# Differential Effects of Leptin on the Invasive  Potential of Androgen-Dependent and -Independent  Prostate  Carcinoma Cells

Authors: Dayanand D. Deo; Ashwin P. Rao; Saideep S. Bose; Allal Ouhtit; Surendra B. Baliga; Shilpa A. Rao; Bruce J. Trock; Rajesh Thouta; Madhwa HG Raj; Prakash N. Rao
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At  a concentration of 200_nm, LNCaP cells showed a significant increase (20% above control;P<.0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P<.01 to P<.0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK  at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity.


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## Body

## 1. Introduction

Prostate cancer is the second leading cause of cancer related
death among American men [1]. Obesity has assumed epidemic  proportions in the United States
and  has been linked with increased risk of prostate, breast, colon, and endometrial
cancers [2–7]. More recently, there have been several studies suggesting that
obesity may impact upon risk, detection, and outcome with regard to prostate
cancer [8].Leptin, one of the major adipose
cytokines controls body weight by regulating food intake and energy expenditure
[9, 10]. Adipose tissue leptin mRNA and circulating leptin levels increase in
obesity. Clinical studies have demonstrated that, blood leptin levels are
associated with clinically relevant prostate cancer [11, 12], although some
studies have questioned these results [13–16]. In vitro, leptin has been
postulated as a growth promoter for androgen-independent DU-145 and PC-3 cells [17–20],
but not in androgen-dependent LNCaP cells [20]. Several reports have
demonstrated that leptin induces the formation of toxic oxygen radicals [21–25].
Oxidant damage has been implicated as a major causative factor in obesity mediated
disease processes. The rationale of the present study is to confirm the role of
leptin on the metastatic potential of prostate carcinoma cells. We tested
androgen-sensitive LNCaP cells as well as androgen-resistant PC-3 and DU-145
cells for their ability to metastasize in the presence and absence of leptin.
Additionally, we investigated the effect of leptin on mitogen-activated protein
kinase (MAPK) phosphorylation, and on the activation of Erk-1 and Erk-2, and
STAT-3.


## 2. Materials and Methods

## 2.1. Reagents

Leptin was obtained from Sigma-Aldrich Inc., St.
Louis, Mo. Antibodies used were
rabbit polyclonal antiphospho-p44/42 MAP kinase, rabbit polyclonal anti-STAT-3,
rabbit polyclonal antiphospho-STAT-3, and horseradish peroxidase-conjugated
goat antirabbit antibodies from Cell Signaling Technology, Beverly, Mass.


## 2.2. Cell Culture

The PC-3 and LNCaP cell lines were propagated in RPMI 1640
medium, whereas DU-145 cell line was propagated in EMEM medium (Invitrogen
Corporation, Carlsbad, Calif) supplemented with 10% FBS (HyClone, Logan, Utah). For protein studies, cells were grown to subconfluent
levels in RPMI or EMEM, respectively, and supplemented with 10% FBS in 6-well culture plates (106 cells/well). The cells were then washed once with basal medium and starved of
growth factors by switching them to basal medium containing 1% FBS for 16 hours. Cells were again washed once with
basal medium and then exposed to various experimental conditions for different
time points. At the end of each time point, cells were washed with ice-cold
phosphate-buffered saline (PBS, Invitrogen), lysed in cell lysis buffer
containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 50 mM NaF, 70 mM 2-mercaptoethanol, 1% v/v Triton X-100, 2% w/v SDS,
and 1μg/ml protease inhibitor mixture (Sigma-Aldrich, Mo). Cell lysates were
used as indicated below.


## 2.3. Proliferation Assay

RPMI or EMEM + 10% FBS was added to each well of a 24-well plate and pretreated for 4   hours with
various concentrations of leptin at37°C in a CO2 incubator. Growth factor
starved PC-3, LNCaP, or DU-145 cells were then transferred to each well (2×104 cells/well) along with
1μM [H3]Thymidine, and incubated in the CO2 incubator.
After 144 hours of incubation, the cells were then washed three times with
ice-cold saline, precipitated with 10% TCA, washed with methanol and air-dried.
The labeled DNA was dissolved in 0.05 N NaOH at 37°C, neutralized with 0.05 N HCl, transferred to scintillation vial containing the
scintillation cocktail and counted.


