Tyr-c[D-Orn-Tyr(Bzl)-Pro-Gly]: A novel antiproliferative acting somatostatin receptor agonist with μ-opioid receptor-sensitizing properties

Article abstract of DOI:10.1038/sj.bjp.0705416

1. Here, we introduce a β-casomorphin-5-derived cyclic pentapeptide, cCD-2 (Tyr-cyclo[D-Orn-Tyr(Bzl)-Pro-Gly]), which inhibits the cell growth of a variety of human cancer cell lines. 2. This opioid-derived peptide possesses only low affinity for μ-receptors, but enhances the agonist binding to μ-receptors in vitro and potentiates the analgesic effect of morphin in vivo. The molecular mechanism of μ-receptor sensitization by cCD-2 is not yet known. 3. The antiproliferative effect of cCD-2 is independent of μ-, δ-, and κ-receptors. 4. Using SH-SY5Y cells as model, we can demonstrate that cCD-2 specifically binds to somatostatin receptors and stimulates the activity of protein tyrosine phosphatases, which are early downstream targets of SST receptors. 5. In SH-SY5Y cells, cCD-2 specifically increases the activity of the cytosolic PTP SHP-2, stimulates the activity of mitogen-activated protein kinase (MAPK), and elevates the expression of the cyclin-dependent kinase inhibitor p21 (WAF1/Cip1), suggesting the involvement of SSTR1 receptor subtype in cCD-2 action in this cell type. 6. In COS-7 cells, for comparison, we found a stimulation of SHP-2 as well as SHP-1 in response to cCD-2. The activation of SHP-1, which is attributed to the SSTR2 receptor and negatively regulates the EGF receptor, corresponds with the ability of cCD-2 to inhibit the EGF-induced MAPK activation in COS-7 cells. 7. Our results show that in SH-SY5Y cells cCD-2 inhibits cell growth via the SSTR1 receptor-signalling pathway but may, in other cells, also use other SSTR subtypes and their signalling mechanisms. 8. cCD-2 represents a novel type of opioid-derived antiproliferative SST receptor agonist, which possesses low μ-receptor affinity but may induce μ-receptor sensitization and is structurally different from the hitherto known SST receptor agonists.

Full text of DOI:10.1038/sj.bjp.0705416

British Journal of Pharmacology (2003) 140, 13–22
&
2003 Nature Publishing Group All rights reserved 0007–1188/03
$25.00
www.nature.com/bjp
Tyr-c[d-Orn-Tyr(Bzl)-Pro-Gly]: a novel antiproliferative acting
somatostatin receptor agonist with l-opioid receptor-sensitizing
properties
1Jorg Stirnweiss, 2Bianka Hartrodt, 3Gisela Greksch, 1Uta Sturzebecher, 4Frank-D. Bohmer, 2Klaus
Neubert & *,1Claus Liebmann
1Institute of Biochemistry and Biophysics, Biological and Pharmaceutical Faculty, Friedrich-Schiller-University Jena,
Philosophenweg 12, Jena D-07743, Germany; Department of Biochemistry and Biotechnology, Institute of Biochemistry,
2
3
University of Halle, Halle D-06120, Germany; Institute of Pharmacology and Toxicology, Faculty of Medicine, University of
4
Magdeburg, Magdeburg D-39120, Germany and Institute of Molecular Cell Biology, Medical Faculty, Friedrich-Schiller-
University Jena, Jena D-07747, Germany
1
Here, we introduce a b-casomorphin-5-derived cyclic pentapeptide, cCD-2 (Tyr-cyclo[d-Orn-
Tyr(Bzl)-Pro-Gly]), which inhibits the cell growth of a variety of human cancer cell lines.
This opioid-derived peptide possesses only low affinity for m-receptors, but enhances the agonist
2
binding to m-receptors in vitro and potentiates the analgesic effect of morphin in vivo. The molecular
mechanism of m-receptor sensitization by cCD-2 is not yet known.
3
The antiproliferative effect of cCD-2 is independent of m-, d-, and k-receptors.
Using SH-SY5Y cells as model, we can demonstrate that cCD-2 specifically binds to somatostatin
4
receptors and stimulates the activity of protein tyrosine phosphatases, which are early downstream
targets of SST receptors.
5
In SH-SY5Y cells, cCD-2 specifically increases the activity of the cytosolic PTP SHP-2, stimulates
the activity of mitogen-activated protein kinase (MAPK), and elevates the expression of the cyclin-
dependent kinase inhibitor p21 (WAF1/Cip1), suggesting the involvement of SSTR1 receptor subtype
in cCD-2 action in this cell type.
6
In COS-7 cells, for comparison, we found a stimulation of SHP-2 as well as SHP-1 in response to
cCD-2. The activation of SHP-1, which is attributed to the SSTR2 receptor and negatively regulates
the EGF receptor, corresponds with the ability of cCD-2 to inhibit the EGF-induced MAPK
activation in COS-7 cells.
7
Our results show that in SH-SY5Y cells cCD-2 inhibits cell growth via the SSTR1 receptor-
signalling pathway but may, in other cells, also use other SSTR subtypes and their signalling
mechanisms.
8
cCD-2 represents a novel type of opioid-derived antiproliferative SST receptor agonist, which
possesses low m-receptor affinity but may induce m-receptor sensitization and is structurally different
from the hitherto known SST receptor agonists.
British Journal of Pharmacology (2003) 140, 13–22. doi:10.1038/sj.bjp.0705416
Keywords: Cyclopeptide; cell growth inhibition; m-receptor sensitization; SH-SY5Y cells; SSTR1 receptors
Abbreviations: b-CM-5, b-casomorphin-5, Tyr-Pro-Phe-Pro-Gly; BRL 52537, (7)-1-(3,4-dichlorophenyl)-2-(1-pyrrolidinyl)
methylpiperidine, k-receptor agonist; cCD, cyclic casomorphin derivative, Tyr-c[d-Orn-Tyr(Bzl)-Pro-Gly];
COS-7, transformed African green monkey kidney cell line; DADLE, d-Ala2-d-Leu-enkephalinamid; DAMGO,
Tyr-d-Ala-Gly-NMe-Phe-Gly-ol; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor;
ERK, extracellular-signal regulated kinase; GPCR, G protein-coupled receptor; ICI-154,129, N,N-Diallyl-Tyr-
Gyl-Tyr-c(CH2)-Phe-Leu-OH, d-receptor antagonist; MAPK, mitogen-activated protein kinase; MBP, myelin
basic protein; p21 (WAF1/Cip1), member of the Cip/Kip family of cyclin-dependent kinase (cdk) inhibitors; PTP,
protein tyrosine phosphatase; RTK, receptor tyrosine kinase; SH, src homology; SHP-1/2, SH2-domain
containing cytoplasmatic protein tyrosine phosphatases; SST, somatostatin; SSTR, somatostatin receptor
Introduction
The majority of peptide hormone and neuropeptide receptors
mediates not only short-term regulation of metabolism,
secretion or contractility, but also stimulation of cell pro-
liferation. The agonists of these mostly G protein-coupled
receptors (GPCRs) may elicit their mitogenic responses by
stimulating the mitogen-activated protein kinase (MAPK)
cascade (reviewed in Gutkind, 1998). Up to now, there are
only few examples suggesting the antiproliferative actions of
peptides (reviewed in Liebmann, 2001). Especially, somatos-
tatin (SST) and a great number of SST-derived analogues,
structurally mainly based on the octapeptide SMS 201-995
*Author for correspondence; E-mail: b9licl@rz.uni-jena.de
Advance online publication: 4 August 2003

14
J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
(Octreotide; d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Thr-Cys]-Thr),
have been demonstrated to induce inhibition of cell growth.
