Effective treatment of experimental U-87MG human glioblastoma in nude mice with a targeted cytotoxic bombesin analogue, AN-215

E ec i e ea men o expe imen al U-87MG human glioblas oma in nude mice wi h a a ge ed cy o oxic bombesin analogue, AN-215 Z Sze eday 1,2 , AV Schally* ,1,2 , A Nagy 1,2 , A Plonowski 1,2 , AM Bajo 1,2 , G Halmos 1,2 , K Szepeshazi 1,2 and K G oo 1 1 Endoc ine, Polypep ide and Cance Ins i u e, Ve e ans A ai s Medical Cen e , 1601 Pe dido S ee , New O leans, Louisiana, LA 70112-1262, USA; 2 Sec ion o Expe imen al Medicine, Depa men o Medicine, Tulane Uni e si y School o Medicine, 1601 Pe dido S ee , New O leans, Louisiana, LA 70112-1262, USA Some b ain umou s, such as glioblas omas exp ess high le els o ecep o s o bombesin/gas in eleasing pep ide. We in es iga ed whe he bombesin/gas in eleasing pep ide ecep o s ound in glioblas oma cell lines can be u ilised o a ge ing o a cy o oxic bombesin analogue, AN-215 consis ing o a po en de i a i e o doxo ubicin, 2-py olino-doxo ubicin (AN-201) linked o a bombesin-like pep ide ca ie . This s udy epo s he e ec o AN-215 on he g ow h o U-87MG human glioblas omas xenog a ed in o nude mice. High a ini y binding o AN-215 o U-87MG umou s was cha ac e ised by an IC 50 alue o 4.0+0.1 nM, as de e mined by adio ecep o assays. mRNA analyses e ealed he p esence o mRNA o BN ecep o sub ypes 1 and 2. T ea men wi h AN-215 signi ican ly (P50.05) ex ended umou doubling ime om 4.54+0.2 days o 8.18+1.8 days and inhibi ed umou g ow h as demons a ed by a 69.6% educ ion in inal umou olume (P50.001) and a 64.6% dec ease in umou weigh as compa ed o con ols. Cy o oxic adical AN-201 a he same dose was ine ec i e. The an i umou e ec o AN-215 could be blocked by p e ea men wi h an excess o a bombesin an agonis , indica ing ha he ac ion o his cy o oxic analogue is ecep o -media ed. Ou esul s sugges ha pa ien s wi h inope able b ain umou s such as malignan gliomas may bene i om a ge ed chemo he apy based on cy o oxic bombesin analogue AN-215. B i ish Jou nal o Cance (2002) 86, 1322 – 1327. DOI: 10.1038/sj/bjc/6600235 www.bjcance .com ª 2002 Cance Resea ch UK Keywo ds: umou a ge ing; doxo ubicin; bombesin ecep o ; RT – PCR Abou 17 000 Ame icans de elop p ima y b ain umou s and 13 000 die om hem each yea (G eenlee e al, 2000). The annual incidence a es o p ima y b ain umou s in he Uni ed Kingdom, F ance, No way and Finland and mo ali ies pe 100 000 popula- ion a e simila o hose in he Uni ed S a es (Mui e al, 1994; Fleu y e al, 1997; P ados and Le in, 2000). The incidence o b ain cance may be inc easing in olde people (Mui e al, 1994). Glio- blas oma mul i o me is he mos common ype o p ima y malignan CNS umou s in adul s and is conside ed incu able (Hochbe g and P ui , 1987). Cu en ea men op ions o malignan gliomas, including su ge y, adia ion, and chemo he apy, a e o limi ed e ec i eness, and no el he apeu ic modali ies mus be explo ed. Ta ge ing o an ineoplas ic agen s o cance cells by linking hem o a ligand wi h high a ini y o umou cells should imp o e umou g ow h inhibi ion and dec ease pe iphe al oxici y (Schally and Nagy, 1999). The p esence o high a ini y ecep o s o bombesin (BN)-like pep ides on a wide a ie y o umou s p omp ed us o employ some o ou powe ul BN/gas in eleasing pep ide (GRP) an agonis s as ca ie molecules o a ge ing cy o oxic agen s o umou cells (Nagy e al, 1997). One o hese cy o oxic BN conju- ga es, AN-215 (Nagy e al, 1997) consis s o a po en cy o oxic de i a i e o doxo ubicin (DOX), 2-py olino-DOX (AN-201) (Nagy e al, 1996), co alen ly linked h ough a glu a ic acid space o he amino e minal o Gln-T p-Ala-Val-Gly-His-Leu-c-Leu- NH 2, a BN-like pep ide ca ie (Nagy e al, 1997). AN-215 displays high a ini y o BN ecep o s on Swiss 3T3 ib oblas s and e ains he an ip oli e a i e e ec o i s cy o oxic moie y (Nagy e al, 1997). This