Oncotarget 2013; 4: 422-432422
Oncotarget, March, Vol.4, No 3
Inhibition of U-87 MG glioblastoma by AN-152 (AEZS-108), a
targeted cytotoxic analog of luteinizing hormone-releasing
Miklos Jaszberenyi1,2,3, Andrew V. Schally1,2,3,4,5, Norman L. Block1,2,3,4, Mehrdad
Nadji3, Irving Vidaurre1,2, Luca Szalontay1,2, Ferenc G. Rick1,2
1 Veterans Affairs Medical Center, Miami, FL
2 South Florida VA Foundation for Research and Education, Miami, FL 33125
3 Department of Pathology, University of Miami, Miller School of Medicine, Miami, FL
4 Division of Hematology/Oncology, University of Miami, Miller School of Medicine, Miami, FL
5 Division of Endocrinology, Department of Medicine, University of Miami, Miller School of Medicine, Miami, FL
Correspondence to: Andrew V. Schally, email: email@example.com
Correspondence to: Ferenc G. Rick, email: firstname.lastname@example.org
Keywords: glioblastoma multiforme, U-87 MG, cytotoxic, LHRHor GnRH receptors, AN-152, AEZS-108, nude mice, targeted therapy
Received: March 5, 2013 Accepted: March 6, 2013 Published: March 7, 2013
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
Glioblastoma multiforme is the most frequent tumor of the central nervous system
in adults and has a dismal clinical outcome, which necessitates the development
of new therapeutic approaches. We investigated in vivo the action of the targeted
cytotoxic analog of luteinizing hormone releasing hormone, AN-152 (AEZS-108) in
nude mice (Ncr nu/nu strain) bearing xenotransplanted U-87 MG glioblastoma tumors.
We evaluated in vitro the expression of LHRH receptors, proliferation, apoptosis
and the release of oncogenic and tumor suppressor cytokines. Clinical and U-87 MG
samples of glioblastoma tumors expressed LHRH receptors. Treatment of nude mice
with AN-152, once a week at an intravenous dose of 413 nmol/20g, for six weeks
resulted in 76 % reduction in tumor growth. AN-152 nearly completely abolished
tumor progression and elicited remarkable apoptosis in vitro. Genomic (RT-PCR) and
proteomic (ELISA, Western blot) studies revealed that AN-152 activated apoptosis,
as reflected by the changes in p53 and its regulators and substrates, inhibited cell
growth, and elicited changes in intermediary filament pattern. AN-152 similarly
reestablished contact regulation as demonstrated by expression of adhesion molecules
and inhibited vascularization, as reflected by the transcription of angiogenic factors.
Our findings suggest that targeted cytotoxic analog AN-152 (AEZS-108) should be
considered for a treatment of glioblastomas.
Malignant tumors are frequently difficult to treat
using conventional chemotherapy treatment. However,
targeted cytotoxic peptide analogs could overcome this
problem. [1, 2] Since various neuropeptides play a pivotal
role in carcinogenesis, their appropriate receptors can be
targeted with cytotoxic peptide complexes.[1, 3] Targeting
increases efficacy, while reducing toxic side effects on
innocent bystander tissues, because, through receptor
internalization, the cytotoxic compounds selectively cross
the cell membrane of the target cells.[1, 2, 4, 5] Our group
has synthesized analogs of luteinizing hormone releasing
hormone (LHRH), somatostatin, and bombesin linked to
doxorubicin (DOX) or 2-pyrrolinodoxorubicin.[1-3, 6-8]
LHRH and its receptor (LHRH-R) are not
confined to the hypothalamic-pituitary axis. In
the periphery, the LHRH system coordinates gonadal
functions and serves as a growth factor of benign
conditions [11-13] and various malignancies.[10, 14]
In the central nervous system (CNS), hypothalamic and
extra-hypothalamic cell populations can be detected [15,
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16], where dense immunostaining for LHRH and LHRH-R
can be demonstrated.[15-17] Beside endocrine functions,
these cells are involved in the regulation of the olfactory
system, feeding, reproductive behavior and circadian
rhythms [15, 16, 18]. The LHRH-positive subventricular
zone, a frequent starting locus of primary glioblastoma
multiforme (GBM) , often shows hypertrophy and
hyperplasia, in the absence of steroid feed-back, in
postmenopausal and andropausal subjects . Since
this age-group has the highest prevalence of GBM ,
and GBM tumors frequently show high expression of
LHRH-R [21, 22], these findings suggest a regulatory role
of the LHRH system in the evolution of brain cancer. The
modulation of the LHRH system is used for the treatment
of several cancers. LHRH agonists are the mainstay in
the therapy of prostate cancer and act through the down-
regulation of LHRH-R. Moreover, our previous
studies showed that the LHRH antagonist, cetrorelix,
and the cytotoxic analog, AN-152 can be successfully
used for the treatment of cancers of the reproductive
system and of other organs.[1, 2, 26-28]
In the present study, we first demonstrated LHRH-R
expression on clinical samples of GBM and U-87 MG
cells by immunohistochemistry (IHC) and Western
blot. In vitro, U-87 MG cells were exposed to AN-152
and viable cells were determined by proliferation assay.
