Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in human breast cancer cell lines, MCF-7 and MDA-MB-468.

Abstract

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF-7 and MDA-MB-468.
Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real-time RT-PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V-FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis.
Treatment of both cell lines with BAY 73-6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP-hydrolytic activity and elevation of the intracellular cGMP response. BAY 73-6691 significantly reduced cell proliferation in a dose- and time-dependent manner and caused marked increase in apoptosis through caspase3 activation.
Our results revealed that BAY 73-6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73-6691 could be utilized as an agent in treatment of breast cancer.

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Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in
human breast cancer cell lines, MCF-7 and MDA-MB-468
R. Saravani*, F. Karami-Tehrani*, M. Hashemi†,‡, M. Aghaei§ and R. Edalat¶
*Clinical Biochemistry Department, Cancer Research Lab, School of Medical Science, Tarbiat Modares University, Tehran, Iran, †Cellular and
Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran, ‡Department of Clinical Biochemistry, School of Medicine,
Zahedan University of Medical Sciences, Zahedan, Iran, §Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Science,
Isfahan University of Medical Sciences, Isfahan, Iran and ¶Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Received 24 December 2011; revision accepted 21 February 2012
Abstract
Objectives: Phosphodiesterase 9 (PDE9) is a major
isoform of phosphodiesterase hydrolysing cGMP
and plays a key role in proliferation of cells, their
differentiationand apoptosis,
cGMP signalling. The study described here was
designed to investigate expression, activity and
apoptotic effect of PDE9 on human breast cancer
cell lines, MCF-7 and MDA-MB-468.
Materials and methods: Activity and expression of
PDE9 were examined using colorimetric cyclic
nucleotide phosphodiesterase assay and real-time
RT-PCR methods respectively; cGMP concentra-
tion was also measured. MTT viability test, annex-
in V-FITC staining, Hoechst 33258 staining and
caspase3 activity assay were used to detect apopto-
sis.
Results: Treatment of both cell lines with BAY 73-
6691 lead to reduction in PDE9 mRNA expression,
PDE9 cGMP-hydrolytic activity and elevation of
the intracellular cGMP response. BAY 73-6691
significantly reduced cell proliferation in a dose-
and time-dependent manner and caused marked
increase in apoptosis through caspase3 activation.
Conclusion: Our results revealed that BAY 73-
6691 induced apoptosis in these breast cancer cell
lines through the cGMP pathway. These data sug-
gest that BAY 73-6691 could be utilized as an
agent in treatment of breast cancer.
via
intracellular
Introduction
Mammalian cyclic nucleotide phosphodiesterases (PDEs)
hydrolysiscyclicadenosine
(cAMP)andcyclicguanosine
(cGMP), which are omnipresent, second messengers and
responsible for transducing extra-cellular signaling path-
ways (1). cGMP is involved in regulation of various
physiological functions including neurotransmission (3),
platelet aggregation(2)
muscle modulation (4). It also plays a key role in cell
proliferation, differentiation and apoptosis (5,6).
Intracellular concentration of cGMP is determined by
net balance between synthesis from guanylylcyclases and
breakdown by 3′, 5′ cyclic nucleotide phosphodiesterases
(7). Eleven different subfamilies of PDE isozymes (PDE1–
PDE11) are known. These enzymes are differentiated in
terms of substrate selectivity, thus PDEs 1, 2, 3, 10, 11
degrade both cAMP and cGMP, PDEs 4, 7 and 8 degrade
merely cAMP, and PDEs 5, 6 and 9 degrade cGMP selec-
tively.PDE9 hasthehighest
(Km = 170 nM) (8). All PDE family members share 25–
50% amino acid sequence identity within the catalytic
domain, suggesting that it should be feasible to identify
isoform selective PDE inhibitors (8). More than 20 splice
variants of PDE9 have been identified (1) but one variant,
PDE9A1, appears to be located within the nucleus (9), sug-
gesting that different variants may have a role in metaboliz-
ing cGMP in different intracellular compartments.
