ICANCER RESEARCH56. 651-657. February 1. 9961
Association of Elevated Levels of Hyaluronidase, a Matrix-degrading Enzyme, with
Prostate Cancer Progression'
Vinata
B. Lokeshwar,2
Bal L. Lokeshwar,
Henri
T. Pham,
and Norman
L. Block
Department of Urology. University of Miami School of Medicine (V. B. L, B. L L, H. T. P., N. L B.), and GRECC', VA Medical Center (B. L Li, Miami, Florida 33101
lumen formation (3). For example, ECM components may sequester
potent angiogenic factors, such as basic fibroblast growth factor,
Components
ofextracellular
matrix and the matrix-degrading
enzymes
which upon release stimulate endothelial cell proliferation and migra
are some of the key regulators of tumor metastasis and angiogenesis.
tion (6, 7). Interestingly, ECM components may also play a more
Hyaluronic acid (HA), a matrix glycosaminoglycan, is known to promote
active role in the process of angiogenesis. For example, HA and more
tumor cell adhesion and migration, and its small fragments are angio
specifically HA fragments are known to be angiogenic (8).
genie. We have compared
levels of hyaluronidase,
an enzyme
that de
HA is a nonsulfated glycosaminoglycan made of repeating disac
grades HA, in normal adult prostate (NAP), benign prostate hyperplasia
(BPH)and prostatecancer(CaP)tissuesand in conditionedmediafrom charide units, D-glucuronic acid, and N-acetyl-D-glucosamine (9). HA
epithelial explant cultures, using a sensitive substrate(HA)-gel assay and is present in body fluids, extracellular matrix, and connective tissues.
an ELISA-like assay. The results show a significant elevation (3—10-fold) It plays an important role in several physiological functions, such as
of this enzyme in tumor tissues compared to that in NAP and BPH tissues.
maintaining cartilage integrity, homeostasis of water and plasma
Furthermore,
the hyaluronidase
levels in tissues correlate well with the
tumor grade. For example, the concentrations in a locally extended CaP proteins in the intercellular matrix, and cell migration and prolifera
lesion (191 ±7.9 milliunits/mg protein) are the highest, followed by tion (9). HA also plays a role in several pathophysiological conditions.
For example, in several tumors (e.g., lung, hepatic, breast, invasive
high-grade tumors (36.6 ±2.9 milliunits/mg protein), and low-grade
tumors (9.4 ±1.4 milliunits/mg protein), respectively. Among the primary
bladder tumor, and others), HA levels are elevated @“9—18-fold,
epithelial explant cultures, CaP cultures secrete at least 10-fold higher suggesting a role for HA in tumor progression and metastasis (10, 11).
levels of hyaluronidase than those secreted by NAP and BPH cultures. The increased amounts of HA in tumor tissues is synthesized by the
Furthermore, among the established prostate cancer cell lines, DU145, an stromal fibroblasts in response to specific signals from the tumor
androgen-unresponsive
metastatic
line, secretes
4-fold more hyaluroni
epithelial cells (10, 12). In tumor tissues, HA expands upon hydration
dase than LNCaP, an androgen-responsive and relatively well-differenti
and opens up spaces for cell migration. Furthermore, HA promotes
ated cell line. We also show that prostatic hyaluronidase has an apparent
tumor cell migration by binding to specific cell surface receptors (e.g.,
Mr @55,000,a pH optimum of 4.6, and is distinct from other well
CD44; Ref. 13). In addition, HA forms a protective halo around the
characterized
hyaluronidases.
tumor cells against immune surveillance (14). More interestingly
however, small fragments of HA (@3—25disaccharide units) have
INTRODUCTION
been shown to promote angiogenesis in vivo (8, 15). We have shown
Tumor metastasis is the correlate of the malignant potential of
recently that HA fragments of 10—15disaccharide units promote
neoplastic growth and dominates the causes of cancer-related deaths.
endothelial cell proliferation (16). Furthermore, HA fragments of
The mechanism(s) that controls the metastatic progression of a local
similar lengths also promote endothelial cell migration and tubule
ized tumor is very complex and involves many biochemical events
formation (17). Thus, a regulated degradation of HA in tumor tissues
(1). Some of the key events in the metastatic process include inter
may be important for both tumor metastasis and angiogenesis.
actions of tumor cells with the host cells (e.g., stromal fibroblasts or
Hyaluromdases are a family of enzymes that degrade HA (18). In
capillary endothelial cells) and various ECM3 components (2, 3). For
vertebrates, hyaluronidases can be categorized into two classes, those
example, in the initial steps of tumor invasion, tumor cells secrete
active at neutral pH (pH optimum, 5.0) and those active at acidic pH
certain ECM-degrading enzymes that degrade the basement mem
(pH optimum, 3.5—4.0;Ref. 19). For example, testicular hyaluroni
brane. This is followed by the tumor-vascular endothelium interac
dase is of the neutral type, whereas the liver enzyme has an acidic pH
tions. These steps allow the tumor cells to enter into the circulation.
optimum (20). Recently, the cloning and sequencing of porcine liver
Following arrest in the target organs, the tumor cells “re-invade―
and
hyaluronidase has revealed that it is identical to hemopexin, a heme
colonize the new tissue by again interacting with the endothelium and
binding j3-glycoprotein that is abundantly present in the serum (21).
stroma (4).
