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Experimental Therapeutics |
Departments of Cell and Developmental Biology [D. L. L., R. K. B., S. M., A. U.], Surgery and Internal Medicine [K. J. P.], and Human Genetics [D. L. A., K. K.], University of Michigan Medical School, Ann Arbor, Michigan 48109-0616
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1 integrin. PHSCN, a competitive
inhibitor, blocked both PHSRN- and serum-induced invasion. Acetylated,
amidated PHSCN (Ac-PHSCN-NH2) was 30-fold more potent;
however, Ac-HSPNC-NH2 was inactive. Rats receiving
injections s.c. with 100,000 MLL cells were treated systemically by
i.v. injection three times weekly with 1 mg of either
Ac-PHSCN-NH2 or Ac-HSPNC-NH2 beginning 24 h later, three times weekly with 1 mg of Ac-PHSCN-NH2
beginning only after surgery to remove large (2 cm) MLL tumors, or were
left untreated. MLL tumors grew rapidly in
Ac-HSPNC-NH2-treated and in untreated rats. MLL tumor
growth in rats treated with Ac-PHSCN-NH2 beginning 1 day
after MLL cell injection was reduced by 99.9% during the first 16 days
of treatment, although subsequent tumor growth occurred. MLL tumor
cryosections immunostained with anti-PECAM-1 showed that
Ac-PHSCN-NH2 inhibited neovascularization by 12-fold during
this time. Whether initiated after MLL cell injection or only after MLL
tumor removal, Ac-PHSCN-NH2 treatment reduced the numbers
of MLL lung colonies and micrometastases by 40- to >100-fold, whereas
Ac-HSPNC-NH2 was inactive. Thus, Ac-PHSCN-NH2
may be a potent antitumorigenic and antimetastatic agent for
postsurgical use prior to extensive metastasis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of
prostate cancers are not organ confined at the time of diagnosis
(2)
. Metastatic disease is treated by androgen ablation
therapy to induce temporary remission. However, relapse after a median
of 18 months is inevitable because of the development of
androgen-independent cancer (3
, 4)
. Because androgen
withdrawal is currently the only effective form of systemic therapy for
metastatic prostate cancer, the appearance of androgen-independent
clones of tumor cells means that there is no effective curative therapy
for patients thereafter (5
, 6)
. Thus, new therapies to
treat invasive or metastatic prostate cancer are necessary.
To develop leads for new antitumorigenic and antimetastatic therapies,
Copenhagen rats engrafted with metastatic MLL cells, a well-tested
animal model of androgen-resistant prostate cancer, have been
successfully used (7
, 8)
. The Dunning (R3327) rat prostate
adenocarcinoma model of prostate cancer was developed from a
spontaneous adenocarcinoma of a male rat (9)
. The MLL
subline is a fast-growing, poorly differentiated metastatic cell line
that, upon injection of a million cells into the thigh of the rat,
leads to animal death within 25 days because of overwhelming primary
tumor burden (10)
; or, if the primary tumor is removed,
results in animals that die of lung metastases 40 days after tumor
inoculation (11)
.
We analyzed the invasion-inducing role of a specific sequence of pFn,3 a soluble extracellular matrix protein present in concentrations of 0.3–0.5 mg/ml in plasma, lymph, and interstitial fluid (reviewed in Ref. 12 ), as well as in serum at reduced concentrations (13) , by using naturally SF basement membranes (SU-ECM) as in vitro invasion substrates (14) . The rationale for using naturally SF basement membranes is that they permit a precise definition of the soluble components of invasion assays. We present results showing that DU 145 cells (15) are induced to invade SU-ECM by pFn, and that this activity maps to the PHSRN sequence of the cell-binding domain. Similar concentrations of the PHSRN peptide stimulate SF DU 145 and MLL cells, as well as normal human prostate epithelial cells, to invade SU-ECM in vitro. Substitution of cysteine forms an efficient, competitive invasion inhibitor, the PHSCN peptide. Ac-PHSCN-NH2 exhibits a substantially increased ability to block serum-induced invasion by DU 145 and MLL cells, but a scrambled peptide (Ac-HSPNC-NH2) is inactive. The Ac-PHSCN-NH2 peptide is a potent antitumorigenic and antimetastatic agent in rats, when treatment begins 1 day after MLL cell injection, whereas Ac-HSPNC-NH2 is ineffective. The antimetastatic activity of Ac-PHSCN-NH2 is attributable to the direct inhibition of metastatic invasion in rats because the initiation of i.v. Ac-PHSCN-NH2 treatment only after the removal of large primary MLL tumors is also very effective in reducing the number of MLL lung metastases. This result, as well as the topology of the SU-ECM invasion substrates used to define its activity, suggests that Ac-PHSCN-NH2 can function as a potent inhibitor of basement membrane invasion in vivo, especially during extravasation by metastatic cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peptide Synthesis.
Peptides were synthesized using standard Fmoc/t-butyl protection
strategies (21)
. Peptides were synthesized at 25 and 100
µmol scales on a Ranin Symphony multiple peptide synthesizer. Larger
scales were synthesized on a Perkin-Elmer/Applied Biosystems model 433
peptide synthesizer or a Ranin Sonata large-scale synthesizer. Peptides
were synthesized on a chlorotrityl resin, and COOH-terminally amidated
peptides were synthesized on Rink resin. The completed peptides were
cleaved from the resin support, and the side chain protecting groups
were simultaneously removed by anhydrous TFA. The peptides were then
precipitated with diethylether, purified by preparative
high-performance liquid chromatography, and then freeze-dried. To
prevent injury to the rats from TFA, a strong acid, residual TFA was
removed by gel permeation chromatography on a Sephadex G-10 column
equilibrated with 1 N acetic acid. Peptide structures were
confirmed by mass spectrometry and amino acid analysis
(21)
. Peptide purities, assessed by reverse phase
high-performance liquid chromatography (22)
, were found to
be 93% for PHSRN, 97% for PHSCN, 96% for
Ac-PHSCN-NH2, and 97% for
Ac-HSPNC-NH2 (not shown).
Zymography and Checkerboard Analysis of Chemotaxis.
Adherent DU 145 cells in SF medium were treated with 1.4
µM PHSRN or with 1.4 µM PHSRN and 14
µM PHSCN, and their media were assayed three times by
gelatin zymography (23)
. The effect of PHSRN on chemotaxis
and chemokinesis was evaluated in standard motility assays by
checkerboard analysis (24)
. Upper and lower chambers were
separated by inserts with 8-µm pores (Becton Dickinson Collaborative
Biomedical Research, Bedford, MA) and coated with sterile 0.2%
gelatin. DU 145 cells were preincubated in SF medium with 1.4
µM PHSRN or with 0.2% BSA for 1 h prior to
suspension. Each assay contained the following combinations: 0.2% BSA
in both upper and lower chambers; 1.4 µM PHSRN in the
upper chamber with 0.2% BSA in the lower chamber (chemokinesis
control); and 0.2% BSA in the upper chamber with 1.4 µM
PHSRN in the lower chamber (to assay chemotaxis). Assays also contained
a well with 0.2% BSA in the upper chamber and 5% FCS in the lower
chamber as a positive control. Cells migrated for 12 h before
microscopic scoring of at least 10 randomly chosen fields per assay.
Analysis of MLL Tumor Vascularization and Metastasis in Rats
Whose Ac-PHSCN-NH2 Treatment Began 1 Day after MLL Cell
Injection.
