Increased chemotactic migration and growth in heparanase-overexpressing human U251n glioma cells.

Abstract

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix (ECM) and basement membrane. Expression of the heparanase gene is associated with the invasion and metastatic potential of a variety of tumor-derived cell types. However, the roles of heparanase in the regulation of gene expression and the subsequent cell function changes other than invasion are not clear. In the current study, we overexpressed the human heparanase gene in a human U251n glioma cell line. We found that heparanase-overexpression significantly increased cell invasion, proliferation, anchorage-independent colony formation and chemotactic migration towards fetal bovine serum (FBS)-supplied medium and stromal cell-derived factor-1 (SDF-1). These phenotypic appearances were accompanied by enhanced protein kinase B (AKT) phosphorylation. Focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1 (ERK1) signaling were not altered by heparanase-overexpression. These results indicate that heparanase has pleiotropic effects on tumor cells.

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BioMed Central
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Journal of Experimental & Clinical
Cancer Research
Open Access
Research
Increased chemotactic migration and growth in
heparanase-overexpressing human U251n glioma cells
Xin Hong1, Feng Jiang2, Steven N Kalkanis1, Zheng Gang Zhang2,
Xuepeng Zhang2, Xuguang Zheng2, Hao Jiang2, Tom Mikkelsen1 and
Michael Chopp*2,3
Address: 1Department of Neurosurgery, Henry Ford Health Science Center, Detroit, MI, USA, 2Department of Neurology, Henry Ford Health
Science Center, Detroit, MI, USA and 3Physics Department, Oakland University, Rochester, MI, USA
Email: Xin Hong - nsxin@neuro.hfh.edu; Feng Jiang - fengj@neuro.hfh.edu; Steven N Kalkanis - kalkanis@neuro.hfh.edu;
Zheng Gang Zhang - zhazh@neuro.hfh.edu; Xuepeng Zhang - xuepeng@neuro.hfh.edu; Xuguang Zheng - xuguang@neuro.hfh.edu;
Hao Jiang - hjiang1@hfhs.org; Tom Mikkelsen - nstom@neuro.hfh.edu; Michael Chopp* - chopp@neuro.hfh.edu
* Corresponding author
Abstract
Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide
constituent of the extracellular matrix (ECM) and basement membrane. Expression of the
heparanase gene is associated with the invasion and metastatic potential of a variety of tumor-
derived cell types. However, the roles of heparanase in the regulation of gene expression and the
subsequent cell function changes other than invasion are not clear. In the current study, we
overexpressed the human heparanase gene in a human U251n glioma cell line. We found that
heparanase-overexpression significantly increased cell invasion, proliferation, anchorage-
independent colony formation and chemotactic migration towards fetal bovine serum (FBS)-
supplied medium and stromal cell-derived factor-1 (SDF-1). These phenotypic appearances were
accompanied by enhanced protein kinase B (AKT) phosphorylation. Focal adhesion kinase (FAK)
and extracellular signal-regulated kinase 1 (ERK1) signaling were not altered by heparanase-
overexpression. These results indicate that heparanase has pleiotropic effects on tumor cells.
Introduction
Tumor cell invasion and metastatic spread depend on the
ability of cancer cells to invade tissue barriers by degrad-
ing extracellular matrix (ECM) and basement membrane
structures [1,2]. The primary components of the basement
membrane and ECM are structural proteins, such as colla-
gen IV, laminin, fibronectin, and heparan sulfate prote-
oglycans (HSPGs). Heparan
glycosaminoglycon (GAG) chain present in HSPGs [3].
sulfate (HS) is a
HS chains interact through specific attachment sites with
the main protein components of basement membrane
and ECM.
