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Azacitidine improves antitumor effects
of docetaxel and cisplatin in
aggressive prostate cancer models
Claudio Festuccia*, Giovanni Luca Gravina
1
*, Anna Maria D’Alessandro
2
,
Paola Muzi, Danilo Millimaggi
2
, Vincenza Dolo
2
, Enrico Ricevuto
3
,
Carlo Vicentini
2
and Mauro Bologna
4
Department of Experimental Medicine, Chair of General Pathology, University of L’Aquila, Via Vetoio, Coppito-2,
67100 L’Aquila, Italy,
1
Division of Radiotherapy, Department of Experimental Medicine,
2
Department of Health Science,
3
Division of Clinical Oncology,
Department of Experimental Medicine and
4
Department of Basic and Applied Biology, University of L’Aquila, L’Aquila, Italy
(Correspondence should be addressed to C Festuccia; Email: festucci@univaq.it)
*(C Festuccia and G L Gravina contributed equally to this work)
Abstract
One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the
development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine
as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of
aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative
effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells
and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells.
In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both
xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and
inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro
treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2
antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer
models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this
agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine
sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were
also tolerable in mice and superior to either agent alone. As DTX is the standard first-line
chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest
in this disease stage. Our results provide a rationale for clinical trials on combination treatments with
azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.
Endocrine-Related Cancer (2009) 16 401–413
Introduction
Prostate cancer represents a global public health
problem. Worldwide, it is the second most common
noncutaneous cancer in men, accounting for w10% of
male cancers (Haas et al. 2008). In recent years, 5 year
survival rates for prostate cancer have been ranked
the third highest of all cancers (Desireddi et al. 2007,
Svatek et al. 2008). Although chemotherapy histori-
cally has had limited utility in treating advanced
prostate cancers, the results from two recent
randomized clinical trials indicated that docetaxel
(DTX)-based chemotherapy improved survival in
patients with hormone-refractory prostate cancer,
HRPC (Patel et al. 2005,Armstrong et al. 2007).
Docetaxel and mitoxantrone are two food and drug
administration – approved chemotherapeutic agents for
clinical treatment of advanced HRPC and DTX is the
standard first-line chemotherapy in this disease stage.
Endocrine-Related Cancer (2009) 16 401–413
Endocrine-Related Cancer (2009) 16 401–413
1351–0088/09/016–401 q2009 Society for Endocrinology Printed in Great Britain
DOI: 10.1677/ERC-08-0130
Online version via http://www.endocrinology-journals.org
Thus, the development of DTX-based combination
therapies is of great interest for HRPC. Some clinical
studies supported taxane-based therapy, a promising
platform to develop new combinational schedule,
which remains a high priority for many ongoing
studies (Patel et al. 2005,Armstrong et al. 2007 and for
a review, see Calabro
`and Stemberg 2007). In addition
a recent report shows that single-agent satraplatin
improves progression-free survival (PFS) as second-
line chemotherapy for patients with HRPC and
provides further evidence that platinum salts may
have a role in the treatment of HRPC (Nakabayashi
et al.2008,Ross et al. 2008). Nevertheless, improvement
in the efficacy of chemotherapy is urgently needed for
patients with chemotherapy-resistant HRPC.
It is now well established that cancer cells exhibit a
number of genetic and epigenetic defects in the
machinery that governs programmed cell death and
that sabotage of apoptosis is one of the principal factors
aiding in the progression and treatment of neoplastic
diseases. Epigenetic modifications, specifically DNA
hypermethylation, are believed to play an important
role in the downregulation of genes important for
protection against apoptosis. In addition to classical
genetic abnormalities, epigenetic modifications have
emerged as a central driving force in the molecular
pathology of prostate cancer (McCabe et al. 2006,
Murphy et al.2008). Several years ago, Plumb et al.
(2000) showed that resistance to carboplatin in ovarian
cancer cells was mediated by hypermethylation and
loss of function of the MLH1 mismatch repair gene.
Of interest, decitabine, a deoxyderivative of aza-CR, a
cytosine analogue with hypomethylating properties,
reverses the downregulation of expression of some
membrane transporters in vitro (Plumb et al. 2000),
raising the untested possibility that repeated treatment
could lead to a progressive increase in efficacy.
In this study, we used azacitidine and its pharma-
cological formulation (Vidaza) to verify if this agent
was able as single agent to alter tumor cell proliferation
and apoptosis, as well as to improve the sensitivity
against DTX and cisplatin in vitro and in vivo using
two models of aggressive prostate cancer. 22Rv1 is an
androgen-responsive human prostate carcinoma cell
line derived from a primary prostate tumor that
expresses mutant (H874Y) androgen receptors (AR),
whereas PC3 is an androgen irresponsive, AR negative
prostate carcinoma cell line. Our results indicate that
azacitidine has antiproliferative effects in both cell
lines and showed pro-apoptotic effects in 22rv1 cells.
In vitro showed synergistic effects with DTX and
cisplatin. Its pharmacological preparation (Vidaza)
caused tumor growth delay without complete stasis or
regression as single agent, whereas improved anti-
tumor effects of DTX and cisplatin resulting these
combinations well tolerable in mice. Therefore, use of
DNA methylation inhibitors might be a suitable
therapeutic tool for the amplification of pharmacologic
responses of HRPC versus first- and second-line
chemotherapies.
