ascorbic acid enhances the effects of 6-hydroxydopamine and … · sk-n-sh cells, grown as...

[CANCER RESEARCH 51.6066-6072. November 15. 1991]

Ascorbic Acid Enhances the Effects of 6-Hydroxydopamine and H2O2 on Iron-

dependent DNA Strand Breaks and Related Processes in the NeuroblastomaCell Line SK-N-SH

Gernot Bruchelt,1 Ingrid U. SchraufstÃ¤tter, Dietrich Niethammer, and Charles G. Cochrane

Department of Immunology, Research Institute ofScripps Clinic, La Jolla, California 92037 [G. B., I. U. S., C. G. C.J, and Department of Hematology and Oncology,Children's Hospital, University of Tuebingen, Ruemelinstrasse 23, D-7400 Tuehingen, Federal Republic of Germany Â¡I).N.j

ABSTRACT

Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastomacells for therapy in combination with reactive oxygen intermediates. Inthe present study the effects of 6-hydroxydopamine (6-OIIDA) and IM),on metabolic parameters critical for cell survival were investigated incells with low and high ferritin content in the presence and absence ofascorbate. Human neuroblastoma SK-N-SH cells were pretrcated with100 n\\ l i'SO, and 10 JÃŒMdesferrioxamine, respectively, for 24 h yieldingcells with different ferritin contents. The effects of 6-OIIDA and H2O2(25 nM-250 m\i ) in the absence and presence of I HIMascorbic acid onDNA strand break formation, activation of poly(ADP-ribose) polymer-ase, and finally decrease in NAD* and ATP concentration were investi

gated. All these parameters were influenced by 6-OHDA and IM), in aconcentration-dependent manner in a similar way. The effects were mostpronounced in ferritin-rich cells and in the presence of ascorbic acid.Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid causedstrand breaks that were prevented in the presence of mannitol or desfer-rithiocine. ! M >rimdialed strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by othersa model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application ofa high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.

INTRODUCTION

Neuroblastoma stage IV has a poor prognosis (1). Therefore,new therapeutic approaches are currently under clinical investigation. Among them is autologous bone marrow transplantation in combination with different purging procedures in orderto remove contaminating neuroblastoma cells (2-4). In one ofthese purging systems bone marrow cells were incubated with100 MM 6-OHDA2 in combination with 1 m\i AA. It was

assumed that neuroblastoma but not bone marrow cells wouldtake up the catecholamine-analogous compound 6-OHDA (5).Inside the cells, H2O2 formed during the oxidation of 6-OHDA(6) would destroy the neuroblastoma cells specifically (7, 8).Furthermore, AA included in a 10-fold molar excess wouldenhance these effects by acting as redox cycler (9. 10). However,some of these theoretical considerations as arguments for purging procedures proved to be incorrect. Since the oxygen concentration present in the purging mixture (about 250 p\i) islimited, most of the AA is not consumed by the redox cycling

Received 8/7/91; accepted 9/9/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Recipient of a grant of FÃ¶rderverein LeukÃ¤miekranker Kindcr.e.V.. Tuebin-gen. Federal Republic of Germany. Supported by the Deutsche Krebshilfe. GrantW54/89/NÃŒ4.To whom requests for reprints should be addressed. Permanentaddress: Department of Hematology and Oncology. Children's Hospital. University of Tuebingen. Ruemelinstrasse 23, D-7400 Tuebingen. Federal Republic ofGermany.

2The abbreviations used arc: 6-OHDA, 6-hydroxydopamine: AA. ascorbicacid: DFO, desferrioxamine: CCC PM2 DNA, closed covalently circular PM2DNA; PBS, phosphate-buffered saline; GSH, glutathione.

