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Recep o - a ge ed he apy o human expe imen al u ina y
bladde cance s wi h cy o oxic LH-RH analog AN-152 (AEZS-
108)
Ka oly Szepeshazi 1,2, And ew V. Schally1,2,3,4,5, Gunhild Kelle 6, No man L. Block1,2,3,4,
Daniel Ben en7, Gabo Halmos1,2,3,8, Luca Szalon ay1,2, I ing Vidau e1,2, Miklos
Jaszbe enyi1,2,3, Fe enc G. Rick1,2,3
1 Ve e ans A ai s Medical Cen e Miami, FL
2 Sou h Flo ida VA Founda ion o Resea ch and Educa ion, Miami, FL
3 Depa men o Pa hology, Uni e si y o Miami, Mille School o Medicine, Miami, FL
4 Di ision o Hema ology/Oncology Uni e si y o Miami, Mille School o Medicine, Miami, FL
5 Di ision o Endoc inology, Depa men o Medicine, Uni e si y o Miami, Mille School o Medicine, Miami, FL
6 Sec ion o Hema ology/Oncology, Uni e si y Clinic, Hambu g, Ge many
7 Depa men o Gas oen e ology Uni e si y Clinic, Hambu g, Ge many
8 Depa men o Biopha macy, School o Pha macy, Uni e si y o Deb ecen, Hunga y
Co espondence o: And ew V. Schally, email: [email p o ec ed]
Co espondence o: Fe enc G. Rick, email: [email p o ec ed]
Keywo ds: u ina y bladde , u o helial cance , a ge ed he apy, LH-RH ecep o , cy o oxic, doxo ubicin
Recei ed: July 11, 2012, Accep ed: July 20, 2012, Published: July 22, 2012
Copy igh : © Szepeshazi e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
ABSTRACT:
Many bladde cance s p og ess o in asion wi h poo p ognosis; new he apeu ic
me hods a e needed. We de eloped a cy o oxic LH-RH analog, AN-152 (AEZS-108)
con aining doxo ubicin (DOX), o a ge ed he apy o cance s exp essing LHRH
ecep o s. We in es iga ed he exp ession o LH-RH ecep o s in clinical bladde
cance s and in HT-1376, J82, RT-4 and HT-1197 human bladde cance lines. The e ec
o analog, AN-152, on g ow h o hese umo lines xenog a ed in o nude mice was
analyzed. Using molecula and unc ional assays, we also e alua ed he di e ences
be ween he e ec s o AN-152, and DOX alone. We demons a ed he exp ession o
LH-RH ecep o s on 18 clinical bladde cance s by immunohis ochemis y and on ou
human u ina y bladde cance lines HT-1376, J82, RT-4 and HT-1197 by Wes e n
blo ing and binding assays. AN-152 powe ully inhibi ed g ow h o hese bladde
cance s in nude mice. AN-152 exe ed g ea e e ec s han DOX and was less oxic.
DOX ac i a ed s ong mul id ug esis ance mechanisms in RT-4 and HT-1197 cance s,
while AN-152 had no o less such e ec . PCR assays and in i o s udies e ealed
di e ences in he ac ion o AN-152 and DOX on he exp ession o genes in ol ed in
apop osis. These esul s sugges ha a ge ed cy o oxic LH-RH analog, AN-152 (AEZS-
108), should be examined o ea men o pa ien s wi h LH-RH ecep o posi i e
in asi e bladde cance s.
INTRODUCTION
Yea ly almos 400,000 new cases o u ina y bladde
cance a e diagnosed in he wo ld and mo e han 150,000
people die o he disease [1]. In he US, bladde cance is
he i h mos equen malignancy and he mos expensi e
umo o ea [2]. App oxima ely 75% o bladde cance s
a e diagnosed a an ea ly s age; hal o hese p og ess o
in asi e umo [2].
Chemo he apy o he ea men o me as a ic o
ecu en ansi ional cell ca cinoma o he u ina y ac
began decades ago. Doxo ubicin (DOX) and cispla in
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as single agen s o in a ious combina ions showed
e icacy in he ea men o ad anced bladde cance and,
in combina ions wi h cyclophosphamide, me ho exa e,
inblas ine, eached esponse a es o 90% [3, 4]. The
combina ion o me ho exa e, inblas ine, ad iamycin
and cispla in (M-VAC) inc eased median su i al o
pa ien s up o 12.5 mon hs [5]. These esponse a es we e
accompanied by signi ican sys emic oxici y and equen
elapse due o esis ance o addi ional he apy [5-7].
