1
J03141
JPP 2003, 55: 000±000
ß2003 The Au ho s
Recei ed May 21, 2003
Accep ed Augus 19, 2003
DOI 10.1211/0022357022359
ISSN 0022-3573
Medical Mycology Uni , Na ional
Ins i u e o Cellula
Bio echnology, Depa men o
Biology, Na ional Uni e si y o
I eland, Maynoo h, Co. Kilda e,
I eland
Joseph O'Kee e, Sean Doyle,
Ke in Ka anagh
Co espondence: K. Ka anagh,
Medical Mycology Uni , NICB,
Depa men o Biology, NUI
Maynoo h, Co. Kilda e, I eland.
E-mail: [email p o ec ed]
Funding: This wo k was
suppo ed by unding om he
Highe Educa ion Au ho i y
unde PRTLI 3. J. O'Kee e is he
ecipien o an En e p ise I eland
Pos -G adua e S uden
Main enance G an .
Exposu e o he yeas Candida albicans
o he an i-neoplas ic agen ad iamycin inc eases
he ole ance o ampho e icin B
Joseph O'Kee e, Sean Doyle and Ke in Ka anagh
Abs ac
Cance pa ien s expe ience a high incidence o ungal in ec ions due o hei immuno-supp essed con-
di ion. This wo k has in es iga ed he in e ac ion o an an i-neoplas ic agen , ad iamycin (doxo ubicin),
wi h he yeas Candida albicans and examined whe he his d ugal e ed he suscep ibili y o he yeas o
ampho e icin BÐan an i- ungal agen used o he ea men o sys emic ungal in ec ions in cance
pa ien s. Exposu e o ad iamycin o 24h inc eased he g ow h o C. albicans and inc eased he ole ance
o ampho e icin B by a small, bu s a is ically signi ican , ex en . G ow h in ad iamycin-supplemen ed
medium supp essed he espi a ion a e o C. albicans, which esul ed in a dec ease in he e gos e ol
con en o he ungal cell memb ane. The ole ance o ampho e icin B was los a e exposu e o
ad iamycin o 48h, which coincided wi h a es o a ion in he espi a ion a e and he e gos e ol con en
o he ungal cell memb ane. This wo k demons a ed ha sho - e m exposu e (24 h) o ad iamycin
inc eased he ole ance o C. albicans o ampho e icin B, which may be media ed by a dec ease in he
e gos e ol con en as a esul o an ad iamycin-induced dis up ion o oxida i e phospho yla ion.
In oduc ion
Cance is a debili a ing, mul i ac o ial disease ha a acks indisc imina ely ac oss all
demog aphic sec o s o socie y. The e a e se e al amilies o an i±neoplas ic agen s used
o ea cance and hese a e sub-di ided on he basis o hei mode o ac ion. One class o
an i-neoplas ic agen , he an h acyclins, a e non±co alen DNA binding agen s and one o
hese, ad iamycin, was isola ed in 1969 om S ep omyces peuce ius (P a & Ruddon
1979). Ad iamycin (also known as doxo ubicin) is used in he ea men o Hodgkin's
disease and Non±Hodgkin's lymphoma and in cance s o he b eas , o a ies and lymph
sys em (Lowen hal & Ea on 1996). Ad iamycin is a opoisome ase II inhibi o (Cummings
e al 1991) and i s s uc u e allows i o in e cala e be ween base pai s o DNA causing
subsequen empla e diso de s, which lead o s and b eakage. Ad iamycin also a ec s
mi ochond ial unc ion by selec i ely binding o ca diolipin in he mi ochond ial
memb ane and dis up ing he ac ion o complex I (Pa ke e al 2001).
