Mu receptor 5 Morphine Cross-Reacts with Somatostatin Receptor.pdf

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

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ICANCERRESEARCH55 .5632 -5636. December 1 , 19951

ABSTRACTIn a previous study, w e found that m orphine decreases, in a dosed ep en de nt m an ne r, th e ce ll g ro wth o f T 47 D hu man b re ast c an ce r c ells ,desp i te the lack of p op io id recep torsand an in terac tion o f morph ine witho th er opioid s it es . We hav e the re fo re exam in ed a pos sibl e int er ac ti on o fmorph ine with o ther membrane recep tor systems of the ce ll . The presen ts tu dy d es cr ib es f or th e fi rs t t im e a n in te ra ct io n b etwe en p -a ct in g o pi oidd ru gs a nd th e s om at os ta ti ne rg ic s ys tem. We h av e f ou nd t ha t [ ta sI] Ty r1 t@soma to st at in b in ds w i th h ig h a ff in it y t o T47D cel ls .An a ly si s o ft he b in di ngdata showed the p re senc e o f two componen ts : one w i th h in ja af fi ni ty bu tlow capac ity (Kd ,0.145 v iM;1450 s ites /ce ll ), and another o f lower a f fini tybut h igher capac ity (Kd ,l .l92 nM; 11920 s ite s/ce ll ). Somatosta tin -14 ands oma to st at in -2 8 s howed mu lt ip ha si c d is pl ac ement c ur ve s, i nd ic at in g h eterogeneity of binding sites. The latter was confirmed by reverse transcript ion -PCR, which revea led the ex is tence o f the somatosta tin recep tor subtypes 2 and 3 (SSTR2 and SSTR3), with a relative mRNA concentrationof 85 and 15%, respect ive ly . Morphineand the morph inomimet ic pept idemor ph ic ep tin e ( Ty r- Pr o- Ph e- Pr o-NH2) d is pl ac e s om at os ta tin f rom it sb inding s it es . Fu rthe r ana ly si s i nd ic at ed tha t p -a ct in g opioids int er ac tw i th the SSTR2 rec ep to r sub type .

INTRODUCTIONT he som atostatinergic and opioid system s are inhibitory and arein vo lv ed in th e d ecrea se o f ho rm on e a nd ne urotran sm itter se cre tio n( 1 6) .F ur th ermo re , t he y h av e b ee n impl ic ate d i n t he c on tr ol o f tumo rgrow th in different organs, including breast (1, 4, 713).Indeed,som atostatin and opioid im munoreactivities have been detected in

tu mor, b ut no t in no rm al b rea st c ells (3 , 1 4, 1 5), a nd s pe cific re ce pto rshave been characterized in prim ary hum an breast tum ors and tum orc el l l in es (4 , 1 2, 1 3, 1 6 23 ),p ro du ci ng a d os e-d ep en de nt in hi bi ti on o fcell prolifera tion .Recently, we have characterized and K opioid receptors in theT47D cell line and have shown that different opioids inhibit cellproliferation (22). N o opioid binding sites w ere found, how ever,w he rea s m orp hin e (th e p ro to ty pe @ .tig and ) sh ow s a d ose-d epe nd en tinhibitory effect on the grow th of cells, w hich is not reversed by theo pio id an tag onist d ip re no rp hin e. M orp hin e d oes n ot cro ss-rea ct w ithany other opioid receptor subtype (@ or K). Our results suggest ano no pio id -m ed iated ac tio n of m orp hine a nd m orp hice ptin e. B eca usesomatostatin and opioid receptors belong to the sam e m embranep ro te in s up er famil y ( 5, 6 ), w e h av e i nv es ti ga te d a p os si ble in te ra ct io no f m orph in e w ith the s om ato statin erg ic sy stem .I n th e p re se nt wor k, w e h av e c ha ra ct er iz ed th e S STRs3 i n th e T47Dcell line, both pharm acologically and by R T-P CR . W e further reportth at m orp hin e a nd th e m orp hin om im etic p ep tid e m orp hic ep tin e in te r