However, the majority of the hyaluronidase activity present in serum
The second key process critical for tumor progression is angiogen
appears to be distinct from hemopexin (21). The concerted actions of
esis. It is well known that the nurturing of tumor mass both at primary
both HA and hyaluronidases are known to play important roles during
and secondary metastatic sites is dependent on neovascularization (5).
embryonic development, vasculogenesis, vascular remodeling, and
Recent evidence suggests that ECM components regulate such key
immune surveillance (22). Although to date the presence of hyalu
steps in angiogenesis as endothelial cell proliferation, migration, and
ronidase in tumor tissues has not been established, it has been shown
that hyaluronidase levels are elevated in the urine of Wilms' tumor
Received 8/21/95; accepted 11/20/95.
(nephroblastoma) patients (23). Therefore, it is possible that hyalu
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
ronida.se levels may be elevated in tumor tissues, and the amount
18 U.S.C. Section 1734 solely to indicate this fact.
secreted may correlate with tumor progression. In this work, we
I This work
was supported
in part by the Weeks
Endowment
Fund,
Department
of
demonstrate that hyaluronidase levels are, in fact, significantly dc
Urology, American Cancer Society Institutional Grant (to V.B.L.), and NIH Grant
CA61038-1R29 (to B.L.L.).
vated in prostate tumor tissues as compared to NAP and BPH tissues.
2 To whom
requests
for reprints
should
be addressed,
at Department
of Urology
Furthermore, primary epithelial cultures of prostate tumor tissue cx
(M-800). University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101.
Phone: (305) 243-6321; Fax: (305) 243-6893.
plant but not of NAP and BPH specimens secrete high levels of
3 The abbreviations
used are: ECM,
extracellular
matrix;
HA, hyaluronic
acid; NAP,
hyaluronidase. Conversely, the amount of hyaluronidase present in
normal adult prostate; BPH, benign prostate hyperplasia; CaP, prostate cancer; CM,
conditioned media.
tumor tissues or secreted by tumor cells in culture appears to correlate
651
ABSTRACT
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
HYALURONIDASELEVELS IN PROSTATE CANCER
with the tumor grade, suggesting that hyaluronidase may play a
pivotal role in the metastatic progression of prostate cancer.
incubation, the degraded HA was washed off, and the HA remaining on the
wells was determined using biotinylated cartilage HA-binding protein, an
avidin-biotin
detection
lin-6-sulfonic
(27). The absorbance
MATERIALS AND METHODS
Tissue
Specimens.
Normal
prostate
assay,
tissues
from adults (21—50 years) were
obtained from organ donors. Tissue procurement from donors was performed
according
to state and federal
regulations.
Neoplastic
tissues
were obtained
from patients (41—72
years) undergoing open prostatectomy for BPH or CaP.
the maximum
and ABTS
(2,2'
azino-bis-3-ethyl-benzthiazo
CA) as described before
was read at 405 nm in a microtiter
absorbance
plate reader.
(Amax)@5 nm was obtained
In each
by incubating
the
HA-coated wells with buffer alone in the absence of any hyaluronidase.
(A,,,,,)@5nmwas obtained by incubation of hyaluronidase in uncoated wells. A
standard graph was prepared by plotting absorbance (405 nm) s'ersus units/mi
of Streptomyces hyaluronidase. Using this standard graph, the hyaluronidase
concentration
A small portion (@ I g) was harvested from the gross surgical specimen. This
tissue specimen was split, and the mirror image segment was fixed in formalin,
(units/ml)
in each dilution
of a tissue extract
or culture
CM was
embedded in paraffin, and sectioned; then the tumor grades of these sectioned
calculated. From several such determinations, the mean hyaluronidase concen
(ration in each sample was determined. The hyaluronidase concentrations were
specimens were analyzed histologically
normalized to the protein content and expressed as milliunits/mg
(24). In this study, we have included
data only from those specimens (or explant cultures) that were confirmed to be
either benign or malignant. Localized tumor and locally extended and meta
static lesions are defined as follows: localized
@
system
acid) substrate (Vector, Burlingame,
prostate;
local extension,
tumor
spread
tumor, tumor confined
to adjacent
organ
(e.g.,
seminal
to
vesi
des); and metastatic lesion, tumor spread to noncontiguous structure (e.g.,
lymph node).
Tissue Extracts.