cy o oxic conjuga e was shown o e ec i ely inhibi BN ecep o -posi i e neoplasms, including H-69 human small-cell lung ca cinoma (Kia is e al, 1999a) and PC-3 human and ogen- independen p os a e cance (Plonowski e al, 2000). In iew o indings ha 85% o human glioblas oma cell lines exp ess unc ional ecep o s o BN/GRP (Moody e al, 1989; S aley e al, 1993; Pinski e al, 1994; Sha i e al, 1997), we e alua ed in his s udy he e icacy o a ge ed chemo he apy based on cy o oxic analogue o BN, AN-215 in U-87MG human glioblas oma xeno- g a ed in o nude mice. MATERIALS AND METHODS Pep ide and cy o oxic agen s Cy o oxic conjuga e AN-215 was made by coupling one molecule o 2-py olino-DOX-14-O-hemiglu a a e o he amino e minus Expe imen al The apeu ics Recei ed 2 No embe 2001; e ised 5 Feb ua y 2002; accep ed 6 Feb ua y 2002 *Co espondence: AV Schally; Endoc ine, Polypep ide and Cance Ins i u e, Ve e ans A ai s Medical Cen e , 1601 Pe dido S ee , New O leans, Louisiana, LA 70112-1262, USA B i ish Jou nal o Cance (2002) 86, 1322 – 1327 ª 2002 Cance Resea ch UK All igh s ese ed 0007 – 0920/02 $25.00 www.bjcance .com o BN-like ca ie analogue Gln-T p-Ala-Val-Gly-His-Leu-c(CH 2 - NH)-Leu-NH 2 as desc ibed (Nagy e al, 1997). GRP(14-27), cy o- oxic adical AN-201 (Nagy e al, 1996) and BN/GRP an agonis RC-3095 [D-Tpi 6 ,Leu 13 c(CH 2 NH)Leu 14 ]BN (6-14) (Radulo ic e al, 1991) we e also syn hesised in ou labo a o y. Fo in a enous (i. .) injec ion, he compounds we e dissol ed in 20 ml o 0.01 N ace ic acid and dilu ed wi h 5% (w 71 ) aqueous D-manni ol (Sigma, S . Louis, MO, USA) solu ion. Cell lines, animals and umou s Human glioblas oma cell line U-87MG was ob ained om Ame ican Type Cul u e Collec ion (Manassas, VA, USA) and cul u ed as desc ibed (Pinski e al, 1994). Male a hymic (Nc nu/nu) nude mice, app oxima ely 6-weeks-old on a i al, we e ob ained om he Na ional Cance Ins i u e (F ede ick Cance Resea ch and De elop- men Cen e , F ede ick, MD, USA), and housed in lamina ai - low cabine s unde pa hogen- ee condi ions wi h a 12 h ligh /12 h da k schedule, and ed au ocla ed s anda d chow and wa e ad libi um. Xenog a s we e ini ia ed by s.c. injec ion o 12610 6 U-87MG cells in o he igh lanks o i e male nude mice. Tumou s esul ing a e 2 weeks we e asep ically dissec ed, mechanically minced, and 3-mm 3 pieces o umou issue we e ansplan ed s.c. wi h a oca needle. The ake a e was 100%. All expe imen s we e pe o med in acco - dance wi h ins i u ional e hical guidelines o animal ca e and we e essen ially in ag eemen wi h UKCCCR guidelines (1998) o he wel a e o animals in expe imen al neoplasia. Expe imen al p o ocols Expe imen 1 was s a ed when umou s had g own o app oxi- ma ely 40 mm 3 in olume. Animals we e andomly di ided in o h ee ea men g oups: g oup 1(10 mice), con ol, which ecei ed ehicle solu ion; g oup 2 (10 mice), injec ed wi h cy o oxic adical AN-201; g oup 3 (11 mice), gi en cy o oxic BN analogue AN-215. Cy o oxic compounds we e injec ed h ough he jugula ein a a dose o 150 nmol kg 71 o body weigh (BW) on days 1, 8, 15 and 22. Tumou olume (leng h6wid h6heigh 60.5236) and BW we e measu ed wice a week. The expe imen was e mina ed on day 29. Blood samples we e collec ed om he in e io ena ca a unde Me o ane (Malink od Ve . Mundelein, IL, USA) anaes- hesia and hen Me o ane was used in o e dose o sac i ice he mice. Tumou s we e excised and weighed. Tumou specimens we e snap- ozen and s o ed a 7708C un il ex ac ion o RNA o e e se ansc ip ion-polyme ase chain eac ion (RT – PCR). Tumou olume doubling ime was calcula ed be ween day 1 and 29 using he o mula: days o ea men ½log ð inal olÞÿlog ðini ial olumeÞ=log2 as desc ibed by Smole e al (1977). Expe imen 2 was s a ed when U-87MG umou s had g own o 70 – 84 mm 3 in olume. The animals we e di ided in o he ollow- ing ea men g oups: g oup 1 (10 mice), con ol, ehicle