the hydroxypyridinone iron chelator cp94 can enhance ppix-induced pdt of cultured human glioma cells

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-inducedPDT of Cultured Human Glioma Cells

Emma Blake* and Alison Curnow

Clinical Photobiology, Peninsula Medical School, University of Exeter, Knowledge Spa, Royal CornwallHospital, Truro, Cornwall, UK

Received 30 March 2010, accepted 17 May 2010, DOI: 10.1111 ⁄ j.1751-1097.2010.00770.x

ABSTRACT

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevu-

linic acid (ALA) or methyl aminolevulinate (MAL) utilizes the

combined interaction of a photosensitizer, light and molecular

oxygen to ablate tumor tissue. To potentially increase accumu-

lation of the photosensitizer, protoporphyrin IX (PpIX), within

tumor cells an iron chelator can be employed. This study

analyzed the effects of ALA/MAL-induced PDT combined with

the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochlo-

ride (CP94) on the accumulation of PpIX in human glioma cells

in vitro. Cells were incubated for 0, 3 and 6 h with various

concentrations of ALA/MAL with or without CP94 and the

resulting accumulations of PpIX, which naturally fluoresces,

were quantified prior to and following light irradiation. In

addition, counts of viable cells were recorded. The use of CP94 in

combination with ALA/MAL produced significant enhancements

of PpIX fluorescence in human glioma cells. At the highest

concentrations of each prodrug, CP94 enhanced PpIX fluores-

cence significantly at 3 h for ALA and by more than 50% at 6 h

for MAL. Cells subsequently treated with ALA/MAL-induced

PDT in combination with CP94 produced the greatest cytotox-

icity. It is therefore concluded that with further study CP94 may

be a useful adjuvant to photodiagnosis and/or PpIX-induced

PDT treatment of glioma.

INTRODUCTION

Malignant glial tumors remain the most common (1) and

problematic primary brain tumors to treat given their mobileand invasive characteristics. Conventional treatments for braintumors include surgery, radiotherapy and chemotherapy.

However, recurrence rates are high with almost 80% of allcases recurring within 2 cm of the resected margin (2). In thelast 15–20 years photodynamic therapy (PDT) has beenincluded in the surgical resection of primary brain tumors (3).

PDT is the interaction of a photosensitizer, molecularoxygen and light of a specific wavelength which whencombined results in the production of cytotoxic species causing

diseased cells to undergo cell death (4). An additional featureof the activated photosensitizer is its ability to fluoresce and asa result photodynamic diagnosis (PDD) has been utilized by

surgeons enabling greater precision in the removal of brain

tumors (5,6). As an adjuvant therapy, PDT can be used todestroy tumor cells unreachable by surgical resection. Improv-ing brain tumor removal is vitally important because

although surgical resection of a primary tumor is potentiallycurative, the 5 year survival rates remain dismal at less than5% (2).

5-Aminolevulinic acid (ALA) and methyl aminolevulinate(MAL) are porphyrin-inducing precursors to the endogenousphotosensitizer protoporphyrin IX (PpIX) produced via theheme biosynthesis pathway in humans. Following PpIX

production the next stage in the pathway is the insertion offerrous iron (Fe2+) under the action of the ferrochelataseenzyme (7), to convert PpIX into heme. Usually, the presence

of free heme acts as a negative feedback mechanism inhibitingALA synthesis (8). Thus, the exogenous administration oflarge amounts of ALA ⁄MAL bypasses this negative feedback

signal and there is a resultant accumulation of PpIX within thecells as the conversion of PpIX to heme by ferrochelatase isrelatively slow making this the secondary rate-limiting step ofthe pathway. In diseased cells some enzymes within the heme

biosynthesis pathway are over- or under-expressed favoringthe continual conversion of ALA into PpIX.