Fresh or frozen tissue specimens (@0.5—lg) were sus
pended in an ice-cold homogenization buffer [5 mtviHEPES (pH 7.2) and 1 msi
benzamidineHCll
and homogenized
for 30 s using a hand-held
tissue homog
enizer. The tissue extracts were clarified by centrifugation at 40,000 X g for 30
mm. The extracts were assayed for protein concentration (Bio-Rad protein
assay reagent; Bio-Rad Labs, Richmond, CA) and hyaluronidase
Immunoprecipitation
of Hyaluronidase
Activity
by Anti-Hemopexin
Antiserum. ThecultureCMof DU145 was incubatedin PBS containing0.1%
BSA with 1:50 and 1:20diluted goat antihuman hemopexin antiserum (kindly
provided by Dr. Ann Smith, University of Missouri, Kansas City, KS) at 4°C
for 6 h, followed by incubation for 90 mm with mouse antigoat IgG agarose
beads. Following incubation, the beads were centrifuged to remove the
immune-complex,
activity.
protein.
To determine the pH activity profile of prostatic hyaluronidase, the HA
coated wells were incubated with aliquots of tissue extracts or culture CM in
formate-NaC1 buffer at various pH (2.0—7.0).The results are expressed as
(A —
nmX 100.The maximum difference is designated as 100%,
and the data are expressed as a percentage of maximum.
and the supernatants
were tested
for hyaluronidase
activity
was collected at 2—4passages, 3 days after subculturing. The culture CM were
by the ELISA-like assay. Various controls used included incubation of DU145
culture CM in the absence of any antiserum or normal goat serum or antigoat
lgG agarose beads alone. In addition, the goat antihuman hemopexin antiserum
was incubated in PBS containing 0.1% BSA. All of the control samples were
also tested simultaneously using the ELISA-like assay.
Immunoblot Analysis. DU145 cultureCM (@5 @xg
protein)and purified
hemopexin (100 ng) were electrophoresed on a 12% SDS-polyacrylamide gel
and blotted on to a polyvinylidene difluoride membrane. Following blocking,
the nonspecific sites on the membrane by incubation in a 3% BSA solution
prepared in the blotting buffer (20 mM TrisHC1, 150 mM NaCI, and 0.05%
concentrated
Tween
Primary Prostate Cultures. Primary prostate cultures were set up as
described
previously
(25). Briefly,
tissue specimens
(@500
mg) were minced
into small pieces (@10 mm3), rinsed several times in PBS, and digested with
collagenase. The digested pieces were washed extensively in PBS and seeded
in T-25 Falcon
Primaria
flasks. Tissue
fragments
were cultured
in a serum-free
epithelial growth medium that supports the growth of prostatic epithelium
(MEOM, Clonetics Corp., San Diego, CA). Medium covering the explant was
renewed
after 24 h. The epithelial
nature of the cell monolayer
was confirmed
by cytokeratin staining as described previously (25). CM from primary cultures
10-fold
and assayed
for protein
concentration
and hyaluronidase
20),
the blot
was
incubated
sequentially
with
goat
antihuman
he
mopexin antiserum (I :500 dilution) at room temperature for 2 h and alkaline
Tissue Culture. EstablishedCaPlines LNCaPandDU145 wereculturedin phosphatase-conjugated mouse antigoat IgG. The blot was developed using
nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate
substrate solu
RPMI 1640 containing 10% fetal bovine serum and gentamicin. At @“60%
confluence, the cultures were washed extensively in PBS and incubated in tions (Bio-Rad Labs, Richmond, CA).
activity.
serum-free
RPM!
1640 containing
insulin,
transferrin,
and selenium
(GIBCO
BRL, Gaithesburg, MD). The serum-free CM were collected after 2—3
days.
RESULTS
The cell viability was >90% at the time of CM collection. The culture CM
were concentrated 10-fold and assayed for protein concentration and hyalu
ronidase activity.
Substrate(HA)-Gel
Assay.
The substrate(HA)-gel
assay for hyaluronidase
activity was performed as described by Gutenhoner el a!. (26). Briefly, tissue
extracts
(10 i.@gprotein)
and culture
CM (@5 xg protein)
were electrophoresed
on a 7.5% polyacrylamide gel under native conditions or on a 12% SDS
polyacrylamide
gel under
denaturing
conditions.
During
polymerization
of
these gels, human umbilical cord HA (0.17 mg/mI; Sigma Chemical Co., St.
Louis, MO) was included as a substrate for hyaluronidase digestion. Following
electrophoresis,
the gels were incubated in formate-NaC1
buffer (0. 1 M sodium
formate and 0.15 M sodium chloride, pH 4.6) at 37°C for 16—20 h to allow the
digestion of HA in the gel. When SDS-PAGE was used, the gel was initially
washed with a 3% solution of Triton X-l00 at room temperature for I h prior
to incubation in formate-NaCI buffer. Following enzymatic digestion, the gels
were stained sequentially with 0.5% Alcian blue and 0.15% Coomassie blue
solutions and destained with 10% methanol and 10% acetic acid solution.