solu ion; g oup 2 (10 mice), analogue AN-215; g oup 3 ( i e mice), uncon- juga ed mix u e o he cy o oxic adical AN-201 and BN an agonis RC-3095. All compounds we e injec ed i. . a 150 nmol kg 71 o BW on days 1, 10, and 17. In addi ion, one g oup o umou -bea - ing mice ecei ed an i. . injec ion o 200 mg o BN an agonis RC- 3095 15 min be o e each adminis a ion o AN-215 a a dose o 150 nmol kg 71 o BW on days 1, 10 and 17 as in he case o g oup 2. The expe imen was e mina ed on day 20 as desc ibed abo e. E alua ion o oxici y Gene al oxici y was e alua ed on he basis o mo ali y a e and changes in BW. Toxici y o BN ecep o -posi i e o gans was assessed by measu ing gas in elease in esponse o GRP s imula- ion (Plonowski e al, 2000). A he end o expe imen 1, blood samples we e collec ed 5 min a e i. . injec ion o 2 mgo GRP(14-27) dissol ed in 100 ml o 5% manni ol. Se um gas in concen a ions we e measu ed by double-an ibody adioimmu- noassay wi h a ki p o ided by ICN Pha maceu icals Diagnos ic Di ision (O angebu g, NY, USA). The in e assay a ia ion was 10.6%, and he in aassay a ia ion was 6%. His ological me hods Samples o umou issues we e ixed in 10% bu e ed o malin. The specimens we e embedded in Pa aplas (Ox o d Labwa e, S . Louis, MO, USA). Six mm hick sec ions we e cu and s ained wi h haema oxylin-eosin. Mi o ic and apop o ic cells we e coun ed in nine s anda d high powe mic oscopic ields con aining an a e age o 330 cells, and hei numbe s pe 1000 cells we e accep ed as he mi o ic and apop o ic indices, espec i ely. To elimina e e o s caused by a iances in hickness o slides and cellula i y o a eas in es iga ed, he a io o apop o ic o mi o ic indices was calcula ed in each umou . Fo he de e mina ion o he ex en o nec osis in umou s, he c ossing poin s o a mic oscope ocula ne ha coin- cided wi h nec osis in he slide made a he la ges c oss-sec ion o each umou we e coun ed. The a io o hese poin s o he numbe o all poin s abo e he umou ep esen ed he pe cen age a ea o nec osis. Fo demons a ion o he nucleola o ganise egion (NOR) in umou cell nuclei, he AgNOR me hod was used (Szepeshazi e al, 1991). NORs a e pa s o DNA closely associa ed wi h nucleoli and encode o ibosomal RNA. They a e also asso- cia ed wi h a gy ophylic p o eins and hus can be isualised by sil e s aining. The amoun o AgNORs is a good indica o o cell p oli e a ion ( an Dies e al, 1998). The sil e -s ained black do s in 50 cells o each umou we e coun ed and he AgNOR numbe pe cell was calcula ed. The da a we e e alua ed by one way analy- sis o a iance (ANOVA) and he ea ed g oups compa ed o he con ol by Dunne ’s es . RNA ex ac ion and RT – PCR To al RNA was ex ac ed om ozen issue samples by using RNAzolB (Tel-Tes , F iendswood, TX, USA) acco ding o he manu ac u e ’s ins uc ions. Concen a ions o o al RNA we e calcula ed by measu ing he OD 260 alue. To al RNA (4 mg) was subjec ed o elec opho esis o 2 h a 70 V in 10 ml o sample bu e con aining 16MOPS bu e (pH 6.5), 6.4% o maldehyde, 48% deionized o mamide, 0.25% w 71 b omophenol blue, 0.05% glyce ol and 0.25 mg ml 71 e hidium b omide. The RNA gel con ained 1.4% aga ose, 16MOPS (pH 6.5) and 1.73% o malde- hyde. The RT – PCR was pe o med using Gene Amp RNA Co e Ki (Pe kin-Elme , Fos e Ci y, CA, USA). To a oid genomic DNA con amina ion, all samples we e subjec ed o DNase diges ion be o e RT – PCR. RNA (2 mg) was ea ed wi h 0.3 uni o RQ1 RNase- ee DNase (P omega, Madison, WI, USA) a 378C o 30 min in 19 ml o mix u e con aining 5 mMMgCl 2 ,16PCR bu e , 1 mMo each dNTP, 1 uni RNase inhibi o and 2.5 mM andom hexame s. The eac ion was e mina ed by enzyme dena- u a ion a 998C o 5 min. A e cooling, 2.5 uni s o mu ine leukaemia i us (MuLV) e e se ansc ip ase was added and RT was ca ied ou a 428C o 30 min. The PCR ampli ica ion o he cDNAs o human glyce aldehyde- 3-phospha e dehyd ogenase (hGAPDH), GRP ecep o (hGRPR, BRS-1) and neu omedin-B ecep o (hNMBR, BRS-2) was pe o med as ollows. Two ml o he cDNA we e ampli ied in a 25-ml solu ion con aining: 2 mMMgCl 2 ,16PCR bu e , 200 mM o each dNTP, 2.5 uni s o Ampli Taq DNA polyme ase and 0.2 mMo each p ime . The p ime s used we e 5’- TCC TCT GAC TTC AAC AGC GAC ACC-3’and 5’-TCT CTC TTC CTC Expe imen al The apeu ics Cy o oxic bombesin analogue AN-215 inhibi s glioblas oma Z Sze eday e al 1323 ª 2002 Cance Resea ch UK B i ish Jou nal o Cance (2002) 86(8), 1322 – 1327 TTG TGC TCT TGG-3’ o hGAPDH, 5’-ATT TGG CAG GAT TGG CTG C-3’and 5’-TGA GGC AGA TCT TCA TCA G-3’ o hGRPR, 5’- CGG ACT CTG CTG GAA AGG A-3’and 5’-GAC GTC TGC ATG TCC ATG G-3’ o hNMBR (Kia is e al, 1999b). Samples we e dena u ed a 948C o 5 min and hen subjec ed o 30 cycles o hGAPDH o 40 cycles o hNMBR o 948C o 30 s, 608C o 30 s and 728C o 40 s o 35 cycles o hGRPR o 948C o 1 min, 588C o 1 min and 728C o 1 min, ollowed by a inal ex ension a 728C o 7 min using a Pe kin- Elme DNA he mal cycle model 2400. The numbe o cycles was de e mined in p elimina y expe imen s o be wi hin he expo- nen ial ange o PCR ampli ica ion. Nega i e con ols using dis illed wa e ins ead o cDNA in he PCR mix u e we e un in pa allel o exclude genomic DNA con amina ion. Aliquo s o each PCR p oduc we e subjec ed o elec opho esis on a 2% aga ose gel, s ained wi h e hidium b omide and isualised unde ul a iole ligh . Fo he quan i a ion o PCR-ampli ied p oduc s, a scanning densi ome e (model GS-700, Bio-Rad) coupled wi h he Bio-Rad pe sonal compu e analysis so wa e was used. All expe imen s we e epea ed a leas wice and simila esul s we e ob ained. The ela i e mRNA le els o each gene we e no malised s he co esponding le els o hGAPDH. Recep o binding assays Binding cha ac e is ics o BN ecep o s on memb ane p epa a ions om U-87MG umou s we e de e mined by ligand compe i ion assays using 125 I-labelled [Ty 4 ]BN, as epo ed ea lie (Halmos and Schally, 1997). Recep o binding a ini y o cy o oxic BN analogue AN-215 o umou memb anes was measu ed in displace- men expe imen s based on compe i i e inhibi ion o 125 I-[Ty 4 ]BN binding, using a ious concen a ions o AN-215 (10 76 –10 712 M). IC 50 alue was calcula ed wi h a compu e ized cu e i ing p og amme and is de ined as he concen a ion o AN-215 causing a 50% inhibi ion o 125 I-[Ty 4 ]BN binding. S a is ical analysis Da a a e exp essed as mean+s.e. Di e ences be ween mean alues we e e alua ed by wo- ailed S uden ’s - es , P50.05 being consid- e ed signi ican . RESULTS Inhibi ion o umou g ow h by AN-215 Expe imen 1 was designed o compa e he an i umou e ec s and oxici y o cy o oxic BN analogue AN-215 and i s cy o oxic adical AN-201. A ea men egimen consis ing o ou i. . injec ions o AN-215 a 150 nmol kg 71 o BW p oduced a s ong umou g ow h inhibi ion (Figu e 1). The inhibi o y e ec o AN-215 was e iden wi hin 6 days and became signi ican om day 11. Fou weeks a e he ini ia ion o he ea men , he umou doubling ime in animals ea ed wi h AN-215 was signi ican ly p olonged om 4.5+0.2 o 8.2+1.8 days (P50.05) (Table 1). In con as , umou doubling ime in mice gi en an equimola dose o AN-201 was 5.9+1.4 days which did no di e signi ican ly om he con ols. Tumou olume and weigh a e shown in Table 1. In expe imen 2, he ea men was ini ia ed when xenog a s o U-87MG glioblas omas had g own o a olume, app oxima ely wice as la ge as ha in expe imen 1. A e 20 days he umou doubling ime in mice ea ed wi h h ee i. . injec ions o AN- 215 was ex ended signi ican ly (P50.05) o 6.6+1.0 days compa ed o he con ol g oup, which came o 4.5+0.2 days. The changes in umou olume a e p esen ed in Figu e 2. E ec o blockade o BN ecep o s As pa o expe imen 2, mice bea ing U-87MG umou s we e p e ea ed i. . wi h a high dose o BN an agonis RC-3095 (200 mg/mouse) o block BN ecep o s p io o each adminis