This is a p e- o pos -p in o an a icle published in
E enbe ge -Neidnich , K., B eye , S., Mahal, K., Sasse,
F., Schobe , R.
Modi ica ion o up ake and subcellula dis ibu ion o
doxo ubicin by N-acylhyd azone esidues as isualised by
in insic luo escence
(2011) Cance Chemo he apy and Pha macology, pp. 1-6.
A icle in P ess.
Modi ica ion o up ake and subcellula dis ibu ion o
doxo ubicin by N-acylhyd azone esidues as isualised by
in insic luo escence
Ka ha ina E enbe ge -Neidnich • Sand a B eye • Ka ha ina Mahal • Flo enz
Sasse • Raine Schobe
Co espondence o R. Schobe , O ganic Chemis y Labo a o y, Uni e si y
Bay eu h, Uni e si ae ss asse 30, NW 1, 95447 Bay eu h, Ge many
e-mail: Raine .Schobe @uni-bay eu h.de
ax: +49(0)921 552671
F. Sasse, Helmhol z Cen e o In ec ion Resea ch (HZI), Depa men o
Chemical Biology, Inho ens asse 7, 38124 B aunschweig, Ge many
Abs ac
Pu pose: Doxo ubicin (1) is commonly used in he ea men o a wide ange o
cance s. Some N-acylhyd azones o 1 we e p e iously ound o ha e an imp o ed
umou and o gan selec i i y. In o de o cla i y he molecula basis o his e ec ,
he cellula up ake in o a ious cance cells and he localisa ion in P K2 po o oo
kidney cells o 1 and i s N-acylhyd azones de i ed om hep adecanoic acid (2)
and 11-(men hoxyca bonyl)undecanoic acid (3) we e s udied d awing on hei
in insic luo escence.
Me hods: The up ake o compounds 1–3 in o human cells o HL-60 leukaemia,
518A2 melanoma, HT-29 colon, and esis an KB-V1/Vbl and MCF-7/Topo
b eas ca cinomas was de e mined luo ime ically om hei esidual amoun s in
he supe na an . Thei ime-dependen accumula ion in P K2 po o oo kidney cells
was isualised by luo escence mic oscopy.
1
Resul s: The up ake, hough no he cy o oxici y, o 2 in mul i-d ug esis an
MCF-7/Topo b eas cance cells was conspicuously g ea e han ha o 1 and 3,
p obably due o an a ac i e lipophilic in e ac ion wi h he lipid- ich memb anes
o hese cells. In non-malignan P K2 cells bo h 1 and 3 accumula ed ini ially in
he nuclei. Upon p olonged incuba ion hei luo escen me aboli es we e
isualised in lysosomes neighbou ing he nuclei. In con as , conjuga e 2 was no
obse ed in he nuclei a any ime. A e wo hou s i had accumula ed in esicles
sca e ed all o e he cells and upon p olonged incuba ion i s luo escen
me aboli es we e concen a ed in he cellula memb ane.
Conclusions: Long unb anched a y acyl esidues when a ached o doxo ubicin
ia a hyd azone can ac as lipophilic memb ane ancho s. This allows an inc eased
up ake o such de i a i es in o lipid- ich memb anes especially o mul i-d ug
esis an cance cells, a e a ded elease om he e in o he cy osol and he
e en ual s o age o hei me aboli es again in he cell memb ane a he han in
lysosomes.
