Multitarget siRNA inhibition of antiapoptotic genes (XIAP, BCL2, BCL-X(L)) in bladder cancer cells.

Abstract

The knockdown of XIAP, BCL2 and BCL-X(L) by siRNAs represents a promising treatment option for bladder cancer (BCa) since the overexpression of antiapoptotic genes is often associated with tumor progression and treatment resistance.
EJ28 BCa cells were transfected with siRNAs--separately and combined--followed by analysis of target expression, viability, clonogenic survival, apoptosis and cell cycle. Furthermore, a possible chemosensitization by siRNA pretreatment was investigated.
The siRNA-mediated inhibition of these targets--either separately or combined--reduced the targets' expression, reduced cell growth and sensitized cells to a subsequent chemotherapy.
Since tumor cells may bypass the inhibition of a single gene by changing their expression profile, e.g. switch from BCL2 to BCL-X(L), the combined knockdown of multiple genes of the same pathway might be more effective in killing cancer cells. The siRNAs used represent appropriate tools for this aim since they reduced their targets' expression significantly and long-lastingly.

Full text

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Abstract. Background: The knockdown of XIAP, BCL2 and
BCL-XLby siRNAs represents a promising treatment option for
bladder cancer (BCa) since the overexpression of antiapoptotic
genes is often associated with tumor progression and treatment
resistance. Materials and Methods: EJ28 BCa cells were
transfected with siRNAs – separately and combined – followed
by analysis of target expression, viability, clonogenic survival,
apoptosis
chemosensitization by siRNA pretreatment was investigated.
Results:The siRNA-mediated inhibition of these targets – either
separately or combined – reduced the targets’ expression,
reduced cell growth and sensitized cells to a subsequent
chemotherapy. Conclusion: Since tumor cells may bypass the
inhibition of a single gene by changing their expression profile,
e.g. switch from BCL2 to BCL-XL, the combined knockdown of
multiple genes of the same pathway might be more effective in
killing cancer cells. The siRNAs used represent appropriate
tools for this aim since they reduced their targets’ expression
significantly and long-lastingly.
andcell cycle.Furthermore,a possible
In 2007, an estimated 50,040 new cases of bladder cancer
(BCa)
approximately 9,630 men will die of the disease (1). These
numbers make BCa the fourth most common malignancy and
the eighth leading cause of cancer deaths in men. At the time
of diagnosis, 70-80% of all BCa are superficial and will be
treated by transurethral resection (TUR-B) (2). However,
50-70% of these BCa will recur and 5-20% will progress to
muscle invasive cancer (2). Instillation therapies, e.g. using
will be diagnosed in men in the US, and
bacillus Calmette-Guérin (BCG) or the chemotherapeutic
agent mitomycin C (MMC), can reduce the recurrence rate,
but MMC does not effect survival and BCG is not active in
about one third of patients (3). Furthermore, BCG and MMC
both cause local (e.g. dysuria, cystitis) and systemic (e.g.
fever, malaise, nausea) side-effects (4).
To improve existing instillation therapies and to reduce their
side-effects, combined treatments with specific nucleic acid
inhibitors such as antisense oligodeoxynucleotides (AS-ODNs)
or small interfering RNAs (siRNAs) might be suitable. siRNAs
are synthetic double-stranded RNA molecules (21-23 basepairs
in length) which reduce their target’s expression by inducing
RNA interference (RNAi) (5). Attractive targets for gene
inhibition should be selectively up-regulated in tumor cells and
should have an essential function in cancer-promoting
pathways (6), such as the selected antiapoptotic targets XIAP,
BCL2 and BCL-XL(7, 8). Furthermore, their overexpression in
BCa and other tumors is often associated with poor prognosis
and resistance to radiotherapy and chemotherapy (CT) (7-12).
XIAP (X-linked inhibitor of apoptosis protein, hILP,
BIRC4), an important member of the inhibitor of apoptosis
protein family, directly binds to and inhibits both initiator
caspase-9 and effector caspase-3 and -7. Thereby, XIAP can
suppress apoptosis triggered by different stimuli such as the
mitochondrial and the death receptor-mediated pathways (8).