Recently, it has been reported that PDE activities are
elevated in tumours (10,11) and overexpression occurs
in many carcinomas (12,13). Phosphodiestrase inhibitors
have been used for treatment of non-malignant condition
such as inflammatory diseases (14), and also have been
suggested for treatment of different types of cancer such
as acute promyelocytic leukaemia (15), and malignant
glioma (16).
3′,5′-monophosphate
3′,5′-monophosphate
andvascular smooth-
affinity forcGMP
Correspondence: F. Karami-Tehrani, Clinical Biochemistry Depart-
ment, Cancer Research Lab, School of Medical Sciences, Tarbiat Mod-
ares University, P.O. Box: 14115-331, Tehran, Iran. Tel.: +98 (21)
82883567; Fax: + 98 (21) 82884555; E-mail: karamitf@modares.ac.ir
© 2012 Blackwell Publishing Ltd
199
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The compound 1-(2-chlorophenyl)-6-[(2R)-3, 3, 3-
trifluoro-2-methylpropyl]-1,5-dihydro-4H-Pyrazolo (3, 4-
d) pyrimidine-4-one (BAY 73-6691, Fig. 1) has been
reported to be a novel and selective PDE9 inhibitor
(17), which (remarkably) has been shown to improve
learning and memory in rodents (18). BAY 73-6691
only moderately inhibits human PDE1 and human
PDE11 with selectivity ratios of 25 and 47 respectively,
and also has only very low potency against other human
PDEs with selectivity ratios of >>73 (17). It could con-
ceivably be used for induction of apoptosis and as a
new potential anti-cancer therapy. In the present study,
PDE9 expression and effects of PDE9 inhibitor, BAY
73-6691 on cell population growth and induction of
apoptosis, were investigated in ER positive and ER neg-
ative, MCF-7 and MDA-MB-468, human breast cancer
cell lines.
Materials and methods
Chemical reagents
Culture media (RPMI 1640) and growth supplements
[trypsin/EDTA, phosphate-buffered saline (PBS), peni-
cillin and streptomycin and foetal bovine serum (FBS)]
were obtained from Gibco (Rockville, Maryland, USA).
cGMP direct immunoassay (ELISA) kit and caspase-3
colorimetric assay kit were purchased from R&D Sys-
tem CO. (Minneapolis, Minnesota, USA). Annexin V-
FITC apoptosis detection kit was procured from Biovi-
sion (San Francisco, California, USA). Bisbenzimide
H33258 (Hoechst 33258), 3-(4,5-Dimethyl thiazol-2-yl)-
2,5diphenyltetrazolium
(dimethyl sulfoxide) and BAY73-6691(1-(2-chlorophe-
nyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-
4H-pyrazolo[3,4-d]pyrimidine-4-one),
inhibitor were purchased from Sigma-Aldrich (St Louis,
MO, USA). Revert Aid M-Mulv Reverse Transcriptase
was obtained from Fermentas (Burlington, ON, Canada).
bromide (MTT),DMSO
selective PDE9
Cell culture
Human breast cancer cell lines MCF-7 and MDA 46-
468 were obtained from the National Cell Bank of Iran
(NCBI). Cell lines were grown adherently in RPMI-
1640 media supplemented with 10% foetal calf serum,
100 U/ml penicillin and 100 mg/ml streptomycin at
37 °C in 5% CO2:95% air.
Gene expression assay
For expression profile analysis, total RNA was extracted
by Trizol reagent (Sigma-Aldrich). RNA concentration
and purity were carried out using UV spectrophotometry
as previously described (19). Total RNA was reverse
transcribed using RevertAid-First-Strand cDNA Synthe-
sis Kit with random hexamer according to the manufac-
turer’sprotocol (Fermentas
Quantitative real-time RT-PCR assays of PDE9 cDNA
were carried out using gene-specific double fluorescently
labelled TaqMan MGB probe (HS00610023-mL) in a
fast real-time PCR system (ABI 7500 fast Real Time
PCR System; Applied Biosystem, Foster, California,
USA) according to the manufacturer’s instructions. Iden-
tical PCR conditions were performed using 1 ll of
cDNA, and relative expression levels of genes were nor-
malized to TaqMan probe GAPDH (HS99999905-m1).