Keywo ds Doxo ubicin • Fluo escence spec oscopy • P K2 cells • Conjuga es •
Memb ane lipids • Fa y acids
Con lic s o disclosu es: NONE
2
In oduc ion
Doxo ubicin (1) – a S ep omyces me aboli e – is used in chemo he apy agains a
wide ange o cance s such as haema ological malignancies, so issue sa comas,
lymphomas and a ious ypes o ca cinomas despi e i s clinical limi a ions such as
ca dio oxici y and induc ion o mul i-d ug esis ance [1–6]. In ea lie s udies, we
had shown ha some N-acylhyd azones o 1 de i ed om long-chain a y acids
o e penes ha e a highe umou and o gan selec i i y han 1 while sha ing wi h i
he same mechanism o apop osis induc ion, cha ac e ised by ele a ed le els o
eac i e oxygen species and apop osis- ele an caspases and a loss o he
mi ochond ial memb ane po en ial. Some o hese new hyd azone conjuga es no
only had a g ea e selec i i y han he pa en d ug 1 [7, 8], bu we e also less good
subs a es o he ABC- anspo e s o mul i-d ug esis an cance cells. In o de o
cla i y he molecula basis o hese e ec s we now s udied he cellula up ake o
1 and i s N-acylhyd azones de i ed om hep adecanoic acid (2) and 11-
(men hoxyca bonyl)undecanoic acid (3) by a ious cance cells and hei
localisa ion in la , easy o isualise P K2 po o oo (Po o ous idac ylis) kidney
epi helial cells by means o hei in insic luo escence (Fig. 1).
((Figu e 1 he e))
Ma e ials and me hods
Gene al
The hyd azones 2 and 3 we e p epa ed as desc ibed p e iously [7]. Fluo escence
spec a we e eco ded on an FP-6500 luo escence spec opho ome e (JASCO,
Tokyo, Japan) be ween 500 and 700 nm. Up ake o he de i a i es was measu ed
using a CM In ini e F200 luo escence pla e eade wi h exci a ion and emission
wa eleng hs o 485/20 and 590/20 nm (TECAN, C ailsheim, Ge many).
3
Fluo escence analyses o ea ed cells we e conduc ed wi h an Axioplan
luo escence mic oscope equipped wi h an Axiocam came a (ZEISS, Jena,
Ge many) and e alua ed wi h he so wa e AxioVision 3.1.
Cell lines and cul u e condi ions
Leukaemia HL-60 cells we e ob ained om he Ge man Collec ion o Biological
Ma e ial (DSMZ), B aunschweig (Ge many), melanoma 518A2 cells om he
Depa men o Oncology and Hema ology o he Ma in Lu he Uni e si y, Halle-
Wi enbe g (Ge many), KB-V1/Vbl ce ix and MCF-7/Topo b eas ca cinoma
cells om he Ins i u e o Pha macy o he Uni e si y Regensbu g (Ge many), and
HT-29 colon ca cinoma cells as well as human o eskin ib oblas s (HF) om he
Uni e si y Hospi al E langen (Ge many). The HL-60 and HT-29 cells we e g own
in RPMI-1640 medium supplemen ed wi h 10% e al cal se um (FCS), 100 IU
mL-1 penicillin G, 100 μg mL-1 s ep omycin sul a e, 0.25 μg mL-1 ampho e icin B
and 250 μg mL-1 gen amycine (all GIBCO). The 518A2, he HF and he KB-
V1/Vbl cells we e cul u ed in Dulbecco’s Modi ied Eagle Medium (DMEM,
GIBCO), con aining 10% FCS, 100 IU mL-1 penicillin G, 100 µg mL-1
s ep omycin sul a e, 0,25 µg mL-1 ampho e icin B and 250 µg mL-1 gen amycine.
The MCF-7/Topo cells we e g own in Eagle’s Minimal Essen ial Medium wi h
Ea le´s sal s (MEM; SIGMA) supplemen ed wi h 2.2 g L-1 NaHCO3, 110 mg L-1
sodium py u a e and 5% FCS. The po o oo kidney cells P K2 we e ob ained om
he Ame ican Type Cul u e Collec ion (ATCC) and cul i a ed in MEM (GIBCO)
supplemen ed wi h 10% FCS and non-essen ial amino acids (GIBCO). They we e
main ained in a mois u e-sa u a ed a mosphe e (5% CO2) a 37°C in 50-mL
cul u e lasks (NUNC, Ge many), and se ially passaged a e ipsinisa ion.
