Suppression of Tumor Recurrence and Metastasis by a Combination of the PHSCN Sequence and the Antiangiogenic Compound Tetrathiomolybdate in Prostate Carcinoma

Article (PDF Available)inNeoplasia 4(5):373-9 · December 2002with16 Reads
DOI: 10.1038/sj.neo.7900258 · Source: PubMed
Abstract
Plasma fibronectin-mediated invasion of human DU145 prostate cancer cell line was efficaciously inhibited in a rat tumor model by treatment with Ac-PHSCN-NH(2) peptide. Invasion of DU145 cells was stimulated by the PHSRN sequence of plasma fibronectin. However, PHSCN acts as a competitive inhibitor of PHSRN-mediated invasion. In the current study, we determined whether PHSCN could inhibit the recurrence and metastasis of DU145 tumors after excision of the primary tumor in an athymic nude mouse model. We demonstrated that mice treated thrice weekly with intravenous Ac-PHSCN-NH(2) peptide survived tumor-free for more than 30 weeks post-primary tumor excision, whereas their untreated counterparts succumbed to recurrence and/or metastatic disease in significantly less time. Because of the universal requirement for angiogenesis in solid tumor growth, we tested the efficacy of copper deficiency induced by tetrathiomolybdate (TM) to retard tumor growth in the Dunning prostate cancer model. Significant reduction in size of the primary tumor was observed in mice rendered copper deficient. We sought to reduce tumor growth at the primary and metastatic sites by combining the anti-invasion Ac-PHSCN-NH(2) peptide with TM. Improved survival, fewer metastatic lesions, and excellent tolerability were observed with the combination therapy.
Suppression of Tumor Recurrence and Metastasis by a
Combination of the PHSCN Sequence and the Antiangiogenic
Compound Tetrathiomolybdate in Prostate Carcinoma
1
Kenneth L. van Golen*, LiWei Bao*, George J. Brewer
y
, Kenneth J. Pienta*
, z
, Jeffrey M. Kamradt*,
Donna L. Livant
x
and Sofia D. Merajver*
Departments of *Internal Medicine, Division of Hematology and Oncology,
y
Human Genetics,
z
Surgery,
x
Radiation
Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109-0948, USA
Abstract
Plasma fibronectinmediated invasion of human DU145
prostate cancer cell line was efficaciously inhibited in a
rat tumor model by treatment with Ac- PHSCN -NH
2
peptide. Invasion of DU145 cells was stimulated by the
PHSRN sequence of plasma fibronectin. However,
PHSCN acts as a competitive inhibitor of PHSRN -
mediated invasion. In the current study, we determined
whether PHSCN could inhibit the recurrence and meta-
stasis of DU145 tumors after excision of the primary
tumor in an athymic nude mouse model. We demon-
strated that mice treated thrice weekly with intravenous
Ac-PHSCN - NH
2
peptide survived tumor-free for more
than 30 weeks post - primary tumor excision, whereas
their untreated counterparts succumbed to recurrence
and/ or metastatic disease in significantly less time.
Because of the universal requirement for angiogenesis
in solid tumor growth, we tested the efficacy of copper
deficiency induced by tetrathiomolybdate (TM) to retard
tumor growth in the Dunning prostate cancer model.
Significant reduction in size of the primary tumor was
observed in mice rendered copper deficient. We sought
to reduce tumor growth at the primary and metastatic
sites by combining the anti - invasion Ac - PHSCN -NH
2
peptide with TM. Improved survival, fewer metastatic
lesions, and excellent tolerability were observed with
the combination therapy.
Neoplasia (2002) 4, 373 379 doi:10.1038/sj.neo.7900258
Keywords: prostate carcinoma, PHSCN, tetrathiomolybdate, metastasis, tumor
recurrence.
Introduction
It is estimated that in the year 2001, approximately 198,100
men will be newly diagnosed with prostate cancer [1]. A large
proportion of those diagnosed with localized disease will
benefit from radical prostatectomy, which tends to be curative
in early-stage disease [ 2 ]. Men who are diagnosed with
locally advanced or metastatic disease will undergo andro-
gen-ablation therapy, and the majority will progress locally or
at distant sites within 18 months [ 3 ]. Hormone-refractory
clones presumably cause relapse, which leads to 31,500
deaths from androgen-independent, metastatic prostate
cancer in the US each year [1 ]. Therefore, novel strategies
are being sought for the treatment of metastatic and high -risk
prostate cancer.
