[Cancer Biology & Therapy 7:4, 603-608; April 2008]; ©2008 Landes Bioscience
Bortezomib (VELCADE®), formerly known as PS-341, is a
novel dipeptide boronic acid proteasome inhibitor with in vitro
and in vivo anti-tumor activity. Bortezomib has been approved for
the treatment of multiple myeloma and mantle cell lymphoma.
In this report, we examined the sensitivity of cell lines derived
from Ewing’s sarcoma-family of tumors (ESFT) to Bortezomib.
Five ESFT-derived cell lines, TC-71, TC-32, SK-N-MC, A4573
and GRIMES, were highly sensitive to Bortezomib (IC50 = 20 to
50 nM), and underwent cell cycle arrest and apoptosis following
drug treatment. Bortezomib-induced apoptosis was associated with
activation of caspase 3, cleavage of PARP and induction of p27
and p21 expression. Moreover, Bortezomib exhibited synergistic
activity against the TC-71 and TC-32 cell lines when combined
with TRAIL. Our results suggest that Bortezomib might be a
useful agent for treatment of ESFT, when used alone or in combi-
nation with TRAIL.
Ewing’s sarcoma family of tumors (ESFT), which include Ewing’s
sarcoma (ES), Askin’s tumor and primitive neuroectodermal tumors
(PNET), arise in bone and soft tissue. They account for 10–15% of
all primary bone tumors and are the second most common malignant
bone tumors occurring in children and young adults.1 Histologically,
ESFT present as small blue round cell tumors with weak neural
differentiation. These tumors are usually associated with a t(11;22)
(q24;q12) chromosomal translocation, which results in the produc-
tion of the EWS-FLI1 fusion protein, a chimeric transcription factor
with oncogenic properties.2 ESFT present with localized disease
in approximately 75% of patients and local therapy, consisting of
surgery and radiotherapy combined with systemic chemotherapy
is able to achieve a 70% cure rate.3 In contrast, the prognosis of
patients with metastases or early relapse remains extremely poor (cure
rates below 25%) despite intensive chemo- and radiation-therapy,
including administration of high dose chemotherapy followed by
stem cell transplantation.3 Moreover, the outcome of patients with
advanced disease has not substantially changed over the last two
decades.3 Thus, there is an urgent need for more effective therapies
for the treatment of these tumors.
Proteasome is a multi-catalytic proteinase complex responsible
for the degradation of most intracellular proteins, including proteins
crucial to cell cycle regulation and apoptosis.4 Bortezomib is a selec-
tive inhibitor of the 26S proteasome and has been approved by
the U.S. Food and Drug Administration for treatment of multiple
myeloma and mantle cell lymphoma.4 Preclinical and early clinical
data suggest that Bortezomib also has significant anti-tumor activity
against several solid tumors, including prostate cancer, ovarian cancer
and squamous cell cancer of the head and neck.4-6
Apo2 ligand (Apo2L) or tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) is one of the members of the
TNF gene superfamily that induce apoptosis through engagement
of its receptors Death receptors 4 and 5 and is currently in clinical
trials for treatment of human malignancies.7 We and others have
previously reported that ESFT-derived cell lines express the TRAIL
receptors and are generally sensitive to TRAIL/Apo2L induced
In this report, we have examined the effect of Bortezomib, when
used alone or in combination with TRAIL/Apo2L, against a group of
five ESFT-derived cell lines. We report that Bortezomib is cytotoxic
to these ESFT-derived cell lines and has a synergistic apoptotic effect
when combined with TRAIL/Apo2L.