Materials and methods
Reagents
All the materials for tissue culture were purchased
from Hyclone (Cramlington, NE, USA). Antibodies
when not otherwise specified were purchased from
Santa Cruz (Santa Cruz, CA, USA). Plasticware was
obtained from Nunc (Roskilde, Denmark). Azacitidine
(Vidaza) was obtained in collaboration with Celgene
Corporation (Summit, NJ, USA). DNA methyltransfer-
ase activity was evaluated in nuclear cell extracts by a
colorimetric EpiQuik DNA methyltransferase Activity
Assay Kit (BioVision, Mountain View, CA, USA).
Cell lines
The two aggressive prostate cancer models (22rv1 and
PC3 cell lines) were obtained from American Tissue
Culture Collection (ATCC, Rockville, MD, USA) and
grown as recommended.
Cell proliferation inhibition assay
Cell proliferation was evaluated by 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma) assay. Briefly, cells were seeded
in 96-well tissue culture plates (Costar, Corning, NY,
USA) at 3000 cells per well per 150 ml. At 24 h after
seeding, cells were treated with 50 ml serial concen-
trations of azacitidine, DTX, cisplatin or the combina-
tion of azacitidine plus DTX and cisplatin. After 72 h
of drug exposure, MTT (100 ml) solution (2 mg/ml)
was added to each well to incubate for 2 h. Absorbance
at 570 nm was recorded using a 96-well plate reader.
Inhibition curves were drawn by means of values
obtained by OD percentages versus control for each
concentration and IC
50
values were calculated by the
GraFit method (Erithacus Software Limited, Staines,
UK). The Chou and Talalay (1984) combination index
(CI) analysis is a well-established index to determine
the pharmacologic interaction of two drugs. When
CIZ1 (represents an additive effect of the two drugs),
the CI formula is in the same form as a traditional
isobologram equation. Synergism is defined as more
than the expected additive effect with CI !1 and
antagonism is defined as CI O1.
C Festuccia and G L Gravina et al.: Azacitidine effects in advanced prostate cancer models
www.endocrinology-journals.org402
Apoptosis analysis by flow cytometry
Treated and untreated cells were collected and fixed
for 30 min by the addition of 1 ml 70% ethanol. After
30 min, the cells were pelleted by centrifugation
(720 g; 5 min), and resuspended in 1 ml DNA staining
solution (PBS containing 200 mg/ml RNase A,
20 mg/ml propidium iodide plus 0.1% Triton X-100)
and stained by incubation at room temperature for
60 min. All cells were then measured on a FACScan
flow cytometer (Becton Dickinson, Bedford, MA,
USA) and analyzed using Cell Quest software (Becton
Dickinson). Resulting DNA distributions were analy-
zed by Mod.t (Verity Software House, Topsham, ME,
USA) for the proportion of cells in sub-G0 (apoptotic
cells), G1, S, and G2-M phases of the cell cycle.
In vivo treatments
Male CD1 nude mice (Charles River, Milan, Italy)
were maintained under the guidelines established by
our Institution (University of L’Aquila, Medical
School and Science and Technology School Board
Regulations, complying with the Italian government
regulation no. 116, January 27 1992, for the use of
laboratory animals). All mice received s.c. flank
injections of 1!10
6
PC3 and 22v1 cells. Tumor
growth was assessed by bi-weekly measurement
of tumor diameters with a Vernier caliper
(length!width). Tumor weight was calculated accord-
ing to the formula: TW (mg)Ztumor volume (mm
3
)Z
d
2
!D/2, where dand Dare the shortest and the longest
diameters respectively. Treatments used in our in vivo
study were started when tumor volumes reached about
80 mm
3
(Day 0) and were stopped after 28 days. Group
1: 10 mice received i.p. injections of 100 l PBS for
consecutive 7 days. Group 2: 10 mice received i.p.
injections of 100 l of aza-CR (0.8 mg/kg) for consecu-
tive 7 days. Group 3: 10 mice received i.p. injections of
DTX (7.5 mg/kg per week) every week for a maximum
of four administrations. Group 4: 10 mice received i.p.
injections of 100 l of aza-CR (0.8 mg/kg) for consecu-
tive 7 days followed by a dose of DTX (7.5 mg/kg per
week) every week for a maximum of three adminis-
trations. Group 5: 10 mice received i.p. injections of
5.0 mg/kg per day cisplatin for 10 consecutive days.
Group 6: mice received i.p. injections of 100 l of aza-
CR (0.8 mg/kg) for consecutive 7 days followed by i.p.
injections of 5.0 mg/kg per day cisplatin for 10
consecutive days. Experiments were stopped at 28th
day. We replicate the experiments in order to analyze a
total of 20 animals for group. The effect of azacitidine
alone or in combination with cisplatin (CP) or DTX on
tumor growth was evaluated measuring, for all groups,
the mean tumor weight at the end of experiments.
Fractional tumor volume (FTV) for each treatment
group was calculated as the ratio between the mean
tumor volumes of treated and untreated animals. This
was performed for treatment a (FTVa), for treatment
b (FTVb), and for treatment aCb(FTVaCb).
Combination indices were assayed as described
above. Animals were killed by spinal cord dislocation
and tumors were subsequently removed surgically. A
part of the tumor was directly frozen in liquid nitrogen
for protein analysis and the other part was fixed in
paraformaldehyde overnight for histochemical evalu-
ations and immunohistochemical analyses including
endothelial (CD31 and vWF; Dako, Glostrup,
Denmark), proliferative (Ki67; Dako), apoptosis
(bcl2, p21, p27, death receptors and their ligands
Santa Cruz), and signaling (p-Akt; Rockland Immuno-
chemicals, Gilbertsville, PA, USA) marker analysis.