process. Nevertheless, the cytotoxic effects of the combination6-OHDA/AA were the more pronounced the more AA waspresent (9). This means that AA must have caused some toxiceffects irrespective of its function as redox cycler. Furthermore,oxidation of 6-OHDA occurs rapidly so that most of the reactive oxygen intermediates are already formed in the incubationmedium before a significant amount of 6-OHDA is taken upselectively by neuroblastoma cells (11). Moreover, only a smallpercentage of neuroblastoma cells can take up catecholaminesselectively (12); therefore both neuroblastoma and hemato-poietic stem cells are equally exposed to the reactive oxygencompounds. Neuroblastoma cells proved to be more vulnerableto 6-OHDA/AA than hematopoietic cells (9, 11) although thereason for this is unknown. We supposed that AA might sensitize neuroblastoma cells in concert with iron. Iron is found inthese cells in large amounts incorporated in ferritin (13, 14).AA is concentrated in sympathetic nervous tissue (15, 16) andshould therefore accumulate in neuroblastoma cells if providedin significant amounts. The potential cytotoxic effects of AAalone or in combination with cytostatic drugs have been described for neuroblastoma and other cells (17-19). However,some confusion exists since AA can act either as antioxidants(e.g. Refs. 20 and 21) or as prooxidants, the latter especially incombination with iron (22-24). Iron stored as ferritin is deprived of its potential cytotoxic effect, but it can be releasedfrom ferritin yielding ferrous iron (Fe2+) by many agents, amongthem 6-OHDA and AA (25-28). Fe2+ catalyzes the formation

of the highly toxic OH radicals from H2O2 in the Fentonreaction (29). In accordance with these findings, a mixture ofH2O: and ferritin was shown to produce OH radicals in thepresence but not in the absence of AA (30). AA and ferritin,acting together, could therefore be an effective enhancer systemfor therapeutic regimens that are operative via generation ofH2O2. The most sensitive cellular target structure for H2O2 isobviously the DNA (31-34). H2O2 causes DNA strand breaksthat are followed by the activation of poly(ADP-ribose) polym-erase; this enzyme consumes NAD"1",a process that is subse

quently associated with a loss of ATP (35, 36).The aim of our study was to investigate the influence of AA

on the effects of 6-OHDA and H2O2 on these DNA-dependentpathways using neuroblastoma cells with high and low ferritincontent in order to find a better rational basis for a potentialapplication of AA in the therapy of this tumor.

MATERIALS AND METHODS

Materials

Ethidium bromide, 6-hydroxydopamine, ascorbic acid, and 1,10-phenanthroline were from Sigma, St. Louis, MO; CCC PM2 DNA wasfrom Boehringer Mannheim. Mannheim, Federal Republic of Germany; mannitol was from Calbiochem-Behring Corp., La Jolla, CA;desferrioxamine and desferrithiocin were from Ciba-Geigy. Basle, Switzerland; |'H]NAD* (specific activity, 25 Ci/mmol) was from ICN,

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ASCORBATE. FERRITIN. AND REACTIVE OXYGEN COMPOUNDS IN NEUROBLASTOMA

Irvine, CA; L-[carboxyl-"C] ascorbic acid (specific activity, 10-30 mCi/mmol) was from Amersham-Buchler, Braunschweig, Federal Republic

of Germany.

Determination of DNA Strand Breaks (Cell-free System)

(a) Ethidium-binding Assay According to the Method of Lown (37).Briefly, CCC PM2 DNA (10 p\\ 2 Mg)was incubated with 10 M!PBS orwith additives dissolved in PBS. After addition of 180 M' of ethidiumbromide-containing alkaline buffer the samples were heated for 4 minat 96Â°C.Twenty M' of this solution were used for electrophoresis (see

below); the remaining 180 M' were mixed with an additional 1 mlalkaline buffer before measurement of ethidium bromide fluorescenceon a Perkin-Elmer model 650-15 (Hitachi-Perkin Elmer Instruments,

Mountain View, CA).(b) Electrophoresis. Twenty M'of DNA containing solution (200 ng,

see a) were mixed with 4 M!loading buffer. Electrophoresis was carriedout using 15 /Â¿Iof this solution on a 0.75% agarose gel [375 mg ofagarose were melted in 50 ml Tris-borie acid-EDTA buffer (8. l g Trisbase plus 4.125 g boric acid plus 0.7 g disodium EDTA-2 H2O/liter)containing 2.5 n\ of ethidium bromide (10 mg/ml)]. Electrophoresiswas carried out using Tris-borie acid-EDTA buffer containing 0.5 mgethidium bromide/liter at 100 V for about 2 h.