MLL cells (100,000) were suspended and injected s.c. into the right
hind legs of 19 Copenhagen rats according to standard procedures
(7)
. Twenty-four h later, 10 of the rats received their
first Ac-PHSCN-NH2 i.v. injection in a tail vein,
whereas the other 9 rats received no i.v. treatment. Injections, at a
dosage of 5 mg/kg, consisted of 1 mg of
Ac-PHSCN-NH2 peptide in 0.5 ml of normal saline
and occurred three times weekly thereafter. As an in vivo
sequence specificity control, four other rats were treated systemically
with Ac-HSPNC-NH2 at the same dosage and on the
same dosage schedule. Three additional rats received no MLL tumor cells
but received injections of the Ac-PHSCN-NH2
peptide at the same dosage and on the same dose schedule as the
treated, MLL tumor-bearing rats. After 16 days of growth and five
systemic peptide treatments, the largest MLL tumor available from the
Ac-PHSCN-NH2-treated rats (1-mm diameter) was
harvested for the immunohistochemical determination of vascularization
and 5ß1 expression, and the rat was euthanized. Normal muscle from
the leg was included as a positive control for vasculature. To provide
a comparable source of metastatic cells, MLL tumors were not removed
from the Ac-PHSCN-NH2-treated rats if they were
smaller than about 2 cm. MLL tumors of untreated rats reached a mean
diameter of 1.8 cm by days 16–18 and were removed surgically with the
hind limb (7)
. The primary MLL tumors in the
Ac-HSPNC-NH2-treated rats reached a mean diameter
of 2.1 cm on day 20 and were surgically removed at this time. Unlike
the primary tumors of the Ac-HSPNC-NH2-treated
and untreated rats, the MLL tumors of all
Ac-PHSCN-NH2-treated rats grew very slowly but
did reach a mean diameter of 1.7 cm 29 days after MLL tumor cell
injection and were removed surgically. All rats were euthanized for
metastasis analysis in their lung tissue 14 days after the surgical
removal of their primary tumors. The lungs of 9
Ac-PHSCN-NH2-treated, 4
Ac-HSPNC-NH2-treated, and 9 untreated MLL
tumor-bearing rats were fixed in 10% formaldehyde in PBS. After
fixation, lung surfaces were scored under 10-fold magnification for the
numbers of lung metastases. At this time, the three
Ac-PHSCN-NH2-treated rats without MLL cells were
euthanized, and their tissues were evaluated grossly and
microscopically for deleterious effects of
Ac-PHSCN-NH2 treatment by veterinary personnel in
the University of Michigan Department of Lab Animal Medicine.
In addition to the 1-mm tumor from the
Ac-PHSCN-NH2-treated rat, a typical 2-cm MLL
tumor from an untreated rat was harvested after 16 days of growth
and analyzed immunohistochemically using the mouse antirat PECAM-1
monoclonal MAB1393 antibody (Refs. 25
and 26
;
Chemicon, Temecula, CA). Immunohistochemistry was also performed with
the anti-5ß1 integrin rabbit AB1928 antiserum raised against a
peptide containing the highly conserved, final 18 amino acids of
the human 5 subunit (27)
in the cytoplasmic domain
(Chemicon). This antiserum has been shown to bind the 5ß1
fibronectin receptors of both rat and human fibroblasts in immunoblots
(27)
and has been used to immunoprecipitate rat 5ß1
receptors from pheochromocytoma PC12 cells (28)
. Tumors
frozen with liquid nitrogen in Tissue-Tek OCT Compound (VWR, Chicago,
IL; Ref. 29
) were cut with a cryostat in sets of 7-µm
serial sections (30)
. One slide of each set was stained
with Gill’s hematoxylin No. 3 and alcoholic eosin (Fisher Scientific,
Pittsburgh, PA) (31)
. Adjacent sections were either fixed
in acetone at -20°C for 30 s prior to incubation with mouse
anti-rat PECAM-1 monoclonal antibody (Chemicon) at 1:200 in 0.5
M Tris (pH 7.6) with 2% normal horse serum overnight at
4°C, followed by incubation with biotinylated horse antimouse
anti-IgG secondary antibody (rat preabsorbed; Vector Laboratories,
Burlingame, CA), diluted 1:200 in Tris with normal horse serum for 45
min, and quenched in
methanol-H2O2; or they were
fixed in methanol at 4°C for 20 min prior to incubation in rabbit
anti-5ß1 AB1928 antiserum (Chemicon) in 0.5 M Tris
with 2% normal goat serum overnight at 4°C, followed by incubation
with biotinylated goat antirabbit IgG (Vector) diluted 1:200 in Tris
with normal goat serum for 45 min and quenching in
methanol-H2O2. All slides
were incubated in avidin-biotin complex (Vectastain ABC kit) for 1 h, then with DAB with nickel (Vector) for 12–15 min. All incubations
were at room temperature unless otherwise noted. All slides were then
stained in Gill’s H&E (Fisher) and DAB substrate kit (Vector) or with
rabbit anti-5ß1 antiserum (Chemicon), biotinylated goat antirabbit
IgG secondary antibody (Vector), and DAB substrate kit (Vector).
Macros were developed for the quantitation of the total area occupied by tumor vasculature using the KS300 image analysis package (Carl Zeiss, Thornwood, NY). Macros of several images from sections of MLL tumors, which had been immunostained with antirat PECAM monoclonal antibody, were digitized and placed on a threshhold. Images were chosen to represent all areas of the tumors in the sections. A number of binary operations were performed on the resultant images to differentiate DAB-stained vessels and their lumens from the surrounding tumor cells. The total area occupied by vasculature was then determined as a fraction of the total field area.
After at least 3 days of fixation, complete lower, left lobes from the
lungs of each of six Ac-PHSCN-NH2 and four
Ac-HSPNC-NH2-treated rats and from six untreated
rats were embedded in paraffin and cut in the plane of the maximal area
of the lobe into eight sections of 5-µm thickness (31)
.
To sample an extensive volume of lung tissue, the eight sections were
each separated by 13 sections, or 65 µm of lung tissue. Thus, a
region 0.5 mm thick of the complete lower lobe from each rat was
analyzed for micrometastasis. All eight sections from each individual
were stained in H&E and scored at 400-fold magnification in their
entirety for MLL micrometastases, using phase contrast optics. To be
scored as a separate micrometastasis, the MLL tumor cells had to have
completed extravasation and had to have been located as a cluster in
the lung tissue instead of in the lumen of a blood vessel. They also
had to have been surrounded by normal lung tissue, instead of by tumor
cells, in the section.
Analysis of MLL Lung Metastasis in Rats Whose
Ac-PHSCN-NH2 Treatment Began 1 Day after the Surgical
Removal of Large MLL Tumors.
MLL (100,000) cells were suspended and injected s.c. into the right
hind legs of 20 Copenhagen rats according to standard protocols
(7)
. MLL tumors grew to a mean diameter of 1.9 cm in the
rats prior to their surgical removal 20 days later. These rats received
no presurgical Ac-PHSCN-NH2 injections.
Twenty-four h after surgery to remove their MLL tumors, 10 of the
previously untreated rats received their first tail vein injections of
1 mg of Ac-PHSCN-NH2 in 0.5 ml of normal saline.
The rats received a total of six i.v.
Ac-PHSCN-NH2 treatments during the next 14 days.
The remaining 10 rats remained untreated. All rats were euthanized for
metastasis analysis in their lung tissue 14 days after surgery. The
lungs of all 20 rats were fixed in 10% formaldehyde in PBS. After
fixation, lung surfaces were scored under 10-fold magnification for
lung metastases. After at least 3 days of fixation, the complete lower
left lobe of lung tissue from each individual was embedded, sectioned,
stained, and scored as described above for MLL micrometastasis analysis
when Ac-PHSCN-NH2 treatment began 24 h after
MLL cell injection.
