# Rosiglitazone Inhibits Adrenocortical Cancer Cell Proliferation by Interfering with the IGF-IR Intracellular Signaling

Authors: Giulia Cantini; Adriana Lombardi; Elisabetta Piscitelli; Giada Poli; Elisabetta Ceni; Sara Marchiani; Tonino Ercolino; Andrea Galli; Mario Serio; Massimo Mannelli; Michaela Luconi
Journal: PPAR Research
 (2008)

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## Abstract

Rosiglitazone (RGZ), a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR)-γ, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R) of human adrenocortical carcinoma (ACC) and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR). We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K)-Akt, and extracellular signal-regulated kinase (ERK1/2) cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.


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## Body

## 1. Introduction

Adrenocortical
carcinoma (ACC) is a rare tumor with an incidence of approximately 1-2 per million population per year. ACC is a very aggressive tumor, characterized by a poor prognosis:
approximately 50% of patients do not survive beyond 2 years from the diagnosis
and the 5-year
mortality rate is between 20% and 60% [1]. Its poor prognosis depends mainly upon the
limited therapeutic resources. At present, a complete surgical removal
following an early diagnosis is the only valuable option for the tumor’s cure.
Moreover, other than improved surgical management, the prognosis for ACC has
not changed significantly over the past three decades [2]. The tumor is, in
fact, resistant to radio- and chemotherapy and medical treatment very rarely
leads to a complete remission in the case of recurrences or metastatic spread.
Although several new medical therapeutic options have been recently proposed [3],
at present the medical treatment of advanced ACC is far from being
satisfactory, due to our poor knowledge of the molecular mechanisms leading to
malignant transformation of adrenocortical cells. In fact, although some
intracellular signaling pathways have been shown to be altered in ACC cells [4],
efforts to identify the events leading to neoplastic transformation and tumor
invasiveness have met with limited success. The role of IGF-I system in
mediating proliferation and progression has been well documented in several
cancers, including adrenocortical carcinoma [5]. In particular, ACC, as well as
the H295R cell line [6], overexpress both IGF-II [7] and its promiscuous
receptor IGF-IR [8] in comparison to adrenal adenomas and normal adrenal tissue.
The overexpressed IGF-II is thought to act in a paracrine fashion through the
IGF-IR to sustain tumor and cell proliferation [6, 9, 10].Peroxisome proliferator
activated receptor (PPAR)-γ is a ligand-activated transcriptor factor and a
member of the nuclear hormone receptors superfamily. Thiazolidinediones (TZDs),
which are a family of PPAR-γ ligands, have been introduced in the therapy of
type 2 diabetes mellitus (T2D) because of their ability to reduce insulin
resistance. In the last few years, an increasing amount of experimental data showing
the ability of these drugs to exert additional pleiotropic actions such as
regulation of inflammatory processes and of cancer cell growth has been
published [11].TZD effects seem to be mainly due to their ability to bind and activate
PPARγ differentially expressed in adipose and
other tissues. Upon ligand binding, PPARγ heterodimerizes with the 9-cis retinoic
acid receptor (RXR) on specific responsive elements in the promoters of genes
involved in glucose and insulin homeostasis, lipid metabolism, and cellular
differentiation. Besides this transactivating activity, a ligand-dependent
transcriptional transrepression mechanism involving PPARγ/RXR complex has been described.
According to such a model, the heterodimerized receptor represses gene
transcription in a DNA-binding independent way by physically sequestering
activated transcriptional factors or their coactivators [12]. More recently, an
increasing number of TZD effects, and in particular the antineoplastic ones, have
been shown to be independent of PPARγ activity [13]. Finally, rapid
nongenomic activities of TZDs, not resulting in modulation of gene
transcription but affecting posttranslational modifications involved in cell
signaling, have been reported [14].In addition to
their action as insulin sensitizers, TZDs inhibit cell growth in breast, colon,
prostate, lung, pancreas, stomach, thyroid, liver and adrenal cancers [15]. However,
the molecular mechanisms underlying such pharmacological activities remain to
be elucidated.Rosiglitazone
(RGZ) and pioglitazone (PGZ), the two most widely used PPARγ agonists, have
been shown to inhibit growth and invasiveness of the human adrenal cancer cell
line H295R [16] as well as to induce cell differentiation and apoptosis [10]. Moreover,
H295R cells and both normal and tumoral adrenal tissue express PPARγ, with no differences in the level of
expression between tumoral and normal tissue and no correlation with clinical
parameters such as tumor size, hormonal profile, and so forth [10, 16].In this study,
we investigate two different cell models of ACC, namely, SW13 and H295R lines, whether
the TZD RGZ may exert its antiproliferative action on human ACC cell lines by
interfering with the intracellular pathways activated by IGF-IR.


