ICANCER RESEARCH55. 5632-5636. December 1, 19951

ABSTRACT

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

tion-PCR, which revealed the existence of the somatostatin receptor subtypes 2 and 3 (SSTR2 and SSTR3), with a relative mRNA concentrationof 85 and 15%, respectively. Morphine and the morphinomimetic peptidemorphiceptine (Tyr-Pro-Phe-Pro-NH2) displace somatostatin from itsbinding sites. Further analysis indicated that p-acting opioids interactwith the SSTR2 receptor subtype.

INTRODUCTION

The somatostatinergic and opioid systems are inhibitory and areinvolved in the decrease of hormone and neurotransmitter secretion(1—6).Furthermore, they have been implicated in the control of tumorgrowth in different organs, including breast (1, 4, 7—13).Indeed,somatostatin and opioid immunoreactivities have been detected intumor, but not in normal breast cells (3, 14, 15), and specific receptorshave been characterized in primary human breast tumors and tumorcell lines (4, 12, 13, 16—23),producing a dose-dependent inhibition ofcell proliferation.

Recently, we have characterized@ and K opioid receptors in theT47D cell line and have shown that different opioids inhibit cellproliferation (22). No@ opioid binding sites were found, however,whereas morphine (the prototype @.tligand) shows a dose-dependentinhibitory effect on the growth of cells, which is not reversed by theopioid antagonist diprenorphine. Morphine does not cross-react withany other opioid receptor subtype (@ or K). Our results suggest anonopioid-mediated action of morphine and morphiceptine. Becausesomatostatin and opioid receptors belong to the same membraneprotein superfamily (5, 6), we have investigated a possible interactionof morphine with the somatostatinergic system.

In the present work, we have characterized the SSTRs3 in the T47Dcell line, both pharmacologically and by RT-PCR. We further reportthat morphine and the morphinomimetic peptide morphiceptine inter

Received 5/2/95; accepted 8/31/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

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

Ministry of Health (KESY), and Hellenic Anticancer Society grants.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: 55Th, somatostatin receptor; RT-PCR, reverse transerip

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

act with the SSTR system, providing a possible explanation for theirobserved inhibitory effect on cell growth.

MATERIALS AND METHODS

Cell Cultures. The human breast cancer cell line T47D (originally isolatedfrom a pleural effusion of breast adenocarcinorna) was obtained at passage 90.Cells were routinely grown in RPMI, supplemented with 10%heat-inactivatedfetal bovine serum. They were cultured at 37°Cin a humidified atmosphere of5% CO2 in air.

Somatostatin Binding Conditions. About 106 cells/well in monolayerwere used for saturation and displacement binding experiments. Before binding, cells were washed twice with 2 ml of PBS. Binding was performed in PBS

in a total volume of 0.5 ml, containing 2]l somatostatin, without (totalbinding) or with (nonspecific binding) a 1000-fold molar excess of somatosta

tin-14. For saturation binding, at least 8—10points with different concentraions of radiolabeled peptide were performed in duplicate. The cells wereincubated for 2 h at room temperature (18—22°C).At the end of the incubationperiod, the unbound radioactivity was eliminated by washing the cells twice

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 threetimes, 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 ELISAplatesat aninitial density of25 X l0@cells/well supplemented with 1ml medium/well. Alldrugs 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 weregrown for a total of4 days, with daily change of the medium containing opioiddrugs or somatostatin analogues. All added drugs were dissolved shortly before

use.Cell Proliferation. Cell growthwas measuredby thetetrazoliumsaltassay

(25). Cells were incubated for 4 h at 37°Cwith the tetrazoliurnsalt [3-(4,5-dimethylthiazol

2-yl)-2,5-diphenyl tetrazolium bromide], and metabolically active cells reduced the dye to purple formazan. Dark blue crystals were dissolved withpropanol. The absorbance was measured at 570 nm and compared against astandard curve of known numbers of T47D cells. All experiments wereperformed a minimum of three times, in triplicate.