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RESULTS |
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shows the mean percentages of invaded DU 145 cells in SF,
FCS, or Fn-FCS media. FCS failed to stimulate
invasion by normal prec cells; however, DU 145 cells rapidly invaded in
the presence of FCS. In contrast, DU 145 cells failed to invade SU-ECM
under SF conditions, even if incubated for up to 24 h before
fixation and scoring (not shown). This suggested that a specific
component of serum induced their invasive behavior. DU 145 cells are
known to express 5ß1 (33)
. Because pFn binding by
5ß1 can induce fibroblast MMP-1 gene expression
in the absence of binding by the 4ß1 fibronectin receptor
(34)
, and metalloproteinases are thought to play a central
role in migration through extracellular matrix, the effect of pFn on DU
145 invasion was tested. As shown in Fig. 1A,
the use of
Fn-FCS completely eliminated DU 145 invasion.
Invasiveness could be fully restored by adding purified pFn to the
Fn-FCS in amounts characteristic of whole serum
(35)
or of serum-containing medium. Thus, pFn was
necessary for DU 145 invasion induction. Furthermore, pFn was also
sufficient, because the addition of identical amounts of purified pFn
in SF medium supported the full extent of DU 145 invasive behavior, as
shown in Fig. 1B
.
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the Mr 120,000 fragment containing
modules 2–11 was sufficient to induce DU 145 invasion in molar
concentrations similar to those of pFn in serum or in serum-containing
medium. Also, the invasive responses of DU 145 cells to varying
concentrations of the Mr 120,000
fragment in serum-free SU-ECM invasion assays closely resembled those
of prec cells (data not shown). This is not surprising because, like
other epithelial cell types and fibroblasts, prec cells express the
5ß1 integrin fibronectin receptor (36)
. At similar
molar concentrations, the Mr 39,000
fragment, containing modules 7–9, was equally active in inducing
invasion, whereas the Mr 11,500
fragment containing module 10 with the RGD sequence and the pure GRGDSP
peptide were inactive at the concentrations tested. Thus, it appeared
that the constitutive invasiveness of DU 145 cells might be
attributable to stimulation by a specific sequence encompassed by the
Mr 39,000 fragment.
In addition to the RGD site, the 5ß1 integrin fibronectin receptor
binds the PHSRN sequence of module 9 (37)
and is known to
be expressed by DU 145 and prec cells (33
, 36)
through
immunostaining with the anti-5ß1 P1D6 function-blocking monoclonal
antibody (Ref. 20
and data not shown). Thus, the ability
of the Mr 39,000 fragment to induce SF
DU 145 invasion suggested that the 5ß1 receptor might be involved.
To test whether invasion induction through 5ß1 occurred during
substrate adhesion or at a prior step, DU 145 cells were bound to the
P1D6 antibody either prior to invasion stimulation by FCS or
concurrently. Although the presence of FCS was varied during antibody
binding, substrate adhesion and invasion always occurred in its
presence. When prebound under SF conditions, the P1D6 antibody reduced
DU 145 invasion by 45% at concentrations as low as 10 µg/ml and
eliminated it completely at 300 µg/ml, whereas P1D6 bound in the
presence of FCS failed to reduce DU 145 invasion at concentrations as
high as 300 µg/ml. Similar results were also obtained for prec cells
induced to invade SU-ECM with the Mr
120,000 pFn fragment instead of serum (data not shown). Thus, invasion
induction by pFn appeared to occur through the 5ß1 receptor prior
to substrate adhesion and to involve the PHSRN sequence of module 9.
These results were consistent with the observation that the
PHSRN-containing synergy region of the ninth type III repeat of pFn
blocks the binding of the P1D6 antibody to 5ß1 (38)
and suggested that the PHSRN sequence might be sufficient to induce SF
DU 145 invasion.
Induction of Invasive Behavior by the PHSRN Peptide.
The ability of the PHSRN peptide to stimulate SU-ECM invasion by SF DU
145 cells or by prec cells was evaluated. PHSRN peptide was present in
the media of SU-ECM invasion assays at concentrations ranging from 14
nM to 1.4 µM (10–1000 ng/ml). The RGD
peptide, GRGDSP, was also tested for its invasion-inducing activity at
a concentration of 1.4 µM. As shown in Fig. 2A,
PHSRN effectively stimulated invasion by DU 145 cells in a
log linear fashion, whereas the RGD peptide was inactive at the highest
peptide concentration tested. As expected from their expression of the
5ß1 receptor (36)
and the invasive responses of other
normal epithelial cells to the PHSRN
peptide,4
very similar dose-response relationships were obtained for prec
cells in SF and FCS media, as shown in Fig. 2B
. Thus, the
PHSRN peptide was as effective at stimulating invasion by prec cells as
it was in stimulating DU 145 invasion in the absence of serum,
suggesting that the dysregulation of the invasive response to the PHSRN
sequence of intact pFn might contribute to the constitutive
invasiveness of these 5ß1-expressing invasive tumor cells.
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5 chain, which
forms a pocket that may contain a divalent cation (38)
.
The arginine of this sequence appears to be crucial for its
invasion-inducing activity in normal epithelial cells and fibroblasts
because replacing it with alanine or glutamic acid eliminates its
activity, even at elevated concentrations.4
Consistent with the importance of the PHSRN arginine in stimulating
basement membrane invasion, observations published previously have
shown that, although the PHSRN sequence in the context of either the
9th or the 8th fibronectin type III repeat could stimulate adherent BHK
cells to de-adhere from pFn substrates, substitution of the arginine
with an amino acid having a neutral or negatively charged side chain
resulted in complete or near-complete loss in activity
(39)
. Replacing the arginine with a cysteine, which has a
large, electron-rich sulfhydryl group on its side chain, formed a
potent competitive inhibitor of invasion induction by the PHSRN
sequence for normal human epithelial cells and
fibroblasts.4
The sulfhydryl group of the
cysteine side chain is known to complex efficiently with divalent
manganese and magnesium cations, either of which might occupy the
PHSRN-binding pocket of the 5 chain, as well as with transition
metal ions (reviewed in Refs. 40
and 41
). It
was hypothesized that the shorter side chain of cysteine and its
ability to coordinate divalent cations might allow it to interact
tightly in the binding pocket without inducing basement membrane
invasion.
The PHSCN peptide was assayed for its ability to inhibit PHSRN-induced
invasion by SF DU 145 cells and by prec cells. A nonsaturating
concentration of the PHSRN peptide (140 nM) was used to
stimulate invasion in the presence of 0–1.4 µM PHSCN
peptide. As shown in Fig. 3
, invasion by DU 145 cells and by prec cells was decreased by slightly
>50% in the presence of equimolar PHSRN and PHSCN. A 10-fold molar
excess of the PHSCN peptide prevented invasion for both cell types.
Although the other four amino acids were identical in the
invasion-inducing and invasion-inhibiting peptides, they might still be
very important for the productive association of the peptide with its
binding pocket on the 5 chain. Furthermore, this association might
even be sufficient for adhesive or migratory effects in the absence of
the arginine side chain. However, these results indicate the importance
of the arginine side chain, specifically for the induction of basement
membrane invasion in normal, as well as in invasive, derivatives of
prec cells.
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PHSCN at 1.4 µM (1 µg/ml)
inhibited the induction of DU 145 invasion by serum. To determine
whether PHSCN functioned as a competitive or allosteric inhibitor of
the PHSRN sequence, DU 145 cells were chilled and prebound to a fully
inhibitory concentration of the PHSCN peptide (1.4
µM) in the presence of serum, prior to being
rinsed thoroughly and placed on SU-ECM in the presence of serum and
increasing amounts of the PHSRN peptide. PHSCN-prebound DU 145 cells
failed to invade, as shown in Fig. 4A
. As shown in Fig. 4B,
increasing concentrations of the PHSRN peptide overcame
PHSCN invasion inhibition very effectively, which would not have been
expected had the PHSCN and PHSRN peptides interacted with distinct
sites in the 5ß1 receptor. Thus, these two peptides appeared to
bind with similar affinities to the same receptor site.