An excessive amount of the PpIX photosensitizer is

clinically useful for PDT and so research is taking place totry to optimize the current PDT treatment regimes bymaximizing the accumulation of PpIX within different cell

types. One such modification, under current investigation, isthe chelation of iron. The theory is that by combiningadministration of a photosensitizer with an iron chelator the

latter can temporarily bind free intracellular ferrous iron thuspreventing its insertion into PpIX, preventing the formation ofheme and therefore further increasing the accumulation ofPpIX within diseased cells (9). Iron chelating agents which

have been investigated in the past include ethylenediaminetet-raacetic acid (EDTA) (10) and desferrioxamine (DFO) (11).DFO has greater specificity for iron than EDTA and research

by our group has shown 1,2-diethyl-3-hydroxypyridin-4-onehydrochloride (CP94) to be significantly superior to DFO inthe enhancement of PpIX accumulation using ALA and MAL

in fetal lung fibroblasts and squamous carcinoma cells (9).This study investigates the effect of the iron chelator CP94

on the PpIX fluorescence produced within human glioma cellsin vitro for the first time using both ALA and MAL as the

porphyrin precursors. Additionally, the effect this noveltreatment has on cytotoxicity following irradiation is alsoinvestigated.

*Corresponding author email: [email protected] (Emma Blake)� 2010TheAuthors. JournalCompilation.TheAmericanSociety of Photobiology 0031-8655/10

Photochemistry and Photobiology, 2010, 86: 1154–1160

1154

MATERIALS AND METHODS

Chemicals and cells. All reagents and chemicals were supplied bySigma-Aldrich Chemical Company (Poole, UK) unless otherwisestated. The U-87 MG (human glioblastoma-astrocytoma, epithelial-like) cell line was purchased from the European Collection of CellCultures (ECACC, Wiltshire, UK). Under aseptic conditions in a classII laminar flow cabinet, cells were cultured in Eagle’s minimumessential medium (EMEM) with 10% fetal calf serum (standardizedto give an iron concentration between 450 and 600 lg ⁄ 100 g), 2%(200 mMM) LL-glutamine and 2% (200 U mL)1) penicillin and(200 lg mL)1) streptomycin solution. Stock solutions of ALA ⁄MALwere prepared in PBS, adjusted to physiological pH (pH 7.4) usingNaOH (0.5 mMM), filter sterilized (0.22 lm Millipore) and stored at)20�C for up to 1 month. Cells were grown in 5% CO2 at 37�C and leftto grow until 70% confluent at which time cells were passaged (every3–5 days). U-87 MG cells were then seeded into 6 · 24-well plates at adensity of 2 · 105 cells per mL (105 cells per well) and left to adhere in5% CO2 at 37�C to reach 70% confluence. After a period of 24 h topermit adherence, diluted solutions of the final concentrations ofALA ⁄MAL prodrugs were prepared with modified EMEM (minusphenol red): ALA: 0–250 lm ⁄MAL: 0–1000 lm. All medium wasaspirated from the wells of the plates and 1 mL of appropriate testsolution (ALA ⁄MAL ± CP94) added to each well, in triplicate, indark room conditions. Concentrations of ALA ⁄MAL alone wereadded to 3 · 24-well plates and concentrations of ALA ⁄MAL incombination with 150 lm CP94 (kindly supplied by Professor Hider,King’s College London, UK) were added to another 3 · 24-well plates.

PpIX fluorescence in U-87 MG cells. After 0, 3 and 6 h incubationwith ALA ⁄MAL ± CP94 all six plates were read for ‘‘pre’’ PpIXfluorescence levels (prior to light irradiation) using a fluorescence platereader (Synergy HT; BIO-TEK, Germany). During the time interval the3 and 6 h plates were kept in the dark in 5%CO2 at 37�C.Measurementswere taken from the bottom of the plate wells with a 400 ± 30 nmexcitation filter and a 645 ± 40 nm emission filter. After each incuba-tion time, following the ‘‘pre’’ fluorescence measurements of the twoplates, one plate was then subjected to red light irradiation (Aktilite,Galderma, UK, 15 J cm)2, 635 ± 2 nm) whilst the other remained inthe dark as a dark control. The most efficacious light dose and fluencerate to be utilized for the treatment of glioma brain tumors withALA ⁄MAL-PDT has yet to be discovered and further light dosimetryoptimization is required. Nevertheless, it is thought that using lowfluence doses compared with high fluence doses will allow greaterselective tumor kill to be achieved (12) which is advantageous partic-ularly when ablating tissue in the brain. Therefore, in our study a totalfluence dose of 15 J cm)2 was employed as it was anticipated fromprevious experiments (data not shown) that this dose would result insufficient PDT damage in these experiments and the delivery of a totallight dose of this order would be feasible for a ‘‘one-shot’’ intraoperativePDT treatment if this intervention is subsequently investigated clini-cally. Finally, both plates were measured for ‘‘post’’ PpIX fluorescencelevels.