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Fluo escence spec oscopy
A JASCO FP-6500 luo escence spec opho ome e was used o luo escence
measu emen s o compounds 1–3 dissol ed in phospha e bu e ed saline (5 µM).
The exci a ion wa eleng h was 490 nm, emission wa eleng hs anged om 500 o
650 nm.
Cellula d ug up ake
The cellula up ake o he es compounds was asce ained using a TECAN CM
In ini e F200 luo escence pla e eade (Exc.: 485/25 nm, Em.: 590/20 nm).
Typically, 500 µL cells o HL-60 leukaemia (5  105 mL-1), HT-29 colon
ca cinoma, 518A2 melanoma, KB-V1/Vbl ce ix ca cinoma and MCF-7/Topo
b eas ca cinoma (each 5  104 mL-1) we e incuba ed in 24-well pla es wi h he
compounds 1, 2 o 3 (5 µM) o 3 h. The medium was emo ed by cen i uga ion
o 5 min a 150 g. The esidual luo escence in ensi y was measu ed in he
supe na an and he co esponding concen a ion o es compounds aken up by
he cells was calcula ed by compa ison wi h iden ically ea ed e e ences [9].
S a is ical analysis o measu emen esul s
The esul s a e exp essed as means ± s anda d de ia ion (SD). The S uden -
Newman-Keuls es was used o de e mine s a is ical signi icance wi h P alue <
0.05 conside ed signi ican (*).
Cellula localisa ion
The dis ibu ion o he es compounds in P K2 cells was isualised ia
luo escence mic oscopy. Typically, he cells we e g own in DMEM (750 µL) in
4-well pla es (Nunc) on glass co e slips and incuba ed wi h he es compounds 1,
2 o 3 (10 µM) o pe iods anging om 2 h o 16 h. The medium was emo ed,
5
he cells we e washed wice wi h phospha e bu e ed saline (PBS) and moun ed
i al in PBS. The Hoechs dye 33342 was op ionally added (5 µg mL-1) o s ain
he nuclei and he cells we e imaged using a ZEISS Axioplan luo escence
mic oscope [9, 10].
Resul s
Fluo escence spec oscopy
Fluo escence spec oscopy has been equen ly employed o s udy he in e ac ion
o doxo ubicin (1), e.g., wi h DNA in he cou se o in e cala ion [11, 12] o wi h
lipid memb anes [13]. We now measu ed he luo escence spec a o he es
compounds 1–3 in phospha e bu e ed saline (PBS; 5 µM) a pH 7.4. Since he
luo opho e o 1 has a known abso p ion maximum be ween 480 nm and 500 nm
[14] we exci ed he es compounds a 490 nm and obse ed h ee cong uen
luo escence spec a (Fig. 2). They a e each cha ac e ised by an o e lay o h ee
bands esul ing in an en elope cu e wi h a maximum a ound 550 nm and a
shoulde a ca. 580 nm. Howe e , he in ensi ies o he spec a o he hyd azone
conjuga es 2 and 3 we e only abou hal o ha o 1 i sel . This is quali a i ely in
keeping wi h a epo by Chou pa e al. on he changes o he in insic
luo escence o 1 upon addi ion o sodium olea e [15]. The au ho s explained his
pH- and a io-dependen e ec by lipophilic in e ac ions o he a oma ic
luo opho e o 1 wi h he long alkenyl chain o oleic acid. A simila and ob iously
mo e in ense lipophilic in e ac ion could esul om a back olding o he
co alen ly a ached hep adecanoyl chain o 2 o o he e he ed men hyl esidue o
3, espec i ely, on op o he an h aquinone luo opho e.