## 2. Materials and Methods

### 2.1. Reagents

Anti-phospho [Akt
(Ser473), ERK1/2 (T202/Y204)] and anti-Akt antibodies
were from Cell Signaling Technology, Inc. (Danvers,
Mass, USA).
Anti-ERK1/2, anti-IGF-IRβ, and anti-actin antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz,
Calif, USA).
Anti-PI3K p85 regulatory subunit  antibody was from Upstate Biotechnology (Lake Placid, NY,
USA). Rosiglitazone
was from Alexis Biochemicals (San
Diego, Calif, USA). MTS solution was purchased
from Promega (Madison, Wis, USA).
IGF-I was from Sigma Aldrich (San
Louis, Mo, USA). [Methyl-3H]thymidine
([3H]TdR) was purchased from NEN Life Science Products (Boston, Mass, USA). NVP-AEW541 was provided by Novartis (Basel, Switzerland).


### 2.2. Tissue Specimens and Cell Cultures

A total of three
normal human adrenal glands, three adrenal carcinomas, and three adenomas were
used in this study. Normal adrenal glands were removed during an expanded
nephrectomy due to renal carcinoma or from organ donors (age 32–72 years).
Approval for the use of human material was given by the Local Ethical
Committee. Informed consent was obtained from each patient. Adrenocortical
fragments, collected immediately after surgery, were snap frozen in liquid
nitrogen and stored at−80°C.The human ACC
cell lines H295R and SW13 were obtained from the American Type Culture
Collection (Manassas, Va, USA).
SW13 were cultured in DMEM/F-12 medium (Sigma-Aldrich) with 10% FBS
(Euroclone), 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin.
H295R need DMEM/F-12 medium enriched with a mixture of
insulin/transferrin/selenium (Sigma-Aldrich). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere.Subconfluent
cells starved 24 hours were treated with different stimuli added to serum-free medium.
Rosiglitazone was added simultaneously with IGF-I, at the doses and for the
time intervals (15 minutes for western blot analysis and from 24 hours up to 7
days for proliferation experiments) indicated in figure legends.Cells were
pretreated 1 hour with NVP-AEW541 before addition of other stimuli. For
incubations longer than 24 hours, media and stimuli were replaced every day.


### 2.3. MTS Assay

SW13 and H295R
were seeded in 96-well plates at the density of3⋅103 and 8⋅103 cells/well, respectively. After 24-hour starvation in serum-free (SF) medium,
cells were treated with the different stimuli in SF medium for the indicated
times (see figure legends) and cell number in each well was evaluated by MTS
assay (Promega), according to the manufacturer’s instructions. The samples were
analyzed by an ELISA plate reader (Wallac 1420 - PerkinElmer) at 490 nm
wavelength to measure optical density (OD). Each experimental point was
performed in quintuplicate in at least three independent experiments.


### 2.4. Cell
Proliferation Assays

#### 2.4.1. Viable Cell Counting

Cells were
seeded in 12-well plates (1,8⋅104 and
5⋅104 cells/well for SW13 and H295R, resp.), and after 24-hour starvation were treated for 2 or 4
days in SF medium, then trypsinizedand counted in the haemocytometer.
Mean cell number was obtained by counting triplicate in three different experiments.
Dead cells were excluded by trypan blue exclusion test.


#### 2.4.2. DNA
Synthesis Assay: [3H]Thymidine Uptake

DNA synthesis
was evaluated according to the amount of [3H]TdR incorporated into
trichloroacetic acid (TCA)-precipitated materials. Cells seeded at different density
(5⋅104 or
2,5⋅104 H295R cells/well in 12 or 24 well plates, and 1,8⋅104 SW13 cells/well in 12 well plates) were grown in 10% FBS complete medium till 70%
confluence. After 24-hour starvation, cells were treated with IGF-I or RGZ in
1% FBS medium for the indicated times (1–7 days) pulsing them with 1.0 μCi/ml [3H]TdR (6.7 Ci/mmol) 4
hours before stopping proliferation in ice-cold 10% TCA. After washing in TCA
and then in methanol, cells were solubilized in 0.2 N NaOH, and radioactivity
was measured in the scintillation counter. Experiments were performed in
triplicate and repeated at least three times.