Previously, Livant et al. [4,5] demonstrated the invasion-
inducing role of plasma fibronectin using a naturally occur-
ring, serum-free extracellular matrix (SU - ECM ) in vitro
system. Plasma fibronectin is a soluble extracellular matrix
protein found in concentrations of 0.3 to 0.5 mg/ml in plasma,
serum, lymph, and interstitial fluid [6]. DU145 prostate
cancer cells were stimulated by the PHSRN peptide
sequence located within the cell - binding domain of plasma
fibronectin, which led to invasion through a process involving
the a
5
b
3
integrin. Subsequently, it was established that the
peptide analog PHSCN acts as a competitive inhibitor of the
PHSRN sequence by efficiently blocking invasion and
metastases of prostate cancer cells in vitro and in vivo [4].
The Ac-PHSCN-NH
2
peptide significantly blocked DU145
invasion in the naturally occurring serum - free SU-ECM
system and inhibited tumor growth and metastasis of highly
aggressive MAT- Ly - Lu prostate cancer cells in a rat model
[4]. In contrast, the scrambled peptide control, Ac-HSPNC -
NH
2
, was completely ineffective in inhibiting invasion and had
no antitumorigenic or antimetastatic activity in the same
systems. These results suggest that Ac-PHSCH-NH
2
is a
potent inhibitor of basement membrane invasion, particularly
in the extravasation step of the metastatic cascade.
To further explore the ability of the Ac -PHSCN-NH
2
sequence to prevent recurrence and metastasis, we used the
Neoplasia . Vol. 4, No. 5, 2002, pp. 373 379
www.nature.com/neo
373
Abbreviations: TM, tetrathiomolybdate; i.v., intravenous; SU - ECM, sea urchin serum - free
extracellular matrix; MLL, Dunning Mat Ly - Lu; PHSCN, peptide proline histadine
serine cysteine asparagine peptide; Ac - PHSCN - NH
2
, acetylated and aminated PHSCN
peptide; Cu, copper
Address all correspondence to: Kenneth L. van Golen, University of Michigan Compre-
hensive Cancer Center, 7216A CCGC, 1500 E. Medical Center Boulevard, Ann Arbor, MI
48109 - 0948, USA. E - mail: kgolen@umich.edu
1
This work was supported by a Specialized Program of Research Excellence Grant P50
CA69568 at the University of Michigan ( K.P. ), by the National Cancer Institute grant R01
CA 77612 ( S.D.M. ), and by a CaP CURE Special Research Award ( D.L. ).
Received 4 February 2002; Accepted 12 March 2002.
Copyright # 2002 Nature Publishing Group All rights reserved 1522-8002/02/$25.00
BRIEF ARTICLE
DU145 prostate cancer cell model injected ectopically in the
hind flank of male athymic nude mice. We found that tumor
growth was retarded by thrice weekly intravenous ( i.v. )
peptide treatment. Moreover, after excision of the primary
tumor, the tumor recurrence rate and appearance of
metastases were completely inhibited in the PHSCN - treated
mice compared with the incidence of aggressive recurrence
and metastases in the control group. The untreated mice
succumbed to either tumor burden from recurrence and / or
metastatic disease by 15 weeks postsurgery. In contrast,
Ac-PHSCN- NH
2
treated mice survived for more than 30
weeks disease free and were tumor and metastasis free at
the time of necropsy. Seeking to test whether inhibition of the
primary tumor could be enhanced by a global inhibitor of
angiogenesis, combination therapy of the copper-lowering
antiangiogenic compound, tetrathiomolybdate ( TM) and
PHSCN was administered and proved to be mildly more
antitumorigenic, especially at the primary site than PHSCN
alone. These data suggest that Ac-PHSCN- NH
2
may
provide a well- tolerated, effective treatment for invasive
and metastatic prostate cancer, alone or in combination with
other modalities.
Materials and Methods
Cell Culture
DU145 cells (American Type Culture Collection, Rock-
ville, MD ) and Dunning MAT- Ly - Lu (MLL ) cells were
cultured according to established conditions. Cells were
harvested before injection into mice by washing with 10 ml
Hanks buffered salt solution ( HBSS; Life Technologies,
Grand Island, NY ) and 0.25% trypsin/1% EDTA (Life
Technologies), rewashed, and suspended in HBSS at a
concentration of 110
6
cells/0.1 ml. The cell suspension
was kept on ice and immediately used for injection.