Bortezomib inhibits cell proliferation and reduces viability
in ESFT cell lines. We examined the effect of Bortezomib on the
viability of five cell lines (TC-71, TC-32, GRIMES, SK-N-MC
and A4573) derived from the ES family of tumors. For comparison,
we included the H460 cell line, which is derived from a human
Proteasome inhibitor Bortezomib induces cell cycle arrest and
apoptosis in cell lines derived from Ewing’s sarcoma family
of tumors and synergizes with TRAIL
Guangrong Lu,1 Vasu Punj2 and Preet M. Chaudhary1,2,*
1Hamon Center for Therapeutic Oncology Research; University of Texas Southwestern Medical Center; Dallas, Texas USA; 2Department of Medicine and University of Pittsburgh
Cancer Institute; Pittsburgh, Pennsylvania USA
Abbreviations: TRAIL, TNF-related apoptosis inducing ligand; Apo2L, Apo2 ligand; ESFT, Ewing’s sarcoma family of tumors
Key words: Ewing’s sarcoma, Bortezomib (PS-341), apoptosis, TRAIL
*Correspondence to: Preet M. Chaudhary; Hillman Cancer Center; 5117 Centre
Avenue; Suite 1.19A; Pittsburgh, Pennsylvania 15213-1863 USA; Tel.: 412.623.7703;
Fax: 412.623.1415; Email: email@example.com
Submitted: 12/19/07; Revised: 01/11/08; Accepted: 01/11/08
Previously published online as a Cancer Biology & Therapy E-publication:
www.landesbioscience.comCancer Biology & Therapy603
Effect of Bortezomib on Ewing’s sarcoma family of tumors
non-small cell lung tumor. Exponentially growing cultures of all
cell lines were treated with various concentrations of Bortezomib
for 48 hours, and cell proliferation and viability were examined
using a non-radioactive cell proliferation assay. The calculated IC50
values for TC-71, A4573, SK-N-MC, TC-32 and GRIMES cell
lines were approximately 15 ng/ml (40 nM), 7.5 ng/ml (20 nM),
7.5 ng/ml (20 nM), 20 ng/ml (50 nM) and 20 ng/ml (50 nM),
respectively (Fig. 1). In comparison, the IC50 for the H460 cell line
was 50 ng/ml (100 nM) (Fig. 1), which is 2 to 5 times higher than
the values obtained for the ESFT cell lines.
Effect of bortezomib on cell cycle in ESFT cell lines. In order to
understand the mechanism by which Bortezomib inhibits the growth
of ESFT cells, we analyzed its effect on the cell cycle distribution
of TC-71 and A4573 cell lines, two representative cell lines of the
group. We treated the two cell lines with a dose of drug equal to their
respective IC50 values of 15.0 ng/ml and 7.5 ng/ml, respectively, and
examined the effect of drug treatment on cell cycle distribution at 4,
8 and 16 h post-treatment (Fig. 2A and B, upper). Bortezomib treat-
ment was found to induce a time-dependent increase in cells in the
pre-G0/G1 phase, which usually contain apoptotic cells. Thus, the
percentage of cells in the pre-G0/G1 region at 16 h post-Bortezomib
treatment increased from 5.3% in the untreated control to 24.3%
in the case of TC-71 cells, and from 3.49% to 21.52% in the case
of A4573 cells (Fig. 2A). Similarly, Bortezomib treatment of TC-71
cells resulted in a dose-dependent increase in the pre-G0/G1 popula-
tion from 5.3% in untreated control to 9.3%, 14.9% and 30.0% in
cells treated with 2.5, 5 and 15 ng/ml of Bortezomib for 12 h, respec-
tively (Fig. 2A, lower). Similar results were obtained upon treatment
of A4573 cells with increasing concentrations of Bortezomib (Fig.
Next, we analyzed the effect of Bortezomib on the cell cycle distri-
bution of non-apoptotic cells. TC-71 and A4573 cells were treated
with Bortezomib at their respective IC50 concentrations of 15 ng/ml
and 7.5 ng/ml, and the distribution of non-apoptotic cells in the non-
cycling (G0/G1) and cycling (S + G2/M)
phases were measured by flow cytometry.
In TC-71 cells, a time-dependent decrease
was observed in the percentage of non-
apoptotic cells in the G0/G1 phase—from
55.7% in the untreated control to 50.7%
and 20.98% in the cells that have been
treated with the drug for 8 h and 16 h,
respectively. This was accompanied by a
corresponding increase in the percentage of
cells in the S + G2/M phase, from 44.2%
in the untreated control to 49.2% and
79.0% at 8 h and 16 h, respectively (Fig.