Subsequently, the effectiveness of the cytotoxic analog,
AN-152 was evaluated, in vivo, on the growth of U-87
MG tumors xenotransplanted into nude mice. To evaluate
its mechanism of action, the most frequently involved
“cancer pathway” genes were screened with real-time PCR
arrays. Also, apoptotic processes and drug resistance were
detected by specific kits. The ability of a chemotherapeutic
drug to pass through the cell membrane and to accumulate
within the cellular compartments of the neoplastic tissue
is one of its most important pharmacodynamic features.
Intracellular accumulation was tested by competition
with a fluorescent test compound on multidrug resistance
(MDR) pumps, by which cells get rid of toxic foreign
molecules. Proteomic verification of functional and
genomic changes was performed by Western blots and
The dense expression of LHRH-R on clinical
samples of GBM was shown by the positive reaction in
the form of brown granules (Fig. 1).
Groups of nude mice bearing U-87 MG tumors
were treated once a week for 6 weeks with AN-152, DOX,
Figure 1: expression of receptors for lHrH in two representative human GbM specimens. The samples were stained by
hematoxylin-eosin (panel HE) and IHC (with affinity-purified goat polyclonal antibody, Santa Cruz, panel LHRH-R). Magnification is 50×.
Oncotarget 2013; 4: 422-432424
D-Trp6-LHRH mimicking the carrier molecule, or with
the combination of DOX and D-Trp6-LHRH. Treatment
with AN-152 produced the greatest inhibition (Fig. 2) and
repeated measure ANOVA revealed significant effects
(Within-Subject F5,62=17.87, p<0.01, Between-Subject
F4,66=78.6, p<0.01). With pair-wise comparison only
the effect of AN-152 was significant compared to the
control (Tukey’s HSD test: p<0.01 vs. control), which
suggests that targeting greatly increased the effectiveness
of therapy. A similar tendency could be observed in the
length of tumor doubling times (given in days; control:
10.02, DOX: 13.75, D-Trp6-LHRH: 13.32, DOX +
D-Trp6-LHRH: 10.32, AN-152: 19.88), although this
effect did not prove to be statistically significant.
Proliferation, Apoptosis and Mdr assays
Single exposure to AN-152 brought about an
almost 70 % inhibition of tumor cell growth (Fig. 3/A,
F4,284=374.49, p<0.01; Tukey’s post hoc p<0.01 vs.
control in both cases). Treatment with DOX or the
unconjugated combination also led to growth suppression
but to a lesser extent. The group treated with AN-152 was
statistically different from the DOX treated one (Tukey’s
post hoc: p<0.01 vs. DOX).