To ensure that the cells were viable and invasive, a trypan
blue (Sigma Chemical, St. Louis, MO) exclusion assay was
performed before final suspension in HBSS. Cells were
visualized and counted using a hemacytometer. Only
cultures with more than 95% viable cells were used for
injection. A Matrigel ( BD Biosciences, Bedford, MA ) invasion
assay was performed as previously described to ensure that
the DU145 cells were invasive [7]. Briefly, cells harvested for
injection were diluted to a final concentration of 3.7510
5
cells/ 0.3 ml in serum-free complete minimal essential
medium (Life Technologies ), and placed into the upper
chamber of a Costar 6.5-mm Transwell ( Corning, Corning,
NY) plate coated with 10 l of 10 mg/ml Matrigel ( BD
Biosciences) with either serum - free (as control) or complete
minimal essential medium supplemented with 10% fetal
bovine serum (Sigma) in the lower chamber. PHSCN
inhibition of invasion was accomplished by adding 1 g/ml
PHSCN to the upper chamber of the Transwell with the cells.
The cells were incubated in 5% CO
2
for 24 hours at 378C.
The filters were removed, fixed, and stained with hematox-
ylin and eosin (H&E). Ten random 40 fields were surveyed
for invaded cells and the number of invaded cells in the
serum-free controls was subtracted from the number of cells
invaded in the 10% fetal bovine serum samples.
Peptide Synthesis
Large-scale peptide synthesis was performed using
standard Fmoc/t-butyl protection strategies on a Perkin -
Elmer/Applied Biosciences (Foster City, CA) model 433
peptide synthesizer as previously described [4 ]. Amidated
peptides were synthesized on Rink resin. Completed
peptides were cleaved from the resin support by anhydrous
TFA. The peptides were precipitated with diethylether,
purified by preparative high-performance liquid chromatog-
raphy, and freeze -dried. To remove residual TFA, gel
permeation chromatography was performed on a Sephadex
G-10 column equilibrated with 1 N acetic acid. Peptide
sequence was confirmed by mass spectroscopy and peptide
purity evaluated by reverse phase high- performance liquid
chromatography.
Athymic Nude Mice
One million DU145 cells in 0.1 ml HBSS (Life Technol-
ogies), were injected subcutaneously in the right hind flank of
8-week-old male nude mice. Treatment with 100 lof
50 mg / kg Ac -PHSCN-NH
2
, administered through tail vein
injection, was begun 2 weeks after injection when tumors
reached 0.5 mm
3
. Ac-PHSCN-NH
2
treatment was given
three times per week, continuously for the duration of the
experiment. Control mice received 100 l normal saline. In a
separate experiment, Ac-PHSCN - NH
2
treatment was
begun 24 hours after s.c. injection of tumor cells. Two
groups of mice were treated daily by gavage with 0.7 mg/
0.250 ml of the antiangiogenic compound TM. Daily treat-
ment with this dose of TM has been previously shown to
effectively render mice copper deficient to a level of 20% of
normal. Mice were monitored four times weekly and tumor
growth checked by measuring the length and the width of the
tumor with a microcaliper. Tumor volumes were calculated
by the formula: (lengthwidth
2
)/2. Tumors were allowed to
progress to approximately 900 mm
3
in size before excision.
To perform excisions, mice were anesthetized by i.p.
injection of 100 mg/kg ketamine and 10 mg / kg xylazine,
tumors were then removed, and the mice kept for up to
30 weeks before sacrificing by CO
2
overdose. Tissues were
collected and fixed in 10% buffered formalin. Gross
metastases were counted on the lung surface under 100
magnification using a Nikon SMZ - U dissecting microscope.
Lung tissue was paraffin embedded, 5- m sections were cut
and stained with H&E. Representative lung sections were
surveyed for evidence of micrometastases under 400
magnification.
Copenhagen Rat Metastasis Model
One million MLL cells were injected into the hind limb of
rats on day 0. Cells were implanted into three groups of 10
Copenhagen rats each,: the control group was untreated
throughout, the copper -depleted group was treated with
1.5 mg/day of TM for 2 weeks before tumor cell injection,
and the delayed treatment group began receiving TM on the
374 Inhibition of Prostate Cancer Recurrence van Golen et al.
Neoplasia . Vol. 4, No. 5, 2002
day of tumor implantation. After 4 weeks, the tumor - bearing
limbs were removed, and the animals were sacrificed
2 weeks later to evaluate metastases.