Determination of Ferritin Content in SK-N-SH Cells

Cells (1 x 106/4 ml) medium were seeded in 6-well plates (Greiner,

Nuertingen, Federal Republic of Germany). After 24 h, 40 M' PBS(control), 40 /il DFO (10 MM),and 40 M!FeSO4 (100 MM),respectively,were added. Ferritin was determined in cell lysates after 24 h using acommercial immunoassay (Tandem-Fer; Hybritech, HÃ¼rth-Effern,Fed

eral Republic of Germany).

Uptake of |'"C]Ascorbic Acid

SK-N-SH cells, grown as monolayers in 12-well plates (Greiner) ata density of 1 x IO6cells, were incubated with 5-100 MM[14C]ascorbic

acid in phosphate-buffered saline plus 1% bovine serum albumin for 30min at 37Â°C.Cells were then washed three times with cold buffer

solution, and cell pellets were lysed with 500 M!0.3 MNaOH and mixedwith 5 ml scintillation cocktail (Unisolve; Zinsser, Frankfurt, FederalRepublic of Germany) before counting.

For calculation of the intracellular AA concentration, cell volumewas determined by centrifugation of SK-N-SH cells in a hematocritcentrifuge; IO6SK-N-SH cells correspond to 2.14 /Â»I.

Generation of SK-N-SH Cells with Different Ferritin Content

SK-N-SH cells were grown as monolayers using RPMI 1640 con

taining glutamine (2 ITIM),gentamicin (50 mg/liter), and 10% fetal calfserum (Gibco) in 75-cm2 flasks. At about 80-90% confluency, flasks

were incubated with either 10 MMDFO or 100 MMFeSO4 for 24 h. Themedium was removed and filtered. After trypsinization, the detachedcells were resuspended in their corresponding media. After 2-3 hincubation at room temperature the cell suspensions were centrifugedand the cell pellets were resuspended in modified Gey's buffer (147 mM

NaCl-5 mM KC1-1.5 mM CaCl2-1.9 mM KH2PO4-0.3 niM MgCl2- 1.1mM Na2HPO4-10 mM HEPES-5 mM glucose, pH 7.3) at a concentration of 1-3 x 106 cells/ml. Cell viability was 90-95% (trypan blueexclusion test). Subsequently, DFO- and FeSO4-pretreated cells wereboth divided and left untreated or were treated with 1 mM AA for 30min. Incubation with H2O2 and 6-OHDA was done for 15 or 30 minat 37Â°C.Samples were further processed according to the test protocols

described below.Determination of DNA strand breaks in SK-N-SH cells was carried

out according to the method of Birnboim and Jevcak (38) using theprotocol described in Ref. 39. In some experiments 1,10-phenanthrolinewas added to the incubation mixture 10 min prior to the addition ofH202.

Determination of poly(ADP-ribose) polymerase (NAD+, ADP ribo-

syltransferase, EC 2.4.2.30) was carried out according to the protocol

described in Ref. 39 with the exception that the permeabilized cellswere incubated for 12 min.

High Performance Liquid Chromatography Analysis of NAD* and ATP

Cells (about 2 x 106/rnl) were incubated for 30 min with 6-OHDA

and H2O2. Supernatants were removed and the cell pellets were treatedwith 220 M!8% perchloric acid. After centrifugation, 200 M' of eachsupernatant were neutralized with 200 M!KOH/Tris. Supernatants werecollected and frozen at â€”¿�20Â°Cfor high performance liquid chromatog-

raphy analysis. Two humdred n\ were injected and separation wascarried out on a Du Pont C,8 column. A gradient of 100% 100 mMpotassium phosphate, pH 6.0, to 60% buffer plus 40% methanol wasapplied with a flow rate of 1.3 ml/min. Absorbance was measured at256 nm; flow rate was 1.3 ml/min. The ATP signal appeared at about5.4 min; the NAD* signal appeared at about 17 min.

