Download Inhibitory Effect of Bombesin Receptor Antagonist RC

[CANCER RESEARCH54, 1035-11141,February 15. 19941
Inhibitory Effect of Bombesin Receptor Antagonist RC-3095 on the Growth of
Human Pancreatic Cancer Cells in Vivo and in Vitro ~
Y u n f e n g Q i n , T i b o r E r t l , R e n - Z h i C a i , G a b o r H a l m o s , a n d A n d r e w V. S e h a l l y 2
Endocrine, Polypeptide and Cancer Institute, Veterans AfJhirs Medical Center, and Section of Experimental Medicine, Department of Medicine, Tulane University School
of Medicine, New Orleans, Louisiana 70146 [Y. Q., T. E., R-Z. C., G. H., A. V. S.]
Pancreatic cancer is one of the greatest challenges for oncologists
(1-5). Carcinoma of the exocrine pancreas is the fifth leading cause of
death from cancer in the United States (1). Most of pancreatic cancers
are histologically ductal cell carcinomas, which constitute about 8090% of the cases (2). In the past two decades, great efforts have been
made to improve the therapies for pancreatic cancers. However, the
overall prognosis of patients with pancreatic cancer is still very poor,
and the 5-year survival rate is only 2-5% (2-4). Less than 15-20% of
pancreatic tumors are resectable, mostly due to the difficulties in early
diagnosis and the frequent occurrence of local or distal metastases,
and fewer than 5% of the patients can survive for over 5 years
postoperatively (3-5). Radiotherapy and chemotherapy are usually
ineffective (2-5). Therefore, an urgent need exists to develop a new
and effective therapy for treatment of patients with pancreatic cancers.
Recently, various investigations have demonstrated that gastrointestinal hormones and growth factors may play important roles in the
regulation of growth of normal and malignant exocrine pancreas (510). In vitro studies have shown that gastrin, CCK, 3 and secretin
stimulate the proliferation of pancreatic adenocarcinoma cells in tissue
cultures (6, 7). Caerulein combined with secretin promotes the in vivo
growth of H-2-T pancreatic ductal adenocarcinoma cells in golden
hamsters (8). Gastrointestinal hormones may also influence the phenotypic transformation of pancreatic cells (6, 8, 9). These findings
suggest that the growth of pancreatic cancers might be possibly controlled by hormonal manipulations (4-10), and various peptide analogues have been suggested for treatment (5-10).
Bombesin and its mammalian counterpart, GRP, are hormonal peptides which can exert diverse physiological or pharmacological actions in various systems (5, 11, 12). In the gastrointestinal tract,
bombesin and GRP stimulate gastric and pancreatic secretions, enhance the release of several gastrointestinal hormones, and promote
the growth of exocrine pancreas (5, 11, 12). Administration of bombesin to rats produces hyperplasia and hypertrophy of pancreatic
acinar cells (13). Bombesin and GRP appear to function as autocrine
or paracrine growth factors and stimulate the growth of some normal
or malignant cells including Swiss 3T3 fibroblast cells (14), human
small cell lung cancer cells (15), human mammary and gastric cancer
cells (16), 4 mouse and human colon cancer cells (17, 18), and CAPAN
human pancreatic tumor cells (19). The trophic effect of bombesin/
GRP has also been demonstrated on the growth of azaserine-induced
pancreatic acinar-cell adenocarcinomas in rats (20, 21) and on the cell
proliferation of acinar tumor in primary cultures (22). The action of
bombesin/GRP is thought to be mediated through its specific receptors
present on the target cells (19, 22, 23). The discovery that bombesin
appears to act as an autocrine growth factor in human small cell lung
carcinoma (15, 23) and may also be involved in other cancers (16-22)
has aroused major interest in the development of competitive
bombesin/GRP receptor antagonists.
During the past few years, various bombesin/GRP antagonists including RC-3095 have been synthesized in our laboratory and evaluated for antitumor activity (24, 25). Receptor studies showed that
these synthetic peptide analogues inhibit the binding of bombesin/
GRP to specific receptors on the membranes of Swiss 3T3 cells,
SCLC cells, and human gastric cancer cells (24, 25). 4 Bombesin
Received 9/20/93; accepted 12/17/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by NIH Grant CA 40077 and the Medical Research Service
of the Veterans Affairs (A. V. S.).
2 To whom requests for reprints should be addressed, at Veterans Affairs Medical
Center, 1601 Perdido Street, New Orleans, LA 70146.
3 The abbreviations used are: CCK, cholecystokinin; GRP, gastrin-releasing peptide;
FCS, fetal calf serum; IMDM, lscove's modified Dulbecco's medium; PBS, phosphatebuffered saline; GHRH, growth hormone-releasing hormone; Tpi, 2,3,4,9-tetrahydro-lHpyrido[3,4-b]indol-3-carboxylic acid; EGF, epidermal growth factor; [D-Trp6]LH-RH,
o-tryptophan-6-1uteinizing hormone-releasing hormone.
4 y. Qin, G. Halmos, R-Z. Cai, B. Szoke, T. Ertl, and A. V. Schally. Inhibition of
specific binding of bombesin and bombesin-stimulated growth of human gastric cancer
cells by bombesin antagonists, submitted for publication.