Cell viability counting. Following ‘‘post’’ PpIX fluorescence mea-surements all media were removed from the wells and the cells washedwith 1 mL PBS. Cells were then trypsinized by the addition of 500 lLTrypsin ⁄EDTA (T ⁄E) to each well. After 3 min, 500 lL modifiedEMEM was added to neutralize the T ⁄E and for each concentration ofALA ⁄MAL ± CP94 (conducted in triplicate) the contents of the threewells were combined and centrifuged. The resulting supernatant wasdiscarded leaving a cell pellet, which was resuspended in 1 mL EMEM.Of this cell suspension 50 lL was added to 50 lL Trypan blue dye,10 lL of this mixture was transferred to a hemocytometer and thenumber of nonstained viable and stained nonviable cells were countedin duplicate and averaged.

Data analysis and statistics. For all 6 · 24-well test plates the firstthree wells contained control cells not incubated with any ALA ⁄MAL(blank controls). The PpIX fluorescence measurements from thesewells were used to remove natural cellular PpIX autofluorescence fromall the other measurements made from the same plate. All data pointsand bars in the figures represent average values calculated from testsolution concentrations being carried out in triplicate. Statisticalsignificance between individual groups was determined using theStudent’s t-test and an ANOVA was employed when consideringdifference between data sets.

RESULTS

ALA ⁄MAL-induced PpIX fluorescence

All PpIX fluorescence measurements of glioma cells incubatedfor 0, 3 or 6 h without ALA ⁄MAL (blank controls) wereconsistent (Fig. 1; 0 and 3 h data not presented). Prior to

irradiation with red light (15 J cm)2) at a wavelength of635 ± 2 nm ‘‘pre’’ measurements of PpIX fluorescence weretaken. Following light irradiation ‘‘post’’ measurements of

PpIX fluorescence were taken. It was observed that asanticipated ‘‘post’’ PpIX fluorescence measurements wereconsistently lower than ‘‘pre’’ fluorescence measurements forglioma cells incubated with either ALA ⁄MAL for 3 or 6 h,

respectively (6 h data presented in Fig. 1a,b) in all but thelowest concentrations of precursors investigated (ALA62.5 lMM; MAL 250 lMM). Additionally, approximately four

times the concentration of MAL was required to produce asimilar level of preirradiation PpIX in this cell type aswas required with ALA (6 h incubation with 1000 lMM MAL

versus 250 lMM ALA) and preirradiation PpIX fluorescenceproduction increased with increased concentrations of eachprodrug.

(a) (b)

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Figure 1. PpIX fluorescence values from U-87 MG glioma cells incubated with (a) 5-aminolevulinic acid (ALA; 6 h) or (b) methyl aminolevulinate(MAL; 6 h) measured both prior to and immediately following light irradiation.

Photochemistry and Photobiology, 2010, 86 1155

ALA ⁄MAL + CP94-induced PpIX fluorescence

Incubation of glioma cells with increasing concentrations ofALA (0–250 lMM) dose-dependently increased levels of PpIX

fluorescence both with (Fig. 2b) and without the iron chelator(Fig. 2a). The greatest levels of PpIX fluorescence wereobserved after 3 h. PpIX fluorescence measurements after 3

and 6 h incubation periods were both found to be significantlyhigher than the initial readings (0 h) both with and without thepresence of CP94. With ALA alone, PpIX fluorescence

measurements after 3 h incubation with three out of the fourALA concentrations investigated (62.5, 125 and 250 lMM) werestatistically higher than those observed with the incubation ofthe same ALA concentrations for 6 h (Fig. 2a). Co-incubation

with ALA and CP94 resulted in all PpIX fluorescencemeasurements being significantly higher following 3 h incuba-tion than those observed after 6 h co-incubation (Fig. 2b).