2C). Bortezomib also resulted in a dose-
dependent accumulation of TC-71 cells in
the S + G2/M phase from 55.59% in the
untreated control to 69.76% in cells treated
with 15 ng/ml of Bortezomib for 12 h (Fig.
2C). The effect of Bortezomib on the accu-
mulation of cells in the S + G2/M phase,
however, was not limited to TC-71 cell
line, but was also observed in the A4573
cells. Thus, the percentage of cells in the
S + G2/M phase increased from 38.45% in the untreated control to
45%, 48.6% and 78.0% following 4, 8 and 16 h treatment of A4573
cells with 7.5 ng/ml of Bortezomib (Fig. 2C). Similarly, treatment
with increasing doses of Bortezomib resulted in a progressive increase
in the S + G2/M phase cells from 42.33% in the untreated control to
60.0%, and 76.5% in cells treated for 12 h with 3.75 and 7.5 ng/ml
of Bortezomib (Fig. 2C). Taken together, the above results suggested
that Bortezomib caused a G2/M arrest of T-71 and A4573 cells,
which was followed by the induction of apoptosis.
Induction of apoptosis in bortezomib treated ESFT cell lines.
Proteasome inhibitors preferentially induce apoptosis in malignant
cells by interfering with degradation of pro-apoptotic molecules,
which are usually cleared through the ubiquitin-proteasome prote-
olytic pathway.4 The appearance of sub G0/G1 cells in the cell
cycle analysis suggested that Bortezomib reduces cell viability by
induction of apoptosis. Therefore, we performed morphological
examination of Bortezomib-treated cells and found typical features
of apoptosis, such as cell rounding, detachment and fragmentation
into small apoptotic bodies (Fig. 3A). Furthermore, nuclear staining
with Hoechst 33342 showed that the nuclei of Bortezomib-treated
cells were condensed and fragmented, which is another hallmark of
apoptotic cells (Fig. 3B).
Nuclear fragmentation during Bortezomib-induced apoptosis
suggested the possible involvement of caspases. Caspase 3 is one
of the executioner caspases of the caspase cascade and is known
to be involved in apoptosis induced by both the intrinsic and
extrinsic apoptosis pathways.12 Therefore, we examined the effect
of Bortezomib treatment on caspase activation in TC-71 cells as a
representative of the group. Caspase 3 was cleaved in a time- and
dose-dependent manner, with significant cleavage observed following
exposure to 15 ng/ml of Bortezomib for 24 h (Fig. 4). This suggested
the activation of caspase 3. Bortezomib treatment also led to PARP
cleavage, one of the substrates for caspase 3, lending more support
for induction of apoptosis (Fig. 4).
Figure 1. Bortezomib inhibits proliferation of ESFT-derived cell lines. 6 x 103 cells from the indicated
ESFT cell lines were plated in each well of 96-well plate and next day treated with a different dose of
Bortezomib in triplicate. Cell viability was measured after 48 hours using the MTS assay. Data shown
(mean ± SEM) is from a representative of two independent experiments performed in triplicate. H460
cells were included for comparison.
604 Cancer Biology & Therapy2008; Vol. 7 Issue 4
Effect of Bortezomib on Ewing’s sarcoma family of tumors
www.landesbioscience.com Cancer Biology & Therapy605
Matsumoto et al. (2001) have recently reported that EWS-Fli1
attenuates the cell cycle inhibitory p27 protein level via activation of
the proteasome-mediated degradation pathway, and forced expres-
sion of p27 inhibits ES cell growth.13 Therefore, we explored whether
Bortezomib-induced cell growth inhibition was associated with p27
induction. We assayed the levels of p27 and p21, a related cell cycle
inhibitory protein, after treatment of TC-71 cells with 15 and 25
ng/ml of Bortezomib and found that they were strongly induced at
24 h following drug treatment. This induction of p27 and p21 was
p53-independent, because no expression of p53 was detected in the
TC-71 cells in their basal state or following
Bortezomib treatment (Fig. 4).