A few of these SST analogues are already in clinical studies
and/or use for cancer treatment (reviewed in Janeck et al.,
2001; Scarpignato & Pelosini, 2001). Interestingly, several
years ago another family of cyclic SST analogues has been
synthesized, possessing relatively low affinity towards SST
receptors but high affinity for m-opioid receptors such as, for
example, CTP (d-Phe-cyclo[Cys-Tyr-d-Trp-Lys-Thr-Pen]-Thr-
NH2), CTAP (d-Phe-cyclo[Cys-Tyr-d-Trp-Arg-Thr-Pen]Thr-NH2),
or CTOP (d-Phe-cyclo[Cys-Tyr-d-Trp-Orn-Thr-Pen]Thr-NH2)
(Pelton et al., 1986). These cyclic octapeptide SST analogues
have been characterized to act as m-receptor antagonists
(Mulder et al., 1991). Based on these and additional results,
Hruby et al. (1998) hypothesized that although SST and opioid
receptor ligands have no structural homology, they obviously
use similar structural elements for the interaction with the
respective receptors. Indeed, even opioid peptides including
enkephalin and casomorphin-derived peptides have been
shown to inhibit cell growth. These peptides mediate their
antiproliferative effects either via opioid receptors or SST
receptors or via both. Thus, in the human breast carcinoma cell
line MCF-7, for instance, m-, d- and k-receptor selective
agonists were shown to decrease cell proliferation in a
naloxone-reversible manner (Maneckjee et al., 1990). In
another breast carcinoma cell line, T47D, which does not
express m-receptors, a-casomorphins mediate their antiproli-
ferative activity mainly via k-opioid binding sites (Kampa et al.,
1996), whereas morphine and the m-receptor preferring
casomorphin derivative morphiceptin were found to inhibit
10 mg mlꢀ1 streptomycin, and 0.25 mg mlꢀ1 amphotericin B in
humified air with 5% CO2 at 371C. SH-SY5Y cells (DSMZ,
Braunschweig, Germany) were grown in Dulbecco’s modified
Eagle’s medium (DMEM supplemented with 10% calf serum
(FCS) and antibiotics. The human lung cancer cell line U1752
was generously provided by Dr J. Bergh, (Uppsala, Sweden).
Cells were treated with the different compounds as indicated in
the figure legends. For preparation of lysates, cells were
washed in cold phosphate-buffered saline (PBS) and lysed at
41C in a buffer containing 20 mm HEPES, pH 7.5, 10 mm
EGTA, 40 mm b-glycerophosphate, 1% Triton X-100, 2.5 mm
MgCl2, 1 mm dithiothreitol (DTT), 2 mm sodium vanadate,
1 mm phenylmethylsulfonylfluoride (PMSF), 20 mg mlꢀ1 apro-
tinin, and 20 mg mlꢀ1 leupeptin. The lysates were centrifuged at
14,000 ꢁ g for 20 min at 41C.
Peptide synthesis
The b-casomorphin analogues were synthesized by conven-
tional solution methods or a combination of solid phase and
solution synthesis. For example, fragment condensation of
Boc-Tyr(tBu)-d-Orn(Fmoc)-OH with H-Tyr(Bzl)-Pro-Gly-
ONB (Boc ¼ tert-butyloxycarbonyl; Fmoc ¼ fluorenylmethy-
orenylmethyloxycarbonyl;
Bzl ¼ benzyl;
tBu ¼ tert-butyl;
ONB ¼ 4-nitrobenzyl ester) resulted in the fully protected
linear precursor of the cyclic peptide cCD-2. After deprotec-
tion of the side chain o-amino group of the d-Orn residue and
the terminal carboxylic group, the ring closure reaction was
carried out using diphenylphosphorylazide as coupling re-
agent. Flash chromatography of the resulting protected cyclic
pentapeptide on silica gel and acidolytic removal of the
tyrosine-protecting groups gave the desired peptide. Alterna-
tively, solid-phase syntheses were carried out using the
multiple peptide synthesizer Symphony/MultiplexTM (Protein
Technologies Inc, U.S.A.). Fmoc-Pro-OH, Fmoc-Tyr(Bzl)-
OH, Fmoc-d-Orn(Mtt)-OH and Fmoc-Tyr-OH were succes-
sively coupled to a H-Gly-2-ClTrt resin using standard solid-
phase protocols. On completion of the synthesis, the peptide
resin was treated with 1% trifluoroacetic acid in dichloro-
methane for cleavage and deprotection of the d-Orn side chain
Mtt (4-methyltrityl) group. After cyclization of Fmoc-Tyr-d-
Orn-Tyr(Bzl)-Pro-Gly-OH and purification of the resulting
cyclopeptide as described above, finally the Fmoc-protecting
group was removed.
cell growth via the SST receptor
2 subtype (SSTR2)
(Hatzoglou et al., 1995). Furthermore, a- and b-casomorphins
were reported to decrease cell proliferation of T47Dcells
(Hatzoglou et al., 1996), as well as of prostatic cancer cell lines
(Kampa et al., 1997) by interaction with both opioid and SST
receptors.
In the course of our studies to prepare cyclic b-casomorphin
analogues with modified opioid receptor affinity profiles as
well as with antiproliferative activity (Schmidt et al., 1991), we
obtained a cyclic pentapeptide, Tyr-cyclo[d-Orn-Tyr(Bzl)-Pro-
Gly] (cCD-2), with unexpected properties. cCD-2 was found to
inhibit the cell growth of tumor cell lines, but the antiproli-
ferative effect was clearly independent of opioid receptors.