### 2.5. Western
Blot Analysis

Treated cells
were extracted in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na3VO4,
1 mM PMSF). Thirty μg of proteins measured by Coomassie reagent (BIO-RAD Labs, Hercules, Calif, USA) were
loaded onto 8–10% reducing SDS-PAGE. After separation, proteins transferred to
nitrocellulose membranes were 1 hour blocked at room temperature in 5% skimmed
milk in TTBS (0.1% Tween-20, 20 mM Tris, 150 mM NaCl) and incubated overnight with primary antibodies
at appropriate dilutions followed by peroxidase-secondary IgG (1:3000).
Proteins were revealed by BM-enhanced chemiluminescence system (Roche
Diagnostics, Milan, Italy). Image acquisition and densitometric analysis were performed with Quantity
One software on a ChemiDoc XRS instrument (BIO-RAD Labs, Hercules, Calif, USA). All western blots were repeated in at least 3
independent experiments. Membrane re-probing was performed after stripping
procedure (Pierce, Rockford, Il, USA).PI3 Kinase Assay
Treated cells were
extracted in lysis buffer A (20 mM Tris, pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1%
NP-40, 1 mM Na3VO4, 1 mM PMSF). After protein measurement, aliquots containing
equal amount of proteins (300 μg) were precleared with 50 μl of Protein G-Sepharose. Precleared
lysates were then immunoprecipitated overnight at 4°C with 3 μg of rabbit anti-p85 PI3K antibody  in the
presence of 50 μl of Protein A-Sepharose. Sepharose
beads washed in a 10 mM Tris-HCl (pH 7.4) containing 0.1 mM EGTA and 5 mM LiCl, were suspended in a kinase buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA) containing 20 μg of L-α-phosphatidyl inositol (Sigma-Aldrich, St. Louis, Mo, USA), 25 mM MgCl2 and 10 μCi of [γ32P]ATP and incubated for 20
minutes at room temperature. The reaction was stopped by the addition of 60 μl of 6 M HCl + 160 μl of chloroform:methanol (1:1). Lipids
were then resolved by thin layer chromatography plates (TLC silica gel 60)
(Merck Laborchimica, Florence,
Italy) in
chloroform, methanol, water and ammonium hydroxide (60:47:11, 3:2). Dried TLC
sheets were developed by autoradiography. Band quantification was performed with
ChemiDoc XRS instrument (BIO-RAD Labs, Hercules, Calif, USA).


### 2.6. Statistical Analysis

Results are
expressed as mean ± SE. The effect of different concentrations of IGF-I and RGZ
on cell proliferation was tested by One-Way ANOVA. MultiplePost
Hoc comparisons were performed by Bonferroni’s correction. Student’s τ-test
for paired or unpaired data was applied when appropriate for comparison of two
sets of data. P<.05 was taken as significant.


## 2.1. Reagents

Anti-phospho [Akt
(Ser473), ERK1/2 (T202/Y204)] and anti-Akt antibodies
were from Cell Signaling Technology, Inc. (Danvers,
Mass, USA).
Anti-ERK1/2, anti-IGF-IRβ, and anti-actin antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz,
Calif, USA).
Anti-PI3K p85 regulatory subunit  antibody was from Upstate Biotechnology (Lake Placid, NY,
USA). Rosiglitazone
was from Alexis Biochemicals (San
Diego, Calif, USA). MTS solution was purchased
from Promega (Madison, Wis, USA).
IGF-I was from Sigma Aldrich (San
Louis, Mo, USA). [Methyl-3H]thymidine
([3H]TdR) was purchased from NEN Life Science Products (Boston, Mass, USA). NVP-AEW541 was provided by Novartis (Basel, Switzerland).


## 2.2. Tissue Specimens and Cell Cultures

A total of three
normal human adrenal glands, three adrenal carcinomas, and three adenomas were
used in this study. Normal adrenal glands were removed during an expanded
nephrectomy due to renal carcinoma or from organ donors (age 32–72 years).
Approval for the use of human material was given by the Local Ethical
Committee. Informed consent was obtained from each patient. Adrenocortical
fragments, collected immediately after surgery, were snap frozen in liquid
nitrogen and stored at−80°C.The human ACC
cell lines H295R and SW13 were obtained from the American Type Culture
Collection (Manassas, Va, USA).
SW13 were cultured in DMEM/F-12 medium (Sigma-Aldrich) with 10% FBS
(Euroclone), 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin.
H295R need DMEM/F-12 medium enriched with a mixture of
insulin/transferrin/selenium (Sigma-Aldrich). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere.Subconfluent
cells starved 24 hours were treated with different stimuli added to serum-free medium.
Rosiglitazone was added simultaneously with IGF-I, at the doses and for the
time intervals (15 minutes for western blot analysis and from 24 hours up to 7
days for proliferation experiments) indicated in figure legends.Cells were
pretreated 1 hour with NVP-AEW541 before addition of other stimuli. For
incubations longer than 24 hours, media and stimuli were replaced every day.


## 2.3. MTS Assay

SW13 and H295R
were seeded in 96-well plates at the density of3⋅103 and 8⋅103 cells/well, respectively. After 24-hour starvation in serum-free (SF) medium,
cells were treated with the different stimuli in SF medium for the indicated
times (see figure legends) and cell number in each well was evaluated by MTS
assay (Promega), according to the manufacturer’s instructions. The samples were
analyzed by an ELISA plate reader (Wallac 1420 - PerkinElmer) at 490 nm
wavelength to measure optical density (OD). Each experimental point was
performed in quintuplicate in at least three independent experiments.


## 2.4. Cell
Proliferation Assays

### 2.4.1. Viable Cell Counting

Cells were
seeded in 12-well plates (1,8⋅104 and
5⋅104 cells/well for SW13 and H295R, resp.), and after 24-hour starvation were treated for 2 or 4
days in SF medium, then trypsinizedand counted in the haemocytometer.
Mean cell number was obtained by counting triplicate in three different experiments.
Dead cells were excluded by trypan blue exclusion test.