ABSTRACT
In this study, we investigated the effect of bombesin/GRP antagonist
RC-3095 on the growth of CFPAC-1 human pancreatic cancer cells transplanted to nude mice or cultured in vitro. Nude mice bearing xenografts of
the CFPAC-1 cell line received s.c. injections of RC-3095 (10/tg twice a
day) or the vehicle (control) for 25 days. Chronic administration of RC3095 inhibited the growth of CFPAC-1 tumors in nude mice as shown by
a significant decrease in tumor volume throughout the period of treatment. Tumor volume doubling time was prolonged by RC-3095 treatment
from 7.2 days to 10 days, and the tumor growth rate was decreased by
49%. In mice treated with RC-3095, the tumor growth delay time was 5.8
days. ~I~reatment with RC-3095 decreased the final tumor weight by 37%
and reduced DNA and protein contents in tumor tissues by 44 and 39.9%,
respectively, compared to the controls. In cultures of the CFPAC-I cell
line, the addition of bombesin(1-14) (1 p~a-0.1 pM) to the medium induced
a dose-dependent increase in cell number. RC-3095 at 1 nM concentration
effectively inhibited the bombesin-stimulated growth of CFPAC-I cells in
cultures. In the presence of 1 btM RC-3095 in the culture medium, the
bombesin-induced growth of CFPAC-1 cells was totally suppressed. Bombesin was also shown to stimulate the DNA synthesis in CFPAC-1 cells in
vitro as based on [3H]thymidine incorporation assay. When the cells were
cultured in the presence of 1-100 nM bombesin, the uptake of [3H]thymidine by the cells was increased by 89-131%. RC-3095 inhibited both the
basal and bombesin-stimulated DNA synthesis of CFPAC-1 cells. Addition
of RC-3095 (10-100 nM) alone to the cultures caused a 39-40% decrease
in the [3H]thymidine incorporation by the cells. Concomitant addition of
RC-3095 (1 p~) and bombesin (1-100 nM) to the cultures induced a significant reduction in the uptake of [aH]thymidine by the cells compared to
the values obtained with bombesin alone. Receptor binding assays showed
the presence of two classes of specific binding sites for bombesin on
CFPAC-1 cells, one with high affinity (Ka = 4.25 _+ 0.77 n~a) and low
capacity (B,,ox = 0.268 +_ 0.052 pmol/106 cells) and the other with low
affinity (K,t -" 321.70 + 68.46 nM) and high capacity (B .... - 3.991 +_0.374
pmoi/106 cells). Antagonist RC-3095 inhibited the binding of ~2SI-Tyr4bombesin to CFPAC-1 cell membranes in a dose-dependent manner. These
observations suggest that bombesin acts as a growth factor and stimulates
proliferation of CFPAC-1 human pancreatic cancer through specific receptors for bombesin/GRP present on the cells. RC-3095 appears to inhibit
the growth of CFPAC-1 cells by blocking the interaction of bombesin with
its receptors. Bombesin/GRP receptor antagonist RC-3095 could be considered for the development of new approaches for treatment of human
pancreatic cancers.
INTRODUCTION
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BOMBESIN ANTAGONIST ON GROWTH OF PANCREATIC CANCER
antagonist RC-3095 has been shown to effectively suppress the
growth o f H T 29 h u m a n colon cancers (26), PC-82 h u m a n prostate
cancers (27), M K N 4 5 h u m a n gastric cancers xenografted in nude
mice (28), M X T breast cancers in mice (29), and nitrosamine-induced
pancreatic cancers in hamsters (30). The inhibitory effect of antagonist
RC-3095 on the cell proliferation in vitro has also been demonstrated
in the cultures o f some o f these cancer cell lines (16, 25, 28). 4
RC-3095 and other novel bombesin antagonists could be considered
for the development o f hormonal therapy for pancreatic cancers. H o w ever, the presence o f specific receptors for b o m b e s i n / G R P has been
reported so far only in C A P A N h u m a n pancreatic cancer cells (19),
and the effects o f b o m b e s i n / G R P and their antagonists on the growth
o f h u m a n pancreatic ductal cancer cells must be evaluated m o r e
extensively.
In the present study, w e investigated the effect of bombesin antagonist RC-3095 on the growth o f CFPAC-1 h u m a n ductal pancreatic
cancer cells implanted in nude mice. Direct effects of bombesin and
RC-3095 on the proliferation o f CFPAC-1 cells w e r e evaluated in cell
cultures. Specific receptors for b o m b e s i n / G R P on C F P A C - I cells
w e r e characterized, and the ability of RC-3095 to inhibit the binding
o f bombesin to the receptors was also studied.
MATERIALS
AND
METHODS
Peptides and Chemicals. Bombesin antagonist D-Tpi6,Leul3aII[CH2NH] Leu14-bombesin(6--14) (RC-3095), originally synthesized by a solid phase
method and characterized in this laboratory (24, 25), was provided by Asta
Pharm Co. (Frankfurt/M, Germany) for this study. Tyrn-bombesin and somatostatin-14 were obtained from Bachem (Torrence, CA) and Wyeth-Ayerst
(Philadelphia, PA), respectively. Bombesin(1-14), GHRH(1-29), cholecystokinin-8, and D-Trp6-LHRH were all synthesized by a solid phase method in this
laboratory (25, 31). 125I-Na and [methyl-3H]thymidine were purchased from
Amersham (Arlington Heights, IL). Enzymobead iodination reagent was obtained from Bio-Rad (South Richmond, CA). FCS, IMDM, PBS, and Hanks'
balanced salt solution were purchased from Gibco (Grand Island, NY). Universol scintillation cocktail was obtained from ICN (Costa Mesa, CA). Other
chemicals were purchased from Sigma (St. Louis, MO).
Cancer Cell Line. CFPAC-1 human pancreatic cancer cell line, originally
established from a well-differentiated ductal pancreatic adenocarcinoma of a
26-year-old white male with cystic fibrosis (32), was purchased from the
American Type Culture Collection (Rockville, MD). The cancer cells were
routinely maintained as a monolayer culture in Costar T75 culture flasks with
IMDM medium containing 10% FCS, 0.5 g/liter L-glutamine, 25 mM HEPES,
3.7 g/liter NaHCO3, 100 units/ml penicillin, 100/xg/ml streptomycin, and 0.25
/xg/ml amphotericin-B under humidified 5% CO2 at 37~ The cells growing
exponentially were harvested by an incubation with 0.25% Trypsin-EDTA in
calcium- and magnesium-free Hanks' balanced salt solution for 5 min at 37~
For tumor implantation, a single cell suspension was prepared in serum-free
IMDM by repeatedly passing the cells through a G-22 needle; then the cells
was diluted to a concentration of 5 • 106 cells/ml.
