Upregulation of DR5 by proteasome inhibitors potently
sensitizes glioma cells to TRAIL-induced apoptosis
Holger Hetschko
1
, Valerie Voss
1
, Volker Seifert
1
, Jochen H. M. Prehn
2
and Donat Ko
¨
gel
1
1 Department of Neurosurgery, Centre for Neurology and Neurosurgery, Johann Wolfgang Goethe University Clinics, Frankfurt ⁄ Main,
Germany
2 Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland
Gliomas are the most common and malignant primary
brain tumors in humans. Glioblastoma multiforme is
the highest-grade as well as the most aggressive and
frequent glioma [1]. Because gliomas are characterized
by a diffuse infiltrative growth into the surrounding
brain tissue, complete surgical resection of glioblas-
toma multiforme tumors is virtually impossible [2]. In
addition, high-grade gliomas exhibit only limited sensi-
tivity to ensuing multimodal treatment with radio-
therapy and chemotherapy [2], which in large part is
Keywords
apoptosis; astrocytoma; death receptor;
proteasome; stress kinase
Correspondence

scription and surface expression of DR5. Transient knockdown of the
transcription factor GADD153⁄ C ⁄ EBP homologous protein and applica-
tion of the synthetic c-Jun N-terminal kinase inhibitor SP600125 indicated
that enhanced DR5 expression occurred independently of GADD153 ⁄
C ⁄ EBP homologous protein, but required activation of the c-Jun N-termi-
nal kinase ⁄ c-Jun signaling pathway. Novel therapeutic approaches using
TRAIL or agonistic TRAIL receptor antibodies in combination with pro-
teasome inhibitors may represent a promising approach to reactivate apop-
tosis in therapy-resistant high-grade gliomas.
Abbreviations
Ac-DEVD-AMC, acetyl-DEVD-7-amido-4-methylcoumarin; CHOP, C ⁄ EBP homologous protein; DR4, death receptor 4; DR5, death receptor 5;
FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; NF-jB,
nuclear factor kappa B; PI, proteasome inhibitor; siRNA, small interfering RNA; TRAIL, tumor necrosis factor-related apoptosis-inducing
ligand.
FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1925
caused by inherent and potent apoptosis resistance [3].
Clearly, overcoming this resistance by approaches
designed to reactivate apoptosis in malignant glioma
has important implications for the development of
novel glioma therapies.
Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL), a member of the tumor necrosis factor
superfamily, has been shown to be a promising candi-
date for novel anticancer therapies [4]. Interestingly,
TRAIL is capable of inducing apoptosis in a wide
range of tumor cells, but not in normal tissue. This
tumor-selective cytotoxicity has been shown for glioma
cells in comparison to non-transformed astrocytes
in vitro [5,6]. The physiological function of TRAIL
remains unclear, although studies suggest that TRAIL

This study reveals that TRAIL-induced apoptosis
can be efficiently reactivated in TRAIL-resistant malig-
nant glioma cell lines by combined treatment with PIs.
Furthermore, we show that reactivation of TRAIL-
induced apoptosis by proteasome inhibition induces
the c-Jun N-terminal kinase (JKN) ⁄ c-Jun stress signal-
ing pathway and requires enhanced JNK ⁄ c-Jun-depen-
dent surface expression of DR5.
Results
Synergistic effects of combined treatment with
TRAIL and gamma irradiation
To analyze the sensitivity of high-grade gliomas to cell
death induced by the death ligand TRAIL, we
employed a panel of six grade III–IV glioma cell lines
(U87, U251, U343, U373, MZ-18, and MZ-54). In an
initial experiment, the cells were treated for 48 h at a
final concentration of 250 ng TRAILÆmL
)1
(Fig. 1A).
Surprisingly, only two of six cell lines (U87 and U251)
significantly responded to TRAIL treatment as
measured by annexin-V–FLUOS ⁄ propidium iodide
staining and flow cytometry (Fig. 1A). Prolonged incu-
bation for up to 96 h also did not induce detectable
cell death in the four TRAIL-resistant cell lines (data
not shown).
As gamma irradiation is an existing component of
current glioma therapies, and as activation of TRAIL
receptors and DNA damage were shown to have syner-
gistic death-inducing effects in other types of cancer