Combina ions o gemci abine and cispla in show esponse
a es simila o he M-VAC [8] wi h less se e e side e ec s
and a e conside ed a s anda d o ca e o pa ien s wi h
me as a ic disease [9].
Elucida ion o he molecula cha ac e is ics o
u o helial cance s in oduced possibili ies o a ge ed
he apies. Recep o s o g ow h ac o s appea o play
a ole in p og ession o u o helial ca cinoma [10]. Thus,
a ge ing ecep o y osine kinases, e.g. EGF, He -2/neu,
o manipula ion o signal ansduc ion pa hways p o ide
new he apeu ic s a egies [11, 12] bu equi e molecula
analyses o selec pa ien s who would bene i [13].
The demons a ion o ecep o s o neu opep ide
ho mones on a ious umo cells [14-16] led o
de elopmen o cy o oxic pep ides and pep ide ho mones
linked o adionuclides o umo diagnosis and he apy
[17-19]. Radiolabeled analogs o soma os a in, bombesin
o asoac i e in es inal pep ide (VIP) a e now inc easingly
used o umo imaging and he apy [17-19]. Ou g oup
syn hesized analogs o LH-RH, soma os a in, and
bombesin linked o DOX which selec i ely a ge umo s
exp essing he speci ic ecep o s while spa ing no mal
issues om oxici y. These analogs inhibi g ow h o
a ious expe imen al human cance s, and a e mo e
e ec i e and ole able han he cy o oxic adical, DOX,
alone [17-19].
Ini ially, ecep o s o LH-RH we e demons a ed on
human b eas , endome ial, o a ian, and p os a ic cance s
[17-20]. Subsequen ly exp ession o hese ecep o s was
also ound on human non-Hodgkin’s lymphomas [21],
enal cell ca cinomas [22] and malignan melanomas [23],
sugges ing he po en ial o a ge ed he apy wi h cy o oxic
analogs o LH-RH. Cy o oxic LH-RH analog, AN-152
(AEZS-108), showed p omising esul s in phase II clinical
ials in women wi h se e al gynecological cance s [24,
25] and is now in phase I/II clinical ials o pa ien s wi h
p os a e [26] and bladde cance s [19, 27]. In his s udy we
in es iga ed he exp ession o LH-RH ecep o s in clinical
human u ina y bladde specimens and in ou human
bladde cance cell lines. We also analyzed he e ec o
cy o oxic LH-RH analog, AN-152 (AEZS-108), on g ow h
o human expe imen al umo s xenog a ed in o nude
mice. We compa ed he e ec o AN-152 and i s cy o oxic
adical, DOX, by molecula and unc ional assays.
RESULTS
LH-RH ecep o exp ession in human bladde
cance
Eigh een human p ima y u o helial cance samples
we e e alua ed by immunohis ochemis y. Posi i e
s aining o LH-RH ecep o s was obse ed in all
specimens (Fig. 1 a-c). Enhanced s aining o he plasma
memb ane as well as cy oplasmic s aining we e de ec ed
in malignan cells and he posi i e con ol (human an e io
pi ui a y) (Fig. 1d). In h ee samples, high le els o LH-
RH ecep o exp ession wi h mo e han 75% posi i ely
s ained malignan cells was ound, wo samples e ealed
weak exp ession; 13 samples we e in e media e.
Exp ession was a iable; a eas o high, dis inc and low
LH-RH ecep o densi y we e some imes seen wi hin one
sample. In hese cases, he domina ing ecep o densi y
was chosen o inal ca ego iza ion. In su ounding
non-malignan issue no o ma ginal LH-RH ecep o
exp ession was ound (Fig. 1a-c).