Implantation of Tumors in Nude Mice. Male athymic BALB/c (nu/nu)
6-week-old mice were obtained from the National Cancer Institute (Bethesda,
MD) and housed in a laminar airflow cabinet under pathogen-free conditions
throughout the experiments. Implantation of CFPAC-1 tumors was performed
according to a method described previously (33). Three nude mice received s.c.
injections in the flanks with 0.2 ml of cell suspension (1 X 106 cells) and
served as tumor donors. After 4 weeks, the implanted tumors grew to a size of
about 5 mm in diameter and were removed from the mice. Tumor samples were
dissected free of necrotic tissue and blood vessels and were cut into small
fragments of about 8 mm 3. Under ether anesthesia, two pieces of tumor
fragments were implanted s.c. by trocars on both sides of the flanks for each
mouse. The mice bearing the implanted tumors were randomly divided into
two groups with 10 mice in each group.
Treatment with Bombesin Antagonist RC-3095. The nude mice with
implanted tumors started to receive injections of bombesin antagonist RC-3095
7 days after the tumor cell injection. RC-3095 solution was freshly prepared by
dissolving the peptide in 200 lxl of 0.01 M acetic acid and was diluted with
0.9% NaCI containing 0.2% bovine serum albumin to a concentration of 125
p.g/ml. Each mouse in the treatment group was given s.c. injections of RC3095 at a dose of 10/.~g twice daily for 25 days. This dose of RC-3095 was
chosen on the basis of a previous report of the inhibitory effect of RC-3095 on
the growth of HT29 human colon cancers in nude mice (26). The animals in the
control group were injected with the same volume of vehicle but without
RC-3095.
Evaluation of Tumor Growth. During the treatment with RC-3095, the
size of the implanted tumors was measured by calipers in each mouse at 3-4
day intervals for 25 days to construct the tumor growth curves in vivo. Tumor
volume was calculated by the formula: Tumor volume = Length • width 2 •
0.5. Tumor growth parameters, Le., tumor volume doubling time, tumor growth
delay time, and tumor growth rate, were calculated from the tumor growth
curves as described previously (34). In this study, the tumor volume doubling
time was defined as the time required for the tumors to grow from 50 mm 3 to
100 mm 3 for the control group and from 35 mm 3 to 70 mm 3 for the treatment
group, respectively. The tumor growth delay time was estimated as the time
difference for the treated tumors and the controls to reach a volume of 70 mm 3.
At the end of the treatment, the animals were sacrificed with an overdose of
ether. The tumors were removed from the animals, weighed, and immediately
frozen in liquid nitrogen for measurements of DNA and protein contents in
tumor tissues.
Determination of DNA and Protein in Tumor Tissues. DNA in tumor
tissues was determined by a method of Labara and Paigen (35, 36), which is
based on the enhancement of fluorescence reaction upon binding bisbenzimidazole Hoechst 33258 (H33258) to DNA in cell nuclei in a high ionic strength
solution. Tumors collected in each group were pooled and homogenized in 10
times their volumes of a buffer consisting of 0.05 MNaH2PO4, 2.0 MNaCl, and
2 mM EDTA (pH 7.4). H33258 was dissolved in the same buffer at a concentration of 1/xg/ml and filtered before use. An aliquot of tumor homogenate (0.4
ml) was suspended in 4 ml of H33258 solution, followed by an incubation in
a dark room for 30 min. The reaction was measured by a fluorescence spectrophotometer at excitation and emission wavelengths of 356 and 492 nm,
respectively. Calf thymus DNA type I was used as a standard reference.
Protein in tumor tissues was measured by a modified method of Bradford
(37) using bovine serum albumin fraction V as a standard.
Measurement of Cell Growth in Vitro. The effects of bombesin and
bombesin antagonist RC-3095 on the growth of CFPAC-1 human pancreatic
cancer cells in vitro was evaluated by direct cell counting and [3H]thymidine
incorporation assay.
Direct Cell Counting. CFPAC-1 cells collected from 60-70% confluent
cultures were used for this study and seeded to 24-well culture plates (1 • 104
cells/well). After the cells were cultured in IMDM containing 10% FCS for 48
h, the medium was replaced by IMDM supplemented with 2.5% FCS and
various concentrations of bombesin(1-14), bombesin antagonist RC-3095, or a
combination of both. The same volume of medium but without peptides was
added to the control wells. Following another 24 h of incubation, the culture
was terminated by aspiration of the medium from the wells and washing with
PBS (0.5 ml/well). The cells were trypsinized by a 10-rain incubation with (0.5
ml/well) 0.25% Trypsin-EDTA. The detached cells were dispersed by repeated
pipeting using a G-22 needle and syringe. The number of cells collected from
each well was counted by an automated electronic cell counter (Coulter
Counter Modei-ZF; Coulter Electronics, Inc., Hialeah, FL).
[3H]Thymidine Incorporation Assay. DNA synthesis in tumor cells was
measured by [3H]thymidine incorporation assay as reported previously (38).
Single cell suspension was prepared in IMDM with 10% FCS and was seeded
to 24-well culture plates (1 • 104 cells/well). After 48 h of culture, the medium
was changed to IMDM (0.5 ml/well) containing 2.5% FCS and various concentrations of bombesin(1-14), bombesin antagonist RC-3095, or a combination of both. The control wells received the same medium without bombesin or
RC-3095. After 24 h of culture, [methyl-3H]thymidine (radioactivity 25 Ci/ml)
was added to each well (1 /zCi/well) to pulse the cells. After a 4-h incubation,
the medium was removed from the wells, and the cells were fixed by Camoy's
solution (1 ml/well; methanol:glacial acetic acid, 3:1 v/v) for 20 min. After
washing three times with PBS, the cells in each well were dissolved with 0.5
ml of 0.3 N NaOH for 15 min at room temperature. The cell lysate was
collected from each well and mixed with 3 ml of Universol scintillation
cocktail. The radioactivity was measured for 1 min by a liquid scintillation beta
counter (Mark III; Searle Analytic, Inc., Des Plaines, IL).