TRAIL: MG132, a potent reversible inhibitor targeting
Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al.
1926 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS
the 26S complex of the proteasome [16], and epoxomi-
cin, a highly specific and irreversible inhibitor of
several hydrolyzing activities of the proteasome [17],
at concentrations of 2.5 lm and 50 nm respectively
(Fig. 2). Whereas treatment with MG132 alone had a
moderate cytotoxic effect in three cell lines (U87,
U373, and MZ-54), treatment with epoxomicin signifi-
cantly induced cell death in only two of six cell lines
(U373 and MZ-54) (Fig. 2). Interestingly, both PIs
potently enhanced TRAIL-induced apoptosis in the
two TRAIL-sensitive cell lines (U87 and U251), and
were able to reactivate apoptosis in four TRAIL-resis-
tant cell lines (U343, U373, MZ-54, and MZ-18). It is
of note that all six investigated cell lines were previ-
ously shown to express DR5, whereas DR4 expression
was undetectable in U251, U373 and MZ-54 cells [18].
Furthermore, reactivation of TRAIL-induced cell
death did not depend on functional p53, as it was also
observed in mutant p53 expressing U373 cells.
PIs potently induce expression of DR5
in glioma cells
To elucidate the underlying molecular mechanisms of
the observed synergistic effects of PIs, we focused on
the transcriptional activation of pro-apoptotic genes
after proteasome inhibition with MG132 and epoxo-
micin. Samples of cells exposed to gamma irradiation
served as a control for p53-dependent gene activation.

)1
TRAIL or were left untreated for an additional 24 h. Cells were
irradiated once in an Elekta SL75 ⁄ 5 linear accelerator (6 MeV). Apoptosis was quantified with annexin-V–FLUOS ⁄ propidium iodide staining
and flow cytometry. Data are means ± SEM from four independent cultures. *P < 0.05 as compared to untreated control.
#
P < 0.05 as com-
pared to treatment of the same cell line with gamma irradiation alone. Similar results were obtained in three separate experiments.
H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors
FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1927
Upregulation of DR5 by PIs contributes to the
reactivation of TRAIL-induced cell death
in glioma cells
Although the PIs prominently upregulated DR5
(Fig. 3), it was not clear whether this elevated expres-
sion was responsible for the dramatic reactivation of
TRAIL-induced apoptosis observed in malignant gli-
oma cells. To address this question, we knocked down
expression of DR5 by small interfering RNA (siRNA)
duplexes targeted against DR5 mRNA and studied the
effect on caspase activation after treatment of the cells
with TRAIL, MG132, and TRAIL in combination
with MG132. In initial experiments, U343 cells were
transfected with DR5 siRNA, treated with or without
2.5 lm MG132, and subjected to western blot analysis
(Fig. 4A). Transfection with siRNA against DR5
resulted in a robust reduction of DR5 activation after
MG132 treatment as compared to MG132-treated con-
trol cells transfected with a nontargeting scrambled
siRNA (Fig. 4A). In contrast, transfection with the
siRNAs had no major effect on expression levels of

iodide staining and flow cytometry. Data are
means ± SEM from four independent cul-
tures. *P < 0.05 as compared to dimethyl-
sulfoxide-treated control.
#
P < 0.05 as
compared to treatment of the same cell line
with the respective PI alone. Similar results
were obtained in three separate experi-
ments.
Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al.
1928 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS
with MG132 was potently attenuated in TRAIL-resis-
tant U343 cells, TRAIL-sensitive U251 cells and
TRAIL-sensitive U87 cells after knockdown of DR5 in
comparison to the respective controls (Fig. 4C). These
results suggest that enhanced surface expression of
DR5 plays a critical role in the reactivation of
TRAIL-induced apoptosis after proteasome inhibition
in glioma cells.
CHOP is not involved in DR5 upregulation by PIs
and cell death induced by proteasome inhibition
plus TRAIL
It was recently reported that CHOP, an endoplasmic
reticulum stress-inducible member of the CCAAT ⁄
enhancer-binding protein family, can act as an
upstream activator of DR5 in certain types of cancer
cells [19,20]. In line with our previous findings [21],
we found CHOP to be strongly activated after pro-
teasome inhibition (Fig. 3), and were therefore inter-

