Morphine Cross-Reacts with Somatostatin Receptor SSTR2 in the T47D Human Breast Cancer Cell Line and Decreases

Cell Growth
Anastassia Hatzoglou, L'Houcine Ouafik, Efstathia Bakogeorgou, et al. Cancer Res 1995;55:5632-5636.

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ICANCER RESEARCH55. 5632-5636. December 1, 19951

Morphine Cross-Reacts with Somatostatin Receptor SSTR2 in the T47D Human Breast Cancer Cell Line and Decreases Cell Growth'
Anastassia Hatzoglou,2 L'Houcine Ouafik, Efstathia Bakogeorgou, Kyriaki Thermos, and Elias Castanas
Laboratories of Experimental Endocrinology (A. H., E. B., E. C] and Phannacology (K. TI, University of Crete, School of Medicine, and University Hospital, P.O. Box 1393, Heraklion GR-71 I 10, Crete, Greece, and Laboratoire de Cancerologie Ezperimentale, CJF INSERM 93-1 1 (A. H.. E. C.], and Neuroendocrinologie Experimentale, U 297 INSERM IL 0.1, Facult de Medecine Nord, Marseille, France

ABSTRACT In a previous study, we found that morphine decreases, in a dose dependent manner, the cell growth of T47D human breast cancer cells, despite the lack of p opioid receptors and an interaction of morphine with other opioid sites. We have therefore examined a possible interaction of morphine with other membrane receptor systems of the cell. The present study describes for the first time an interaction between p-acting opioid drugs and the somatostatinergic system. We have found that [tasI]Tyr1t@ somatostatin binds with high affinity to T47D cells. Analysis ofthe binding data showed the presence of two components: one with hinjaaffinity but low capacity (Kd,0.145 viM;1450 sites/cell), and another of lower affinity but higher capacity (Kd,l.l92 nM; 11920 sites/cell). Somatostatin-14 and somatostatin-28 showed multiphasic displacement curves, indicating het
erogeneity of binding sites. The latter was confirmed by reverse transcrip

act with the SSTR system, providing a possible explanation for their observed inhibitory effect on cell growth. MATERIALS AND METHODS
was obtained at passage 90.

Cell Cultures. The human breast cancer cell line T47D (originally isolated
from a pleural effusion of breast adenocarcinorna)

Cells were routinely grown in RPMI, supplemented with 10% heat-inactivated fetal bovine serum. They were cultured at 37C in a humidified atmosphere of
5% CO2 in air.

Somatostatin

Binding

Conditions.

About 106 cells/well

in monolayer
in PBS

were used for saturation and displacement binding experiments. Before bind
ing, cells were washed binding) twice with 2 ml of PBS. Binding was performed

in a total volume of 0.5 ml, containing

or with (nonspecific binding)

2]l
a 1000-fold

somatostatin, without (total

molar excess of somatosta

tion-PCR, which revealed the existence of the somatostatin receptor sub

types 2 and 3 (SSTR2 and SSTR3), with a relative mRNA concentration

of 85 and 15%, respectively. Morphine and the morphinomimetic peptide morphiceptine (Tyr-Pro-Phe-Pro-NH2) displace somatostatin from its binding sites. Further analysis indicated that p-acting opioids interact with the SSTR2 receptor subtype.

tin-14. For saturation binding, at least 810 points with different concentra ions of radiolabeled peptide were performed in duplicate. The cells were incubated for 2 h at room temperature (1822C). At the end of the incubation
period, the unbound radioactivity was eliminated by washing the cells twice

INTRODUCTION The somatostatinergic and opioid systems are inhibitory and are involved in the decrease of hormone and neurotransmitter secretion (16). Furthermore, they have been implicated in the control of tumor growth in different organs, including breast (1, 4, 713).Indeed, somatostatin and opioid immunoreactivities have been detected in tumor, but not in normal breast cells (3, 14, 15), and specific receptors have been characterized in primary human breast tumors and tumor cell lines (4, 12, 13, 1623), producing a dose-dependent inhibition of cell proliferation. Recently, we have characterized and K opioid receptors in the T47D cell line and have shown that different opioids inhibit cell proliferation (22). No opioid binding sites were found, however, whereas morphine (the prototype @.t ligand) shows a dose-dependent inhibitory effect on the growth of cells, which is not reversed by the opioid antagonist diprenorphine. Morphine does not cross-react with any other opioid receptor subtype (@ or K). Our results suggest a nonopioid-mediated action of morphine and morphiceptine. Because somatostatin and opioid receptors belong to the same membrane protein superfamily (5, 6), we have investigated a possible interaction of morphine with the somatostatinergic system. In the present work, we have characterized the SSTRs3 in the T47D cell line, both pharmacologically and by RT-PCR. We further report that morphine and the morphinomimetic peptide morphiceptine inter
Received 5/2/95; accepted 8/31/95.