Rec ei ve d 5 /2 /9 5 ; a cc ep te d 8 /3 1/ 95 .The cos ts o f p ub li ca ti on o f t hi s a rt ic le we re d ef rayed i n p ar t b y t he p aymen t o f p ag ec ha rg es . T his a rt ic le m us t t he re fo re b e h er eb y m ar ke d a dv er ti se me nt i n a cc or da nc e w it h1 8 U .S .C . S ec tio n 1 73 4 s olely to in dica te th is f ac t.I This work was partially supported by the University of Crete Research Committee,Min is tr y o f H ea lt h ( KESY ), a nd H ell en ic A nt ic an ce r S oc ie ty g ra nt s.2 To whom requests for reprints should be addressed.3 The abbreviations used are: 55Th, somatostatin receptor; RT-P CR, reverse transeript ion -PCR ; B IM 23034C . o -Phe -c [Cys-Ty r -o -Trp -Lys-Val -Cys]Na l-NH2 .

act w ith the S ST R system , providing a possible explanation for theirob serv ed in hib ito ry e ffec t o n c ell g row th .MATERIALS AND METHODS

Cell Cu lture s. The human breast cance rce l l l in e T47D (o riginal ly i so la tedfro m a p le ura l e ff usio n o f b re ast a de no ca rc in or na ) w as o bta in ed a t p assa ge 9 0.Cell s were rout ine ly g rown in RPMI , supp lemented wi th 10%hea t- inact iva tedfetal bovine serum.They werecu l tu red a t 37Cn a humid if ied a tmosphe re o f5% CO2 in air.Som atostatin Binding C onditions. About 106 cells/well in m onolayerwere used for sa tu rat ion and d isp lacemen t b ind ing expe riments. Be fore b inding, cells were washed tw ice with 2 ml of PBS. Binding was performed in PBSin a total volume of 0.5 m l, containing 2]l som atostatin, without (totalbinding) or with (nonspecific binding) a 1000-fold m olar excess of som atostat in -14 . Fo r satu ra tion b ind ing , a t l ea s t 810o in t s wi th d if fe ren t concent raio ns o f r ad io la be le d p ep ti de w er e p er fo rm ed i n d up li ca te . T he c ell s w er eincubated fo r 2 h a t room tempera tu re (1822C) .t t he e nd o f t he i nc ub at io nperiod, the unbound radioactivity was elim inated by washing the cells twicew it h 2 m l c ol d PBS. C el ls w er e r emov ed f rom p la te s w it h 0 .4 m l 2 N NaOH ,a nd th e b oun d ra dio activ ity w as co un te d in a -y c oun ter (T nc arb S eries;Pa ck ar d) , w it h a 9 5% e ff ic ie nc y f or 1 251,B in di ng wa s r ep ea te d a t l ea st t hr eetim es , an d th e re su lts w ere a na ly zed b y th e O rig in V ersio n 3 .5 p ac kag e(MicroCal Co .) u s ing equa tions desc ribed by Munson and Rodba rd (24) .Cell Growth Cond it ions. Ce ll s werep lated in 24 -we ll ELISApla tesa t anin it ia ldens ity o f25 X l0@ce ll s/wel l supp lemented wi th 1ml med ium/wel l.A l ldrugs were added to cultures 1 day after seeding (designated as day 0) toensu re un ifo rm a ttachmen t o f ce ll s a t the onse t o f the expe riments. Ce ll s wereg rown for a total o f4 days , wi th da ily change o f the med ium contain ing op io iddrugs or som atostatin analogues. A ll added drugs were dissolved