MG132 are capable of inducing the JNK ⁄ c-Jun path-
way [23]. To address the issue of whether activation of
B
A
Fig. 3. PIs enhance expression of DR5. (A)
DR5, CHOP and c-Jun are transcriptionally
induced after proteasome inhibition. Cells
were either treated with 2.5 l
M MG132,
50 n
M epoxomicin or vehicle (dimethylsulf-
oxide) for 16 h or subjected to gamma irradi-
ation (20 Gy) and subsequently lysed 24 h
after exposure. Expression of DR4 (28
cycles), DR5 (25 cycles), CHOP (28 cycles)
and GAPDH (25 cycles) was determined by
semiquantitative RT-PCR. For detection of
c-Jun expression, RT-PCR with GAPDH
serving as an internal control (in the same
PCR reaction) was performed (30 cycles for
c-Jun and 30 cycles for GAPDH). Similar
results were obtained in at least three sepa-
rate experiments. (B) Western blot analysis
of DR4, DR5 and CHOP expression levels.
Forty micrograms of protein were loaded
onto each lane, with a-tubulin serving as a
loading control. Similar results were
obtained in two separate experiments.
H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors
FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1929

C
Fig. 4. RNA interference against DR5 effi-
ciently protects cells from TRAIL-induced
apoptosis after proteasome inhibition.
Twenty-four hours after transfection with
scrambled control siRNA or DR5 siRNA,
TRAIL-resistant U343 cells were treated
with 2.5 l
M MG132 or dimethylsulfoxide for
16 h. (A) Analysis of DR5 and DR4 expres-
sion levels by western blotting. a-Tubulin
served as a loading control. Similar results
were obtained in two separate experiments.
(B) Analysis of cell surface expression of
DR5 after RNA interference against DR5
(DR5) or treatment with control siRNA (scr),
subsequent treatment with 2.5 l
M MG132
or dimethylsulfoxide (16 h), and staining
with a specific goat anti-DR5 IgG. Unspecific
goat IgG served as isotype control. The
experiment was repeated twice with similiar
results. (C) Knockdown of DR5 inhibits
apoptosis after treatment with TRAIL and
MG132. Following transfection with DR5
siRNA (DR5) and control siRNA (scr), cells
were treated with dimethylsulfoxide (con-
trol), 2.5 l
M MG132, TRAIL (250 ngÆmL
)1

ing gliomas. Despite the described tumor-selective pro-
apoptotic properties of TRAIL, many cancer cells are
innately resistant to TRAIL. In line with previous
studies [6,25], all 18 glioma cell lines investigated in
this study exhibited expression of DR5, but four of six
cell lines showed no significant response to TRAIL.
In order to identify synergistic treatments to modu-
late TRAIL sensitivity and to reactivate apoptosis in
glioma cells, we compared the effects of already exist-
ing treatment modalities (gamma irradiation) with PIs
(MG132 and epoxomicin), a new class of chemothera-
peutic drugs with tremendous therapeutic potential [9].
The correct functioning of the ubiquitin–proteasome
pathway is essential for the degradation of the major-
ity of intracellular proteins and is implicated in many
cellular processes, including regulation of apoptosis.
Both epoxomicin and MG132 were able to potently
enhance apoptosis in the TRAIL-sensitive cell lines.
Both PIs increased apoptosis  5–6-fold in the
TRAIL-sensitive cell lines U87 and U251, and even at
subtoxic levels they were able to reactivate apoptosis
in both TRAIL-resistant cell lines (U343 and U373).
It is widely accepted that PIs can act on mutiple cellu-
lar targets implicated in regulation of apoptosis
[20,21,26,27]. However, despite abundant evidence for
the therapeutic potential of PIs in a variety of malig-
nancies, the relevant signaling pathways leading to
apoptosis triggered by proteasome inhibition seem to
vary substantially between different types of cancer. In
some types of cancer, such as prostate cancer and leu-