a ion o AN-215. As shown in Figu e 2, he p e ea men wi h RC-3095 a enua ed he an i umou e ec o he cy o oxic BN analogue AN- Expe imen al The apeu ics Table 1 The e ec s o cy o oxic analogue o BN AN-215 and i s cy o oxic adi- cal AN-201 on umou g ow h o U-87MG glioblas omas xenog a ed in o nude mice Final umou Tumou Tumou G oup and Ini ial umou olume (mm 3 ) doubling weigh (mg) ea men a olume (mm 3 ) (% inhibi ion) ime (days) (% inhibi ion) Con ol 39.7+3.8 3201.5+375.7 4.5+0.2 3050+0.4 AN-201 37.7+6.3 2353.7+506.7 5.9+1.4 2650+0.5 d (26%) (13.2%) AN-215 35.5+5.2 971.5+227.9 b 8.2+1.8 c 1080+0.3 b (69.6%) (64.6%) a In a enous, injec ions o 150 nmol kg 71 o BW o each compound we e adminis e ed on days 1, 8, 15 and 22. The expe imen was e mina ed on day 29. The alues a e means+s.e. b P50.001 s con ol. c P50.05 s con ol. d P50.05 s AN-215. e8 e7 e6 e5 e4 Na u al log o umou olume 0 5 10 15 20 25 30 Days o ea men Con ol AN-215 4×150 nmol kg–1 AN-201 4×150 nmol kg–1 Figu e 1 The e ec s o cy o oxic BN analogue AN-215 and cy o oxic adical AN-201 on he g ow h o s.c. xenog a s o U-87MG human glio- blas oma in nude mice (expe imen 1). The ea men consis ing o ou i. . injec ions o he espec i e compounds a 150 nmol kg 71 o BW, was s a ed when umou olume eached app oxima ely 40 mm 3 (a ows indica e he days o injec ions; e ical ba s show s.e.). Cy o oxic bombesin analogue AN-215 inhibi s glioblas oma Z Sze eday e al 1324 B i ish Jou nal o Cance (2002) 86(8), 1322 – 1327 ª 2002 Cance Resea ch UK 215, he g ow h inhibi ion being educed o only 22% and he umou doubling ime was 4.7+02 days, close o con ol da a. Toxici y In expe imen 1, wo o 10 animals (20%) died in he g oup ha ecei ed AN-201, bu only one o 11 animals (9%) died a e ea - men wi h AN-215. To es ima e he side e ec s caused by a ge ing he cy o oxic agen o no mal o gans ha exp ess GRP/BN ecep- o s, we e alua ed he e ec o AN-201 and AN-215 on he se um concen a ion o gas in ollowing i. . s imula ion wi h GRP(14-27) a he end o he expe imen . Nei he AN-215 no AN-201 a ec ed GRP-s imula ed gas in elease (Table 2). In expe imen 2, wo o i e animals died (40%) in he g oup ha ecei ed an unconjuga ed mix u e o AN-201 and RC-3095 and one o nine animals (11%) died in he g oup ha ecei ed an i. . injec ion o 200 mg o BN an agonis RC-3095 abou 15 min be o e adminis a ions o AN-215, bu no oxici y- ela ed dea hs occu ed du ing he expe imen in he AN-215- ea ed g oup. One o 11 animals (9%) died in he con ol g oup on he day when he expe imen was e mina ed. His ology His ologically, U-87MG glioblas omas a e highly cellula umou s consis ing o ela i ely la ge undi e en ia ed cells showing a mode a e polymo phism. The cells ha e na ow cy oplasms and ound o o al nuclei wi h loose ch oma in s uc u e and p omi- nen nucleoli. A some a eas, he e a e mo e elonga ed cells a anged in la ge bundles. The quan i a i e his ological da a a e shown in Table 3. The ex en o nec osis in he umou s ea ed wi h AN-215 was signi ican ly la ge han in he con ols, while AN-201 caused only a sligh and no signi ican change in nec osis. The mean mi o ic and apop o ic indices we e no app eciably changed by he ea men s. Howe e , a sligh dec ease in mi osis and an inc ease in apop osis in he umou s ea ed wi h AN-215 esul ed in a signi ican ele a ion o he a io o apop o ic o mi o- ic indices. Exp ession o mRNAs o GRP/BN ecep o s The o al RNA isola ed om con ol and ea ed samples was un on o maldehyde aga ose gel. As expec ed, he e we e only wo in ense bands, co esponding o he 28S and 18S RNAs indica ing ha he o al RNA was in ac (da a no shown). The le els o mRNA o BN/GRP ecep o sub ypes hGRPR/BSR1 and hNMBR/BRS-2 we e analysed in U-87MG umou s o all g oups in expe imen 1 (Figu e 3). Densi ome ic analyses o RT – PCR p oduc s e ealed ha hGRPR/BRS-1 mRNA exp ession was 15.1+1.5 a bi a y uni s (a.u.) in he con ol g oup and 21.6+3.4 a.u. and 25.4+5.1 a.u. in he AN-201- and AN-215- ea ed g oups, espec i ely; he inc ease in bo h ea ed g oups being no signi ican . The hNMBR/BRS-2 mRNA exp ession was 7.6+3.5 a.u. in he con ol g oup and 10.9+3.6 a.u. and 3.3+1.0 a.u. in he AN-201- and AN-215- ea ed g oups, espec- i ely. The dec ease in he AN-215 g oup was again no s a is ically signi ican . No co esponding PCR p oduc s we e de ec ed om he nega i e con ol, indica ing ha he PCR p oduc s gene a ed om cDNA, and no om genomic DNA con amina ion. PCR p oduc s wi h he expec ed size o 207 bp Expe imen al The apeu ics e8 e7 e6 e5 e4 Na u al log o umou olume 0 5 10 15 20 Days o ea men Con ol AN-215 3×150 nmol kg–1 Recep o blockade 3×(200 µg RC-3095 + 150 nmol kg–1 AN-215) AN-201 + RC-3095 3×150 nmol kg–1 Figu e 2 E alua ion o he an i umou e ec o AN-215 in animals p e- ea ed o no wi h 200 mg o BN an agonis RC-3095 15 min be o e ad- minis a ion o AN-215 a a dose o 150 nmol kg 71 o block he ecep o - media ed up ake o he conjuga e. E ec s o an unconjuga ed mix u e o cy o oxic adical AN-201 and RC-3095 a e also shown. (A ows indica e he days o injec ions; e ical ba s show s.e.). Table 2 Tole ance o nude mice bea ing s.c. xenog a s o U-87MG hu- man glioblas omas o cy o oxic BN analogue AN-215, cy o oxic adical AN-201 and he mix u e o RC-3095 and AN-201 G oup Final BW, GRP(17-24)-e oked and g (% o se um gas in le el ea men Mo ali y a con ol) (pg ml 71 ) Con ol (Expe imen 1) 0/10 29.3+1.0 223.0+17.9 AN-201 2/10 25.5+0.8 (86%) 251.5+22.1 AN-215 1/11 25.4+0.6 (87%) 257.9+11.6 Con ol (Expe imen 2) 1/10 30.2+0.8 ND b AN-215 0/10 27.3+0.9 (90%) ND b Mix u e o AN-201 and 2/5 27.1+1.6 (89%) ND b RC-3095 RC-3095 and AN-215 c 1/9 29.6+0.8 (98%) ND b a Numbe o dead animals/numbe o o al. b ND=No de e mined. c RC-3095 was injec ed 15 min p io o AN-215 o block he ecep o s o BN. Table 3 The e ec s o ea men wi h cy o oxic BN analogue AN-215 and cy o oxic adical AN-201 on he his ological cha ac e is ics o U-87MG human glioblas omas in nude mice Numbe o Ra io o Numbe o umou s % a ea o Mi o ic Apop o ic apop o ic o AgNORs G oup e alua ed nec osis index index mi o ic indices pe cell 1 Con ol 9 60.0+5.8 16.6+1.4 5.9+0.7 0.38+0.06 4.93+0.18 2 AN-201 7 69.6+7.0 15.2+1.5 5.3+0.6 0.36+0.04 4.31+0.09* 3 AN-215 9 81.7+6.7* 10.9+2.2 6.2+0.8 0.85+0.25* 4.16+0.12* The alues a e means+s.e. *P50.05 s con ol. Cy o oxic bombesin analogue AN-215 inhibi s glioblas oma Z Sze eday e al 1325 ª 2002 Cance Resea ch UK B i ish Jou nal o Cance (2002) 86(8), 1322 – 1327 o hGAPDH gene we e p esen in all samples (Figu e 3) con i m- ing ha no RNA deg ada ion occu ed du ing he p epa a ions. Radio ecep o assays In he con ol g oup, adiolabelled [Ty 4 ]BN was bound o a single class o speci ic binding si es wi h high a ini y (K d =5.36+ 0.69 nM), and low capaci y (B max =362.7+11.3 mol mg 71 memb ane p o ein). Speci ic high a ini y binding si es o BN/ GRP we e also ound on he AN-215- ea ed umou s (K d = 5.98+0.91 nM,B max =362.6+8.6 mol mg 71 memb ane p o ein). Thus, he ea men wi h AN-215 did no a ec he binding cha - ac e is ics o ecep o s o BN/GRP in U-87MG human glioblas oma. The concen a ion o unlabelled AN-215 equi ed o inhibi 50% o he speci ic 125 I-[Ty 4 ]BN binding (IC 50 ) was 4.0+0.1 nM. This IC 50 alue o AN-215 ep esen s a high binding a ini y o BN/GRP ecep o p o ein exp essed on U-87MG umou s. DISCUSSION Chemo he apy is s ill one o he majo modali ies o he ea men o inope able malignancies o o adju an he apy a e su ge y. Howe e , he use o an ineoplas ic agen s is hampe ed by hei non-speci ic oxici y o no mal, heal hy issues and in insic o acqui ed chemo esis ance o cance ous cells. The a ge ing o chemo he apeu ic agen s speci ically o umou si es is a mode n app oach ha may help o e come some o hese d awbacks (Schally and Nagy, 1999). Recen