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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