Statistical Analysis
The University of Michigan Biostatistics Department
performed statistical analysis of the data. Incidence of lung
metastasis and tumor growth rates were compared using the
Wilcoxon signed rank test and the Kruskal - Wallis test.
Results
The PHSCN Peptide Can Effectively Reduce DU145
Invasion In Vitro
It was previously demonstrated that DU145 prostate
cancer cells were invasive in the SU-ECM assay and their
invasive capabilities could be inhibited by the addition of the
antagonist PHSCN peptide. To ensure that the DU145 cells
used in our in vivo experiments were invasive in vitro,we
performed a Matrigel ( BD Biosciences ) invasion assay to
quantitate the invasive abilities of the cells before implanta-
tion. Figure 1 demonstrates that the cells were invasive
through a Matrigel - coated filter in response to 10% serum
containing medium. In the presence of the PHSCN peptide,
the cells were six- fold less invasive than their untreated
counterparts. These data demonstrate that DU145 prostate
cancer cells are invasive in vitro and their ability to invade a
Matrigel-coated filter can be significantly reduced by treat-
ment with the PHSCN peptide.
PHSCN Treatment Can Effectively Block Tumor
Recurrence, Metastasis, and Micrometastasis
The ability of the acylated and amidated PHSCN peptide
to affect prostate tumor growth, recurrence, and metastasis
was tested. Male nude mice were injected s.c. in the rear,
right flank with DU145 cells. Approximately 2 weeks later
mice began thrice weekly treatments with 50 mg / kg Ac-
PHSCN-NH
2
, administered through tail vein injection once
the tumors reached palpable mass size ( 0.5 mm
3
). Tumor
growth was monitored by measuring tumor size with calipers
and calculating approximate tumor volume. Tumor growth
rate was not significantly effected by Ac -PHSCN-NH
2
treatment compared with saline controls. Tumors were
excised from control and treated mice at the same time,
approximately 8 to 9 weeks postinjection, when they reached
a volume of approximately 900 mm
3
. Interestingly, we noted
that tumors from the Ac -PHSCN-NH
2
treated animals
tended to be less invasive and appeared to be encapsulated
compared with the controls. Treatment continued with the
same dose schedule postsurgery.
Tumors began to recur in the control group between 2 and
3 weeks postexcision of the primary tumor. As demonstrated
in Figure 2, all of the control mice (n=20 ) exhibited tumor
recurrences between 3 weeks and 15 weeks postsurgery.
Strikingly, none of the Ac -PHSCN-NH
2
treated mice
(n=18) had tumor recurrences. Furthermore, the treated
animals survived disease free, out to 30 weeks postsurgery,
a time point arbitrarily chosen as an end -point for the
evaluation of disease-free survival. In contrast, all of the
untreated animals had evidence of recurrent disease by 17
weeks postsurgery. The volumes of the recurring tumors
tended to be larger and grew at a faster rate than the primary
tumors, with 5/15 tumors reaching 900 mm
3
within 5 weeks
post-primary tumor excision. As shown in Table 1, the
average tumor volume of the recurrences was 728 mm
3
at
15 weeks postsurgery. All of the untreated control mice
eventually succumbed to large recurrences and / or meta-
static disease, with a median number of lung metastases of
10 visible nodules (with a range of 0 to 22 visible nodules )
in contrast to a median number of zero visible nodules
(range of 0–0) in the Ac-PHSCN-NH
2
treated group.
Upon examination of representative H&E- stained sections
from the lungs of each of the animals, it was found that the
Ac-PHSCN- NH
2
animals had significantly fewer lung
Figure 1. Results of a Matrigel invasion assay comparing untreated with Ac -
PHSCN - NH
2
treated DU145 prostate cancer cells. Ac - PHSCN - NH
2
treated cells were coincubated with 1 g / ml Ac - PHSCN - NH
2
during the
24 - hour invasion period. Addition of the Ac - PHSCN - NH
2
peptide signifi-
cantly reduced the ability of the DU145 cells to invade.
Figure 2. Percentage of mice that were recurrence - free after primary DU145
tumor excision. Mice treated thrice weekly with 100 l 50 mg / kg Ac-PHSCN -
NH
2
i.v. remained recurrence - free up to 30 weeks post - primary tumor
excision. In contrast, 100% of control mice treated with normal saline had
recurrences out to 15 weeks post - primary tumor excision.