Cance pa ien s a e a an inc eased isk o a ange o oppo unis ic ungal in ec ions
by i ue o hei debili a ed s a e and he immuno-supp essi e na u e o an i-neoplas ic
egimes (Sa al 1991; De Pauw 1997). The yeas Candida albicans is an impo an oppo -
unis ic ungal pa hogen, a ec ing indi iduals whose immune sys ems become immuno-
comp omised due o diseases such as leukaemia o an i-neoplas ic he apy (Schule &
Haag 1997). The incidence o in ec ions caused by C. albicans has inc eased in ecen
yea s among immuno-comp omised pa ien s due, in pa , o ine icien o incomple e
he apies and by ad ances in medicine allowing he su i al o immuno-comp omised
pa ien s o p olonged pe iods. C. albicans accoun s o be ween 52 and 63% o all
nosocomial ungal in ec ions, and yeas o he genus Candida a e he ou h mos
common o ganisms isola ed om blood (P alle e al 1998; Ve duyn Lunel e al 1999)
T ea men o ungal in ec ions is gene ally achie ed by using azole o polyene an i-
ungal agen s such as ampho e icin B (Schule & Haag 1997; P en ice e al 1999;
La e die e e al 2000). Ampho e icin B is employed o ea ad anced sys emic ungal
in ec ions, bu i s e icacy can be low (Sa al 1991; Walsh e al 1999). In addi ion,
ampho e icin B can ha e se e e side e ec s, which may limi
i s use in ce ain pa ien g oups. Howe e , ampho e icin B
s ill emains an impo an an i- ungal agen o he ea -
men o ecalci an sys emic ungal in ec ions (Ha sel &
Bola d 1996). The an i- ungal ac i i y o ampho e icin B
lies in i s abili y o bind o e gos e ol in he ungal cell
memb ane and c ea e po es h ough which cy oplasmic
con en s leak and p o ons en e , leading o cy oplasmic
acidi ica ion and subsequen cell dea h (Abu Salah 1996).
The aim o his wo k was o de e mine whe he expo-
su e o C. albicans o he an i-neoplas ic agen ad iamycin
al e ed he suscep ibili y o he yeas o ampho e icin B.
We pos ula ed ha in ce ain ins ances an i-neoplas ic
he apy may inad e en ly al e he ole ance o C. albi-
cans o selec ed an i- ungal d ugs employed o a es he
de elopmen o in ec ions in cance pa ien s.
Ma e ials and Me hods
G ow h condi ions
C. albicans ATCC 10231 was o iginally ob ained om he
Ame ican Type Cul u e Collec ion (Manasas, VA) and
was g own o he s a iona y phase (app oxima ely
1.5 10
8
cells mL
1
)a 30
C o e nigh in yeas ex ac -
pep one-D-glucose b o h (YEPD; 1% (w/ ) yeas ex ac
(Sigma Ald ich Chemical Co., Do se , UK), 2% (w/ )
bac e iological pep one (Sigma Ald ich) and 2% (w/ )
glucose (Sigma Ald ich)) in an o bi al incuba o a 200
e min
1
. Whe e app op ia e, media we e solidi ied by
he addi ion o 2% (w/ ) aga (Oxoid). Yeas cul u es
we e main ained on YEPD aga a 4 C and sub-cul u ed
e e y ou o six weeks.
An i ungal and an i±neoplas ic agen s
Ad iamycin was pu chased as Doxo ubicin (Ebewe
A zneimi el Gmbh., Un e ach, Aus ia). Ampho e icin B
(Sigma Ald ich) was dissol ed in dime hyl sul oxide
(DMSO, Sigma Ald ich) and dilu ed o wo king concen a-
ions in s e ile phospha e-bu e ed saline (PBS, pH 7.2)
(Li e Technologies, Paisley, UK) be o e use. The maximum
DMSO concen a ion employed wi h cells was 2% ( / ).
Ampho e icin B suscep ibili y es ing
Yeas cul u es we e g own o he s a iona y phase in an i-
bio ic medium 3 (AB 3, Oxoid) supplemen ed wi h 2% (w/ )
glucose o e nigh a 30 Cand200 e min
1
. Cul u es we e
ha es ed by cen i uga ion (2220 g o 5 min in a Beckmann
GS-6 cen i uge) and dilu ed o 1 10
6
cells mL
1
.Cells
(110
5
in 100 L) we e added o each well o a 96-well
pla e (Sa sd ed , Wex o d, I eland) con aining ampho e icin
B (Sigma Ald ich) dissol ed in an ibio ic medium 3 (AB3) in
se ial dilu ions om 2.5 o 0.0048 gmL
1
. The pla es we e
incuba ed a 37 C o 24 h and he op ical densi y was ead
a 450 nm using a MRX spec ome e (Dynax Technology).
The MIC90 was de e mined o be he lowes concen a ion
o ampho e icin B equi ed o educe g ow h by 90%, ela-
i e o he con ol (Mo an e al 1997).
E ec o p e-g ow h in ad iamycin on he g ow h o
C. albicans in ampho e icin B AB3 medium (supplemen-
ed wi h 2% (w/ ) glucose and ad iamycin (20 gmL
1
))
was inocula ed wi h C. albicans a a densi y o 5 10
5
mL
1
and g own a 37 C o 24 h. Cells we e ha es ed by
cen i uga ion and esuspended in AB3 con aining
ampho e icin B (0.625 gmL
1
) a a densi y o 5 10
5
cells mL
1
. A d ug ee con ol consis ed o cells ha
had no been exposed o ad iamycin. Cell densi y was
de e mined a e g ow h o 24 h using a PAMAS SVSS±
C Pa icle Coun e (Ru esheim, Ge many).