Furthermore, whereas cCD-2 itself has only a very low affinity
to m-receptors, this compound is able to increase the agonist
binding to m-receptors in vitro and to potentiate the analgesic
effect of the m-receptor agonist morphine in vivo. Using the
human neuroblastoma cell line SH-SY5Y as cellular model, we
demonstrate that the antiproliferative activity of cCD-2 is
exclusively mediated via SST receptors and their signalling
pathways.
Determination of analgesic activity
At 1 week prior to the experiments, chronic microcannula were
implanted under deep nembutal anesthesia (40 mg kgꢀ1) into
the lateral ventricle of male Wistar rats (8–9-weeks old). The
peptide was dissolved in physiological saline and injected
intracerebroventricularly in a volume of 5 ml. The determina-
tion of pain threshold was carried out by electrical tail
stimulation as described previously (Greksch et al., 1981).
Briefly, incremental current pulses (rectangular pulses, 50 Hz,
4 ms) were delivered to the tail via a stainless-steel electrode
subcutaneously implemented in the tail. The minimum current
strength (in mA) evoking vocalization was taken as the
analgesic threshold. An index of analgesia (IA) was calculated
according to Cox et al. (1968).
Methods
Cell culture and preparation of cell lysates
The cell lines used (either from the German Collection of
Microorganisms and Cell Culture, Braunschweig, Germany, or
from the American Type Culture Collection, Rockville,
U.S.A.) were grown in RPMI 1640 medium supplemented
with 10% (v vꢀ1) fetal calf serum, 100 U mlꢀ1 penicillin,
British Journal of Pharmacology vol 140 (1)

J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
15
Binding studies
10 ml assay buffer (50mm HEPES, pH 7.5, 0.1 mm EDTA,
0.015% Brij 35, 0.1 mg mlꢀ1 BSA, and 0.2% b-mercaptoethanol),
10 ml ATP solution (buffer, containing 1 mm ATP and 15 mCi mlꢀ1
[g-32P]ATP), and 2 ml (50Umlꢀ1) active Src kinase. The reaction
mixture was incubated for 3 h at 301C and then applied to a
0.5 ml Dowex AG 1X8 column. [32P]Raytide was eluted with 30%
acetic acid and stored at ꢀ801C not longer than 4 weeks.
For measuring PTP activity, the reaction mixture contained
assay buffer, labelled Raytide (approximately 50,000 cpm per
assay), and diluted samples of cell lysates or PTP immuno-
precipitates in a total volume of 50 ml. After incubation for
30 min at 371C, reaction was terminated by adding 750 ml of
ice-cold stop solution, containing 0.9 m HCl, 90 mm sodium
pyrophosphate, 2 mm NaH2PO4, and 4% activated charcoal.
After centrifugation for 5 min at 10,000 ꢁ g, 400 ml of the
supernatants were counted for radioactivity.
For opioid receptor binding, crude membranes were prepared
from Wistar rat brains (without cerebella and medullae) by
homogenization in 50 mm Tris-HCl, pH 7.4. The homogenate
was filtered through two layers of medical gauze, centrifuged
at 40,000 ꢁ g for 30 min, resuspended in buffer to about 5 mg
protein mlꢀ1, and stored at ꢀ801C. Protein concentration was
determined by the method of Lowry et al. (1951). Binding
affinities were estimated by coincubation of the unlabelled
substances with either 0.5 nm [3H]DAMGO for 60 min at 41C
or 1 nm [3H]DPDPE for 180 min at 251C in 50 mm Tris-HCl,
pH 7.4 (displacement studies). The data were analyzed by
using a nonlinear regression program (GraphPad Prism).
SST receptor binding studies using whole cells were
performed as previously described for the human breast
carcinoma cell line T47D(Hatzoglou et al., 1995). Briefly,
cells (5 ꢁ 105 SH-SY5Y cells per well) were washed with 2 ml
PBS and then incubated for 45 min at 221C in a total volume of
0.5 ml PBS containing 10 fmol [125I]Tyr1-SST and increasing
concentrations of SST or cCD-2 as indicated. After incuba-
tion, cells were washed twice with 2 ml cold PBS and removed
from plates with 0.4 ml 2 n NaOH. The bound radioactivity
was estimated using a Gammacounter. The binding data were
analyzed by nonlinear regression analysis (GraphPad Prism).
Measurement of DNA synthesis
Subconfluent cells were deprived of serum for 24 h and then
treated with the different compounds as indicated. The cells
were incubated for another 24 h, followed by the addition of
[3H]thymidine (1 mCi mlꢀ1) for 2 h. Incorporation of [3H]thy-
midine was measured by washing of cells sequentially twice
with ice-cold PBS, 5% trichloracetic acid (TCA), and 5%
ethanol. Then, the DNA was extracted with 1 n NaOH and the
[3H]thymidine incorporation into the TCA precipitate was
assayed by liquid scintillation counting.
Immunoprecipitation and Western blotting
For immunoprecipitation of tyrosine phosphatases, polyclonal
anti-SHP-1 or anti-SHP-2 (2 mg mlꢀ1) were used. Antigen–
antibody complexes were recovered using protein A–sepha-
rose. The immunoprecipitates were washed three times with
PBS containing 1% Triton X-100 and 2 mm orthovanadate
(omitted in the case of PTP immunoprecipitates). For ERK1
immunoprecipitates (MAPK assay), a separate washing proto-
col was used. The samples were boiled in 50 ml Laemmli buffer
and subjected to SDS–PAGE using 7.5% gels (13% gels for
MAPK assay samples). Separated proteins were transferred to
PVDF membranes, blocked overnight with 3% nonfat dried
milk in TBST (Tris-HCl, 10 mm, pH 7.5/0.5 m NaCl/0.1%
Tween 20). For immunoblotting analysis, blots were probed
with the respective antibodies. Tyrosine phosphorylation of
EGFR was detected with a monoclonal anti-phosphotyrosine
antibody 4G10. In this case, the filters were blocked in bovine
serum albumin in TBST. For reblotting, a polyclonal anti-
EGFR antibody was used. Horseradish peroxidase-conjugated
anti-rabbit IgG was used as secondary antibody and detected
using a chemiluminescence (ECL) Western blotting detection
system by exposure to Biomax films.