ELISA-like Assay for Hyaluronidase. The quantitativeestimationof hy
aluronidase in culture CM and tissue extracts was performed by using an
ELISA-like assay essentially as described by Stem and Stem (27). Briefly,
96-well microtiter wells (Corning, Corning, NY) were coated with human
umbilical cord HA (200 p@g/ml)dissolved in 0.1 M sodium bicarbonate solu
tion,
pH 9.2. The
HA-coated
wells
were
washed
three
times
in PBS
and
incubated with serial dilutions of either tissue extracts or culture CM or
purified Streptomyceshyaluronidase (Calbiochem, San Diego, CA) in formate
NaC1 buffer containing 0.2 mg/ml BSA at 37°Cfor 16—18h. Following
Detection of Hyaluronidase
Activity in Prostate Tissue Ex
tracts. To detect the presence of hyaluronidase activity in various
prostate tissue extracts prepared from NAP, BPH, and CaP, we used
a substrate(HA)-gel assay technique (26). As shown in Fig. 1, Lanes
1 and 2, a band representing HA digestion is observed in both of the
CaP tissue extract lanes, suggesting the presence of hyaluromdase in
these samples. However, no substrate digestion is observed in BPH
(Fig. 1, Lanes 3 and 4) and NAP (Fig. 1, Lanes 5 and 6) tissue extract
lanes, suggesting that very low hyaluronidase activity may be present
in these specimens. Interestingly, between the two CaP tissue extract
samples, the extract prepared from a locally extended CaP lesion (e.g.,
CaP metastasized to seminal vesicles; Fig. I, Lane 1 ) appears to
contain higher hyaluronidase activity than that present in a high-grade
(Gleason sum 7/10; TNM stage, T1NØM0)but localized tumor spec
imen (Fig. 1, Lane 2).
To accurately quantitate the levels of hyaluronidase activity present
in various tissue extracts, we used an ELISA-like assay and used the
commercially available Srreptomvces hyaluronidase as a standard. As
shown in Fig. 2A, the hyaluronidase activity present in NAP (n
5)
and BPH (n = 5) tissue extracts is very similar. For example, NAP
and BPH tissue extracts contain 2.5 ±0.52 milliunits/mg protein and
3.4 ±0.42 milliunits/mg protein of hyaluronidase activity, respec
tively. The tissue extracts prepared from low-grade prostate tumors
652
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
HYALURONIDASELEVELS IN PROSTATE CANCER
that a tiny focus of tumor could exist in a benign sample; however, the
estimated low hyaluronidase levels in BPH samples suggest that such
a possibility is either low or noncontnbutory to the resultant data. The
differences between hyaluronidase levels present in various tissue
extracts (NAP, BPH, low-grade CaP, and high-grade CaP) are statis
tically significant (P < 0.0001 ; ANOVA test). For a paired compar
ison between any two categories, we performed the Tukey-Kramer
multiple comparison test. As shown in Table 1, this test predicted that
the differences observed in the mean hyaluronidase activity present in
NAP and BPH tissue extracts are not statistically significant at the
95% confidence limit (P > 0.05). But such differences in hyaluron
idase activity between NAPIBPH tissue extracts and low-grade CaP
extracts are statistically significant (P < 0.05; Table 1). In addition,
the observed differences in the enzyme levels among NAPIBPHIIow
grade CaP and high-grade CaP are also statistically significant
(P < 0.001). These results suggest that the elevated levels of hyalu
ronidase activity in prostate tumor tissues may be indicative of a more
aggressive disease. We next analyzed hyaluronidase activity present
in extracts prepared from the normal seminal vesicles and a locally
extended CaP lesion (e.g., seminal vesicles). As shown in Fig. 2B, the
locally extended lesion contains °°°75-fold
(191 ±7.9 milliunits/mg
protein), higher levels of hyaluronidase activity than that present in
the normal seminal vesicles (2.6 ±0.3 milliunits/mg protein). It is
interesting to note that the amount of enzyme activity present in the
locally extended lesion is °‘°S-fold
higher than that present in the
Fig. 1. Detection of hyaluronidase activity in prostate tissue extracts by substrate
localized high-grade CaP tissues (36.6 ±2.9 milliunits/mg protein).
(HA)-gel assay. Extracts were prepared from NAP. BPH, and CaP tissues. The extracts
To corroborate this finding, we examined hyaluronidase levels in the
(@ 10 p@gprotein) were electrophoresed on a 7.5% HA-substrate gel. The gel was
extracts of another metastatic lesion (a regional lymph node colonized
incubated in buffer to allow HA digestion and stained and destained as described in
“Materialsand Methods.―Lane 1, a locally extended CaP lesion from seminal vesicles;
completely with CaP). The results indicate that the extract of this
Lane 2, high-grade CaP; Lanes 3 and 4. BPH; Lanes 5 and 6. NAP.
metastatic lesion also contains 2.5-fold (86.6 ±7.8 milliunits/mg
protein) higher hyaluronidase levels compared to those in localized
high-grade tumor.
