apoptosis (<15% at

5.0 mM; Figure 1B).

448 Valproic Acid Potentiates Apo2L/TRAIL Cytotoxicity Ziauddin et al.


Synergistic Induction of Cytotoxicity and Apoptosis

by VA and Apo2L/TRAIL Combinations

We next wished to determine if treating thoracic cancer

cells with the combination of VA (at concentrations that are

clinically achievable and have HDAC inhibitory activity) and

Apo2L/TRAIL would mediate synergistic induction of cyto-

toxicity and apoptosis, similar to other more familiar HDACIs.

Intrinsic sensitivity to Apo2L/TRAIL varied greatly between

the six cultured thoracic cell lines used in this study, with

H460, H322, H211, and TE12 cells being sensitive to this

ligand (IC50 values <100 ng/ml) and TE2 and H513 cells

being more refractory to the cytotoxic effect of Apo2L/TRAIL

(IC50 values >150 ng/ml) (Figure 2A). The cytotoxic effect of

high concentrations of Apo2L/TRAIL (100 or 200 ng/ml) in

TE2, TE12, or H513 cells was totally abrogated by Bcl2,

indicating that these were type II cells (Figure 2B). Concur-

rent exposure of cultured thoracic cancer cells to VA (0.5

to 5.0 mM) and Apo2L/TRAIL (5 to100 ng/ml) for 36 hours

resulted in significant supra-additive suppression of cell

viability in MTT assays (representative data for H513,

H460, and TE12 cells are shown in Figure 3A). VA alone,

at the treatment conditions used, mediated little reduction of

viability (15–20% at 5.0 mM). When normalized for this mild

VA-mediated cytotoxic effect, there was substantial further

suppression of cell viability in combination-treated cells

(Figure 3A). Using Apo2L/TRAIL IC50 values as indicators

of cellular sensitivity to this ligand, we found that there

was 1.5- to >10-fold reduction of Apo2L/TRAIL IC50 values

in VA-treated cells in a VA dose–dependent manner

(Figure 3B). This effect was observed in all cultured thoracic

Figure 1. (A) VA-mediated reduction of cell viability of cultured thoracic cancer cells. Data are presented as means ± SEM of four independent experiments.

Accumulation of hyperacetylated histone H3 or H4 in H322, H513, and TE12 cells after 24-hour exposure to VA (either 0.5 or 5.0 mM); blotting for �-actin indicated

equal protein loadings. Representative data of two independent experiments with similar results are shown here (B). Mild induction of apoptosis by VA (1.0 or

5.0 mM � 48 hours). Apoptosis was quantified by TUNEL-based Apo-BrdU assay. Data are presented as means ± SEM of four independent experiments.



cancer cells regardless of their intrinsic sensitivity to Apo2L/

TRAIL and particularly most pronounced in Apo2L/TRAIL-

resistant (TE2, TE12, and H513 cells) cell lines. Moreover,

VA treatment did not affect the intrinsic susceptibility of the

Apo2L/TRAIL-sensitive cells H322, H460, and H211 to this

ligand. Most importantly, the VA + Apo2L/TRAIL combina-

tion, although very toxic to cancer cells, was not harmful to

primary cultured normal cells (Figure 3C). Treating cancer

cells with either concurrent (VA + Apo2L/TRAIL) or sequen-

tial VA followed by Apo2L/TRAIL in the presence of VA (VA/

Apo2L/TRAIL) schedule similarly sensitized cancer cells to

Apo2L/TRAIL (Figure 3D). Concurrent VA + Apo2L/TRAIL

combination profoundly and synergistically induced apop-

tosis in thoracic cancer cells, even at VA concentration as

low as 0.5 mM (Figure 4).

The Effect of VA on Expression of TRAIL Receptors

and Proapoptotic Proteins

We next sought to determine if VA increased the expres-

sion of TRAIL receptors DR4, DR5 andDcR1, DcR2, as other

HDACIs have been shown to upregulate TRAIL receptor

expression in cultured cancer cells of different histology

[56–61]. Treating H460, H513, or TE12 cells with VA (5 mM

for 12 hours) did not substantially change the basal expres-

sion of TRAIL receptors in these cells except a two-fold re-

duction of DR4 in H460 cells (Figure 5A). As significant

cytotoxicity was observed after concurrent exposure of can-

cer cells to VA and Apo2L/TRAIL, and knowing that Apo2L/

TRAIL would bind to its cognate receptors and initiate the

signaling cascade within minutes of exposure to receptor-

bearing cells, it is conceivable that alteration of TRAIL re-

ceptor repertoire by VA would not play any significant role

in the process of enhancing the cytotoxic effect of Apo2L/

TRAIL in VA-treated cells. We thus further investigated the

effect of VA on other components of the apoptosis-inducing

apparatus, namely, members of the DISC (FLIP, FADD, pro-

caspase 8) and the mitochondria-associated pro- or anti-

apoptotic proteins (Bax, Bak, Bcl2, BclXL). Time-course

experiments indicated that there was no discernible alteration

of the levels of these proteins in H513, H460, or TE12 cells

continuously treated with VA (1 mM) and harvested at indi-

cated timepoints after the onset of drug exposure (Figure 5B).