Materials
[g-32P]ATP (3000 Ci mmolꢀ1), [3H]DAMGO (50Ci mmolꢀ1),
[3H]DPDPE
(42Ci mmolꢀ1),
and
[
125I]Tyr1-SST
(2200 Ci mmolꢀ1) were obtained from NEN (Perkin-Elmer life
sciences, Koln, Germany). [3H]thymidine (2.0 Ci mmolꢀ1) and
all reagents for SDSFPAGE were purchased from Amersham
Biosciences (Europe, Gmbh, Freiburg, Germany). SST, epi-
dermal growth factor (EGF), DAMGO, DADLE, aprotinin,
leupeptin, b-glycerophosphate, myelin basic protein, sodium
orthovanadate, phenylmethylsulfonyl fluoride, DTT, EGTA,
ATP, Triton X-100, protein A–sepharose, HEPES, MOPS,
naloxone, and diagnostic film (Biomax, Eastman Kodak Co.)
were obtained from Sigma (Deisenhofen, Germany). Raytide
was obtained from Oncogene Research Products (Cambridge,
U.S.A.). Polyclonal antibodies against p44 MAPK (ERK1),
SHP-1, SHP-2, p21 (WAF1/Cip1), and the monoclonal
phospho-ERK specific antibody were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The mono-
clonal anti-phosphotyrosine specific antibody 4G10 was
obtained from Upstate Biotechnology (Lake Placid, U.S.A.).
BRL 52537 hydrochloride, b-funaltrexamine hydrochloride,
ICI-154,129, and nor-binaltorphimine dihydrochloride were
obtained from Tocris Cookson Ltd, Bristol, U.K.
MAP kinase assay
MAPK activity was measured using the myelin basic protein
(MBP) assay as previously described (Graness et al., 2000).
Phosphorylated MBP was visualized by autoradiography and
quantified with a phosphoimager.
Results
Determination of PTP activation
The b-casomorphin-derived cyclopeptide, cCD-2, inhibits
the proliferation of various tumor cell lines
For substrate labelling, the synthetic peptide Raytide was
dissolved in 50 mm HEPES, pH 7.5, containing 0.1 mm EDTA
and 0.05% Triton X-100 at a concentration of 1 mg mlꢀ1. The
reaction mixture was prepared by mixing 10 ml Raytide solution,
As shown in Figure 1, cCD-2 inhibits the growth of very
different tumor cell lines between 20 and 50%. The influence
British Journal of Pharmacology vol 140 (1)

16
J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
on DNA synthesis was studied in serum-deprived cells to see
not only inhibitory, but also stimulatory effects of cCD-2. In
none of the affected cell lines did the opioid receptor
antagonist naloxone prevent the inhibitory effect of cCD-2
on cell growth, but rather stabilized the inhibitory effect of
cCD-2 in most cell lines. Naloxone alone did not significantly
alter DNA synthesis. In Figure 2, it is shown that in different
cell lines, cCD-2 may inhibit [3H]thymidine incorporation in a
concentration-dependent manner to different degrees. Taken
together, these findings suggest a broad but, nevertheless, cell-
specific antiproliferative effect of cCD-2, which apparently
does not involve its interaction with opioid receptors.
cCD-2 modulates the binding properties of m-opioid
receptors
We considered that cCD-2 may exert antiproliferative activity
either via opioid receptors, SST receptors, or both. We first
examined the opioid receptor affinity of cCD-2. Compared
with the m-opioid receptor-specific agonist DAMGO
(IC50 ¼ 5 nm), cCD-2 displayed a weak m-receptor affinity with
an IC50 value higher than 1000 nm (Figure 3a). Interestingly, in
contrast to other opioids, we found that low concentrations of
this cyclopeptide increased the specific binding of [3H]DAM-
GO nearly up to 150%. The apparent EC50 value for elevating
the specific binding of [3H]DAMGO was approximately 10 nm.
In agreement with the enhanced binding of the m-receptor
Figure 2 Effects of cCD-2 on DNA synthesis in different cell lines
in dependency on peptide concentration. Different serum-deprived
tumor cells were treated with increasing concentrations of cCD-2
and assayed for [3H]thymidine incorporation as described under
Methods. The results shown represent the mean7s.e.m. of 12 wells
in three independent experiments.
agonist [3H]DAMGO, cCD-2 also increased the analgesic
potency of the m-receptor agonist morphine (Figure 3b).
cCD-2 alone did not exert any antinociceptive effect.
Furthermore, at a concentration of 1.25 mg/kg, morphine
itself is unable to induce analgesia. However, this subeffective
dose of morphine becomes increasingly analgesic in combina-
tion with increasing concentrations of cCD-2.
The inhibitory effect of cCD-2 on cell proliferation is not
mediated via opioid receptors
Using the human neuroblastoma cell line SH-SY5Y as cellular
model, we evaluated several derivatives of cCD-2 for their
ability to inhibit [3H]thymidine incorporation. Figure 4 shows
that only analogues containing either Tyr(Bzl) or Tyr in
position 3 may act antiproliferative, whereas other modifica-
tions in position 3 or the linearization of the cyclopeptide
resulted in the loss of the antimitogenic potency. These
findings indicate
a structure–activity relationship which
additionally demonstrates the specificity in the antiprolifera-
tive action of cCD-2. To clarify whether opioid receptors
might play a role in the antimitogenic signalling pathway of
cCD-2, we measured its effect on [3H]thymidine incorporation
in the absence and presence of opioid receptor subtype-specific
antagonists. In Figure 5, it is shown that these antagonists did
not significantly block the antiproliferative effect of cCD-2 in
SH-SY5Y cells. In contrast, the m-receptor-selective agonist
DAMGO, the d-receptor-selective agonist DADLE as well as
the k-receptor-selective agonist BRL 52537 significantly
decreased DNA synthesis, suggesting that in SH-SY5Y cells
all opioid receptor subtypes may mediate the inhibition of cell
growth. The cell growth-inhibiting effects of the respective
receptor subtype-specific agonists were significantly attenuated
by the respective antagonists. These results confirm our
assumption that in SH-SY5Y cells the antiproliferative action
of cCD-2 is independent of m-, d-, and k-receptors.
Figure 1 Effects of cCD-2 (1 mm) on DNA synthesis in various cell
lines: MCF-7, EFM-192A ¼ human breast carcinoma cell lines;
U1752 ¼ human squamous lung cell carcinoma; H-69 and COLO-
677 ¼ human small cell lung carcinoma; A-427 ¼ human lung
carcinoma; A431 ¼ human epidermoid carcinoma cells; SW-
480 ¼ human colon carcinoma cells; SH-SY5Y ¼ human neuroblas-
toma cells; NG 108-15 ¼ mouse neuroblastoma ꢁ rat glioma hybrid
cells; C6 ¼ rat glioma cells. DNA synthesis was assessed by
measuring [3H]thymidine incorporation using the TCA precipitation
technique. Each value is the mean7s.e.m. of three to six
independent experiments in six-fold determinations. *Significantly
lower compared with the respective basal [3H]thymidine incorpora-
tion (controls) of each cell line (Student’s t-test; Po0.05).