Relative mRNA expression level was determined using
the 2?DDCtanalysis method.
Cat. No. K1631).
PDE9 activity assay
After treatment, MCF-7 and MDA-MB-468 cells were
harvested for total protein, in lysis buffer [(50 mM Tris–
HCl (pH 7–8), 150 mM NaCl, 100 mM EDTA, Triton
X-100 1%, 0.1% (w/v) sodium dodecyl sulphate (SDS)
10 ml 10X and PMSF, with protease and phosphatase
inhibitors)]. Total protein was determined using the
Bradford method (20). cGMP hydrolytic activity phos-
phodiesterase 9 was determined using a colorimetric
cyclic nucleotide phosphodiesterase assay kit (Biomol
Life Science, Exeter, UK) as described previously (21).
Briefly, protein was purified using column and P6 DG
desalting resin (BML-KI 100) to remove excess phos-
phate. The basis for the assay is the cleavage of cGMP
by a cyclic nucleotide phosphodiesterase, such as PDE9,
followed by the release
nucleotidase. Phosphate released was assessed using Bio-
mol Green reagent in a modified Malachite Green assay.
Optimal time reactions were obtained. To measure
PDE9-specific cGMP hydrolytic activity, each sample
per time-dependent manner was read, with and without
PDE9 inhibitor (BAY 73-6691). Samples were incubated
of free phosphate by a 5'
Figure 1. Selective PDE9 inhibitor BAY 73-6691.
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at 30 °C for 30 min and appropriately stopped by addi-
tion of the Biomol Green reagent. Results were measured
using an automated plate reader Synergy HT (Bio Tek
instrument, Winooski, Vermont, USA) at 620 nm and
compared to a standard curve drawn with cGMP and 5′-
nucleotidase. Differences between pmol cGMP hydroly-
sed per mg total protein per minute without and with
BAY 73-6691 PDE9 inhibitor, showed PDE9-specific
cGMP-hydrolytic activity.
Cyclic GMP assay
Cyclic guanosine 3′, 5′-monophosphate was quantita-
tively determined using an immunoassay cGMP kit pro-
vided by R&D Pharmacia Biotech, according to the
manufacturer’s instructions, as described previously
(22). In brief, cells were cultured for 24 h. At 80%
confluence they were treated with PDE9 inhibitor (BAY
73-6691) in a time-dependent fashion. Cells were lysed
by cell lysis buffer and mixtures were placed in wells of
a 96-well plate pre-coated with goat anti-rabbit antibody.
Following washing to remove excess conjugate and
unbound sample, substrate solution was added to the
wells and incubated for 30 min at room temperature.
Then measurement of cGMP was carried out using a
microplate reader (Bio-Rad., Hemel Hempstead, UK) set
at 450 nm.
MTT viability assay
Cell viability was determined by MTT assay as described
previously (23). Briefly, approximately 5000 cells/well
were seeded in 96-well plates and incubated for 24 h. On
reaching 60–80% confluence, used medium was replaced
by fresh medium containing appropriate concentrations
of specific compound, and was incubated for 24, 48 and
72 h. Then, 20 ll MTT (5 mg/ml in PBS) was added to
each well and incubated at 37 °C for 4 h, culture medium
was then removed carefully and dye dissolved in 200 ll
dimethyl sulphoxide (DMSO). Plates were incubated in
the dark for an additional 10 min and absorbance was
measured 570 nm wavelength, using a microplate reader
(Tekan Sunrise Instruments, Wiesbaden, Austria). All
assays were performed in triplicate.
Flow cytometry assay using annexin V/PI staining
Apoptotic cell death induced by BAY 73-6691 was
quantified by flow cytometry using an annexin-V–FITC
kit, according to the manufacturer’s protocol. Briefly,
cells were plated to 3 9 105/well density in six-well
plates and incubated with or without appropriate concen-
trations of BAY 73-6691, for 48 h. Treated and
untreated cells were harvested and washed twice in cold
PBS. Cell pellets were re-suspended in 500 ll 19 bind-
ing buffer, and 5 ll annexin V-FITC and 5 ll of PI
were added into the cell suspension, followed by gentle
vortexing. Staining samples were incubated for 15 min
at room temperature in the dark. Samples were analysed
using a FACScalibur flow cytometer (BD Biosciences,
San Jose, CA, USA) using software supplied with the
instrument. This allows discrimination of living cells
(unstained with either fluorochrome) from early apopto-
tic cells (stained with annexin V only) and late apoptotic
cells (stained with both annexin V and PI).