AS-ODN-mediated inhibition of XIAP reduced protein
expression and sensitized T24 BCa cells to doxorubicin (13).
Similar sensitization effects after XIAP down-regulation
were described for several cancer types including prostate,
lung and ovarian, as well as for melanoma cells (8, 14).
BCL2 (B-cell lymphoma 2) and BCL-XL(BCL2-like 1,
BCL2L1), two antiapoptotic members of the BCL2 family, are
mitochondrial membrane proteins which act by preventing the
release of mitochondrial cytochrome c into the cytoplasm (15).
Release of cytochrome c would lead to apoptosome assembly
and thereby to the activation of caspase-9 (15). Studies in BCa
cell lines describeAS-ODN-mediated down-regulation of BCL2
or BCL-XL, which was frequently accompanied by apoptosis
induction and chemosensitization (7, 9, 11, 13, 16). In tumor
cells expressing both BCL2 and BCL-XL, the prediction which
2259
*Both authors contributed equally to this work.
Correspondence to: Doreen Kunze, Department of Urology,
Technical University of Dresden, Fetscherstrasse 74, D-01307
Dresden, Germany. Tel: +49 (0) 351 4584544, Fax: +49 (0) 351
4585771, e-mail: doreen.kunze@uniklinikum-dresden.de
Key Words: BCL2, BCL2L1, BIRC4, siRNA, chemosensitization,
inhibition of apoptosis.
ANTICANCER RESEARCH 28: 2259-2264 (2008)
Multitarget siRNA Inhibition of Antiapoptotic Genes
(XIAP, BCL2, BCL-XL) in Bladder Cancer Cells
DOREEN KUNZE1*, DANIELA WUTTIG1*, SUSANNE FUESSEL1, KAI KRAEMER1, MATTHIAS KOTZSCH2,
AXEL MEYE1, MARC-OLIVER GRIMM1, OLIVER W. HAKENBERG3and MANFRED P. WIRTH1
1Department of Urology and2Institute of Pathology, Technical University of Dresden, Dresden;
3Department of Urology, Rostock University, Rostock, Germany
0250-7005/2008 $2.00+.40

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of these two proteins is more important for cell survival is
difficult (10). Furthermore, tumor cells have been found to
switch the expression from BCL2 to BCL-XL(17). Therefore, a
combined inhibition of both targets seems to be promising.
To date, no results of siRNA-mediated inhibition of selected
targets in BCa cells have been published. However, their efficacy
was shown in other tumor entities, e.g. BCL2 and XIAP were
down-regulated in MCF-7 human breast cancer cells using
RNAi, thereby sensitizing cells to chemotherapeutics (18).
In the present study, the expression of XIAP, BCL2 and
BCL-XLwas reduced by siRNAs in EJ28 BCa cells. The
resulting effects on cell growth were analyzed – comparing
the seperate inhibition of one gene with the combined
reduction of two or three targets. Furthermore, effects of
siRNA pretreatment on a subsequent CT were examined.
Materials and Methods
Cell culture, siRNAs and transfection. The human BCa cell lines EJ28
(University of Frankfurt, Frankfurt, Germany), J82 and T24 (ATCC,
Manassas, VA, USA) were cultured under standard conditions (37˚C,
humidified atmosphere containing 5% CO2) without antibiotics in
Dulbecco’s modified Eagle’s medium (4.5 g/l glucose) containing
10% fetal calf serum, 1% MEM and 1% HEPES (all from
Invitrogen, Karlsruhe, Germany). The target-directed siRNAs (Table
I) were synthesized by Eurogentec (Seraing, Belgium) and the non-
silencing-siRNA
normalization was purchased from Qiagen (Hilden, Germany). After
seeding and adherence for 24 or 72 h, cells were transfected in serum-
free OptiMEM with 200 nM of one siRNA, or with combinations of
two or three siRNAs (each 100 or 67 nM) using DOTAP liposomal
transfection reagent (ratio 1:4, w/w) according to the manufacturer’s
instructions (Roche, Mannheim, Germany). Following transfection
(4 h, 37˚C), cells were washed with phosphate buffered saline (PBS)
and incubated in serum-containing medium for 24-72 h. For further
analyses, cells were harvested by trypsin treatment (0.05%
trypsin/0.02% EDTA, 5 min, 37˚C). Detached and adherent cells
were pooled and analyzed together.
control(ns-siRNA)whichwasusedfor
Chemotherapeutics
concentration 2.1 μg/ml) and MMC (final concentration 0.9 μg/ml)
were diluted in culture media. Twenty hours after siRNA
transfection, cells were incubated with the chemotherapeutics for
2 h (MMC) or 24 h (CDDP) followed by PBS washing and further
cultivation. The ns-siRNA+CT combination was used as control to
evaluate siRNA-mediated effects of the treatment.
andtreatment.