Anti-DNMT1, DNMT3a, and DNMT3b antibodies
were purchased from Biocarta (Hamburg, Germany).
Indirect immunoperoxidase stain of tumor xenografts
was performed on paraffin embedded with four
micrometer tissue sections. Briefly, sections were
incubated with primary antibodies for a night at 4
o
C.
Next, avidin–biotin assays were done using the
Vectastain Elite kit obtained from Vector Laboratories.
Mayer’s hematoxylin was used as nuclear counterstain.
Slides were analyzed separately from CF and PM.
Martius yellow-brilliant crystal scarlet blue
technique
Stains for these techniques were purchased from HD
Supplies (Aylesbury, UK). This technique was used to
analyze the presence of red cells dispersed in tumor
tissue and present in blood vessels as well as to
evaluate better the presence of micro-thrombi and
bleeding zones. Martius yellow, a small molecule dye,
together with phosphotungstic acid in alcoholic
solution stains red cells. Early fibrin deposits may be
colored, but the phosphotungstic acid blocks the
staining of muscle, collagen, and connective tissue
fibers. Brilliant crystal scarlet, a medium-sized
molecule, stains muscle, and mature fibrin. Phospho-
tungstic acid removes any red stain from the collagen.
The large molecule dye aniline blue stains the collagen
and old fibrin.
Hemoglobin assay
Tumor hemoglobin levels were quantified as described
elsewhere (14). Tumors were homogenized in double-
distilled water. Eighty microliters of homogenates
were mixed with 1 ml Drabkin’s solution and
Endocrine-Related Cancer (2009) 16 401–413
www.endocrinology-journals.org 403
incubated for 15 min at room temperature. After
centrifugation at 400 gfor 5 min, the supernatants
were subjected to absorbance measurement at 540 nm.
The absorption that is proportional to hemoglobin
concentration, was divided by tumor weight.
Assessment of global methylation levels
Proteins were extracted from untreated and in vivo
Aza-CR-treated tumors and cells at 7, 14, 21, and
28 days and DNMT activity were assayed. As
described, frozen tumors were pulverized in a liquid
nitrogen-cooled Thermovac pulverizer. The resulting
powders were homogenized in 10 volumes of a 10 mM
Tris–HCl buffer, pH 7.4, containing 1 mM EDTA,
0.5 mM dithiothreitol, 10 mM sodium molybdate,
phosphatase inhibitor cocktail 2 with a dilution 1/100,
and protease inhibition cocktail 2 with a dilution 1/100,
both from Sigma-Aldrich. The homogenates were
centrifuged for 1 h at 105 000 g(C48C) and the
supernatants (cytosols) were used for protein
determination by immunoblotting. Total protein con-
tent was measured using the bicinchoninic acid assay.
Western blot analysis
Cells were washed with cold PBS and immediately
lysed with 1 ml lysis buffer (50 mM HEPES, pH 7.5,
150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM
EDTA, 1 mM EGTA, 50 mM NaF, 1 mM sodium
orthovanadate, 30 mM p-nitrophenyl phosphate,
10 mM sodium pyrophosphate, 1 mM phenylmethyl-
sulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml
leupeptin). Flash-frozen tissue samples were crushed
using liquid nitrogen pre-chilled mortar and pestles.
Upon addition of lyses buffer, tissues were further
homogenized with an electric homogenizer and
centrifuged in a microfuge a top spin. Supernatants
were collected and analyzed. Cell lysates and tissue
extracts were electrophoresed in 7% SDS-PAGE, and
separated proteins transferred to nitrocellulose and
probed with the appropriate antibodies using the
conditions recommended by the antibody suppliers.
Statistics analysis
Continuous data were expressed as mean and S.D. and
were compared using an umpired Student’s t-test.
Categorical data were analyzed by the exact Fisher’s
test. Pvalue less than 0.05 were considered statistically
significant.
Results
In vitro experiments: effects of azacitidine
as single agent
Initially, we analyzed the effects of azacitidine on
DNA methylation and expression of DNMT1,
DNMT3a, and DNMT3b. After 48 h of treatment
with azacitidine, the decrease in the DNMT activity
was maximal after 10 days and it was maintained up to
2 weeks (Fig. 1A). Simultaneously, a significant
decrement in DNMT1 and DNMT3b expression levels
occurred with a peak observed at 5–10 days (Fig. 1B).
Conversely, azacitidine treatment did not affect the
protein expression levels of DNMT3a. Antiprolifera-
tive effect of azacitidine treatment was higher in 22rv1
cells than in PC3 cells with IC50 of 0.5 and 2.5 mM
respectively. Treatment of with azacitidine resulted in
a dose- and time-dependent inhibition of growth with
G1 phase arrest associated to apoptosis in 22rv1 cells,
whereas this drug arrested PC3 cells in G2/M phase
without acute cell killing. Also, azacitidine increased
the levels of p21, p27, p53, Bcl-Xs, and Bax and to
reduce Bcl2 and Bcl-Xl expression in 22rv1 cells
(Fig. 2A). This was also associated with an increased
expression of cleaved caspase 3 and PARP. The
expression of p21, Bcl2, caspase 3, and PARP was
Figure 1 DNMT activity (A) and expression of DNMT1,
DNMT3a, and DNMT3b (B) measured at different time after a
48 h 1.0 mM Aza-CR treatment in 22rv1 and PC3 cells. In PC3
cells, a decrement of about twofold in DNMT1 and fivefold in
DNMT3b expression was observed after 5–10 days of
treatment with azacitidine. The levels of DNMT1 were restored
after 15 days, whereas the levels of DNMT3b were maintained
lower up to 20 days (about twofold). In 22rv1 cells, the levels of
DNMT1 and DNMT3b were weakly reduced also if the overall
DNMT activity was significantly decreased.