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and a representative zymogram is shown
in Fig. 4D
. When compared with untreated cells,
PHSRN-treated DU 145 cells were found to secrete a mean of
225 ± 10% more Mr
70,000 MMP-1. Also, PHSCN treatment of PHSRN-stimulated DU 145 cells
was found to reduce MMP-1 secretion by a mean of 50 ± 32% with respect to untreated cells. Although secreted in larger
amounts, similar levels of Mr 92,000
MMP-2 were expressed by DU 145 cells, irrespective of the peptides
added. Thus, although the expression, localization, and function of
metalloproteinases in DU 145 invasion is likely to be very complex, the
effects of the PHSRN and PHSCN peptides on MMP-1 secretion were
consistent with their effects on SU-ECM invasion.
Because the PHSRN peptide induced DU 145 invasion of SU-ECM basement
membranes, it was likely that it would also function as a
chemoattractant in gelatin-coated migration chambers (24)
.
This was tested by prebinding DU 145 cells to 0.2% BSA or to 1.4
µM PHSRN peptide in SF medium and placing them in
standard checkerboard assays in gelatin-coated migration chambers
containing no PHSRN peptide or containing 1.4 µM PHSRN in
either the top or the bottom chambers. As shown in Fig. 4E,
the presence of 1.4 µM PHSRN in the lower
chambers of 10 Boyden chamber assays stimulated the chemotactic
migration of BSA-pretreated or PHSRN-pretreated DU 145 cells by means
of >30-fold as compared with the presence of 0.2% BSA in the lower
chambers. Because at least 3 SDs separate the means, these results are
significant to >99% confidence. In contrast, the amount of
chemokinesis was negligible, as shown by the observation that the
presence of 1.4 µM PHSRN in the upper chambers
failed to stimulate migration to the lower chambers by either
PHSRN-prebound or BSA-prebound DU 145 cells. Also, PHSRN pretreatment
alone was shown to be insufficient to stimulate the observed
chemotactic migration because neither PHSRN-prebound nor BSA-
prebound DU 145 cells migrated to the lower chambers when these
chambers contained 0.2% BSA, instead of 1.4 µM
PHSRN. Finally, PHSRN prebinding was not necessary for DU 145
chemotaxis because BSA-prebound DU 145 cells had an equally high
chemotactic response to PHSRN. Thus, the ability of the PHSRN peptide
to stimulate the motility of DU 145 cells, as suggested by the
experiments on SU-ECM invasion substrates, was confirmed in
conventional gelatin-coated chambers.
Inhibition of MATLyLu Metastatic Rat Prostate Carcinoma Cell
Invasion and Metastasis by PHSCN Peptides.
To test whether MLL tumor cells express the 5ß1 fibronectin
receptor and thus might be inhibited by PHSCN peptides, 100,000 MLL
cells were injected s.c. into a rat hindlimb, and the MLL tumor was
grown for 16 days. Cryostat sections of the MLL tumor were
immunostained for 5ß1 using a commercially available rabbit
antiserum raised against a synthetic peptide derived from the highly
conserved last 18 amino acids of the 5 chain. This antiserum has
been demonstrated to react with rat 5ß1 by both immunoblotting and
immunoprecipitation (27
, 28)
. Fig. 5A
shows a section of an untreated MLL tumor that was reacted
to the rabbit anti-5 antiserum, to biotinylated goat anti-rabbit IgG
secondary antibody, and then stained with DAB. Fig. 5B
shows
a section of the same tumor immunostained in parallel without the
primary antibody. Although the amount of anti-5ß1 immunostaining
evident on the surfaces of MLL cells varied slightly because of the
plane of section, all MLL cells appeared to stain with anti-5ß1
and biotinylated secondary antibody, whereas no nonspecific reactivity
was evident. Because the Ac-PHSCN-NH2 peptide is
a competitive ligand for the 5ß1 receptor, it was possible that
systemic administration of it down-regulated 5ß1 expression on the
surfaces of the MLL tumors in the treated rats. However, as shown by
the example in Fig. 5C,
sections of a 1-mm MLL tumor
obtained from a rat treated with five i.v. 1-mg
Ac-PHSCN-NH2 injections over a period of 16 days
appeared to react equally well with the rabbit anti-5ß1 antiserum
as did the MLL tumors from the untreated rats. Thus, exposure to the
Ac-PHSCN-NH2 peptide in vivo did not
appear to affect the cell surface expression of the 5ß1 receptor
on the tumors.
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5ß1 integrin fibronectin receptor by MLL tumors
suggested that MLL cells should be stimulated to invade in serum-free
medium by the PHSRN peptide. To verify that the PHSRN peptide
stimulated MLL invasion in vitro, MLL cells were placed on
SU-ECM invasion substrates in SF medium in the presence of 1.4
µM PHSRN peptide or in its absence, in FCS
medium. The results of these invasion assays are shown in Fig. 5D
. Similar to the human DU 145 cells, rat MLL cells failed
to invade in SF medium but were induced to invade when 1.4
µM PHSRN peptide or FCS was added.
The dose responses of DU 145 and MLL cells to PHSCN invasion inhibitors
in FCS media were also evaluated. Because acetylation and amidation of
the termini of the invasion-stimulating PHSRN peptide significantly
increased its potency both in vitro and in vivo
(4)
, the activities of the PHSCN and the
Ac-PHSCN-NH2 peptides were compared for DU 145
and MLL cells in SU-ECM invasion assays containing FCS. As a sequence
specificity control, the Ac-HSPNC-NH2 scrambled
peptide was also tested. The results of these assays are shown in Fig. 6
. As can be seen in Fig. 6A,
the inhibitory activity of the
PHSCN peptide on DU 145 cells was increased log linearly with
increasing PHSCN concentration from 4.2 nM to 1.4
µM. The presence of 1.4
µM PHSCN peptide completely inhibited
serum-induced DU 145 invasion of SU-ECM.
Ac-PHSCN-NH2 exhibited a 30-fold increased
invasion-inhibitory activity, perhaps because removing charges at the
NH2 and COOH termini allowed the peptide to
assume a more active conformation or increased its resistance to
exoproteinases. In contrast, the Ac-HSPNC-NH2
peptide had no detectable invasion-inhibitory activity on DU 145 cells
in the presence of serum, even when present in concentrations as high
as 14 µM. The histidine residue of the PHSRN
peptide is replaced in rats and mice by proline (reviewed in Ref.
37
). Nevertheless as shown in Fig. 6B,
the
PHSCN and Ac-PHSCN-NH2 peptides also had very
significant invasion-inhibitory activities in rat MLL cells on SU-ECM
invasion substrates in serum-containing medium, suggesting extensive
conservation of function in the other four amino acids of the sequence.
As in DU 145 cells, the Ac-PHSCN-NH2 peptide was
at least 30-fold more active at inhibiting serum-induced invasion than
was the unblocked peptide. Although the relative potencies of PHSCN and
Ac-PHSCN-NH2 were very similar in MLL cells and
in DU 145 cells, both peptides showed slightly more invasion-inhibitory
activity in the human cells, probably because they correspond to the
human sequence. Also, the sequence specificity of invasion inhibition
by the PHSCN sequence in rat cells was demonstrated because MLL cell
invasion failed to be inhibited by concentrations as high as 14
µM of the scrambled
Ac-HSPNC-NH2 peptide.
|
after 16 days of tumor growth and a total of five i.v.