Morphological analysis using Hoechst 33258 staining
To confirm cells undergoing apoptosis, Hoechst 33258
staining was performed as described previously (24).
Briefly, Cells were grown in eight well chamber slides
and allowed to adhere. After treatment with 75 lM of
BAY 73-6691 for 48 h, cells were fixed in 4% parafor-
maldehydefor30 minat
washed twice in PBS. Hoechst 33258 (50 ng/ml) was
added to the fixed cells, incubated for 30 min at 37 °C
in dark, then washed in PBS. Cells were examined by
fluorescence microscopy. Apoptotic cells were identified
by their characteristic nuclear condensation, whereas
nuclei of non-apoptotic cells had normal uniform chro-
matin.
roomtemperaturethen
Measurement of caspase-3 activity
Caspase-3 activity was measured using Colorimetric
Assay Kit (R&D systems Co., Grodig, Germany) accord-
ing to the manufacturer’s instructions, as described previ-
ously (19). Briefly, cells were treated with PDE9
inhibitor (BAY 73-6691) for 24 h, then were washed in
PBS and lysed in 50 ll lysis buffer for 10 min; then they
were centrifuged (10 min at 10 000 g). Supernatants
were incubated in 50 ll reaction buffer and 5 ll of cas-
pase-3 substrate, at 37 °C for 60 min. Absorbance was
measured at 405 nm using a Tekan Sunrise microplate
reader.
Statistical analysis
Results were expressed as mean ± SD and statistical
analyses were performed by nonparametric analysis of
variance between groups (ANOVA), followed by Dun-
nett’s post-hoc test. All experiments were repeated at
least three times. Statistical analyses were performed
using software package
Chicago, IL, USA). Differences were considered statisti-
cally significant at P < 0.05.
SPSS version 17 (SPSS Inc.,
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Results
Quantitative RT-PCR assay
Differential expressions of PDE9 in both cell lines
were investigated by qRT-PCR using TaqMan probe
and PDE9 mRNAs were detected in both MCF-7 and
MDA-MB-468 cell lines (Fig. 2a). Effects of PDE9
inhibitor BAY 73-6691, on expression of the PDE9-
coding gene had been performed in a time-dependent
manner and results indicated that PDE9 expression
level gradually reduced in MCF-7 cells in the presence
of PDE9 inhibitor (200 lM), over times of 30, 60,
120, 240 and 480 min compared to that of the
untreated group (P < 0.05). Subsequently, a significant
increase was observed in mRNA expression level at
720 min (Fig. 2b) (P < 0.05). Similar effects were also
observed in MDA-MB-468 cells. As shown in Fig. 2b,
mRNA expression level of PDE9 decreased signifi-
cantly at times 30, 60, 120, 240 min (P < 0.05). How-
ever, gene expression level increased at 480 min
(P < 0.05) in comparison to that of the untreated
group.
PDE9 activity assay
Treatment of MCF-7 cells with BAY 73-6691 signifi-
cantly decreased PDE9 activity at 30–240 min, but
PDE9activityincreased
(P < 0.05). Similarly, BAY 73-6691 reduced PDE9
activity at 30–480 min in the MDA-MB-468 cell line,
compared to thatof controls
whereas this activity was elevated by 24 h. Thus, PDE9
activity correlated significantly with PDE9 mRNA
expression in both cell lines (data not shown).
significantly by 12–24 h
(P < 0.05) (Fig. 3),
cGMP assay
Effects of PDE9 inhibition on intracellular levels of
cGMP in MCF-7 and MDA-MB-468 cells had been
assessed. As shown in Fig. 4, BAY 73-6691 signifi-
cantly increased cGMP levels at 480 and 720 min in
MCF-7 and MDA-MB-468 respectively, compared to
those of controls (P < 0.05). These results demonstrated
that inhibition of PDE9 promoted cGMP levels, and
suggests that PDE9 may be a major regulator of basal
cGMP levels in these breast cancer cells.