## 2.4. In Vitro Migration and Invasion Assay

A modified Boyden chamber method using
Transwell membrane inserts (Costar, Corning,
NY) was used to measure PC-3 and
DU-145 migration and invasion. For invasion studies, Matrigel (BD Biosciences,
Bedford, Mass) was added to the upper surface of the Transwell membrane and
allowed to solidify in a sterile environment for 24 hours. PC-3 or DU-145 cells
(14×104 cells/ml) were suspended in basal medium supplemented with
2% FBS, and 0.02% BSA, incubated with
various concentrations of leptin, and plated onto the upper surface of the
Transwell membrane inserts in a total volume of 100 μL. Basal medium supplemented with 2% FBS and 0.02% BSA was added to the lower chamber in
a total volume of 400 μL. The cells were then incubated at 37°C in a
CO2 incubator. After 10 hours of incubation, cells present on the
upper surface of the membrane were scraped off, and the cells that had migrated
to the lower surface of the membrane were fixed, stained and counted using an
inverted Axiovert 25 microscope (Carl Zeiss, Thornwood, NY).


## 2.5. Western Blotting

Prostate cancer cells, pretreated for 16 hours with basal
medium supplemented with 1% FBS, were treated with 25, 50, 100, and 200 ng of
leptin for various time points. After the completion of each time point, the
cells were lysed in the cell lysis buffer containing 0.1% w/v bromophenol blue.
The samples were boiled for 5 minutes and separated using 7.5% SDS-PAGE (40μg
of total protein/lane). The separated proteins were then transferred onto
nitrocellulose membranes (Bio-Rad, Hercules, Calif), blocked with Tris-buffered
saline containing 5% nonfat dry milk for 1 hour at room temperature, and
incubated with the respective antibodies against the proteins in Tris-buffered
saline containing 5% nonfat dry milk and 0.5% Tween 20, overnight at 4°C. After
washing and incubation with horseradish peroxidase-conjugated secondary
antibody, the proteins were revealed using the enhanced chemiluminescent
detection kit (Pierce Biotechnology, Biotechnology, Rockford, Ill).


## 2.6. Statistical Analysis

The data were analyzed using the Statview software to obtain the
SE values and the significance (P) values for the various results from 3
separate experiments.


## 3. Results

## 3.1. Leptin Attenuates Cellular Invasion of Androgen-Resistant Prostate Cancer Cells

Androgen-resistant
PC-3 cells and DU-145 cells were suspended in media along with increasing
concentrations of leptin and plated onto the upper surface of the Transwell
inserts that were previously coated with Matrigel, as described in Material and
Methods section. As seen in Figure1(a), leptin attenuated the invasion of
Matrigel by PC-3 cells as well as DU-145 cells in a concentration dependent
manner, with significant attenuation observed at 50 ng, 100 ng, and 200 ng concentrations of leptin,
indicating that nanogram concentrations of leptin inhibit the invasive
capacity of these androgen-independent prostate carcinoma cell lines.Cellular invasion and migration
of androgen-resistant prostate cancer cells are attenuated by leptin. PC-3 and
DU-145 cells were suspended in basal medium supplemented with 2% FBS, and 0.02% BSA, incubated with various
concentrations of leptin, and plated onto the upper surface of the Transwell
membrane inserts. For invasion studies, Matrigel was added to the upper surface
of the Transwell membrane and allowed to solidify in a sterile environment for
24 hours. After 10 hours of incubation at 37°C in a CO2 incubator,
cellular invasion (a) and migration (b) were measured
by counting the cells that had migrated to the lower surface of the membrane.
Values represent the mean ±S.E. of triplicate samples of a representative
experiment.
(a)(b)


## 3.2. Migration of PC-3 Cells and Du-145 Cells  is Attenuated by Leptin

Androgen-independent
PC-3 cells and DU-145 cells were incubated with increasing concentrations of
leptin (see Figure1(b)) and allowed to migrate through the pores of the
membrane in the modified Boyden chamber  containing transwell inserts.
Attenuation of migration of both PC-3 and Du-145 cell lines was observed in the
presence of increasing
concentrations of leptin, with significant attenuation being observed at 100 ng
and 200 ng concentrations
of leptin for PC-3 cells and at 200 ng concentration of leptin for DU-145 cells,
respectively, suggesting that leptin at nanogram concentrations inhibits the
mechanism regulating the migration of these cell lines.