C Festuccia and G L Gravina et al.: Azacitidine effects in advanced prostate cancer models
www.endocrinology-journals.org404
not changed in PC3 cells following azacitidine
treatment, whereas the expression of protein associated
to G2/M arrest such as p16INKA, cyclin B and cdk1,
cdk2 and cdk4 were differentially modulated (Fig. 2B).
The treatment with HA-14-1, a Bcl2 antagonist, was able
to induce more apoptotic events when co-administrated
with Aza-CR in PC3 (Fig. 3A) and 22rv1 cells
(Fig. 3B). These results suggest that the amount of
apoptosis is related to Bcl2 levels and the low levels of
apoptosis observed in PC3 cells might be partially
associated with the higher expression of Bcl2. Aza-CR
treatment in PC3 cells was also associated with the
phosphorylation of p38 MAP kinase and with the
reduced expression of activated Erk (Fig. 5B) which it
is required for the induction of cell cycle arrest in the
G2/M phase. However, the induction of p38 MAPK
activity was able to start the apoptotic program by
downmodulation of Bcl2 when Aza-CR treated PC3
cells were grown in polyhema-covered Petri dishes
(Fig. 3C). The apoptosis mediated by absence of
adhesion (anoikis) was induced by a p38 MAPK-
dependent Bcl-2 downmodulation (Fig. 3D) because
SB203580, a p38 MAPK inhibitor, treatment caused a
significative slow down in Aza-CR/polyhema-dependent
Bcl2 downmodulation.
In vitro effects of azacitidine in combination with
DTX or cisplatin
We analyzed the effects of combined treatments with
azacitidine (0.5–1 mM for PC3 and 0.1–0.5 mM for
22rv1), DTX (0.5, 1.0, and 10 nM for both cells) and
cisplatin (1, 2.5, and 10 mg/ml for both cells).
Azacitidine sensitized versus DTX and cisplatin in
PC3 and 22rv1 cells (Tables 1 and 2). In PC3 cells, the
stronger antitumor effect was observed with azacitidine
(1.0 mM) and DTX (1.0 nM (CIZ0.29)) and
with azacitidine (1.0 mM) and cisplatin (10 mg/ml
(CIZ0.27)). In 22rv1 cells, the highest antitumor
effects were observed with azacitidine (0.5 mM) and
Figure 2 Western blot analysis on the expression of pro-
apoptotic and antiapoptotic elements in 22rv1 (A) and PC3 (B)
cells treated with 1, 2.5, and 5.0 mM aza-CR. In 22rv1 cells, aza-
CR increased the expression of pro-apoptotic elements such as
p21 (twofold with 2.5 and 5.0 mM aza-CR), p53 (four- to five-fold
with 2.5 and 5.0 mM aza-CR), Bax (two- to four-fold with 2.5 and
5.0 mM aza-CR) and Bcl-XS (twofold with both 2.5 and 5.0 mM
aza-CR) as well as the activity of caspase 3 (four- to six-fold
with 2.5 and 5.0 mM aza-CR). The antiapoptotic element Bcl2
and Bcl-XL was reduced (about tenfold with both 2.5 and 5.0 mM
aza-CR). In PC3 cells, no caspase 3 and PARP activity was
showed and this was associated to no significant modulation of
Bcl2 and p21 expression. Modulation in check point proteins
of cell cycle was, however, shown. P16INK4A levels were
significantly increased (about twofold with 5.0 mM aza-CR),
CdK1 levels were increased of about two with both 1.0 and
2.5 mM aza-CR and of about fourfold with 5.0 mM aza-CR, CdK2
levels were weakly reduced (1.5-fold with 5.0 mM
aza-CR), CdK4 levels were significantly reduced of about
fourfold with 2.5 and 5.0 mM aza-CR and cyclin B1 levels were
not modified. Each lane was loaded with 40 mg proteins and
normalized versus actin. This experiment is representative of
three individual experiments.
Endocrine-Related Cancer (2009) 16 401–413
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DTX (1.0 nM (CIZ0.50)) and with azacitidine
(1.0 mM) and cisplatin (2.5 mg/ml (CIZ0.44)).