Both DOX and AN-152 elicited a significant
increase in apoptosis (F4,91=110.61, p<0.01, Tukey’s post
hoc test: p<0.01 vs. control). Again, AN-152 was the most
effective (with almost 250 % increase) and its effect was
statistically more significant than that of DOX (Tukey’s
post hoc: p<0.01 vs. DOX; Fig. 3/B). These assays,
measuring viable cell count and cell death, confirmed
our in vivo findings. Further, in both cases, the targeted
cytotoxic compound was significantly more effective than
In the MDR assay, only AN-152 caused a greater
increase in calcein retention than the Cyclosporin-A
positive control (F4,117=46.8, p<0.01; Tukey’s post hoc
test: p<0.01 vs. positive control). Treatment with AN-152
proved to be significant even compared to DOX (Tukey’s
post hoc test: p<0.01 vs. DOX; Fig. 3/C). Presumably
receptor mediated internalization leads to significantly
higher intracellular concentrations of DOX, which, in turn
leads to overload of MDR transporters that try to eliminate
toxic, foreign compounds from the cell. The increased
competition on the MDR transporters, eventually results
in increased calcein retention.
PCR array studies revealed a significant antitumor
effect of AN-152 on the marker regulators of cell
proliferation and cell death (nuclear factor κB (NF-
κB), platelet derived growth factor (PDGF), metastasis
associated 1 family, member 2 (MTA2)), contact and
humoral control (integrins, tumor necrosis factor
receptor 10β (TNF-R10β)), invasion (MMP-9, urokinase
plasminogen activator (uPA)) and metastasis formation
(melanoma cell adhesion molecule (MCAM), MTA2).
Compared to the parallel treatments AN-152 generally
elicited more profound changes in gene expression and its
activity on invasion, angiogenesis and metastasis markers
(uPA, MMP-9, MCAM, MTA2) was especially significant
Western blot and elIsA experiments
Western blot studies (Fig. 4) verified the expression
of LHRH-R expression in U-87 MG xenograft samples.
Figure 2: the effect of the cytotoxic lHrH analog, An-152 (AeZs-108), on the growth of xenotransplanted u-87 MG,
human glioblastoma tumors. The pooled standard errors of the groups: control: 313.9; D-Trp6-LHRH: 645.3; doxorubicin (DOX):
267.8, D-Trp6-LHRH + DOX: 308.9; AN-152: 172.1. Numbers in brackets are the number of successfully grafted tumors. Numbers at
the end of each line represents the tumor doubling times. * = p < 0.05 vs. control for the repeated measure evaluation of tumor growth
Oncotarget 2013; 4: 422-432425
Importantly, AN-152 treatment did not induce any down-
regulation of LHRH-R. These experiments also verified
the remarkable up-regulation of the tumor suppressor and
pro-apoptotic p53 by the cytotoxic analog (IDVs: control:
4102.7±1096.7, AN-152: 14895.0±1153.4). Further, AN-
152 reestablished contact inhibition through up-regulation
of E-cadherin (IDVs: control: 6636.6±2042.0, AN-152:
21925.7±163.7) and down-regulation of β-catenin (IDVs:
control: 16392.5±2155.3, AN-152: 1349.6±757.2). One
of the most important results of the Western blot studies
was that the cytotoxic analog inhibited the expression of
the primordial, neuroectodermal stem cell marker, nestin
(IDVs: control: 3149.1±157.1, AN-152: 739.5±143.5)
and stimulated the synthesis of the maturation antigen,
GFAP (IDVs: control: 12007.7±2209.8, AN-152:
The level of several oncogenic cytokines and
tumor suppressor molecules was modified by the AN-152
treatment as shown by ELISA (Fig. 5). First, these studies,
using homogenized cell culture samples, confirmed the
increase of p53 and the decrease of β-catenin observed
in the Western blot experiments. The statistical analyses
showed significant changes in both cases (F4,14=7.2,
p<0.01, Tukey’s post hoc test: p<0.01 vs. control (n=4)
for p53 and F4,23=32.48, p<0.01, Tukey’s post hoc test:
p<0.01 vs. control (n=5) for β-catenin). In addition, FGFβ,
one of the decisive markers of glial growth, and VEGF,
the most important factor of tumor vascularization and
nutrition, were significantly down-regulated by AN-152
treatment (F4,19=7.8, p<0.01, Tukey’s post hoc test:
p<0.01 vs. control (n=5) for FGFβ; F4,11=4.8, p<0.05,
Tukey’s post hoc test: p<0.05 vs. control (n=3) for
VEGF). The expression of the maturation marker, GFAP,
was reduced by the treatment with DOX (F4,26=14.67,
p<0.01, Tukey’s post hoc test: p<0.01 vs. control (n=6)).