### 2.4.2. DNA
Synthesis Assay: [3H]Thymidine Uptake

DNA synthesis
was evaluated according to the amount of [3H]TdR incorporated into
trichloroacetic acid (TCA)-precipitated materials. Cells seeded at different density
(5⋅104 or
2,5⋅104 H295R cells/well in 12 or 24 well plates, and 1,8⋅104 SW13 cells/well in 12 well plates) were grown in 10% FBS complete medium till 70%
confluence. After 24-hour starvation, cells were treated with IGF-I or RGZ in
1% FBS medium for the indicated times (1–7 days) pulsing them with 1.0 μCi/ml [3H]TdR (6.7 Ci/mmol) 4
hours before stopping proliferation in ice-cold 10% TCA. After washing in TCA
and then in methanol, cells were solubilized in 0.2 N NaOH, and radioactivity
was measured in the scintillation counter. Experiments were performed in
triplicate and repeated at least three times.


## 2.4.1. Viable Cell Counting

Cells were
seeded in 12-well plates (1,8⋅104 and
5⋅104 cells/well for SW13 and H295R, resp.), and after 24-hour starvation were treated for 2 or 4
days in SF medium, then trypsinizedand counted in the haemocytometer.
Mean cell number was obtained by counting triplicate in three different experiments.
Dead cells were excluded by trypan blue exclusion test.


## 2.4.2. DNA
Synthesis Assay: [3H]Thymidine Uptake

DNA synthesis
was evaluated according to the amount of [3H]TdR incorporated into
trichloroacetic acid (TCA)-precipitated materials. Cells seeded at different density
(5⋅104 or
2,5⋅104 H295R cells/well in 12 or 24 well plates, and 1,8⋅104 SW13 cells/well in 12 well plates) were grown in 10% FBS complete medium till 70%
confluence. After 24-hour starvation, cells were treated with IGF-I or RGZ in
1% FBS medium for the indicated times (1–7 days) pulsing them with 1.0 μCi/ml [3H]TdR (6.7 Ci/mmol) 4
hours before stopping proliferation in ice-cold 10% TCA. After washing in TCA
and then in methanol, cells were solubilized in 0.2 N NaOH, and radioactivity
was measured in the scintillation counter. Experiments were performed in
triplicate and repeated at least three times.


## 2.5. Western
Blot Analysis

Treated cells
were extracted in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na3VO4,
1 mM PMSF). Thirty μg of proteins measured by Coomassie reagent (BIO-RAD Labs, Hercules, Calif, USA) were
loaded onto 8–10% reducing SDS-PAGE. After separation, proteins transferred to
nitrocellulose membranes were 1 hour blocked at room temperature in 5% skimmed
milk in TTBS (0.1% Tween-20, 20 mM Tris, 150 mM NaCl) and incubated overnight with primary antibodies
at appropriate dilutions followed by peroxidase-secondary IgG (1:3000).
Proteins were revealed by BM-enhanced chemiluminescence system (Roche
Diagnostics, Milan, Italy). Image acquisition and densitometric analysis were performed with Quantity
One software on a ChemiDoc XRS instrument (BIO-RAD Labs, Hercules, Calif, USA). All western blots were repeated in at least 3
independent experiments. Membrane re-probing was performed after stripping
procedure (Pierce, Rockford, Il, USA).PI3 Kinase Assay
Treated cells were
extracted in lysis buffer A (20 mM Tris, pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1%
NP-40, 1 mM Na3VO4, 1 mM PMSF). After protein measurement, aliquots containing
equal amount of proteins (300 μg) were precleared with 50 μl of Protein G-Sepharose. Precleared
lysates were then immunoprecipitated overnight at 4°C with 3 μg of rabbit anti-p85 PI3K antibody  in the
presence of 50 μl of Protein A-Sepharose. Sepharose
beads washed in a 10 mM Tris-HCl (pH 7.4) containing 0.1 mM EGTA and 5 mM LiCl, were suspended in a kinase buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA) containing 20 μg of L-α-phosphatidyl inositol (Sigma-Aldrich, St. Louis, Mo, USA), 25 mM MgCl2 and 10 μCi of [γ32P]ATP and incubated for 20
minutes at room temperature. The reaction was stopped by the addition of 60 μl of 6 M HCl + 160 μl of chloroform:methanol (1:1). Lipids
were then resolved by thin layer chromatography plates (TLC silica gel 60)
(Merck Laborchimica, Florence,
Italy) in
chloroform, methanol, water and ammonium hydroxide (60:47:11, 3:2). Dried TLC
sheets were developed by autoradiography. Band quantification was performed with
ChemiDoc XRS instrument (BIO-RAD Labs, Hercules, Calif, USA).