shortly beforeuse.Cell Proliferation. Cellgrowthwas measuredby thetetrazoliumsaltassay(25) . Ce ll s were incubated for 4 h a t 37Cwi th the tet ra zol iurnsal t [3 -(4,5-dimethylthiazol2 -yl )-2 ,5 -diphenyl tet razol ium bromide ] , and me tabol ica lly ac tive ce ll s red uc ed t he d ye t o p urp le f orma za n. D ar k b lu e c ry st als w ere d is so lv ed w ithp ro pa no l. T he a bs orb an ce w as m ea su re d a t 5 70 nm a nd c omp ar ed a ga in st astan dard c urv e of k now n nu mb ers o f T 47 D ce lls. A ll ex perim en ts w erep er fo rmed a m inimum o f t hr ee t ime s, i n t ri pl ic at e.De te ct ion o f SSTR mRNA by RT-PCR. To ta lRNA was p repa redf romthe T47D cell l ine using the guanidin ium thiocyanate/phenol/chloroform proc ed ur e ( 26 ). T ot al RNA ( 5 @xg)rom T47D ce ll s was reve rse t ran sc ribed in tocDNA usi ng 1 @xgl ig o( dT )1 2_18 s p rimer i n 2 0- pi r ea ct io n vol ume [50 m@iTris (pH 8.0)-75 mM KC1-5 mM M gC12-5 @xMDIT-SO @ xg/mlBSA-20 units ofRNasin-0.5 mM each of four dNTPs-400 units of Moloney murine leukemiavirus reverse transcriptase) at 37C for 90 mm. A negative control wasperfonned with the first strand synthesis reaction and where the RNA samplewas omi tte d. RT m ix tu re s w ith ou t re ve rs e t ra ns cr ip ta se w er e i nc lu de d a scont ro ls fo r each sample to de tec t any genomic DNA contaminat ion .Af ter thei nc ub at io n, cDNA d eri ve d f rom 5 00 n g t ota l RNA was amp li fie d in a 5 0-p Jr ea ct io n c on ta in in g 50 mMKC1 , 1 0 mMTr is ( pH 8 .3 ), 1 .5 mMMgCl2, 1 0 nm iD li', 1 mMo f e ac h o lig on uc le ot id e, a nd 2 .5 u ni ts o f T aq DNA p olyme ra se .Op timal t emper at ur es a nd cyc li ng c ondi ti on s we re e st ab li sh ed f or a ll f iv eSSTRs ,a s fo llows : SSTR1 ,denaturat ion a t 95Cor S mm, and then 35 cycle sat 95 Cor 6 0 s, 6 5 C or 9 0 s, an d 7 2 C or 1 20 s [se nse, 5'-A AA -T GCGTC-CCA-GAA-CGG-GAC-CT-(C )- 3'; a nt is en se , 5 '-CAG-GTf-CTCAGQ-TTG-GAA-GTC-TT-(C)-3']; SSTR2, denaturation at 95C for S mm,a nd t he n 35 cyc le s a t 9 5Co r 6 0 s , 6 0Co r 9 0 s , a nd 72Co r 1 20 s ( se ns e,5'-CAT-ATA-GGA-TAG-GQT-TGG-CAC-AGC-TGT-T-3'; antisense, 5'-

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M orphine Cross-Reacts with Somatostatin Receptor SSTR2 in the T47D HumanBreast Cancer Cell Line and Decreases Cell Growth'A nastassia H atzoglou,2 L 'H oucine O uafik , Efstath ia B akogeorgou, K yriaki Th erm os, and E lias C astanasL aboratories of E xperim ental E nd ocrinology (A . H ., E . B ., E . C ] a nd P hannacology (K. T I, U niversity o f C rete, School of M edicine, and U niversity H ospital, P .O . B ox 1393,H eraklion G R-71 I 1 0, C rete, G reece, and L aboratoire de C ancerologie E zperim entale, C JF IN SE RM 93-1 1 (A . H .. E . C .], and N euroendocrinologie E xp erim entale, U 297IN SE RM IL 0 .1 , F acu lt d e M ed ecin e N or d, M ar se ille , F ra nc e