E ec o ea men s on umo g ow h in nude
mice
In Expe imen 1, AN-152 s ongly inhibi ed HT-
1376 cance s. Tumo olume and umo weigh s we e
signi ican ly less han con ol (Fig. 2a) (Table 1). In
con as , DOX, Ce o elix and [D-T p6]LH-RH had no
e ec on umo g ow h. The median g ow h a e alues
o he umo s ea ed wi h AN-152 and DOX di e ed
signi ican ly (P=0.018). Body and o gan weigh s we e
Figu e 1:Exp ession o LH-RH ecep o s in
human bladde ca cinomas. The issue was s ained by
immunohis ochemis y wi h p ima y LH-RH ecep o an ibody.
A-C: u o helial bladde ca cinoma; D: posi i e con ol, an e io
pi ui a y.
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simila in all g oups, excep o o a ian weigh s, which
we e lowe in he AN-152 g oup (also in Expe imen s 3
and 4) (da a no shown).
In Expe imen 2 (Fig. 2b), bo h AN-152 and DOX
a i s powe ully inhibi ed g ow h o J82 umo s. The
umo s ea ed wi h DOX s a ed eg owing a day 80
while hose ea ed wi h AN-152 con inued inhibi ion. A
he end o he expe imen , AN-152 p oduced a signi ican
(84%) educ ion in olume. The DOX dec ease was less
(76%) and no signi ican ly di e en om con ol. Tumo
weigh s and body weigh s we e lowe han con ol in bo h
ea ed g oups (Table 1). The mice ha ecei ed DOX
we e emacia ed; hei mean weigh was signi ican ly
lowe han ha o con ols o animals ea ed wi h AN-
152 (Table 1).
In Expe imen 3, AN-152 s ongly inhibi ed
g ow h o RT-4; he umo s did no esume g ow h a e
ea men cessa ion (Fig. 2c). DOX ini ially educed RT-4
p oli e a ion, he umo s s a ed g owing mo e in ensely
a e ea men cessa ion. Tumo weigh s we e also lowe
a e ea men wi h AN-152, bu no DOX (Table 1).
DOX again signi ican ly lowe ed animal body weigh s
(Table 1).
In Expe imen 4, bo h AN-152 and DOX p oduced
a subs an ial olume educ ion o HT-1197 cance s; he
e ec o AN-152 was signi ican ly g ea e han ha o
DOX (Fig. 2d). Tumo weigh s we e lowe only in he
g oup ecei ing AN-152 (Table 1). DOX signi ican ly
educed he weigh s o mice a day 29, hese emained
signi ican ly lowe o he end o he expe imen (Table
1). Thus, G oup 2 ecei ed ano he AN-152 injec ion on
day 30, bu DOX was discon inued because o sys emic
Figu e 2:E ec o ea men wi h cy o oxic LH-RH analog, AN-152 (AEZS-108), and doxo ubicin (DOX), on g ow h o
human bladde cance s in nude mice. The e ical ba s ep esen SE. Solid a ows show ea men s wi h bo h cy o oxic compounds,
dashed a ow shows ea men only wi h AN-152 (2d).
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oxici y.
Recep o assays and Wes e n blo s
Radiolabeled [D-T p6]LH-RH was bound o a single
class o speci ic binding si es on all ou cance lines.
The concen a ions o LH-RH ecep o s and he binding
a ini y a ied sligh ly among he umo models, as shown
in supplemen a y Table S1. LH-RH ecep o p o ein (38
KD) was de ec ed in all ou umo s by Wes e n blo ing,
he le els o ecep o p o ein being no signi ican ly
di e en be ween ea ed g oup and con ol (Fig. 3).
Molecula analysis
Using he Cance D ug Resis ance and Me abolism
PCR A ay, we analyzed he exp ession o 84 genes ha
may in luence esponse o chemo he apy. The assays
in ol e genes ela ed o d ug esis ance, d ug me abolism,
DNA epai , ansc ip ion, and cell cycle egula ion, as
well as hose encoding ecep o s o g ow h ac o s and
ho mones. Th ee bladde cance lines (J82, RT-4 and
HT-1197) we e in es iga ed and he esul s a e p esen ed
in Figu es 3-4. The deg ee and pa e n o changes in
gene exp ession we e di e en in each o he h ee. J82
showing he leas and HT-1197 he s onges , al e a ions.