Oxygen consump ion
Cells we e g own in YEPD supplemen ed wi h ad iamycin
(20 gmL
1
)a 30
C o 24 o 48 h, ha es ed by cen i-
uga ion and esuspended a a densi y o 1 10
8
mL
1
in
PBS (pH 7.2). A Cla k Type oxygen elec ode (Rank
B o he s L d, Camb idge, UK) was employed o de e -
mine he espi a ion a e o cells and he a e o oxygen
consump ion was exp essed as he numbe o moles o
oxygen consumed pe minu e pe 1 10
8
cells.
S e ol ex ac ion and gas ch oma og aphy analysis
S e ol ex ac ion was pe o med as desc ibed by
A hing on-Skaggs e al (1999). Equi alen we weigh
samples (2.28 g) o s a iona y phase cells we e esuspended
in 2 mL dena u ing solu ion (20% w/ po assium hyd o-
xide (Sigma Ald ich), 60% / e hanol) and placed in a
90 C shaking wa e ba h (190 e min
1
) o 1.5 h.
Hep ane (600 L) was added o each o he samples.
Samples we e le in he da k un il a dis inc hep ane
laye o med. S e ols we e iden i ied using a Va ian
Se ies II Gas Ch oma og aph (Va ian L d, Vic o ia,
Aus alia). An e gos e ol s anda d cu e was cons uc ed
o e he ange 100±0.25 gmL
1
and he lowes le el o
de ec ion o e gos e ol was 0.1 gmL
1
.
S a is ical analysis
Using ei he he K uskal±Wallis es o Mann-Whi ney U
es (Sigma S a S a is ical Analysis Package Ve sion 1.00;
SPSS Inc., Chicago, IL). P alues less han 0.05 deno ed
signi icance (Wa dlaw 2000). All expe imen s we e pe -
o med on h ee independen occasions and he means
we e exp essed s.e.
Resul s
E ec o ad iamycin exposu e on C. albicans
g ow h and suscep ibili y o ampho e icin B
Ad iamycin did no display a signi ican ungis a ic o un-
gicidal e ec on C. albicans o e he concen a ion ange
0.1±40 gmL
1
(da a no p esen ed). Expe imen s we e pe -
o med o es ablish he esponse o C. albicans o ampho e -
2Joseph O'Kee e e al
icin B, and o ampho e icin B when co-cul u ed in he p e-
sence o ad iamycin (2.0 gmL
1
). Exposu e o C. albicans
inAB3supplemen edwi had iamycin oampho e icinB
yielded an ampho e icin B MIC90 o 0.13 gmL
1
com-
pa ed wi h he con ol which ga e an ampho e icin B
MIC90 alue o 0.07 gmL
1
(Figu e 1). These da a we e
signi ican a P0.05 and indica ed ha 24-h exposu e o C.
albicans o ad iamycin al e ed he suscep ibili y o he yeas
o ampho e icin B. No al e a ion in ole ance o he azoles,
ke oconazole and miconazole, o he polyene, nys a in, was
obse ed ollowing p e-g ow h o C. albicans in ad iamycin
(da a no p esen ed).
Expe imen s we e pe o med o de e mine whe he a
co ela ion exis ed be ween he enhanced ole ance o C.
albicans o ampho e icin B and cell g ow h. The cell den-
si y a ained in he cul u e ha had been p e-g own in
ad iamycin was 1.55 10
7
mL
1
whe eas he cell numbe
a ained in he con ol was 3.57 10
6
mL
1
(Figu e 2).
Cul u es ha had been exposed o ad iamycin o 48 h
showed a lowe inal cell densi y (1.28 10
6
mL
1
) han
he con ol.
In es iga ion o he e ec o ad iamycin on
espi a ion o C. albicans
The oxygen consump ion o C. albicans p e-g own in ad ia-
mycin (20 gmL
1
) o 24 o 48h was de e mined. Oxygen
consump ion o con ol cells was 9.6 mol oxygen/10
8
cells
min
1
while cells p e-g own in ad iamycin o 24 h dis-
played a espi a ion a e o 5.4 mol oxygen/10
8
cells
min
1
, which was signi ican ly less (56%) han he con ol
a P0.05 (Figu e 3). Cells p e-g own in ad iamycin o
48 h demons a ed a 23% educ ion in oxygen consump ion
(7.4 mol oxygen/10
8
cells min
1
) ela i e o he con ol.