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MORPH IN E A N D S S TR s IN T 47 D B R EA S T C AN CE R C EL L STAC-ATC-CTC-AAC-CTG-GCC-ATC-GCA-GAT-GA-3 ' ) ;SSTR3 ,dena turation at 95 C for 5 m m, and then 40 cy cles at 95 C f or 60 s and 67 C for 4 m m(sense, 5'-A GA -A CG-CCC-T GT -CCT -A CT -GGC-C-3'; antisense, 5'-T GAA GC-GGT-AGG-AGA -GGA-A GC-C-3 ') ;S STR 4 , denaturat ion a t 95Cor Smm , an d th en 3 5 cy cle s at 9 5 Co r 6 0 s , 6 0 Co r 9 0 s, an d 72 Co r 1 20 s(sense, 5'-A TG-GT C-GCT -A TC-CA G-T GC-3'; antisense, 5'-GGG-CT CCTC-AGA -AGG-TGG-T-3 ') ; and SSTR 5 ,denatura tiona t 95CorS 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 '; an ti sense , 5 '-CAG-GTF-GA CGAT-GTF-GAC-GGT-GAA-G-3 ' ) .

S ou th er n B lo ts. Tw en ty p A of t he PCR p r od uct w e re fr ac tio na ted onag aro se g el s i n TBE b uf f er ( 89 mM T ris , 2 mM EDTA ) . A f t er s tain in g w i thethidium brom ide, the gels w ere photographed, and then treated and transf erred by capillarity to a Hy bond-N m em brane (A m ersham , A m ersham , UnitedK ingdom ). T he specif icity of the PCR product w as checked by S outhern blothy bridization by using an internal sequence as probe for all S ST Rs separately .Radiochem ical s and Chem ical s. [ 5 1 -so m ato statin ( 20 00 C i lmm o l)w as obtained f rom A mersham . M orphine w as a gif t from Farm acopia, anddiprenorphine w as f rom R eck it and Colem an Co. Cell culture m edia w ere fromGIBCO. The sornatostatinpeptides were obtained from Bissendorff Biochemicals (Hanover, Germ any ). S andostatin w as a gif t from Sandoz (B asel, Sw itz erland). A ll other peptides w ere from S igm a Chem ical Co. (S t. L ouis, M O).A l l c hem ical s we re f rom Merc k (Darms tad , Ge rmany ) . Mo l ecul ar B io lo gyproduc ts were from BoehringerM annhe im (Mannheim, Germany).RESULTSId en t if ic at ion and Cha r ac te r iz a tion o f SSTRS on T47D Br ea stCance r Ce lls

C har act er iz at ion of Bind ing Sit es. Fig. 1 p resent s t he b ind ingi so th erm o f [ 251 ]T y r' - somatos tat int o who le T47D cel ls . A b ip has ics atu ratio n cu rv e w as o btain ed w ith a h ig h-af fin ity c om p on en t re ac hing saturation (Fig. 1, inset A ) and a low -af finity com ponent that didnot reach saturation. N onspecif ic binding w as 27 5% of totalb in din g. A n aly sis of th e data in S catc hard c oo rd in ates (Fig . 1, in set B )show ed that the binding isotherm could be best f itted in a tw o-sitem odel, w ith af finities of 0.145 0.017 and 1.192 0.093 nr@ i,andb in din g c ap acities o f 1 45 0 2 35 an d 1 192 0 1 14 7 sites /c ell. T hish et erogene it y o f b in di ng o f [ 251 ]T y r' - somatos tat in c ould i nd ic at e