(n
5; Gleason sum 2—6/10;TNM stage, T1_2NØMØ)contain “@‘3123456
p
fold higher levels of hyaluronidase (9.4 ±1.4 milliunits/mg protein)
as compared to those in NAP and BPH tissues. The high-grade CaP
(n
5; Gleason sum 7—10/10;TNM stage: T2_3N0_1MØ_1,with and
without local and distant spread) tissue extracts contain very signifi
cantly
elevated
levels
of hyaluronidase
(36.6
± 2.9 milliunits/mg)
protein. Due to the multifocal nature of prostate cancer, it is possible
Examination of Hyaluronidase Activity in Prostate Tissue Ex
plant Cultures. In this set of experiments, we have tested the pres
ence of hyaluronidase activity in culture CM from the epithelial
outgrowth of prostate tissue explants. As shown in Fig. 3, the sub
strate(HA))-gel assay reveals that CM from CaP tissue explant cul
tures contain significant hyaluronidase activity (Fig. 3, Lanes 1 and
>@
40
.r-I
>
@
Fig. 2. Quantitative determination of hyaluron
idase activity in various prostate tissue extracts by
an ELISA-like assay. Various concentrations of
tissue extracts were incubated with HA-coated
microtiter wells at 37°Cfor 16 h in formate@NaCl
buffer (pH 4.6). Following incubation, wells were
washed to remove the degraded HA. The HA
remaining on the wells was determined as de
scribed in “Materialsand Methods.―The units of
hyaluronidase in each sample were calculated
from a standard curve. The results are represented
as milliunits/mg protein of hyaluronidase activity
as the means from five samples in each category;
bars, SEM. Inset. hyaluronidase activity in the
extracts of normal seminal vesicle and a locally
extended CaP lesion in seminal vesicle.
j—.'
‘r-4 C
_1_J ‘i-I
00)
30
<-I-)
0
11)
‘r-I
0@
E
20
C
OD
LE
:@
‘I
10
>@
:i:
NAP
BPH
Low grade
CaP
High grade
CaP
653
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HYALURONIDASELEVELS IN PROSTATE CANCER
Table I Tukey-Kramer multiple comparison test of hyaluronidase levels in NAP, BPH,
extractsThe
and CaP tissue
data presented in Fig. 2 were analyzed statistically using the Tukey-Kramer
multiple0.05.Tissue
comparison test. The test dictates that if q > 3.997, then P <
PNormal comparison
Mean difference
>0.05Normal
vs. BPH
—0.9875
q
0.05935
<0.05Normal
vs. low-grade
-6.937
4. 170
>0.001BPH
vs. high-grade
—
34. 138
20.5 18
>0.05BPH
vs. low-grade
—5.950
3.576
<0.001Law-grade
vs. high-grade
—33.150
19.924
—27.200
16.348
vs. high-grade
<0.001
2). However, BPH (Fig. 3, Lanes 3 and 4) and NAP (Fig. 3, Lanes 5
and 6) explant cultures secrete very little hyaluronidase.
Quantitation of the amount of hyaluronidase secreted in the CM of
various prostate epithelial explant cultures was performed using the
ELISA-like assay. As shown in Fig. 4A, the epithelial explant cultures
of NAP and BPH tissues secrete small amounts of hyaluronidase in
their culture CM. For example, the hyaluronidase levels secreted in
NAP and BPH explant culture CM (n = 5/category) are 2.3 ±0.4
milliunits/mg
protein
and 1. 1 ± 0.42 milliunits/mg
protein,
DU145 cell line, primary prostate tumor epithelial culture, and pros
tate tumor tissue extracts have a distinct pH optimum °°°4.6
for HA
degradation. The pH optimum for prostatic hyaluronidase is thus
different from those reported for hyaluronidases from other sources
such as serum, liver, kidney, and testis (20, 21, 23, 30, 31).
To test the possibility that prostatic hyaluronidase may be related to
the liver enzyme that is identical to hemopexin (21), we examined
whether the hyaluronidase activity present in culture CM of DUI45
cells can be immunoprecipitated using an antihuman hemopexin an
tiserum. As shown in Fig. 7A, the antihuman hemopexin antiserum
fails to remove the hyaluronidase activity from the DU145 culture CM
by immunoprecipitation. This suggests that the prostatic hyaluroni
dase is probably distinct from hemopexin. To further corroborate this
result, we performed an immunoblot analysis using the antihuman
hemopexin antiserum. As shown in Fig. 7B, Lane 1, the antiserum
does not cross-react with any protein secreted in DU145 culture CM
but reacts with purified human hemopexin of Mr @70,000(Fig. 7B,
Lane 2), suggesting that prostatic hyaluronidase is distinct from
hemopexin.
DISCUSSION
respec
tively. Furthermore, as observed in the CaP tissue extracts, the epi
thelial explant cultures (n = 7) set up from low-grade tumor tissues
secrete hyaluronidase activity (8.1 ±1.2 milliunits/mg protein) only
3.5-fold higher than the NAP and BPH explant cultures (Fig. 4A).