British Journal of Pharmacology vol 140 (1)

J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
17
Figure 4 Influence of different analogues of cCD-2 (cCD-4 ¼ H-
Tyr-c[d-Orn-Tyr-Pro-Gly]; cCD-6 ¼ H-Tyr-c[d-Orn-Phe-Pro-Gly];
cCD-12 ¼ H-Tyr-c[d-Orn-1-Nal-Pro-Gly]; V-2 ¼ H-Tyr-d-Orn-Tyr
(Bzl)-Pro-Gly) on the rate of DNA synthesis in the human
neuroblastoma cell line SH-SY5Y. Serum-deprived cells were
treated with the compounds, as indicated, in a concentration of
1 mm for 24 h and then with [3H]thymidine (1 mCi mlꢀ1) for 4 h of
incubation. The results are expressed as the mean7s.e.m. from 12
wells in three independent experiments. *Significantly different
compared with the basal value (Po0.05; Student’s t-test).
played an IC50 value of 20 nm towards the binding sites of
[
125I]Tyr1-SST. The affinity of cCD-2 with an IC50 of
approximately 250 nm is about one order of magnitude lower
than that of SST. Thus, our results suggest a specific binding of
cCD-2 to SST receptors in SH-SY5Y cells. To obtain
indications for the SSTR subtype involved in the cCD-2
actions, we analysed the effect of cCD-2 on MAPK activity.
MAPK is differentially affected by SSTR receptor subtypes.
Among the five SST receptor subtypes known, only SSTR1
has been demonstrated to mediate a stimulation of ERK
activity (Florio et al., 1999; 2000). Indeed, a small amount of
endogenously expressed SSTR1 was immunochemically de-
tectable in SH-SY5Y cells (Figure 7a), and we observed a
stimulation of MAPK activity by SST as well as cCD-2
(Figure 7b). When both peptides were added together no
additive effect was observed, suggesting that the peptides act
via the same receptor. For comparison, the levels of SSTR1 in
COS-7 cells were measured, which are highly expressing
SSTR1 (Stetak et al., 2001). Activation of SSTR1 receptors
has also been shown to result in G1 arrest, which is specifically
induced by the cdk/cyclinDcomplex inhibitor p21 (WAF1/
Cip1) (Florio et al., 1999; 2000). When SH-SY5Y cells were
stimulated by SST or cCD-2, we detected in both cases a
higher amount of the mitotic inhibitor p21 than in non-
stimulated cells (Figure 7c). Taken together, these findings
suggest that in SH-SY5Y cells the SSTR1 receptor mediates
the cell growth-inhibiting effect of cCD-2.
Figure 3 The cyclopeptide cCD-2 is able to increase agonist
binding to m-opioid receptors. (a) Binding of the m-receptor agonist
[3H]DAMGO (0.5 nm) to rat brain membranes was measured in the
presence of increasing concentrations of cCD-2. For comparison,
the inhibition curves of [3H]DAMGO binding by unlabelled
DAMGO is shown. Inset: cCD-2 does not modulate the binding
of the d-receptor agonist [3H]DPDPE. Data for cCD-2 are
mean7s.e.m. of six independent experiments in duplicate determi-
nations. (b) cCD-2 is capable of increasing the analgesic potency of
morphine in vivo. Male Wistar rats were pretreated with either saline
(open symbols) or 1.25 mg kgꢀ1 morphine (s.c.) (filled symbols)
20 min prior to intracerebroventricular injection of the peptide. The
effect of cCD-2 was determined 40 min after peptide application as
described under Methods. The mean index of analgesia7s.e.m.
calculated for the different peptide concentrations used in two
independent series of experiments is shown. Statistical analysis was
performed using the two-way ANOVA program.
cCD-2 interacts with SST receptors of the SSTR1
subtype
The proliferation of SH-SY5Y cells is not only inhibited by
opioid peptides (Figure 5) but also by SST (Figure 6a). To
investigate a putative affinity of cCD-2 towards SST receptors,
SH-SY5Y cells were labelled with [125I]Tyr1-SST and assayed
for the competition of the bound radioligand by increasing
concentrations of cCD-2 or of SST. In Figure 6b, the
respective displacement curves are demonstrated. SST dis-
cCD-2 stimulates protein tyrosine phosphatase (PTP)
activity
PTPs have been reported to be essentially involved in the SST
receptor signalling pathways (Lopez et al., 1997; Florio et al.,
1999; 2000; Pages et al., 1999). When SH-SY5Y cells were
British Journal of Pharmacology vol 140 (1)

18
J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
Figure 6 In SH-SY5Y cells both SST and cCD-2 inhibit cell
growth and compete for [125I]Tyr1-SST binding. (a) Serum-deprived
SH-SY5Y cells were incubated with 1 mm SST or cCD-2 for 24 h,
followed by treatment with [3H]thymidine (1 mCi mlꢀ1) for 4 h. The
results are expressed as the mean7s.e.m. from 12 wells in three
independent experiments. *Significantly different compared with the
basal value (Po0.05; Student’s t-test). (b) Displacement of
[
125I]Tyr1-SST binding to SH-SY5Y cells by SST and cCD-2. Cells
were labelled with approximately 50,000 cpm [125I]Tyr1-SST and
incubated with increasing concentrations of either SST or cCD-2 for
45 min at 221C. The specific binding was determined as described
under Methods. The results are the mean7s.e.m. from two separate
experiments in quadruplicate determinations. þ Value from a single
experiment.
Figure 5 Effects of opioid receptor subtype-specific antagonists on
cCD-2-induced inhibition of DNA synthesis in SH-SY5Y cells.
Serum-starved cells were pretreated for 30 min with 1 mm of the m-
receptor-selective antagonist b-funaltrexamine hydrochloride (a),
the d-receptor-selective antagonist ICI-154,129 (b), or the k-
receptor-selective antagonist nor-binaltorphimine dihydrochloride
(c). Then, cCD-2 (100 nm) or, for comparison, 100 nm of the
respective receptor subtype-specific agonists DAMGO (m-receptors),
DADLE (d-receptors), or BRL 52537 (k-receptors) were added. The
incorporation of [3H]thymidine was measured as described under
Methods. The results are expressed as the mean7s.e.m. of 12 wells
representative of two independent experiments. *Significantly lower
compared with the basal values; þ significantly higher compared
with the basal value; **no significant difference compared with the
effect of cCD-2 (Po0.05; Student’s t-test).
treated with cCD-2, the cyclopeptide moderately but signifi-
cantly increased PTP activity in total cell lysates in a
concentration-dependent manner, as measured by the depho-
sphorylation of [32P]Raytide (Figure 8a). Corresponding with
the lower affinity of cCD-2 towards [125I]Tyr1-SST binding
sites, the total increase in PTP activity in response to cCD-2
was lower than that induced by SST. In Figure 8b, the effects
of several cCD-2 analogues on PTP activity are demonstrated.