Anti-proliferative effect of PDE9 inhibitor, BAY 73-
6691, on human breast cancer cells
Effects of selective PDE9 inhibitor (BAY 73-6691) on
the breast cancer cell lines’ viability was examined.
MCF-7 and MDA-MB-468 cells were treated with dif-
ferent concentrations of PDE9 inhibitor (10–200 lM) for
24, 48 and 72 h. BAY 73-6691 significantly inhibited
viability of both MCF-7 (Fig. 5a) and MDA-MB-468
(Fig. 5b) cells in a time- and dose-dependent manner
(P < 0.05). The most significant inhibitory effects of
BAY-73-6691 on MCF-7 cells were 55.3 ± 5.5% and
46.9 ± 3.5% at 200 lm, after treatment for 48 and 72 h
respectively. Similar effects were observed on MDA-
MB-468 cells; treatment with 200 lM BAY-73-6691
caused reduction of 70.3 ± 3.2% and 58.8 ± 5.4% on
cell viability after 48 and 72 h respectively.
BAY 73-6691 induction of apoptosis in breast cancer
cell lines
To study whether BAY 73-6691-induced cell population
growth inhibition was related to apoptosis, effects of
(a)
(b)
Figure 2. (a) PDE9 mRNA was detected on human breast cancer cell lines, MCF-7 and MDA-MB-468. (b) Quantitative real-time PCR analysis of
PDE9 mRNA expression in MCF-7 and MDA-MB-468 cells following BAY 73-6691 treatment in a time-dependent manner. Expression levels
were normalized to human GAPDH mRNA. Error bars represent standard deviation.*P < 0.05; **P < 0.01 compared to untreated control groups.
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BAY 73-6691 on apoptosis were evaluated. MCF-7 and
MDA-MB- 468 cells were exposed to various concentra-
tions (50–200 lM) of BAY 73-6691 for 48 h, and analy-
sed by flow cytometry, using annexin-V and PI double
staining. A significant increase was shown in percentage
of both early (annexin-V positive, PI negative) and late
(annexin-V positive, PI positive) apoptosis, in a dose-
dependent manner (P < 0.01) (Fig. 6). To confirm that
BAY 73-6691 had induced apoptosis in the cells, an
additional apoptotic marker, Hoechst 33258 staining,
was evaluated. As shown in Fig. 7, treatment with
75 lM of BAY 73-6691 for 48 h caused increased
changes in nuclear morphology such as chromatin con-
densation and fragmentation, in both cell lines.
Caspase-3 activation assay
To examine any contribution of caspases in PDE9 inhibitor
-induced apoptosis, the role of caspase-3 was investigated.
Treatment of MDA-MB-468 cells with various concentra-
tions of BAY 73-6691 induced significant increases in
activity of caspase-3, in a dose-dependent manner (Fig. 8)
(P < 0.05). Unlike MDA-MB-468 cells, activity of cas-
pase-3 remained unchanged in MCF-7 cells after treatment
with BAY 73-6691.
Discussion
In recent studies, it has been reported that inhibition of
PDE reduced cancer cell proliferation and induced apop-
tosis, through the cGMP pathway. Thus, selective PDE
inhibitors have been considered for treatment of various
human diseases such as cancer (11). BAY 73-6691, as
a selective PDE9 inhibitor, has been implicated in
Figure 4. Effect of PDE9 inhibitor (BAY 73-6691) on cGMP levels
in MCF-7 and MDA-MB-468 cells. Cells were treated with 200 lM
BAY 73-6691 over a time course. Measurement of cGMP was per-
formed using a colorimetric competitive ELISA kit. Each point,
mean ± SD of three experiments. Each experiment was repeated three
times. *P < 0.05; **P < 0.01 compared to untreated control groups.