## 3.3. Leptin Induces the Proliferation of  Androgen-Dependent Prostate Cancer Cells

Proliferation of androgen-dependent and independent prostate
cancer cell lines was observed in the presence of nanogram concentrations of
leptin. After achieving the basal level of growth by overnight incubation in
basal medium, increasing concentration of leptin was added to the cells,
respectively. As seen in Figure2, a dose-dependent increase in proliferation
was observed when the androgen-dependent LNCaP cells were incubated with
leptin, with significant growth being observed at 50 ng, 100 ng, and 200 ng
concentrations of leptin, respectively. There was no change in growth of
androgen-independent PC-3 cells and Du-145 cells in the presence of increasing
concentration of leptin (data not shown), suggesting that leptin induces the
proliferative capacity of only the androgen-dependent prostate carcinoma cell
lines.Figure 2
Proliferation of
androgen-dependent prostate cancer cells is induced by leptin. Growth
factor starved LNCaP cells were incubated with increasing concentrations of
leptin, and proliferation was measured by the incorporation of [H3]thymidine
after 144 hours. Values represent the
mean ±S.E. of triplicate samples of a representative experiment.


## 3.4. Leptin Stimulated MAPK Phosphorylation

We hypothesized that binding of leptin to its receptors in
the prostate cancer cell lines would lead to the early phosphorylation of MAPK.
To test this hypothesis, androgen- dependent LNCaP cells and androgen-independent
PC-3 and DU-145 cells were starved of growth factors overnight (basal medium
supplemented with 1% FBS), following
which they were exposed to increasing concentration of leptin for various time
points. Phosphorylation of MAPK was observed at the end of each time point for
all cell lines. Interestingly, leptin did not induce the phosphorylation of
MAPK in the androgen-dependent LNCaP cells (data not shown). However, in
androgen-independent prostate cancer cell lines, phosphorylation of MAPK was
observed to be maximal at 15 minutes (PC-3 cells, see Figures3(a) and 3(b)) and
10 minutes (DU-145 cells, see Figures 3(c) and 3(d)) upon exposure to leptin.
Maximal increase in MAPK phosphorylation was observed upon incubating these
cells with 100 ng and 200 ng of leptin, respectively, suggesting that binding of
leptin to its receptors induces an intracellular signaling cascade leading to
the phosphorylation of MAPK in the androgen-independent prostate cancer cell
lines.Leptin stimulates MAPK
phosphorylation in PC-3 and DU-145 cells. Growth factor-starved PC-3 (a) or DU-145 cells (c) were exposed to increasing
concentration of leptin for various time points. Cell lysates obtained after
each time point were separated on a 7.5% SDS-PAGE and Western-blotted. Phosphorylated
MAPK levels were obtained after incubation with p44/42 MAPK antibody and
chemiluminescent detection. (b) and
(d), levels of p44/42 MAPK obtained after densitometric analysis and
represented as percentage of control against time (control being set at 100%).
The results of one of the three independent experiments are shown.
(a)(b)(c)(d)


