RESEARCH ARTICLE

Neoplasia . Vol. 7, No. 7, July 2005, pp. 631 637

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Endothelin-1 Inhibits Apoptosis in Prostate Cancer1

Joel B. Nelson, Michael S. Udan, Georgi Guruli and Beth R. Pflug*
Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Abstract
Endothelin-1 (ET-1), produced by the prostate epithelia,
likely plays an important role in the progression of
prostate cancer. ET-1 can bind two receptor subtypes;
generally, binding of the endothelin receptor A (ETA)
induces a survival pathway, whereas binding of the
endothelin receptor B (ETB) mediates clearance of
circulating ET-1 as well as promotes apoptosis. In
prostate carcinoma, hypermethylation of the ETB promoter results in repression of ETB expression, thereby
eliminating the negative growth response that ET-1
binding elicits through this receptor. Therefore, activation of ETA exclusively provides a pathway for survival advantage. Our current studies examine the
mechanisms by which activation of the ETA may allow
growth/survival. ET-1 treatment of prostate tumor
cells significantly decreased paclitaxel-induced apoptosis through activation of the ETA subtype. The antiapoptotic effects of ET-1 are mediated, at least in
part, through the Bcl-2 family. Although no significant
changes in Bcl-2 expression occurred with ET-1 treatment, the pro-apoptotic family members Bad, Bax, and
Bak all decreased significantly. Further analysis of
the survival pathway demonstrated that phosphorylation of Akt occurs with ET-1 treatment in a time- and
dose-dependent manner through phosphatidyinositol
3-kinase activation. These data support the combination of ETA antagonists and apoptosis-inducing therapies for prostate cancer treatment.
Neoplasia 7, 631 637
Keywords: Endothelin-1, apoptosis, prostate cancer, endothelin receptors, Akt.

Introduction
Prostate cancer is characterized by low rates of cell proliferation coupled with diminished rates of cell death [1]. This
pattern has made prostate cancer among the most resistant
of malignancies to cytotoxic chemotherapeutic agents.
Furthermore, the cornerstone of the management of advanced
prostate cancer, androgen deprivation therapy, relies on the
effective induction of apoptotic pathways. The emergence of
androgen refractory prostate cancer, which leads to the
lethal form of the disease, indicates that these cells likely
have developed survival mechanisms to escape death.
The potent vasoconstrictor endothelin-1 (ET-1) has
been implicated in prostate cancer disease progression
[2 4]. ET-1 expression occurs in almost every human

prostate cancer tissue studied [4,5]. Moreover, patients with

metastatic prostate cancer have elevated levels of plasma ET-1
compared with patients with organ-confined cancer as well as
healthy individuals [4]. ET-1 binds to two receptor subtypes.
Activation of the endothelin receptor A (ETA) can lead to induction of a survival pathway, whereas activation of the endothelin receptor B (ETB) can result in clearance of circulating
ET-1 as well as in stimulation of apoptosis. However, the response to the binding of either receptor remains cell type
dependent. In prostate cancer, the expression of the endothelin
receptors, ETA and ETB, is altered compared to the pattern seen
in normal prostatic tissues [6,7]. The ETB, predominant on
benign prostatic epithelial cells, has a much lower expression
on prostate cancer cells, owing, at least in part, to frequent
hypermethylation of the ETB gene, EDNRB [8]. Increased ET-1
expression, coupled with the increased ETA expression that
occurs with higher prostate tumor stage and grade, may produce a survival advantage for the prostate cancer cells. Indeed,
in a phase II clinical trial of the ETA antagonist, atrasentan, there
was a significant delay in time to disease progression compared
to placebo in men with hormone refractory disease [9,10].
In studies of endothelial and stromal cell populations, ET-1
acting through the ETA inhibited apoptosis induced by a cytotoxic agent [11]. Given that endothelin receptor expression
in prostate cancer favors the ETA and the compelling results
from the atrasentan clinical trials, it is our hypothesis that
ET-1 can act as a survival factor for prostate cancer. Therefore, we studied ET-promoted survival in prostate cancer, and
demonstrated that ET-1, acting through ETA and the phosphatidyinositol 3-kinase (PI3-kinase)/Akt pathway, inhibited
paclitaxel-induced apoptosis.