This suggests dedifferentiation or the survival of an
immature aggressive proliferation prone phenotype. AN-
152 treatment caused a much lesser reduction (p<0.01 vs.
DOX), which suggests that the cytotoxic analog, most
importantly, may inhibit the survival of resistant stem cell-
Our IHC and Western blot analyses clearly
demonstrated that LHRH-R is expressed on human GBM
cells, suggesting a role of intrinsic LHRH secretion
in the autocrine/paracrine control of GBM cells (Fig.
1, 4). The treatment with ligands of LHRH-R did not
down-regulate the LHRH receptors, which augurs well
for continuing long term therapy with AN-152. D-Trp6-
LHRH representing the carrier molecule exerted only a
weak effect on in vivo and in vitro tumor growth (Fig.
2 Fig. 3/A) and the expression of adhesion molecules
and growth factors (Table 1, Fig. 4-5). However, in the
case of some oncoproteins such as MAPK1, the MAPK
activator, V-raf-1 murine leukemia viral oncogene
homolog 1 (Raf-1), phosphoinositide-3-kinase (PI3K) and
the differentiation marker, GFAP, the changes reached the
level elicited by the AN-152 treatment.
AN-152 elicited profound inhibition of tumor
growth both in vivo (Fig 2) and in vitro (Fig. 3/A), and
the effect of the cytotoxic analog exceeded that of DOX
(Fig. 2-5). This intensified antitumor potential appears
to be related to the “homing property” of the cytotoxic
analog, which can lead to an increased competition on
the MDR transporter proteins reflected by the increased
calcein retention in the MDR studies. However, according
to the PCR experiments, D-Trp6-LHRH mimicking the
carrier molecule, may also act as an inhibitor of the MAPK
Figure 3: the effect of the treatment with An-152 (AeZs-108) on the proliferation (A), apoptosis (b) and calcein
retention (c) of u-87 MG cells. Sample numbers at the bottom of each column refer to the seeded wells, which underwent the given
treatment. Abbreviations: DOX: doxorubicin, D-Trp6 + DOX: D-Trp6-LHRH + doxorubicin. * = p < 0.05 vs. control (n=24); + = p < 0.05
Oncotarget 2013; 4: 422-432 426
pathway (Table 1). This may lead to a direct inhibition of
MDR-1 protein translocation/activation and decreased
resistance, which confirms our previous findings.[4, 30]
According to the signal transduction studies, AN-
152 influenced the cell-cycle, differentiation, contact and
humoral control, angiogenesis and invasion. The cytotoxic
analog increased levels of the pro-apoptotic tumor
suppressor, p53, which molecule is frequently mutated
in GBMs[31, 32] (Fig. 4-5). The increase of p53 can be
related to the down-regulation of MTA2, which plays a
role in the deacetylation and breakdown of p53. The
apoptosis stimulating potential of AN-152 is supported by
the increased expression of the death domain containing
TNF-R10β (Table 1). The cytotoxic analog also
suppressed the antiapoptotic NF-κB expression.
AN-152 mitigated the release of several glial growth
factors, such as insulin-like growth factor I (IGF-I), FGF,
and PDGF and also blocked the interwoven MAPK and
PI3K-protein kinase B (PKB)/Akt survival pathways (Fig.
5, and Table 1). The cytotoxic analog decreased the
expression of MAPK-1, Raf-1 and the regulator subunit 1
(PIK3R1) of PI3K. Since growth promoting cytokines
are able to stimulate the survival cascades, both direct
and indirect inhibition can explain the antiproliferative
activity of AN-152.