Furthermore, comparison of ALA + CP94 with ALA aloneindicated that higher levels of PpIX fluorescence were detectedin the iron chelated group and this difference was found to be

statistically significant at both 3 and 6 h (ANOVA, P < 0.05).Incubation of glioma cells with increasing concentrations of

MAL (0–1000 lMM) also resulted in a dose-dependent increasein levels of PpIX fluorescence both without (Fig. 3a) and with

the iron chelating agent (Fig. 3b). Up to 750 lMM

MAL ± CP94 PpIX fluorescence was higher after 3 h incu-bation compared with measurements taken after 6 h although

these results were not found to be statistically different.However, at a concentration of 1000 lMM MAL a greater levelof PpIX fluorescence was recorded following 6 h incubation

(compared with 3 h incubation) and this difference was foundto be statistically significant both with and without thepresence of CP94. All 3 and 6 h measurements with concen-trations of MAL of 250 lMM and above were found to be

statistically higher than those recorded at 0 h. In general,similar levels of PpIX fluorescence were observed in theMAL ± CP94 groups (Fig. 3) when compared with the

ALA ± CP94 groups (Fig. 2). However, when the MALalone and MAL + CP94 data sets were compared usingANOVA to detect any significant difference (P < 0.05) none

was found (unlike with ALA ± CP94, Fig. 2). Using aStudent’s t-test between the MAL alone and MAL + CP94data sets collected at 6 h with the individual concentrationsemployed indicated however a statistical significance

(P < 0.05) at this level at all concentrations except 0 lMM

MAL.At the highest concentrations of ALA (250 lMM) and MAL

(1000 lMM) employed very little PpIX fluorescence was observed

(a)

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Figure 2. Protoporphyrin IX (PpIX) fluorescence values from U-87MG glioma cells incubated with various concentrations of (a)5-aminolevulinic acid (ALA) or (b) ALA + 150 lMM CP94 for 0, 3 and6 h prior to light irradiation. *, + and D indicate respectivelya statistically significant difference (P < 0.05) in PpIX fluorescencebetween the 0, 3or 6 hdatawhen compared tobetween the3 and6 hdataand between the ALA ± CP94 groups observed at the same time point.

(a)

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∆

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Figure 3. Protoporphyrin IX (PpIX) fluorescence values from U-87MG glioma cells incubated with various concentrations of (a) methylaminolevulinate (MAL) or (b) MAL + 150 lMM CP94 for 0, 3 and 6 hprior to light irradiation. *, + and D indicate respectively a statisticallysignificant difference (P < 0.05) in PpIX fluorescence between the 0, 3or 6 h data when compared to between the 3 and 6 h data and betweenthe MAL ± CP94 groups observed at the same time point.

1156 Emma Blake and Alison Curnow

at 0 h, but this was significantly increased at both 3 and 6 hwith each congener (Fig. 4a,b). There was also no significantdifference observed in the PpIX fluorescence of cells co-incubated with ALA + CP94 for 0 or 6 h when they were

compared with cells incubated for the same time periods withALA alone (Fig. 4a). However, there was a significant differ-ence in cells co-incubated with ALA and CP94 for 3 h when

compared with cells incubated with ALA alone (Fig. 4a).Conversely, at the highest concentration of MAL used(1000 lMM) cells co-incubated with MAL and CP94 for 0 and

6 h showed statistically significant increases in PpIX fluores-cence (even though the change observed at 0 h was minimal).Notably however, those cells incubated at 6 h with the

combined treatment of MAL + CP94 produced PpIX fluo-rescence measurements which had increased by more than 50%(Fig. 4b). At 3 h, although some enhancement was producedby co-incubation of the iron chelator with MAL, this difference

was not found to be statistically significant (Fig. 4b).