We next turned our attention on analyzing the functional

aspect of the caspase cascades in combination-treated cells.

Involvement of the Mitochondria-Dependent Death Signal

Cascade in VA-Mediated Enhancement of Apo2L/TRAIL

Sensitivity

The specific proteolytic activities of caspases 8, 9, and 3

were assayed at intervals after drug treatment in H513 cells

(parental cells or Bcl2-overexpressing stable transfectants

H513Bcl2 cells) after exposure to VA, Apo2L/TRAIL, or

sequential VA/Apo2L/TRAIL combination. Whereas no or

little (zero to four-fold) activation of caspases 8, 9, or 3 was

observed after exposure of H513 cells to VA (0.5 mM) or

Apo2L/TRAIL (20 ng/ml), a supra-additive five- to eight-fold

increase in the activity of these caspases was observed in

cells treated with the VA + Apo2L/TRAIL combination. More

interestingly, treatment-induced activation of not only cas-

pase 9 but also of caspase 8 and caspase 3 was totally

abrogated by overexpression of Bcl2 (Figure 6). Similarly,

complete suppression of the high levels of caspases 8, 9,

and 3 was noted in similarly treated cells incubated with the

selective caspase 9 inhibitor Z-LEHD-fmk (data not shown).

In both experiments, inhibition of caspase 9 activation served

as an internal control, whereas complete inhibition of cas-

pase 3 indicated the exclusive role of the mitochondria

pathway in activating this downstream executioner caspase,

and inhibition of caspase 8 implied activation of this apical

caspase was downstream of caspase 9 and the result of the

amplification feedback loop mediated by the mitochondria-

dependent caspase activation cascade.

Bcl2-mediated inhibition of caspases 8, 9 and 3 activity

was translated to complete abrogation of the intense cyto-

toxic effect of the VA + Apo2L/TRAIL combination in Bcl2

stable transfectants H513Bcl2 and TE12Bcl2 (Figure 7A). The

mild growth-inhibitory effect of Apo2L/TRAIL (20% or 30%

reduction of cell viability in TE12 or H513, respectively) was

Figure 2. (A) Intrinsic susceptibility of cultured thoracic cancer cells to Apo2L/

TRAIL. Cell viability was determined by MTT assay. Apo2L/TRAIL IC50

values of each cell line were derived from the respective cell viability dose-

response curves. Data are presented as means ± SEM of four independent

experiments. (B) Abrogation of cytotoxicity mediated by high concentrations

of Apo2L/TRAIL (100 or 200 ng/ml � 36 hours) by overexpression of Bcl2.

Vector control stable transfectants were functionally similar to parental control

cells (data not shown). Data are presented as means ± SEM of four

independent experiments.



also sensitive to Bcl2 overexpression, indicating that these

were type II cells. The essential role of the mitochondria-

mediated death signal cascade in combination-treated cells

was further evidenced by the complete abrogation of apop-

tosis by the selective caspase 9 inhibitor (Figure 7B).

Discussion

Primary cancers of the thoracic cavity (cancers of the lung,

the esophagus, or the pleura) when presented with re-

gional or systemic metastasis are notoriously refractory to

standard-of-care therapy regimes of cytotoxic chemo-

therapy, external beam irradiation, and surgical resection in

various combinations [62,63]. Better understanding of the

molecular basis of carcinogenesis and elucidation of signal

transduction pathways regulating cell growth and death in

normal cells and their roles in the process of malignant

transformation offers great opportunities for the development

of novel, molecularly targeted anticancer therapy. Within

this context, therapeutic strategies aiming at direct induction

of cell death by activation of the TRAIL receptor–mediated

signal transduction pathways has attracted a great deal of

attention and, in fact, recently entered early-phase clinical

development. Recombinant protein (such as Apo2L/TRAIL

[19] or recombinant human agonistic TRAIL-R1 monoclonal

antibody [64] are commonly used to activate TRAIL receptor

for the induction of apoptosis of cancer cells. It became clear

to investigators in this field that a significant proportion of

cultured malignant cells, although expressing the functional

TRAIL receptors DR4/DR5, are refractory to the cytotoxic

effect of recombinant soluble TRAIL [21]. The molecular

basis of this intrinsic or acquired resistance to TRAIL-

induced cytotoxicity in various cancer cell lines is complex

and multifactorial [21,22]. Moreover, in vitro experimental

conditions can significantly influence the intrinsic suscepti-

bility of cultured cancer cells to TRAIL, thus making deter-

mination of cellular sensitivity to this death-inducing ligand

or others like FasL somewhat arbitrary and subjected to a

wide range of variability between laboratories. We defined

Apo2L/TRAIL-sensitive cells in our study using the described

Figure 3. (A) Dose-dependent enhancement of Apo2L/TRAIL-mediated cytotoxicity by VA (0.5 to 5 mM) in representative cultured thoracic cancer cells H460, TE12,

and H513. Cell viability is expressed as percentages of viable cells in untreated controls (for Apo2L/TRAIL treatment alone) or cells exposed to VA alone (in VA +

Apo2L/TRAIL– treated cells to normalize for the very mild growth-inhibitory effect of VA). Data are presented as means ± SEM of four independent experiments.