Cisplatin (CDDP;final
Viability and proliferation assays. Using the cell proliferation
reagent WST-1 (Roche) cellular viability was examined in
quadruplicates 72 h or 96 h after transfection. Colony formation
assay was performed by seeding 100 cells 24 h after treatment in
triplicates in 6-well plates. Cells were incubated for 9-12 days. After
Giemsa staining, clonogenic survival was determined by counting
macroscopically visible colonies.
Apoptosis detection and cell cycle analysis. Apoptosis was assessed
72 h and 96 h after transfection by staining cells with annexin
V/propidium iodide and analyzed using flow cytometry (Annexin
V-FITC Apoptosis Detection Kit I; FACScan; BD Biosciences,
Heidelberg, Germany). Cell cycle analysis was performed 72 h and
96 h after transfection by flow cytometry using the CycleTest Plus
DNA Reagent Kit (BD Biosciences).
RNA isolation, cDNA synthesis and quantitative PCR. Total RNA was
isolated from cell lines and tissue samples according to the
manufacturer’s instructions (Invisorb Spin Cell/Tissue RNA Mini Kit;
Invitek, Berlin, Germany) and transcribed into cDNA (SuperScript™ II
Reverse Transkriptase; Invitrogen). Transcript amounts of XIAP and the
reference gene TBP (TATA box binding protein) (19) were determined
by quantitative PCR (qPCR) using the LightCycler (LC) FastStart DNA
Master Hybridization Probes Kit and the LC instrument (both from
Roche). For XIAP, the following primers and probes were used:
GTGATAAAGTAAAGTGCTTTCACTGT, GTAGTTCTTACCA GACA
CTCCTCAA, GTGAAGAC CCTTGGGAACAACAT-fluorescein, LC
Red640-ctaaatggtatccagggtgcaa atatctg-PH. BCL2 and BCL-XLwere
quantified by Roboscreen (Leipzig, Germany).
Western blot. A total of 5×104cells per sample were lysed in
loading buffer (20% glycerol, 2% SDS, 125 mM Tris pH 6.8, 5%
β-mercaptoethanol, bromophenol blue), incubated at 95˚C for 5 min
and separated on 4-12% NuPAGE Bis-Tris gels (Invitrogen).
Proteins were transferred to a Hybond-P PVDF membrane (GE
Healthcare, Munich, Germany). Membranes were incubated with
monoclonal primary antibodies against BCL2 oncoprotein (1:200;
clone 124; Dako, Glostrup, Denmark), BCL-XL(1:100; clone 2H12;
QED Bioscience Inc., San Diego, California, USA), or XIAP
(1:250; clone 28; BD Biosciences). β-Actin detected by a
monoclonal anti-β-actin antibody (1:15,000; Sigma, St. Louis,
Missouri, USA) served as a loading control. The secondary
polyclonal
peroxidase (HRP)-linked antibody (1:1,000; Dako) and the
Enhanced Chemiluminescence Kit (GE Healthcare) were used for
visualization. Quantification was performed using Kodak 1D image
analysis software (V 3.6; Fisher Scientific, Schwerte, Germany).
Statistics. An unpaired Student’s t-test was used to compare the
rabbit anti-mouseimmunoglobulinshorseradish
ANTICANCER RESEARCH 28: 2259-2264 (2008)
2260
Table I. Sequences of siRNAs.