ALA ⁄MAL ± CP94 PDT cytotoxicity

Following ALA ⁄MAL-induced PDT ± CP94 the number of

viable cells in each treatment group was recorded and cellsurvival was displayed as a percentage of the 0 lMM ALA ⁄MAL ± CP94-treated cells (Fig. 5). PDT with increasing

concentrations of ALA ⁄MAL alone, incubated for 3 h, wasfound to lead to a decrease in cell survival. Incubation withincreasing concentrations of ALA alone for 6 h did notdecrease cell survival; in fact there was a slight increase up to

and including 187.5 lMM ALA (Fig. 5a). A small but noteddecrease in cell survival was observed when cells were incubatedwith increasing concentrations of MAL alone for 6 h (Fig. 5b).

When increasing concentrations of ALA ⁄MAL were com-bined with CP94 incubation, the cell survival decreasedconcurrently with both prodrugs. This pattern was found to

be statistically significant at both 3 and 6 h for ALA and at 0and 6 h for MAL (ANOVA, P < 0.05). In addition, in the1000 lMM MAL + CP94 treatment group no live cells were

recorded and so 100% cytotoxicity was produced by theseparticular parameters in these cultured human glioma cells.

DISCUSSION

The third most common cause of cancer deaths in 15- to 35-year-olds is attributed to primary brain tumors with malignant

glial tumors remaining the most common (13). Between 1986and 2000, in a study by Stylli et al. (14), 136 patients with‘‘high-grade’’ gliomas were treated with HpD-PDT followingsurgical resection and in a similar study by Muller and Wilson

(1) 96 patients with malignant gliomas were treated withPhotofrin-PDT following surgical resection. Both studiesreported prolonged survival rates for patients with malignant

gliomas who had surgical resection of their tumor with eitherHpD- or Photofrin-PDT as an adjuvant treatment (1,14).Although these photosensitizers have been successfully used in

clinical PDT trials of the brain in the past, other photosen-sitizers with greater tumor-to-normal tissue PpIX localizationand shorter photosensitization periods are favored (2). For

example, ALA has a shorter photosensitization period andincreased brain tumor selectivity (15) and in 2007 the Euro-pean Medicines Agency (emeA) authorized Gliolan (activesubstance ALA-induced PpIX) as a medicinal product for

human use. In a study of 415 patients who had their malignantgliomas removed either with or without Gliolan assistance, at6 months it was reported that 20.5% of patients who had

received Gliolan administration remained alive compared withonly 11%, of whom had not received Gliolan administration(16). As well as its use as a fluorescent marker in PDD, ALA-

PDT as an adjuvant therapy in the surgical resection ofmalignant gliomas has also been investigated (17). In arandomized controlled trial of 27 patients (13 in the studygroup and 14 in the control group), the control group had

standard surgical resection of their glioblastoma multiforme(GBM) ‘‘high-grade’’ glioma tumor, whilst the study groupwas treated with ALA and Photofrin fluorescence-guided

resection (FGR) and repetitive PDT (17). Between the twogroups there was no difference in the number of complicationsreported or hospital stays and the mean survival of the control

group was 24.6 weeks compared with 52.8 weeks for the studygroup. Therefore, Eljamel et al. (17) concluded that ALA andPhotofrin FGR and repetitive PDT offered a worthwhile

survival advantage without any added risk to these patients.Bearing in mind the median survival of patients with GBM

is 15 months (18) it is recognized that further research toincrease survival rates for these patients and improve quality

of life is vitally important and various ways to try and enhance

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Figure 4. Stacked bar graphs of mean protoporphyrin IX (PpIX)fluorescence from U-87 MG glioma cells incubated for 0, 3 and 6 hwith (a) 250 lMM 5-aminolevulinic acid (ALA) ± 150 lMM CP94 or(b) 1000 lMM methyl aminolevulinate (MAL) ± 150 lMM CP94. Astatistically significant difference (P < 0.05) in PpIX fluorescencewith the addition of CP94 is indicated by *.