In vivo effects of azacitidine as single drug or
followed by DTX or cisplatin
The i.p. administration of Vidaza (0.8 mg/kg for 7
consecutive days) on intact nude male mice inoculated
subcutaneously with 22rv1 and PC3 cells resulted in a
statistically significant tumor growth decrement
(P!0.001) in both xenograft models (Fig. 4). In PC3
xenografts (Table 3), following azacitidine treatment
we demonstrated a 36.7% of reduction in tumor
weight respect to controls (P!0.001) with a tumor
growth delay (TGD) of 5.5 days. The proliferation
index was also significantly reduced to 42.6% in
azacitidine treated tumors (P!0.001) with tumor
apoptosis of about 5%. The vessel count was also
reduced to 18.7% (P!0.05). In 22rv1 xenografts
(Table 4), we demonstrated a 65.5% reduction of tumor
weight in aza-CR treated mice respect to controls
(P!0.001), with a tumor growth delay (TGD) of
25.5 days. The proliferation index was also reduced to
74.3% (P!0.001) and apoptosis was evident in about
15% of cells. The vessel count was reduced to 70.4%
Figure 3 Role of Bcl2 in the absence of aza-mediated PC3 cell apoptosis. We used the Bcl2 inhibitor HA-14.1 to demonstrate that
bcl2 overexpression in PC3 cells could be responsible for the absence of acute apoptosis induced by aza-CR treatment. HA-14.1
(10 mM) triggered apoptosis of PC3 cells both in basal culture condition and in the presence of aza-CR pretreatment (1.0 mM).
HA-14.1 was synergistic with aza-CR in PC3 cells, whereas this drug was only additive in 22rv1 cells. These results are
representative of three individual experiments. Each lane was loaded with 40 mg proteins.
Table 1 Pharmacological parameters of combination treatment with azacitidine and docetaxel in PC3 and 22rv1 cells
Treatment A (DTX) Treatment B (aza-CR) Combination treatment
Cells Dose (nM) GI Dose (mM) GI Expected GI Observed GI Increment (CI)
PC-3 0.5 0.95 0.5 0.90 0.85 0.54 1.57 (0.64)
1 0.75 0.70 0.25 2.80 (0.36)
1.0 0.90 0.5 0.90 0.80 0.31 2.58 (0.39)
1 0.75 0.70 0.20 3.50 (0.29)
10.0 0.75 0.5 0.90 0.70 0.25 2.80 (0.36)
1 0.75 0.50 0.20 2.50 (0.40)
22rv1 0.5 0.90 0.1 0.75 0.65 0.40 1.62 (0.62)
0.5 0.50 0.40 0.30 1.33 (0.75)
1.0 0.70 0.1 0.75 0.45 0.25 1.80 (0.55)
0.5 0.50 0.20 0.10 2.00 (0.50)
10.0 0.55 0.1 0.75 0.30 0.20 1.50 (0.67)
0.5 0.50 0.05 0.10 0.50 (2.00)
GIZinhibition (percent versus control).
C Festuccia and G L Gravina et al.: Azacitidine effects in advanced prostate cancer models
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(P!0.001). Similarly to that observed in vitro, the
levels of DNMT1 and DNMT3b were also lower in
azacitidine treated mice when compared with controls
both in PC3 and 22rv1 xenograft models (data not
shown).
The treatment with azacitidine followed by DTX or
CP was also studied in vivo. An increase in the
effectiveness of DTX and cisplatin was observed after
7 days of co-administration with azacitidine both in
PC3 and 22rv1 xenograft models (Fig. 4 and Tables 3
and 4). The results are as follows:
(i) Docetaxel and cisplatin alone reduced tumor
weight of PC3 xenografts by 41.0% (P!0.001) and
22.2% (P!0.01), with a tumor growth delay of 5.1 and
2.1 days respectively when compared with controls.
The proliferation index was also reduced to 49.9%
(P!0.001) and 23.6% (P!0.01) respectively in DTX-
and cisplatin-treated tumors. Apoptosis was evident
only in DTX-treated tumors at levels of about 7%
(P!0.01). In this xenograft model, 7 days of treatment
with azacitidine followed by DTX or cisplatin resulted
in a significant decrease in tumor weight of 76.4%
Table 2 Pharmacological parameters of combination treatment with azacitidine and cisplatin in PC3 and 22rv1 cells
Treatment A (CP) Treatment B (aza-CR) Combination treatment
Cells Dose (mg/ml) GI Dose (mM) GI Expected GI Observed GI Increment (CI)
PC-3 1 1.00 0.5 0.90 0.90 0.60 1.50 (0.67)
1 0.75 0.75 0.45 1.67 (0.60)
2.5 0.90 0.5 0.90 0.80 0.40 2.00 (0.50)
1 0.75 0.65 0.32 2.03 (0.49)
10.0 0.80 0.5 0.90 0.70 0.25 2.80 (0.36)
1 0.75 0.55 0.15 3.67 (0.27)
22rv1 2.5 0.90 0.1 0.85 0.75 0.60 1.25 (0.80)
0.5 0.67 0.57 0.44 1.30 (0.77)
10.0 0.90 0.1 0.85 0.75 0.45 1.67 (0.60)
0.5 0.67 0.57 0.25 2.28 (0.44)
10.0 0.85 0.1 0.85 0.70 0.35 2.00 (0.50)
0.5 0.67 0.52 0.25 2.08 (0.48)
GIZinhibition.
Figure 4 Tumor proliferation in PC3 and 22rv1 xenografts in the presence of azacitidine alone or in combination with docetaxel (DTX)
and cisplatin (CP). Group 1: 10 mice received (i.p.) injections of 100 ml PBS for consecutive 7 days. Group 2: 10 mice received i.p.
injections of 100 ml of aza-CR (0.8 mg/kg) for consecutive 7 days. Group 3: 10 mice received i.p. injections of docetaxel (7.5 mg/kg
per week) every week for a maximum of four administrations. Group 4: 10 mice received i.p. injections of 100 ml of aza-CR
(0.8 mg/kg) for consecutive 7 days followed by a dose of docetaxel (7.5 mg/kg per week) every week for a maximum of three
administrations. Group 5: 10 mice received i.p. injections of 5.0 mg/kg per day cisplatin for 10 consecutive days. Group 6: mice
received i.p. injections of 100 ml of aza-CR (0.8 mg/kg) for consecutive 7 days followed by i.p. injections of 5.0 mg/kg per day cisplatin
for 10 consecutive days. Proliferation was measured considering the variation of tumor volume (mm
3
) in the time (days). Controls
were administered with saline and treated xenografts were administered with 0.8 mg/kg for the first 7 days of treatment. Animals
were killed at 28th day of treatment.