ly, we de eloped a se ies o a ge ed cy o oxic conjuga es based on analogues o pep ide ho mones such as lu einizing ho mone- eleasing ho mone(LH-RH), soma os a in and BN/GRP, o which high a ini y ecep o s a e exp essed on a a ie y o umou s (Schally and Nagy, 1999; Nagy and Schally, 2001). DOX o i s in ensely po en de i a i e, 2-py olino-DOX (AN-201), which is 1000 imes mo e po en han DOX in i o, we e linked h ough hei 14-O-glu a yl es e s o a ee amino g oup in he pep ide ca ie s o o m conjuga es which e ained cy o oxic ac i - i y and high binding a ini y o espec i e ecep o s. LH-RH analogue AN-207 con aining cy o oxic adical AN-201 also s ongly inhibi s DOX- esis an MX-1 human b eas cance s in i o (Kahan e al, 1999), indica ing ha 2-py olino-DOX is non-c oss- esis an wi h DOX, in analogy wi h simila highly po en de i a i es o DOX such as Nemo ubicin (Nagy and Schally, 2001). In addi ion, we demons a ed ha he p esence o ecep o s o soma os a in on U-87MG human glioblas omas can be u ilised o an imp o emen in he apeu ic e icacy when he a ge ed cy o oxic soma os a in analogue AN-238 which con ains AN-201 is used, bu no i s coun- e pa con aining DOX (Kia is e al, 2000). I has been shown ha a high pe cen age o b ain umou cell lines, including U-87MG, exp ess ecep o s o BN/GRP (Sha i e al, 1997). In ou s udy we e alua ed he e ec s o a ge ed he apy o U-87MG human glioblas oma xenog a ed in o nude mice u ilising one o ou cy o oxic BN-like conjuga es, AN-215 con aining AN-201. The esul s o expe imen 1 indica e, ha simila ly o soma os a in ecep o s, BN/GRP ecep o s on b ain umou s can also be employed o he a ge ing o conjuga es con aining he highly po en de i a i e o DOX, AN-201 in o de o imp o e he e icacy and lowe i s oxici y. This inding is in ag eemen wi h ou p e ious esul s on PC-3 human and ogen independen p os a e cance s and H-69 small cell lung cance s, which indica ed ha imp o ed e icacy can be achie ed by a ge - ing AN-201 o ecep o s o soma os a in o BN/GRP. To co obo a e he concep ha AN-215 ac s h ough he binding o ecep o s o BN on U-87MG umou s, we designed a second expe imen , in which an excess o he BN an agonis RC-3095 was injec ed i. . 15 min be o e he adminis a ion o AN-215 in o de o block he ecep o s o BN. As an icipa ed, he blockade o BN ecep o s signi ican ly lowe ed he an i umou ac i i y o AN-215 esul ing only in a non-signi ican g ow h inhibi ion o U-87MG glioblas omas, simila o ha p oduced by AN-201. This insigni - ican e ec is p obably due o he ac ha he blockade o he BN ecep o s caused a longe exposu e o AN-215 o non-speci ic ca boxyles e ase enzymes (EC3.1.1.) in he ci cula ion, which can elease he cy o oxic adical be o e he a ge ing is comple ed (Nagy e al, 2000). P eclinical and clinical esul s wi h new adi- oligands, de eloped o BN- ecep o scin ig aphy, also indica e ha BN analogues can accumula e in BN ecep o -posi i e umou s, u he suppo ing he heo y ha BN ecep o s can be used o a ge ed chemo he apy (B eeman e al, 1999; Van de Wiele e al, 2000). Binding assays using 125 I-labelled[Ty 4 ]BN con i med he p esence o BN/GRP ecep o s on U-87MG umou memb anes (Pinski e al, 1994) and demons a ed a high a ini y binding o AN-215 o hese ecep o s cha ac e ised by an IC 50 alue o 4.0+0.1 nM. RT – PCR analyses also con i med p e ious indings ha hese umou s exp ess mRNAs o BN ecep o sub ype 1 and 2 bu no o he o phan ecep o sub ype 3 (Kia is e al, 1999b) His ological analysis showed ha ea men wi h AN-215, bu no wi h cy o oxic adical AN-201, inc eased signi ican ly he a ea o nec osis and he a io o apop o ic o mi o ic indices in U- 87MG umou s. This can be explained by a highe accumula ion o he cy o oxic adical in umou issue a e ea men wi h AN-215, as a esul o a ge ing. The his ological da a also sugges ha such accumula ion o AN-201 can cause cell dea h h ough nec