Detection of SSTR mRNA by RT-PCR. Total RNA was preparedfromthe T47D cell line using the guanidinium thiocyanate/phenol/chloroform procedure (26). Total RNA (5 @xg)from T47D cells was reverse transcribed intocDNA using 1 @xgoligo(dT)12_18as primer in 20-pi reaction volume [50 m@iTris (pH 8.0)-75 mM KC1-5 mM MgC12-5 @xMDIT-SO @xg/mlBSA-20 units of

RNasin-0.5 mM each of four dNTPs-400 units of Moloney murine leukemia

virus reverse transcriptase) at 37°C for 90 mm. A negative control was

perfonned with the first strand synthesis reaction and where the RNA sample

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

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

5632

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 297INSERM IL 0.1, Facultéde Medecine Nord, Marseille, France

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TAC-ATC-CTC-AAC-CTG-GCC-ATC-GCA-GAT-GA-3'); SSTR3, denaturation at 95°C for 5 mm, and then 40 cycles at 95°C for 60 s and 67°C 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 95°Cfor Smm, and then 35 cycles at 95°Cfor 60 s, 60°Cfor 90 s, and 72°Cfor 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 95°Cfor S mm, andthen 40 cycles at 95°C for 40 s, 65°C for 30 s, and 72°C for 90 s (sense,

5'-CTC-TFG-GTG-TFC-GCG-GAC-GT-3'; antisense, 5'-CAG-GTF-GACGAT-GTF-GAC-GGT-GAA-G-3').

Southern Blots. Twenty pA of the PCR product were fractionatedonagarose gels in TBE buffer (89 mMTris, 2 mMEDTA). After staining withethidium bromide, the gels were photographed, and then treated and trans

ferred by capillarity to a Hybond-N membrane (Amersham, Amersham, United

Kingdom). The specificity of the PCR product was checked by Southern blot

hybridization by using an internal sequence as probe for all SSTRs separately.

Radiochemicals and Chemicals. [‘251 -somatostatin (2000 Cilmmol)was obtained from Amersham. Morphine was a gift from Farmacopia, and

diprenorphine was from Reckit and Coleman Co. Cell culture media were from

GIBCO. The sornatostatinpeptides were obtained from Bissendorff Biochemicals (Hanover, Germany). Sandostatin was a gift from Sandoz (Basel, Swit

zerland). All other peptides were from Sigma Chemical Co. (St. Louis, MO).

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

RESULTS

Identification and Characterization of SSTRS on T47D BreastCancer Cells

Characterization of Binding Sites. Fig. 1 presents the bindingisotherm of [‘251]Tyr'‘-somatostatinto whole T47D cells. A biphasicsaturation curve was obtained with a high-affinity component reaching saturation (Fig. 1, inset A) and a low-affinity component that didnot reach saturation. Nonspecific binding was 27 ±5% of totalbinding. Analysis of the data in Scatchard coordinates (Fig. 1, inset B)showed that the binding isotherm could be best fitted in a two-sitemodel, with affinities of 0.145 ±0.017 and 1.192 ±0.093 nr@i,andbinding capacities of 1450 ±235 and 11920 ±1147 sites/cell. Thisheterogeneity of binding of [‘251]Tyr'‘-somatostatincould indicate

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the presence of multiple somatostatin binding sites in T47D breastcancer cells.

The specificity of the [‘251]Tyr'â€s̃omatostatin binding to SSTRsis shown in Fig. 2A. Displacement of [‘251}Tyr'‘-somatostatinfrom its receptor sites by somatostatin-14 and somatostatin-28, atconcentrations ranging from l0 ‘â€t̃o lO_6 M, produced paralleldisplacement curves, indicating an interaction with the same receptor populations. Both curves were multiphasic, suggesting, asdid the saturation data, the presence of multiple somatostatinbinding sites. No displacement was found by corticotrophin-releasing 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 depictedan IC50 of 5 nM. Comparing Figs. 2A and 2B, it appeared that thefirst site detected in the somatostatin-l4 and somatostatin-28 displacement curves, and with IC50s of 0.8 and 0.9 nM, respectively,is the SSTR2 subtype. This was further confirmed by displacementof [‘ ‘-somatostatin by somatostatin- 14 and somatostatin-28 in the presence of BIM 23034C (not shown). These data aresummarized in Table 1.

Characterization of SSTR Subtypes by RT-PCR. To analyze theheterogeneity of the binding sites, we have investigated further theidentity of the SSTR subtypes present in T47D cells by performing ananalysis of their mRNA by RT-PCR. As shown in Fig. 3, in thesecells, only SSTR2 and SSTR3 mRNA were found. The most prominent receptor mRNA found in T47D cells was SSTR2, representingabout 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 cellproliferation of T47D human breast cancer cells by morphine and themorphinomimetic peptide morphiceptine. Both p.-acting drugs inhibitcell proliferation in a dose-dependent manner, with apparent IC50s of1.08 and 1.25 nM, respectively. This effect was not inhibited by theopioid antagonist diprenorphine.