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the presence of m ultiple som atostatin binding sites in T 47D breastcancer ce ll s.T he sp ecif ic ity o f the [2 51 ]T y r' o m ato statin bin din g to S S T R sis show n in Fig. 2A . D isplacem ent of [251} T yr'-som atostatinf rom its receptor sites by som atostatin-14 and som atostatin-28, atconcentrations ranging from l0 o lO _6 M , produced paralleldisplacem ent curv es, indicating an interaction w ith the sam e receptor populations. B oth curv es w ere m ultiphasic, suggesting, asdid the saturation data, the presence of m ultiple som atostatinb in di ng s it es . N o d is pl ac eme nt was f ou nd b y c orti co tro ph in -re le asing horm one. O n the contrary , the displacem ent of [25I]T yr'-som atostatin-14 by B IM 23034C, a selective S ST R2 analogue,produced a m onophasic displacem ent curv e (Fig. 2B ) and depictedan IC50 of 5 nM . C om paring Figs. 2A and 2B , it appeared that thef irst site detected in the som atostatin-l4 and som atostatin-28 displacem ent curv es, and w ith IC 50s of 0.8 and 0.9 nM , respectiv ely ,is the S S TR 2 subty pe. T his w as f urther conf irm ed by displacem entof [ som atostatin by som atostatin- 14 and som atostatin-28 in the presence of B IM 23034C (not show n). T hese data aresum m arized in T able 1.Characterization of S ST R S ubty pes by R T-PCR . T o analy ze theheterogeneity of the binding sites, w e hav e inv estigated f urther theidentity of the S ST R subty pes present in T 47D cells by perf orm ing ananaly sis of their m RN A by R T-PCR . A s show n in Fig. 3, in thesecells, only SS TR 2 and SST R3 m RN A w ere found. The m ost prom inent receptor m R NA f ound in T 47D cells w as S ST R2, representingabout 85% of the w hole SST R m RN A .Ef fect of p-acting O pioids and S om atostatin on C ell G row th

Ef fect of il-acting opioids. Fig. 4 show s the inhibition of cellpro lif eratio n o f T 47D hum an b re as t can cer ce lls b y m o rp hin e an d th emo rp hi nom im e tic p ep tid e mo rp hi ce pt in e. B o th p .- ac ti ng d ru gs in hib itce ll pro lif eratio n in a d ose -de pen den t m an ner, w ith ap pare nt IC 50 s o f1.08 and 1.25 nM , respectiv ely . T his ef fect w as not inhibited by theop io id an tagonist d iprenorph ine .

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ruFig . 1 . B in din g o f [ 2 5I lT y r'-somatostatin toT 4 7D c el ls. S at urat io n b in di ng w as p erf orm e d f or 2h at ro om t em p erat ure o n w h ol e c el ls, as i nd ic at edin Materialsand Methods .The saturat ion bindingiso th erm is p rese nted . A , m ag nif icatio n o f th e m itial p art o f th e c urv e; B , rep rese ntatio n o f d ata inScatchard coordinates.

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F ig. 3. E xpression of SS TR m RN A in T 47 D hum an breast cancer cells by R T-P CR .R es ults o f R T-P CR o f th e fiv e 5 5T h m RN A in T 47 D c ells . S ec Ma te ria ls nd M eth od s f or d et ai ls . Re su lt s a re exp re ss ed a s a p e rc en tage o f t he whol e SSTR mRNA det ec ted.

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Fig .2 . Specif ici tyof [25I]Tyr 'somatostatinbindingon T47D cells. Displacement o f [ 2 5l JTy r' -somatos ta t inby d i ffe ren t analogues. A , 12 fmol o f [25I ]Tyr '-s om ato statin (ab ou t 5 0,0 00 c pm ) w er e in cu ba ted w ith th e in dica ted c on ce ntra tio ns o fc ortic otro ph in -rele as in g h orm on e (A ), s om ato statin -1 4 (s ), o r s om ato statin -2 8 ( );B , incubated with BIM 23034C, a selective SSTR2 agonist. Points, mean of threeexper imen ts in t ripl ica te ; bars , SEM. See Mater ia lsnd Methodso r de ta i ls o f theb ind ing expe r imen ts .

TableI IC50ofdifferentsubstanceson somatostatineceptorsof T47DcellsD ata w er e o bta in ed fro m r es ults p res en ted in F ig . 3 .