Endocrine-Related Cancer (2009) 16 401–413
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(P!0.001 versus both Vidaza and DTX single
treatments) and of 58.3% (P!0.01 versus Vidaza
and P!0.001 versus cisplatin) respectively. Moreover,
sequential regimens of azacitidine CDTX or
azacitidine Ccisplatin were respectively associated
with tumor growth delay values of 25.5 and 15.2 days:
reductions in the proliferation index of 96.5%
(P!0.001 versus both single treatments) and 81.5%
(P!0.001 versus both single treatments); significant
increases in apoptosis of 18% (P!0.001 versus both
single treatments) and 14.3% (P!0.001 versus both
single treatments); and reductions in vessel counts of
w75% (P!0.001 versus both single treatments) and
55% (P!0.001 versus Vidaza and P!0.01 versus
cisplatin treatments). Additionally, 15.0% (3/20, NS)
and 25.0% (5/20, (PZ0.056, NS)) of mice were tumor-
free after 28 days of treatment.
(ii) In 22rv1 xenografts, DTX or cisplatin alone
reduced tumor weight of 46.8% (P!0.001) and 30.9%
(P!0.001), with a tumor growth delay of 17.3 and
7.5 days respectively when compared with controls.
The proliferation index was also reduced of 66.1%
(P!0.001) and 53.2% (P!0.001) respectively in
DTX- and cisplatin-treated animals. Apoptosis was
evident in 10% (P!0.05) and 25% (P!0.01) of cells
in DTX- and cisplatin-treated animals respectively.
In this xenograft model, 7 days of treatment with
azacitidine followed by DTX or cisplatin resulted
in a significant decrease in tumor weight of 68.5%
(P!0.05 versus DTX treatment, whereas not signi-
ficant versus Vidaza single treatment) and 77.5%
(P!0.001 versus both single treatments) respectively.
Additionally, sequential regimens of azacitidine CDTX
or azacitidine Ccisplatin were respectively associated
with tumor growth delay values of 28.0 and 30.2 days;
reductions in the proliferation index of 79.6% (P!0.01
versus Vidaza and P!0.001 versus DTX single
treatments) and 83.5% (P!0.001 versus both single
treatments); significant increases in apoptosis of
18.2 and 34.3% (P!0.001 versus all single treat-
ments); reductions in vessel counts of about
75% (P!0.05 versus Vidaza and P!0.001 versus
Table 3 Antitumor activity of Vidaza in PC-3 tumors alone or in combination with docetaxel (DTX) or cisplatin (CP)
Treatment
Drug
Dose
mg/kg Mice
Weight
of mice
(grGS.D.)
Tumor
weight
(mgGS.D.)
TGD
(days)
PI
(Ki67 %) Apoptosis Vessels
Tumor-free
mice
Saline 20 25.0G2.1 585G135 37.3G4.5 !2 30.0G4.3 0/20
Vidaza 0.8 20 23.4G2.3 370G183 5.5 21.4G2.7 5.4G2.6 24.4G2.7 0/20
DTX 7.5 20 25.0G1.7 345G132 5.1 18.7G3.3 7.2G2.7 27.5G4.5 0/20
VidazaCDTX 0.8C7.5 20 21.8G2.6 138G84 25.5 1.3G1.7 18.2G3.2 7.5G2.5 3/20
CP 5 20 25.1G1.5 455G161 2.1 28.5G3.1 !2 28.5G3.5 0/20
VidazaCCP 0.8C5 20 20.8G2.8 244G58 15.2 6.9G0.6 14.3G2.1 13.4G4.4 5/20
TGD (tumor growth delay) was determined by the difference in time taken for the mean tumor volume to double in treated versus
control animals. PI (proliferation index) was measured considering the mean of Ki67 positive cell percentageGS.D. measured on five
random fields at !100. Apoptosis was measured as the percentage of tunnel positive cells GS.D. measured on five random fields
(!400). Vessels were measured as the vessel number per field at !100.
Table 4 Antitumor activity of Vidaza in 22rv1 tumors alone or in combination with docetaxel (DTX) or cisplatin (CP)
Treatment
Drug
Dose
mg/kg Mice
Weight of
mice
(grGS.D.)
Tumor
weight
(mgGS.D.)
TGD
(days)
PI
(Ki67 %) Apoptosis Vessels
Tumor-free
mice
Saline 20 25.3G1.8 652G220 45.5G6.5 !2 38.5G5.0 0/20
Vidaza 0.8 20 24.1G2.0 225G55 25.5 11.7G0.7 15.4G3.0 11.4G0.8 1/20
DTX 7.5 20 25.0G1.7 305G121 17.3 15.4G5.1 10.2G1.5 17.5G3.0 0/20
VidazaCDTX 0.8C7.5 20 21.8G2.6 205G125 28.0 9.3G1.4 18.2G2.5 9.5G1.0 2/20
CP 5 20 25.1G1.5 450G101 7.5 21.3G3.9 25.5G2.5 7.5G1.5 1/20
VidazaCCP 0.8C5 20 20.8G2.8 147G66 30.2 7.5G2.0 34.3G8.0 7.4G4.4 6/20
TGD (tumor growth delay) was determined by the difference in time taken for the mean tumor volume to double in treated versus
control animals. PI (proliferation index) was measured considering the mean of Ki67 positive cell percentage GS.D. measured on
five random fields at !100. Apoptosis was measured as the percentage of tunnel positive cells GS.D. measured on five random
fields (!400). Vessels were measured as the vessel number per field at !100.