with 2 ml cold PBS. Cells were removed from plates with 0.4 ml 2 N NaOH, and the bound radioactivity was counted in a -y counter (Tncarb Series; Packard), with a 95% efficiency for 1251, Binding was repeated at least three times, and the results were analyzed by the Origin Version 3.5 package (MicroCal Co.) using equations described by Munson and Rodbard (24). Cell Growth Conditions. Cells were platedin 24-well ELISAplates at an initial density of25 X l0@ cells/well supplemented with 1 ml medium/well. All
drugs were added to cultures 1 day after seeding (designated as day 0) to

ensure uniform attachment of cells at the onset of the experiments. Cells were grown for a total of4 days, with daily change of the medium containing opioid
drugs or somatostatin analogues. All added drugs were dissolved shortly before

use. Cell Proliferation. Cell growthwas measuredby the tetrazoliumsaltassay (25). Cells were incubated for 4 h at 37C with the tetrazoliurnsalt [3-(4,5dimethylthiazol

@ @

2-yl)-2,5-diphenyl tetrazolium bromide], and metabolically active cells re duced the dye to purple formazan. Dark blue crystals were dissolved with propanol. The absorbance was measured at 570 nm and compared against a standard curve of known numbers of T47D cells. All experiments were performed a minimum of three times, in triplicate. Detection of SSTR mRNA by RT-PCR. Total RNA was preparedfrom the T47D cell line using the guanidinium thiocyanate/phenol/chloroform pro cedure (26). Total RNA (5 @xg) from T47D cells was reverse transcribed into cDNA using 1 @xg oligo(dT)12_18 as primer in 20-pi reaction volume [50 m@i
Tris (pH 8.0)-75 RNasin-0.5 mM KC1-5 mM MgC12-5 @xM DIT-SO @xg/mlBSA-20 murine units of leukemia mM each of four dNTPs-400 units of Moloney

virus reverse transcriptase) at 37C for 90 mm. A negative control was perfonned with the first strand synthesis reaction and where the RNA sample

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.
I This work was partially supported by the University of Crete Research Committee,

was omitted. RT mixtures without reverse transcriptase were included as controls for each sample to detect any genomic DNA contamination. After the incubation, cDNA derived from 500 ng total RNA was amplified in a 50-pJ reaction containing 50 mMKC1, 10 mMTris (pH 8.3), 1.5 mMMgCl2, 10 nmi Dli', 1 mMof each oligonucleotide, and 2.5 units of Taq DNA polymerase. Optimal temperatures and cycling conditions were established for all five SSTRs, as follows: SSTR1, denaturation at 95C for S mm, and then 35 cycles at 95C for 60 s, 65C for 90 s, and 72C for 120 s [sense, 5'-AAA-TGC GTC-CCA-GAA-CGG-GAC-CT-(C)-3'; antisense, 5'-CAG-GTf-CTC
AGQ-TTG-GAA-GTC-TT-(C)-3']; SSTR2, denaturation at 95C for S mm, antisense, 5'-

Ministry of Health (KESY), and Hellenic Anticancer Society grants.

2 To 3 The whom requests for reprints should be addressed. receptor; RT-PCR, reverse transerip abbreviations used are: 55Th, somatostatin

and then 35 cycles at 95C for 60 s, 60C for 90 s, and 72C for 120 s (sense,
5'-CAT-ATA-GGA-TAG-GQT-TGG-CAC-AGC-TGT-T-3';

tion-PCR; BIM 23034C. o-Phe-c[Cys-Tyr-o-Trp-Lys-Val-Cys]Nal-NH2.

