Original Paper
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file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 304-310
doi:10.1111/j.1745-7270.2008.00402.x
Angiotensin II suppresses
adriamycin-induced apoptosis through activation of phosphatidylinositol
3-kinase/Akt signaling in human breast cancer cells
Yanbin
Zhao1, Xuesong Chen1, Li Cai1, Yanmei Yang2, Guangjie Sui1*, and Jin Wu1*
1 Department of Medical Oncology, The Third
Affiliated Hospital of Harbin Medical University, Harbin 150040, China
2 Cancer research
institute of Heilongjiang province, Harbin 150040, China
Received: October
8, 2007
Accepted: January
24, 2008
* Corresponding
authors:
Jin Wu: Tel,
86-451-86298730; E-mail, [email protected]
Guangjie
Sui: Tel, 86-451-86298279; E-mail, [email protected]
Angiotensin II
(Ang II) stimulates tumor growth and angiogenesis in some solid cancer cells,
but its anti-apoptosis role in breast cancer remains unclear. To address this
issue, we investigated the effect of Ang II on adriamycin-induced apoptosis in
breast cancer MCF-7 cells. Treatment of human breast cancer MCF-7 cells with
adriamycin, a DNA topoisomerase II inhibitor, caused apoptosis. However, cells
pretreated with Ang II were resistant to this apoptosis. Ang II significantly
reduced the ratio of apoptotic cells and stimulation of phospho-Akt-Thr308 and
phospho-Akt-Ser473 in a dose-dependent and time-dependent manner. In addition,
Ang II significantly prevented apoptosis through inhibiting the cleavage of
procaspase-9, a major downstream effector of Akt. The Ang II type 1 receptor
(AT1R) was responsible for these effects. Among the signaling molecules
downstream of AT1R, we revealed that the phosphatidylinositol 3-kinase/Akt
pathway plays a predominant role in the anti-apoptotic effect of Ang II. Our
data indicated that Ang II plays a critical anti-apoptotic role in breast
cancer cells by a mechanism involving AT1R/phosphatidylinositol 3-kinase/Akt
activation and the subsequent suppression of caspase-9 activation.
Keywords angiotensin II; angiotensin II type 1 receptor; PI3-kinase/Akt;
anti-apoptosis; breast cancer
Angiotensin II (Ang II), a multifunctional bioactive octapeptide of
the rennin-angiotensin system, plays a fundamental role as a vasoconstrictor in
controlling cardiovascular function and renal homeostasis. Ang II acts as a
potent growth factor and cytokine in vascular smooth muscle cells [1], cardiac
myocytes [2], and cardiac fibroblasts [3]. Recently, it has been shown that Ang
II stimulates tumor growth and angiogenesis, including choriocarcinoma [4],
breast cancer [5], ovarian cancer [6], and pancreatic cancer [7], suggesting
that Ang II might also be involved in cancer development.Ang II mediates its biological effects through binding to two
subtypes of receptors, the Ang II type 1 receptor (AT1R) and the Ang II type 2
receptor (AT2R), which belong to the superfamily of G-protein-coupled receptors
[8]. The major functions of Ang II in tumor growth and angiogenesis are
mediated through AT1R [9–12].Inhibition of apoptosis and cellular proliferation are important
mechanisms in carcinogenesis. Ang II also activates multiple signaling pathways
related to cell apoptosis and proliferation, including protein kinase C [4,13]
and mitogen-activated protein kinase [14]. Recently, stimulation of AT1R has
been shown to trigger the activation of phosphatidylinositol 3 (PI3)-kinase and
Akt [15]. It has been reported that the PI3-kinase pathway is an important
anti-apoptotic signal pathway that is frequently activated in cancer cells
[16,17]. The most important downstream effector of PI3-kinase is the
serine/threonine kinase Akt or protein kinase B. The induction of Akt activity
is primarily under the control of phosphoinositide products of PI3-kinase,
PIP2, and PIP3, which bind to the pleckstrin homology domain of
membrane-associated Akt. Subsequently, full activation of Akt requires
phosphorylation of two amino acids in Akt, one within the activation loop
(Thr308 ) and one at the C-terminus hydrophobic domain (Ser473) [18,19].
Activated Akt phosphorylates pro-apoptotic proteins, including Bad, caspase-9,
and forkhead transcription factors [20–22], thereby inhibiting apoptosis. Ang II has
been reported to significantly prevent cisplatin-induced apoptosis through
nuclear factor-kB activation and the subsequent production of anti-apoptotic
molecules, including survivin and Bcl-XL, in pancreatic cancer cells [7].