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SubstanceIC@ 1 (nM)IC50 2(nM)Somatostatin140.822.6Somatostatin280.96.37BIM

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Results of RT-PCR of the five 55Th mRNA in T47D cells. Sec “Materialsand Methods―for details. Results are expressed as a percentage of the whole SSTR mRNA detected.

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DisplacerFig.2. Specificityof [‘25I]Tyr'-somatostatinbindingon T47D cells. Displace

ment of [‘25lJTyr'‘-somatostatinby different analogues. A, 12 fmol of [‘25I]Tyr'‘-somatostatin (about 50,000 cpm) were incubated with the indicated concentrations ofcorticotrophin-releasing hormone (A), somatostatin-14 (s), or somatostatin-28 (•);B, incubated with BIM 23034C, a selective SSTR2 agonist. Points, mean of threeexperiments in triplicate; bars, SEM. See “Materialsand Methods―for details of thebinding experiments.

TableI IC50ofdifferentsubstanceson somatostatinreceptorsof T47DcellsData were obtained from results presented in Fig. 3.

Effect of Somatostatin-28 and Sandostatin. Fig. 5 presents theeffects of somatostatin-28 and SMS 201-995 (Sandostatin, a stablesomatostatin analogue) on cell proliferation. Both agents produce adose-dependent inhibition on cell growth. It was of interest thatmaximal suppression of the growth of the cultures was obtained at theconcentration of 0.1 ni@i.At higher concentrations, the inhibition ofcell growth was reversed. The concomitant addition of morphine andsomatostatin did not result in an additive effect, indicating that,probably, both substances might share the same receptor site (notshown).

Cross-reaction of Morphine with SSTRS

To evaluate the possibility that morphine, morphiceptine, and somatostatin interact with the same population of receptor sites, weexamined the ability of morphine and morphiceptine to displace[‘25I]Tyr'‘-somatostatinbinding in T47D cells. Morphiceptine sharesthe same spectrum as morphine on opioid binding sites in othersystems. Our results are presented in Fig. 6. It can be seen that both

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Fig. 4. Effect of @z-actingopioids on cell proliferation of T47D cells. Opioid agonists(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 T47Dcells for 4 days, with daily change of the medium. Cell proliferation was estimated by thetetrazolium salt assay, as indicated in “Materialsand Methods.―Columns, mean of fourexperimentsin triplicate;bars, SEM.

5634

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MORPHINE AND SSTRs IN T47D BREAST CANCER CELLS

further substantiated in the present study by using RT-PCR, showingthe expression of SSTR2 (—85%)and SSTR3 (—15%) in T47D cells.

Previous investigations have shown that inhibition of cell proliferation by somatostatin analogues might be mediated by SSTR sybtypesSSTR1 and SSTR2 (30). No SSTR1 has been found in the presentstudy. On the contrary, somatostatin-28 and sandostatin, sharing thesame affinity for the SSTR2 subtype, decrease the proliferation inT47D cells by the same extent (50 and 55%, respectively), indicatinga probable involvement of SSTR2 on the cell growth of T47D cells.The action of somatostatin on cell growth is biphasic. Indeed, maximal suppression of growth was obtained at 0. 1 flM.At higher concentrations, the inhibition of cell growth is reversed. The same result hasbeen reported previously on the MCF7 breast cancer cell line bysomatostatin (12), antiestrogens (31), and epidermal growth factor(32). Although the underlying mechanism of this biphasic effects isnot known, different hypotheses have been proposed, implicatingeither a desensitization/intemalization/conformational change of thereceptor 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 ethylketocyclazocine) selective toward different subtypes of the opioid receptor produce a dose-dependent, reversible, inhibitory effect on cellproliferation. Morphine, the p-selective agonist, presenting no affinityfor the other opioid receptor subtypes in the T47D cells, produces amajor inhibition of cell proliferation, whereas no @topioid receptorshave been detected (22). In the present work, we show that this effectis shared by the amidated tetrapeptide morphiceptine (derived fromthe enzymatic degradation of j3 casein), which shares, in other systems, the same pharmacological selectivity as morphine. In view ofthe structural homology between the opioid and the SSTRs, whichwere proposed to derive from the same ancestor gene (5, 6), we haveinvestigated a possible interaction of morphine with the somatostatin

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Fig. 6. Displacement of [1251]Tyrt ‘-somatostatin from its binding sites by jx-actingopioids. Displacement of [‘@I]Tyr'‘-somatostatinbinding by the indicated concentrationsof morphine (B) or morphiceptine (•).See “Materialsand Methods―for details.