5632

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MORPHINE AND SSTRs IN T47D BREAST CANCER CELLS

TAC-ATC-CTC-AAC-CTG-GCC-ATC-GCA-GAT-GA-3'); SSTR3, denatur

ation at 95C for 5 mm, and then 40 cycles at 95C for 60 s and 67C for 4 mm (sense, 5'-AGA-ACG-CCC-TGT-CCT-ACT-GGC-C-3'; antisense, 5'-TGA

AGC-GGT-AGG-AGA-GGA-AGC-C-3'); SSTR4, denaturation at 95C for S mm, and then 35 cycles at 95C for 60 s, 60C for 90 s, and 72C for 120 s
(sense, 5'-ATG-GTC-GCT-ATC-CAG-TGC-3'; antisense, 5'-GGG-CTC

CTC-AGA-AGG-TGG-T-3'); and SSTR5, denaturation at 95C for S mm, and

then 40 cycles at 95C for 40 s, 65C for 30 s, and 72C for 90 s (sense,

5'-CTC-TFG-GTG-TFC-GCG-GAC-GT-3'; antisense, 5'-CAG-GTF-GAC GAT-GTF-GAC-GGT-GAA-G-3'). Southern Blots. Twenty pA of the PCR product were fractionatedon agarose gels in TBE buffer (89 mMTris, 2 mMEDTA). After staining with
ethidium Kingdom). hybridization was obtained diprenorphine cals (Hanover, zerland). bromide, the gels were to a Hybond-N photographed, membrane sequence Morphine and then treated (Amersham, was checked and trans United blot ferred by capillarity Amersham,

The specificity

of the PCR product

by Southern

by using an internal from Amersham.

as probe for all SSTRs was a gift from Co. Cell culture

separately. and

Radiochemicals and Chemicals. [ 251

was from Reckit and Coleman Germany). Sandostatin

-somatostatin (2000 Cilmmol)

Farmacopia, media were from (Basel, Swit MO).

GIBCO. The sornatostatin peptides were obtained from Bissendorff Biochemi

was a gift from Sandoz Chemical All other peptides were from Sigma Co. (St. Louis,

All chemicals were from Merck (Darmstad, Germany). Molecular Biology products were from Boehringer Mannheim (Mannheim, Germany).

RESULTS Identification and Characterization of SSTRS on T47D Breast Cancer Cells

Characterization of Binding Sites. Fig. 1 presents the binding isotherm of [251]Tyr' -somatostatinto whole T47D cells. A biphasic saturation curve was obtained with a high-affinity component reach ing saturation (Fig. 1, inset A) and a low-affinity component that did not reach saturation. Nonspecific binding was 27 5% of total binding. Analysis of the data in Scatchard coordinates (Fig. 1, inset B) showed that the binding isotherm could be best fitted in a two-site model, with affinities of 0.145 0.017 and 1.192 0.093 nr@i, and binding capacities of 1450 235 and 11920 1147 sites/cell. This heterogeneity of binding of [251]Tyr' -somatostatincould indicate

the presence of multiple somatostatin binding sites in T47D breast cancer cells. The specificity of the [251]Tyr' somatostatin binding to SSTRs is shown in Fig. 2A. Displacement of [251}Tyr' -somatostatin from its receptor sites by somatostatin-14 and somatostatin-28, at concentrations ranging from l0 to lO_6 M, produced parallel displacement curves, indicating an interaction with the same re ceptor populations. Both curves were multiphasic, suggesting, as did the saturation data, the presence of multiple somatostatin binding sites. No displacement was found by corticotrophin-releas ing hormone. On the contrary, the displacement of [25I]Tyr'somatostatin-14 by BIM 23034C, a selective SSTR2 analogue, produced a monophasic displacement curve (Fig. 2B) and depicted an IC50 of 5 nM. Comparing Figs. 2A and 2B, it appeared that the first site detected in the somatostatin-l4 and somatostatin-28 dis placement curves, and with IC50s of 0.8 and 0.9 nM, respectively, is the SSTR2 subtype. This was further confirmed by displacement of [ -somatostatin by somatostatin- 14 and somatosta tin-28 in the presence of BIM 23034C (not shown). These data are summarized in Table 1. Characterization of SSTR Subtypes by RT-PCR. To analyze the heterogeneity of the binding sites, we have investigated further the identity of the SSTR subtypes present in T47D cells by performing an analysis of their mRNA by RT-PCR. As shown in Fig. 3, in these cells, only SSTR2 and SSTR3 mRNA were found. The most promi nent receptor mRNA found in T47D cells was SSTR2, representing about 85% of the whole SSTR mRNA. Effect of p-acting Opioids and Somatostatin on Cell Growth

Effect of il-acting opioids. Fig. 4 shows the inhibition of cell proliferation of T47D human breast cancer cells by morphine and the morphinomimetic peptide morphiceptine. Both p.-acting drugs inhibit cell proliferation in a dose-dependent manner, with apparent IC50s of 1.08 and 1.25 nM, respectively. This effect was not inhibited by the opioid antagonist diprenorphine.