In J82 umo , genes ela ed o d ug esis ance we e no
changed, excep ABCC2 (ATP-binding casse e, sub-
amily B, membe 1), which was educed by DOX. In
RT-4 cance s, DOX p oduced a mode a e inc ease o 5
and a mino dec ease o 4 genes, while AN-152 caused a
sligh dec ease in 6. P ac ically all d ug esis ance genes
we e o e exp essed in HT-1197 umo s ea ed wi h ei he
agen ; he inc ease was much g ea e a e DOX (Fig.
4a). Nea ly all genes in ol ed in d ug me abolism we e
inc eased by DOX in all umo s, while ea men wi h
AN-152 caused only sligh ampli ica ions o hese genes
in HT-1197. (Fig. 4b). Genes engaged in DNA epai
we e no a ec ed by ei he ea men in J82, mode a ely
changed in RT-4 and s ongly inc eased in HT-1197
umo s; DOX he apy induced s onge al e a ions (Fig.
4c). Fig. 4d demons a es ha genes encoding cyclines and
Figu e 3:De ec ion LH-RH ecep o p o ein (38 KD) by
Wes e n blo ing. All ou human u ina y bladde cance lines
g own in nude mice exp essed LH-RH ecep o s. Rep esen a i e
blo s o h ee independen expe imen s a e shown.
Table 1: G ow h cha ac e is ics o human u ina y bladde cance s in nude mice and
changes in body weigh s o he animals a e ea men wi h LH-RH analogs AN-152 o
DOX
G oups Tumo olume (mm3) Tumo weigh s (mg) Body weigh s (g)
Expe imen 1 HT-1376
1. Con ol 999 ± 375 900 ± 340 27.3 ± 1.5
2. Ce o elix 1154 ± 438 623 ± 124 28.6 ± 0.6
3. [D-T p6]LH-RH 1007 ± 349 648 ± 161 24.6 ± 0.6
4. AN-152 377 ± 240 256 ± 139* 26.2 ± 0.9
5. DOX 858 ± 365 396 ± 96 24.2 ± 0.6*
Expe imen 2 J82
1. Con ol 1456 ± 493 1256 ± 384 29.2 ± 0.9
2. AN-152 238 ± 111* 360 ± 181 24.3 ± 0.9*
3. DOX 351 ± 131 386 ± 181 21.6 ± 0.6*†
Expe imen 3 RT-4
1. Con ol 2824 ± 961 1353 ± 532 24.6 ± 0.6
2. AN-152 416 ± 313* 475 ± 339* 24.3 ± 1.1
3. DOX 1613 ± 764 868 ± 673 22.0 ± 1.6*
Expe imen 4 HT-1197
1. Con ol 563 ± 152 527 ± 173 28.9 ± 1.5
2. AN-152 60 ±20* 54 ± 30* 25.3 ± 0.9
3. DOX 150 ± 38*† 70 ± 23 23.1 ± 1.1*
Values a e means ± SE. *P<0.05 s. Con ol †P<0.05 s. AN-152 g oup
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Figu e 4:Human u ina y bladde cance s g own in nude mice we e analyzed wi h he Human Cance D ug Resis ance
& Me abolism RT P o ile PCR A ay The e ical ba s on he le shows 10- old change compa ed o con ol. *=
P<0.05 s. con ol.
(A)Changes in genes in ol ed in d ug esis ance. ABCB1: ATP-binding casse e, sub amily B, membe 1; ABCC1-6: ATP-binding
casse e, sub amily C, membe s 1-6; ABCG2: ATP-binding casse e, sub amily G, membe 2; BAX: BCL2-assicia ed X p o ein; BCL2:
B-cell CCL/lymphoma 2; BCL2L1: BCL2-like 1; MVP: Majo aul p o ein; RB1: Re inoblas oma 1; TOP1: Topoisome ase (DNA) I;
TOP2A: Topoisome ase (DNA) II alpha; TOP2B: Topoisome ase (DNA) II be a; TP53: Tumo p o ein p53.
(B)Changes in genes in ol ed in d ug me abolism. ARNT: A yl hyd oca bon ecep o nuclea ansloca o ; BLMH: Bleomycin
hyd olase; CLPTM1L: Cle lip and pala e ansmemb ane p o ein 1-like p o ein (cispla in esis ance- ela ed p o ein); CYP1A1,
CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5: Cy och ome P450, amily 1-3, sub amily
A-D, polypep ide 1-19; DHFR: Dihyd o ola e educ ase; EPHX1: Epoxide hyd olase , mic osomal (xenobio ic); GSK3A: Glycogen
syn hase kinase 3 alpha; GSTP1: Glu a hione S- ans e ase pi 1; NAT2: N-ace yl ans e ase 2; SOD1: Supe oxide dismu ase 1; SULT1E1:
Sul o ans e ase amily 1E, es ogen-p e e ing, membe 1; TPMT: Thiopu ine S-me hyl as e ase.