(b)
(a)
Figure 5. Effect of BAY 73-6691 PDE9 inhibitor, in inhibition of
cell proliferation of the breast cancer cell lines. Cells were treated
with different concentrations of BAY 73-6691 for 24, 48 and 72 h,
and proliferation was measured by MTT assay. BAY 73-6691 reduced
cell proliferation in MCF-7(5A) and MDA-MB-468(5B) breast cells in
a time- and dose-dependent manner. Each value is presented as
mean ± SD of threeexperiments
**P < 0.01 are significant compared to untreated control groups.
(Eachtriplicate).
*P < 0.05,
Figure 3. BAY 73-6691 reduced PDE9 activity in MCF-7 and
MDA-MB-468 cells. Cells were incubated to concentration of PDE9
inhibitor; BAY 73-6691 (200 lM) in a time-dependent manner and
total protein was harvested. PDE9-specific activity was measured as
BAY 73-6691-inhabitable fraction of total cGMP hydrolysis, normal-
ized to total milligrams of protein. Each point, mean ± SD of three
experiments. Each experiment was repeated three times. *P < 0.05;
**P < 0.01 compared to untreated control groups.
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suppression of tumour growth both in vitro and in vivo
and it has been used in clinical trials for treatment of
Alzheimer’s disease (17,25). In a previous study by our
group, it was demonstrated that activation of PKG by
cGMP induced cell population growth inhibition and
apoptosis in human breast cancer cell lines, MCF-7 and
MDA-MB-468 (22). Biological roles of cGMP-specific
phosphodiesterase have been poorly investigated in
human breast cancer cells yet in a preliminary study,
Tinsley et al. showed that Sulindac sulphide (a PDE5
inhibitor), inhibited proliferation of SK-BR-3 and MDA-
MB-231 breast cancer cell lines (13). However, the
molecular mechanisms and apoptotic pathway of cGMP-
specific phosphodiesterase9, PDE9, have not been eluci-
dated in breast cancer cell lines. Thus, the effect of
BAY 73-6691 on expression level and activity of PDE9,
as well as the cGMP signalling pathway, has been stud-
ied in the both ER positive and ER negative human
breast cancer cell lines, MCF-7 and MDA-MB-46
respectively. Cell population growth inhibitory effect of
BAY 73-6691 and possible mechanisms of activity have
been investigated.
cGMP-specific PDE9 mRNAs were expressed in
both cell lines and PDE9 expression level was found to
gradually decrease, in a time-dependent manner, by
BAY 73-6691 (Fig. 2b). There was significant differ-
ence between MDA-MB-468 and MCF-7 cells in
mRNA expression of PDE9 at 720 and 480 min after
inhibitor administration, as well (Fig. 2b). Elevation was
maximum after 720 min in both cell lines, although
increase in PDE9 mRNA expression in MDA-MB-468
(ER negative breast cancer cell line) occurred earlier
than in MCF-7s (ER positive breast cancer cell line).
To verify activity of PDE9 and regulation of second
messenger cGMP in human breast cancer cells, we
Figure 6. Flow cytometric evaluation of apoptosis in MCF-7 and
MDA-MB-468 cells, using annexin-V and propidium iodide (PI)
staining. After 48 h treatment, PDE9 inhibition resulted in a signifi-
cant increase of early apoptotic cells and of late apoptotic cells, in a
concentration-dependent manner. Results, mean ± SD three indepen-
dent experiments. *P < 0.05; **P < 0.01 compared to control.
(b)
(a)
MCF-7 Control
(d)
75 µ?
75 µ?
(c)
MDA-MB-468 Control
Figure 7. Detection of typical features of
apoptotic nuclear condensation by Hoechst
33258staining(after
MCF-7 control (a), MCF-7 treated with
75 lM of BAY73-6691 (7b), MDA-MB-468
control(7c),MDA-MB-468
75 lM of BAY 73-6691 (d).
48 htreatment).
treatedwith
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investigated the ability of BAY 73-6691 to modulate
PDE9 activity and the cGMP signalling pathway. Our
data indicated that BAY 73-6691 significantly reduced
PDE9 activity, and increased levels of intracellular
cGMP, in the both cell lines. PDE9 mRNA expression
significantly correlated with PDE9 activity and intracel-
lular cGMP levels.