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BOMBESIN ANTAGONIST ON GROWTH OF PAN('RkAT[(" CANCER
Receptor Binding Assay. Receptor binding assay was performed using
intact CFPAC-I cells in monolayer cultures. Tyr4-bombesin was radiolabeled
with '25I-Na using a Bio-Rad enzymobead iodination kit. Mono-J25I-Tyr4bombesin was purified by high performance liquid chromatography as described (39). The specific activity of '25I-Tyr4-bombesin was about 2,000
Ci/mmol. CFPAC-1 cells were seeded to 24-well culture plates (1 • 10 4
cells/well) and cultured with IMDM containing 10% FCS for 48 h. The cells
in subconfluent culture were washed once with serum-free IMDM supplemented with 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 10
mM MgCI2, 1 mM EGTA, 10 mM monothioglycerol, 0.25 mM phenylmethylsulfonyl fluoride, aprotinin 10,000 kallikrein inactivator units/liter, and 0.1%
bovine serum albumin (pH 7.5), followed by an incubation for 2 h at 22~ with
the same medium containing (0.5 riM) '25I-Tyr4-bombesin in the presence or
absence of various concentrations of unlabeled bombesin(1-14), bombesin
antagonist RC-3095, or structurally unrelated peptides somatostatin-14, cholecystokinin-8, GHRH(1-29), and D-Trp6-LHRH. The binding reaction was
terminated by adding 0.5 ml of ice-cold medium to each well. After washing
four times with ice-cold PBS (pH 7.4), the cells in each well were dissolved
with 0.5 ml of 0.3 N NaOH. The resultant cellular lysate was collected from
each well for measurement of radioactivity by a gamma counter (APEX;
Microdemic System, Inc., Huntsville, AL).
Statistical Analysis. All data are expressed as the mean _+ SEM of duplicate or triplicate observations from at least 2-3 repeated experiments. Mean
values between the RC-3095 treatment group and the control group were
analyzed by the Student t test, Mann-Whitney U test, or one-way analysis of
variance. The difference was statistically considered to be significant when a
two-tailed value of P was less than 0.05.
Data from receptor binding assays were analyzed by a ligand-PC computerized curve-fitting program created by Munson and Rodbard as modified by
McPherson (40) to determine the types of binding sites, the dissociation
constants (Kd), and the maximal binding capacity of receptors (B,,,,=).
measured at intervals of every 3--4 days during the treatment. A
significant decrease in tumor volumes was observed in the RC-3095treated mice throughout the treatment. At the end of the treatment, the
final tumor volume in the RC-3095 treated mice was decreased by
38% compared to the controls (P < 0.01). Administration of RC-3095
resulted in a 49.3% reduction of the growth rate of CFPAC-1 tumors
in the treated mice (Table 1). Tumor volume doubling time was
prolonged by RC-3095 treatment from 7.2 days in the control group
to 10 days for the treated group. The tumor growth delay time in the
RC-3095-treated mice was estimated to be 5.8 days. The final tumor
weight in the RC-3095-treated mice was decreased by 37% compared
to the controls. (P < 0.01; Table 1).
Effect of RC-3095 on DNA and Protein Contents in CFPAC-1
Tumors. The inhibitory effect of bombesin antagonist RC-3095 on
the growth of CFPAC-1 tumors was further examined by the determinations of DNA and protein contents in tumor tissues. As shown in
Fig. 2, administration of RC-3095 induced a significant decrease in
the amount of both DNA and protein in the RC-3095-treated tumors.
After 25 days of RC-3095 treatment, DNA and protein contents of
tumor tissues were decreased by 44.0 and 39.9%, respectively, in the
treated mice compared to the controls (P < 0.01; Fig. 2).
Effects of Bombesin and RC-3095 on the Growth of CFPAC-1
Cells in Vitro. Bombesin stimulated the growth of CFPAC-1 human
pancreatic cancer cells in vitro. Fig. 3 shows the cell number after
CFPAC- 1 cells were cultured in the presence or absence of bombesin(1-14) for 24 h. Addition of bombesin(1-14) at the doses of i pM-0.1
/xM to the culture induced a dose-dependent increase in the number of
cultured cells. Maximal stimulation of the cell growth by bombesin
was observed at a close of 0.1 /~M, where the number of cells in the
culture with bombesin was 53% higher than that of the control (P <
0.01).
RESULTS
Bombesin antagonist RC-3095 alone did not show any significant
Effect of RC-3095 on Growth of CFPAC-1 Tumors in Nude
effect on the growth of CFPAC-1 cells in the culture (Fig. 3). Addition
Mice. Nude mice with implanted CFPAC-1 human pancreatic cancers
of RC-3095 alone at the doses of 1 pM-0.1 /XM to the medium only
started to receive injections of bombesin antagonist RC-3095 7 days
induced a slight (6.1-11.8%) but not significant decrease in the numafter the tumor inoculation when all animals had palpable tumors in
ber of cells in the cultures (P > 0.05). However, RC-3095 powerfully
the flanks. Injections of RC-3095 at a dose of 10/~g twice a day for
inhibited the bombesin-stimulated growth of CFPAC-1 cells in vitro
25 days did not show any obvious side effects on the growth of the
(Fig. 4). Addition of RC-3095 at a dose of 1 nM or 1 /XMto the cultures
treated animals. There was no significant difference in body weights
reduced or nullified the stimulatory effect of bombesin(1-14) on cell
between the RC-3095-treated and the control groups (P > 0.05).
growth. In the cultures with both (1 riM) RC-3095 and (1 pM-0.1 p~M)
Chronic administration of RC-3095 inhibited the growth of imbombesin(1-14), the number of cells was significantly lower than that
planted CFPAC-1 tumors in nude mice. Fig. 1 shows the change in
with bombesin(1-14) alone (P < 0.01). This inhibitory effect of RCtumor volumes in the RC-3095 treated mice and the controls as
3095 on the bombesin-stimulated growth of CFPAC-1 cells appeared
to be dependent on the doses of the antagonist added to the cultures.