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cycline dependen kinases we e simila ly changed by he
wo compounds, while DOX caused a s onge inc ease
in kinase inhibi o s. Rega ding g ow h ac o s and hei
ecep o genes, DOX inc eased EGF ecep o (EGFR ),
E bB2, E bB4 (V-e b-b2 e y h oblas ic leukemia i al
oncogene homolog 2 and 4), ib oblas g ow h ac o 2
FGF2 and IGF-1 ecep o (IGF-1R) in a ious umo s,
while AN-152 he apy esul ed in small inc eases in
E bB2, E bB4 and FGF2 in HT-1197 cance s only (Fig.
4e). Inc eased exp ession in a a ie y o ho mone ecep o
genes was he s onges in HT-1197 umo s, wi h DOX
ha ing a much g ea e e ec han AN-152 (Fig. 4 ).
Genes ela ed o ansc ip ion ac o s we e mos ly down-
egula ed in RT-4 and up- egula ed in HT-1197 cance s;
DOX had a mo e powe ul e ec in he la e (Fig. 4g).
We also used he Human Apop osis PCR A ay
o de ec changes in exp ession o 84 genes in ol ed in
p og ammed cell dea h. An i-apop o ic gene exp ession
was inc eased by DOX in all h ee umo s, a ec ing a ew
genes only in J82, mo e in RT-4 and he mos in HT-1197.
AN-152 esul ed in a mode a e inc ease in an i-apop osis
genes in HT-1197 cance s (Supplemen a y Fig. S1a).
Rega ding p o-apop o ic genes, DOX p oduced s onge
inc eases han AN-152 in RT-4 umo s (Supplemen a y
Fig. S1b).
P o ein analysis by Wes e n blo showed inc eases
in an i-apop o ic p o eins, Bcl2 and BclX, in all ea ed
umo s ( esul s no shown), mo e in hose ea ed wi h
DOX. The di e ences we e s a is ically no signi ican .
Mul i-d ug esis ance and apop osis assays in
i o
The MDR s udy showed ha ea men wi h ei he
cy o oxic compound esul ed in a e en ion o calcein in all
h ee umo cell lines, bu he e en ion was signi ican ly
highe a e ea men wi h AN-152 han DOX. The
g ea es di e ences be ween he e ec o AN-152 and
DOX we e in RT-4 and HT-1376 and he leas in J82 (Fig.
5a). Apop osis assay in i o e ealed ha AN-152 had a
s onge apop ogenic e ec han DOX on RT-4 and HT-
1376 umo cells, while bo h compounds ac ed simila ly
on J82 cells (Fig. 5b).
DISCUSSION
Siegel e al [28] es ima e ha 73,510 new cases
o u ina y bladde cance will be diagnosed in he US
in 2012 wi h 14,880 es ima ed dea hs. The numbe s o
newly diagnosed cases and dea hs a e high and equi e
imp o emen [28]. The es ima ed 5-yea cos o ca e o
Medica e is app oxima ely one billion dolla s [29].
Pla inum based egimens a e cu en ly he s anda d
o he apy bu me as a ic u o helial cance emains a
deadly and cos ly disease [5, 29, 30]. Mo e esea ch wi h
no el, a ge ed agen s is needed o imp o e ou comes [29].
Ta ge ed he apies a e being widely in es iga ed
and inc easingly used o ea men o a ious umo s.
Ta ge ing p oduces an imp o emen in umo e ec and
diminishes sys emic oxici y [17-19, 27]. Pep ide ho mone
ecep o s p esen on a ious cance cells, can unc ion as
a ge s o speci ic compounds composed o cy o oxic
agen s conjuga ed o pep ide analogs. The pep ide
ho mone se es as a ca ie molecule o homing he
cy o oxic agen o a ge cells con aining speci ic ecep o s.