(B) Lack of VA + Apo2L/TRAIL– induced cytotoxicity in either primary normal skin fibroblasts or HUVEC. Significant accumulation of acetylated H4 histone

protein was observed in these primary normal cells treated with VA (1.0 or 5.0 mM for 24 hours). Data are presented as means ± SEM of three independent

experiments. (C) Apo2L/TRAIL IC50 values of cultured cancer cells treated with the VA + Apo2L/TRAIL combinations. These values were estimated from the

respective dose-response curves and used as indicators of cellular sensitivity to Apo2L/TRAIL. Data are presented as means ± SEM of four independent

experiments; #P < .001 and *P < .05 versus Apo2L/TRAIL alone. (D) Treatment schedules (concurrent VA and Apo2L/TRAIL or VA pretreatment for 12 hours

before adding Apo2L/TRAIL) have no impact on the enhancement of cellular sensitivity to Apo2L/TRAIL in cell treated with drug combinations. Data are presented

as means ± SEM of four independent experiments.



Figure 3. (continued)

Figure 4. Profound and synergistic dose-dependent induction of apoptosis by the VA + Apo2L/TRAIL combinations in representative cultured thoracic cancer cells.

Data are presented as means ± SEM of four independent experiments; #P < .001 versus Apo2L/TRAIL alone or VA treatment alone.



experimental conditions as those with IC50 values less than

100 ng/ml. We have screened about 15 cultured thoracic

cancer cell lines and identified 7 that are intrinsically sen-

sitive to Apo2L/TRAIL (2 NSCLC, 3 EsC, and 2 MPM cell

lines). The cancer cells selected for this study repre-

sented both Apo2L/TRAIL-sensitive as well as Apo2L/

TRAIL-resistant lines.

Previous studies have demonstrated the synergistic

cytotoxicity mediated by HDACIs and TRAIL combinations

[56–60,65–69]. Both leukemic cell lines and those derived

from solid tumors were evaluated in these studies using

different classes of HDACIs. Enhanced TRAIL cytotoxicity

in cells treated with such drug combinations was frequently

attributed to upregulation of DR4/DR5 expression or DISC

assembly leading to increased caspase 8 activation and

apoptosis [56–61,65]. Modulation of the expression of pro-

and antiapoptotic proteins of the Bcl2 superfamily was also

observed in HDACI-treated cells and thought to play a role in

sensitizing cultured cancer cells to the apoptosis-inducing

effect of TRAIL [61,70,71]. Others have demonstrated the

involvement of the intrinsic pathway in mediating enhanced

cytotoxicity after treatment with the HDACI + TRAIL combi-

nation [61,67]. In direct contrast to previously mentioned

publications, we did not observe any alteration of DR4/DR5

expression in VA-treated cells. As profound cytotoxicity was

observed in cells concurrently exposed to TRAIL and HDACI

and HDACI-induced upregulation of DR4/DR5 expression

occurred after latent periods ranging from 4 to 6 hours in

many reported studies, it is hard to attribute receptor up-

regulation as having a direct impact on the enhancement of

cellular sensitivity to TRAIL. Even if there was a delayed up-

regulation of TRAIL receptors in VA-treated cells that was not

readily detected by the flow cytometry method that we used

in our study, such a change would not have an impact on the

enhanced cytotoxicity of this drug combination, as similar

degrees of growth inhibition were observed in cancer cells

treated with either concurrent VA + Apo2L/TRAIL or sequen-

tial VA/Apo2L/TRAIL combinations. Enhancement of Apo2L/

TRAIL tumoricidal activity by VA, either in ligand-sensitive

or -resistant cells, was totally abrogated by either Bcl2 over-

expression or by the selective caspase 9 inhibitor, implying

that the intrinsic pathway was essential in regulating this

process. Data from the caspase activity experiments defined

the regulatory role of the mitochondria-derived caspase 9

(and possibly caspase 3) in amplifying the caspase cascade

by potentiating the activation of caspase 8 after Apo2L/TRAIL

exposure, particularly in the presence of VA. The strong

caspase 8 activation in combination-treated H513 cells may

be the result of increased DISC activity or secondary to ac-

tivation by downstream executioner caspases such as cas-

pase 3. Processing of caspase 3 can bemediated by caspase

8 (extrinsic pathway) or by caspase 9 (intrinsic pathway).