Effect of S om atostatin-28 and Sandostatin. Fig. 5 presents theeffects of som atostatin-28 and S MS 201-995 (S andostatin, a stablesom atostatin analogue) on cell proliferation. B oth agents produce adose-dependent inhibition on cell growth. It was of interest thatm axim al suppression of the grow th of the cultures w as obtained at thec on cen tratio n of 0 .1 ni@ i.A t h igh er co nce ntration s, th e in hib itio n o fcell grow th w as reversed. T he concom itant addition of m orphine andsom atostatin did not result in an additive effect, indicating that,probably, both substances m ight share the sam e receptor site (notshown) .C ross-reaction of M orphine w ith SS TR S

T o evaluate the possibility that m orphine, m orphiceptine, and som atostatin interact w ith the sam e population of receptor sites, w eexamined the ability of m orphine and morphiceptine to displace[ 25 I]Tyr ' -somatos ta tinb ind ing in T47D cel ls . Morph icep tine sharesthe same spectrum as morphine on opioid binding sites in othersystem s. O ur results are presented in F ig. 6. It can be seen that both

L o g O p lo i dF ig . 4 . E ff ec t o f @ z- ac tin go pi oi ds o n c el l p ro li fe ra ti on o f T 47 D c ell s. O pi oi d a go ni st s( mo rp hi ne i n A a nd m or ph ic ep tin e in B ) w er e a pp lie d a lo ne i n t he i nd ic ate d c on ce ntr at io ns

(filled colum ns) or in the presen ce of l06 M diprenorphine (sh aded colum ns) on T 47Dce lls f or 4 d ay s, w ith d aily ch an ge o f th e m ed iu m. C ell p ro life ra tio n w as es tim ate d b y th ete tr az ol iu m s al t a ss ay , a s i nd ic at ed i n Mat er ia ls n d Me th od s. Co lu mn s, m ea n o f f ou rexperimentsn triplicate;bars,SE M.5634

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M OR PH INE AND SST Rs I N T 47D BR EAST C ANC ER C EL LS

f urthe r s ubs tanti ate d i n the pre se nt s tudy by us ing RT-PCR, s howingthe expre ss ion o f SSTR2 (85%)and SSTR3 (15%) in T47D cel ls .Pre vi ous inv es ti gati ons hav e shown that inh ib iti on o f c el l pro li fe r

ati on by somato statin analogue s m ight be med iated by SSTR sybtype sSSTR1 and SSTR2 (30). No SSTR1 has been found in the presents tudy . On the con trary , s omato statin- 28 and sandos tatin, s haring thesame affinity fo r the S STR2 s ubty pe , de cre ase the pro life ratio n inT47D c ell s by the s am e e xte nt ( 50 and 5 5%, re spe ctiv ely ), i ndic atinga probable involvement of S STR2 on the cell grow th of T47D cells.The ac tio n o f somato statin o n ce ll gro wth is biphasic. Inde ed, max imal s uppre ss io n o f g rowth was o btai ne d at 0 . 1 f lM .At hig he r c onc entratio ns , the inhi bi tio n o f c el l g rowth i s re ve rs ed. The s ame re sul t hasbeen reported previously on the MCF7 breast cancer cell line bys omato stati n (1 2) , anti es tro ge ns ( 31 ), and e pide rmal g rowth f ac to r(3 2). A ltho ug h the underly ing mec hanism o f this biphasic e ffe cts isno t know n, differe nt hy po the ses hav e bee n propo se d, implic atinge ither a des ensi ti zati on /intemal iz ati on/ conformati onal change o f there ce pto r o r a po ss ibl e ag oni st/ antag onis t e ff ec t o f the ag ents .In a prev io us study , w e hav e sho wn that T4 7D c ance r c ells e xpre ssand K opioid receptors. D ifferent opioid agonists (ED-Ala2,D-Leu5]-