The treatment with AN-152, according to the
proteomic studies, also influenced the expression of the
glial differentiation markers, nestin and GFAP (Fig. 4-5).
[39, 40] During astrocyte maturation, nestin as a common
neuroectodermal marker progressively disappears, while
GFAP becomes the characteristic intermediary filament.
In our experiments, DOX treatment elicited an increase in
the expression of nestin and a decrease in the transcription
table 1: relative expression of genes related to tumor growth
Insulin-like growth factor 1
Integrin, α4 (α4 subunit of VCAM-1 receptor)
Integrin, αV (domain of vitronectin receptors)
Integrin, β5 (domain of vitronectin receptor)
Mitogen-activated protein kinase 1
Melanoma cell adhesion molecule
Matrix metalloproteinase 9 (gelatinase B)
Metastasis associated 1 family, member 2
Nuclear factor κB
Phosphoinositide-3-kinase, regulatory subunit 1α
Plasminogen activator (urokinase)
Platelet-derived growth factor α
Serpin, clade B, member 5 (maspin)
Tumor necrosis factor receptor superfamily, 10β
V-raf-1 murine leukemia viral oncogene homolog 1 0.74
In vivo glioblastoma specimens were evaluated by Cancer Pathway RT2 Profiler PCR Array system. Only genes with at
least three-fold or statistically significant changes are represented. Four tumor samples from each group were analyzed.
Relative expressions are compared to the control. *p < 0.05 vs. control. Abbreviations: DOX: doxorubicin, D-Trp6 +
DOX: D-Trp6-LHRH + doxorubicin, VCAM: vascular cell adhesion molecule.
Figure 4: Western blot analyses of lHrH receptor and
tumor marker expression following An-152 treatment.
Abbreviations: LHRH-R: luteinizing hormone releasing
hormone receptor, GFAP: glial fibrillary acid protein, DOX:
doxorubicin, D-Trp6 + DOX: D-Trp6-LHRH + doxorubicin. * =
p < 0.05 vs. control.
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of GFAP suggesting that, an undifferentiated, pluripotent
clone became dominant upon treatment. Such cancer
stem-like cell populations may retain the capability of
expressing a wide array of resistance factors, which later
would normally disappear, through differentiation.
Concersely, the cytotoxic analog elicited opposite changes,
perhaps due to its “homing” neuropeptide molecule (Fig.
AN-152 also exerted a beneficial effect on contact
and humoral regulatory factors, which restrain tumor
growth at a population level (Fig. 4-5, and Table).
The increase in E-cadherin expression, and β-catenin
degradation (Fig. 4-5) may inhibit the so-called cadherin-
switch, when the expression pattern from E-cadherin
changes to N-cadherin, resulting in loss of adhesion
and stimulation of invasion.[42, 43] Moreover, AN-152
decreased the transcription of three (α4, αV, β5) contact
activator integrin domains (Table 1), which are frequently
over-expressed in GBM tumors.
Beside the pronounced inhibition of VEGF
secretion (Fig. 5), the cytotoxic analog decreased the
expression of angiopotein-1, and MCAM (Table 1),
which are beneficial effects, since the suppression of
angiogenesis is one of the most important complementary
therapeutic approaches in the case of GBMs. Beside
neovascularisation, MCAM regulates cell proliferation
through the PKB/Akt pathway, stimulates cell migration,
increases the expression of MAPK and the proteolytic
MMPs. AN-152 also suppressed the expression
of MMP-9 and the uPA and augmented the local tumor
suppressor, maspin, level thus inhibiting the invasive
capability activity of tumor cells (Table 1).[48, 49]
The present results, taken together with our
previous findings promote the concept of a new, multi-
faceted chemotherapeutic paradigm in the treatment of
GBM, based on targeted peptide analogs. This concept
is enhanced by the fact that analogs of hypothalamic
peptide-hormones can cross the blood brain barrier.