((Figu e 2 he e))
Cellula d ug up ake
6
Nex , we asce ained he up ake o 1 and i s de i a i es 2 and 3 by i e human
cance cell lines o en i ies ha a e ypically ea ed wi h 1, namely leukaemia,
melanoma as well as b eas , ce ix and colon ca cinomas. The cells we e
incuba ed wi h 5 µM o 1–3 o 3 h and he esidual luo escence in ensi y in he
supe na an ollowing incuba ion was measu ed a e a cen i uga ion s ep. The
pe cen al cellula d ug up ake ela i e o e e ence solu ions was calcula ed and
plo ed as shown in Figu e 3 [9]. All es compounds accumula ed s ongly in he
518A2 and he HL-60 cells which co esponds well wi h hei p e iously epo ed
high cy o oxici y agains hese cell lines [7]. In con as , hei accumula ion in he
emaining h ee mul i-d ug esis an cance cell lines was compa a i ely low,
which ac ma ches hei low cy o oxici y in hese cells. Howe e , a y acid
conjuga e 2 is an excep ion since i was selec i ely and signi ican ly (> 20%)
accumula ed in mul i-d ug esis an MCF-7/Topo b eas ca cinoma cells.
((Figu e 3 he e))
Cellula localisa ion
Finally, o analyse he cellula localisa ion o 1 and i s de i a i es 2 and 3, non-
malignan P K2 po o oo kidney cells we e incuba ed wi h 10 µM concen a ions o
hem o up o 16 h, hen moun ed i al in PBS and imaged using luo escence
mic oscopy. The esul s o hese au o luo escence analyses a e shown in Figu es 4
and 5.
((Figu e 4 he e))
While a e 2 h o incuba ion 1 was localised only in P K2 nuclei (Fig. 4 / A, B),
upon p olonged exposu e i o i s luo escen me aboli es we e accumula ed in
esicles which we e ini ially neighbou ing he nuclei (Fig. 4 / C, D) bu la e we e
dis ibu ed h oughou he cells (Fig. 4 / E, F). This is in line wi h he p e iously
7
epo ed up ake and subcellula dis ibu ion o 1 in o he cells, e.g., a emb yo
ib oblas s and human myeloid leukaemia cells [9, 10]. Fo he men hyl-
e mina ed hyd azone 3 we ound a subcellula localisa ion simila o ha o 1,
albei p oceeding mo e slowly. The accumula ion in he nuclei equi ed wice as
long (4 h) o comple ion as in he case o he pa en d ug. Howe e , again some
di e ences eme ged o conjuga e 2. He e, no d ug was ound in P K2 nuclei a
any ime bu in esicles h oughou he cell ye a e incuba ion pe iods as sho as
2 hou s (Fig. 5 / A, B). This was con i med by co-localisa ion expe imen s wi h
he nuclei-s aining Hoechs dye 33342. Upon p olonged incuba ion 2, o i s
luo escen me aboli es, pene a ed in o he cellula memb ane gi ing ise o
in ensi e luo escence he e (Fig. 5 / C, D). We assume ha he long linea a y
acyl esidue ac s as a lipophilic ancho hus immobilising he conjuga e o a
me aboli e o i a he memb ane. This would no be possible o 1 o 2 which lack
such a s aigh lipophilic e he .
((Figu e 5 he e))
Discussion
The cell memb ane o esis an cance cells is ich in lipids wi h sa u a ed
hyd ophobic acyl chains, neu al and sphingomyelin lipids, which can a ac i ely
in e ac wi h doxo ubicin [16]. Ve y ecen ly, Labhase wa e al [17] epo ed ha
doxo ubicin pene a es in o he highly o ganised lipid monolaye s o esis an
MCF-7 b eas cance cell memb anes and is e ained he e. This s ong lipid-d ug
in e ac ion is also belie ed o educe he abili y o he d ug o di use ac oss he
cell memb ane and in o he cy osol, and o enhance he likelihood o nea -by
ABC-e lux anspo e s expelling he d ug [18]. The a y acyl conjuga e 2 can
in e wine e en mo e e ec i ely han 1 wi h he memb ane lipids o esis an
8