## 2.6. Statistical Analysis

Results are
expressed as mean ± SE. The effect of different concentrations of IGF-I and RGZ
on cell proliferation was tested by One-Way ANOVA. MultiplePost
Hoc comparisons were performed by Bonferroni’s correction. Student’s τ-test
for paired or unpaired data was applied when appropriate for comparison of two
sets of data. P<.05 was taken as significant.


## 3. Results

Adrenal cancer
is characterized by an increased expression of the IGF-IR compared to both nontumoral
and adenomal adrenal tissues as demonstrated by western blot analysis with a
specific antibody against theβ subunit of IGF-IR (Figure 1(a)). A marked
expression of this receptor is also present in SW13 and in particular in H295R
adrenocortical cancer cells (Figure 1(a)), suggesting that both tissue and cell
systems may be highly responsive to the effects of IGF-I and IGF-II.Adrenocortical cancer is
responsive to IGFs. (a) Western blot analysis of adrenocortical tissues
(adrenocortical carcinoma: ACC; normal adrenal tissue: NOR; adrenocortical
adenoma: ADE) and adrenocortical cancer cell lines (SW13 and H295R) reveals a
high expression of IGF-IR in cancer tissue and cells and lower levels in normal
and adenoma tissues. Western blot for actin was performed for lane protein normalization.
Molecular weight (Mw) markers are indicated. The effects of cell incubation
with increasing doses of IGF-I (1, 10 and 50 nM) for the indicated times on
cell proliferation were evaluated by MTS in SW13 (b) and H295R (c) and
confirmed in H295R by evaluation of [3H]TdR incorporation in cell
DNA content (d). Data represent mean ± SE OD (b), (c) or mean ± SE of [3H]TdR
incorporation (counts per minute, cpm) (d). Statistical significance versus
respective control: P*<.05, °P<.01, §P<.005, #P<.001.
(a)(b)(c)(d)In order to
study the role of IGF-IR and of its downstream intracellular signaling pathways
on cell proliferation, we stimulated both SW13 and H295R cells with increasing
concentrations of the receptor’s elective ligand, IGF-I. IGF-I is able to
induce cell proliferation in a time- and dose-dependent manner as demonstrated
by evaluating cell viability through MTS assay in both SW13 (Figure1(b)) and
H295R (Figure 1(c)) cells stimulated with increasing concentrations of IGF-I (1
to 50 nM) for 24 hours up to 7 days. Such a stimulatory effect is confirmed by
[3H]thymidine uptake experiments in H295R cells (Figure 1(d)).Following 24-hour
treatment, RGZ reduces cell viability in a dose-dependent manner in SW13 cells treated (Figure2(b)) or not (Figure 2(a)) with 10 nM IGF-I, showing an IC50 of 22.48±1.54μM (coefficient of variation 6.9%) as
calculated with ALLFIT program [17], Figure 2(c).
Interestingly, the IC50s derived from the two RGZ dose response
viability curves obtained for IGF-I-treated and untreated cells are not
statistically different, suggesting that the effect of RGZ is similar
independently of exogenous IGF-I stimulation. Thus, we chose to use 20 μM RGZ in all the experiments. The
inhibitory effect of RGZ on cell proliferation in basal conditions is
significantly increased with the time of incubation as evaluated by [3H]thymidine
uptake experiments in both SW13 (Figure 2(d)) and H295R (Figure 2(e)) cells. Again,
the negative effect on cell viability exerted by RGZ is similar in IGF-I
treated and untreated SW13 cells (Figures 3(a)–3(e)). Conversely, in H295R cells
where the effect of RGZ requires longer times to become evident, RGZ is also able
to revert IGF-I-stimulation (Figures 3(f)–3(h)). Both IGF-I-stimulated proliferation
and the inhibitory effect of RGZ are confirmed by SW13 (Figure 3(i)) and H295R (Figure
3(j)) cell counting at 2-
and 4-day incubation, respectively.Dose dependent effects of RGZ on
adrenocortical carcinoma cell proliferation. The dose dependent effect of
RGZ on SW13 cell proliferation was evaluated by MTS assay at 48-hour stimulation
in the absence (a), (c) or presence (b) of 10 nM IGF-I. A complete dose response
curve for RGZ (10−3 to 2⋅102 μM) on cell proliferation in
SW13 is reported (c), showing the IC50 for RGZ calculated by ALLFIT
program [17]. Data represent mean ± SE percentage of OD over controls (RGZ
0 μM). Statistical significance versus respective control (RGZ 0 μM):
P*<0.05, #P<0.001. The time dependent effect of RGZ administration (20 μM) on cell proliferation was evaluated
at the indicated times in SW13 (d) and H295R (e) by [3H]TdR
incorporation. Data represent mean ± SE percentage of cpm over untreated control.
Statistical significance versus respective Ctrl: °P<0.01, §P<0.005, #P<0.001.
(a)(b)(c)(d)(e)IGF-induced cell proliferation is
differently affected by RGZ in SW13 and H295R cell lines. SW13 (a)–(e) and
H295R (f)–(h) proliferation was evaluated by MTS assay following stimulation with
increasing doses of IGF-I in the presence or absence of 20 μM RGZ for the indicated times. Mean ± SE
percentage of OD over respective control. Statistical significance: P*<0.05,
§P<0.005, #P<0.001 versus the corresponding points with RGZ, n = 6. Total number/well of SW13 (i) and H295R (j) cells
treated with 10 nM IGF-I and 20 μM RGZ for 2 and 4 days, respectively, was
evaluated by cell counting under optical microscope. Data are expressed as mean ± SE
of cell number evaluated in triplicate in three independent experiments. °P<.01, #P<.001 versus
Ctrl; P*<.001 versus IGF-I.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)To elucidate the
intracellular mechanism by which RGZ affects adrenal cancer cell proliferation,