Neoplasia . Vol. 4, No. 5, 2002
Inhibition of Prostate Cancer Recurrence van Golen et al. 375
micrometastases (P>.001) than the untreated controls
(Table 1).
Taken together, these data suggest that treatment of
DU145 prostate tumors with Ac -PHSCN-NH
2
has minimal
effect on primary tumor growth. However, this peptide can
nearly abrogate tumor recurrences and the formation of
macroscopic and microscopic metastases.
TM has Efficacy to Decrease Tumor Growth in the Dunning
Prostate Cancer Tumor Model
We investigated the activity of TM in the Dunning rat
model. The cell lines derived from the original rat tumor have
diverse phenotypes such as nonmetastatic (AT.1) and
highly aggressive ( MAT - Ly-Lu; MLL ). Arguably, the MLL
model is a more aggressive prostate tumor in rodents than
DU145. Previous experiments demonstrated efficacy of Ac-
PHSCN-NH
2
. Therefore, we wished to determine if treat-
ment of MLL tumors with TM would lead to decreased
primary tumor growth. They can be injected subcutaneously
in the groin of syngeneic previously decoppered rats to test
our hypotheses.
To test the hypothesis that copper (Cu) reduction to 20%
of normal levels would be efficacious in controlling tumor
growth in a prostate cancer implant model, we studied three
groups of 10 Copenhagen rats each, implanted with the MLL
prostatic tumorigenic cell line: the control group was
untreated throughout, the copper-depleted group was
treated with 1.5 mg/day of TM for 2 weeks before tumor
cell injection, and the delayed - treatment group began
receiving TM on the day of tumor implantation. After
4 weeks, the tumor- bearing limbs were removed, and the
animals were sacrificed 2 weeks later to evaluate meta-
stases. Figure 3, A and B depict the tumor weights and the
average number of lung metastases, respectively, for the
three groups. A statistically significant decrease in tumor
weight was observed for the copper - depleted group in
comparison to either the control or the delayed- treatment
groups (P = .0313 ). Interestingly, both the copper-depleted
and the delayed - treatment groups showed a trend toward
lower numbers of metastases that did not reach statistical
significance in this study. These results support the
conclusion that TM interferes with the growth of implanted
tumors, and does so most effectively after Cu deficiency is
achieved. TM also appears to decrease metastatic growth
under the same conditions. The safety, potency, and speed
of action of TM as an anti - Cu agent in this model suggest
that it may be synergistically efficacious in combination with
anti-invasive therapies, such as the Ac-PHSCN-NH
2
peptide.
Inhibition of Tumor Growth, Recurrence and Metastasis by a
Combination of Ac - PHSCN - NH
2
and TM
Because primary DU145 primary tumor growth was only
moderately effected by Ac- PHSCN - NH
2
treatment, we
sought to potentiate the effect on primary tumor growth by
the peptide with TM. The effect of TM on prostate tumor
growth in nude mice has not been previously determined. For
these experiments, as before, treatment with peptide and TM
began 24 hours after 1 million DU145 cells were implanted
s.c. into the hind flank of male nude mice. Mice were treated
with either thrice weekly Ac - PHSCN - NH
2
alone, daily TM
alone, a combination of daily TM and thrice weekly Ac-
PHSCN-NH
2
, or normal saline as a control.
Table 1. Comparison of DU145 Tumor Bearing Mice Left Either Untreated or Treated with 50 mg / kg Ac - PHSCN - NH
2
.
Treatment
Group
Avg. Recurrence
Volume ( mm
3
)
Median No. ( Range )
of Lung Metastases
Avg. No. of Lung
Micrometastases
Untreated ( n = 20 ) 728 10 ( 0 22 )* 308 ( n =5)*
Ac - PHSCN - NH
3
( n =18) 0 0 (0–0) 17 (n =5)
Treated mice had no tumor recurrence and significantly less gross and micrometastases than untreated mice ( *P < .001, as determined by a Wilcoxon signed
rank test ).
Figure 3. Tumor development in Copenhagen rat following implantation with
MLL cells in a limb. Control animals were treated with saline. Copper - depleted
animals were treated with TM for 2 weeks before tumor cell injection.
Concurrent treatment animals began TM treatment at the time of injection;
these animals were not copper deficient until 5 to 7 days postinjection. ( A )
Tumor weight in grams at the time of necropsy. ( B ) Average number of lung
metastases in each group.
Table 2. Number of Weeks Postinjection to Tumor Excision ( Tumor Volume
900 mm
3
).