However, the primary epithelial cultures (n
6) derived from high
grade CaP tumors secrete significantly high levels (47.1 ±7.5 mu
units/mg protein) of hyaluronidase (Fig. 4A). The ANOVA test re
vealed that the differences between various categories (e.g., NAP,
BPH, low-grade CaP, and high-grade CaP) are significant
(P < 0.0001). Furthermore, as shown in Table 2, the Tukey-Kramer
multiple comparison test indicates that the differences observed in the
hyaluronidase secretion by NAPIBPH and high-grade CaP are statis
tically significant (P < 0.001). Thus, it is possible that CaP tumor
cells with more aggressive potential secrete more hyaluronidase than
those with less aggressive or benign potential. This is further con
firmed by examining the hyaluronidase levels secreted by established
prostate cancer cell lines LNCaP and DU145 and comparing them
with those secreted by an epithelial explant culture established from a
locally extended CaP lesion (e.g., seminal vesicles). Although both
LNCaP and DU145 cell lines were derived from metastatic lesions,
the former is a relatively well-differentiated, androgen-responsive cell
line compared to the latter, which is highly metastatic in xenograft
models and unresponsive to androgens (28, 29). In addition, the
LNCaP cell line, unlike DU145, secretes prostate-specific antigen and
contains functional androgen receptors, representing a less advanced
CaP line as compared to DU145 (28, 29). As shown in Fig. 4B,
LNCaP cells secrete 72 ±5.6 milliunits/mg protein hyaluronidase
activity in the CM. Conversely, DU145 cells secrete hyaluronidase
levels (@233 ±3.4 milliunits/mg protein) 4-fold higher than those
secreted by LNCaP cells. Interestingly, the primary epithelial explant
culture set up from a metastatic CaP lesion secretes hyaluronidase
activity (136 ±2.1 millliunits/mg protein) higher than LNCaP but
lower than DU145.
Characterization
of Hyaluronidase
Activity. A substrate(HA)
SDS-PAGE was used to determine the relative molecular mass of
prostatic hyaluronidase (26). As shown in Fig. 5, the hyaluronidase in
a CaP tissue extract (Fig. 5, Lane 1 ) and the serum-free culture CM
of primary tumor epithelial cells (Fig. 5, Lane 2) and DU145 (Fig. 5,
Lane 3) has an apparent Mr of “@‘55,000.
We next determined the pH activity profile of the prostatic hyalu
ronidase. As shown in Fig. 6, the hyaluronidase secreted by the
In the present study, we examined whether the hyaluronidase levels
present in prostate tissues can be correlated with the aggressiveness of
the prostate cancer. Hyaluronidases are a class of enzymes that de
grade HA, a free nonsulfated glycosaminoglycan (18). The HA con
centration in serum has been an indicator of several disorders, includ
ing rheumatoid arthritis, liver dysfunction, psoriasis, and scleroderma
(9). Similarly, HA concentrations are elevated in several tumor tissues
and in the serum of patients with cancers such as the Wilms' tumor
(23). The presence of HA in tumor tissues appears to play a role in
tumor metastasis and angiogenesis (32, 33). Conversely, it is impli
123456
Fig. 3. Examination of hyaluronidase activity in the prostate tissue explant culture CM.
Prostate tissue aliquots were grown in serum-free MEGM that supports the outgrowth of
epithelial cells. Serum-free culture CM (5
@gprotein) were electrophoresed
on a 7.5%
HA-substrate gel. The gel was processed as described in “Materials
and Methods.―
Lanes
I and 2, CaP; Lanes 3 and 4, BPH; Lanes 5 and 6, NAP.
654
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HYALURONIDASELEVELS IN PROSTATE CANCER
>@
6
>
.1-I
@
Fig. 4. Determination of hyaluronidase levels
in the culture CM of various primary prostate
epithelial cultures. Serum-free CM were collected
5@
C
.
0
-i-'
@
from the primary epithelial cultures set up from
0
@
NAP
L
@
low-grade and n = 6 for high-grade) tissues. Inset,
hyaluronidase activity present in established CaP
cell lines LNCaP and DU145 and a locally cxtended CaP lesion (e.g., seminal vesicle). The
hyaluronidase activity in the culture CM was determined by the ELISA-like assay as described in
“Materialsand Methods.―The results are means
expressed as milliunits/mg protein of culture CM;
bars, SEM.
@
(n = 5),BPH
(n
5),and CaP (n = 7 for
0..
-@
,
0')
C
E
o
L
j
E
,__@
(0
>@
I
4
3@
2@
10
NAP
Table 2 Turkey-Kramer cultureCMfrom
multiple comparison test of hyaluronidase
explantsThe
NAP, BPH, and CaP
levels in the
Tukey-Kramermultiple
data presented in Fig. 4 were analyzed statistically using the
comparison
0.05.MeanCM
test. The test dictates that if q > 3.977 then P <
PNormal comparison
difference
>0.05Normal
vs. BPH
>0.05Normal
vs. low-grade
<0.001BPH
vs. high-grade
0.2780
—5.793
1.525
—44.783
11.364
—7.018
1.640
<0.001Low
vs. high-grade
—46.008
10.442
—38.990
10.267
or metastatic
High grade
CaP
levels of this enzyme (Fig. 4). Thus, the increased secretion of
hyaluronidase may be a direct correlate of the aggressive potential of
the tumor cells.
Since hyaluronidase levels correlate with tumor grade, it would be
kD
123
200
121
80
<0.001
cated that hyaluronidase may also play a role in tumor progression.
However, to date, a direct correlation between the hyaluronidase
levels and tumor progression is lacking.