Heparanase is a mammalian endo-β-D-glucuronidase
responsible for HS degradation [4-7]. Heparanase activity
may therefore play an important role in fundamental bio-
logical processes associated with ECM remodeling and
cell invasion [8,9]. Increased expression of heparanase
Published: 22 July 2008
Journal of Experimental & Clinical Cancer Research 2008, 27:23
23
doi:10.1186/1756-9966-27-
Received: 7 May 2008
Accepted: 22 July 2008
This article is available from: http://www.jeccr.com/content/27/1/23
© 2008 Hong et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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mRNA and protein has been reported in a variety of met-
astatic cell lines and human tumor tissues, whereas adja-
cent normal-looking tissue does not exhibit detectable
levels of heparanase [10-15]. Moreover, increased hepara-
nase mRNA expression correlates with reduced postoper-
ative survival of cancer
Overexpression of heparanase cDNA in tumor cells with
low metastatic potential confers a high metastatic poten-
tial after injection of these cells into the experimental ani-
mals [4]. Heparanase has also been shown to elicit an
angiogenic response by releasing heparan sulfate-bound
angiogenic factors sequestered in the ECM, such as vascu-
lar endothelial growth factor (VEGF) and basic fibroblast
growth factor (bFGF) [17,18].
patients [12,15,16].
Recently, it was reported that heparanase is translocated
into the cell nucleus where it may degrade the nuclear
heparan sulfate and thereby affect nuclear functions (i.e.
regulation of gene expression and signal transduction)
that are thought to be regulated by heparan sulfate [19].
Thus, the function of heparanase seems not to be only
limited to degrading extracellular matrix. Cellular func-
tion may be affected by heparanase through regulating
gene expression. There are accumulating evidences that
certain signal transduction cascades are altered under
heparanase stimulation [20-22]. However, genes and cel-
lular functions involved in heparanase regulation, other
than cell invasion, is still unclear. In the present study, we
demonstrate that heparanase overexpression in stably
transfected human U251n glioma cells results in a marked
increase of cell chemotactic migration toward fetal bovine
serum (FBS)-supplied medium and stromal cell-derived
factor-1 (SDF-1). Cell proliferation and anchorage-inde-
pendent growth are also increased in heparanase-overex-
pressing cells. These phenotypic changes are combined
with elevated protein kinase B (AKT) phosphorylation.
Materials and methods
Antibodies and reagents
The following antibodies were purchased from Santa Cruz
Biotechnology: anti-heparanase (H-80, sc-25825), anti-
phospho-FAK and anti-actin. Antibodies for ERK1, phos-
pho-ERK1, AKT, phospho-AKT (ser473), phosphor-
GSK3β (ser9) and phosphor-eNOS (ser1177) were
obtained from Cell Signaling Technology (Beverly, MA).
Other antibodies included anti-FAK (44624G) (Bio-
source, Camarillo, CA) and integrin β1 (AB1952)
(Chemicon, Temecula, CA). Growth factor reduced
matrigel was purchased from Becton Dickinson (San
Diego, CA). Heparin (H3149), dimethyl formamide
(D4551) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) (M2128) were the prod-
ucts of Sigma (St. Louis, MO).
Cell culture and transfection
U251n and U87 glioma cells originally were obtained
from the American Type Culture Collection (ATCC). Cells
were cultured at 37°C with 5% CO2 and maintained in
DMEM containing 10% (v/v) fetal bovine serum (FBS), 4
mM glutamine, 100 IU/ml penicillin, 100 μg/ml strepto-
mycin, and 1% nonessential amino acid (Invitrogen). Pri-
mary cultured glioma tumor cells (HF2303) were
obtained from Hermelin Brain Tumor Center, Henry Ford
Hospital (Detroit, MI, USA) with written consent in
accordance with institutional guidelines. For stable trans-
fection, U251n cells were transfected with the full-length
human heparanase cDNA (kindly provided by Dr. Ian N.
Hampson, University of Manchester, United Kingdom) or
a control pcDNA3 vector, using the Lipofactamine 2000
reagent (Life Technologies). Cells were selected with G418
(800 μg/ml) for 3 weeks, expanded, pooled and further
selected for high heparanase-expressing cells, as evaluated
by real-time reverse transcription-PCR. The pool with the
highest heparanase expression levels was labeled as
"U51n-hpa" throughout the manuscript, whereas, the
parental pcDNA3 transfected cells was referred to as
"U251n-pc". U251n cells were also transfected with
pcDNA3-Flag-HA-AKT1 (Addgene Inc. Cambridge, MA)
for 48 hours. AKT overexpressing U251n cells (U251n-
AKT) will be used as control for Western blot.