Treatment Group
Animal
12345
Control 11 11 11 12 12
Ac - PHSCN - NH
2
11 11 12 12 15
TM 11 12 13 15 + 22*
TM + Ac - PHSCN - NH
2
11 12 15 15 + 22*
Number of weeks postinjection of DU145 tumor cells into nude mice until
tumor excision. Although no significant differences were observed in the
tumor growth rates, tumors in treated animals appeared to take longer to
reach an excisable size. Furthermore, two treated animals had tumors that
never grew larger than 500 mm
3
.
*These animals never grew tumors greater than 500 mm
3
.
376 Inhibition of Prostate Cancer Recurrence van Golen et al.
Neoplasia . Vol. 4, No. 5, 2002
As shown in Table 2, the time to tumor excision ( 900
mm
3
) was faster for the untreated control animals compared
with the animals in the treated groups. However, as
described previously, there was not a significant difference
in tumor growth rate between the untreated group and Ac-
PHSCN-NH
2
only treated group (P=.18). The time to
tumor excision in the TM-treated groups ( TM alone and
TM+Ac-PHSCN - NH
2
) was significantly longer compared
with the untreated controls. Interestingly, one mouse from
each of the TM-treated groups never grew tumors greater
than 500 mm
3
. In fact, the tumor volumes for these mice
fluctuated around a mean of only 200 mm
3
. There was a
slight difference in tumor growth between the TM and
TM+Ac-PHSCN - NH
2
groups. These data indicate that TM
is effective in significantly slowing primary tumor growth in
nude mouse xenografts; in addition, its growth-retarding
effect may be enhanced by cotreatment with the PHSCN
peptide.
Tumor recurrences were measured in both the untreated
and TM- only treated groups between 5 and 6 weeks post -
primary tumor excision ( Figure 4). All of the untreated
animals had recurrences by 13 weeks postsurgery. In
contrast, only one of the TM-alone treated mice had a
recurrence and none of the Ac-PHSCN-NH
2
-only or
TM+Ac-PHSCN - NH
2
group had recurrences. The average
volume of recurrence was, as expected, significantly higher
(P=.01) in the untreated group compared with all other
treatment groups ( Table 3 ). All of the control mice were
sacrificed 16 weeks after excision of the primary tumor due to
recurrence and metastatic disease.
As previously observed, the untreated mice had
significantly more gross lung metastases ( P = .01) than all
the treated animals with a median number of eight lung
metastases ( range of 0 to 13) for the controls and 0 lung
metastases ( ranges of 0 0, 0 1, and 0–1) for the treated
animals. In each of the TM -treated groups, there was a
single animal with one visible lung metastasis. Similarly,
the average number of lung micrometastases was found to
be highest in the untreated group with an average of 235
micrometastases. This was significantly higher (P<.001)
than the treatment groups. The TM-treated mice had an
average of 42 mircrometastases. Although this was higher
than the Ac-PHSCN- NH
2
or TM + Ac -PHSCN-NH
2
treated groups (with an average of 27 and 19 micro-
metastases, respectively ), the difference was not signifi-
cant. These data suggest that TM and Ac- PHSCN - NH
2
can be combined in vivo to effectively retard DU145
prostate tumor growth, and to drastically inhibit tumor
recurrence and metastasis after debulking surgery of the
primary tumor.
Discussion
It has been demonstrated that invasive prostate cancer cells
have a distinct integrin and cell adhesion molecule profile
that allows for increased metastatic capabilities [810].
Expression of these integrins is thought to mediate cell
adhesion with specific extracellular basement membranes
such as laminin, vitronectin, and fibronectin and facilitate cell
migration and invasion through specific pathways such as
the focal adhesion kinase pathway [11]. Specific integrin
pairs are also implicated in forming cell to cell ( tumor cell to
tumor cell or tumor cell to endothelial cell) interactions,
required for tumor cell metastasis [ 12,13 ]. Integrins that
recognize the canonical tripeptide sequence, RGD, found in
a variety of extracellular matrix proteins, are thought to
potentially mediate prostate cell interactions with endothelial
cells [12 ]. The DU145 cell has specifically been shown to
express the a
5
b
1
integrin pair, which recognizes the RGD
sequence [ 9 ].
Table 3. Comparison of DU145 Tumor Bearing Mice Left Untreated or Treated with Ac - PHSCN - NH
2
Alone, Tetrathiomolybdate ( TM ) Alone or TM + Ac -
PHSCN - NH
2
.