Materials and Methods

Cell Lines
Prostate cell lines DU145, PC3, LNCaP (American Type
Culture Collection, Manassas, VA) PPC-1 [12], and TSU [13]
were grown in RPMI 1640, and LAPC4 (gift from Dr. Robert
Abbreviations: ABT-627, atrasentan; ET, endothelin; ETA, endothelin receptor A; ETB, endothelin receptor B; EDNRB, endothelin receptor B gene; PI3-kinase, phosphatidyinositol 3-kinase
Address all correspondence to: Beth R. Pflug, Department of Urology, University of Pittsburgh,
5200 Centre Avenue, G-35, Pittsburgh, PA 15232. E-mail: pflugbr@upmc.edu
1
Support for the data analysis was provided by the Pharsight Academic License Program (for
the WinNonlin software). This work was supported, in part, by the Mellam Family Foundation.
Received 16 December 2004; Revised 8 February 2005; Accepted 9 February 2005.
Copyright D 2005 Neoplasia Press, Inc. All rights reserved 1522-8002/05/$25.00
DOI 10.1593/neo.04787

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Reiter, UCLA, Los Angeles, CA) cells were grown in Iscoves

modified Dulbeccos medium supplemented with 10% FBS
and penicillin/streptomycin.
Apoptosis Assay
Prostate cell lines were pretreated with 1.0 mM ABT-627
or A127722 (ETA antagonists; Abbott Laboratories, Abbott
Park, IL), 1.0 mM RES-701 (ETB antagonist; American Peptide,
Sunneyvale, CA), or A-192621 (ETB antagonist; Abbott Laboratories), 200 nM wortmannin (Sigma Chemical Co., St. Louis,
MO), 10 mM LY294002 (Sigma Chemical Co.), or 20 mM
PD98059 (Calbiochem, La Jolla, CA) for 1 hour prior to ET-1
treatment in serum-free medium. ET-1 (100 nM) was added
followed by 100 nM paclitaxel (Bristol-Myers Squibb, Princeton, NJ) or an antibody to fas (10 ng/ml; Signal Transduction
Laboratories, Lexington, KY) and the cells were incubated
for 4 to 24 hours. The cells were scraped from the plates and
pelleted by centrifugation (200g). The cell pellet was then
lysed using apoptosis lysis reagent and centrifuged at
10,000g for 10 minutes. A spectrophotometric ELISA-based
assay was used to quantify histone-associated DNA fragments present in the cell lysates according to the manufacturers instructions (Roche Diagnostics, Indianapolis, IN).
Immunoblot Analysis
Prostate cells were plated in 100-mm dishes and treated
with ET-1 (0.1 nM 1.0 mM) for 5 minutes to 24 hours in
the presence or absence of: 100 nM ABT-627 or A127722;
PI3-kinase inhibitors, 200 nM wortmannin and 10 mM
LY294002; MEK inhibitor, 20 mM PD98059; and p70 S6
kinase inhibitor, 5 nM rapamycin. The cells were lysed in
20 mM Tris HCl buffer (pH 8.0) containing 10% glycerol, 1%
Triton X-100, and 135 mM NaCl with fresh protease inhibitors. The proteins (40 mg) were separated by 10% or 12%
SDS-PAGE and electrotransferred onto PVDF membranes.
The membranes were blocked and incubated in primary
antibody [phospho-Akt, Akt, phospho-p44/42 MAP kinase,
BAD, 1:1000 dilution (NEB, Beverly, MA); Bcl-2, Bcl XL
phospho-Raf, 1:500 dilution (Transduction Laboratories);
Bax, Bak, caspase3, caspase 9, 1:200 dilution (Oncogene,
Boston, MA)] in TBST (20 mM Tris HCl, pH 8.0, 150 mM
NaCl, 0.1% Tween 20) overnight at 4jC. After washing, the
blots were incubated in secondary antibody (goat anti-mouse
or goat anti-rabbit HRP, 1:20,000; Roche Diagnostics) for
1 hour and washed in TBST. Immunoreactive proteins were
visualized by ECL (Amersham, Piscataway, NJ) and Kodak
(Rochester, NY) XAR film. Densitometry was performed
using a Molecular Imager FX phosphorimaging system with
Quantity One 4.1 quantitation software (Bio-Rad, Hercules,
CA). Band densities were normalized to corresponding b-actin
bands. EC50 for ET-1 induced Akt phosphorylation was
determined from the nonlinear regression analysis of the
densitometry data using WinNonlin Pro Academic v4.0.1
(Mountain View, CA).
Annexin V Staining and Flow Cytometry
PPC-1 cells were grown in 100-mm tissue culture dishes
(Becton Dickinson, San Jose, CA). When cells were sub-