Fig. 5. Effect of somatostatin analogues on cell proliferation of T47D cells. Somatostatin-28 (SS28, shaded columns) and SMS 201-995 (Sandostatin, filled columns) wereapplied in the indicated concentrations on T47D cells for 4 days, with daily change of themedium. Cell proliferation was estimated by the tetrazolium salt assay, as indicated in“Materialsand Methods.―Columns, mean of three experiments in triplicate; bars, SEM.

morphine and morphiceptine displace the specific binding of[1 1-somatostatin in a dose-dependent and monophasic man

ner, similar to BIM 23034C (compare Figs. 2B and 6), with IC50s of1.67 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 inculture media produces a dose-dependent inhibition of cell growth(1 1, 12, 21). In addition, 30% of breast cancers express somatostatinimmunoreactivity; preneoplastic lesions express less, whereas normaltissue does not show any somatostatin immunoreactivity (14). Fromthe five cloned subtypes of the SSTR, only SSTR2 was found in 5primary breast tumors, whereas both SSTR2 and SSTR3 were expressed in 1 case (16). Furthermore, three breast cancer cell lines withdistinct steroid and growth factor receptor patterns (i.e., MCF7,MDA-MB23 1, and ZR-75- 1) express only the SSTR2 subtype, whichseems to be the most widely expressed SSTR in different human androdent tumors or tumor cell lines (27). Using a chemical cross-linkingassay, four distinct sites have been identified in the MCF7 cell line inmonolayer, one of which binds the somatostatin analogue BIM23014C (20). In the same study, BIM-23014C identifies one site inMCF7 and T47D cells, three in MDA-MB23 1, and none in the HBL100 cell line. Finally, the BIM-23014C somatostatin analogue identifies at least 1 site in 90% of 30 human breast biopsies. The percentage of somatostatin-positive tumors varies between 14 and 48% (13,28—30).This percentagebecomes67% when only small tumorsareselected (29), and 90% with the use of the chemically cross-linkedsomatostatin analogue and electrophoresis (20). The existence ofSSTRs has been reported to be of prognostic value for the evolutionof breast cancer (19).

The results of the present study indicate the presence of multipleSSTR subtypes in T47D human breast cancer cells. Scatchard analysisof [‘25IIJTyr'‘-somatostatin-14 binding identified two components:one with high affinity but low capacity (Kd, 0. 145 revs;1450 sites/cell),and another oflower affinity but higher capacity (Kd, 1.192 nr@i;11920sites/cell). These results are comparable with those reported bySeytono-Ham et a!. (12) and Prevost et al. (20) on MCF7 cells for thehigh-affinity site, and by Weckbecker et al. (21) on the ZR-75-1breast cancer cell lines for the lower affinity site. These results were

5635

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MORPHINE AND SSTRs IN T47D BREAST CANCER CELLS

ergic system. It was surprising that cross-displacement experiments ofsomatostatin by morphine and morphiceptine indicated that theseopioids compete for somatostatin binding in a similar manner, inhibiting cell proliferation through an interaction with SSTR2. The exposure of cells to both somatostatin and morphine does not produceadditive effects, enhancing the assumption that these substances mightinteract with the same receptor site. From these data, we have concluded that morphine may exert its antiproliferative action throughSSTR2.

Previous studies have indicated a possible interaction betweensomatostatin analogues and the opioid receptor system. Indeed,Maurer et a!. (33) and Walker et a!. (34) have reported that theoctapeptide analogues of somatostatin act, with high affinities, asantagonists to @.topioid receptors in the central nervous system.Although the high degree of homology between the opioid and SSTRssuggests that some ligands might be able to interact with both receptors (6), to our knowledge, no cross-reaction of opiates with SSTRshas been described previously. Furthermore, from our studies andthose of others (30), it appears that SSTR2 may be responsible for themediation of the observed antiproliferative effect. Together, the datapresented here provide evidence for a functional interaction of the twomajor inhibitory neuroendocrine systems (opioids and somatostatin),which may involve peptide competition and may have implicationsfor the biology and pathophysiology of tumor cells and disease states.In addition, the fact that morphiceptine, a casomorphin produced from13casein(a normalproductof breastepithelium),bindsto SSTRsanddecreases cell growth, raises the question of a possible interaction ofother a and f3 casomorphins in cell proliferation. This is beinginvestigated in our laboratory.

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1995;55:5632-5636. Cancer Res   Anastassia Hatzoglou, L'Houcine Ouafik, Efstathia Bakogeorgou, et al.   Growththe T47D Human Breast Cancer Cell Line and Decreases Cell Morphine Cross-Reacts with Somatostatin Receptor SSTR2 in

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