enkephal in , [D-Ser@,Leu5] -enkephal in , Thr6 e torphine , and e thylketo cy clazo cine) s elec tiv e to ward diffe rent subty pes o f the o pioid rec ep to r produc e a dos e-dependent, re ve rs ib le , i nh ib ito ry e ff ec t on c el lpro l if era tion. Morphine , the p-selective agonist, presenting no a ff in ityfo r the o the r o pioid re ce pto r subty pes in the T4 7D c ells , pro duc es amajo r i nhibi ti on o f c ell pro li fe rati on, whe re as no @ tpio id re ce pto rshav e bee n de te cted (2 2). In the prese nt w ork, w e show that this effe ctis s hare d by the am idate d te trape ptide morphi ce pti ne ( de ri ve d f romthe enz ym atic deg radatio n o f j3 case in), w hic h shares, in o the r sy stems, the same pharm ac olo gic al s elec tiv ity as m orphine . In v iew o fthe structural homology betw een the opioid and the S STRs, w hichw ere pro po sed to deriv e from the s ame ance sto r g ene (5 , 6 ), w e hav einv es ti gated a po ss ib le inte rac ti on o f morphine w i th the s omato statin

C,aC,)

Fi g. 6 . D i sp lac emen t o f [ 12 5 1]Ty rt -s omato sta ti n f rom i ts b indi ng s ite s by jx -a cti ngopioids. Displacement of [@I]Tyr'-somatostatinbinding by the indicated concentrationsof morphine (B) or morphiceptine () .See Materialsnd Methodsor details.

Fi g. 5 . E ff ec t o f s omato stati n an al og ue s o n c el l pro li fe rati on o f T4 7D c el ls . S omato sta ti n- 28 (SS28 , s haded c ol umns ) and SMS 201 -9 95 (Sandos tati n, f il le d c ol umns ) we reappl ie d in th e in dic ate d c onc entratio ns o n T4 7D c el ls f or 4 day s, w ith dai ly c hang e o f themedi um . C el l pro lif erati on w as e stimate d by the te traz ol ium s al t as say , as i ndi cate d i nMaterial sand Methods .Co lumns , mean of three experiments in tripl icate; bars , SEM.

morphine and morphiceptine displace the specific binding of[1 1-som a tost a t in in a d ose-d ep end en t a n d m onoph a sic m anner, similar to B IM 23034C (compare Figs. 2B and 6), w ith IC50s of1 .67 and 1 .71 f lM,respec tive ly .DISCUSSIONVari ous bre as t c anc er c ell li ne s hav e be en f ound to e xpre ss SSTRs

(1 1 , 1 2, 2 0, 2 1) . Add iti on o f s omato statin o r s omato statin analogue s inculture me dia produc es a do se -de pendent inhibition o f c ell g ro wth(1 1 , 1 2, 2 1) . In addi tio n, 3 0% o f bre as t c anc ers e xpre ss s omato stati nimmunoreac tiv ity ; preneoplas tic le sions express les s, whereas normalti ss ue do es no t s how any s omato stati n immuno re ac tiv ity ( 14 ). Fromthe five cloned subtypes of the SSTR, only SSTR2 was found in 5primary breast tumors, w hereas both S STR2 and S STR3 w ere expre ss ed in 1 cas e ( 16 ). Furthermore , thre e breas t c anc er c el l l ines w ithdistinct steroid and grow th factor receptor patterns (i.e., MCF7,MDA -MB23 1, and ZR-7 5- 1 ) e xpres s o nly the S STR2 subty pe, w hichseems to be the mo st w idely e xpre sse d S STR in diffe rent human androdent tumors o r tumor c el l l ines ( 27 ). U s ing a chemical c ro ss -l inkingass ay , fo ur distinc t site s hav e be en identified in the MCF7 c ell line inmonolayer, one of w hich binds the somatostatin analogue BIM23014C (20). In the same study, B IM -23014C identifies one site inMCF7 and T47D cells, three in MD A-MB 23 1, and none in the HB L1 00 ce ll line. Finally , the B IM -23 01 4C s om ato statin analo gue identifies at least 1 site in 9 0% o f 30 hum an breast bio psies. The perc entag e o f s omato stati n-po si ti ve tumors v arie s be tw ee n 1 4 and 4 8% (13 ,28 30).his percentagebecomes67% when only small tumorsarese le cted (2 9), and 9 0% w ith the us e o f the c hemically cro ss -linke dsomato statin analo gue and e le ctro pho res is (2 0). The ex iste nce o fS STRs has bee n repo rte d to be o f pro gnos tic v alue fo r the e vo lutio no f bre as t c anc er ( 19 ).The results o f the pre se nt s tudy indic ate the pres ence o f m ultiple