Synergistic growth inhibition and apop-
tosis in ESFT cell lines upon combined
treatment with bortezomib and TRAIL/
Apo2L. TRAIL (also known as Apo2L)
is a promising agent for the treatment of
drug-resistant cancers as it has been shown
to selectively induce apoptosis in cell lines
derived from diverse tumors without affecting
the normal cells.14,15 We, as well as others,
have previously reported that cell lines derived
from ES are sensitive to TRAIL-induced
apoptosis.8-10 Since proteasome inhibitors
are known to enhance TRAIL-induced
apoptosis of cancer cells,16 we compared the
effects of combining TRAIL and Bortezomib
on the ESFT cell lines. For this purpose, we
treated TC-71 cells with different concen-
trations of Bortezomib (1–10 ng/ml) and
TRAIL (20–100 ng/ml) either alone or in
combination for 24 h, and measured cell
viability using the MTS assay. Combination
index (CI) values for each fraction affected
were calculated by median drug effect anal-
ysis according to the method of Chou and
Talalay.17 In this method, CI <1.0 indicates
synergy; CI = 1.0 indicates an additive effect;
and CI >1.0 indicates antagonism. A strong
synergy between Bortezomib and TRAIL
was observed in experiments involving
higher doses (10 ng/ml) of Bortezomib (CI =
.003) (Fig. 5A). A microscopic examination
revealed that the synergistic cytotoxic effect
of TRAIL and Bortezomib combination was
due to the induction of apoptosis (Fig. 5C).
A synergistic cytotoxic interaction between
Bortezomib and TRAIL was, however, not
limited to the TC-71 cells. As shown in
Fig. 5B, combined treatment of TC-32 cells
with varying concentrations of Bortezomib
(2.5–10 ng/ml) and TRAIL (20–100 ng/
ml) yielded combination indices of <1.0,
indicating synergistic interactions.
Bortezomib is a novel dipeptide boronate
proteasome inhibitor, which has shown potent anticancer activity
against a variety of cancer cell lines both in vitro and in vivo.4-6 Here
we present evidence that cell lines derived from ESFT were also sensi-
tive to Bortezomib (Fig. 1). The IC50 for the ESFT cell lines were
about 2 to 5 times less than the IC50 for H460 cells (100 nM), a non-
small cell lung cancer cell line with neuroendocrinie features, which
was one of the most sensitive lung cancer cell lines in a recently
reported study of Bortezomib.5 Additionally, the IC50 values of the
ESFT cell lines were between 2 to 14 times less than the IC50 values
of >25 ng/ml and >100 ng/ml reported previously for two multiple
Figure 2. Effect of Bortezomib on cell cycle in ESFT cell lines. (A and B) Exponentially growing TC71 and
A4573 cells were treated for the indicated time intervals with 15 ng/ml and 7.5 ng/ml of Bortezomib
(upper), or with indicated concentrations of the drug for 12 h (lower). Cells were subsequently fixed
in 70% ethanol, stained with PI and the cell cycle distribution analyzed using a flow cytometer. The
numbers represent the percentage of cells in the different phases of cell cycle. M1, Sub-G0/G1; M2,
G0/G1; M3, S and M4, G2/M phases. (C) Relative distribution of non-apoptotic cells between the
non-cycling (G0/G1) and cycling (S + G2/M) phases following exposure of TC-71 and A4573 cells to
different concentrations of Bortezomib or to different durations of Bortezomib treatment.
Effect of Bortezomib on Ewing’s sarcoma family of tumors
606 Cancer Biology & Therapy2008; Vol. 7 Issue 4
myeloma cell lines U266 and ARH-77, respectively.16 Thus, ESFT-
derived cell lines were even more sensitive to Bortezomib than those
derived from multiple myeloma, a disease for which Bortezomib has
been approved for clinical use.18
The ubiquitin-proteasome pathway plays a pivotal role in the
regulated degradation of intracellular damaged and misfolded
proteins as well as rapid degradation of a number of short-lived
proteins involved in the control of cell cycle, tumor growth and
metastasis, such as cyclins and cyclin-dependent kinase inhibi-
tors.4 Consistent with previous reports with prostate and lung
cancer cell lines,5,19 treatment of ESFT cell lines with Bortezomib
resulted in cell cycle arrest at the G2/M phase and an induction of
apoptosis (Fig. 3). Apoptosis induction was also supported by our
observed cleavage of caspase 3 and PARP (Fig. 4).