However, with regard to breast cancer cells, little is known about the effect
of Ang II in apoptosis. In this article, we studied the potential roles of Ang
II on adriamycin (ADR)-induced apoptosis, and found that Ang II plays an
anti-apoptotic role in MCF-7 cells. We further revealed the underlying
molecular mechanisms by examining the effects of Ang II on signaling pathways
related to apoptosis. The signaling molecules include AT1R, PI3-kinase, and
Akt. We further showed that caspase-9 participates in this anti-apoptotic
effect of Ang II downstream of Akt.
Materials and methods
Reagents and antibodies
Human ang II was
purchased from AnaSpec (San Jose, USA). AT1R antagonist, losartan (DuP 753),
was purchased from DuPont Merck (Hangzhou, China). AT2R
antagonist, PD123319, was purchased from sigma-Aldrich
(St. Louis, USA). LY294002, Akt, phospho-Akt-S473, phospho-Akt-T308, and
procapase-9 antibodies were from Cell Signaling Technology (Boston, USA).
Cell lines and culture
MCF-7 cell lines were the generous gift of Heilongjiang Cancer
Institute (Harbin, China). Cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum and 50 U/ml penicillin/50 mg/ml
streptomycin, then incubated at 37 ?C in a humidified atmosphere of 5% CO2.
Apoptosis morphological
analysis
Serum-starved MCF-7 cells were incubated with or without Ang II
and/or losartan and the chemotherapeutic agent ADR for 24 h. Cells were
collected, washed with phosphate-buffered saline (PBS), and stained with an
equal volume of Hoechst 33342 and propidium iodide (PI) for 20 min at 4 ?C.
Then the cells were washed gently with PBS. The morphology of the nuclei was
examined using a fluorescence microscope (Olympus, Tokyo, Japan).
DNA fragmentation analysis
DNA fragmentation analysis
Fragmented DNA was extracted with a DNA purification kit (Beyotime
Biotechnology, Hangzhou, China). A total of 107 cells were collected and suspended in 200 ml PBS, 4 ml RNase A, and
20 ml
Proteinase K, and incubated for 2 min at room temperature. Then 200 ml sample lysis
buffer B was added and incubated for 10 min at 70 ?C. The sample was mixed with
200 ml dehydrated alcohol and pipetted into a filter tube. The mixture
was centrifuged at 6000 g for 1 min and the flow-through was discarded.
Washing buffer I (500 ml) was added to the filter tube, then centrifuged as above. Washing
buffer II (600 ml) was added to the filter tube and centrifuged at 18,000 g
for 1 min, then the flow-through was discarded. The washing step was repeated,
then a final high speed spin (18,000 g) was carried out for 1 min. One
hundred microliters of warm elution buffer was added to the filter tube. the eluted DNA was collected by
centrifugation at 18,000 g for 1 min, and analyzed electrophoretically
on 1% agarose gels containing 0.1% ethidium bromide. The DNA band patterns were
visualized under ultraviolet illumination.
Flow cytometry
MCF-7 cells were incubated with or without Ang II and/or losartan
and the chemotherapeutic agent ADR. The adherent cells were collected by
trypsin, combined with floating cells, and centrifuged at 1000 g for 5
min. After being washed with PBS, cells were added into 500 ml of annexin V
binding buffer (Keygen, Nanjing, China), and incubated for 10 min with 5 ml
fluorescein-isothiocyanate-conjugated annexin V and 5 ml PI (Keygen), followed by
two-color flow cytometric analysis (Beckman Coulter, Fullerton, USA).
Fluorescence was measured with a minimum of 10,000 events for each sample in a
fluorescence-activated cell sorter according to the method suggested by the
manufacturers. For each cell, fluorescence channels 1 (annexin
V-fluorescein-isothiocyanate) and 3 (PI) were recorded. Annexin V-positive
cancer cells that did not take up PI were identified as early apoptotic cells,
whereas doubly positive cancer cells were classified as late apoptotic cells.
Western blot analysis
MCF-7 cells were lysed in a lysis buffer consisting of 20 mm Tris-HCl (pH 7.5), 2 mm EDTA, 150 mm NaCl, 1% Triton X-100, and protease inhibitors. After
centrifugation at 12,000 g for 5 min at 4 ?C, the supernatant was
obtained. The supernatant was used as a total cell lysate and analyzed for
protein concentration by the Bradford method (BioRad, Hercules, USA). Equal
amounts of cellular proteins (30 mg/lane) were separated by SDS-PAGE and
transferred to a nitrocellulose membrane. After blocking with 1% skimmed milk
in Tris-buffered saline/Tween 20 overnight at 4 ?C, the blots were incubated
with rabbit anti-human Akt, phospho-Akt-S473, phospho-Akt-T308, or procapase-9
antibody at a dilution of 1:600 for 2 h at room temperature. The blots were
subsequently washed three times (10 min for each wash) with Tris-buffered saline/Tween
20 then incubated with the appropriate alkaline phosphatase-conjugated
anti-rabbit secondary antibody (dilution 1:5000; Promega, Madison, USA) for 1 h
at room temperature. The bands were visualized using the BCIP/NBT (Promega)
coloration method.