confluent, growth media were removed, cells were washed

with RPMI, and serum-free media were added. Six groups
were formed according to administered treatment: 1) serumfree media only; 2) ET-1 (10 7 M); 3) paclitaxel (10 7 M); 4)
ET-1 for 1 hour followed by paclitaxel; 5) ABT-627 (10 6 M),
followed by ET-1 for 1 hour, and then followed by paclitaxel; 6) A192621 (10 6 M), followed by ET-1 for 1 hour, and
then followed by paclitaxel. Each group was treated for a
total of 4 hours. In addition to paclitaxel, these studies were
carried out using a fas antibody (10 ng/ml for 4 hours) to
induce cell death with and without ET-1 treatment. Cells
were collected in 24 hours and evaluated for apoptosis by
Annexin V staining.
For flow cytometry using the Annexin V assay, cells were
collected and double-stained with fluorescein isothiocyanate
conjugated Annexin V (PharMingen, San Diego, CA) and propidium iodide (PI) (Sigma Chemical Co.). Cells were counted
and 105 cells for each condition (in 100 ml of Annexin V binding buffer) were placed in 5-ml round-bottom tubes (Becton
Dickinson). Each condition was done in duplicate. Annexin V
was added according to the manufacturers recommendations. PI was used at a final concentration of 5 mg/ml.
Annexin V positive cells were considered apoptotic and
their percentage of the total number of cells was calculated.
Cells taking up vital dye PI were considered dead. Samples
of 10,000 cells were analyzed by FACScan flow cytometer
with LYSYS II software package (Becton Dickinson).

Results
ET-1 Inhibits Prostate Cancer Apoptosis In Vitro
Treatment of the prostate cancer cell PPC-1 with paclitaxel induces apoptosis as demonstrated by an ELISA-based
apoptosis assay as well as by flow cytometric analysis of
Annexin V staining (Figures 1 and 2). In the presence of
ET-1, however, there is a significant reduction in the amount
of cell death. The addition of the selective ETA antagonist,
A-127722, or its racemic compound, ABT-627, reversed
the ability of ET-1 to inhibit apoptosis. The selective ETB
antagonist, A192621, did not significantly affect the ability
of ET-1 to inhibit apoptosis (Figure 2). The cells treated
with A192621 + ET-1 + paclitaxel showed a slightly higher
rate of apoptosis (31.63%) compared with ET-1 + paclitaxel
(23.44%); however, this increase was not statistically significant (P = .558) and may be the result of partial nonspecific blockade of ETA by using 10 mM ETB antagonist in
these studies. A similar pattern was seen in the other prostate cancer cell lines tested, all of which have no detectable ETB expression due to EDNRB methylation. The antiapoptotic actions of ET-1 were also blocked by LY294002,
an inhibitor of the PI-3 kinase pathway, but not by PD98059,
an inhibitor of the MEK1 pathway (Figure 1). To confirm
these results, prostate cancer cell lines were exposed to
agonistic antibodies to fas, a potent inducer of apoptosis [14,15]. The addition of ET-1 significantly inhibited fasinduced apoptosis, whereas ETA antagonists blocked this
effect (data not shown).