of CHOP expression has no effect on TRAIL-induced apoptosis
after proteasome inhibition. U343 cells were treated as described
in (A), and caspase-3-like activity was measured by Ac-DEVD-AMC
cleavage. Data are means ± SEM from four to eight independent
cultures. *P < 0.05 as compared to dimethylsulfoxide-treated con-
trol. Similar results were obtained in two separate experiments.
H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors
FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1931
surface expression levels and apoptosis induced by
TRAIL in combination with MG132 in U343 and
U251 cells. These data suggest that, in contrast to
other cancer types, such as hepatocellular carcinoma
[30], transcriptional upregulation of DR5 plays a piv-
otal role in potentiation and reactivation of TRAIL-
induced apoptosis in glioma cells.
We then addressed the question of which upstream
signaling pathways might lead to enhanced DR5 sur-
face expression after PI treatment, and in line with
previous observations in other types of cancer cells
[21], we demonstrated that the pro-apoptotic transcrip-
tion factor CHOP was potently activated on the tran-
scriptional level by PIs in glioma cells. Although
CHOP has been previously described as a putative
upstream regulator of DR5 in different types of cancer
cells [19,20], we did not observe any significant CHOP-
dependent effects on DR5 expression and cell death,
indicating that CHOP is not required for DR5 induc-
tion triggered by PIs in glioma cells.
Two other apoptosis-regulating transcription fac-
tors whose activity is known to be modulated by PIs

SP600125. Protein levels of phosphorylated c-Jun, DR5 and CHOP were analyzed by western blotting. a-Tubulin served as a loading control.
(C) SP600125 inhibits apoptosis induced by TRAIL plus MG132 in three different glioma cell lines. U87, U251 and U343 cells were treated
as described in (B). Caspase-3-like activity was measured by Ac-DEVD-AMC cleavage. Data are means ± SEM from four to eight indepen-
dent cultures. *P < 0.05 as compared to dimethylsulfoxide-treated control. Similar results were obtained in three separate experiments.
Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al.
1932 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS
changes of antiapoptotic NF-jB target genes such as
Bcl-2, Bcl-xL, and the inhibitors of apoptosis (IAPs)
[21]. In addition, it was recently reported that most
glioblastoma cell lines exhibit only low constitutive
NF-jB activity, and inhibition of NF-jB did not sig-
nificantly influence apoptosis induced by DNA dam-
age, TRAIL and PIs in glioma cells in this study [35].
Although inactivation of NF-jB has been suggested to
play a major role in the antitumorigenic effect of the
PI bortezomib (Velcade ⁄ PS-341) in multiple myeloma
[36] and melanoma cells [26], inhibition of NF-kB is
not required to sensitize hepatocellular carcinoma cells
and lymphoma cells to apoptosis [37,38], again empha-
sizing the cancer type-specific effects of PIs.
As PIs are also known to trigger the stress-induced
JNK ⁄ c-Jun pathway [23,33], we finally asked the
question of whether this pathway might be involved
in the observed DR5 induction triggered by PIs.
RT-PCR analysis revealed a potent upregulation of
c-Jun at the transcriptional level, suggesting upstream
activation of JNKs, phosphorylation of c-Jun and
enhanced c-Jun expression by an autoregulatory feed-
forward mechanism [39]. Indeed, application of the
synthetic JNK inhibitor SP600125 significantly