15000
--

rn

....

ecx,
@ ..,........

U) w C, I 0000
Fig. 1. Binding of [25IlTyr' -somatostatin to T47D cells. Saturation binding was performed for 2 h at room temperature on whole cells, as indicated in Materialsand Methods.The saturation binding isotherm is presented. A, magnification of the mi tial part of the curve; B, representation of data in Scatchard coordinates.

2t@
0

.s@
@. I I I

0
Cs)

2500

5O1X@mOO 10000
2.0

ru
i.e @0

E
0. S
0

5000

u_ 1.0
0.5 0

@T'@@: a
@n i@m 16000
Bound (pmclesllO c&1s)

0 0 50000 100000

Total (pmoles)
5633

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MORPHINE ANDSSTRsIN T47D BREASTCANCERCELLS

A
100!

I-.

C,)

3
0

C,)

40.

20

@[1@
3 4 5

SSTRtype
Fig. 3. Expression of SSTR mRNA in T47D human breast cancer cells by RT-PCR. Results of RT-PCR of the five 55Th mRNA in T47D cells. Sec Materials and Methods

10.11

10b0

i-@

10.8

j@-l

10.

for details. Results are expressed as a percentage of the whole SSTR mRNA detected.

Displacer
Fig. 2. Specificityof [25I]Tyr' -somatostatinbindingon T47D cells. Displace
ment of [25lJTyr' -somatostatin by different analogues. A, 12 fmol of [25I]Tyr' somatostatin (about 50,000 cpm) were incubated with the indicated concentrations of corticotrophin-releasing hormone (A), somatostatin-14 (s), or somatostatin-28 (); B, incubated with BIM 23034C, a selective SSTR2 agonist. Points, mean of three

L@1
.I-@

experiments in triplicate; bars, SEM. See Materials and Methods for details of the
binding experiments.

TableI IC50ofdifferent substances on somatostatin receptorsof T47Dcells

Data were obtained from results presented in Fig. 3.

(nM)Somatostatin SubstanceIC@
140.822.6Somatostatin 280.96.37BIM 23034C5.05

1 (nM)IC50

@,
1@

x
I-

0.60.5 0.4 0.3 0.2 0.1

0 -12 -11

Effect of Somatostatin-28 and Sandostatin. Fig. 5 presents the effects of somatostatin-28 and SMS 201-995 (Sandostatin, a stable somatostatin analogue) on cell proliferation. Both agents produce a dose-dependent inhibition on cell growth. It was of interest that maximal suppression of the growth of the cultures was obtained at the concentration of 0.1 ni@i. At higher concentrations, the inhibition of cell growth was reversed. The concomitant addition of morphine and somatostatin did not result in an additive effect, indicating that, probably, both substances might share the same receptor site (not shown). Cross-reaction of Morphine with SSTRS

z
C,

1,11:1.11
-10 -9 -8 -7 -6

LogOploid To evaluate the possibility that morphine, morphiceptine, and so matostatin interact with the same population of receptor sites, we Fig. 4. Effect of @z-acting opioids on cell proliferation of T47D cells. Opioid agonists examined the ability of morphine and morphiceptine to displace (morphine in A and morphiceptine in B) were applied alone in the indicated concentrations (filled columns) or in the presence of l06 M diprenorphine (shaded columns) on T47D [25I]Tyr' -somatostatinbinding in T47D cells. Morphiceptine shares cells for 4 days, with daily change of the medium. Cell proliferation was estimated by the the same spectrum as morphine on opioid binding sites in other tetrazolium salt assay, as indicated in Materials and Methods.Columns, mean of four experiments in triplicate; bars, SEM. systems. Our results are presented in Fig. 6. It can be seen that both 5634
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MORPHINE AND SSTRs IN T47D BREAST CANCER CELLS

-12

-11

-10

Log Peptide
Fig. 5. Effect of somatostatin analogues on cell proliferation of T47D cells. Soma tostatin-28 (SS28, shaded columns) and SMS 201-995 (Sandostatin, filled columns) were applied in the indicated concentrations on T47D cells for 4 days, with daily change of the medium. Cell proliferation was estimated by the tetrazolium salt assay, as indicated in Materials and Methods.Columns, mean of three experiments in triplicate; bars, SEM.