E ec o ad iamycin on he e gos e ol con en o
C. albicans
E gos e ol is a majo componen o he ungal cell mem-
b ane which helps main ain s uc u e and egula es he
in lux and e lux o ex a/in acellula componen s.
Ampho e icin B unc ions by binding o e gos e ol leading
o he c ea ion o po es h ough which in acellula con-
s i uen s leak and p o ons en e he cell (Abu Salah 1996).
The combined e ec o hese wo p ocesses is he acidi i-
ca ion o he cy oplasm and he dea h o he cell.
Resis ance o ungal cells o ampho e icin B is a e bu ,
whe e encoun e ed, is cha ac e ized by a educ ion in he
amoun o e gos e ol in he cell memb ane (Kelly e al
1997; Whi e e al 1998).
E gos e ol quan i ica ion by GC analysis demons a ed
(Figu e 4) ha ollowing g ow h o 24 h in ad iamycin-
supplemen ed medium he e gos e ol con en o C. albicans
was educed om 58.5 o 29.6 gmL
1
(g cells)
1
(we w ),
a dec ease o 49% (signi ican a P0.05). G ow h o
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Con ol Ad iamycin (2.0 µ g mL–1)
MIC90 ampho e icin B (µ g mL–1)
∗
T ea men
Figu e 1 Ampho e icin B MIC90 o C. albicans g own in AB3
supplemen ed wi h ad iamycin B (2.0 gmL
1
). *P0.05 compa ed
wi h con ol.
0.00E + 00
2.00E + 06
4.00E + 06
6.00E + 06
8.00E + 06
1.00E + 07
1.20E + 07
1.40E + 07
1.60E + 07
1.80E + 07
2.00E + 07
Con ol
T ea men
P e-g ow h in
ad iamycin
20 µ g mL–1, 24 h
P e-g ow h in
ad iamycin
20 µ g mL–1, 48 h
∗
Cell densi y (mL–1)
Figu e 2 G ow h o C. albicans in AB3 supplemen ed wi h ampho-
e icin B (0.625 gmL
1
) ollowing p e-g ow h in ad iamycin
(20 gmL
1
) o 24 o 48 h. *P0.05 compa ed wi h con ol.
0
∗
1
2
3
4
5
6
7
8
9
10
Con ol
µmole oxygen consumed / 108 cells min–1
Ad iamycin
20 µ g mL–1, 24 h
Ad iamycin
20 µ g mL–1, 48 h
Figu e 3 Oxygen consump ion o C. albicans ollowing g ow h in
ad iamycin (20 gmL
1
) o 24 o 48 h. *P0.05.
Ad iamycin inc eases he ole ance o
C. albicans
o ampho e icin B 3
C. albicans o 48 h in ad iamycin esul ed in a cellula
e gos e ol con en simila o he con ol.
Discussion
The an h acycline an ibio ics a e one o he mos widely
used g oups o an i-neoplas ic agen s o he ea men o
solid and non-solid malignancies (P a & Ruddon 1979;
Lowen hal & Ea on 1996). Ad iamycin is an an h acycline
which unc ions by inhibi ing he ac ion o opoisome ase
II (Cummings e al 1991), inducing DNA s and b eakage
and kills mammalian cells by inducing apop osis (p o-
g ammed cell dea h) (Skladanowski & Konopa 1993;
Husch scha e al 1995). In addi ion, i in e ac s wi h a
phospholipid (ca diolipin) o he mi ochond ial mem-
b ane leading o an al e a ion in he pe meabili y o he
mi ochond ion wi h a concomi an ad e se e ec on oxi-
da i e phospho yla ion (Pa ke e al 2001).
Fungal in ec ions in cance pa ien s a e a se ious, and
in some cases li e- h ea ening, p oblem which a ise due o
he debili a ed s a e o he pa ien o as a consequence o
immuno-supp ession induced by an i-neoplas ic he apy
(De Pauw 1997). Ampho e icn B is one o he an i- ungal
agen s used o he ea men o sys emic ungal in ec ions
equen ly seen in leukaemic and solid umou pa ien s
(Ha sel & Bola d 1996) and ac s by binding o e gos e ol
in he ungal cell memb ane and c ea ing po es h ough
which in acellula cons i uen s escape (Abu Salah 1996).