The hierarchical ordering of caspase activation downstream

of caspase 9 has been well described [72]. Caspases 3, 8, 7,

and 10 are all substrates of activated caspase 9; whether

caspase 9 directly processes caspase 8 is not completely

clear, but it is well known that caspase 3 does activate

caspase 8. It is entirely possible that, via caspase 3, cas-

pase 9 indirectly regulates caspase 8 activation. Blocking of

caspase 3 activity using a selective inhibitor would abrogate

caspase 8 activation if this was indeed secondary to a down-

stream caspase-dependent feedback loop, but caspase 3

blockade would not discriminate which pathway downstream

from the DISC (intrinsic versus extrinsic) is responsible for

perpetuation of caspase 8 activation. However, inhibition of

caspase 9 activation by using the selective inhibitor Z-LEHD-

fmk or overexpression of Bcl2 would block both caspase 3

and caspase 8 activation if proteolytic processing of cas-

pase 8 was secondary to caspase 3 activation through

the mitochondria-mediated intrinsic pathway. This was ex-

actly what we observed in that caspase 8 activation in

combination-treated cells was completely suppressed by

either Bcl2 overexpression or the selective caspase 9 inhib-

itor. VA, therefore, through a molecular mechanism yet to be

Figure 5. (A) The effect of VA treatment on the expression of DR4/DR5 and

DcR1/DcR2 on representative H460 NSCLC, TE12 EsC, and H513 MPM

cells. TRAIL receptors were quantified by flow cytometry using biotinylated

primary antibodies plus streptavidin-PE and expressed as mean fluorescence

intensity index. Representative data of two independent experiments with

similar results are shown here. (B). Expression of key components of DISC or

the pro-/antiapoptotic proteins of the Bcl2 superfamily in H513 or H460 cells

at time points after VA (1.0 mM) exposure. Representative data of two

independent experiments with similar results are shown here.



fully elucidated further, activates the intrinsic pathway of

type II cells to increase the efficacy of Apo2L/TRAIL. As we

have not identified type I cells in our panel of cultured tho-

racic cancer cells, it is not known if VA would activate the

intrinsic signaling cascade to induced Apo2L/TRAIL sensiti-

zation, thus invoking the type II characteristic to potentiate

the cytotoxicity of this ligand in such cell lines. Our attempt to

evaluate the effect of VA on altering phenotypic expressions

of key DISC proteins or some well-described mitochondria-

related pro- and antiapoptotic proteins was not fruitful. This

was in contrast to previous findings of other investigators

as well as of our own observation of profound upregulation of

Bax and/or Bak in conjunction with reduction of the levels of

Bcl2/BclXL shortly after treating the same cancer cells with a

more potent HDACI trichostatin A (Reddy et al., unpublished

data). Functionally, we made similar observations with VA +

Apo2L/TRAIL combinations but were unable to reproduce the

phenotypic alterations of members of the Bcl2 superfamily

after VA treatment. Ongoing studies are being conducted to

define the molecular mechanism by which VA instigates the

mitochondria to sensitize cancer cells to Apo2L/TRAIL. Un-

like other studies in which mitochondria activation after

chemotherapy or TRAIL treatments was evaluated by mito-

chondrial release of cytochrome c, apoptosis-inducing factor,

and SMAC/DIABLO and depolarization of mitochondria

membrane potential (Dcm), we concentrated on evaluating

the catalytic activity of caspase 9 as the functional marker

(‘‘readout’’) of activation of the proapoptotic activity of mito-

chondria.Weevaluated VA in combination with Apo2L/TRAIL

based on its biologic property as an HDACI; yet, the VA-

mediated enhancement effect observed in this drug combi-

nation may be secondary to a separate mechanism entirely

independent of its HDAC inhibitory activity.

In summary, VA, at clinically achievable drug concentra-

tions, profoundly enhances the intrinsic sensitivity of cultured

thoracic cancer cells to Apo2L/TRAIL in vitro. As VA is a

Food and Drug Administration–approved antiepileptic drug

with well-described pharmacokinetics and toxicity profiles

and Apo2L/TRAIL is currently undergoing clinical testing,

this drug combination has a good translational potential and

should be considered for clinical development.

Acknowledgements

J. B. Maxhimer is a Howard Hughes Medical Institute Re-

search Scholar. A. Baras and A. Chua are recipients of a

Figure 6. Time course of caspase 8 and caspase 9 activation in H513 cells treated with VA (1 mM) + Apo2L/TRAIL (20 ng/ml). Not only the activity of caspase 9 but

also that of the apical caspase 8 was totally abrogated by Bcl2-overexpressing stable transfectant H513Bcl2. Respective cells treated with either VA, Apo2L/TRAIL,

or VA + Apo2L/TRAIL were harvested at indicated time points after the onset of treatment and assessed for caspase 8 or 9 activity using the fluorimetric assay kit.

Representative data of two independent experiments with similar results are shown here.