we investigated the effects of the drug on the two main intracellular signaling
pathways engaged by the activated IGF-IR, namely, the phosphatidyl inositol 3
kinase (PI3K)-Akt cascade and the extracellular signal-regulated (ERK) signaling
[18].Rapid
stimulation (15 minutes) of SW13 (Figure4(a)) and H295R (Figure 4(b)) cells with
10 nM IGF-I determines an increased phosphorylation of Akt in Ser473, resulting
in the activation of the enzyme (Figure 4, upper and lower panels). Concomitant
addition of 20 μM RGZ for 15 minutes in the presence or
absence of IGF-I interferes with Akt phosphorylation/activation in SW13 (Figure
4(a)) and H295R (Figure 4(b)) cells. The inhibitory effect of RGZ is statistically
significant versus IGF-I only, but this trend is also present on active Akt in
basal conditions (Figure 4 lower panels). The in vitro immunokinase assay for PI3K performed on SW13 (Figure 5(a))
and on H295R (Figure 5(b)) lysates demonstrates a rapid activation (15 minutes)
of the enzyme by IGF-I, which is reverted by co-incubation with RGZ. The
inhibitory effect of RGZ is evident in basal conditions in H295R cells only (Figures
5(a), 5(b)). Similarly, RGZ interferes with IGF-I-rapid stimulation of
phosphorylation/activation of ERK1/2 in SW13 (Figure 6(a)) and H295R (Figure 6(b)),
but, conversely, the effect of RGZ in basal conditions is evident in SW13 cells
only (Figures 6(a), 6(b)).RGZ blunts IGF-I stimulation of
Akt phosphorylation/activation. Western blot analysis of SW13 (a) and H295R
(b) cell lysates following 15-minutestimulation with 10 nM IGF-I and 20 μM RGZ, reveals an increased
phosphorylation of Akt in Ser473 following IGF-I, which is reverted by RGZ.
Lane protein normalization for Akt is shown in the middle panels. Molecular
weight markers are indicated. Mean ± SE of phospho-Akt band intensity over
Ctrl is shown in 4 independent experiments for SW13 (a) and H295R (b) cells (lower
panels). Statistical
significance: P*<.05 versus Ctrl or IGF + RGZ.
(a)
SW13(b)
H295RRGZ inhibits IGF-I stimulated PI3K. PI3K activity in SW13 (a) and
H295R (b) treated or not for 15 minutes with 10 nM IGF-I and 20 μM RGZ was evaluated by an in vitro assay after immunoprecipitation
with an antibody against the PI3K regulatory subunit p85. The spots correspond
to PI3K catalytic product [32P]phosphatidyl inositol phosphate
(PIP). Representative of two similar experiments. Mean ± SE (n = 2) of
phospho-Akt band intensity (arbitrary units) is shown in the table under each
blot.
(a)
SW13(b)
H295RRGZ dampens IGF-I stimulated
ERK1/2 phosphorylation. Western blot analysis of SW13 (a) and H295R (b)
cell lysates following 15-minutes stimulation with 10 nM IGF-I and 20 μM RGZ, reveals an increased
phosphorylation of ERK1/2 following IGF-I, which is reverted by RGZ. Lane
protein normalization for ERK1/2 is shown in the middle panels. Molecular
weight markers are indicated. Mean ± SE of phospho-ERK1/2 band intensity
over respective Ctrl is shown in 3 independent experiments for SW13 (a) and
H295R (b) cells (lower panels). P*<0.05
versus respective Ctrl; °P<.05 and #P<.005 versus IGF-I.
(a)
SW13(b)
H295RIn order to
further demonstrate the involvement of Akt and ERK signaling downstream of the
IGF-IR in mediating cell proliferation, we use the NVP-AEW541 (NVP)
inhibitor of the IGF-IR tyrosine kinase activity. As shown in Figure7(a), NVP is
able to block phosphorylation of Akt  (upper
panel) and ERK1/2 (middle panel) both in the basal conditions and following 15-minute
IGF-I stimulation. The block of the IGF-IR system by NVP results in inhibition of IGF-I-stimulated cell
proliferation evaluated at 2 and 4 days of treatment in SW13 (Figure  7(b)) and H295R, respectively, (Figure 7(c)). Moreover, a further
addition of RGZ to the inhibitor results in no significant reduction of cell
proliferation in the presence of IGF-I compared to NVP + IGF-I (Figures 7(b), 7(c))
suggesting that RGZ growth inhibition is mediated via IGF-IR signaling.ERK and Akt activation downstream
of the IGF-IR mediates IGF-I stimulation of cell proliferation. Western
blot analysis of H295R cell lysates following 15-minute stimulation with 10 nM
IGF-I in the presence or absence of 1 μM NVP-AEW541 (NVP) inhibitor of the tyrosine
kinase activity of IGF-IR (a). The inhibitor blocks phosphorylation of Akt
(upper panel) and of ERK1/2 (middle panel) both in basal conditions and
following IGF-I stimulation. Lane protein normalization for actin is shown in
the lower panel. Molecular weight markers are indicated. Cell
proliferation was evaluated by MTS assay following 2 or 4 day stimulation with
the indicated treatments (10 nM IGF-I, 20 μM RGZ, 1 μM NVP) in SW13 (b) or H295R (c) cells,
respectively. Mean ± SE
percentage of OD over respective control IGF-I taken as 100%. #P<.001 versus IGF-I, n = 6.
(a)(b)(c)Finally, we
investigated whether RGZ could affect not only the downstream signaling of IGF-IR,
but also the levels of the receptor itself. Figure8 shows that the IGF-IR levels
do not change following up to 4-day stimulation of SW13 (a) d H295R (b) cells
with 20 μM RGZ.RGZ does not affect IGF-IR levels.  Western blot analysis of SW13 (a) and H295R (b) cell lysates following 2, 3,
and 4 day cell incubation in the absence (Ctrl) or in the presence of 20 μM RGZ reveals no significant change in
IGF-IR (upper panels). Lane protein normalization for actin is shown in the
lower panels. Molecular weight markers are indicated. Representative of two
independent experiments.
(a)
SW13(b)
H295R