Ac-PHSCN-NH2 injections, the mean diameter of the
tumors in the treated rats was <0.5 mm. In contrast, the mean diameter
of the MLL tumors in the untreated rats was 1.8 cm at this time.
Because spherical volume is proportional to the radius cubed, the mean
volume of the MLL tumors in the treated rats was about 2000-fold less
than the mean volume of the MLL tumors in the untreated rats. This
suggested that systemic Ac-PHSCN-NH2 treatment
could significantly reduce the formation of blood vessels in MLL
tumors. This was verified by sectioning and immunostaining the largest
MLL tumor (1-mm diameter) from an
Ac-PHSCN-NH2-treated rat after 16 days of tumor
growth. A typical 1.8-cm MLL tumor from an untreated rat after 16 days
of growth was also sectioned and immunostained. Cryosections of the
treated and the untreated MLL tumors were immunostained with
rat-specific, anti-PECAM-1 monoclonal antibody (25
, 26)
,
the appropriate biotinylated secondary antibody, and DAB staining
reagents. PECAM-1 is a member of the immunoglobulin superfamily, which
is expressed at high levels on the cell surfaces of endothelial cells,
as well as on platelets, monocytes, neutrophils, and subsets of T
lymphocytes (reviewed in Ref. 42
). Thus, it is a standard
method for evaluating vascularization histochemically. Some of the
normal muscle of the hind limb was retained in the sections of treated
MLL tumor as a positive control for vasculature. The rat that was the
source of the Ac-PHSCN-NH2-treated MLL tumor
sections for anti-PECAM staining was sacrificed instead of being
maintained for later metastasis analysis because of the small size of
the tumor removed. Scoring of 20 randomly chosen fields from each of
four sections of MLL tumors from treated and from untreated rats
suggested that the density of blood vessels in the MLL tumors of
Ac-PHSCN-NH2-treated rats was reduced by 8- to
10-fold relative to that of MLL tumors from untreated rats. Fig. 7B
shows the typical appearance of an anti-PECAM-stained MLL
tumor section from a treated rat. No blood vessels are evident in the
field shown. The typical appearance of H&E-stained MLL cells in an
adjacent section of the treated tumor is shown in the inset
to panel B. Consistent with lower nutrition because of the
paucity of blood vessels, the MLL cells in the sections analyzed
exhibited more heterochromatic nuclei and less extensive cytoplasm than
the cells of untreated MLL tumors, reflecting more limited
transcription and translation. Fig. 7C
shows the typical
appearance of the normal anti-PECAM stained muscle. The muscle shown is
from the same section of tumor tissue that appears in Fig. 7B
. Many anti-PECAM immunostained vessels were evident in
this section and in the other sections analyzed, as would be expected
because leg muscle is a highly vascularized tissue. Fig. 7D
shows the typical appearance of an anti-PECAM immunostained MLL tumor
section from an untreated rat. A significant number of blood vessels
were evident in this and other sections, indicating that the MLL tumors
from untreated rats were highly vascularized after 16 days of growth.
The inset to panel D shows the typical appearance
of H&E-stained MLL cells in an adjacent section of the untreated tumor,
which was immunostained with anti-PECAM antibody. These cells were
photographed at the same magnification, and the same size area was cut
from the image as for the inset in Fig. 7B
.
Hence, the relative differences in the cell sizes apparent in these two
insets reflect those in the MLL tumor sections. Consistent with their
extensive vascularization, the MLL cells of the untreated tumors also
appeared to have large euchromatic nuclei with prominent nucleoli, as
well as much more extensive cytoplasm, suggesting that these cells had
significant transcriptional and translational activities after 16 days
of growth.
|
Although unlikely, it was possible that Ac-PHSCN-NH2 treatment stimulated MLL cell apoptosis, thereby slowing the growth of the primary MLL tumors in the rats. To insure that Ac-PHSCN-NH2 treatment did not affect the MLL cell cycle or stimulate apoptosis, MLL cells were cultured for 72 h (the time required for at least two cell cycles) in serum-containing medium with 0, 1.4, or 14 µM Ac-PHSCN-NH2. After labeling their DNA with propidium iodide, the percentages of the MLL cells in G1, G2, or S phase, as well as the fraction of sub-G1 (apoptotic) cells were determined by flow cytometry (43) . No sub-G1 cells were observed, even at 14 µM, a concentration of Ac-PHSCN-NH2 significantly higher than that to which the MLL tumors were likely exposed in vivo. Also, the percentages of MLL cells in G1, G2, or S phase were unaffected by the presence of Ac-PHSCN-NH2 at 1.4 or 14 µM (not shown). Thus, the slow growth of the MLL tumors in the Ac-PHSCN-NH2-treated rats was probably attributable to the paucity of their vasculature, rather than to direct effects of systemic Ac-PHSCN-NH2 on cell cycle or on apoptosis.
This experiment, in which three times weekly
Ac-PHSCN-NH2 i.v. treatment commenced 1 day after
the injection of MLL cells, was continued to quantitate the numbers of
metastatic MLL colonies in the lungs. To control for possible
nonspecific effects of Ac-PHSCN-NH2 treatment,
four Ac-HSPNC-NH2-treated rats were also
included. These four rats received 100,000 MLL cells s.c. in the right
flank 24 h before the initiation of three times weekly, 1-mg
Ac-HSPNC-NH2 i.v. injections. Primary tumors in
Ac-HSPNC-NH2-treated rats reached a mean diameter
of 2.0 cm prior to surgery after 17 days of growth. Because the
Ac-PHSCN-NH2 dosage was not increased, primary
MLL tumors eventually grew in the
Ac-PHSCN-NH2-treated rats to a mean diameter of
1.7 cm 2 days prior to euthanization for lung metastasis analysis.
Thirty-one days after MLL cell injection or 14 days after surgery, all
rats were sacrificed, and the numbers and sizes of MLL colonies on the
surfaces of their fixed lungs were determined by examination under
10-fold magnification. After scoring surface metastases, an entire lobe
was removed from the fixed lungs for the histological analysis of MLL
micrometastasis. At this time, the
Ac-PHSCN-NH2-treated rats that received no MLL
tumor cells were also sacrificed, and necropsies were performed.
Particular attention was paid to the lung tissue because, having been
introduced in the tail vein, the lungs were the first organ encountered
by the injected peptide. No obvious side effects, including lung
infections, were observed in the rats that received
Ac-PHSCN-NH2 treatment without MLL tumor cells.
Fig. 8
compares the numbers of lung metastases and micrometastases in the
Ac-PHSCN-NH2-treated, the
Ac-HSPNC-NH2-treated, and the untreated rats.