Heparanase enzyme activity assay
Heparanase activities were assayed in cell lysates by using
a heparan-degrading enzyme assay kit (TaKaRa Bio Inc.).
One million cells were lysed with 1 ml extraction buffer,
centrifuged at 10,000 × g for 5 min at 4°C, and then
supernatants were collected as samples. Heparanase activ-
ities in all samples were interpolated from a standard
curve performed by using an unlabeled HS as a standard
substitute. The absorbance was read by a microplate
reader (Multiskan MCC/340, Labsystems, Finland) at the
wavelength of 450 nm.
Real-time RT-PCR
Total RNAs were extracted using a RNeasy mini kit with
DNase digestion (Qiagen, Santa Clarita, CA). Two step
real-time PCR was performed as described previously
[23]. Housekeeping gene TATA box binding protein (TBP)
was used for each RNA sample as control. The mRNA
expression was expressed as the fold change related to TBP
mRNA expression. Primers included 5'-TGA TCT TGA
CCA GAA TAC CAT CGA-3' and 5'-GGC TTG CGA GGG
AAG AAG TT-3' for MMP-2 [GenBank: NM_004530], 5'-
GAC AAG CTC TTC GGC TTC TG-3' and 5'-TCG CTG GTA
CAG GTC GAG T-3' for MMP-9 [Gene bank::
NM_004994], 5'-CCT TGC TAT CCG ACA CCT TT-3' and
5'-CAC CAC TTC TAT TCC CAT TCG-3' for heparanase
[GenBank: NM_006665], 5'-TGC ACA GGA GCC AAG
AGT GAA-3' and 5'-CAC ATC ACA GCT CCC CAC CA-3'

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for TBP [GeneBank: NM_003194]. Specificity of the pro-
duced amplification product was confirmed by examina-
tion of dissociation reaction plots. A distinct single peak
indicated that a single DNA sequence was amplified dur-
ing RT-PCR. Each sample was tested in triplicate and sam-
ples obtained from three independent experiments were
used for analysis of relative gene expression.
Western blot analysis
Cell cultures were washed twice with ice-cold PBS, and
scraped in lysis buffer (50 mM Tris pH 7.4, 250 mM NaCl,
5 mM EDTA, 1% NP40, 0.1% SDS, 0.5% sodium deoxy-
cholate, 1 mM phenylmethylsulphonyl fluoride) contain-
ing 1% protease inhibitor cocktail (Calbiochem). Lysates
were obtained by centrifugation at 13,000 rpm for 10 min
at 4°C and protein concentration was determined using
the BCA protein assay kit (Pierce, Rockford, IL). Twenty to
forty μg of total protein were subjected to SDS-PAGE,
transferred to polyvinylidene fluoride (PVDF) membrane,
and probed with various primary antibodies, followed by
HRP-conjugated secondary antibodies. Specific proteins
were detected by enhanced chemiluminescence (Pierce).
The experiments were repeated in triplicate. β-actin was
used as the internal protein control.
Matrigel invasion assay
Invasion of cells through matrix membrane was deter-
mined using 24-well BD invasion chambers (8.0-μm pore
size with polycarbonate membrane; BD Biosciences, Cow-
ley, United Kingdom) in accordance with the manufac-
turer's instructions with the following modifications. The
cells were detached by using 2 mM EDTA and 5 × 104 cells
were placed into the upper compartment of the invasion
plates in duplicate in a 0.5 ml serum-free volume. Subse-
quently, the lower compartment was filled with 750 μl of
10%FBS medium. After 22 h of incubation at 37°C with
5% CO2, cells remaining on the upper membrane surface
were removed with a cotton swab. Cells on the lower sur-
face of the membrane were stained with CellTracker
Green (Molecular Probes, Eugene, OR) and fixed in meth-
anol. The invasive cells on the lower surface of membrane
were counted under the fluorescence microscope at × 4
magnification. Each experiment was repeated three times.