serum, 100 U ÆmL
)1
penicillin, and 100 mgÆmL
)1
strepto-
mycin. Human glioma cell lines MZ-18 and MZ-54 were
established from a primary glioblastoma and a recurrent
grade IV tumor, respectively. To isolate glioma cells, tumor
specimens were homogenized, suspended in NaCl ⁄ P
i
and
centrifuged at 400 g. Pellets were resuspended in 10 mL of
medium, and plated into Petri dishes. Cultivation was per-
formed under standard conditions at 37 °C and a humidi-
fied 5% CO
2
atmosphere. When confluent monolayers had
been obtained, tumor-derived cells were trypsinized and
replated in new Petri dishes for serial passaging. All newly
established cell lines were analyzed for glial fibrillary acidic
protein expression by immunostaining with a monoclonal
glial fibrillary acidic protein IgG (R&D Systems,
Wiesbaden, Germany). Isotypic primary antibody (Serotec,
Du
¨
sseldorf, Germany) was used as control.
RT-PCR
Extraction of total cellular RNA, reverse transcription and
PCR were performed as previously described [40]. Primer
sequences were as follows: CHOP-sense, 5¢-GGT

instructions. For analysis of DR5 surface expression, cells
were stained with goat polyclonal anti-DR5 IgG (Axxora,
Gru
¨
nberg, Germany) and a goat IgG isotype control
(SouthernBiotech, Birmingham, AL, USA), respectively,
according to the manufacturer’s instructions. In all cases, a
minimum of 10
4
events per sample were acquired. Flow
cytometric analyses were performed on a FACScan (BD
Biosciences; Heidelberg, Germany) followed by analysis
using cellquest and winmdi software.
Gene silencing using siRNA
The following annealed double-stranded siRNAs from
Dharmacon (Chicago, IL, USA) were used: CHOP siGe-
nome duplexes D-004819-01-0005 and D-004819-02-0005;
and DR5 siGenome duplexes D-004448-01-0005 and
D-004448-03-0005. Scrambled siRNA siCONTROL from
Dharmacon was used as a negative, nonsilencing control.
Cells were transfected with 100 nm siRNAs using siM-
PORTER (Biomol, Hamburg, Germany) as described by
the manufacturer.
Statistics
Data are given as means ± SEM. For statistical compari-
son, a t-test or one-way ANOVA followed by a Tukey test
were employed using spss software (SPSS GmbH Software,
Munich, Germany). P-values smaller than 0.05 were consid-
ered to be statistically significant.
Acknowledgements

Smac agonists sensitize for Apo2L ⁄ TRAIL- or antican-
cer drug-induced apoptosis and induce regression of
malignant glioma in vivo. Nat Med 8, 808–815.
6 Hao C, Beguinot F, Condorelli G, Trencia A, Van Meir
EG, Yong VW, Parney IF, Roa WH & Petruk KC
(2001) Induction and intracellular regulation of tumor
necrosis factor-related apoptosis-inducing ligand
(TRAIL) mediated apotosis in human malignant glioma
cells. Cancer Res 61, 1162–1170.
7 Takeda K, Smyth MJ, Cretney E, Hayakawa Y,
Kayagaki N, Yagita H & Okumura K (2002) Critical
role for tumor necrosis factor-related apoptosis-induc-
ing ligand in immune surveillance against tumor
development. J Exp Med 195, 161–169.
8 Song JH, Song DK, Pyrzynska B, Petruk KC,
Van Meir EG & Hao C (2003) TRAIL triggers
apoptosis in human malignant glioma cells through
extrinsic and intrinsic pathways. Brain Pathol 13, 539–
553.
9 Adams J (2004) The proteasome: a suitable antineoplas-
tic target. Nat Rev Cancer 4, 349–360.
10 Richardson PG, Barlogie B, Berenson J, Singhal S,
Jagannath S, Irwin D, Rajkumar SV, Hideshima T,
Xiao H, Esseltine D et al. (2005) Clinical factors pre-
dictive of outcome with bortezomib in patients with
relapsed, refractory multiple myeloma. Blood 106,
2977–2981.
11 Aghajanian C, Soignet S, Dizon DS, Pien CS, Adams J,
Elliott PJ, Sabbatini P, Miller V, Hensley ML, Pezzulli
S et al. (2002) A phase I trial of the novel proteasome