## 4. Discussion

Although
adrenocortical carcinomas are very rare tumors, they are very aggressive and
highly resistant to chemo- and radiotherapy. Moreover, the use of the
adrenolytic agent, mitotane (o,p-DDD), is the only medical therapy available at present. Thus, a better knowledge of
the molecular mechanisms underlying the tumor growth and progression is
mandatory in order to develop more selective and specific treatments.Recently, PPARγ ligands have been described as suppressing
tumor cell proliferation as well as inducing apoptosis and a more
differentiated phenotype in several types of cancers [15], including adrenocortical carcinoma [10, 16], thus suggesting the use of these drugs as a potential new anticancer therapy.
However, all these studies have been performed either in vivo, on animal models,
or in vitro, on human cancer cells. Moreover, these anticancer effects have
been observed with concentrations of PPARγ agonists which are not only higher than
the clinical doses used for T2D treatment but also affect PPAR α and δ isoforms, being no longer selective for PPARγ. However, treatment with RGZ doses
higher than the therapeutical 8 mg/die has been well tolerated and did not
result in any increase in the percentage of adverse events compared to the
placebo group in a double-blind clinical trial [19].At present, only
three papers address the effects of PPARγ ligands in ACC using H295R cells as an in vitro model [10, 16, 20]. Both RGZ
and pioglitazone (PIO), the most used PPARγ TZD ligands, inhibit cell growth by
affecting key cell cycle elements and inducing apoptosis [10, 16], also
dampening cell invasiveness through reduction of metalloproteinase 2 (MMP2) expression and activity [16]. Moreover,
in these cells, RGZ and PIO induce a more differentiated phenotype where
steroidogenesis is increased due to a significant upregulation of MC2-R and
Star expression [10].In this paper,
we use two different cell models of ACC, H295R, and SW13. H295R cells retain
the ability to synthesize steroid hormones, while SW13, derived from a stage IV
tumor, do not, thus suggesting them to be less differentiated than H295R. Both
cell lines seem to be suitable models for studying the effects of RGZ on IGF-I/IGF-IR
axis, since they express high levels of the IGF-IR, similarly to ACC tissue. In
order to stimulate IGF-IR and its downstream signaling cascade, we added to the
cells increasing concentrations of its elective ligand, IGF-I. Indeed, IGF-IR
binds IGF-I with a 15-fold higher affinity than IGF-II [21]. However, H295R
cells have been described as producing high levels of IGF-II, which acts in an
autocrine-paracrine loop stimulating the IGF-IR axis even in basal conditions [8].
For this reason, in our experimental procedure, we changed cell incubation
media every day to remove the endogenously produced IGF-II thus making the
receptor more responsive to the exogenously added IGF-I.In this
condition, IGF-I was able to stimulate cell proliferation in a dose- and a
time-dependent manner in both cell models through activation of the two main
downstream intracellular signaling cascades involving phosphatidyl inositol
3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (ERK1/2) [21], as
described also for other tumors [18]. However, these two pathways are already
active even in basal conditions, as demonstrated by the rather high levels of ERK
and Akt phosphorylation as well as of PI3K activity found in the absence of IGF-I
stimulation, probably due to the paracrine-autocrine effects of endogenously
produced IGF-II [8].RGZ has been
demonstrated to impair IGF-I system both in
vivo and in vitro by reducing IGF-I production in bone marrow cells and
in the liver [22]. Moreover, troglitazone inhibits IGF-I tumor-promoting
activity in mouse skin by affecting the intracellular pathway involving AMP
kinase [23].In SW13 as well
as in H295R cells, RGZ was able to significantly reduce cell proliferation in a
dose- and time-dependent manner as evaluated by different techniques (MTS,
thymidine uptake and cell counting), with a calculated IC50 of 22.48±1.54 μM. This concentration is higher than the
RGZ plasma levels estimated from the total area under the plasma
concentration-time curve (Cmax 1.33 μM) obtained by pharmacokinetic studies