Treatment Group Avg. Recurrence
Volume ( mm
3
)
Median No. ( Range )
of Lung Metastases
Avg. Lung
Micrometastases
Untreated ( n = 5 ) 882 8 ( 0 13 )* 235**
TM ( n = 5 ) 540 0 ( 0 1 ) 42
Ac - PHSCN - NH
3
( n =5) 0 0 (0–0) 27
Ac - PHSCN - NH
3
+TM (n =5) 0 0 (0–1) 19
Untreated and TM - treated mice had large tumor recurrences. However, only the untreated mice had significantly more gross lung and micrometastases ( *P = .01
and **P < .001 ) than any of the treatment groups.
Figure 4. Comparison of tumor recurrences after primary DU145 tumor
excision. All mice treated with Ac - PHSCN - NH
2
, either alone or in
combination with the antiangiogenic compound tetrathiomolybdate ( TM ),
remained recurrence free up to 30 weeks post - primary tumor excision,
whereas 90% of mice treated with TM alone remained recurrence free up to
30 weeks postexcision. In contrast, 100% of control mice had sizeable
tumor recurrences at 13 weeks post - primary tumor excision.
Neoplasia . Vol. 4, No. 5, 2002
Inhibition of Prostate Cancer Recurrence van Golen et al. 377
Plasma fibronectin is a soluble extracellular matrix
protein found in plasma, lymph, and interstitial fluids [6].
DU145 and MAT-Ly-Lu cells in serum - free medium can
be stimulated to invade through a naturally occurring
serum-free basement membrane, SU- ECM, by the addi-
tion of plasma fibronectin [ 4 ]. Livant et al. demonstrated
that the PHSRN sequence of plasma fibronectin, which
maps to the cell-binding domain, was sufficient for inducing
invasion of these cells [ 4 ]. Furthermore, they demonstrated
that this is due to PHSRN binding to and stimulating the cell
through the a
5
b
1
receptors on the cell surface. In vivo,
prostate cells may also be induced to invade locally by
liberation of fibronectin by prostate- specific antigen ( PSA)
[14].
Substitution of the arginine residue in the PHSRN
sequence with cysteine forms a competitive inhibitor of
basement membrane invasion. The PHSCN peptide can
effectively block serum-free, PHSRN-induced invasion of
human DU145 and rat MAT-Ly-Lu prostate carcinoma cells
in vitro. Blocking the amino and carboxyl ends of the peptide
by acetylation and amidation, respectively, increases its
inhibitory activity by 30 -fold in serum-containing medium.
Taken together, these data led to the hypothesis that Ac-
PHSCN-NH
2
may be useful in preventing prostate carci-
noma metastasis in vivo, due to its anti - invasive properties.
Rats that were systematically administered peptide begin-
ning 24 hours after inoculation with MLL cells had signifi-
cantly fewer lung metastases compared with rats treated
with either saline alone or Ac-HSPNC - NH
2
scrambled
peptide [ 4 ].
We, thus, surmised that administration of Ac-PHSCN -
NH
2
after resecting a primary human prostate xenograft
might reduce tumor recurrence and metastases by blocking
invasion. Athymic nude mice that had tumors of approx-
imately 900 mm
3
began Ac-PHSCN-NH
2
24 hours after
tumor cell injection. None of the Ac-PHSCN-NH
2
had
tumor at the site of surgery. All of the untreated animals
succumbed to either tumor recurrence and/or metastasis
within 17 weeks postsurgery. The peptide - treated animals
remained disease free 30 weeks after surgery and had no
evidence of disease at either the primary or secondary
sites. Microscopic analysis of representative lung sections
demonstrated 18-fold greater numbers of micrometastases
in the untreated animals compared with the treated
animals.
As suggested in previous experiments, the presence of
micrometastasis suggest that metastasis could be occurring
before surgery. However, metastases could also occur due
to, or be enhanced by the ‘‘spilling’’ of tumor cells into the
blood or lymph during surgical intervention. Given the
observation that tumor recurrences reached a large volume
much faster than the primary tumors, it is possible that
tumor growth is enhanced by physical injury and proximity
to a wound-healing process where brisk angiogenesis is
occurring. Likewise, circulating factors could also stimulate
tumor cell invasion [ 15 17]. Ac-PHSCN- NH
2
treatment is
effective in controlling the growth of recurrences and distant
metastases, even if the above processes are in effect.