This s udy demons a ed ha exposu e o C. albicans
o he an i-neoplas ic agen ad iamycin, led o an inc ease
in ole ance o ampho e icin B (Figu e 1). P e-exposu e o
ad iamycin esul ed in highe cell g ow h in he p esence
o ampho e icin B (Figu e 2). While ad iamycin did no
a ec he iabili y o C. albicans o a signi ican ex en i
did educe oxygen consump ion (Figu e 3). Cells exposed
o ad iamycin o 24 h showed a 56% educ ion in espi a-
ion and demons a ed a educed amoun o e gos e ol
(Figu e 4). E gos e ol biosyn hesis equi es a unc ional
mi ochond ion o he p o ision o NADPH which is
necessa y o one o he in e media e s ages in i s bio-
syn hesis (Daum e al 1998). The deple ed e gos e ol con-
en o ad iamycin- ea ed cells migh ha e been a
consequence o hei dep essed espi a ion a e. In con-
as , he e gos e ol con en o cells g own in he p esence
o ad iamycin o 48 h was simila o he con ol, which
e lec ed he highe espi a ion a e in hese cells ela i e
o ha in cells exposed o ad iamycin o 24 h.
Exposu e o C. albicans o ad iamycin o 24 h caused a
small, bu s a is ically signi ican , inc ease in ole ance o
ampho e icin B, which migh ha e been due o he
deple ed e gos e ol le els as a esul o he educed
espi a ion a e. Ad iamycin a ec s espi a ion by binding
o he phospholipid ca diolipin (diphospha idylgly-
ce ol)Ða componen o he mi ochond ial memb ane
(Cummings e al 1991; Pa ke e al 2001). This has he
e ec o dis up ing memb ane pe meabili y and inhibi ing
he ac ion o complex I (P ebble 1981; Das & Mazumda
2000), which is a c i ical componen o he elec on ans-
po chain o oxida i e phospho yla ion in C. albicans
(Helme ho s e al 2002). I is pos ula ed ha ad iamycin
binds o ca diolipin which dis up s he ac ion o complex I
leading o a dep ession in he espi a ion a e. This leads
o deple ed le els o NADPH (which is equi ed o e gos-
e ol biosyn hesis), educed le els o e gos e ol and an
inc ease in ole ance o ampho e icin B. While ole ance
o ampho e icin B is clinically a e (Van den Bossche e al
1998) i has been obse ed in cases whe e e gos e ol le els
a e deple ed lea ing ewe binding si es o ampho e icin
B (Kelly e al 1997; Whi e e al 1998; Kon oyiannis &
Lewis 2002). Reduced le els o e gos e ol ha e been impli-
ca ed in luconazole ole ance in C. albicans (Lo le e al
2002). In his case i was pos ula ed ha e gos e ol de i-
cien mu an s had an al e ed memb ane pe meabili y ha
e a ded he en y o luconazole.
The eappea ance o suscep ibili y o ampho e icin B
a e g ow h o C. albicans in ad iamycin o 48 h was
accompanied by a simul aneous es o a ion in he espi a-
ion a e and e gos e ol con en o he cell. The esump-
ion o espi a ion migh ha e been due o he cells
eplacing ca diolipin damaged by ad iamycin in he ini ial
24 h. As a consequence, espi a ion esumed a nea no -
mal le els wi h a concomi an inc ease in he e gos e ol
le el leading o a es o a ion in he suscep ibili y o
ampho e icin B.
This wo k demons a ed ha ad iamycin an i-neoplas ic
he apy had he po en ial o inc ease he ole ance o
C. albicans o ampho e icin B by dis up ing espi a ion,
which had an ad e se e ec on he e gos e ol con en o he
ungal cell memb ane. This may ha e dele e ious con-
sequences o cance pa ien s and could lead o he appea -
ance o ampho e icin B- ole an C. albicans in ec ions in
pa ien s ecei ing ad iamycin an i-neoplas ic he apy.
Conclusion
Sho - e m exposu e (24 h) o ad iamycin inc eased he
ole ance o C. albicans o ampho e icin B, which may
be media ed by a dec ease in he e gos e ol con en as a
esul o an ad iamycin-induced dis up ion o oxida i e
phospho yla ion.
0.00E + 00
1.00E – 02
2.00E – 02
3.00E – 02
4.00E – 02
5.00E – 02
6.00E – 02
7.00E – 02
8.00E – 02
Con ol
E gos e ol (µg mL–1 (g cells)–1 (we w ))
∗
Ad iamycin
20 µ g mL–1, 24 h
Ad iamycin
20 µ g mL–1, 48 h
Figu e 4 E gos e ol con en o C. albicans ollowing g ow h in
ad iamycin (20 gmL
1
) o 24 o 48 h. *P0.05.
4Joseph O'Kee e e al
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o ampho e icin B 5