Figure 7. (A) Overexpression of Bcl2 totally suppressed apoptosis induced by VA + Apo2L/TRAIL combinations in cultured thoracic cancer cells. Parental or Bcl2-

overexpressing stable transfectants H513Bcl2 and TEBcl2 were concurrently treated with VA (5.0 mM) and apo2L/TRAIL (20 ng/ml). Cell viability was evaluated by

MTT assay. Vector controls behaved exactly like parental cells. Data are presented as means ± SDs of four independent experiments. (B) Profound inhibition of

VA + APo2L/TRAIL– induced apoptosis by the selective caspase 9 inhibitor Z-LEHD-fmk (40 �M). Cells were pretreated with the selective caspase 9 inhibitor for

1 hour before addition of VA (5.0 mM) and Apo2L/TRAIL (10 or 20 ng/ml). Cells were harvested 48 hours later and apoptosis was determined by the TUNEL-based

Apo-BrdU assay. Representative data of three independent experiments with similar results are shown here.



Cancer Research Training Award, National Cancer Institute,

National Institutes of Health.

References

[1] Almasan A and Ashkenazi A (2003). Apo2L/TRAIL: apoptosis signaling,

biology, and potential for cancer therapy. Cytokine Growth Factor Rev

14, 337–348.

[2] Yagita H, Takeda K, Hayakawa Y, Smyth MJ, and Okumura K (2004).

TRAIL and its receptors as targets for cancer therapy. Cancer Sci 95,

777–783.

[3] Hymowitz SG, O’Connell MP, Ultsch MH, Hurst A, Totpal K, Ashkenazi

A, de Vos AM, and Kelley RF (2000). A unique zinc-binding site re-

vealed by a high-resolution X-ray structure of homotrimeric Apo2L/

TRAIL. Biochemistry 39, 633–640.

[4] LeBlanc HN and Ashkenazi A (2003). Apo2L/TRAIL and its death and

decoy receptors. Cell Death Differ 10, 66–75.

[5] Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, and

Ashkenazi A (1996). Induction of apoptosis by Apo-2 ligand, a new

member of the tumor necrosis factor cytokine family. J Biol Chem

271, 12687–12690.

[6] Ashkenazi A and Dixit VM (1999). Apoptosis control by death and decoy

receptors. Curr Opin Cell Biol 11, 255–260.

[7] Nicholson DW (1999). Caspase structure, proteolytic substrates, and

function during apoptotic cell death. Cell Death Differ 6, 1028–1042.

[8] Salvesen GS and Dixit VM (1999). Caspase activation: the induced-

proximity model. Proc Natl Acad Sci USA 96, 10964–10967.

[9] Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM,

Ricci JE, Edris WA, Sutherlin DP, Green DR, et al. (2003). A unified

model for apical caspase activations. Mol Cell 11, 529–541.

[10] Kroemer G, Dallaporta B, and Resche-Rigon M (1998). The mitochon-

drial death/life regulator in apoptosis and necrosis. Annu Rev Physiol

60, 619–642.

[11] van Loo G, Saelens X, van Gurp M, MacFarlane M, Martin SJ, and

Vandenabeele P (2002). The role of mitochondrial factors in apop-

tosis: a Russian roulette with more than one bullet. Cell Death Differ

9, 1031–1042.

[12] Ruffolo SC, Breckenridge DG, Nguyen M, Goping IS, Gross A,

Korsmeyer SJ, Li H, Yuan J, and Shore GC (2000). BID-dependent

and BID-independent pathways for BAX insertion into mitochondria.

Cell Death Differ 7, 1101–1108.

[13] Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, and Schlesinger PH

(2000). Pro-apoptotic cascade activates BID, which oligomerizes BAK

or BAX into pores that result in the release of cytochrome c. Cell Death

Differ 7, 1166–1173.

[14] Scorrano L and Korsmeyer SJ (2003). Mechanisms of cytochrome c

release by proapoptotic BCL-2 family members. Biochem Biophys Res

Commun 304, 437–444.

[15] Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin

KM, Krammer PH, and Peter ME (1998). Two CD95 (APO-1/Fas) sig-

naling pathways. EMBO J 17, 1675–1687.

[16] Saelens X, Festjens N, Vande WL, van Gurp M, van Loo G, and

Vandenabeele P (2004). Toxic proteins released from mitochondria in

cell death. Oncogene 23, 2861–2874.

[17] Ozoren N and el Deiry WS (2002). Defining characteristics of types I

and II apoptotic cells in response to TRAIL. Neoplasia 4, 551–557.

[18] Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B,

Hillan K, Totpal K, DeForge L, Schow P, et al. (2001). Differential hepa-

tocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 7,

383–385.

[19] Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA,

Blackie C, Chang L, McMurtrey AE, Hebert A, et al. (1999). Safety and

antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104,

155–162.

[20] Kelley SK, Harris LA, Xie D, DeForge L, Totpal K, Bussiere J, and Fox

JA (2001). Preclinical studies to predict the disposition of Apo2L/tumor

necrosis factor – related apoptosis-inducing ligand in humans: charac-

terization of in vivo efficacy, pharmacokinetics, and safety. J Pharmacol

Exp Ther 299, 31–38.