When a higher dose (1 /XM) of RC-3095 was added to the culture, the
cell growth was totally suppressed even though these cells were culv
Control
160
tured simultaneously with effective doses (1 pM-1 /.ZM) of bombesin9
RC-3095
lO/~:j/bid
(1-14). The number of cells in the cultures in which both (1 /~M)
d-"
RC-3095 and (1 pM-1 /XM) bombesin(1-14) were present was much
120
lower than that with bombesin alone or that with a combination of a
lower dose (1 riM) of RC-3095 with bombesin (P < 0.01).
Effects of Bombesin and RC-3095 on D N A Synthesis of CF80
PAC-1 Cells in Cultures. Using the [3H]thymidine incorporation
O
~>
assay, bombesin was shown to have mitogenic effects on the proliferation of CFPAC-1 human pancreatic cancer cells in vitro (Fig. 5).
O
40
Addition of bombesin (1-100 nM) to the cultures induced a 89-131%
[.-,
increase in the uptake of [3H]thymidine by the cells as compared to
the controls (P < 0.01). In contrast, bombesin antagonist RC-3095
I
,
, I
,
I
J
l
,
I
0
i
inhibited both the basal and bombesin-stimulated proliferation of
0
5
10
15
20
25
30
CFPAC-1 cells in cultures (Fig. 5). Addition of RC-3095 alone to the
cultures at doses of 10-100 nM decreased the [3H]thymidine uptake by
Day of R C - 3 0 9 5 T r e a t m e n t
Fig. 1. Growthof implantedtumorsof CFPAC-1humanpancreaticcancercells in nude 39-40% as compared to the controls (P < 0.05). When the cells were
incubated with a combination of (1 /XM) RC-3095 and (1-100 riM)
mice duringthe treatmentwith bombesinantagonistRC-3095.
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BOMBESIN ANTAGONIST ON GROWTtt OF PANCREATIC CANCER
Table 1 Effect of bombesin antagonist RC-3095 on the growth parameters of CFPAC-1 human pancreatic cancers in nude mice
Treatment
group"
Body
weight
(g)
Final tumor
weight
(g)
Tumor growth
rate
(mm3/day)
Tumor volume
doubling time
(days)
Tumor growth
delay time
(days)
Control
RC-3095
28.2 • 0.78
26.35 • 0.31
0.133 • 0.012
0.084 t' • 0.009
6.9
3.5
7.2
10.0
-5.8
a Nude mice with implanted tumors (10 in each group) received injections of RC-3095 (10 ~g twice a day) or vehicle (control) for 25 days.
b Versus control, Student's t test, P < 0.01.
300
1600
DISCUSSION
The present study demonstrates that pseudonanopeptide bombesin/
GRP antagonist RC-3095 can effectively inhibit the growth of
CFPAC-1 human pancreatic ductal cancers xenografted to nude mice.
Chronic administration of RC-3095 induced a significant decrease in
the tumor volume throughout the treatment. At the end of the treat-
250
3
1200
==
8oo
.~
400
~
200
=
~,
15o
IO0
200
50
9 Bombesin
180
0
Control
RC-3095
Control
9 RC-3095
RC-3095
Fig. 2. DNA (A) and protein (B) contents in implanted tumors of CFPAC-1 human
pancreatic cancer cells in nude mice after 25 days of treatment with bombesin antagonist
RC-3095.
.
16o
o
14.0
""
120
,ID
I::I
:::I
bombesin(1-14), the amount of [3H]thymidine incorporated by the
treated cells was significantly lower than that with bombesin alone
(e
<
100
Z
d
0.05).
8o
60
Specific Binding of lZSI-Tyr4-bombesin to CFPAC-1 Cells. The
binding o f 125I-Tyr4-bombesin to the m e m b r a n e s o f C F P A C - 1 human
i
40
pancreatic cancer cells w a s studied by competitive binding assays
after the cells w e r e incubated with 0.5 nM 125I-Tyr4-bombesin in the
i
.
-12
0
i
,
-11
,
I
i
-10
.
-9
i
i
,
-8
.
-7
Concentrations of Peptides (log M)
presence or absence o f (1 pM-1 /xM) unlabeled b o m b e s i n ( 1 - 1 4 ) . Fig.
6,4 s h o w s that b o m b e s i n ( 1 - 1 4 ) had a high affinity to the binding sites
for 12SI-Tyr4-bombesin on CFPAC-1 cells. Addition o f increasing
doses o f unlabeled b o m b e s i n ( 1 - 1 4 ) to the cultures induced a dosedependent displacement o f the specific binding o f 12SI-Tyr4-bombesin
to the receptors on CFPAC-1 cells. In contrast, addition of structurally
unrelated peptides G H R H ( 1 - 2 9 ) , D-Trp6-LHRH, somatostatin-14, and
C C K to the cultures did not affect the binding o f 125I-Tyr4-bombesin
to C F P A C - 1 cells (Table 2), indicating the specificity o f these binding
sites for b o m b e s i n . Scatchard analysis s h o w e d that for the data of the
dose-inhibition curve of 125I-Tyr4-bombesin binding by b o m b e s i n
(1-14), a two-site m o d e l provided the best fit (Fig. 6B), indicating the
presence o f two classes o f b o m b e s i n / G R P receptors on the cells. One
class o f receptors s h o w e d high affinity (Ka = 4.25 _ 0.77 nM) and low
capacity (Bmax : 0.268 ___ 0.052 pmol/106 cells) and the other had low
affinity ( K a = 321.70 ___ 68.46 riM) and high capacity (Bmax --- 3.991
- 0.374 pmol/106 cells).