Zymographic analysis of matrix metalloproteinase
Total cell proteins were prepared in the same way as for
Western blot analysis. Twenty micrograms of each lysate
or 15 μl of cell culture supernatant were mixed with non-
reducing electrophoresis loading buffer and subjected to
electrophoresis on an 8% SDS-PAGE copolymerized with
gelatin (1 mg/ml). After electrophoresis, gels were washed
with 2.5% Triton X-100 for 1 h (3 times, 20 min each) and
incubated for 24 h in enzyme assay buffer (25 mM Tris,
pH 7.5, 5 mM CaCl2, 0.9% NaCl, and 0.05% Na3N) for
the development of enzyme activity bands. After incuba-
tion, the gels were stained with 0.25% coomassie brilliant
blue R-250 and destained in 10% methanol with 5% ace-
tic acid. The gelatinolytic activities were detected as trans-
parent bands against the blue background of the
coomassie brilliant blue-stained gelatin. The experiment
was repeated three times. Human MMP-2 and MMP-9
gelatinase zymography standard (Chemicon) were used
as markers.
Chemotactic Migration Assay
A Chemicon QCM™ 96-well migration assay kit was used
to study cell chemotactic migration. Cells were detached
by 2 mM EDTA/PBS and resuspended with serum-free
medium. Approximately 5 × 104 cells were seeded in the
upper chamber of the plate. Medium with 10%FBS or 100
ng/ml SDF-1 was added to the lower chamber as a chem-
oattractant, and serum-free medium was also used as con-
trol. After 6 h incubation, cells migrated through the 8 μm
pore size membrane, were detached, and treated with lysis
buffer/dye solution supplied by the kit. Aliquot mixes
were read using fluorescence reader with 485/525 nm fil-
ter set. Cell numbers were correlated with the optical den-
sity values and expressed as Relative Fluorescence Unit
(RFU). The experiments were repeated three times with at
least four duplicate of each treatment.
Cell proliferation assays
MTT assay was used to determine the cell proliferation.
U251n-pc and U251n-hpa cells were initially grown to
confluence before seeding in a 96-well plate at the density
of 4 × 103 cells/well. Viable cells were determined by add-
ing 0.5 mg/ml MTT into each well and incubated for addi-
tional 2 h. The cells were then solubilized in 200 μl
detergent (50% dimethyl formamide and 10%SDS). The
absorption was determined at the wavelength of 540 nm.
Observed optical density is directly correlated with the cell
numbers. The experiments were repeated three times with
duplicates.
Colony formation in soft agar
Three ml of DMEM containing 0.5% Low Melt agarose
(Bio-Rad, Hercules, CA) and 10% FCS was poured into a
6-well plate. The layer was covered with cell suspension (1
× 104 cells) in 1.5 ml of DMEM containing 0.3% Low Melt
agarose and 10% FCS, and the dish was covered with 2 ml
of DMEM containing 10% FCS. Cells were seeded in trip-
licate and medium was changed every 3 days. After 3
weeks, colonies were visualized by MTT staining (0.5 mg/
ml) for 4 h and counted under a microscope. The experi-
ment was repeated three times.
Statistics
Data are presented as mean ± SD. Statistical significance
was analyzed by one-way ANOVA. The value of P < 0.05
was considered statistically significant.

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Results
Heparanase increases U251n cell invasion
To understand the role of heparanase on the regulation of
cell function, the full length human heparanase gene was
stably transfected into U251n cells. Western blot analysis
using cell lysates from heparanase-overexpressing U251n-
hpa cells showed a great increase of protein expression of
both latent (65 kDa) and active (50 kDa) forms of hepara-
nase. Heparanase expression was confirmed by analyzing
U87 and HF2303 cells (Figure 1A). U251n-hpa cells
exhibited a 100 fold increase of mRNA expression (Figure
1B) and a 12-fold increase of heparanase activity in cell
lysates compared to U251n-pc cells (Figure 1C). Hepara-
nase cleaves HS residues, participates in ECM degrada-
tion, and thereby may facilitate tumor cell invasion [4].