on subjects undergoing oral RGZ administration with the therapeutical dosage of
8 mg/die [24]. However, it is in perfect agreement with the doses currently used
for in vitro studies, in particular in cancer cells [10, 16]. Although the RGZ
inhibitory effect on cell proliferation and viability has already been reported
in H295R cells [10, 16], this is the first time that RGZ IC50 and
maximal effect have been calculated with an appropriate statistical analysis [17],
thus validating the current dose of 20 μM used in cancer studies and suggesting
that the anti-proliferative effects of RGZ are obtained at higher
concentrations than the ones effective on insulin sensitivity [13]. Toxic
studies associated with a pharmacokinetic studies to define the RGZ oral dose capable
of resulting in a range of 20 μM circulating concentration of RGZ and
to investigate the possible toxic effect associated to such doses, are
mandatory in order to hypothesize a possible therapeutic use of RGZ and other
TZD for ACC treatment.In addition to
its effect on cell proliferation in basal conditions, RGZ was also effective on
cell growth induced by addition of increasing concentrations of IGF-I. Interestingly,
such an inhibitory effect was similar in the presence or in the absence of IGF-I
only in SW13 cells, while in H295R, RGZ also prevents the stimulatory effect of
IGF-I. However, the inhibitory effect of RGZ was reached more slowly in H295R (4–7 days) than in
SW13 (1-2 days), probably due to the differences in the kinetic of duplication
between the two cell lines. Moreover, in H295R RGZ inhibition increases with
the dose of IGF-I, while in SW13, the effect was similar independently of the
dose of the growth factor. Consequently, the IC50 calculated for RGZ
curve on SW13 cell proliferation was similar in the presence or absence of IGF-I.In order to
elucidate at which level RGZ interferes with the activated IGF-I axis both in
basal conditions (IGF-IR activated by endogenously produced IGF-II) and
following a further activation of the receptor by exogenous addition of IGF-I,
we investigated the signaling pathways acting downstream of the receptor. RGZ
was able to block the rapid activation of the PI3K-Akt axis induced by IGF-I.
In agreement with the experiments on cell proliferation, we found that in H295R
cells only, RGZ was more effective on IGF-I-treated than on untreated cells.
RGZ also affected the ERK pathway through inhibition of the rapid
phosphorylation-activation of ERK1 and 2 isoforms. A similar effect of RGZ on
rapid phosphorylation of ERKs has been recently described in the inflammatory
response of endothelial cells [14]. Such an effect is too rapid (15 minutes) to
be ascribed to the classical transactivation mechanism of PPARγ on specific target genes, and suggests
a PPARγ independent mechanism or a nongenomic
activity of the receptor. Interestingly, among the anticancer action exerted by
RGZ in H295R, both PPARγ dependent and  independent effects seem to coexist in these
cells, since the PPARγ antagonist GW9662 has been demonstrated
to block RGZ induction of MC2R expression and cortisol secretion but not RGZ inhibition
of cell proliferation  (Betz et al., 2005).
Further studies are required to elucidate the precise mechanism by which PPARγ ligands affect rapid signaling in these
cells. Interestingly, no effect of RGZ was detected up to 4 day stimulationon the level of IGF-IR in both cell
lines. In accordance, Betz and colleagues described that IGF-II production
increased with time despite the clear growth-suppressive effects of RGZ and PIO
in H295R [10]. Our findings suggest that RGZ inhibition acts on the signaling
pathways downstream but not at the level of the IGF-IR.In conclusion,
our result shed new light on the molecular mechanisms underlying cell
proliferation and progression in adrenal carcinoma, contributing to demonstrate
that the inhibitory effect exerted by RGZ on cell growth is due to the TDZ
interferences with the two main signaling pathways downstream of the activated
IGF-IR. RGZ ability to block the IGF-IR axis suggests the potential application
of this molecule for the treatment of ACC.



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*Source: 904041-2008-07-28.xml*