I n h i b i t i o n o f 12SI-Tyr4-bombesin B i n d i n g to C F P A C - I Cells by
Fig. 3. Effects of bombesin and antagonist RC-3095 on the growth of CFPAC-1 human
pancreatic cancer cells in vitro. CFPAC-1 cells (1 • 104 cells/well) were cultured in
24-well plates in the presence or absence of (1 pM-0.1 ~M) bombesin(1-14) or RC-3095.
After 24 h, the cells in each well were directly counted. The cell growth is expressed as
the percentage of the cell number in the cultures in the absence of bombesin or RC-3095
(control). Points, the mean - SEM of triplicate observations from 2-3 separate experiments.
200
180
m e m b r a n e s o f C F P A C - 1 cells w a s estimated to be 0.018 + 0.002 p,M,
w h i c h w a s higher than that o f b o m b e s i n ( 1 - 1 4 ) (Table 2).
Bombesin
alone
9
Bombesin
§
~c-ao95 ( - 9 u)
A
o
"~
o
160
"~
Bombesin +
RC-3095
(-6
M)
140
120
~ , ~
100
z
RO
R C - 3 0 9 5 . The ability o f b o m b e s i n antagonist R C - 3 0 9 5 to inhibit the
binding o f 125I-Tyr4-bombesin to CFPAC-1 cells w a s also studied by
displacement binding assay. Fig. 6A s h o w s that b o m b e s i n antagonist
RC-3095 could effectively inhibit the binding of 1 2 5 I-Tyr4 -bombesin
to the receptors on CFPAC-1 cells. When the cells were incubated
with 0.5 nM of 1 2 5 I-Tyr4 -bombesin in the presence of increasing doses
of RC-3095 (1 pr~-I/xM), the amount of 1 2 5 I-Tyr4 -bombesin bound to
CFPAC-1 cells was reduced in a dose-dependent manner. The dose
which causes 50% inhibition of 1 2 5 I-Tyr4 -bombesin binding to the
9
60
4.0
,
i
0
,
i
-12
,
i
-11
Concentrations
,
i
-10
,
i
-9
of Bombesin
,
i
-8
,
i
,
-7
( L o g M)
Fig. 4. Inhibitory effect of bombesin antagonist RC-3095 on bombesin-stimulated
growth of CFPAC-1 human pancreatic cancer cells in vitro. CFPAC-1 cells (1 • 1 0 4
cells/well) were cultured in 24-well plates with a combination of (1 pM-0.1 tXM)bombesin(1-14) and (1 nM or 1 /xr~) RC-3095. After 24 h, the cells in each well were directly
counted. The cell growth is expressed as the percentage of the cell number in the cultures
in the absence of bombesin or RC-3095 (control). Points, the mean •
of triplicate
observations from 2-3 separate experiments.
1038
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
BOMBESIN A N T A G O N I S T
ON GROWTH
250
r
IZ, ~
o
-B o
~
200
150
loo
.=
I
CAN(ER
genic effect of bombesin on the proliferation of CFPAC-I cells was
also confirmed by an enhanced DNA synthesis in bombesin-treated
cells. Bombesin was observed to significantly increase the uptake of
[3H]thymidine into DNA by the tumor cells in the culture. These
results provide strong evidence that bombesin is a mitogen for the
growth of human ductal pancreatic cancer cells in vitro.
The action of bombesin/GRP to stimulate cell growth is thought to
be mediated through specific membrane receptors on the target cells
(17, 18, 22-25). 4 Our recent study 4 has demonstrated that high affinity
receptors for bombesin/GRP are present on the membranes of Hs746T
human gastric cancer cells, which are coupled with G-protein in the
signal transduction pathway. Although several previous studies have
300
o
OF P A N C R E A T I C
50
-9 -8
C -9 -8 -7
Bombesin
RC-3095
C
C -9 -8 -7
Bombesin +
RC-3095 (-6 M)
A
Fig. 5. Effects of bombesin and bombesin antagonist RC-3095 on [3H]thymidine
incorporation in CFPAC-1 human pancreatic cancer cells in cultures. CFPAC-1 cells (1 x
104 cells/well) were cultured in 24-well plates in the presence or absence of (1 nM-0.1 #.M)
bombesin(1-14) and RC-3095 or a combination of both. After 24 h, the cells were pulsed
with [methyl-3H]thymidine (1 ~Ci/well) for 4 h. The DNA synthesis activity is expressed
as the percentage of the [3H]thymidine incorporated by the ceils in the absence of
bombesin (control). Points, the mean +- SEM of triplicate observations of 2-3 separate
experiments.
ment with RC-3095, the final tumor volume was reduced by 38% in
the treated mice as compared to the controls. The inhibition of growth
of CFPAC-1 tumors by RC-3095 was also shown by other tumor
growth parameters, including the prolongation in tumor volume doubling time, the lowered tumor growth rate, and the decrease in the
final tumor weight. Chemical analyses further revealed that treatment
with RC-3095 resulted in an apparent reduction in DNA and protein
contents in the CFPAC-1 tumor tissues. Our results are in agreement
with the previous observations from this laboratory on the inhibitory
effect of RC-3095 on tumor growth in several experimental models
including nitrosamine-induced pancreatic cancers in Syrian golden
hamsters (30), MXT breast cancers in mice (29), and xenografts of
PC-82 human prostate cancer (27), HT-29 colon cancer (26), and
MKN-45 gastric cancer in nude mice (28). These observations suggest
that bombesin/GRP antagonist RC-3095 is a potent inhibitor of the
growth of pancreatic and other cancers in vivo.