Similar results were observed when Ac-PHSCN - NH
2
was combined with the antiangiogenic compound TM. TM
exerts its antiangiogenic effects by sequestering dietary and
circulating copper, forming a tripartite complex with copper
and protein that is eliminated in the bile and urine. Because
copper appears to be required for activation of the an-
giogenic switch in nascent tumors as well as for sustained
angiogenesis in bulky tumors, TM-treated animals produced
a significant difference in tumor growth rates relative to the
untreated tumors reaching resectable size ( 900 mm
3
), with
the latter reaching the target size by 12 weeks whereas 1 of 5
of the TM-treated tumors never reached the target resect-
able size at all.
Most strikingly, the time to tumor resection was very
different between the treatment groups and the control.
The times to tumor resection increased in the following
order: TM + Ac - PHSCN-NH
2
>TM>Ac-PHSCN-NH
2
>untreat-
ed. Interestingly, in each of the TM- treated groups there
was one animal whose tumor never reached a resectable
size. Also, the Ac - PHSCN-NH
2
tumors appeared to be
more defined than the untreated or TM-treated tumors. The
untreated tumors in particular had more diffuse contours and
were visibly vascularized. TM alone was protective against
tumor recurrence, albeit to a slightly lesser extent than Ac-
PHSCN-NH
2
alone, with 80% of the TM - treated animals
being recurrence free compared with 100% of the Ac-
PHSCN-NH
2
treated animals. Most significantly, all of the
treatment groups survived disease free for longer than
30 weeks, compared with the control group that succumbed
to disease within 16 weeks postsurgery.
Taken together, these data strongly suggest that Ac-
PHSCN-NH
2
alone, or in combination with TM, may provide
a highly effective, tolerable, long-term treatment for the
metastatic and recurrence events of prostate cancer in
human xenografts postsurgery. Postsurgical administration
of these compounds, each alone or in combination, can
effectively prevent prostate cancer recurrence and meta-
stasis. Because the Ac-PHSCN-NH
2
is so far available only
in injectable form, it is very useful to consider combining or
alternating therapy with an oral, nontoxic global inhibitor of
angiogenesis such as TM, which alone has significant
activity in the prevention of metastasis, has the added
advantage of impairing primary tumor growth, and is
amenable to long-term administration.
Acknowledgements
We thank Lisa Robbins for help in preparing this manu-
script, Satoru Hayasaka for performing statistical analysis,
and Kelli A. Sullivan for assistance in preparing histologic
samples.
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    • "In preclinical and Phase-I trials using lewis lung carcinoma and HT 29 colon carcinoma model a U-shaped dose response was observed for ATN-161 peptide with rapid clearance from serum [77]. The therapy with ATN-161 administration in combination with tetrathiomolybdate and also with 5-flurouracil also suggested the ATN-161 as more anti-tumorigenic agent [18,78]. Few other analogues of ATN-161 like ATN-453, PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP), PhScN (where Histidine and Cysteine were replaced with D-isomers), PHSC(S-OAc)N, PHSC(S-Me)N, PHSC(Sacm )N reported to be more potent in metastatic human prostate cancer cells [20]. "
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    • "The acetylated amidated PHSCN peptide was even more potent than PHSCN peptide [146], and was developed by Attenuon LLC (San Diego, CA, USA) under the name ATN-161. ATN-161 treatment blocks prostate tumor recurrence, metastasis and micrometastasis [149], reduces colorectal liver metastasis and improves survival when given in addition with chemotherapy [150], and blocks breast cancer growth and metastasis [151] in preclinical mouse models. Targeting α5β1 integrin with ATN-161 in combination with radiotherapy enhanced apoptosis of breast cancer cells grown in 3D culture [91]. "
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    • "Notably, ATN-161 suppressed the development of metastases when treatment was started after resection of primary MLL tumours [183], a model highly relevant to adjuvant treatment in the clinic. Treatment started prior to the removal of DU145 tumours completely prevented recurrence of the primary tumour and significantly reduced metastasis [185]. A polylysine dendrimer, Ac-PHSCNGGK-MAP, bearing 8 PHSCN peptides has been shown to be significantly more potent than ATN-161, with synergistic enhancements of inhibition of fibronectin-induced invasion of PC-3 or DU145 cells in vitro, and inhibition of cell extravasation and development of micrometastases in the lungs in vivo [186] To date, studies on PHSCN peptides have focussed on combating lung metastasis. "
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