Among them, only the cyclic peptides with Tyr(Bzl) or Tyr in
position 3 are capable of stimulating PTP activity. These are
the same derivatives that have antiproliferative activity. This
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J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
19
finding suggests that the effect of cCD-2 on PTP might be
involved in its antimitogenic action.
SHP-1 and SHP-2 were immunochemically detected in SH-
SY5Y cells and, for comparison, in COS-7 cells. In both cell
lines high levels of SHP-2 could be detected, whereas SHP-1
could only be detected in COS-7 cells but not in SH-SY5Y cells
(Figure 9c). These findings might explain why in SH-SY5Y
cells SHP-2 was selectively activated by cCD-2 or SST. Indeed,
in COS-7 cells both SHP-1 and SHP-2 were significantly
stimulated in response to SST or cCD-2 (Figure 9b). In
addition, in COS-7 cells cCD-2 is able to inhibit the EGF-
induced activation of ERKs (Figure 9d). The EGF receptor is
known to be a target of SHP-1 but not of SHP-2. These results
suggest that in SH-SY5Y cells cCD-2 stimulates SHP-2, which
is selectively coupled to the SSTR1 subtype. In dependency on
their expression patterns, cCD-2 appears to activate even other
PTPs preferentially via other SST receptor subtypes such as
SHP-1 via SSTR2 receptors.
We next investigated whether SHP-1 and/or SHP-2, which
are well-known targets of SST receptors, might be differen-
tially activated by cCD-2. For that purpose, lysates from SH-
SY5Y cells triggered with either SST or cCD-2 were subjected
to immunoprecipitation with the respective antibodies. As
shown in Figure 9a, increased PTP activity was detectable only
in immunoprecipitates of SHP-2 but not in those of SHP-1.
The expression patterns of the types of PTP may vary in
different cells. Therefore, the endogenous expression levels of
Discussion
Opioids including morphine, enkephaline-like peptides as well
as casomorphins have been demonstrated to inhibit cell growth
via opioid receptors, SST receptors or both (Maneckjee et al.,
1990; Hatzoglou et al., 1995; 1996; Kampa et al., 1996; 1997).
Here, we introduce a b-casomorphin-5-derived cyclic penta-
peptide, cCD-2, which inhibits the DNA synthesis in a variety
of tumor cell lines via SST receptors. In contrast to morphin or
antimitogenic acting opioid peptides, cCD-2 possesses only
weak affinity towards m-receptors and no affinity at d-
receptors. Most interestingly, in the presence of cCD-2,
m-receptors were sensitized towards their agonists in vitro
and in vivo. The potency of cCD-2 to induce m-receptor
sensitization strongly depends on Tyr(Bzl) in position 3,
whereas its antimitogenic activity is dependent on either
Tyr(Bzl) or Tyr in position 3 within the cyclopeptide-ring
structure. Therefore, m-opioid receptor sensitization and cell
growth inhibition in response to cCD-2 appear to be mediated
by different mechanisms.
We focussed our further investigations on the molecular
mechanism of the antimitogenic activity of cCD-2. We have
chosen the human neuroblastoma cell line SH-SY5Y as
cellular model, because these cells endogenously express opioid
receptors of the m-, d- and k-subtype (Cheng et al., 1995; Toll
et al., 1997; Wang & Sadee, 2000) as well as SST receptors
——————————————————————————
:
Figure 7 In SH-SY5Y cells, cCD-2 activates the SSTR1 receptor
signalling pathway. (a) Western blots from SH-SY5Y cell lysates or,
for comparison, COS-7 cell lysates using anti-SSTR-1 receptor
antibodies. (b) Effects of cCD-2 and SST on MAPK activity in SH-
SY5Y cells. Lysates from SH-SY5Y cells differently stimulated with
SST (10 nm), cCD-2 (100 nm), or both together were analysed for
MAPK (ERK1) activity using the myelin basic protein (MBP) assay
as described under Methods. The representative autoradiograms of
four independent experiments are shown. Control blots of im-
munoprecipitated ERK1 after Western blotting with anti-ERK1 are
presented in the panel below the MBP blot. (c) Influence of SST and
cCD-2 on the expression of the cdk inhibitor p21 (WAF1/Cip1) in
SH-SY5Y cells. Cells were treated with either SST (10 nm) or cCD-2
(100 nm) for 4 h. Then, lysates were prepared and analysed using
anti-p21 (WAF1/Cip1). A representative Western blot from three
separate experiments is shown. (b, c). *Significantly higher
compared with the basal values; þ no significant difference
compared with the single effects (Po0.05; Student’s t-test).
British Journal of Pharmacology vol 140 (1)

20
J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
(Connor et al., 1997). Furthermore, SH-SY5Y cells were most
sensitive to growth inhibition among a panel of cell lines.
The principle finding of the present study is that cCD-2 acts
via SST receptors but not via opioid receptors. This is
supported by two lines of evidence: (i) specific antagonists of
m-, d-, and k-receptors did not significantly attenuate the effect
of cCD-2 in SH-SY5Y cells, and (ii) cCD-2 specifically binds
to SST receptors in these cells. Binding studies with [125I]Tyr1-
SST reveal a single IC50 value of 20 nm for SST-14 and an IC50
value of approximately 250 nm for cCD-2. In contrast, in
T47Dcells the presence of multiple SST binding sites with IC 50
values of 0.8 and 22.6 nm has been reported (Hatzoglou et al.,
1995). The discrepancy might be explained by the use of a
different cell line and/or the use of different labels. In addition,
Figure 8 Effects of cCD-2 on PTP activity in SH-SY5Y cells. (a)
The labelled PTP substrate, [32P]Raytide, was incubated with lysates
prepared from untreated SH-SY5Y cells (basal) or from cells after
treatment with increasing concentrations of cCD-2 or, for compar-
ison, SST. (b) The [32P]Raytide assay was performed with lysates
from SH-SY5Y cells after treatment with cCD-2 (1 mm) or different
cCD-2 analogues (1 mm) as indicated. The PTPase assay was
performed as described under Methods. Data are expressed as
percentage of released [32P]Pi from the respective controls (basal).
Each value is the mean7s.e.m. of three (a) or two (b) separate
experiments in quadruplicate determinations. *Significantly differ-
ent, compared with the respective basal values (Po0.05; Student’s
t-test).