Thus, we ha e de eloped cy o oxic compounds con aining
DOX conjuga ed o an LH-RH agonis , es ed hem on a
a ie y o expe imen al umo models, and showed ha
hey a e mo e e ec i e and less oxic han unconjuga ed
DOX [17-19, 25, 27]. Besides he pi ui a y, ecep o s o
LH-RH ha e been de ec ed in a ious human cance cell
Figu e 4:Human u ina y bladde cance s g own in nude mice we e analyzed wi h he Human Cance D ug Resis ance
& Me abolism RT P o ile PCR A ay The e ical ba s on he le shows 10- old change compa ed o con ol. *=
P<0.05 s. con ol.
(C)Changes in genes in ol ed in DNA epai . 1: APC: Adenoma ous polyposis coli; ATM: A axia eleangiec asia mu a ed; BRCA1,
BRCA2: B eas cance 1, 2; ERCC3: Excision epai c oss-complemen ing oden epai de iciency, complemen a ion g oup 3 (xe ode ma
pigmen osum g oup B complemen ing); MSH2:Mu S homolog 2, colon cance , nonpopyposis ype 1; XPA, XPC: Xe ode ma pigmen osum,
complemen a ion g oup A, C.
(D)Genes in ol ed in cell cycle. CCND1, CCNE1: Cyclin D1, E1; CDK2, CDK4: Cyclin dependen kinase 2, 4; CDKN1A, CDKN1B,
CDKN2A: CDKN2D: Cyclin-dependen kinase inhibi o 1A, 1B, 2A, 2D.
(E)Changes in g ow h ac o genes. EGFR: Epide mal g ow h ac o ecep o ; ERBB2, ERBB3, ERBB4: V-e b-b2 e y h oblas ic
leukemia i al oncogene homolog 2, 3, 4:FGF2: Fib oblas g ow h ac o 2; IGF1R, IGF2R: Insulin-like g ow h ac o 1, 2 ecep o ; MET:
Me p o o-oncogene (hepa ocy e g ow h ac o ecep o ).
(F)Ho mone ecep o genes. AR: And ogen ecep o ;ESR1, ESR2: Es ogen ecep o 1, 2; PPARA, PPARD, PPARG: Pe oxisome
p oli e a o -ac i a ed ecep o alpha, be a, gamma; RARA, RARB, RARG: Re ioic acid ecep o alpha, be a, gamma; RXRA, RXRB:
Re inoid X ecep o alpha, be a.
(G)Genes ela ed o ansc ip ion ac o s. AHR: A yl hyd oca bon ecep o ; AP1S1: Adap o - ela ed p o ein complex1, sigma
1 subuni ; ELK1: ELK1, membe o ETS oncogene amily; FOS: FBJ mu ine os eosa coma i al oncogene homolog; HIF1A: Hypoxia
inducible ac o 1, alpha subuni ; MYC: V-myc myelocy oma osis i al oncogene homolog; NFKB1, NFKB2, NFKBIB, NFKBIE: Nuclea
ac o o kappa ligh polypep ide gene enhance in B cells1, 2, inhibi o be a, epsilon; RELB: V- el e iculoendo heliosis i al oncogene
homolog B; TNFRSF11A: Tumo nec osis ac o ecep o supe amily, membe 11a.
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lines and human cance specimens. These include p os a e,
b eas , o a ian and endome ial cance s and o he cance s,
which a e ou side o he ep oduc i e sys em, such as
enal cell ca cinoma, melanoma, Hodgkin’s lymphoma
and colo ec al cance s [18, 19]. The exp ession o pep ide
ho mone ecep o s on bladde cance s has been minimally
in es iga ed. Only one g oup e i ied he exp ession o
LH-RH ecep o s in human bladde epi helium , bladde
cance s and bladde cance cell lines, bu did no de ec
any e ec o LH-RH on bladde cance cells in i o [31].
This s udy e eals he p esence o LH-RH ecep o s
(LH-RH R) in all 18 specimens o bladde cance pa ien s.