osis, which may be p e e able in cance he apy o ha caused by apop osis (Kia is and Schally, 1999). In ou p e ious s udy (Plonowski e al, 2000) we epo ed ha a e he inal injec ion o AN-215, AN-201 o he mix u e o AN- 201 and RC-3094 he e was an app oxima e 50% educ ion in WBC coun in nude mice bea ing PC-3 human p os a e cance s. This dec ease in WBC was accompanied by a ansien dec ease in BW. In he p esen s udy no measu emen s o WBC we e ca ied ou . Ins ead, he assessmen o oxici y was based on changes in BW and mo ali y which indica ed, in bo h expe imen s, ha a ge ed cy o oxic BN analogue AN-215 is no oxic (Table 2). In expe imen 1, wo o 10 animals (20%) died a e ou conse- cu i e injec ions o AN-201 a 150 nmol kg 71 o BW dose bu , one o 11 mice (9%) also died ecei ing he same dose o AN- 215. This oxici y o AN-215 can be explained by he ac ha he ac i i y o es e ase enzymes in he se um o nude mice is e y high, abou six imes highe han in humans, causing a pa ial elease o AN-201 be o e he a ge ing is comple ed (Nagy e al, 2000). This phenomenon can also accoun o he dea h o one o nine animals in expe imen 2, in which a high dose o BN an agonis RC-3095 was injec ed in o nude mice o block BN Expe imen al The apeu ics M – 1 2 3 4 5 6 7 8 9 hGRPR — hNMBR — hGAPDH — Figu e 3 Aga ose gel elec opho esis o e e se- ansc ibed and PCR- ampli ied mRNAs o hGRPR/BRS-1 and hNMBR/BRS-2 in U-87MG u- mou s in nude mice. The bands o he co esponding hGAPDH a e also shown. M, DNA size ma ke ; 7, nega i e con ol; 1 – 3, un ea ed umou s; 4 – 6, AN-201- ea ed umou s; 7 – 9, AN-215- ea ed umou s. All PCR- ampli ica ion eac ions yielded p oduc s o he expec ed size, which we e 158 bp o hGRPR/BRS-1, 484 bp o hNMBR/BRS-2 and 207 bp o hGAPDH. Cy o oxic bombesin analogue AN-215 inhibi s glioblas oma Z Sze eday e al 1326 B i ish Jou nal o Cance (2002) 86(8), 1322 – 1327 ª 2002 Cance Resea ch UK ecep o s, be o e each o he h ee injec ions o AN-215 a 150 nmol kg 71 o BW. The blockade o BN ecep o s would inhi- bi ecep o -media ed accumula ion o AN-215 in BN ecep o - posi i e issues, and he ex ended ime o AN-215 in he ci cula- ion can cause he elease o a high pe cen age o AN-201 om he conjuga e. In his expe imen , no dea hs occu ed in he g oup ea ed wi h he same dose o AN-215 wi hou p e ea - men wi h RC-3095. In u he suppo o his iew, a signi ican ly highe ole ance o AN-215 by nude mice was demons a ed when ca boxyles e ase enzymes we e inhibi ed (Nagy e al, 2000; Plonowski e al, 2000). Because ecep o s o BN a e also exp essed in a ious no mal issues such as he CNS, lungs, panc eas, b eas , p os a e o he gas oin es inal ac , i is assumed ha he e is a compe i ion o a ge ed cy o oxic conjuga e AN-215 be ween no mal and cance ous issues. Ou p e ious esul s indica e ha despi e his compe i ion, AN-215 is highly e icacious e en in nude mice wi h supp essed es e ase ac i i y (Plonowski e al, 2000). In his s udy and also in ou p e ious wo k (Plonowski e al, 2000), we ound ha AN-215 caused no speci ic damage o he gas oin es inal cells as de e - mined by GRP-s imula ed gas ic sec e ion. In conclusion, he p esen s udy demons a es ha cy o oxic BN analogue AN-215 is no oxic and signi ican ly mo e e ec i e in inhibi ing he g ow h o U-87MG human glioblas omas han i s non- a ge ed cy o oxic adical AN-201. Because ecep o s o BN/ GRP a e p esen on a high pe cen age o human glioblas oma cell lines, i is likely ha u he s udies wi h AN-215 ollowed by he - apeu ic ials could esul in he de elopmen o a new he apeu ic app oach o he managemen o pa ien s su e ing om ad anced glioblas omas. ACKNOWLEDGEMENTS The wo k desc ibed in his pape was suppo ed by he Medical Resea ch Se ice o he Depa men o Ve e ans A ai s and g an s om Zen a is (F ank u /Main, Ge many) o Tulane Uni e si y (all o AV Schally). 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