morphine
[1

and morphiceptine
1-somatostatin in

displace

the specific
and

binding
man

of

a dose-dependent

monophasic

ner, similar to BIM 23034C (compare Figs. 2B and 6), with IC50s of 1.67 and 1.71 flM, respectively. DISCUSSION Various breast cancer cell lines have been found to express SSTRs (1 1, 12, 20, 21). Addition of somatostatin or somatostatin analogues in culture media produces a dose-dependent inhibition of cell growth (1 1, 12, 21). In addition, 30% of breast cancers express somatostatin immunoreactivity; preneoplastic lesions express less, whereas normal tissue does not show any somatostatin immunoreactivity (14). From the five cloned subtypes of the SSTR, only SSTR2 was found in 5 primary breast tumors, whereas both SSTR2 and SSTR3 were ex pressed in 1 case (16). Furthermore, three breast cancer cell lines with distinct steroid and growth factor receptor patterns (i.e., MCF7, MDA-MB23 1, and ZR-75- 1) express only the SSTR2 subtype, which seems to be the most widely expressed SSTR in different human and rodent tumors or tumor cell lines (27). Using a chemical cross-linking assay, four distinct sites have been identified in the MCF7 cell line in monolayer, one of which binds the somatostatin analogue BIM 23014C (20). In the same study, BIM-23014C identifies one site in MCF7 and T47D cells, three in MDA-MB23 1, and none in the HBL 100 cell line. Finally, the BIM-23014C somatostatin analogue iden tifies at least 1 site in 90% of 30 human breast biopsies. The percent age of somatostatin-positive tumors varies between 14 and 48% (13,

further substantiated in the present study by using RT-PCR, showing the expression of SSTR2 (85%)and SSTR3 ( 15%) in T47D cells. Previous investigations have shown that inhibition of cell prolifer ation by somatostatin analogues might be mediated by SSTR sybtypes SSTR1 and SSTR2 (30). No SSTR1 has been found in the present study. On the contrary, somatostatin-28 and sandostatin, sharing the same affinity for the SSTR2 subtype, decrease the proliferation in T47D cells by the same extent (50 and 55%, respectively), indicating a probable involvement of SSTR2 on the cell growth of T47D cells. The action of somatostatin on cell growth is biphasic. Indeed, maxi mal suppression of growth was obtained at 0. 1 flM.At higher concen trations, the inhibition of cell growth is reversed. The same result has been reported previously on the MCF7 breast cancer cell line by somatostatin (12), antiestrogens (31), and epidermal growth factor (32). Although the underlying mechanism of this biphasic effects is not known, different hypotheses have been proposed, implicating either a desensitization/intemalization/conformational change of the receptor or a possible agonist/antagonist effect of the agents. In a previous study, we have shown that T47D cancer cells express and K opioid receptors. Different opioid agonists (ED-Ala2, D-Leu5]enkephalin, [D-Ser@,Leu5]-enkephalin, Thr6 etorphine, and ethylke tocyclazocine) selective toward different subtypes of the opioid re ceptor produce a dose-dependent, reversible, inhibitory effect on cell proliferation. Morphine, the p-selective agonist, presenting no affinity for the other opioid receptor subtypes in the T47D cells, produces a major inhibition of cell proliferation, whereas no @t opioid receptors have been detected (22). In the present work, we show that this effect is shared by the amidated tetrapeptide morphiceptine (derived from the enzymatic degradation of j3 casein), which shares, in other sys tems, the same pharmacological selectivity as morphine. In view of the structural homology between the opioid and the SSTRs, which were proposed to derive from the same ancestor gene (5, 6), we have investigated a possible interaction of morphine with the somatostatin

C,

2830). This percentage becomes67% when only small tumors are

selected (29), and 90% with the use of the chemically cross-linked somatostatin analogue and electrophoresis (20). The existence of SSTRs has been reported to be of prognostic value for the evolution of breast cancer (19). The results of the present study indicate the presence of multiple SSTR subtypes in T47D human breast cancer cells. Scatchard analysis of [25IIJTyr' -somatostatin-14 binding identified two components: one with high affinity but low capacity (Kd, 0. 145 revs;1450 sites/cell), 0 @t0.h1 lcTbO 10-s 10@8 and another oflower affinity but higher capacity (Kd, 1.192 nr@i; 11920 sites/cell). These results are comparable with those reported by Log Oploid Seytono-Ham et a!. (12) and Prevost et al. (20) on MCF7 cells for the Fig. 6. Displacement of [1251]Tyrt -somatostatin from its binding sites by jx-acting high-affinity site, and by Weckbecker et al. (21) on the ZR-75-1 opioids. Displacement of [@I]Tyr' -somatostatinbinding by the indicated concentrations breast cancer cell lines for the lower affinity site. These results were of morphine (B) or morphiceptine (). See Materials and Methods for details. 5635