Although trophic effect of bombesin/GRP has been demonstrated in
various normal and malignant cells, the role of this class of peptides
in the regulation of the proliferation of pancreatic cancer cells has not
been fully established. In vivo studies have shown that bombesin/GRP
enhanced the tumor growth of azaserine-induced pancreatic acinar
cells in rats (20-22) but inhibited the development of nitrosamineinduced pancreatic ductal cell type lesions in hamsters (41) and
growth of SKI human pancreatic cancers in nude mice (42). These
contradictory observations were suggested to be related to the origin
of tumors, i.e., acinar versus ductal cell types (22). Hajri et al. (22)
reported that GRP increased the [3H]thymidine incorporation in azaserine-induced rat pancreatic acinar carcinoma cells in primary cultures, indicating a stimulatory effect of bombesin/GRP on the proliferation of pancreatic acinar cancer cells in vitro. However, bombesin
was found to stimulate the mitogenesis of both acinar and ductal cells
in rat pancreas (43) and growth of CAPAN human ductal pancreatic
cancer in vitro (19). A possible down-regulation of EGF receptors
after chronic administration of bombesin may have been also involved
in these phenomena (42, 44; also see below), and it is difficult to draw
unambiguous conclusions from in vivo investigations since endogenous release of various hormones and growth factors may affect
tumor growth in animals. In the present study, we observed that
bombesin stimulated the growth of CFPAC-1 human ductal pancreatic
cancer cells in cultures. Addition of bombesin(1-14) to cell cultures
induced a dose-dependent increase in the number of cells. The mito-
~
100
9
-9
9
9
9
9
Bombesln
9 RC-3095
k
~" "~
~0
m
N
5o
25
o
-12
-11
-10
-9
-8
-7
-6
-5
Concentrations of Peptides (Log M)
0.006 .
0.005 f
0,004
"r
0.003
o
0.002
0.001
0.000
0.0
i , , 9 , 9 , 9 , 9
0.5
1,0
1.5
2.0
2.5
3,0
3.5
118
4
l
Bound l-Tyr - B o m b e s i n ( p m o l / l O c e l l s )
Fig. 6. (A) Displacement of 1251.Tyr4.bombesin binding to CFPAC-1 human pancreatic
cancer cells by unlabeled bombesin(l-14) and bombesin antagonist RC-3095. CFPAC-I
cells (1 x 104 cells/well) were incubated with 0.5 nM of ]25I-Tyr4-bombesin in the
presence or absence of various concentrations of bombesin(1-14) or RC-3095 for 2 h at
22~ Binding is expressed as the percentage of maximum specific binding of 125I-Tyr4bombesin to the cells in the absence of unlabeled ligand (100% binding). Points, the mean
-+ SEM of triplicate observations from 2 separate experiments. (B) Scatchard plot of the
specific binding of 12-Sl-Tyr4-bombesin indicating the presence of two classes of specific
binding sites on CFPAC-1 cells, one with high affinity (Ka = 4.25 +- 0.77 riM) and low
capacity (Bmax = 0.268 -+ 0.052 pmol/106 cells) and the other with low affinity (Ka =
321.70 • 68.46 riM) and high capacity (Bmax = 3.991 _+ 0.374 pmol/106 cells).
Table 2 Displacement of 1251-Tyr4-bombesin binding to CFPAC-1 human pancreatic
cancer cells by bombesin, antagonist RC-3095, and unrelated peptides
CFPAC-1 cells were incubated with 0.5 nM of 125I-Tyr4-bombesin and unlabeled
bombesin and other peptides at the doses of 10-12-10 -5 M for 2 h at 22~
Peptides
IC5o"
Bombesin(1-14)
RC-3095
GHRH(I-29)
Cholecystokinin-8
Somatostatin- 14
D-Trp6-LHRH
9.5 • 10-10M
1.8 • 10-8 M
NB
NB
NB
NB
'~ IC5o, the dose causing 50% inhibition of specific binding of 1251-Tyr4-bombesin to
CFPAC-1. Structurally unrelated peptides GHRH(1-29), CCK-8, SS-14, and D-Trp6LHRH could not displace the binding of 125I-Tyr4-bombesin to CFPAC-1 cells. NB, no
binding.
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Research.
BOMBESINANTAGONISTON GROWTtt OF I'AN('REATI('CANCER
shown that bombesin/GRP receptors are widely distributed on the
membranes of many benign and malignant tumor cells including rat
pancreatic acinar cancer cells (17, 18, 22-26), 4 the presence of
bombesin/GRP receptors in human pancreatic cancer cells has been
reported so far only by Avis et al. (19). In our previous study, antagonist RC-3095 did not inhibit the growth of MIA PaCa-2 human pancreatic cancer xenograft in nude mice (45). It is possible that our MIA
PaCa-2 line has undergone some changes in morphology and receptor
content. The absence of receptors may have been a contributing factor
as specific binding sites for bombesin were not found in our cultures
of MIA PaCa-2 cells and neither bombesin nor bombesin antagonist
RC-3095 influenced cell growth in vitro (45). In the present study, we
demonstrated that specific receptors for bombesin/GRP are present on
the membranes of CFPAC-1 human ductal pancreatic cancer cells. In
competitive receptor binding studies, we observed that bombesin
(1-14) was able to effectively displace 125I-Tyra-bombesin from the
binding sites on CFPAC-1 cells. In contrast, structurally unrelated
peptides such as somatostatin-14, cholecystokinin-8, o-Trp6-LHRH,
and GHRH(1-29) did not affect the binding of 125I-Tyr4-bombesin to
the cell membranes. Scatchard analysis indicated that two types of
bombesin/GRP receptors are present on the membranes of this cancer
cell line, one class having a high affinity and low capacity and the
other class having a low affinity and high capacity. Characteristics of
bombesin receptors on CFPAC-1 cells are similar to those on MC-26
mouse colon cancer cells, which also show two classes of binding
sites for bombesin/GRP (17). These receptors are apparently different
from those on Swiss 3T3 cells (24) and many other tumor cell lines
(18, 22, 23, 25) 4 in which only one class of binding sites is found.