Fig. 8A
compares the mean number of MLL lung metastases per
Ac-PHSCN-NH2-treated rat, as well as the total
number of MLL colonies in all
Ac-PHSCN-NH2-treated rats to the number/rat and
the total number in the untreated group of rats. A total of nine
metastatic colonies were observed in a total of four
Ac-PHSCN-NH2-treated rats. The remaining five
rats in this group had no detectable MLL metastatic colonies in their
lungs when the tissue was examined at 10-fold magnification. Thus, the
mean number of lung metastases/rat in the
Ac-PHSCN-NH2-treated group was 1. In contrast, a
total of 357 metastatic colonies were observed in the group of nine
untreated rats, for a mean of 39.7 lung colonies/individual. The number
of metastases observed per rat varied from 1 to 243. Because it does
not assume a standard distribution, the significance of these results
was evaluated by the nonparametric Mann-Whitney method
(44)
. As shown in Fig. 8A,
P0, the probability that the incidence
of MLL metastasis was the same in the
Ac-PHSCN-NH2-treated and in the untreated groups
is <0.002, implying a significance of >99.8%. Fig. 8B
compares the mean number of MLL lung metastases per
Ac-PHSCN-NH2-treated rat, and the total number of
MLL colonies in the Ac-PHSCN-NH2-treated group to
the number/rat and the total number in the
Ac-HSPNC-NH2-treated group. Although nine
metastatic colonies were observed in the group of nine
Ac-PHSCN-NH2-treated rats, a total of 927
metastases were observed in the group of four
Ac-HSPNC-NH2-treated rats, for a mean number of
231.8 metastases/rat. The number of metastases/rat ranged from 21 to
602 in this group. As indicated, P0 is
<0.005, implying a significance of >99.5%. Fig. 8C
compares MLL lung micrometastasis in the
Ac-PHSCN-NH2-treated and in the untreated rats.
The MLL lung micrometastases observed in eight longitudinal sections of
the complete lower left lobes from six rats treated i.v. with
Ac-PHSCN-NH2 beginning 1 day after MLL cell
injection are shown. Each of these sections was separated from the next
by 65 µm of lung tissue. Thus, a total thickness of about 0.5 mm was
sampled. No micrometastases were detected in the sections from four of
these individuals. Of the other two rats, one had a single
micrometastasis and one had two micrometastases, for a metastasis
frequency of 0.5/rat. In contrast, a total of 789 micrometastases were
observed in eight longitudinal sections of the lower left lobes
obtained from each of the six untreated rats, for a micrometastasis
frequency of 131.5/rat. As indicated,
P0 is <0.005, indicating a
significance of >99.5%. Fig. 8D
compares MLL lung
micrometastasis in the Ac-PHSCN-NH2-treated and
in the Ac-HSPNC-NH2-treated rats. The MLL lung
micrometastases observed in six
Ac-PHSCN-NH2-treated rats are compared with those
observed in four Ac-HSPNC-NH2-treated
individuals. The total MLL lung micrometastases observed in eight
longitudinal sections spanning about 0.5 mm of the complete lower left
lobes from each rat of the two treatment groups are shown. Although a
metastasis frequency of 0.5/rat was observed in the
Ac-PHSCN-NH2-treated group, 107.0/rat were
observed in the Ac-HSPNC-NH2-treated group, for a
total of 428 micrometastases in the four rats. As indicated,
P0 is <0.005, indicating a
significance of >99.5%. Fig. 8E
shows a typical example of
sectioned and stained lung tissue from an
Ac-PHSCN-NH2-treated rat. Normal alveolar lung
tissue and vasculature is seen. Fig. 8F
shows a typical
section of lung tissue from an untreated rat. Three micrometastases are
indicated with arrows, one of which is seen to surround a
blood vessel. Sections of lung tissue from the
Ac-HSPNC-NH2-treated rats had a very similar
appearance (data not shown). Thus, a total of 13
Ac-PHSCN-NH2 injections administered over a
31-day period appeared to prevent the formation of visible MLL colonies
in the lungs of 55% of the treated rats and reduced the total number
of MLL lung metastases in all nine treated rats by >97%, with respect
to the untreated rats, and by >99% with respect to the
Ac-HSPNC-NH2-treated rats. Greater than 100-fold
reductions in the total numbers of micrometastases were observed when
the sections of lung tissue from the
Ac-PHSCN-NH2-treated rats were compared with
those from untreated or from Ac-HSPNC-NH2-treated
animals.
|
As shown in Fig. 9
, i.v. therapy with Ac-PHSCN-NH2 was effective at
reducing MLL metastasis and micrometastasis in rats which grew large
MLL tumors and underwent surgery prior to its initiation. Fig. 9A
compares the frequencies of MLL lung metastasis and the
total numbers of metastases in the
Ac-PHSCN-NH2-treated and in the untreated groups.
A total of eight lung metastases were observed in the 10
Ac-PHSCN-NH2-treated rats, for a metastasis
frequency of 0.8/rat. Forty % of these rats had no detectable surface
metastases on their lungs, and 60% had small numbers of surface
metastases, ranging from 1 to 3. In contrast, all untreated rats had
surface MLL metastases on their lungs, ranging from 4 to 495 per rat. A
total of 1188 metastases were observed on the lungs of the 10 untreated
rats, for a metastasis frequency of 118.8/rat. As indicated in Fig. 9A,
P0, the chance
probability of these results, is <0.0005; thus, they are significant
to >99.9%. Fig. 9B
compares the frequencies of MLL
micrometastasis in the longitudinally sectioned left lower lobes of the
Ac-PHSCN-NH2-treated and the untreated rats.
Eight sections, each separated by 65 µm, were scored for each rat.
From 0 to 38 micrometastases/rat were observed in eight sections of
lung tissue from each of the 10
Ac-PHSCN-NH2-treated rats, for a total of 78
micrometastases and a mean micrometastasis frequency of 7.8/rat. In
contrast, from 2 to 441 micrometastases/rat were observed in eight
sections of lung tissue obtained from each of nine untreated rats, for
a total of 1506 micrometastases and a mean micrometastasis frequency of
167.3/rat. As indicated in Fig. 9B,
P0 is 0.001, implying a significance
of 99.9%. Thus, at a dosage level of 5 mg/kg, i.v.
Ac-PHSCN-NH2 administered three times weekly
reduced MLL metastasis by 99% and micrometastasis by 95%, although
treatment did not commence until 24 h after the removal of large
primary tumors.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Replacing the arginine of the PHSRN peptide with cysteine forms a
competitive inhibitor of basement membrane invasion in
vitro. This PHSCN inhibitor blocks PHSRN-induced invasion both by
SF DU 145 cells and by prec cells with similar activities. The PHSCN
peptide also inhibits the serum-induced invasion of DU 145 cells. This
suggested that the PHSCN peptide might be an effective inhibitor of
metastatic tumor cell invasion in vivo. Metastatic rat MLL
prostate carcinoma cells also require serum for in vitro
invasiveness, and the PHSRN peptide is sufficient to induce their
invasive behavior under SF conditions. Blocking the ends of the PHSCN
peptide by acetylation and amidation increases its invasion inhibitory
activity in serum-containing medium by 30-fold for both DU 145 and
MLL cells, perhaps because removal of the charges at the
NH2 and COOH termini allows the peptide to assume
a more active conformation or results in increased stability.
Furthermore, the invasion-inhibitory activity of
Ac-PHSCN-NH2 is shown to be attributable to the
specific spatial arrangement of its amino acid side chains because the
scrambled peptide Ac-HSPNC-NH2 fails to inhibit
serum-induced invasion by either DU 145 or MLL cells.