Our results show that AN-152 is a versatile multi-pronged
hybrid molecule which, beside direct antiproliferative and
pro-apoptotic activity, elicits maturation. These features
establish AN-152 as a very promising therapeutic option
against brain cancers which express LHRH-R. In view of
ongoing encouraging clinical phase I//II/III trials with AN-
152 (also denoted by its commercial designation, AEZS-
108) in gynecological, prostatic and urothelial cancers[1,
3, 26, 28], our findings strongly suggest a significant step
forward in the successful therapy of malignant gliomas,
curative treatment of which is not yet available.
Investigation has been conducted in accordance with
the ethical standards and according to the Declaration of
Helsinki and according to national and international
guidelines and has been approved by the authors’
institutional review board.
Peptides and chemicals
The cytotoxic LHRH conjugate, AN-152, and the
LHRH agonist D-Trp6-LHRH were synthesized in our
laboratory as described. DOX hydrochloride was
Figure 5: the effect of the treatment with An-152 (AeZs-108) on the release of cytokines and signal transducers
verified by ELISA experiments. Sample numbers at the bottom of each column refer to the seeded wells, which underwent the given
treatment. Abbreviations: GFAP: glial fibrillary acid protein, DOX: doxorubicin. * = p < 0.05 vs. control; + = p < 0.05 vs. DOX
Oncotarget 2013; 4: 422-432 428
obtained from Chemex Export-Import Gmbh (Vienna,
Austria). The compounds were dissolved for injection in
5% (w/v) aqueous D-mannitol solution as vehicle.
Six-week-old female nude mice (Ncr nu/nu) were
obtained from the NCI (Bethesda, MD). The animals were
maintained according to the guidelines of the Institutional
Animal Care and Use Committee, as described previously.
In vivo study design
The animal studies with the U-87 MG GBM cell
line (American Type Culture Collection, Manassas, VA)
were performed as described previously . Donor
mice were injected in the flanks with 1×106 glioblastoma
U-87 MG cells. After 4 weeks, tumor tissue grown in
these donor animals was minced and passed through a
wire mesh and a 150 μl suspension of this was injected
s.c. into experimental nude mice. The experiment was
initiated when U-87 MG tumors had reached a volume
of approximately 70 mm3. Mice bearing xenografts were
randomized into 5 groups of 10 mice each with a random
number generator function of Microsoft Excel (Microsoft
Corp. Redmond, WA). The groups received, once
weekly, the following intravenous treatment for 6 weeks,
respectively: group 1: (control, 14 tumors, 100 μl vehicle
solution); group 2: (DOX, 14 tumors); group 3: (D-Trp6-
LHRH, 14 tumors); group 4: (DOX + D-Trp6-LHRH, 14
tumors); group 5: (AN-152, 15 tumors). The concentration
of the compounds was equimolar (413 nmol/20g) and was
adjusted to the maximum tolerable dose of the cytotoxic
drugs, which had proved the most effective in our previous
oncological studies.[5-7, 52] Tumor dimensions were
measured with microcalipers once a week and volume
was calculated using the formula: (length × width ×
height × π)/6. Tumor doubling time was calculated using
the formula: (study duration × LOG 2)/(LOG final tumor
volume - LOG initial tumor volume).
In vitro experiments
Formalin-fixed, paraffin-embedded, surgically
removed tissue samples were used for IHC. Three micron
paraffin sections were stained by hematoxylin and eosin
to confirm the presence of GBM. Adjacent serial sections
were utilized for immunoperoxidase staining following
standard protocols as previously described and using
a polyclonal antibody to LHRH-R (GnRHR-N20, Santa
Cruz Biotechnology, Santa Cruz, CA). The sections were
then counterstained with hematoxylin. Human pituitary
glands (anterior lobe) obtained from autopsy were used
as positive controls. The use of archival samples of GBM
was approved by the institutional review board.
U-87 MG cells were cultured in EMEM (ATCC)
medium (supplemented with 10% FBS, (ATCC) and
0.1% penicillin/streptomycin) at 37°C and 5% CO2
atmosphere. This cell line is classified as grade IV GBM
and was characterized and deposited by J. Ponten and
associates. As a treatment, those doses (100 nM) of
AN-152 were used which had proved most effective in our
previous studies. Other compounds were administered
in equimolar concentrations.