Interestingly, Matsumoto et al. (2001) have recently
reported that EWS-Fli1 attenuates p27 protein
level via activation of the proteasome-mediated
degradation pathway, and forced expression of p27
inhibits ES cell growth.13 Therefore, it is conceiv-
able that Bortezomib exerts its anti-proliferative
effect on ES cell lines by blocking EWS-FLi1-
induced stimulation of proteasome activity, thereby
upregulating the expression of p27 and its related
cell cycle regulatory proteins that are normally
degraded by the proteasome. We found support
of this hypothesis in our experimental results of
p53-independent induction of the cell cycle inhibi-
tors p27 and p21 on Bortezomib treatment (Fig.
4). Bortezomib has been also shown to activate
apoptosis by inducing endoplasmic stress,20 and it
is possible that this mechanism also contributes to
its anti-proliferative and cytotoxic activity against
ESFT-derived cell lines.
A recent phase II study of Bortezomib as a single
agent in recurrent or metastatic sarcomas showed
minimal activity and excessive toxicity.21 However,
this study included only two patients with Ewing’s sarcoma as
the arm enrolling patients with Ewing’s sarcoma had to be closed
early due to poor accrual. Although the limited number of Ewing’s
sarcoma patients accrued in this study do not entirely rule out a
role for single-agent Bortezomib against this disease, the authors
of the study suggested that if this drug is to be developed in the
future for sarcoma patients, it should be used in combination with
agents with demonstrated preclinical synergy.21 In this context, our
results showing a synergistic interaction between Bortezomib and
TRAIL/Apo2L in inducing apoptosis in ESFT cell lines gain added
significance. Bortezomib is known to sensitize tumor cells to TRAIL-
induced apoptosis via multiple mechanisms including upregulation
of the pro-apoptotic members of the Bcl2 family (Bik and Bim),
downregulation of cFLIP and Mcl-1 and release of Smac/DIABLO,
and it is possible that some or all of the above mechanisms also
contribute to its sensitizing effect on the ESFT cell lines.22-25 Finally,
TRAIL is known to activate the anti-apoptotic NFκB pathway,26 and
inhibition of this pathway by Bortezomib may contribute to its sensi-
tization of TRAIL-induced apoptosis. However, we have previously
reported that TRAIL failed to activate the NFκB pathway in ESFT
cell lines,8 and, therefore, it is unlikely that Bortezomib sensitized
ESFT cell lines to TRAIL-induced apoptosis via NFκB inhibition.
In conclusion, we demonstrated that Bortezomib, a protea-
some inhibitor approved for cancer therapy, was cytotoxic to five
ESFT-derived cell lines in vitro. The two possible mechanisms of
cytotoxicity were found to be induction of cell cycle arrest and
apoptosis. Bortezomib was also found to synergistically enhance the
cytotoxicity of TRAIL against ESFT cell lines. Thus, Bortezomib,
alone or together with TRAIL, might be a promising anti-cancer
drug for this family of difficult-to-treat tumors.
Materials and Methods
Cell lines and reagents. TC-71, TC-32, SK-N-MC, A4573,
GRIMES and H460 cell line have been described previously.8 Among
Figure 3. Bortezomib induces apoptosis in ESFT cell lines. Approximately 5000 cells of each
cell line were plated in each well of 96-well plate and next day treated with the IC50 concentra-
tions of Bortezomib in triplicate. Cells were stained with Hoechst 33342 and examined under
phase-contrast and fluorescence microscopes 48 h after drug treatment.