[21] Shankar S and Srivastava RK (2004). Enhancement of therapeutic po-

tential of TRAIL by cancer chemotherapy and irradiation: mechanisms

and clinical implications. Drug Resist Updat 7, 139–156.

[22] Zhang L and Fang B (2005). Mechanisms of resistance to TRAIL-

induced apoptosis in cancer. Cancer Gene Ther 12, 228–237.

[23] Kondo K, Yamasaki S, Sugie T, Teratani N, Kan T, Imamura M, and

Shimada Y (2005). Cisplatin-dependent upregulation of death receptors

4 and 5 augments induction of apoptosis by TNF-related apoptosis-

inducing ligand against esophageal squamous cell carcinoma. Int J

Cancer 118, 230–242.

[24] Lacour S, Micheau O, Hammann A, Drouineaud V, Tschopp J, Solary E,

and Dimanche-Boitrel MT (2003). Chemotherapy enhances TNF-

related apoptosis-inducing ligand DISC assembly in HT29 human colon

cancer cells. Oncogene 22, 1807–1816.

[25] Kim JH, Ajaz M, Lokshin A, and Lee YJ (2003). Role of antiapoptotic

proteins in tumor necrosis factor-related apoptosis-inducing ligand and

cisplatin-augmented apoptosis. Clin Cancer Res 9, 3134–3141.

[26] Jin H, Yang R, Fong S, Totpal K, Lawrence D, Zheng Z, Ross J,

Koeppen H, Schwall R, and Ashkenazi A (2004). Apo2 ligand/tumor

necrosis factor – related apoptosis-inducing ligand cooperates with che-

motherapy to inhibit orthotopic lung tumor growth and improve survival.

Cancer Res 64, 4900–4905.

[27] Liu W, Bodle E, Chen JY, Gao M, Rosen GD, and Broaddus VC (2001).

Tumor necrosis factor – related apoptosis-inducing ligand and chemo-

therapy cooperate to induce apoptosis in mesothelioma cell lines. Am J

Respir Cell Mol Biol 25, 111–118.

[28] Muhlethaler-Mottet A, Bourloud KB, Auderset K, Joseph JM, and Gross

N (2004). Drug-mediated sensitization to TRAIL-induced apoptosis in

caspase-8-complemented neuroblastoma cells proceeds via activation

of intrinsic and extrinsic pathways and caspase-dependent cleavage of

XIAP, Bcl-xL and RIP. Oncogene 23, 5415–5425.

[29] Evdokiou A, Bouralexis S, Atkins GJ, Chai F, Hay S, Clayer M, and

Findlay DM (2002). Chemotherapeutic agents sensitize osteogenic sar-

coma cells, but not normal human bone cells, to Apo2L/TRAIL-induced

apoptosis. Int J Cancer 99, 491–504.

[30] Kim DM, Koo SY, Jeon K, Kim MH, Lee J, Hong CY, and Jeong S (2003).

Rapid induction of apoptosis by combination of flavopiridol and tumor

necrosis factor (TNF)-alpha or TNF-related apoptosis-inducing ligand

in human cancer cell lines. Cancer Res 63, 621–626.

[31] Sayers TJ, Brooks AD, Koh CY, Ma W, Seki N, Raziuddin A, Blazar BR,

Zhang X, Elliott PJ, and Murphy WJ (2003). The proteasome inhibitor

PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by re-

ducing levels of c-FLIP. Blood 102, 303–310.

[32] Fulda S, Jeremias I, and Debatin KM (2004). Cooperation of betulinic

acid and TRAIL to induce apoptosis in tumor cells. Oncogene 23,

7611–7620.

[33] Ravi R and Bedi A (2002). Requirement of BAX for TRAIL/Apo2L-

induced apoptosis of colorectal cancers: synergism with sulindac-

mediated inhibition of Bcl-x(L). Cancer Res 62, 1583–1587.

[34] LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P,

Fong S, Schwall R, Sinicropi D, and Ashkenazi A (2002). Tumor-cell

resistance to death receptor – induced apoptosis through mutational

inactivation of the proapoptotic Bcl-2 homolog Bax. Nat Med 8,

274–281.

[35] Rosato RR and Grant S (2004). Histone deacetylase inhibitors in clinical

development. Expert Opin Investig Drugs 13, 21–38.

[36] Schrump DS and Nguyen DM (2005). Targeting the epigenome for the

treatment and prevention of lung cancer. Semin Oncol 32, 488–502.

[37] Jaboin J, Wild J, Hamidi H, Khanna C, Kim CJ, Robey R, Bates SE, and

Thiele CJ (2002). MS-27-275, an inhibitor of histone deacetylase, has

marked in vitro and in vivo antitumor activity against pediatric solid

tumors. Cancer Res 62, 6108–6115.