TargetSequence
siRNA (sense)siRNA (antisense)
BCL2
BCL-XL
XIAP
ns-siRNA
GGC GCA CGC UGG GAG AAC AdTdT
GCA GCU UGG AUG GCC ACU UdTdT
GUG GUA GUC CUG UUU CAG CdTdT
UUC UUC GAA CGU GUC ACG UdTdT
UGU UCU CCC AGC GUG CGC CdTdT
AAG UGG CCA UCC AAG CUG CdAdG
GCU GAA ACA GGA CUA CCA CdTdT
ACG UGA CAC GUU CGG AGA AdTdT

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differences in cell viability between target-directed-siRNA+CT and
ns-siRNA+CT (*p≤0.05, **p≤0.01 and ***p≤0.001).
Results
The mRNA expression levels of BCL2, BCL-XLand XIAP in
EJ28, T24 and J82 BCa cells are shown in Table II. Since
EJ28 cells express all targets at a high or moderate level, this
cell line was chosen as model for the in vitro inhibition of
the targets.
The siRNAs used reduced the target’s mRNA expression
down to 54-61% compared to ns-siRNA-treated cells 48 h after
transfection (data not shown). Even 96 h after transfection, a
comparable significant reduction of the targeted mRNA levels to
37-59% was detected (Table III). Western blot analysis showed
protein reduction of XIAP to 52%, of BCL2 to 26% and of
BCL-XLto 34% in the single target treatments compared to ns-
siRNA treated cells 96 h after transfection. The combined
treatment of EJ28 cells with two (BCL2+BCL-XL) or three
(BCL2+BCL-XL+XIAP) siRNAs caused mRNA down-
regulation of all targets and showed a rate of mRNA inhibition
comparable to the mono-target therapies (Table III). At the
protein level, combined knockdown of BCL2+BCL-XLreduced
BCL2 to 29% and BCL-XLto 55%. Simultaneous inhibition
of BCL2+BCL-XL+XIAP down-regulated XIAP protein to
53%, BCL2 to 57% and BCL-XLto 28%.All siRNAs – either
separately or combined – sufficiently reduced the mRNA and
protein expression of their targets.
The siRNA-mediated inhibition of the three targets notably
decreased cell count 96 h after transfection (Table III). This
was caused by the induction of apoptosis which was already
prominent 72 h after transfection (Figure 1). However, the
reduction of XIAP only affected the cell count, not apoptosis.
None of the siRNA treatments (mono-target or combinations)
significantly influenced cell viability (Figure 2), cell cycle
distribution or clonogenic survival (data not shown).
Nevertheless, combined treatment with target-directed siRNAs
and chemotherapeutics provoked greater antiproliferative
effects than the application of ns-siRNA+CT. Specific
combinations of siRNA+MMC caused a significant reduction
of the cellular viability compared to the ns-siRNA+MMC
control (Figure 2). The combination of target-directed siRNAs
with CDDP reduced viability compared to ns-siRNA+CDDP,
however, the changes were not significant (Figure 2).
Moreover, subsequent CT with CDDP after blockage of the
antiapoptotic genes (single or combined, except XIAP) further
increased apoptosis (Figure 1).
Discussion
Since apoptosis is frequently dysregulated in tumor cells, for
instance by up-regulation of antiapoptotic proteins, the
manipulation of these pathways may represent an appropriate
course in treating primary and recurrent cancer (6, 10).
Overexpression of the selected targets BCL2, BCL-XLand
XIAP is associated with treatment resistance in various tumor
entities and therefore presents a limitation for existing
therapies (7, 8, 11, 14). Due to the induction of natural RNAi,
siRNAs represent attractive tools for sequence-specific gene
inhibition. With the siRNAs directed at BCL2, BCL-XLand
XIAP, a significant and long-lasting reduction of the target
mRNA
Furthermore, the tumor cell count was reduced and apoptosis
increased,
Combinational
BCL2+BCL-XL+XIAP with lower siRNA concentrations per
target (100 or 67 nM) caused similar target-mRNA reduction
and growth inhibiting rates as the mono-target therapies with
200 nM of a single siRNA. Though siRNA-monotreatment
was not able to reduce cell viability, the combination of target-
directed siRNAs with a subsequent CT with MMC or CDDP
provoked a viability decrease. Furthermore, the apoptosis
increasing effect of the target-directed siRNAs was intensified.