The data presented here indicate that there exists a correlation
between tumor grade and tissue hyaluronidase levels. For example,
high-grade prostate tumor contains significantly high (lO-fold)
1ev
els of hyaluronidase as compared to those found in NAP and BPH
tissues (Figs. 1 and 2). The levels are also elevated, although only
moderately (“3-fold),in low-grade CaP tissues, which further sup
ports the thesis that hyaluronidase secretion may be related to the
tumor grade. The reason why the low-grade CaP (Gleason sum
2—6/10)show only a moderate increase in hyaluronidase levels could
be that the elevated hyaluronidase secretion is a distinct attribute of
highly aggressive prostate tumor cells. Furthermore, it is known that
the percentage of highly aggressive and metastatic cells present in a
tumor correlates with the tumor volume as well as the disease pro
gression. This is further supported by our findings that hyaluronidase
levels, at least in locally extended
Low grade
CaP
q
1.225
>0.05BPH
vs. low-grade
grade vs. high-grade
BPH
0
50
33
29
19
8
CaP lesions (n = 1/per
category), contain 2.5—5-fold higher levels of hyaluronidase than
those in the high-grade primary tumors (Fig. 2). The correlation
between hyaluronidase secretion and the tumor grade is strengthened
by our observations that the explant cultures of epithelial cells from
CaP tumor tissues also display the same attribute. For example, the
low-grade CaP cultures secrete only moderately (3-fold) higher levels
of hyaluronidase than NAP and BPH cultures. But the high-grade
tumor explants, either primary or metastatic, secrete
10-fold higher
Fig. 5. Molecular weight determination of prostatic hyaluronidase by SDS substrate
(HA)-gel assay. A CaP tissue extract and serum-free culture CM of primary high-grade
CaP and DU145 cells were electrophoresed on a 12% SDS HA-substrate gel. Following
electrophoresis, the gel was processed as described in “Materialsand Methods.―Lane I,
Cap tissue extract; Lanes 2 and 3, culture CM. Lane 2, primary high-grade CaP; Lane 3,
DU145. Numbers on the right, molecular weights of known protein standards (Bio-Rad
prestained broad range markers); k D, kilodaltons.
655
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
HYALURONIDASELEVELS IN PROSTATE CANCER
100
E
80
3
Fig. 6. Determination of the pH activity profile
of prostatic hyaluronidase. CaP tissue extract, CaP
culture CM, and DUI45 culture CM were incu
bated on the HA-coated wells at different pH.
Following incubation, HA remaining on the wells
was estimated as described in “Materialsand
Methods.―The results are calculated as described
in “Materialsand Methods.―
E
.x
(0
60
4-
0
“@@0
0@―@
40
20
0
2
3
4
5
6
7
pH
interesting to investigate whether there is a correlation between hya
luronidase levels in CaP and patient survival. In our study, all samples
were collected between 1993 and early 1995. All patients with local
ized prostate tumor are alive, but CaP is a slow disease to progress.
The patients with locally extended or metastatic CaP lesions, which
also showed the highest levels of hyaluronidase among all the samples
that were examined, have progressed further in the disease and present
with a grim prognosis. Currently, we are investigating a correlation
between hyaluronidase activity and patient survival using archival
prostate tissue specimens.
Currently, our understanding of hyaluronidase is based upon the
testicular and hepatic forms of this enzyme. The liver is a major site
for HA clearance (4 g/day) from the blood circulation (9, 34). It has
been shown that in the liver, the sinusoidal endothelial cells internal
ize and degrade HA via the lysosomal pathway. Therefore, liver
hyaluronidase is most likely synthesized by the sinusoidal endothelial
cells and is of lysosomal origin (32). Since, the prostatic hyaluroni
dase is synthesized and secreted by the epithelial cells, it is not
surprising that this enzyme is different from the liver hyaluronidase
that has been demonstrated to be hemopexin (2 1). Furthermore, since
the interstitial environment in malignant tumors is known to be acidic,
the secreted enzyme can degrade HA present in the tumor matrix (35).
Hyaluronidase has been used clinically to augment the efficacy of
several chemotherapeutic drugs such as mitomycin C, cisplatin, yin
desine, and doxorubicin in the treatment of bladder cancer, head and
neck tumors, and others (13, 36, 37). This is because the increased
Fig. 7. Immunoprecipitation and immunoblot
analysis of DU145 culture CM using goat anti
human hemopexin antiserum. Immunoprecipita
tion analysis: the DU145 culture CM was incu
bated alone or immunoprecipitated
using
antihuman hemopexin antiserum as described in
“Materialsand Methods―. After removing the
immune-complex, the supematants were tested
for hyaluronidase activity. The results are pre
sented as absothance@5 nm versus protein con
centration in culture CM; bars, SEM. 0, no
E
C
In
0
0)
U
C
(0
..0
treatment; & immunoprecipitation using 1:50 di
L
lution of the antiserum; U, immunoprecipitation
using 1:20 dilution of the antiserum. inset, im
munoblot analysis: DU145 culture CM (@5 @xg
protein) and purified hemopexin were electro
phoresed on a 12% SDS-polyacrylamide
gel,
blotted, and probed with goat antihuman he
mopexin antiserum as described in “Materials
and Methods.―Lane I, DU145 culture CM he
mopexin; Lane 2. purified hemopexin.