British Journal of Pharmacology vol 140 (1)

J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
21
cCD-2 is able to induce a main downstream signalling event of
SST receptors, such as the stimulation of PTP activity. As we
demonstrated in cell lysates, the dephosphorylation of the
specific PTP substrate [32P]Raytide was increased by cCD-2 in
a concentration-dependent manner and with a structure–
activity relationship that corresponds with the effects of cCD-2
analogues on DNA synthesis.
available PTPs in a given cell type. However, SSTR1 and
SSTR2 elicit cell cycle arrest (Lopez et al., 1997; Florio et al.,
1999; 2000; Pages et al., 1999). Their identification as targets of
cCD-2 suggests a cytostatic rather than a cytotoxic effect of
cCD-2. When we studied the effect of cCD-2 in an apoptosis
assay, no cleavage of poly-ADP-ribose-polymerase (PARP)
was found. In contrast, in a positive control with cytosine b-d-
arabino-furanoside, the 89 kDa PARP fragment as marker of
apoptosis was detectable (not shown). Therefore, at least in
SH-SY5Y cells, a possible apoptotic effect of cCD-2 may be
excluded.
The identification of the SSTR1 subtype to mediate the
antimitogenic effect of cCD-2 in SH-SY5Y cells was based on
three lines of evidence. First, treatment of SH-SY5Y cells with
both SST and cCD-2 specifically increased the activity of SHP-
2, but not of SHP-1. Secondly, SST as well as cCD-2
stimulated the activity of MAPK (ERK1) in SH-SY5Y cells.
The effects are not additive, suggesting that both peptides act
via the same type of receptor. Thirdly, both SST and cCD-2
increased the expression level of the cyclin-dependent kinase
(cdk) inhibitor p21 (WAF1/Cip1). All of them indicate
signalling mechanisms which have been specifically attributed
to the SSTR1 subtype (Florio et al., 1999; 2000). Indeed, SH-
SY5Y cells express SSTR1 receptors endogenously, but it
cannot be excluded that other SSTR subtypes may be also
expressed. Thus, even the existence of a SSTR2-like receptor
has been postulated in SH-SY5Y cells (Connor et al., 1997). It
may be assumed that the specific stimulation of SHP-2 by SST
as well as cCD-2 depends on the high expression level of SHP-
2 and the absence of SHP-1 in SH-SY5Y cells. This hypothesis
is supported by our finding that in COS-7 cells expressing
comparable amounts of SHP-1 as well as SHP-2, both PTPs
were equally activated by SST and cCD-2. SHP-1 belongs to
the few PTP isoforms that are able to negatively modulate
EGF receptor (reviewed in Ostman & Bohmer, 2001) and has
been attributed to the SSTR2 subtype (Lopez et al., 1997;
Pages et al., 1999). We found, indeed, that cCD-2 inhibits the
EGF-induced MAPK activation in COS-7 cells. These findings
suggest that in dependency on the cell type, cCD-2 is able to
interact with different SSTR subtypes and, subsequently,
different types of PTP. One could speculate that SSTRs may
modulate PTP activity by some general mechanism as for
example, local modulation of redox conditions (Ostman &
Bohmer, 2001). This might lead to activation of different
Taken together, in this study we present a b-casomorphin-
derived cyclopeptide as a novel type of compound with two
interesting properties: inhibition of tumor cell growth and
sensitization of m-opioid receptors towards agonists such as
morphine. Both effects are independent of each other. Whereas
the molecular mechanism of m-receptor sensitization is not yet
understood, we demonstrate that the antiproliferative effect of
cCD-2 is selectively mediated via SST receptors and their
signalling pathways. In the human neuroblastoma cell line SH-
SY5Y, the molecular mechanisms of cCD-2 action can be
related to the signalling pathway of SSTR1 subtype. In other
cells, such as COS-7, signalling events of cCD-2 were
identified, which are mediated via SSTR2 receptors. Therefore,
the signalling of cCD-2, like that of SST, appears to depend on
the cell-specific expression patterns of SSTR subtypes, of
different PTPs, and of other putative effector molecules. Thus,
cCD-2 represents a novel type of opioid peptide-derived SST
receptor agonist with low affinity towards m-receptors, but m-
opioid receptor-modulating properties that are structurally
different compared with the hitherto existing peptide SST
receptor ligands. The combination of two therapeutically
interesting properties, such as inhibition of tumor cell growth
and stimulation of the analgesic potency of morphine within
the structure of a single pentapeptide may be useful in the
development of new anticancer drugs.
We thank C. Mertens, B. Haarseim, and C. Langer for excellent
technical assistance. This work was supported by a grant from the
Stiftung Deutsche Krebshilfe to C.L. and K.N.
References
——————————————————————————
:
CHENG, J., STANDIFER, K.M., TUBLIN, P.R., SU, W. & PASTERNAK,
G.W. (1995). Demonstration of kappa 3-opioid receptors in the SH-
SY5Y human neuroblastoma cell line. J. Neurochem., 65, 170–175.
CONNOR, M., YEO, A. & HENDERSON, G. (1997). Neuropeptide Y Y2
receptor and somatostatin sst2 receptor coupling to mobilization of
intracellular calcium in SH-SY5Y human neuroblastoma cells. Br.
J. Pharmacol., 120, 455 –463.
COX, B.M., GINSBURG, M. & OSMAN, O.H. (1968). Acute tolerance to
narcotic analgesic drugs in rats. Br. J. Pharmacol., 33, 245 –256.
FLORIO, T., THELLUNG, S., ARENA, S., CORSARO, A., BAJETZTO,
A., SCHETTINI, G. & STORK, P.J. (2000). Somatostatin receptor 1
(SSTR1)-mediated inhibition of cell proliferation correlates with the
activation of MAP kinase cascade: role of the phosphotyrosine
phosphatase SHP-2. J. Physiol. (Paris), 94, 239–250.
Figure 9 Effects of SST and cCD-2 on the PTPs SHP-1 and SHP-2.
Lysates from untreated cells (basal) and SH-SY5Y cells (a) or
COS-7 cells (b) stimulated for 5 min with either 10 nm SST or 100 nm
cCD-2 were immunoprecipitated with anti-SHP-1 or anti-SHP-2
antibodies (2 mg mlꢀ1). The immunoprecipitates were then subjected
to the [32P]Raytide assay as described under Methods. The results
are expressed as a percentage of released [32P]Pi obtained with the
respective immunoprecipitates from untreated cells (basal). For
control, immunoprecipitation was performed with nonimmune
serum and assayed for dephosphorylation of [32P]Raytide. The
nonspecific release of [32P]Pi varied between 0.5 and 1.0% of the
basal values and was neglected. The values shown are the
mean7s.e.m. of six (a) or four (b) separate experiments in duplicate
determinations. *Significantly different from the respective control
values (Po0.05; Student’s t-test).). (c) Western blots from SH-SY5Y
cell lysates as well as COS-7 cell lysates using polyclonal antibodies
against SHP-1 and SHP-2. (d) COS-7 cells were stimulated with
EGF (10 nm) or costimulated with EGF (10 nm) and cCD-2
FLORIO, T., YAO, N., CAREY, K.D., DILLON, T.J. & STORK, P.J.