C Festuccia and G L Gravina et al.: Azacitidine effects in advanced prostate cancer models
www.endocrinology-journals.org408
DTX single treatments) and 81% (P!0.001 versus
Vidaza and NS versus cisplatin single treatments).
Moreover, 10.0% (2/20) and 30.0% (6/20) of mice
treated with the Vidaza plus DTX and Vidaza plus
cisplatin combined treatments were tumor-free after
28 days, although only the second combined treatment
was statistically significant versus control (P!0.05)
and the statistic analysis showed not significant values
for the comparisons versus Vidaza or cisplatin
(PZ0.096 for both comparisons).
(iii) Sequential treatments with either azacitidine
CDTX or azacitidine Ccisplatin resulted in a
significant reduction in tumor blood vessels compared
with controls (Tables 3 and 4) both in PC3 and 22rv1
xenografts. This antiangiogenetic effect was further
supported by quantification of hemoglobin content in
Figure 5 (A) Western blot analysis showing the increment of DNMT1 andDNMT3b expression without modulation of Akt activity after
CP treatment in PC3 and 22rv1 tissues. Forty micrograms of proteins were loaded in each lane. (B). Immunohistochemical appearance
of PC3 tumors harvested from control andCP treated mice showing increased DNMT1 expression after CP treatment. (C). Microscopic
appearance of PC3 and 22rv1 tumors stained withMartius yellow-brilliant crystal scarlet blue technique. We show the presence of large
blood vessels (stained in yellow/orange)near to cancer cell nests of untreated 22rv1 tumors. Untreated PC3 tumors show the presence
of numerous and small blood vessels scatteredin the tumor mass and delineating single tumor cell nests. Vidaza reduces blood vessel
number both in 22rv1 and PC3 xenografts. Vessels appear dilated with unstructured capillary bed dispersed in collagen I deposits
(azure staining) primarily in 22rv1 tumors. DTX treated 22rv1 tissues appear with large unstructured capillary bed and abundant
hemorrhagic areas. The presence of numerousphagocytes (neutrophilic granulocytes and monocytes) dispensed in collagen I deposit
suggests that a previous colliquative necrosis was present. In the combined treatment (Vidaza plus DTX), the above-mentioned
appearance was amplified with deposition also of fibrin clots (pale pink-staining) evident in 22rv1 tissues and formation of dense
collagen I deposits (fibrosis) which envelop tumor cell nests. Single pictures are at !400. (B) Angiogenesis was evaluated measuring
hemoglobin presence in tissue extracts or counting CD34/von Willebrand double positive endothelial cells forming blood vessels. Full
colour version of this figure available via http://dx.doi.org/10.1677/ERC-08-0130.
Endocrine-Related Cancer (2009) 16 401–413
www.endocrinology-journals.org 409
tumor tissues, used as a marker of the number of
blood vessels. To specifically assess vascular density
within the tumors, sections were stained with Masson
trichromic and BMBS. Blue-stained spiral strands of
collagen were detected in peripheral areas, whereas
PC3 cells grow to form large cell masses that were
surrounded by red-stained blood vessels that often
seemed dilated. Control xenografts exhibited numer-
ous dilated and even sinusoid-looking vessels as
compared with treated xenografts. The antiangiogenic
effects of azacitidine followed by either DTX or
cisplatin were further confirmed by immunohisto-
chemical localization of von Willebrand factor that is
a marker for endothelial cells and the presence of
microvessels. However, a reduction in microvessel
density was only observed in tumors treated with
azacitidine Ccisplatin compared with controls or
with azacitidine alone. Results also showed anti-
angiogenetic effects with the combination of azaciti-
dine and DTX, including significant reductions in the
vessels analyzed with the presence of fibrin clots and
red dispersion in the tumor parenchyma, and the
amount of hemoglobin analyzed in tissue extracts
(Fig. 5A and B).
(iv) In addition, we found that treatments with
cisplatin were able to increase the levels of DNMT1
in PC3 but not in 22rv1 cells. This is in agreement
with the higher effects of combination azacitidine
plus cisplatin in PC3 when compared with those
observed in 22rv1. In Fig. 5C, we show western blot
analysis performed on PC3 and 22rv1 tissue extracts
whereas in Fig. 5D, we show the expression of
DNMT1 in PC3 xenografts with or without cisplatin
treatment. DTX was not able to modify DNMT
expression in PC3 and 22rv1 xenografts (data not
shown) supporting the high effects of combination
treatment with azacitidine and cisplatin observed in
both PCa models.
Discussion
One of the major obstacles in curing locally advanced
and metastatic prostate cancer is the development of
resistance to therapy. Historically, chemotherapy has
had limited utility in treating castration-resistant
locally advanced and metastatic prostate cancer.