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633

anti-apoptotic and pro-apoptotic proteins was examined

after exposure of PPC-1 cells to ET-1 for 0 to 6 hours. The
expression of the anti-apoptotic proteins, Bcl-2 (Figure 3)
and Bcl-xL (data not shown), was not altered by exposure
to ET-1. However, the expression of the pro-apoptotic protein, Bad, was significantly reduced by ET-1, as were the
pro-apoptotic proteins, Bax and Bak, although to a lesser
degree than Bad (Figure 3). Density measurements of the
immunoblot bands were obtained using a phosphorimaging system and normalized to corresponding b-actin bands.
Densitometric analysis indicated a 3.2-fold reduction in Bak,
a 14.3-fold reduction in Bax, and an undetectable expression of Bad after 6 hours of ET-1 treatment. Expression of
caspase 3 did not change (Figure 3). In addition, expression
of BclXL, caspase 9, phospho-p44/42 MAP kinase, and
Raf remained constant in cells treated with ET-1 from 4 to
72 hours (data not shown).
Figure 1. ET-1 treatment of the prostate tumor cell line (PPC-1) significantly
reduced paclitaxel-induced apoptosis. An ETA antagonist (ABT-627) and PI3kinase inhibitor (LY924002) blocked the survival effect of ET-1. No inhibition
of ET-1 promoted cell survival effects was observed with a MEK inhibitor
(PD98059) (P < .05). Each condition was performed in triplicate and results
are shown as the mean SE. This figure is representative of four independent experiments.

ET-1 Reduces Pro-Apoptotic Protein Expression

To understand the mechanisms whereby ET-1 inhibits
apoptosis in prostate cancer cells, the expression of both

ET-1 Induces Phosphorylation of Akt

The serine/threonine kinase Akt (protein kinase B) is a
point of signaling convergence for growth and survival pathway signaling through PI3-kinase and PDK1. Once activated, the kinase Akt inhibits apoptosis by phosphorylating
a variety of substrates including pro-apoptotic proteins Bad,
forkhead transcription factors (FKHR), and glycogen synthase kinase (GSK-3) [16 18]. Phosphorylation of Akt also
induces MDM2 activity, which leads to the degradation of

Figure 2. Annexin V staining and flow cytometric analysis for apoptosis. PPC-1 cells were treated with: (A) media only; (B) ET-1 (10 7 M); (C) paclitaxel (10 7 M);
(D) ET-1 + paclitaxel; (E) ABT-627 (10 6 M) + ET-1 + paclitaxel; and (F) A-192621 (10 6 M) + ET-1 + paclitaxel. After 24 hours, cells were stained with fluoresceinconjugated Annexin V and PI, and analyzed by flow cytometry. Paclitaxel-induced apoptosis was significantly reduced in ET-1 treated cells (P < .001). This effect
was abrogated with ETA antagonist (ABT-627). The ETB antagonist had no significant effect on the survival-promoting effects of ET-1. Transparent histograms:
unstained cells; shaded histograms: Annexin V treated cells. Numbers depict the percentage of positively stained cells. Results are representative of four
independent experiments.

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inhibitors [22 25] and indirect effects on cyclin D1 and p53

[18,19]. To investigate which pathways were involved in the
ET-1 activation of Akt in prostate cancer cells, we used
inhibitors of PI3-kinase (wortmannin, LY294002), MEK1
(PD98059), and p70 S6 kinase (rapamycin) (Figure 5). Incubation with PI3-kinase inhibitors prior to ET-1 stimulation
resulted in loss of Akt phosphorylation. The p70 S6 and
MEK1 inhibitors, rapamycin and PD98059, had no effect
on ET-1 induced phosphorylation of Akt, further indicating
that ET-1 survival signaling occurs through a PI3-kinase
pathway and not a p44/42 MAP kinase pathway and does
not involve p70 S6 kinase signaling.

