PurposeObjective:

1
Antisense MDM2 oligonucleotides sensitizes
androgen-insensitive human LNCaP prostate cancer
cells to androgen deprivation in vitro and in
vivo
Zhaomei Mu, Paul Hachem, Harvey Hensley,
Radka Stoyanova, Alexandra L Hanlon, Sudhir.
Agrawal, Alan Pollack Departments of Radiation
Oncology and Basic Science, Fox Chase Cancer
Center, Department of Public Health ,Temple
University, Philadelphia, PA, USA Idera
Pharmaceuticals, Cambridge, MA, USA
Purpose/Objective We have previously shown that
antisense MDM2 (AS) sensitizes wild type LNCaP
cells to androgen deprivation (AD), radiation
(RT) and the combination in vitro. As an
extension of these studies, a growth-inhibition
resistant human LNCaP cell line (LNCaP-Res) was
developed. These cells are representative of the
early transition to androgen dependence and
likely are present in many patients with
locally-advanced tumors. The objectives are to 1)
determine if LNCaP-Res cells continue to respond
to AS, with and without AD and 2) compare these
responses to those in bcl-2 overexpressing LNCaP
(LNCaP-BST) cells. The latter comparison was made
because LNCaP-Res cells overexpress bcl-2 when
grown in AD medium.

Figure 3. Effect of AS-MDM2 on orthotopically in
vivo grown LNCaP-Res cells.
Table 1. Effects of AS-MDM2 on apoptotic cell
death Caspase-37 activity assay
Figure 1. Cell growth and BrdU incorporation in
Complete (CM), AD or ADR1881 medium
MM
AS
A
B
LNCaP
LNCaP-Res
Tumor volume (mm3)
Tumor volume (mm3)
LNCaP
LNCaP-Res-------------------
n5
n4
AD--------
AD MM AS MM AS
MDM2
MMAD
ASAD
AR
C
D
LNCaP
LNCaP-Res
LNCaP-BST
p53
LNCaP-BST
Tumor volume (mm3)
Tumor volume (mm3)
Bcl-2
Bax
n7
n7
ß-actin

Days after first treatment
Days after first treatment
Western blot analysis of proteins from tumor
homogenates in the normal or castrated (AD) mice
bearing LNCaP (control) and LNCaP-Res xenografts.
AS or MM (25 mg/kg/per day) was given by i.p.
injection for 7 consecutive days.
CM
CM
CM
AD
AD
AD
Antitumor activity of AS, MM or in combination
with AD (castration) in nude mice bearing
LNCaP-Res xenografts. LNCaP-Res cells were
injected orthotopically into the prostates of
intact and castrated mice. Tumor volume (mm3) was
directly measured by MRI. When tumor volume was
between 10 and 30 mm3, AS or MM (25 mg/kg/per
day) was given by i.p. injection, 5 days/week for
15 days.
ADR1881
ADR1881
ADR1881
Cells were cultured in complete (CM),
charcoal-stripped serum (AD), or ADR1881
(synthetic androgen) medium for 3 days and the
number of viable cells counted at the indicated
time (A,B,C), or incubated with BrdU for 1 h and
stained with anti-BrdU antibody. The percentages
of BrdU-labeled cells were quantified by flow
cytometry. The bars represents the mean of three
experiments with SEM (D).
Materials/Methods The LNCaP-Res
line was established by long-term culture of
LNCaP cells in AD medium for gt12 mo. Cell death
was quantified by Annexin V and Caspase 37
activity. For the in vivo studies, LNCaP-Res
cells were implanted orthotopically into the
prostates of intact and castrated nude mice.
Tumor growth was monitored by magnetic resonance
microscopy imaging (MRI). AS and the mismatch
control (MM) were given by i.p. injection at
doses 25 mg/kg per day, 5 days /week for 15 days.
Statistical comparisons between groups were
accomplished with Students t-Test for the tumor
volume doubling times and Fisher Exact Test for
freedom from tumor volume failure (FFTVF
endpoint of lt100mm3 at 30 days).
Cells were treated with AS-MDM2 (200 nM) alone or
in combination with ADR1881. Abbreviations
RFLU relative fluorescent units CM complete
medium AD androgen-deprivation medium LC
lipofectin control AS antisense MDM2 MM
antisense mismatch R1881 synthetic androgen
used for replacement. Compared to group above ,
one way Anova, Bonferroni test (n 9 treatment
groups). The data shown represent the average
values (? SEM) from three independent
experiments. Other LNCaP-Res comparisons
ADAS versus AS (plt0.0001). Other LNCaP-BST
comparisons (n 9 treatment groups) ADAS
versus AS (plt0.0001).
Figure 2 . Effects of AS-MDM2 on protein levels
of MDM2, p53, p21, AR, Bcl-2 and Bax in LNCaP-Res
and LNCaP-BST cells
Table 3. Sammary of the tumor baseline size,
doubling time and freedom from MRI-based tumor
volume failure
CM-----------------
AD----------------- ADR1881--------
LNCaP-Res
LC MM AS LC MM
AS LC MM AS
MDM2
Table 2. Effects of AS-MDM2 on apoptotic
cell death Annexin V-assay
p53
p21
AR
Bcl-2
Bax
ß-actin
AS antisense MDM2 MM antisense mismatch.
The oligonucleotides were given at 25 mg/kg
i.p. for 5 days /week for 15 days. Tumor
doubling time was calculated from the linear
regression analysis of log volume and time.
FFTVF Freedom from MRI-based tumor volume
(lt100 mm3 at 30 days). Tumor doubling time
for the ASAD group is significantly longer than
for AS, MM and MMAD groups at p lt 0.05
(Students t-Test). FFTVF for ASAD versus AS is
significantly greater at p lt 0.05 (Fisher Exact
Test).
CM--------------- AD----------------
ADR1881--------
LNCaP-BST
LC MM AS LC MM AS LC MM
AS
Results LNCaP-Res cells displayed no growth
and proliferation inhibition to AD (Fig. 1).
AS caused a significant reduction in MDM2
expression, a slight decrease in AR levels in
complete and AD medium, and an increase in p53
and p21 expression in both cell lines (Fig.
2). ASAD caused significantly higher levels of
apoptosis, compared to AS or AD alone (Table 1,
2). Apoptotic response of ASAD translated into
a significant tumor growth inhibition of
LNCaP-Res cells grown in vivo (Fig. 3 and Table
3).
MDM2
p53
p21
Summary/Conclusions AS enhanced androgen
insensitive LNCaP-Res apoptotic cell death in
vitro and anti-tumor activity in vivo when
combined with AD. MDM2 knockdown has promise
for the treatment of men with high risk
clinically localized prostate cancer, as well as
those with early hormone refractory disease.
AR
Bcl-2
Bax
Cells were treated with AS-MDM2 (200nM) alone or
in combination with ADR1881. Abbreviations CM
complete medium AD androgen-deprivation
medium LC lipofectin control AS antisense
MDM2 MM antisense mismatch R1881 synthetic
androgen used for replacement. Compared to group
above , one way Anova, Bonferroni test (n 9
treatment groups). The data shown represent the
average values (? SEM) from three independent
experiments. Other LNCaP-Res comparisons
ADAS versus AS (plt0.0001). Other LNCaP-BST
comparisons ADAS versus AS (p1.000).
ß-actin