clinical PDT is currently under investigation. Researchincludes using different photosensitizers, adjusting the lightdosimetry (total fluence and fluence rates) (19), light fraction-ation (20,21), manipulating oxygen levels (22), and as in this

study, supplementing PpIX prodrugs with iron chelators(11,23). Normally, within cells via the heme biosynthesispathway PpIX is converted to heme through the addition of

ferrous iron. The addition of an iron chelator removes thisferrous iron and PpIX accumulates within the cells. It isaccepted that if adequate amounts of photosensitizer can be

delivered to tumor cells and the two other requirements aremet—sufficient molecular oxygen availability and activationby light of a specific wavelength—then successful ablation oftumor cells can be achieved. The aim of increasing photosen-

sitizer levels within human glioma cells with an iron chelator aspresented here is therefore to investigate the possibility of thisapproach to increase malignant cell visualization in PDD and

assist in FGR and ⁄ or cellular destruction of brain tumorsfollowing PDT.

The initial objective of this study was to evaluate the effects

of two different prodrugs separately in combination with theiron chelating agent CP94 on the accumulation of thephotosensitizer PpIX in human glioma cells. The effects this

treatment had on glioma cell viability following irradiation

were also subsequently investigated. Although previous studiesconducted with human glioma spheroids and ALA ⁄MAL-PDT have demonstrated that fluorescence can be successfullyquantified in this manner in vitro (24), we have demonstrated

for the first time that the iron chelator CP94 combined withALA- or MAL-induced PDT significantly increased PpIXfluorescence levels in human glioma cells and subsequently

decreased cell viability on illumination.Our experiments have demonstrated that glioma cells

incubated with ALA ⁄MAL alone produced similar levels of

PpIX fluorescence even though ALA was administered at aconcentration four times lower than that of MAL. This findingis in disagreement with some of the existing literature whichsuggests the use of ALA esters may result in enhanced PDT

efficacy in some cell types ⁄ tumors due to increased membranepenetrance (24). This is because although ALA has a goodtumor-to-normal brain tissue localization (25), it is highly

hydrophilic and has poor transport across cell membranes(24). This led to the development of lipophilic ALA esters,such as MAL and hexyl aminolevulinate (HAL). This theory is

supported by the results reported by Hirschberg et al. (24):they found that PDT of human glioma spheroids with ALAlipophilic esters had an equivalent response compared to ALA

but obtained at concentrations 10–20 times lower. In addition,

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Figure 5. Counts of viable U-87 MG glioma cells presented as percentage cell survival (of the blank control) at 0, 3 and 6 h following incubationwith various concentrations of (a) 5-aminolevulinic acid (ALA) ± 150 lMM CP94 photodynamic therapy (PDT)-treated cells or (b) methylaminolevulinate (MAL) ± 150 lMM CP94 PDT-treated cells.


Perotti et al. (26) found a seven-fold increase in brain porphyrinlevels after injection with HAL compared to ALA. Investiga-tions into ALA uptake systems have reported that different celltypes appear to have distinctive uptake mechanisms. For

example, the LM3 mammary adenocarcinoma cell line takesup ALA via BETA transporters, MAL by an active transportmechanism andHAL is via simple diffusion. This led Rodriguez

et al. (27) to suggest that to understand and improve ALA-mediated PDT, knowledge of the mechanisms of ALA deriv-atives entry into different cell types is essential. In a study to

investigate the transport of ALA between blood and brain,Ennis et al. (28) reported the movement of ALA at the blood–brain barrier (BBB) to be a diffusional process and reported no

evidence for the presence of a carrier-mediated transportmechanism. Notably, the study indicated that the permeabilityof ALAat the BBBwas very low and suggestedALAderivativeswith higher lipid solubilities than ALA may have greater

diffusion across the BBB (28). So although our in vitroexperimentation in U-87 MG human glioma cells has indicatedthat lower doses of ALA are required to produce the same level

of PpIXfluorescencewithMAL, due to the effect of the BBB it isunlikely that this same trend would be observed in clinicalpractice. It is interesting to note thatCP94 is known fromanimal