Figure 3. Immunoblot analysis of regulation of apoptotic proteins by ET-1.

PPC-1 cells were serum-starved overnight and treated for 30 minutes to
6 hours with ET-1. The pro-apoptotic proteins Bad, Bax, and Bak decreased
with ET-1 treatment. Bcl-2 and caspase-3 expression did not change. Bcl-xL,
caspase-9 phospho-p44/42 MAP kinase, and phospho-Raf (not shown)
expression also did not change. Immunoblots were analyzed by densitometry, normalized to corresponding -actin bands and reported as relative level
of expression to untreated cells. Results are representative blots from five
separate experiments.

p53 [19]. In both a dose- and time-dependent fashion, ET-1

induced phosphorylation of Akt in the prostate cancer cell
line PPC-1 (Figure 4A). Immunoblots were reprobed with
b-actin (to confirm that equal loading was achieved) (Figure 4)
and Akt antibody (to ensure that ET-1 treatment did not affect
overall Akt expression levels) (data not shown). Densitometry was carried out on the immunoblots, and EC50 for ET-1
was calculated using WinNonlin software (Pro Academic
v4.0.1; Mountain View). The EC50 was 220 pM for ET-1
induced Akt phosphorylation calculated from the dose response experiments. A 2.33-fold increase in Akt activation
occurred within 30 minutes of ET-1 treatment and remained
elevated for the duration of the experiment (3 hours). In both
androgen-sensitive (LNCaP and LAPC-4) and androgeninsensitive cell lines (PC3 and PPC-1), ET-1 increased
phosphorylation of Akt, whereas ET-1 did not produce a
significant effect on Akt phosphorylation in the bladder cell
line, TSU (Figure 4B).
Akt Phosphorylation by ET-1 Is Mediated by the
PI-3 Kinase Pathway
Cell survival requires active repression of apoptosis
through the inhibition of pro-apoptotic proteins or the induction of anti-apoptotic factors. Many cell survival signaling
cascades trigger the activation of PI-3 kinase, leading to
phosphorylation of Akt. Akt can regulate cell growth through
its effects on the p70 S6 kinase pathway [20,21] as well as
by negative regulation of p21WAF1 and p27KIP1 cell cycle

Discussion
Following the initial description in prostate cancer [7,8], loss
of ETB expression through hypermethylation of EDNRB has
been reported in a variety of malignancies [26 31]. As the
receptor responsible for ligand clearance and pathways
counterregulatory to ETA signaling, a decrease in ETB
expression results in unfettered ET-1/ETA activity. However,
it remains unknown whether silencing of the ETB is crucial
for carcinogenesis and/or disease progression. Although
ETA and ETB are coupled to many of the same signaling
molecules, ETB activation can uniquely mediate divergent
responses such as vasodilation through nitric oxide production and apoptosis in opposition to the vasoconstriction and
cell survival response of ETA signaling. This study demonstrated an anti-apoptotic effect of ET-1/ETA in prostate
cancer cell lines that occurs through a PI3-kinase/Akt
dependent pathway. Other studies have demonstrated that
ET-1 may also signal through Erk1/2 to induce Akt phosphorylation and promote cell survival in other cell types
[32 36]. The Erk1/2 pathway appears to be involved in ET1 induced responses primarily in stromal cell populations
or through ETB mediated signaling. Similar to our findings,
Del Bufalo et al. [37] demonstrated that ET-1 signaling
through the ETA leads to activation of Akt through PI3kinase in ovarian carcinoma cells. Clearly, ET-1 has diverse
signaling cascades and cellular responses depending on
cell type and receptor subtype expression. Interestingly,
a recent study demonstrated a correlation between high
levels of Akt phosphorylation and an increased probability
for recurrence of prostate cancer [38]. Inactivation of proapoptotic proteins through phosphorylation by Akt activation
may represent a direct mechanism by which ET-1 promotes
cell survival. Akt can also induce MDM2 activity, resulting in
enhanced degradation of p53 and subsequent abrogation
of p53-mediated gene expression [19]. Indeed, ET-1 treatment of prostate cancer cells resulted in a decrease in the
pro-apoptotic proteins Bad, Bax, and Bak, all of which are
members of the Bcl-2 family of proteins whose expression
is positively regulated by p53. The profile of expression of
the ET axis in prostate cancer cells may therefore promote
a unique survival pathway. Although we previously demonstrated that ET-1/ETA can act as a weak mitogen for prostate cancer cells [5], we believe that the inhibition of
apoptosis through the upregulation of Akt activity accounts