0
U)
-Q
DU145CM (@..tg
protein/well)
656
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research.
HYALURONIDASELEVELS IN PROSTATE CANCER
I
@
I
I
levels of HA in tumors probably form a halo around the tumor cells,
preventing the penetration of the chemotherapeutic agents (14). How
ever, the secretion of elevated levels of hyaluronidase by tumor cells
not
paradoxical
levels
present
protein)
are
with
these
in the tumor
l03-106-fold
less
observations.
tissues
than
Because
(e.g.,
those
36.6
the
14. Hobarth, K., Maier, U., and Marberger, M. Topical chemoprophylaxis
hyaluronidase
± 2.9 miuliunits/mg
administered
to
the
patients
(e.g., intravesical administration) of 200,000 IU in bladder (14),
peritumoral injection into the breast tissue (36), and 200,000 lU/kg in
I
@
thetreatment
of headandnecktumors(37).Thus,thetumorcells
levels of hyaluronidase
in tumor tissues
are very likely the
ACKNOWLEDGMENTS
We thank Dr. Ann Smith (University of Missouri, Kansas City, KS) for her
gift of goat antihuman
hemopexin
antiserum
and purified
J. Whelan (ed), The Biology of Hyaluronan, pp. 41—59.New York: Wiley, Clii
J.,205: 69—74,1982.
indicators of a malignant and aggressive disease.
generous
15. West, D. C., and Kumar, S. The effect of hyaluronate and its oligosaccharides on
endothelial cell proliferation and monolayer integrity. Exp. Cell Res., 183: 179—196,
1989.
16. Lokeshwar, V. B., and Bourguignon, L. Y. W. Hyaluronic acid receptor GP1 16 is a
new CD44 variant in endothelial cells. Mo1. Cell Biol., 4: 1030, 1993.
17. Banarjee, S., and Toole, B. P. Hyaluronan binding protein in endothelial cell mor
phogenesis. J. Cell Biol., 119: 643—652, 1992.
18. Fraser, J. R. E., and Laurent, T. C. Turnover and metabolism of hyaluronan. in:
chester (Ciba Foundation Symposium), 1989.
19. Roden, L., Campbell, P., Fraser, J. R. E., Laurent, 1. C., Pertoff, H., and Thompson,
J. N. Enzymatic pathways of hyaluronan catabolism. in: J. Whelan (ed), The Biology
of Hyaluronan, pp. 60—86.New York: Wiley, Chichester (Ciba Foundation Sympo
sium), 1989.
20. Gold, E. W. Purification and properties of hyaluronidase from human liver. Biochem.
probably secrete adequately elevated levels of hyaluronidase to allow
a controlled degradation of HA that would promote angiogenesis and
the invasive phenotype of tumor cells. A direct proof of this hypoth
esis would be to clone prostatic hyaluronidase, overexpress in less
aggressive, well-differentiated prostate tumor cell lines (e.g., LNCaP),
and test the invasive and angiogenic potential of these transfected
lines. Currently, we are purifying and cloning prostatic hyaluronidase
such that these experiments can be performed. Nevertheless, the
increased
of superficial
bladder cancer by mitomycin C and adjuvant hyaluronidase. Eur. Urol., 21: 206—210,
1992.
hemopexin.
21. Zhu, L., Hope, T. J., Hall, J., Davies, A., Stem, M., Muller- Eberhard, U., Stem, R.,
and Parslow, 1. G. Molecular cloning of a mammalian hyaluronidase revels identity
with hemopexin, a serum heme-binding protein. J. Biol. Chem., 269: 32092—32097,
1994.
22. Toole, B. P. Glycosaminoglycans in morphogenesis. in: E. D. Hay (ed), Cell Biology
of the Extracellular Matrix, pp. 1384—1386.New York: Plenum Publishing Corp.,
1991.
23. Stem, M., Longkar, M. T., Adzick, N. S., Harrison, H. R., and Stem, R. Hyaluron
idase levels in urine from Wilms' tumor patients. J. NatI. Cancer Inst., 83: 1569—
1574, 1991.
24. Gleason, D. F. Classification of prostate carcinomas. Cancer Chemother. Rep., 50
(Part I): 125—130,1966.
25. Lokeshwar, B. L., Selzer, M. G., Block, N. L, and Smith, Z. G. Secretion of matrix
metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by
human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase
secretion by malignant tissues. Cancer Res., 53: 4493—4498, 1993.
26. Gutenhoner, N. W., Pogrel, M. A., and Stem, R. A. Substrate gel assay for hyalu
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Association of Elevated Levels of Hyaluronidase, a
Matrix-degrading Enzyme, with Prostate Cancer Progression
Vinata B. Lokeshwar, Bal L. Lokeshwar, Henri T. Pham, et al.
Cancer Res 1996;56:651-657.
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