Because bombesin was observed to have direct stimulatory effects on
the proliferation of CFPAC-1 cells in cultures, our receptor studies
suggest that the specific binding sites for bombesin/GRP on CFPAC-1
cells may represent functional receptors which mediate the action of
bombesin on the growth of these tumor cells.
RC-3095 [D-Tpi6,Leu13~[CH2-NH]Leu14-bombesin(6-14)] is a
potent bombesin antagonist, which was synthesized in our laboratory
specially for antitumor therapy. Previous studies have shown that this
synthetic peptide inhibits the binding of a25I-Tyr4-bombesin to the
membranes of Swiss 3T3 cells (24) and H-345 human small cell lung
cancer cells (25) and suppresses the basal and GRP-induced amylase
release from rat pancreatic acini in a superfusion assay (24). RC-3095
was proven to be a potent tumor growth inhibitor in experimental
models of nitrosamine-induced pancreatic cancers in hamsters (30),
MXT breast cancers in mice (29), PC-82 human prostate cancers (27),
MKN 45 gastric cancers (28), and HT-29 colon cancers in nude mice
(26). The direct inhibitory effect of RC-3095 was observed on the cell
proliferation of mammary (16) and gastric cancer cell lines (28). 4 The
results of the present study further demonstrate that the addition of
bombesin antagonist RC-3095 alone to the medium produced only a
slight but not significant decrease in the number of cells in the culture.
This suggests that RC-3095 itself does not affect the growth of
CFPAC-1 cells in vitro, but it can effectively inhibit the bombesinstimulated growth of CFPAC-1 cells in cultures. The presence of
RC-3095 in culture medium reduced or nullified the stimulatory action of bombesin on the proliferation of CFPAC-1 cells as evidenced
by a lower cell count in cultures treated with both RC-3095 and
bombesin as compared to that with bombesin alone. Furthermore,
RC-3095 inhibited DNA synthesis in CFPAC-1 cells during the cell
proliferation. Addition of RC-3095 to the cultures reduced the bombesin-enhanced DNA synthesis in the CFPAC-1 cells as measured by
[3H]thymidine incorporation assay. The inhibitory effect of RC-3095
on bombesin-stimulated proliferation of CFPAC-1 cells was apparently dependent on the doses of RC-3095 added to the cultures. On the
basis of both cell number and [3H]thymidine incorporation assay,
addition of RC-3095 at a higher dose of 1 /xM completely suppressed
the bombesin-induced growth of CFPAC-I cells in cultures, suggesting that the action of this antagonist is competitive. This view is
supported by the observations of the inhibitory effect of RC-3095 on
the binding of bombesin to its receptors on CFPAC-1 cells. In displacement binding assays, we noticed that bombesin antagonist RC3095 blocked, in a dose-dependent manner, the binding of 125I-Tyr4bombesin to CFPAC-1 cells. Our findings indicate that the inhibitory
effect of RC-3095 on bombesin-induced growth of CFPAC-1 cells is
probably due to competitive occupation of the binding sites for
bombesin/GRP on the cells.
However, the suppressive effect of RC-3095 on the growth of
CFPAC-1 tumors in nude mice cannot be solely explained by the
competitive action of this antagonist to displace the binding of
bombesin/GRP to the receptors on the tumor cells. Several other
regulatory mechanisms, such as a major down-regulation of EGF
receptors, may also mediate the action of RC-3095 (26-30, 44). It has
been found that in vivo treatment with RC-3095 induced a downregulation of receptors for both bombesin/GRP and EGF on the membranes of MKN45 gastric cancers in nude mice (28) and greatly
reduced concentration of EGF receptors in pancreatic, mammary,
colorectal, and prostatic cancers (27, 29, 30, 44). RC-3095 may also
affect EGF binding through an action on the protein kinase C system
(46) and inhibition of up-regulation of EGF receptors produced by
bombesin/GRP (47). Whether the effect of RC-3095 on the growth of
CFPAC-1 tumors is related to the down-regulation of bombesin/GRP
and EGF receptors on the membranes of CFPAC-1 cancer cell line or
the interference with the function of EGF receptors (46, 47) needs to
be elucidated. In addition, since bombesin/GRP can powerfully stimulate the release of gastrointestinal hormones that may have trophic
effects on the growth of pancreas and pancreatic cancers (5-12), the
possible involvement of RC-3095 not only in the inhibition of action
of bombesin/GRP but in the suppression of the release of these trophic
hormones should be also considered. It is possible that the inhibitory
action of RC-3095 on tumor growth of CFPAC-1 cells in vivo is
mediated through several mechanisms.
In summary, our study shows that bombesin/GRP antagonist RC3095 can powerfully suppress the growth of CFPAC-1 human ductal
pancreatic cancer cells xenografted into nude mice as well as cultured
in vitro. The effect of RC-3095 appears to be mediated by the interference with the binding of bombesin to its receptors on this cancer
cell line. These findings suggest the merit of continued evaluation of
bombesin/GRP antagonist RC-3095 for the possible development of
new approaches for the treatment of pancreatic cancers.
ACKNOWLEDGMENTS
We express our thanks to Drs. Balazs Szoke, Kate Groot, and Herta Reile,
as well as Patti Armatis, for their help with iodination and cell cultures and to
Harold Valerio and Nancy Hsi for excellent technical assistance.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer
Research.
Inhibitory Effect of Bombesin Receptor Antagonist RC-3095
on the Growth of Human Pancreatic Cancer Cells in Vivo and
in Vitro
Yunfeng Qin, Tibor Ertl, Ren-Zhi Cai, et al.
Cancer Res 1994;54:1035-1041.
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