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635

Figure 4. (A) Immunoblot analysis of ET-1 activation of the cell survival protein, Akt, in PPC-1 cells demonstrated phosphorylation of Akt occurred in a time- and dosedependent manner. The immunoblots were reprobed for -actin to control for protein loading. Representative immunoblot from three separate experiments. (B)
Immunoblot analysis demonstrated ET-1 induced phosphorylation of the cell survival protein, Akt, in prostate tumor cell lines. The LNCaP and LAPC-4 cells
demonstrated high endogenous Akt activation. Cells were treated for 1 hour with 100 nM ET-1. Immunoblots were analyzed by densitometry, normalized to corresponding -actin bands, and reported as relative level of expression to untreated cells. Results are representative immunoblots from three separate experiments.

for the majority of the effects seen with ET-1 treatment. In

this study, we saw a clear dose response with a halfmaximal effect of ET-1 on Akt phosphorylation at a 220 pM
concentration. These results are similar to the EC50 values
of ET-1 on other functional responses in cells, including
arachidonic acid release, Ca2+ mobilization, and glucose
uptake ranging from 300 pM to 2.9 nM ET-1 [39 43]. We
had found previously that circulating plasma ET was elevated in men with hormone refractory prostate cancer
(13.2 pg/ml) compared with normal controls (5.1 pg/ml) [4].
It is also likely that ET-1 concentrations in the circulation
would be lower than local tissue concentrations wherein
the prostate cancer cells are eliciting an autocrine mechanism of ET-1 signaling through the ETA. The identification
of the ETA survival signaling mechanisms may lead to
new insights into the function of ET-1 and its receptors in
malignant prostate tissues, as well as provide a potential
therapeutic target.
Although ET-1 is produced by a variety of malignancies,
including those arising from breast, lung, colon, pancreas,
kidney, and ovary cancers, many normal tissues also produce abundant ET-1. Indeed, endothelial cells perhaps make
the majority of ET-1 found in the body. If the findings in
prostate cancer extend to other malignancies expressing
the ETA, ET-1 from any source may act as a survival factor for these malignancies. Thus, blockade of the ETA may

Neoplasia . Vol. 7, No. 7, 2005

enhance the efficacy of therapies based on cytotoxic agents

as well as hormone ablation. For androgen-sensitive tissues,
such as the prostate, androgen deprivation induces a wave
of apoptosis. Androgen deprivation may also stimulate the
expression of the ETA receptor. In canine and rat models,
expression of the endothelin receptors increased following
castration [44,45]. Increased ETA expression, coupled with

Figure 5. Akt phosphorylation by ET-1 was eliminated by pretreatment of the

PPC-1 cells with PI3-kinase inhibitors (wortmannin and LY294002) but not
with the MEK inhibitor (PD98059) or the p70 S6 kinase inhibitor (rapamycin)
in prostate cancer cell lines by immunoblot analysis. Results are representative of three separate experiments.

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the hypermethylation of the ETB promoter, may provide

a mechanism whereby prostate cancer cells can survive
androgen deprivation. The use of ETA antagonists at the
initiation of androgen deprivation may potentiate prostate
epithelial cell death by removing a survival pathway. Given
the minimal toxicity profile of ETA antagonists, the combination of these agents with other therapeutic agents
merits investigation.

Acknowledgements
The authors are very grateful to Moira Hitchens for the critical
review of the manuscript and the members of the University
of Pittsburgh flow cytometry core facility for assistance with
the apoptosis studies.

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