These results demonstrate that inhibition of PDE9
promotes cGMP accumulation, and suggest this phos-
phodiesterase to be a major regulator of basal cGMP
levels in breast cancer cells. Recent studies have shown
that inhibition of phosphodiesterases elevates intracellu-
lar cGMP (17,26). Other studies have indicated that
other PDEs are involved in the negative feedback con-
trol of cyclic nucleotides pathways (14) and our obser-
vations indicate that inhibition of PDE9 by BAY 73-
6691 leads to accumulation of intracellular cGMP and
maybe, activates the cGMP signalling pathway.
The results here indicate that PDE9 activity in MCF-
7 cells is higher than in MDA-MB-468 by 480 min after
using the inhibitor. This difference is probably due to
differences in cell type. PDE activities are regulated
through a variety of mechanisms including protein
expression and negative feedback regulation (14). Nega-
tive feedback regulation is one of the most fundamental
of biological regulatory processes and, in mammals, can
occur at several levels. The most common mechanism
for negative feedback control of PDEs is by mass
action, that is, elevation of cAMP or cGMP increasing
activity of PDE. Most studies have been focused on
negative feedback regulation of PDE5 and according to
them it would be expected that other PDEs within the
PDE superfamily would be involved in negative feed-
back control of cAMP or cGMP pathways, in addition
to their roles by mass action (14).
We have also demonstrated that BAY73-6691 inhib-
ited proliferation of these breast cancer cells through
induction of apoptosis via cGMP signalling. These find-
ings are consistent with those of previous studies using
MCF-7 and MDA-MB-468 breast cancer cells (22) and
human colon tumour HT29 cells (27), showing that
cGMP can play an important role in inhibition of cell
proliferation and induction of apoptosis. Moreover, the
results have shown that anti-proliferative and apoptotic
effects of BAY73-6691 on MCF-7s (ER positive breast
cancer cell line) were higher than on MDA-MB-468s
(ER negative human breast cancer cell line).
In addition, induction of apoptosis by treatment of the
cells with various concentrations (50–200 lM) of BAY73-
6691 was observed to externalize phosphatidylserine, to
activate caspase3 and to cause morphological changes.
Recently, studies have shown that inhibition of selective
PDEs induced apoptosis and cell cycle arrest in malignant
cells; thus, PDE inhibitors may be used as novel therapeu-
tic agents for tumour cell population growth inhibition
and induction of apoptosis (11,13,14,28,29).
Activation of a caspase cascade is a critical event in
induction of apoptosis in mammalian cells (30) and
recently, a number of studies has demonstrated that cas-
pase 3 plays an important role in the apoptotic response
(22,31,32). It has been found that activation of PKG by
cGMP induces cell population growth inhibition and
apoptosis in breast cancer cell lines (22). We have
shown that in MDA-MB-468 cells, apoptosis is caspase-
3 dependent. Our findings indicated that treatment of
these with various concentrations of BAY 73-6691
resulted in a significant increase in caspase-3 activity,
while activation of caspase-3 was not observed in MCF-
7 cells. MCF-7 cells do not express caspase-3 protein
due to presence of a 47-bp deletion in exon 3 of the
caspase-3 gene (33).
Conclusion
This study introduces a possible mechanism for control
of breast cancer cell population growth through PDE9
and shows that BAY73-6691 can suppress MCF-7 and
MDA-MB468breastcancer
induces apoptosis via the cGMP signalling pathway.
Apoptosis induced by BAY73-6691 was also determined
by characteristic morphological changes and activation
of caspase3, which indicated that the caspase pathway
also, was involved in the apoptotic signalling. Thus, our
results provide valuable new information concerning a
novel therapeutic target for breast cancer, using a selec-
tive PDE9 inhibitor.
cellproliferation,and
Figure 8. Colorimetric assay of caspase -3 activty after treatment
with BAY73-6691 (10-200µM). Activity of caspase-3 increased in a
concentration-dependent manner in MDA-MB-468, but in MCF-7,
activity of caspase-3 remained unchanged. *P < 0.05; **P < 0.01 sig-
nificance compared to control.
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