SSTR subtype s in T47D human breas t c ance r c el ls . Sc atchard analys isof [25IIJTyr' -somatos tatin-14 binding identi fi ed two components :one w ith h igh af fi ni ty but l ow capac ity (Kd , 0 . 1 4 5 revs ;1 450 s ite s/ ce ll ),and another o fl owe r af fi ni ty but h ighe r c apac ity (Kd , 1 .1 92 nr@i ; 1920sites/cell). These results are comparable w ith those reported byS ey to no -Ham et a!. (1 2) and Prev ost et al. (2 0) o n MCF7 ce lls for thehigh-affinity site, and by W eckbecker et al. (21) on the ZR-75-1bre as t c anc er c ell l ine s f or the lowe r af fini ty s ite . The se re sul ts w ere

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-12 -11 -10Log Peptide

0 @ t0.h 1 lcT bO 10-s 10@8L og O p loid



6/6

MO RP HI NE A N D S STRs I N T 47 D B REAS T C AN C ER C ELLSe rg ic s ys tem. I t w as s ur pri si ng t ha t c ro ss -d is pl ac em en t e xp er im en ts o fsom atostatin by m orphine and m orphiceptine indicated that theseo pio id s c om pe te fo r so ma to statin b ind in g in a sim ila r m an ner, in hibitin g c ell p ro lifera tio n th rou gh an in tera ctio n w ith S ST R2 . T he ex posure of cells to both somatostatin and m orphine does not producea dd it iv e e ff ec ts , e nh an cin g t he a ss umpt io n th at th es e s ub st an ce s m ig htinteract w ith the sam e receptor site. From these data, w e have concluded that m orphine m ay exert its antiproliferative action throughSSTR2.

Previous studies have indicated a possible interaction betw eensom atostatin analogues and the opioid receptor system . Indeed,Maurer et a!. (33) and Walker et a!. (34) have reported that theoctapeptide analogues of som atostatin act, w ith high affinities, asantagonists to @ .topioid receptors in the central nervous system .A lth ou gh the h ig h d eg re e o f h om olo gy b etw een th e o pio id an d S ST Rssuggests that som e ligands m ight be able to interact w ith both receptors (6), to our know ledge, no cross-reaction of opiates w ith S ST Rshas been described previously. F urtherm ore, from our studies andthose of others (30), it appears that S ST R2 m ay be responsible for them ediation o f th e o bs erv ed an tip roliferative effe ct. T og eth er, th e d atap rese nte d he re pro vide e vid en ce fo r a fu nc tio nal in tera ctio n o f th e tw omajo r in hi bi to ry n eu ro en do cr in e s ys tems ( op io id s a nd s om ato st at in ),w hich m ay involve peptide com petition and m ay have im plicationsfo r th e b io log y an d p ath oph ysio lo gy o f tu mo r ce lls a nd d ise ase state s.In addition, the fact that morphiceptine, a casomorphin produced from13ca s e in (a no rm a l p rodu c t o f b re a s te p ith e l ium ) ,b in d s to S S TR sandd ec rea ses ce ll g ro wth , raise s th e q ue stio n o f a p ossible in tera ctio n o fother a and f3 casomorphins in cell proliferation. This is beingi nv es ti ga te d i n o ur l ab or at or y.REFERENCESI . Re ich lin,S.Somatostatin.n:D.T.Krieger,M.J. Brownstein,ndi. B.M artin(eds.),B ra in P ep ti de s, p p. 7 1 1 75 2.N ew Y ork: John W iley & S ons, Inc., 19 83.2 . S ch al ly , A . V . O nc ol og ic al a pp li ca ti on s o f s om at os ta ti n a na lo gu es . C an ce r R es ., 4 8:

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