The Ac-PHSCN-NH2 peptide appears to function as
an effective, nontoxic, antitumorigenic agent in Copenhagen rats
bearing metastatic MLL tumors because its systemic administration
reduces the growth of primary MLL tumors by >2000-fold and the density
of their vasculature by >10-fold during the first 16 days of growth in
rats. As shown by comparing sections of rat anti-PECAM-stained tumors
from Ac-PHSCN-NH2-treated and from untreated rats
and quantitating the relative volumes occupied by the vasculature in
their tumors, as well as by the inability of
Ac-PHSCN-NH2 to affect the MLL cell cycle or to
stimulate apoptosis in vitro at similar or higher
concentrations, its pronounced antitumorigenic effects are likely to be
attributable to the inhibition of neovascularization. The appearance of
MLL cells in rats treated systemically with
Ac-PHSCN-NH2 is also consistent with its ability
to inhibit nourishment of the tumor by new blood vessels. The effects
of systemic treatment with the Ac-PHSCN-NH2
peptide could be attributable to a direct anti-invasive effect on the
5ß1 receptors known to be expressed by rat endothelial cells
(45)
. Consistent with this possibility, we have observed
that the Ac-PHSRN-NH2 peptide can stimulate
invasion in vitro by normal human microvascular cells, which
can be inhibited by Ac-PHSCN-NH2 (data not
shown). Also, the presence of Ac-PHSCN-NH2 in the
circulation could have inhibited proteinase secretion by MLL tumor
cells and hence reduced the fragmentation of host pFn by secreted MLL
tumor proteinases. Thus, the potential effect on endothelial 5ß1
receptors might have also been mediated, in part, through the
inhibition of PHSRN sequence binding by the 5ß1 receptors shown to
be present on the surfaces of MLL tumor cells. Invasion by host
endothelial cells in response to fibronectin fragments produced in
wounds is believed to be crucial to the angiogenic response necessary
for healing (reviewed in Ref. 46
). The constitutive
activation of this pathway by an aggressive tumor such as MATLyLu could
thus result in neovascularization and rapid tumor growth, as well as in
tumor invasion and metastasis.
Systemic Ac-PHSCN-NH2 peptide has a very significant and sequence-specific antimetastatic effect on rats bearing metastatic MLL tumors, irrespective of whether systemic treatment is initiated 1 day after MLL cell injection or not until 1 day after the surgical removal of large MLL tumors. i.v. injections of Ac-PHSCN-NH2, beginning 1 day after MLL tumor cell injection and continuing for the following month, prevent the formation of detectable lung metastases or micrometastases in the majority of treated rats and reduce the mean number of lung metastatic colonies or micrometastases/rat in the treated cohort by 40–200-fold, with respect to untreated, MLL-bearing rats. Consistent with its lack of invasion inhibitory activity in vitro, the Ac-HSPNC-NH2 peptide also lacks antitumorigenic or antimetastatic activity in vivo.
The antimetastatic effects of systemic Ac-PHSCN-NH2 are nearly as pronounced in treated rats that do not begin systemic therapy until 1 day after their 2-cm MLL tumors are removed, as they are in rats that begin to receive treatment 1 day after MLL cell injection. Because metastases are scored under conditions that permit the detection of single MLL cells in lung tissue, and because Ac-PHSCN-NH2 does not appear to affect MLL cell cycle progression or to stimulate apoptosis at the concentrations used, it is very likely that the inhibition of postsurgical MLL metastasis in rats is a consequence of the invasion-inhibitory activity of Ac-PHSCN-NH2 on MLL cells in vivo. Because neither the sensitivity of the assay nor the growth of metastases is likely to have limited their detection, the paucity of MLL metastases observed in these rats suggests that the metastatic potential of individual MLL cells remaining in a rat increases significantly after surgery. This could be because some MLL cells are released into the blood or lymph during surgery, thus reducing the requirement for intravasation in this system. It is interesting to note that specific proteinases may be required for intravasation, a process that requires invading cells to traverse the basement membrane in an orientation opposite from that required during extravasation (47) . If MLL cells were less adept at intravasation than at extravasation because of their proteinase expression pattern, then perhaps Ac-PHSCN-NH2 may be functioning primarily as an extravasation inhibitor. This implies that a slow rate of MLL intravasation may permit the pronounced antimetastatic activity of systemic Ac-PHSCN-NH2 therapy to be observed, even when initiated only after the surgical removal of a large tumor. In this context, it is interesting to note the topology of the SU-ECM in vitro invasion substrates used to evaluate the activity of Ac-PHSCN-NH2. In this assay, invading cells are placed on the extracellular matrix that surrounded ectodermal cells located on the apical side of the basement membrane. Thus, cells encounter the extracellular matrix of the ectoderm and the underlying lamina lucida prior to encountering the lamina densa (32) , as they do when crossing the endothelial cell layer and its underlying basement membrane during extravasation. Therefore, the SU-ECM invasion assay may be especially well suited for defining effective inhibitors of extravasation.
Proteolytic fragments of pFn containing the cell-binding domain are
present in wounds and have been shown to stimulate monocyte
extravasation (48)
and fibroblast chemotaxis through the
extracellular matrix (49)
. The constitutive induction of
invasive behavior by this intact and prevalent serum protein is a
possible mechanism for stimulating tumor cell invasion and metastasis,
as well as angiogenesis. The ability to stimulate prec cell invasion
in vitro by the same PHSRN pFn sequence and to inhibit it by
the PHSCN competitive inhibitor suggests that metastatic prostate
carcinoma cells may constitutively express an invasive behavior latent
in normal prec cells and necessary for migration through the
extracellular matrix during wound healing. This hypothesis is
consistent with the fibronectin receptor expression patterns of these
two cell types. Both DU 145 and prec cells express the 5ß1
integrin fibronectin receptor (33
, 36)
, which is known to
bind the PHSRN sequence of the cell-binding domain (39)
.
It has been shown that the interaction of fibroblast 5ß1 with the
pFn cell-binding domain induces interstitial collagenase MMP expression
in the absence of 4ß1 binding (34)
. Loss of the
4ß1 receptor, which is present on the surfaces of prostate
epithelial cells, from the cell membranes of prostate cancer cells
(Refs. 33
and 36
and data not shown) is a
possible mechanism for generating constitutive invasive responses. The
indistinguishable in vitro invasive behaviors of prostate
epithelial and DU 145 cells in the presence of the PHSRN sequence of
the pFn cell-binding domain are consistent with this hypothesis.
Specific therapeutic strategies for the treatment of androgen-resistant
prostate carcinomas in Copenhagen rats have been devised by a number of
labs to control the growth of the primary tumors or to limit metastasis
without necessarily limiting tumor growth. For example, systemic
treatments with a peptide growth hormone-releasing hormone antagonist
(50)
, with interleukin-2 and radiation (51)
,
with somatostatin analogue (RC-160) and the
bombesin/gastrin-releasing-peptide antagonist (RC-3095; Ref.
52
), or with the cytotoxic drug methotrexate coupled to a
luteinizing hormone-releasing hormone receptor ligand (53
, 54)
all displayed some effectiveness in slowing the growth of
AT-1 tumors in rats. Paclitaxel and quinacrine have also been used in
combination to inhibit microtubule function in DU 145 prostate
carcinoma cells implanted in athymic mice. DU 145 tumors in the treated
nude mice were shown to be reduced in volume by 2.5-fold relative to
the untreated tumors during 2 weeks of treatment (55)
. In
contrast to these results, the systemic therapy with the
Ac-PHSCN-NH2 peptide reduces tumor volume by
>2000-fold during the first 16 days of growth.
In a strictly antimetastatic therapy, modified citrus pectin, which inhibits the adhesive interactions of metastatic cells with endothelium by competing with the natural ligand(s) for binding to tumor cell surface galectins, was tested in Copenhagen rats bearing MLL primary s.c. tumors. Oral administration of this agent resulted in a 40–50-fold reduction in the mean number of metastases/lung without affecting the rate of growth of the primary tumor (7) . Thus, the antimetastatic effects of this agent were comparable with those of Ac-PHSCN-NH2 treatment; however, therapy with the Ac-PHSCN-NH2 peptide was uniquely effective in that it also had significantly more pronounced effects on the growth of the primary tumors than did the other antitumorigenic therapies reported.