## 3.5. STAT-3 Phosphorylation is Induced by Leptin

After an
overnight incubation in basal medium with 1% FBS,
androgen-dependent (LNCaP) and -independent (PC-3) prostate cancer cells were
incubated with a dose-dependent increase in leptin levels for various time
points. STAT-3 phosphorylation was investigated over time for both cell lines.
As seen in Figures4(a) and 4(b), leptin induced the time-dependent
phosphorylation of STAT-3 in PC-3 cells, reaching maximal levels after 15 minutes
of exposure, and returning to baseline after 60 minutes of incubation. Exposure
of PC-3 cells to increased leptin levels led to a correspondent increase in the
level of maximal STAT-3 phosphorylation at the 15 minutes time point. Leptin
exposure also induced the phosphorylation of STAT-3 in LNCaP cells (see Figures
4(c) and 4(d)), reaching maximal levels after 30 minutes of stimulation.
However, in LNCaP cells, STAT-3 remained phosphorylated even after prolonged
(60 minutes) exposure to leptin, suggesting that leptin induces the
phosphorylation of STAT-3 only at an early time point in androgen-independent
cells, while in androgen-dependent prostate cancer cells, STAT-3
phosphorylation is a delayed but prolonged event.Leptin stimulates early
phosphorylation of STAT-3 in PC-3 cells. PC-3cells (a) or LNCaP cells (c), devoid of growth factors by an overnight exposure to basal medium, were
exposed to increasing concentrations of leptin for various time points. 25μg of
the cell lysates obtained after each time point were separated on a 7.5%
SDS-PAGE and Western-blotted, and the phosphorylated STAT-3 proteins were
revealed by incubating with phospho-STAT-3 antibody followed by
chemiluminescent detection. The membranes were stripped and reprobed with
STAT-3 antibodies to verify the protein levels. In (b) and (d), densitometric analysis of phospho-STAT-3 and STAT levels was
determined. A time-course, dose-response curves of phosphor-STAT-3/STAT-3
(PSTAT/STAT) ratios for the effect of increasing leptin concentrations are
depicted for both (b) PC3 and (d) LNCaP. The original western blots are shown
in (a) and (c). Results shown are representative of three separate experiments.
(a)(b)(c)(d)