studies to be able to cross the BBB (29) and we suggest thatfuture studies should explore PpIX accumulation and subse-quent PDT cytotoxicity in cultured human glioma cells with thecombined use of HAL + CP94 as HAL has also been shown to

be able to cross the BBB.Once combined with CP94 greater levels of PpIX were

observed to be produced with MAL than with ALA and

incubation with MAL exceeded the maximum PpIX fluores-cence produced by ALA co-incubated with CP94 by more than20%. These results were not unexpected as the literature from

previous studies points toward an increasing PpIX accumula-tion with increasing concentrations of exogenous ALA ⁄MALand an even more enhanced effect with the addition of CP94

(9); however, this is the first time this has been demonstrated inglioma cells as the majority of previous experimentation hasemployed cells of dermatological origin. The reason whyMAL + CP94 produced greater enhancement in PpIX accu-

mulation when compared with ALA + CP94 requires furtherinvestigation. MAL affects the enzymes of the heme biosyn-thesis pathway slightly differently from ALA, and these subtle

differences may be emphasized when free iron within cells islimited (by the presence of CP94).

Addition of sufficient amounts of exogenous ALA ⁄MAL

results in the normal feedback mechanism of the hemebiosynthesis pathway being bypassed leading to a temporaryaccumulation of PpIX. CP94 chelates cellular free iron,inhibiting PpIX conversion to heme leading to the further

increased accumulation of PpIX observed here. Furthermore,cell viability was greatly reduced when either ALA or MALwere combined with CP94 indicating that the increased levels

of PpIX fluorescence produced via iron chelation could beactivated on irradiation to produce an increased cytotoxicresponse. Cell viability was only assessed at one time point in

this investigation (immediately after irradiation) and so it islikely that only immediately induced necrotic cell death wasdetected at this time point and so future experimentation

should consider longer time points following irradiation todetect any apoptotic-mediated cell death. It should also be

noted that although it was demonstrated that complete celldeath could be achieved in this cell type, parameters wereemployed that did not routinely kill all the cells so that theeffects of the different compounds could be observed and

compared with one another.To our knowledge, this study is the first to investigate the

combined interaction of ALA ⁄MAL-induced PDT with CP94

in human glioma cells. The hydroxypyridinone iron chelatorCP94 has advantages over other widely available iron chela-tors such as EDTA and DFO. Firstly, CP94 is a bidentate

hydroxypyridinone and unlike hexadentate DFO which has tobe administered by long infusion, it is orally active. Secondly,CP94 has a molecular weight of 167 meaning that more than

70% absorption from the gastrointestinal tract is achievable.EDTA may have a molecular weight of only 292 but itsresulting iron complex is toxic. In addition, in clinicalinvestigations (30) CP94 has been demonstrated at an intra-

venous dose of 100 mg kg)1 to be effective at chelating iron inconditions of iron overload without producing significanttoxicity whereas another related hydroxypyridinone CP20 at

the same dose daily for many weeks was observed to producesome toxicity (although iron chelation was observed to bemore effective with this compound) (31). Investigations of

CP94 and CP20 in the normal rat colon found CP94 to bemore effective in enhancing ALA-PDT than CP20 (23),producing double the PpIX fluorescence and triple the necrosison illumination compared with the ALA-only control group.

Subsequently CP94 has now been administered topicallyclinically in humans to enhance ALA (32) and MAL (33)PDT of nodular basal cell carcinoma without significant side

effects, although CP94 is yet to be administered orally toenhance PpIX-induced PDT.

In conclusion, our study has demonstrated that the iron

chelator CP94 significantly enhanced PpIX accumulation andsubsequent PDT cytotoxicity in cultured human glioma cells.The application of these findings to clinical ALA ⁄MAL-

induced PDT ⁄PDD, used alongside conventional options, maywith further experimentation be able to help improve survivalrates in this difficult-to-treat malignancy.

Acknowledgement—This work has been supported in part by the

Duchy Health Charity Limited.

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the hydroxypyridinone iron chelator cp94 can enhance ppix-induced pdt of cultured human glioma cells

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