Nevertheless, DTX is the standard first-line chemo-
therapy for this disease. The responses to DTX are,
however, partial and associated with increased resis-
tance to apoptosis, thus indicating a clear need for new
therapies in HRPC patients. A recent report shows that
single-agent satraplatin improves PFS as second-line
chemotherapy for patients with HRPC (Nakabayashi
et al. 2008,Ross et al. 2008). The resistance of tumor
cells to anticancer agents remains a major cause of
failure in the treatment of patients with cancer. The
classic mechanism for the evolution of a multidrug-
resistant phenotype was believed to involve a single
molecular mechanism, such as overexpression of
P-glycoprotein. However, it now seems that the
multidrug-resistant phenotype represents a complex,
multifactor process in which epigenetic mechanisms
may play a crucial role. Epigenetic alterations,
including aberrant promoter methylation, are respon-
sible for the silencing of tumor suppressor genes and
for increased resistance to chemotherapy. This high-
lights a potential role for DNMT as potential molecular
targets in cancer therapy (Mishra et al. 2008).
Exploiting gene reactivation by epigenetic acting
agents in combination with cytotoxic therapies has
been a strategy that holds much clinical promise (for
review, see Lu et al. 2006). Although azacitidine has
shown significant promise in the treatment of hemato-
poietic malignancies, and is approved for the treatment
of myelodysplastic syndrome (MDS; Issa et al. 2005,
Abdulhaq & Rossetti 2007), research into its activity in
solid tumors has been less impressive and have been
similarly disappointing (Schwartsmann et al. 2000,
Pohlmann et al. 2002,Appleton et al. 2007). However,
it is important to note that the majority of these studies
used dose and schedule combinations that are now
recognized to be suboptimal. In addition, the major
issue involves either drug uptake or tumor cell
proliferation rate because the analysis of tumor tissues
showed a disappointingly small decrease of methyl-
ation (3%). This has been shown to be below the
threshold necessary for resensitization to chemother-
apy (Appleton et al. 2007). Our research demonstrates
that tumor levels of DNMT1 and DNMT3b were lower
in azacitidine-treated conditions both in vitro and in
PC3 and 22rv1 xenograft models. Our in vitro data
indicate that DNA methylation activity reaches
maximum inhibition of inhibition at 10 days after a
48 h of azacitidine treatment. This observed inhibition
remained at high levels for a maximum 2 weeks.
In parallel, a significant downregulation in DNMT1
and DNMT3b expression occurred and peaked at
5–10 days, and persisted even after 14 days. This
reduction of DNMT activity was associated with the
in vitro and in vivo re-expression of markers of
differentiation, including AR (Jarrard et al. 1998,
Gravina et al.2008), and is responsible for the
restoration of responsiveness to hormonal therapy
(Izbicka et al. 1999,Sonpavde et al. 2007,Gravina
et al. 2008). This reduction in DNMT activity is also
likely responsible for the delay in progression to
C Festuccia and G L Gravina et al.: Azacitidine effects in advanced prostate cancer models
www.endocrinology-journals.org410
androgen independent disease and for the improvement
in survival in the transgenic adenocarcinoma of mouse
prostate mouse model of prostate cancer (Zorn et al.
2007). In the present report, we observed also that
DNMT inhibition with azacitidine was able to induce
the in vitro and in vivo expression of p16INKA, Bax,
Bak, p21/WAF1, and p27/KIP1. This was also
responsible for inhibiting the activation of Akt, the
expression of cyclin D1, Bcl-2, and Bcl-XL associated
with the processing of caspases-3 with increased
apoptosis. Nevertheless, we observed an increased
Bcl2 mediated apoptosis by culturing aza-CR treated
cells in presence of polyhema which impedes cell
adhesion. This condition induces a specialized apopto-
tic mechanism termed anoikis that is normally
suppressed in tumor cells (Diaz-Montero et al. 2006,
Glinsky 2006,Kupferman et al. 2007) including PC3
cells. Although the reactivation of anoikis by aza-CR
can represent an important therapeutic approach to
increase tumor cell death present in the blood stream
and could slow down the metastatic process and
contribute to antimetastatic treatments of advanced
tumors, ad hoc experiments are required.
In addition, the downmodulation of p-Akt and
members of Bcl-2 family could indeed sensitize PC3
and 22rv1 cells versus DTX and cisplatin increasing
the antiproliferative and pro-apoptotic effects of single
drugs both in vitro and in vivo. The in vivo experiments
revealed synergistic effects with DTX and cisplatin
both in PC3 and 22rv1 xenografts. Immunocyto-
chemistry and enzymatic assays revealed also that
in vivo DNMT1 expression was significantly increased
in cisplatin treated 22rv1 and PC3 xenografts, whereas
the induction of DNMT1 levels was lower in DTX
treated tumors. This is in agreement with previous
reports showing that an increased DNMT1 expression
is a common event in a multimodality-resistant
phenotype in tumor cells (Misrha et al. 2008), in
cisplatin and taxane (Segura-Pacheco et al. 2006)
resistance mechanisms. Taken together, our study
indicate the DNMT downmodulation represents an
useful therapeutic approach for enhancing chemosen-
sitivity of prostate cancers to DTX and cisplatin and
provide a rationale for clinical trials on combination
treatments with azacitidine in patients with advanced
prostate tumors.
Declaration of interest
There is no conflict of interest that could be perceived as
prejudicing the impartiality of the research reported.
Funding
This research did not receive any specific grant from any
funding agency in the public, commercial or not-for-profit
sector.
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