Proliferation and apoptosis assays in vitro
For proliferation studies, 104 cells/well were
seeded in 100 μl medium, in a 96-well plate and were
then incubated for 24 h in a humidified incubator at 37
°C. Next, culture medium was replaced with FBS free
medium (starvation) for 24 h. After another 24 h, the cells
were exposed to complete medium containing 100 nM
of AN-152, DOX, D-Trp6-LHRH, or the combination of
DOX and D-Trp6-LHRH. The cells were then incubated
for 48 h. The effect of the compounds on cell proliferation
was evaluated by using the 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay (CellTiter
96® Non-Radioactive Cell Proliferation Assay, Promega,
Madison, WI, USA), according to the manufacturer’s
instructions with the help of a Victor3 multilabel counter
(Perkin-Elmer, Waltham, MD, USA)(5). Determination of
apoptosis was performed on freshly seeded cell samples
(104 cells/well, in 100 μl media, in a 96-well plate) by
the Multi-Parameter Apoptosis Assay Kit (Cayman
Chemical Company, Ann Arbor, MI), according to the
Multidrug resistance assay
This assay was performed according to the
manufacturer’s instructions (Cayman Chemical Company,
Ann Arbor, MI). U-87 MG cells were seeded in 5×104
cells/well density in 100 μl medium, to 96-well, black,
clear bottom plates and were grown overnight in a
humidified incubator at 37 °C. The next day the medium
was discarded, and the cells were treated with medium
containing 100 nM of AN-152, DOX, D-Trp6-LHRH, or
the combination of DOX and D-Trp6-LHRH. As a positive
control Cyclosporin-A solution was used in 1/1000 dilution
according to the manufacturer’s description. Afterwards,
the cells were incubated for 1 h, then calcein AM/Hoechst
dye combined staining solution was added. Fifteen
Oncotarget 2013; 4: 422-432429
minutes later both cell density (at excitation and emission
wavelengths of 355 nm and 465 nm, respectively) and
calcein retention (at excitation and emission wavelengths
of 485 nm and 535 nm, respectively) were detected with
the help of a Victor3 multilabel counter (Perkin-Elmer,
Waltham, MD, USA). Relative calcein retention values
were expressed as a function of cell density.
total rnA isolation and reverse transcription
Total RNA was isolated from representative, DNAse
treated, U-87 MG tumor samples using a NucleoSpin kit
according to the manufacturer’s instructions (Macherey-
Nagel Inc., Bethlehem, PA). Four tumor samples from
each group were analyzed. The yield and the quality of
RNA samples were determined spectrophotometrically
using 260 nm, and 260/280 and 260/230 nm ratios. The
synthesis of cDNA was performed as described.
Briefly, 1 µg of RNA from each sample was reverse-
transcribed into cDNA by RT First Strand kit (Qiagen).
Reverse transcription was done in a Veriti 96-well thermal
cycler (Applied Biosystems).
cancer Pathway Finder quantitative Pcr array
The Human Cancer Pathway Finder quantitative
PCR array (PAHS-033A, Qiagen) used in our study
contains 84 unique genes related to cell proliferation,
apoptosis, cell cycle, angiogenesis, invasion and
metastasis. All PCR arrays were studied using iQ5
Multicolor Real-Time Detections System (Bio-Rad). All
genes represented by the array showed a single peak on
the melting curve characteristic of the specific products.