Statistical analysis
Data are expressed as the mean±SD. Statistical analysis was carried
out using Student’s t-test for unpaired samples and ANOVA. A P
value less than 0.05 was considered statistically significant.
Results
Ang II blocks apoptosis in MCF-7
cells through AT1R
To study the effect of Ang II on anti-apoptosis, MCF-7 cells were
exposed to 0.1 mM ADR, a chemotherapeutic agent commonly used to treat patients with
breast cancer. It was anticipated that approximately 30% of the cells would be killed.
We detected apoptosis through observation of nuclear morphology by staining
cells with Hoechst 33342 and PI. The cells showed a distinct condensed and
fragmented chromatin in the nuclei after treatment with 0.1 mM ADR for 24 h,
but the characteristics for apoptosis were markedly suppressed by Ang II
pretreatment in a dose-dependent manner [Fig. 1(A)]. One hundred
nanomoles of Ang II had the maximal inhibition effect (P<0.01). Cells were further subjected to nuclear DNA fragmentation analysis [Fig. 1(B,c)]. Ang II pretreatment clearly
inhibited ADR-induced DNA fragmentation. These results were further confirmed
by PI/annexin V [Fig. 1(D,E)]. The percentage of cancer cells in the
apoptotic phase was (30.172.17)% with 0.1 mM ADR. However, the
percentage of apoptotic cells was significantly reduced by pretreatment with
100 nM Ang II (P<0.01). To determine the receptor responsible for mediating the anti-apoptotic effects of Ang II, we used receptor-type specific inhibitors. Losartan, an AT1R antagonist, completely suppressed the anti-apoptotic effect of Ang II, whereas PD123319, an AT2R antagonist, had no effect (Fig. 1). These results indicated that Ang II exerts its
anti-apoptotic effect in MCF-7 cells through the AT1R. Therefore, cells were
not pretreated with PD123319 in subsequent experiments.
Anti-apoptotic effect of Ang
II in MCF-7 cells is mediated by PI3-kinase/Akt pathway
To further study the mechanisms underlying the anti-apoptotic effect
of Ang II in MCF-7 cells, we investigated the signaling molecules in pathways
downstream of AT1R. PI3-kinase/Akt is a key regulatory pathway that controls
the cellular response to apoptosis. Therefore, we hypothesized that the
anti-apoptotic effect of Ang II in MCF-7 cells might be one of the biological
consequences of PI3-kinase/Akt activation. To address this possibility, MCF-7
cells were pretreated for 30 min with LY294002, a PI3-kinase inhibitor, and
then stimulated with Ang II for 24 h. At 50 mM LY294002, the
anti-apoptotic effect of Ang II was completely reversed (Fig. 1). These
results indicated that PI3-kinase plays a predominant role in the
anti-apoptotic effect of Ang II and prompted us to further examine the
potential link between Ang II and Akt, a major downstream effector of
PI3-kinase.Western blot analyses revealed that Ang II stimulates
phospho-Akt-Thr308 and phospho-Akt-Ser473 in MCF-7 cells in a dose-dependent
and time-dependent manner. As shown in Fig. 2(A), 100 nM Ang II caused
maximal phosphorylation of Akt and induced a significant increase of Akt
phosphorylation at 15 min. The peak level of phospho-Akt persisted for at least
6 h [Fig. 2(B)]. Therefore, these results suggested that Ang II exerts
its anti-apoptotic effect in MCF-7 cells through PI3-kinase and the subsequent
Akt activation.
Ang II stimulates
phosphorylation of Akt through AT1/PI3-kinase
We also investigated the molecular mechanisms leading to Akt
activation in Ang II signaling. It was revealed that the AT1R inhibitor losartan
completely blocked the Ang II-induced phosphorylation of Akt. This indicated
that AT1R is responsible for Ang II-induced Akt activation. Because PI3-kinase
was reported to mediate Akt activation, we then tested the effects of LY294002.
PI3-kinase inhibitors completely abolished Akt phosphorylation in response to
Ang II (Fig. 3).
Ang II inhibits caspase-9
activation through AT1/PI3-kinase
Cardone et al [20] reported that Akt induced the
phosphorylation of procaspase-9, thereby inhibiting its protease activity.