Figure 4. Bortezomib induces caspase activation in TC-71 cells. TC-71 cells
were treated with 15 ng/ml of Bortezomib for the indicated time intervals
(left) or with the indicated concentrations of Bortezomib for 24 h (right).
Cell lysates were prepared from the drug-treated and untreated cells and
the lysates were analyzed by Western blots with antibodies against cleaved
caspase 3, PARP, p27, p21 and p53. Actin blots served as loading controls.
BC-3, a human primary effusion lymphoma cell line, served as a positive
control for p53 expression.
Effect of Bortezomib on Ewing’s sarcoma family of tumors
www.landesbioscience.com Cancer Biology & Therapy607
these, the TC-71, A4573 and GRIMES cell lines were derived from
ES, and the TC-32 and SK-N-MC cell lines were derived from
PNET. H460 cell line was derived from a human a non small cell
lung tumor. All cells were grown in RPMI 1640 supplemented with
10% fetal bovine serum in a humidified atmosphere of 5% CO2
and 95% air at 37°C. Bortezomib was obtained from Millennium
Pharmaceuticals Inc. (Cambridge, MA), dissolved in DMSO and
diluted to the required concentration in PBS. Recombinant human
TRAIL was purchased from R&D systems (Minneapolis, MN).
Cleaved PARP and cleaved Caspase3 antibodies were purchased from
Cell signaling Technology (Danvers, MA). HRP-conjugated anti-
mouse and anti-rabbit secondary antibodies were bought from Bio
Rad Laboratories (Hercules, CA). Hoechst 33342 and propidium
iodide were purchased from Sigma (St. Louis, MO).
Cell viability assay. Cell viability was measured using CellTiter
96® Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega,
Madison WI). This assay used the soluble tetrazolium salt, MTS
sulfophenyl)-2H-tetrazolium, inner salt], and the electron coupling
reagent, phenazine methosulfate (PMS). MTS and PMS detection
reagents were mixed, using a ratio of 20:1 (MTS:PMS) immedi-
ately prior to addition to the cell culture at a ratio of 1:5 (detection
reagents: cell culture medium). The microplates were incubated for
4 hours under identical conditions before absorbance was measured
at 492 nm.
Hoechst 33342 staining. For the measurement of apoptosis, cells
were stained with Hoechst 33342 (10 μg/ml) for 30 minutes at 37°C
and then photographed under a Fluorescence microscope.
Western blot analysis. Western blot analysis was performed
essentially as described previously.11 Primary antibodies used in these
experiments included cleaved caspase 3 and cleaved PARP (1:5000
dilution), p53, p27, p21, Bcl2, (1:2000 dilution) and anti-mouse and
anti-rabbit HRP-conjugated secondary antibodies (1:5000 dilution).
Cell cycle analysis. In order to assess the effect of Bortezomib
on cell cycle, cells (1 x 106) were either treated for 12 hours with
different doses (2.5, 5, 15 ng/ml for TC71 and 1, 3.75, 7.5 ng/ml
for A4573) or treated for different time intervals (4, 8 and 16 h) with
a fixed concentration of Bortezomib (15 ng/ml for TC-71 and 7.5
ng/ml for A4573). Cells were harvested, washed with cold PBS, fixed
with cold 75% ethanol at 4°C overnight, incubated with 5 μg/ml of
RNase A at 37°C for 30 minutes, washed 3 times with cold PBS,
and then stained with 500 μl of 1 μg/ml of propidium iodide (PI)
at room temperature for 3 hours in the dark. The number of cells
in different stages of cell cycle and the apoptotic cells (sub-G1) were
measured by flow cytometry (BD Biosciences, San Jose, CA) using
the CellQuest software.
This work was supported by a grant from the Nearburg Family
Foundation (to P.M.C.). We would like to thank Dr. Siddhartha Kar
for a critical read of the manuscript and helpful suggestions.
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apoptosis in TC-71 cells. Cells were treated in triplicate with varying concen-
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or in combination, and cell viability was measured 24 h post-treatment using
the MTS assay. Synergy was quantified by median-dose effect analysis using
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