Using the BD matrigel invasion assay, we evaluated the
invasion ability of U251n cells. Matrigel, a reconstituted
basement membrane containing HSPG, was used as a rel-
evant barrier. As expected, the heparanase-overexpressing
U251n-hpa cells exhibited a 10-fold increase in cell inva-
sion as compared to control U251n-pc cells, and the
increased cell invasion was significantly blocked by the
heparanase inhibitor heparin at a concentration of 40 μg/
ml (P < 0.01) (Figure 2A). In order to distinguish the role
of heparanase on cell invasion, MMP-2 and MMP-9,
members of the MMP family that is also involved in gli-
oma invasion [24], was measured using zymography
assay and real-time PCR in both heparanase-overexpress-
ing and control cells. No significant changes were
observed in both enzyme activity and mRNA expression
levels between U251n-pc and U251n-hpa cells (Figure 2B
and 2C).
Overexpression of heparanase in U251n cells
Figure 1
Overexpression of heparanase in U251n cells. (A) Heparanase expression of U251n, U87 and primary cultured glioma
cells (HF2303) were detected by Western blot. Both U251n and U87 exhibited baseline level of heparanase expression. Latent
form of heparanase was detected in HF2303 cells. Increased heparanase expression, both the latent (65 kDa) and active (50
kDa) forms, was observed in cell lysate derived from U251n-hpa cells, as compared to U251n-pc control cells. (B) U251n-hpa
cells expressed about 100 times more mRNA than U251n-pc cells. (C) Increased heparanase activity was detected in U251n-
hpa cells.

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Heparanase enhances U251n cell chemotactic migration
Apart from the traditional function of heparanase on cell
invasion, cell migration towards chemoattractants was
detected. Our results showed that migration of U251n-
hpa cells was significantly increased towards DMEM
medium supplied with 5% and 10% FBS as compared to
U251n-pc cells (P < 0.01), while cell migration was signif-
icantly blocked by 40 μg/ml heparin (Figure 3A) (P <
0.01). These results indicate that increased migration is
heparanase-induced cellular function change which is
independent of ECM degradation. Since SDF-1 and its
receptor CXCR4 (CXC chemokine receptor) system was
reported to be correlated with cancer cell migration [25-
27], SDF-1 was also tested as a chemoattractant. More
cells were migrated when SDF-1 was added into the 10%
FBS medium than 10% FBS medium alone (P < 0.01), and
total number of migrating cells of U251n-hpa group was
significantly higher than that of U251n-pc group (P <
0.01). The number of migrating cells was not increased
when SDF-1 was added into 0% FBS medium (Figure 3B),
indicating that SDF-1 induced migration needs the activa-
tion of other cellular signals.
Heparanase overexpression increases U251n cell growth
To test the possibility of heparanase on cell growth, cell
proliferation and anchorage-independent growth was
therefore evaluated. Growth curve was measured during 4
days in culture. Initially 4 × 103 cells were plated in each
well of a 96-well plate, and cell numbers were determined
daily by MTT assay. Four days after seeding, the cell
number of the U251-hpa group was significantly higher
than that of the U251n-pc group (P < 0.01) (Figure 4A).
Furthermore, the anchorage-independent cell growth was
determined by colony formation in soft agar. U251n-hpa
Heparanase overexpression increases cell invasion
Figure 2
Heparanase overexpression increases cell invasion. (A) Cell invasion of U251n-hpa was significantly increased as com-
pared to U251n-pc cells, measured by BD invasion assay. Increased cell invasion was greatly inhibited by 40 μg/ml heparin. **, P
< 0.01, compared with U251n-pc cells. #, P < 0.01, compared with U251n-hpa group of no heparin treatment. (B) mRNA levels
of MMP-2 and MMP-9 showed no significant difference between U251n-pc and U251n-hpa cells. (C) No significant differences
were detected in MMP-2 and MMP-9 activity between U251n-hpa cells and U251n-pc cells as measured by zymography assay.