Exposure of the yeast Candida albicans to the anti-neoplastic agent adriamycin increases the tolerance to amphotericin B

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 Re e ences Abu-Salah, K. M. (1996) Ampho e icin B: an upda e. B . J. Biomed. Sci.53: 122±133 A hing on-Skaggs, B. A., Wa nock, D. W., Mo ison, C. J. 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