[38] Schrump DS, Nguyen DM, Kunst TE, Hancox A, Figg WD, Steinberg

SM, Pishchik V, and Becerra Y (2002). Phase I study of sequential

deoxyazacytidine/depsipeptide infusion in patients with malignancies

involving lungs or pleura. Clin Lung Cancer 4, 186–192.

[39] Kelly WK, Richon VM, O’Connor O, Curley T, MacGregor-Curtelli B,

Tong W, Klang M, Schwartz L, Richardson S, Rosa E, et al. (2003).

Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide

hydroxamic acid administered intravenously. Clin Cancer Res 9,

3578–3588.

[40] Piekarz R and Bates S (2004). A review of depsipeptide and other

histone deacetylase inhibitors in clinical trials. Curr Pharm Des 10,

2289–2298.

[41] Yu X, Guo ZS, Marcu MG, Neckers L, Nguyen DM, Chen GA, and

Schrump DS (2002). Modulation of p53, ErbB1, ErbB2, and Raf-1 ex-

pression in lung cancer cells by depsipeptide FR901228. J Natl Cancer

Inst 94, 504–513.

[42] Ruefli AA, Ausserlechner MJ, Bernhard D, Sutton VR, Tainton KM,

Kofler R, Smyth MJ, and Johnstone RW (2001). The histone deacety-

lase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic

acid (SAHA) induces a cell-death pathway characterized by cleavage of



Bid and production of reactive oxygen species. Proc Natl Acad Sci USA

98, 10833–10838.

[43] Marks PA and Jiang X (2005). Histone deacetylase inhibitors in pro-

grammed cell death and cancer therapy. Cell Cycle 4, 549–551.

[44] Garcia-Morales P, Gomez-Martinez A, Carrato A, Martinez-Lacaci I,

Barbera VM, Soto JL, Carrasco-Garcia E, Menendez-Gutierrez MP,

Castro-Galache MD, Ferragut JA, et al. (2005). Histone deacetylase

inhibitors induced caspase-independent apoptosis in human pancreatic

adenocarcinoma cell lines. Mol Cancer Ther 4, 1222–1230.

[45] Doi S, Soda H, Oka M, Tsurutani J, Kitazaki T, Nakamura Y, Fukuda M,

Yamada Y, Kamihira S, and Kohno S (2004). The histone deacetylase

inhibitor FR901228 induces caspase-dependent apoptosis via the mi-

tochondrial pathway in small cell lung cancer cells. Mol Cancer Ther 3,

1397–1402.

[46] Dunant A, Pignon JP, and Le Chevalier T (2005). Adjuvant chemo-

therapy for non–small cell lung cancer: contribution of the International

Adjuvant Lung Trial. Clin Cancer Res 11, 5017s–5021s.

[47] Blaheta RA and Cinatl J Jr (2002). Anti-tumor mechanisms of valproate:

a novel role for an old drug. Med Res Rev 22, 492–511.

[48] Brodie MJ and Dichter MA (1996). Antiepileptic drugs. N Engl J Med

334, 168–175.

[49] Gurvich N, Tsygankova OM, Meinkoth JL, and Klein PS (2004). Histone

deacetylase is a target of valproic acid–mediated cellular differentia-

tion. Cancer Res 64, 1079–1086.

[50] Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, and Klein PS

(2001). Histone deacetylase is a direct target of valproic acid, a potent

anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 276,

36734–36741.

[51] Kramer OH, Zhu P, Ostendorff HP, Golebiewski M, Tiefenbach J, Peters

MA, Brill B, Groner B, Bach I, Heinzel T, et al. (2003). The histone

deacetylase inhibitor valproic acid selectively induces proteasomal deg-

radation of HDAC2. EMBO J 22, 3411–3420.

[52] Takai N, Desmond JC, Kumagai T, Gui D, Said JW, Whittaker S,

Miyakawa I, and Koeffler HP (2004). Histone deacetylase inhibitors

have a profound antigrowth activity in endometrial cancer cells. Clin

Cancer Res 10, 1141–1149.

[53] Seynaeve CM, Kazanietz MG, Blumberg PM, Sausville EA, and

Worland PJ (1994). Differential inhibition of protein kinase C isozymes

by UCN-01, a staurosporine analogue. Mol Pharmacol 45, 1207–1214.

[54] Gottlicher M, Minucci S, Zhu P, Kramer OH, Schimpf A, Giavara S,

Sleeman JP, Lo CF, Nervi C, Pelicci PG, et al. (2001). Valproic acid

defines a novel class of HDAC inhibitors inducing differentiation of

transformed cells. EMBO J 20, 6969–6978.

[55] Charo J, Finkelstein SE, Grewal N, Restifo NP, Robbins PF, and

Rosenberg SA (2005). Bcl-2 overexpression enhances tumor-specific

T-cell survival. Cancer Res 65, 2001–2008.