Kim et al. 2007 (20) describe an enhanced radiosensitivity of
grade II chondrosarcoma cells after the combined knockdown
of two of the three antiapoptotic targets BCL2, BCL-XLand
XIAP compared with the mono-target inhibition. However, not
all combinations intensified the level of radiosensitivity
obtained with the single target inhibitions. Lei et al. (21)
report that a simultaneous BCL2 and BCL-XLinhibition by
siRNA expression vectors in human hepatoblastoma cells
andprotein expressionlevelswas achieved.
indicating
treatment
theantiproliferative
against
potency.
BCL2+BCL-XL
or
Kunze et al: Multitarget Gene Inhibition in Bladder Cancer Cells
2261
Table II. Target expression normalized to TBP (zmole/zmole) in BCa
cell lines.
Cell line XIAP/TBP BCL2/TBPBCL-XL/TBP
EJ28
T24
J82
6.85
7.76
7.43
0.18
0.28
0.02
16.5
9.9
21.7
Table III. Cell count and mRNA expression relative to ns siRNA control
of EJ28 cells 96 h after siRNA transfection. Expression values are
normalized to TBP (zmole/zmole).
siRNA targeted atTotal cell count
(cell count relative
to ns-siRNA)
mRNA expression
XIAP/
TBP
BCL2/ BCL-XL/
TBP TBP
BCL2
BCL-XL
XIAP
BCL2+BCL-XL
BCL2+BCL-XL+XIAP 6.7×106(74%)
ns-siRNA
Untreated
5.3×106(58%)
6.2×106(69%)
5.0×106(55%)
5.1×106(56%)
91%
95%
46%
100%
62%
59%
111%
93%
58%
53%
100%
107%
85%
37%
96%
69%
61%
100%
83%
9.1×106(100%) 100%
8.4×106(93%) 101%

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ANTICANCER RESEARCH 28: 2259-2264 (2008)
2262
Figure 1. Rate of early and late apoptotic cells 72 h after treatment of EJ28 cells with siRNAs with or without subsequent chemotherapy with
cisplatin. Abbrevations: ns, ns-siRNA; B2, BCL2; BXL, BCL-XL; X, XIAP.
Figure 2. Relative viability of EJ28 cells 96 h after transfection with siRNA with or without subsequent chemotherapy (CT) with mitomycin C (MMC)
or cisplatin (CDDP). Except for CT monotherapies (normalized to untreated cells), values are normalized to ns-siRNA treated cells (=100%).
Asterisks indicate a significant difference compared to ns-siRNA+CT (p<0.05). Abbrevations: ns, ns-siRNA; B2, BCL2; BXL, BCL-XL; X, XIAP.

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further increased the sensitivity of the cells towards 5-
fluorouracil and 10-hydroxycamptothecin than the single
target knockdown. These results demonstrate the potential of
the simultaneous targeting of apoptosis inhibitors for
anticancer treatment. Like the switch from BCL2 to
BCL-XLexpression in human leukemia HL-60 cells (17), a
takeover of the function of one antiapoptotic protein by
another, e.g. because of the simultaneous expression of
multiple apoptosis inhibitors or changes in the expression
profile, is possible. Therefore, it is important to examine the
long-term effects of the combined silencing of apoptosis
inhibitors in in vivo studies. Furthermore, if only one gene is
targeted, determining its expression before treatment would be
required to avoid unnecessary therapy – using siRNAs against
different genes together might make this unnecessary. Of
course, it would be advantageous to add more siRNAs against
further inhibitors of apoptosis, e.g. survivin, or other tumor-
associated genes which are involved in cell cycle regulation,
angiogenesis or immortalization.
Achieved antiproliferative effects after the combined
down-regulation of all three targets – which, despite lower
siRNA concentrations used per target, were comparable to
the mono-target treatments – confirm the possibility of a
multitarget inhibition of antiapoptotic proteins.
Acknowledgements
The authors would like to express their gratitude to Joerg Hofmann
(Department of Urology) and Antje Zobjack (Institute of Pathology)
for their excellent technical assistance. This study was supported by
a grant of the Dr. Robert Pfleger-Stiftung (to S.F. & A.M.).
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and therapeuticactivityof XIAPantisense
Received January 23, 2008
Revised April 4, 2008
Accepted April 21, 2008
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