Although the role of Ac-PHSCN-NH2 as an
antimetastatic agent, i.e., as an inhibitor of the induction
of a constitutive invasive response in tumor cells by a ligand of the
5ß1 receptor, is distinct from the competitive inhibition of the
adhesive interaction between tumor cells and the extracellular matrix
by an excess of the matrix adhesion sequence, the adhesion of tumor
cells to the extracellular matrix plays a very significant role in
metastasis formation. This is shown by a number of in vitro
and in vivo experiments using peptides as competitive
inhibitors. For example, peptides containing the RGD sequence of the
cell-binding domain in fibronectin, which binds to the 5ß1
integrin, or the Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence of laminin, which
binds to a high-affinity Mr 67,000
glycoprotein receptor, showed marked inhibition of lung metastasis when
coinjected with melanoma cells and of invasion through reconstituted
basement membrane in vitro (56
, 57)
. In a model
of spontaneous lung metastasis, in which tumors of B16-BL6 melanoma
were allowed to grow 21 days prior to treatment, repeated
administration of the poly(RGD) or poly(RGDT) polypeptides before or
after surgical excision of the primary tumor resulted in a 5-fold
reduction of metastatic lung colonies without affecting primary tumor
growth, thus substantially prolonging the survival time of mice
(58)
. However, the significantly more pronounced
antimetastatic activity of systemic Ac-PHSCN-NH2
therapy, even when begun after the removal of a large primary tumor,
suggests that therapeutic intervention in the dysregulation of an
integrin-mediated invasive response may be a promising avenue for
treating androgen-resistant prostatic neoplasms.
ACKNOWLEDGMENTS
We thank Dr. Kathleen Woods-Ignatoski in the laboratory of
Stephen Ethier for performing the fluorescence-activated cell sorter
analysis of propidium iodide-labeled MLL cells to verify their lack of
apoptosis after Ac-PHSCN-NH2 treatment. We also
thank Dr. Jeremy Taylor, Director of the University of Michigan
Comprehensive Cancer Center Biostatistics Core Facility, for helpful
suggestions regarding the statistical analysis of the metastasis and
micrometastasis results. We gratefully acknowledge the expert technical
assistance of Robert Crawford, Susan Brumfield, and LaJuanda
Haygood-Carter.
|
FOOTNOTES |
|---|
1 Supported in part by a Basil O’Connor Award
from the March of Dimes, NIH Award 1P50CA69568 entitled "SPORE in
Prostate Cancer" and NIH Grant HL53713 (to K. K.), and an award from
the Office of the Vice President for Research at the University of
Michigan (to D. L. L.). D. L. A. is the recipient of a postdoctoral
training fellowship from the University of Michigan Cardiovascular
Research Center. 
2 To whom requests for reprints should be
addressed, at University of Michigan Medical School, Department of Cell
and Developmental Biology, 1150 West Medical Center Drive, Ann Arbor,
MI 48109-0616. Phone: (734) 764-0313; Fax: (734) 763-1166; E-mail: dlivant{at}umich.edu
3 The abbreviations used are: pFn, plasma
fibronectin; SF, serum free; Ac-PHSCN-NH2, acetylated
amidated PHSCN; TFA, trifluoroacetic acid; PECAM-1, platelet
endothelial cell adhesion molecule-1; DAB, 3,3'-diaminobenzidine; prec,
prostate epithelial (cells); MMP, matrix metalloproteinase; FCS, fetal
calf serum; BSA, bovine serum albumin; PBS, phosphate buffered saline;
H&E, hematoxylin and eosin.
4 D. L. Livant, R. K. Brabec, K. Kurachi, D.
Allen, Y. Wu, R. Haaseth, P. Andrews, S. Ethier, and S. Markwart. The
PHSRN sequence induces invasion of the extracellular matrix and
accelerates wound healing in obese diabetic mice, submitted for
publication.
Received 7/20/99. Accepted 11/11/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
/ß heterodimeric receptors for laminin and type IV collagen. J. Cell Biol., 107: 1241-1252, 1988.5ß1 and 4ß1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J. Cell Biol., 129: 867-879, 1995.5ß1-fibronectin interactions using inhibitory anti-5 and anti-ß1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the 5 subunit. J. Biol. Chem., 272: 17283-17292, 1997.5ß1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles. J. Clin. Investig., 100: 1647-1653, 1997.[Medline]
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Z.-Z. Zeng, Y. Jia, N. J. Hahn, S. M. Markwart, K. F. Rockwood, and D. L. Livant Role of Focal Adhesion Kinase and Phosphatidylinositol 3'-Kinase in Integrin Fibronectin Receptor-Mediated, Matrix Metalloproteinase-1-Dependent Invasion by Metastatic Prostate Cancer Cells Cancer Res., August 15, 2006; 66(16): 8091 - 8099. [Abstract] [Full Text] [PDF]
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A. Maglott, P. Bartik, S. Cosgun, P. Klotz, P. Ronde, G. Fuhrmann, K. Takeda, S. Martin, and M. Dontenwill The Small {alpha}5{beta}1 Integrin Antagonist, SJ749, Reduces Proliferation and Clonogenicity of Human Astrocytoma Cells. Cancer Res., June 15, 2006; 66(12): 6002 - 6007. [Abstract] [Full Text] [PDF]
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A. Abdollahi, D. W. Griggs, H. Zieher, A. Roth, K. E. Lipson, R. Saffrich, H.-J. Grone, D. E. Hallahan, R. A. Reisfeld, J. Debus, et al. Inhibition of {alpha}v{beta}3 Integrin Survival Signaling Enhances Antiangiogenic and Antitumor Effects of Radiotherapy Clin. Cancer Res., September 1, 2005; 11(17): 6270 - 6279. [Abstract] [Full Text] [PDF]
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Y. Jia, Z.-Z. Zeng, S. M. Markwart, K. F. Rockwood, K. M. W. Ignatoski, S. P. Ethier, and D. L. Livant Integrin Fibronectin Receptors in Matrix Metalloproteinase-1-Dependent Invasion by Breast Cancer and Mammary Epithelial Cells Cancer Res., December 1, 2004; 64(23): 8674 - 8681. [Abstract] [Full Text] [PDF]
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 C. G. Kleer, Q. Cao, S. Varambally, R. Shen, I. Ota, S. A. Tomlins, D. Ghosh, R. G. A. B. Sewalt, A. P. Otte, D. F. Hayes, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells PNAS, September 30, 2003; 100(20): 11606 - 11611. [Abstract] [Full Text] [PDF]
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 E. S. White, D. L. Livant, S. Markwart, and D. A. Arenberg Monocyte-Fibronectin Interactions, Via {alpha}5{beta}1 Integrin, Induce Expression of CXC Chemokine-Dependent Angiogenic Activity J. Immunol., November 1, 2001; 167(9): 5362 - 5366. [Abstract] [Full Text] [PDF]
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C. R. Cooper, L. McLean, M. Walsh, J. Taylor, S. Hayasaka, J. Bhatia, and K. J. Pienta Preferential Adhesion of Prostate Cancer Cells to Bone Is Mediated by Binding to Bone Marrow Endothelial Cells as Compared to Extracellular Matrix Components in Vitro Clin. Cancer Res., December 1, 2000; 6(12): 4839 - 4847. [Abstract] [Full Text]
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, DU
145 cells in SF medium and various concentrations of PHSRN. 
, DU 145
cells in SF medium with 1.4 µM GRGDSP. B,
Prec cells. X axis, log peptide concentration
(nM); Y axis, mean percentages of invaded
prec cells. Bars, SD. •, prec cells in SF medium or in
medium containing 10% FCS, both with various concentrations of PHSRN
peptide. 
, prec cells in 10% FCS with 1.4 µM GRGDSP.
,
1.4 µM PHSRN peptide in top chambers only,
SF medium in bottom chambers. 