We also demons a ed LH-RH R exp ession by molecula
me hods and binding assays o LH-RH ecep o s in all
ou human bladde cance lines in es iga ed. The ou cell
lines used a e ansi ional cell ca cinomas o igina ing om
bladde , bu wi h a ious deg ees o di e en ia ion and
di e ing in cha ac e is ics and beha io . Thus, HT-1376
o igina ed om a g ade 3 ca cinoma wi h a unc ional
loss mu a ion in p53; RT-4 is a ansi ional cell papilloma
wi h wild- ype p53. Clinical ele ance o a ious umo
cell lines is linked o he clinical umo beha io [32].
RT-4 cance is associa ed wi h long su i al, low g ade,
and low in asi eness. In con as , J82 co esponds o high
g ade and in asion; HT-1376 has he sho es su i al.
The di e si y o he ou umo lines in es iga ed makes
he s udy mo e clinically ele an . These cell lines also
ha e a ying sensi i i y o DOX, as ea men wi h
DOX inhibi ed g ow h o J82 and HT-1197 umo s, bu
no HT-1376 and RT-4 cance s. The ou umo s showed
consis en inhibi ion in esponse o AN-152, which had a
s onge e ec and was less oxic han DOX.
To damage umo cells, an i-cance d ugs mus
en e he cell h ough he cell memb ane, and emain
o he ime necessa y o hei ac ion, a oiding se e al
de ensi e mechanisms. Resis ance a ec s many un ela ed
d ugs and is he e o e called mul id ug esis ance [33-
35]. Some cance s a e in insically esis an o speci ic
d ugs, o he s ini ially espond, bu de elop esis ance
du ing ea men . D ug esis an cells may o e g ow
du ing he apy and seconda y gene ic changes induced
by a d ug can lead o inc eased he apeu ic esis ance
Figu e 5:E ec s o AN-152, and DOX on HT-1376, J82 and RT-4 human u ina y bladde cance cells in i o. 5a:
calcein e en ion in he cells analyzed wi h MDR Assay Ki and 5b: apop osis in es iga ed wi h he Mul i-Pa ame e Apop osis Assay.
Onco a ge 9
www.impac jou nals.com/onco a ge
[34]. One mechanism in umo cells is he d ug-e lux
sys em ha consis s o a ious molecules belonging o
he ABC anspo e amily, and which can ejec d ugs
such as DOX om he cells. ABC anspo e s include
P-glycop o eins, (MDR1, mul i d ug esis ance p o ein
1; ABCB1, ATP-binding casse e sub- amily B, membe
1), MRP1 (mul i-d ug esis ance associa ed p o ein 1)
and o he ela ed compounds [33, 35]. Gene exp ession
analysis can elucida e speci ic esis ance pa hways [36];
iden i ying di e ences o d ugs such as DOX, cispla inum,
o pacli axel [37].
Mos bladde ca cinomas a e ini ially sensi i e o
chemo he apy, bu he majo i y de elop esis ance [38].
Tada e al [6] clea ly demons a ed ha , a e elapse,
he esponse a e o he apy in e sely co ela es wi h
he exp ession o genes ela ed o mul id ug esis ance
(MDR1, MRP1, MRP3). Thus, a no el app oach has been
de eloped o a ge ing esis ance ela ed molecules in
umo cells [39, 40].
In his s udy, he esul s o he Cance D ug
Resis ance and me abolism PCR A ay e ealed impo an
di e ences be ween he e ec s o AN-152 (AEZS-108)
and DOX on MDR ela ed genes. Ch onic ea men wi h
DOX esul ed in o e exp ession o mos genes in ol ed
in d ug e lux and d ug me abolism in RT-4 and HT-1197
cance s, while AN-152 (AEZS-108) caused educ ions
o smalle inc eases in hese genes. The gene al e a ions
seem o pe sis a e he apy cessa ion. The in i o
unc ional MDR assay also clea ly showed di e ences
among he bladde cance cell lines in hei eac ion o
a single ea men wi h DOX o AN-152 (AEZS-108).
Calcein e en ion was signi ican ly highe in all cell lines
a e ea men wi h AN-152, compa ed o ha caused
by DOX. These di e ences be ween he wo compounds
may be explained by an inc eased up ake by he cells o
DOX inco po a ed in he molecule o AN-152 compa ed
o unconjuga ed DOX, and also by a likely dec eased
anspo e ac i i y in he p esence o AN-152 compa ed
o DOX.