(1999). Somatostatin activation of mitogen-activated protein kinase
via somatostatin receptor 1(SSTR1). Mol. Endocrinol., 13, 24–37.
GRANESS, A., HANKE, H., BOEHMER, F.-D., PRESEK, P.
&
LIEBMANN, C. (2000). Protein-tyrosine-phosphatase-mediated
epidermal growth factor (EGF) receptor transinactivation and
EGF receptor-independent stimulation of mitogen-activated pro-
tein kinase by bradykinin in A431 cells. Biochem. J., 347, 441–447.
(100 nm). Lysates were assayed for MAPK activity using
a
phospho-ERK specific antibody. A Western blot from a represen-
tative experiment in duplicate determinations, which was repeated
twice with similar results, is shown.
British Journal of Pharmacology vol 140 (1)

22
J. Stirnweiss et al
A novel antiproliferative acting somatostatin receptor agonist
GREKSCH, G., SCHWEIGERT, C.H. & MATTHIES, H. (1981). Evidence
for analgesic activity of b-casomorphin in rats. Neurosci. Lett., 27,
325 –328.
MANECKJEE, R., BISWAS, R. & VONDERHAAR, B. (1990). Binding of
opioids to human MCF-7 breast cancer cells and their effects on
growth. Cancer Res., 50, 2234 –2238.
GUTKIND, J.S. (1998). The pathways connecting G protein-coupled
receptors to the nucleus through divergent mitogen-activated
protein kinase cascades. J. Biol. Chem., 273, 159 –161.
HATZOGLOU, A., BAKAGEORGOU, E., HATZOGLOU, C.,
MARTIN, P.M. & CASTANAS, E. (1996). Antiproliferative and
receptor binding properties of alpha- and beta-casomorphins in
the T47Dhuman breast cancer cell line. Eur. J. Pharmacol., 310,
217 –223.
MULDER, A.H., WARDEH, G., HOGENBOOM, F., KAZMIERSKI, W.,
HRUBY, V.J. & SCHOFFELMEER, A. (1991). Cyclic somatostatin
analogues as potent antagonists at mu-, but not delta- and kappa-
opioid receptors mediating presynaptic inhibition neurotransmitter
release in the brain. Eur. J. Pharmacol., 205, 1–6.
OSTMAN, A. & BOHMER, F.-D. (2001). Regulation of receptor tyrosine
kinase signaling by protein tyrosine phosphatases. Trends Cell.
Biol., 11, 258–266.
HATZOGLOU, A., OUAFIK, L., BAKOGEOGOU, E., THERMOS, K. &
CASTANAS, E. (1995). Morphine cross-reacts with somatostatin
receptor SSTR2 in T47Dhuman breast cancer cell line and
decreases cell growth. Cancer Res., 55, 5632 –5636.
PAGES, P., BEKALI, N., SAINT-LAURENT, N., ESTEVE, J.P.,
SCHALLY, A.V., TKACZUK, J., VAYSSE, N., SUSINI, C.
&
BUSCAIL, L. (1999). Sst2 somatostatin receptor mediates cell
cycle arrest and induction of 227 (Kip1). J. Biol. Chem., 274,
15186–15193.
HRUBY, V.J., HAN, G.
bioactivities of melanotropic peptide agonists and antagonists:
design based on conformationally constrained somatostatin
& HADLEY, M.E. (1998). Design and
PELTON, J.T., KAZMIERSKI, W., GULYA, K., YAMAMURA,
H.I. & HRUBY, V.J. (1986). Design and synthesis of confor-
a
template. Lett. Pept. Sci., 5, 117–120.
JANECK, A., ZUBBRZYCKA, M. & JANECKI, T. (2001). Somatostatin
analogs. J. Pept. Res., 58, 91 –107.
mationally constrained somatostatin analogues with high potency
and specificity for mu opioid receptors. J. Med. Chem., 29,
2370 –2375.
KAMPA, M., BAKAGEORGOU, E., HATZOGLOU, A., DAMIANSKI,
A., MARTIN, P.-M. & CASTANAS, E. (1997). Opioid alkaloids and
casomorphin peptides decrease the proliferation of prostatic cancer
cell lines (LNCaP, PC3 and DU145) through a partial interaction
with opioid receptors. Eur. J. Pharmacol., 335, 255–265.
KAMPA, M., LOUKAS, S., HATZOGLOU, A., MARTIN, P., MARTIN,
P.-M. & CASTANAS, E. (1996). Identification of a novel opioid
peptide (Tyr-Val-Pro-Phe-Pro) derived from human alpha S1 casein
(alpha S1-casomorphin, and alpha S!-casomorphin amide).
Biochem. J., 319, 903 –908.
LIEBMANN, C. (2001). Regulation of MAP kinase activity by peptide
receptor signalling pathway: paradigms of multiplicity. Cell Signal.,
13, 777–785.
LOPEZ, F., ESTEVE, J.P., BUSCAIL, L., DELESQUE, N.,
SAINT-LAURENT, N., THEVENIAU, M., NAHMIAS, C., VAYSSE,
N. & SUSINI, C. (1997). The tyrosine phosphatase SHP-1 associates
with the sst2 somatostatin receptor and is an essential component of
sst2-mediated inhibitory growth signaling. J. Biol. Chem., 272,
24448 –24454.
SCARPIGNATO, C. & PELOSINI, I. (2001). Somatostatin analogs
for cancer treatment and diagnosis: an overview. Chemotherapy, 47,
1–29.
SCHMIDT, R., NEUBERT, K., BARTH, A., LIEBMANN, C., SCHNITTLER,
M., CHUNG, N.N. & SCHILLER, P.W. (1991). Structure –activity
relationships of cyclic b-casomorphin-5 analogues. Peptides, 12,
1175 –1180.
STETAK, A., LANKENAU, A., VANTUS, T., CSERMELY, P.,
ULLRICH, A. & KERI, G. (2001). The antitumor somatostatin
analogue TT-232 induces cell cycle arrest through PKCdelta and
c-SRC. Biochem. Biophys. Res. Commun., 285, 483 –488.
TOLL, L., POLGAR, W.E. & AUH, J.S. (1997). Characterization of the
delta-opioid receptor found in SH-SY5Y neuroblastoma cells. Eur.
J. Pharmacol., 323, 261 –267.
WANG, Z.
& SADEE, W. (2000). Tolerance to morphine at the
mu-opioid receptor differentially induced by cAMP-dependent
protein kinase activation and morphine. Eur. J. Pharmacol., 389,
165–171.
LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J.
(1951). Protein measurement with the Folin phenol reagent. J. Biol.
Chem., 193, 265 –275.
(Received March 31, 2003
Revised May 26, 2003
Accepted June 16, 2003)
British Journal of Pharmacology vol 140 (1)