Therefore, we further studied the effect of Ang II on caspase-9 activation
after exposure of MCF-7 cells to ADR. As shown in Fig. 4(A), ADR
treatment decreased the procaspase-9 level and concomitantly caused the
appearance of a cleaved caspase-9 fragment. However, Ang II pretreatment
suppressed the ADR-induced cleavage of procaspase-9. The suppressive effects of
Ang II on the cleavage of procaspase-9 were completely inhibited by
pretreatment with losartan and LY294002, suggesting that this suppression is
through a mechanism dependent on AT1R and PI3-kinase [Fig. 4(B)].
Discussion
Recent studies have shown that normal and cancerous human breast
tissue express both AT1R and AT2R [23], but that AT1R is overexpressed in
breast ductal carcinoma in situ [24]. Therefore, Ang II/AT1R could be
involved in abnormal breast tissue function [25]. Our studies also show that
Ang II significantly promotes MCF-7 cell growth in a dose-dependent and
time-dependent manner. Losartan decreased the level of Ang II-induced
proliferative effects. However, treatment with losartan alone had no effect on
cell viability (data not shown). These results are consistent with the report
by Muscella et al [5]. Moreover, Ang II could modulate integrin
expression in MCF-7 cells through AT1R [26]. However, the role of Ang II on
ADR-induced apoptosis in breast cancer is not clear.We first revealed that Ang II protects MCF-7 cells from ADR-induced
apoptosis in a dose-dependent manner by Hoechst 33342/PI staining and nuclear
DNA fragmentation analysis. These results were further confirmed by flow
cytometry analysis using PI/annexin V. This finding is the first indication of
an anti-apoptotic role for Ang II in breast cancer cells. Recently, reports
have shown that Ang II stimulates the growth of tumor cells through AT1R [9–12]. In this
study, we clearly showed that Ang II plays an anti-apoptotic role in MCF-7
cells and that AT1R, but not AT2R, is the receptor that mediates this
mechanism.We next investigated apoptosis-related signaling pathways and
PI3-kinase, previously reported to be downstream of AT1R [19]. Ang II is
reported to prevent apoptosis in microvascular endothelial cells through the
PI3-kinase pathway [27]. In this study, Hoechst 33342/PI staining, nuclear DNA
fragmentation analysis, and flow cytometric analysis revealed that 50 mM PI3-kinase
inhibitor completely reversed the anti-apoptotic effect of Ang II in MCF-7
cells. Akt has been identified as a major downstream target of PI3-kinase,
involved in Ang II-induced proliferation in rat aortic smooth muscle cells
[28]. So we used Western blot analyses to determine the phosphorylation status
of Thr308 and Ser473. Ang II increased phospho-Akt-Thr308 and
phospho-Akt-Ser473 in MCF-7 cells in a dose-dependent and time-dependent manner.
These results indicated that the PI3-kinase/Akt pathway has an essential role
in anti-apoptosis signaling by Ang II. Because the PI3-kinase/Akt pathway in
breast cancer cells has also been shown to play a critical role in
chemoresistance [29,30], our results might further support a general concept
that this pathway is indispensable for protection against apoptosis.We further studied the pathways leading to Akt phosphorylation in
Ang II/AT1/PI3-kinase signaling. A previous report indicated that PI3-kinase is
required for Ang II-induced Akt phosphorylation in vascular smooth muscle cells
[15]. In MCF-7 cells, our study showed that losartan and LY294002 significantly
inhibit Akt phosphorylation. These data suggest that AT1/PI3-kinase signaling
is the mechanism for Akt activation by Ang II.The intracellular machinery responsible for apoptosis depends on a
family of caspases. Caspase-9 is a key caspase involved in ADR-induced
apoptosis [31]. Another mechanism whereby Akt functions to promote survival is
through the phosphorylation and inactivation of procaspase-9, because Akt has
been found to phosphorylate procaspase-9 and thereby inhibit its protease
activity [20]. Therefore, we focused on the effect of Ang II on procaspase-9.
In our studies, Ang II pretreatment suppressed the ADR-induced cleavage of
procaspase-9, and this inhibition was suppressed in the presence of losartan or
LY294002. Thus, it seems that AT1R and PI3-kinase/Akt pathways could be
essential for Ang II to transmit its protective signal against ADR-induced
apoptosis.In summary, our results show that Ang II is a prominent
anti-apoptotic molecule in breast cancer MCF-7 cells and the underlying
molecular mechanism for this effect involves AT1R and PI3-kinase/Akt. We
further uncovered that a novel signaling pathways responsible for Ang
II-induced anti-apoptosis was inhibition of caspase-9 activation. Moreover, De
Paepe et al [24] proved that normal mammary epithelial HMec cells showed
little effect when treated with increasing concentrations of Ang II and
losartan. Thus, the present observations suggest that clinical benefits in
treating patients with breast cancer could be obtained with appropriate
combinations of novel AT1R inhibitors and conventional chemotherapeutic drugs.
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