## 4. Discussion

It has been
shown that leptin influences the proliferation and migration of endothelial and
epithelial cells and is capable of promoting angiogenesis [21]. In vitro studies on prostate cancer
cell lines showed that leptin promotes proliferation of androgen-independent
prostate cancer [20]. Leptin also induces cell migration and the expression of
growth factors in prostate cancer cells, suggesting that studies undertaken to
ascertain leptin and/or obesity association to prostate cancer should
differentiate patients according to androgen resistance [19]. Leptin has been
demonstrated to potently induce the invasion of endometrial cancer cells [22] and
also contributes to the invasive phenotype of colonic and kidney epithelial
cells at various stages of the neoplastic progression [23]. Our results
demonstrate that the invasive and migratory capabilities of
androgen-insensitive prostate cancer cells are attenuated upon exposure to nanogram
concentrations of leptin for a short duration of 10 hours, and that this
attenuation increases in a dose-dependent manner. Our observations are in
contrast to those of Frankenberry et al. [19] who demonstrated that a longer 24
hour exposure of leptin increased the migration of androgen-independent
prostate cancer cells.  We did not
observe any change in the proliferation of androgen-independent prostate cancer
cells upon exposed to nanogram concentrations of leptin (data not shown). In
contrast, the proliferation of androgen-sensitive prostate cancer cells is
increased in a dose-dependent manner when they are exposed to nanogram
concentrations of leptin, suggesting leptin’s ability to differentiate between
the androgen-sensitive and -insensitive prostate carcinoma cells. Our results are in
contrast to those observed by Onuma et al. [20] who showed an increased proliferation of
androgen-independent prostate cancer cells using high (microgram) levels of
leptin. Our contrasting observations using very low (nanogram) levels of leptin
suggest that prostate cancer cells are very sensitive and behave differently at
varying levels of leptin exposure.As
prostate cancer progresses, decreased androgen levels promote the growth of
androgen-resistant prostate cells. These cells are hypersensitive to growth
factors and cell regulators, especially mitogenic factors. The source of these
growth factors and cytokines, in addition to outside factors affecting their
expression, is important to understanding the progression of prostate cancer
disease [21]. The leptin signal is transduced through the leptin receptor
(LEPR). The different isoforms of this receptor arise from alternate splicing
of the precursor mRNA [24]. The “long form” LEPRb leptin receptor is the only
isoform that forms an IL-6-type homodimer and associates with the Janus kinase
2 (JAK2) tyrosine  kinase to mediate intracellular signaling. JAK2
phosphorylates itself and the tyrosine residue (Tyr 985) on the intracellular
tail of the LEPRb receptor. This phosphorylated tyrosine residue on the
receptor then recruits the adaptor protein Grb-2 and p21ras, ultimately leading
to the phosphorylation and activation of MAPK [25]. Our observations
demonstrate that exposure of androgen-independent prostate cancer cells to
leptin does indeed stimulate the phosphorylation of MAPK at an early time
point, suggesting the stimulation of the intracellular signaling cascade  upon leptin binding to its cognate receptor
on the cell surface of these cells. Our observations are in accordance with those
reported earlier by Banks et al. [25] suggesting involvement of MAPK activation
upon binding of leptin to its surface receptor.
Interestingly, we did not observe MAPK phosphorylation when the
androgen-sensitive prostate cancer cells were exposed to leptin, suggesting the
involvement of a different signaling cascade in these cells.Activated
JAK2 induces the phosphorylation of another tyrosine residue (Tyr 1138) on the
intracellular tail of the LEPRb receptor, which binds and mediates the
phosphorylation-dependent activation of signal transducer and activator of
transcription 3 (STAT3). STAT proteins regulate gene expression by affecting
transcription. They are part of the signal transduction pathway of many growth
factors and cytokines and are activated by phosphorylation of tyrosine and
serine residues by upstream kinases [26]. In benign cells, the signaling by
STAT3 is under tight regulation so that the signal is transient. However,
aberrant signaling by STAT3 is observed in many types of malignancies, such as
myeloma, head and neck cancer, breast cancer, and prostate cancer [27]. Malignant
cells expressing persistently activated STAT3 become dependent on it for
survival; disruption of activation or expression of STAT3 resulted in apoptosis
[28]. Lou, et al. have shown that  STAT3 signaling is
critical for the growth and survival of prostate cancer cells [29]. We observed the
involvement of STAT3 phosphorylation in prostate cancer cells, which is
concurrent with earlier published reports, thus suggesting that this
transcription factor is an integral part in the leptin signal-transduction
cascade.  Our observations further
suggest that STAT3 phosphorylation can differentiate between the androgen
sensitivity of prostate cancer cells when they are exposed to nanogram levels
of leptin. In androgen-independent PC-3 prostate cancer cells, leptin induces
an early phosphorylation of STAT-3, which then decreases with time to the
baseline level, whereas in androgen-sensitive LNCaP cells, a delayed and
prolonged STAT3 phosphorylation is observed upon exposure to leptin. It has
been previously demonstrated that part of leptin action is mediated through the
generation of toxic oxygen radicals [30]. Although, in this study we did not measure
a direct cause-effect relationship between free radical generation, and the
changes in metastatic potential of prostate cancer cells, indirect evidence of
the role of free radicals in this process is available in studies investigating
the use of antioxidants in prostate cancer [31].In
conclusion, binding of leptin to its surface receptor induces a signal
transduction cascade that has slight variations depending on the androgen
sensitivity of the prostate cancer cells. These variations in the intracellular
signaling events are translated into biological consequences such as the
invasive, migratory, and proliferative capabilities of prostate carcinoma cells
that are drastically different between the androgen-independent and -sensitive
prostate cancer cells. Thus, differentiation of the metastatic potential of
prostate cancer cells on the basis of their androgen sensitivity can be
observed when they are exposed to low levels of leptin. Since a majority of
leptin's effects (at least on metabolism) are centrally mediated, the in vivo
effects of leptin on the metastatic potential of prostate cancer cells may
differ from the in vitro observations. However, mechanism-dependent variations
in the effects of leptin treatment on the basis of androgen sensitivity of
prostate cancer cells can give us a vital insight into the possible in vivo
effects of leptin.



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