ama Y, Tomita K, Yamamoto H, Konishi M & Oki T
(1992) Epoxomicin, a new antitumor agent of microbial
origin. J Antibiot (Tokyo) 45, 1746–1752.
18 Hetschko H, Voss V, Horn S, Seifert V, Prehn JH &
Ko
¨
gel D (2008) Pharmacological inhibition of Bcl-2
family members reactivates TRAIL-induced apoptosis
in malignant glioma. J Neurooncol 86, 265–272.
19 Yamaguchi H & Wang HG (2004) CHOP is involved in
endoplasmic reticulum stress-induced apoptosis by
enhancing DR5 expression in human carcinoma cells.
J Biol Chem 279, 45495–45502.
20 Yoshida T, Shiraishi T, Nakata S, Horinaka M,
Wakada M, Mizutani Y, Miki T & Sakai T (2005)
Proteasome inhibitor MG132 induces death receptor 5
through CCAAT ⁄ enhancer-binding protein homologous
protein. Cancer Res 65, 5662–5667.
21 Concannon CG, Koehler BF, Reimertz C, Murphy
BM, Bonner C, Thurow N, Ward MW, Villunger A,
Strasser A, Kogel D et al. (2007) Apoptosis induced by
proteasome inhibition in cancer cells: predominant role
of the p53 ⁄ PUMA pathway. Oncogene 26, 1681–1692.
22 Wu GS, Burns TF, McDonald ER III, Jiang W, Meng
R, Krantz ID, Kao G, Gan DD, Zhou JY, Muschel R
et al. (1997) KILLER ⁄ DR5 is a DNA damage-inducible
p53-regulated death receptor gene. Nat Genet 17,
141–143.
23 Meriin AB, Gabai VL, Yaglom J, Shifrin VI &
Sherman MY (1998) Proteasome inhibitors activate

11, 1175–1193.
30 Ganten TM, Haas TL, Sykora J, Stahl H, Sprick MR,
Fas SC, Krueger A, Weigand MA, Grosse-Wilde A,
Stremmel W et al. (2004) Enhanced caspase-8
recruitment to and activation at the DISC is critical for
sensitisation of human hepatocellular carcinoma cells to
TRAIL-induced apoptosis by chemotherapeutic drugs.
Cell Death Differ 11(Suppl. 1), S86–S96.
31 Qin JZ, Ziffra J, Stennett L, Bodner B, Bonish BK,
Chaturvedi V, Bennett F, Pollock PM, Trent JM,
Hendrix MJ et al. (2005) Proteasome inhibitors trigger
NOXA-mediated apoptosis in melanoma and myeloma
cells. Cancer Res 65, 6282–6293.
32 Wagenknecht B, Hermisson M, Eitel K & Weller M
(1999) Proteasome inhibitors induce p53 ⁄ p21-indepen-
dent apoptosis in human glioma cells. Cell Physiol
Biochem 9, 117–125.
33 Yin D, Zhou H, Kumagai T, Liu G, Ong JM, Black
KL & Koeffler HP (2005) Proteasome inhibitor PS-341
causes cell growth arrest and apoptosis in human glio-
blastoma multiforme (GBM). Oncogene 24, 344–354.
34 Karin M & Ben-Neriah Y (2000) Phosphorylation
meets ubiquitination: the control of NF-[kappa]B activ-
ity. Annu Rev Immunol 18, 621–663.
35 La Ferla-Bruhl K, Westhoff MA, Karl S, Kasperczyk
H, Zwacka RM, Debatin KM & Fulda S (2007) NF-
kappaB-independent sensitization of glioblastoma cells
for TRAIL-induced apoptosis by proteasome inhibition.
Oncogene 26, 571–582.
36 Hideshima T, Chauhan D, Richardson P, Mitsiades C,