Many enzymes con ibu e o his in acellula
me abolism and inac i a ion o cy o oxic agen s; hese
enzymes can be induced o ac i a ed by he speci ic
d ugs. Ou in es iga ion e ealed impo an di e ences
be ween he e ec s o DOX and AN-152 on exp ession
o genes ela ed o d ug me abolism. A simila pa e n was
obse ed in DNA epai genes which ha e impo an oles
in esponses o he apy. Thus, ea men wi h pla inum
sal s is mo e e ec i e on umo s which exp ess low le els
o DNA epai ela ed genes. In he p esen s udy, DOX
caused g ea e changes han AN-152 in DNA epai ela ed
genes making umo s less sensi i e o he apy. DOX
esis ance can be also associa ed wi h changes in p o eins
in ol ed in cell cycle egula ion [41]. In ou s udy, cell
cycle associa ed genes in h ee umo lines we e a iously
a ec ed by he ea men s.
Rega ding g ow h ac o ecep o genes, ea men
wi h DOX inc eased some o hese genes, such as E bB2,
E bB4 and FGF2 in he umo s, which a e up- egula ed in
bladde cance s and esponsible o disease p og ession
[42-44]. Mo eo e , E bB4 may play a ole in DOX-
induced myoca dial DNA damage [45]. Ou esul s
showing ha DOX causes a g ea e inc ease in E bB4 han
AN-152 may explain he lack o ca dio oxici y so a seen
in p eclinical and clinical s udies wi h AEZS-108 [24, 25,
27].
Mos ho mone ecep o ela ed genes analyzed
we e inc eased by DOX and o a lesse deg ee by AN-
152. Es ogen ecep o s ha e a well-es ablished ole in
bladde cance p og ession [46, 47]. Simila ly, and ogen
ecep o s a e implica ed in bladde ca cinogenesis [48,
49]. The o he ho monal ecep o s in es iga ed, PPARs
(pe oxisome p oli e a o -ac i a ed ecep o s), RARs
( e inoic acid ecep o s) and RXRs ( e inoid X ecep o s)
can egula e se e al p ocesses impo an in umo
de elopmen , including cell p oli e a ion, di e en ia ion
and apop osis.
T ansc ip ion ac o s a e in ol ed in many
p ocesses, such as g ow h, di e en ia ion, umo igenesis
and apop osis. T ea men wi h DOX inc eased he
exp ession o many genes ela ed o ansc ip ion ac o s,
especially in HT-1197 umo s. Since Ka ashima e al
[50] e i ied ha NFkBs (nuclea ac o kappB) ha e a
ole in angiogenesis and me as asis o bladde cance s,
we selec ed NFkBs and showed ha NFkB was s ongly
inc eased by DOX bu no by AN-152.
P o- and an i-apop o ic genes in he umo s we e
a ec ed di e en ly by ch onic adminis a ion o he
wo d ugs. DOX changed p o-apop o ic genes, mo e
a o ably pa icula ly in RT-4 cance s. DOX also s ongly
inc eased he exp ession o many an i-apop o ic genes;
AN-152 caused ewe changes in hese. Ou sho in i o
s udy e ealed ha bo h DOX and AN-152 signi ican ly
inc eased apop osis in all h ee umo s. They ac ed
simila ly on J-82 cells and he apop ogenic ac ion o AN-
152 was much s onge han ha o DOX on RT-4 and
HT-1376 cance cells.
The PCR a ays used in his s udy highligh essen ial
di e ences be ween he ac ion o AN-152 and DOX on
a ious bladde cance s. The PCR a ays in es iga e
a wide spec um o umo cha ac e is ics; ou s udy
analyzed o e 150 genes. The pa e n o changes, a he
han indi idual changes in exp ession o single genes
seemed o be mo e impo an . The unc ional pa hways o
g oups o genes a e in e connec ed. Thus, we sepa a ely
in es iga ed genes ha a ec cell cycle and ansc ip ion
ac o s o hose in ol ed in DNA epai , bu he combined
e ec de e mines whe he a d ug such as DOX will induce
cell cycle a es , epai , p oli e a ion o apop osis [51].
Summa izing he esul s o ou s udy, we showed
he exp ession o LH-RH ecep o s in umo s o 18
pa ien s. We hen demons a ed ha cy o oxic LH-RH
analog, AN-152, powe ully inhibi s he g ow h o HT-