C,)

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MORPHINE AND SSTRs IN T47D BREAST CANCER CELLS

ergic system. It was surprising that cross-displacement experiments of somatostatin by morphine and morphiceptine indicated that these opioids compete for somatostatin binding in a similar manner, inhib iting cell proliferation through an interaction with SSTR2. The expo sure of cells to both somatostatin and morphine does not produce additive effects, enhancing the assumption that these substances might interact with the same receptor site. From these data, we have con cluded that morphine may exert its antiproliferative action through SSTR2. Previous studies have indicated a possible interaction between somatostatin analogues and the opioid receptor system. Indeed, Maurer et a!. (33) and Walker et a!. (34) have reported that the octapeptide analogues of somatostatin act, with high affinities, as antagonists to @.t opioid receptors in the central nervous system. Although the high degree of homology between the opioid and SSTRs suggests that some ligands might be able to interact with both recep tors (6), to our knowledge, no cross-reaction of opiates with SSTRs has been described previously. Furthermore, from our studies and those of others (30), it appears that SSTR2 may be responsible for the mediation of the observed antiproliferative effect. Together, the data presented here provide evidence for a functional interaction of the two major inhibitory neuroendocrine systems (opioids and somatostatin), which may involve peptide competition and may have implications for the biology and pathophysiology of tumor cells and disease states. In addition, the fact that morphiceptine, a casomorphin produced from

effects of somatostatin

(analogues)

on the growth of human breast cancer cells.

Cancer Res., 47: 1566-1570, 1987.

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551558, 1987. 14. Ciocca, D. R., Puy, L. A., Fasoli, L. C., Tello, 0., Aznar, J. C., Gago, F. E., Papa, S. I.,

and Sonego, R. Corticotropin releasing hormone, luteinizing hormone-releasing hor mone, growth hormone-releasing hormone, and somatostatin-like immunoreactivities
in biopsies from breast cancer patients. Breast Cancer Res. Treat.. 15: 175-184, 1990. 15. Scopsi, L., Balslev, E., Brunner, N., Skovgaart-Poulsen, H., Andersen, J., Rank, F.,

and Larson, L-I. Immunoreactive opioid peptides in human breast cancer. Am. J.
Pathol., 134:473479, 1989. 16. Reubi, J. C., Schaer, J. C., Waser, B., and Mengod, G. Expression and localization of somatostatin receptor SSTRI, SSTR2, and SSTR3 messenger RNA in primary human tumors using in situ hybridization. Cancer Res., 54: 34553459, 1994. 17. Reubi, J. C., Krenning, E., Lamberts, S. W. J., and Kvols, L. In vitro detection of somatostatin receptors in human tumors. Metabolism, 41 (Suppl. 2): 1041 10, 1992. 18. Srkalovic, G., Cai, R. Z., and Schally, A. V. Evaluation of receptors for somatostatin in various tumors using different analogs. J. Clin. Endocrinol. Metab., 70: 661669,

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19. Foekens, J. A., Portengen, H., van Putten,W. L. J., Trapman.A. M. A. C., Reubi,
J-C., Alexieva-Figusch, J., and Klijn, J. G. M. Prognostic value of receptors for insulin-like growth factor I, somatostatin, and epidermal growth factor in human breast cancer. Cancer Res., 49: 70027009, 1989. 20. Prevost, G., Lanson, M., Thomas, F., Veber, N., Gonzalez, W., Beaupain, R., Stance,

A., and Bogden,A. Molecular heterogeneity of somatostatin analogueBIM-230'4C

receptors in human breast carcinoma cells using the chemical cross-linking assay. Cancer Res., 52: 843850, 1992. 21. Weckbecker, G. Liu, R., Tolcsvai, L., and Bruns, C. Antiproliferative effects of the somatostatin analogue octreotide (SMS 201995)on ZR-75-l human breast cancer cells in vivo and in vitro. Cancer Res., 52: 49734978, 1992. 22. Hatzoglou, A., Bakogeorgou, E., and Castanas, E. The antiproliferative effect of opioid agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors. Eur. J. Pharmacol., in press, 1995. 23. Maneckjee, R., Biswas, R., and Vonderhaar, B. K. Binding of opioids to human MCF-7 breast cancer cells and their effects on growth. Cancer Res., 50: 22342238,

13 casein(a normalproductof breastepithelium),bindsto SSTRsand

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