[56] Vanoosten RL, Moore JM, Ludwig AT, and Griffith TS (2005). Depsi-

peptide (FR901228 enhances the cytotoxic activity of TRAIL by redistrib-

uting TRAIL receptor to membrane lipid rafts. Mol Ther 11, 542–552.

[57] Kim YH, Park JW, Lee JY, and Kwon TK (2004). Sodium butyrate sen-

sitizes TRAIL-mediated apoptosis by induction of transcription from the

DR5 gene promoter through Sp1 sites in colon cancer cells. Carcino-

genesis 25, 1813–1820.

[58] Chopin V, Slomianny C, Hondermarck H, and Le Bourhis X (2004).

Synergistic induction of apoptosis in breast cancer cells by cotreatment

with butyrate and TNF-alpha, TRAIL, or anti-Fas agonist antibody involves

enhancement of death receptors’ signaling and requires P21(waf1).

Exp Cell Res 298, 560–573.

[59] Nakata S, Yoshida T, Horinaka M, Shiraishi T, Wakada M, and Sakai T

(2004). Histone deacetylase inhibitors upregulate death receptor

5/TRAIL-R2 and sensitize apoptosis induced by TRAIL/APO2-L in hu-

man malignant tumor cells. Oncogene 23, 6261–6271.

[60] Guo F, Sigua C, Tao J, Bali P, George P, Li Y, Wittmann S, Moscinski L,

Atadja P, and Bhalla K (2004). Cotreatment with histone deacetylase

inhibitor LAQ824 enhances Apo-2L/tumor necrosis factor – related

apoptosis inducing ligand-induced death inducing signaling complex

activity and apoptosis of human acute leukemia cells. Cancer Res 64,

2580–2589.

[61] Singh TR, Shankar S, and Srivastava RK (2005). HDAC inhibitors en-

hance the apoptosis-inducing potential of TRAIL in breast carcinoma.

Oncogene 24, 4609–4623.

[62] Enzinger PC and Mayer RJ (2003). Esophageal cancer. N Engl J Med

349, 2241–2252.

[63] Spira A and Ettinger DS (2004). Multidisciplinary management of lung

cancer. N Engl J Med 350, 379–392.

[64] Pukac L, Kanakaraj P, Humphreys R, Alderson R, Bloom M, Sung C,

Riccobene T, Johnson R, Fiscella M, Mahoney A, et al. (2005). HGS-

ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces

cell death in multiple tumour types in vitro and in vivo. Br J Cancer 92,

1430–1441.

[65] Inoue S, MacFarlane M, Harper N, Wheat LM, Dyer MJ, and Cohen GM

(2004). Histone deacetylase inhibitors potentiate TNF-related apoptosis-

inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies.

Cell Death Differ 11 (2), S193–S206.

[66] Neuzil J, Swettenham E, and Gellert N (2004). Sensitization of meso-

thelioma to TRAIL apoptosis by inhibition of histone deacetylase: role

of Bcl-xL down-regulation. Biochem Biophys Res Commun 314,

186–191.

[67] Rosato RR, Almenara JA, Dai Y, and Grant S (2003). Simultaneous

activation of the intrinsic and extrinsic pathways by histone deacetylase

(HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing

ligand (TRAIL) synergistically induces mitochondrial damage and apop-

tosis in human leukemia cells. Mol Cancer Ther 2, 1273–1284.

[68] Zhang XD, Gillespie SK, Borrow JM, and Hersey P (2003). The histone

deacetylase inhibitor suberic bishydroxamate: a potential sensitizer of

melanoma to TNF-related apoptosis-inducing ligand (TRAIL) induced

apoptosis. Biochem Pharmacol 66, 1537–1545.

[69] Inoue H, Shiraki K, Ohmori S, Sakai T, Deguchi M, Yamanaka T, Okano

H, and Nakano T (2002). Histone deacetylase inhibitors sensitize hu-

man colonic adenocarcinoma cell lines to TNF-related apoptosis induc-

ing ligand-mediated apoptosis. Int J Mol Med 9, 521–525.

[70] Wajant H, Haas E, Schwenzer R, Muhlenbeck F, Kreuz S, Schubert G,

Grell M, Smith C, and Scheurich P (2000). Inhibition of death receptor–

mediated gene induction by a cycloheximide-sensitive factor occurs at

the level of or upstream of Fas-associated death domain protein

(FADD). J Biol Chem 275, 24357–24366.

[71] Fandy TE, Shankar S, Ross DD, Sausville E, and Srivastava RK

(2005). Interactive effects of HDAC inhibitors and TRAIL on apoptosis

are associated with changes in mitochondrial functions and expres-

sions of cell cycle regulatory genes in multiple myeloma. Neoplasia 7,

646–657.

[72] Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD,

Wang HG, Reed JC, Nicholson DW, Alnemri ES, et al. (1999). Ordering

the cytochrome c– initiated caspase cascade: hierarchical activation of

caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

J Cell Biol 144, 281–292.



valproic acid, an antiepileptic drug with histone ... · valproic acid, an antiepileptic drug with...

Documents