Four tumor samples from each group were analyzed. Data
analysis of gene expression was performed using Excel
based PCR Array Data Analysis Software provided by the
manufacturer (Qiagen): fold-changes in gene expression
were calculated using the ΔΔCt method and five stably
expressed housekeeping genes (B2M, HPRT1, RPL13A,
GAPDH, and ACTB) were used for normalization of the
Western blot analyses
Proteins from the tumor tissue were isolated
using the Macherey-Nagel NucleoSpin kit. The
protein concentrations of the supernatant were
determined by NanoDrop (NanoDrop Technologies
Inc., Wilmington, DE). Equal amounts of protein were
resuspended in sample loading buffer (0.25 M Trizma
Base, 8% SDS, 40% glycerol, 0.004% bromophenol
blue, 4% b-mercaptoethanol; pH 6.8), boiled for 3
min and separated by 12% SDS-polyacrylamide gel
electrophoresis. Proteins from the gel were transferred
onto nitrocellulose membranes, which were blocked with
50-50% Tris-buffered saline (TBS) (20 mM Tris-HCl
pH:7.5, 150 mM NaCl): Odyssey blocking buffer for 1 h
at room temperature, followed by an overnight incubation
at 4°C with the following primary antibodies: nestin
(cat. no.: ab92391), glial fibrillary acid protein (GFAP)
(cat. no.: ab48050, both from AbCam Inc., Cambridge,
MA), LHRH-R (sc-13944), β-actin (cat. no.: sc-47778,
both from Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), β-catenin (cat. no.: 9562), E-cadherin, (cat. no.:
3195), p21 (cat. no.: 2947), p53 (cat. no.: 9282, all from
Cell Signaling Technology Inc. (Danvers, MA). The
signals were developed by incubating the membrane for
1 h at room temperature with the appropriate Infrared
IRDye-labeled secondary antibodies (1:10000; LI-
COR Biosciences, Lincoln, NE) and were then visualized
with the Odyssey Infrared Imaging System (LI-COR
Biosciences, Lincoln, NE). The protein bands were
quantified using V3.0 software (LI-COR Biosciences,
Lincoln, NE) and integrated densitiy values (IDV)s of
duplicate experiments were calculated.
elIsA assays for the determination of
oncoprotein and tumor suppressor expression.
U-87 MG cells (105 cells per well) were seeded
onto 6-well plates, cultured overnight, and then exposed
to the previously outlined treatments (100 nM of DOX,
D-Trp6-LHRH, AN-152 or the combination of DOX and
D-Trp6-LHRH) for 24 hours. Concentrations of specific
proteins in cell lysates were determined according to the
manufacturer’s instructions. Human p53 and vascular
endothelial growth factor (VEGF) ELISA kits were
purchased from Biovendor, LLC (Candler, NC), fibroblast
growth factor (FGF) basic human ELISA kit was obtained
from AbCam Inc., total β-Catenin ELISA kits were
received from Cell Signaling Technology, and GFAP
human ELISA kit was purchased from Cayman Chemical
Company. One or two plates were run and readings were
normalized to protein concentrations determined by
NanoDrop (NanoDrop Technologies Inc., Wilmington,
Statistical analyses were performed using either
t-test for independent samples (two-sided, for PCR
assays), univariate analysis of variance (ANOVA) (in
vitro studies) or repeated measure ANOVA (in vivo
experiment). ANOVA was followed by Tukey’s post hoc
tests for group-wise comparisons. Results are expressed
either as the means ± SEM (in vitro studies) or as means
and pooled standard errors (in vivo studies). Differences
with p<0.05, compared to the control, were considered
statistically significant. Data reductions and statistical
Oncotarget 2013; 4: 422-432 430
analyses were performed by SigmaPlot 12.0 (Systat
Software, Inc., Chicago, IL) and IBM SPSS Statistics 20.0
(IBM Corporation, Armonk, NY).
The work described in this paper was supported
by the Medical Research Service of the Veterans Affairs
Department, University of Miami, Miller School of
Medicine, Departments of Pathology and Medicine,
Division of Hematology/Oncology, the South Florida
Veterans Affairs Foundation for Research and Education
(both to A.V.S.) and the L. Austin Weeks Endowment
for Urologic Research (to N.L.B.). This work was also
supported in part by a grant from the Urology Care
Foundation Research Scholars Program and the AUA
Southeastern Section (to F.G.R.). M.J. is on leave from
the Department of Pathophysiology, University of Szeged,
Conflicts of Interest and Source of Funding:
Dr A.V. Schally is listed as co-inventor on the
Tulane University patent on AN-152 (AEZS-108). Dr.
Schally receives a grant for research on AN-152 from
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