RESULTS

SK-N-SH cells grown as monolayers were split and treatedeither with 10 MMDFO, 100 /Â¿MFeSO4, or no further additionsfor 24 h. As shown in Fig. 1, DFO treatment reduced andFeSO4 treatment strongly enhanced the ferritin content in thesecells. The mean ferritin content of untreated cells was 19.1 Â±7.0 (SD) ng/106 cells. After treatment with DFO, ferritin content dropped to 6.10 Â±3.67 ng/106 (P < 0.01), after treatmentwith FeSO4 it increased to 62.62 Â±15 ng/106 cells (P < 0.01;4 independent experiments in duplicate, Student's i test). Fer

ritin content in iron-pretreated cells was 13.5 Â±8.7 times higherthan in the correspondent DFO-pretreated cells.

Fig. 2 shows the uptake of 5-100 MM[I4C] ascorbic acid after30 min incubation time into SK-N-SH cells. Similar to neutro-phils, the uptake system is composed of two compounds: a highaffinity transport system (in the example given: Km, 7.1 MM;Vmax:480 pmol/106 cells/30 min, calculated according to meth

ods given in Ref. 40); and a less active uptake system whichproved to be linear for an AA concentration of at least 1 mivi(data not shown). A more detailed analysis is given elsewhere(41).

DNA Strand Breaks in Isolated PM2 Phage DNA. In orderto characterize the effects of H2O2,6-OHDA, and AA on DNA,first experiments were carried out on isolated CCC PM2 DNA.Fig. 3A shows that 6-OHDA as well as AA generate singlestrand breaks in CCC PM2 DNA. H2O2 caused DNA strand

100

90

80

70

60

50

40

30

20

10

oÂ«DO

ete

DFO control FeSO4Fig. 1. Ferritin levels in SK-N-SH cells after 24 h incubation with 10 ^M DFO

and 100 ^M FeSO4 compared to untreated controls [4 independent experiments,each in duplicate (SEM, 5.1-8.9%)]. , changes of ferritin content in correspondent cells after treatment with DFO or FeSO4.

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1600

I 1200

800

400

20 O 60

ascorbic acid

80 100

Fig. 2. Uptake of |'"C] ascorbic acid into SK-N-SII cells. About IO6cells wereincubated at 37'C in 12-well plates for 30 min with 5-100 /iM ["CJascorbic acid.

Mean of triplicate measurements; bars, SEM.

Fig. 3. A, strand break formation of CGC PM2 DNA. Lane I, control; Lane2, H2O2; Lane 3, 6-OHDA; Lane 4, ascorbic acid; Lane 5, H2O2 plus ascorbicacid; Lane 6. 6-OHDA plus ascorbic acid. Incubation was carried out for l husing 250 MMH2O2 or 6-OHDA and 10 mM ascorbic acid. B, determination ofCGC PM2 DNA strand breaks after 20 min incubation with 1 m,\i ascorbic acidand 100 MM6-OHDA in the absence and presence of mannitol (100 mM) anddesferrithiocine (10 mM). In parallel. DNA strand breaks were determined usingthe fluorescence assay according to the method of Lown (numbers in parentheses,percentage of DNA strand breaks measured in the fluorescence assay). Lane 1,control (0%); Lane 2, 1 misi ascorbic acid (45.7%); Lane 3, 6-OHDA (100%);Lane 4, 6-OHDA plus mannitol (22.9%); Lane S, 6-OHDA plus desferrithiocine(10.5%); Lane 6, ascorbic acid plus mannitol (5.8%); Lane 7, ascorbic acid plusdesferrithiocine (5.8%).

breakage only in the presence of AA. Fig. 3Ã„shows that DNAstrand breaks caused by 6-OHDA or AA could be prevented byadding mannitol (an OH radical scavanger) or desferrithiocine(an iron chelator), suggesting that in both cases DNA strandbreakage is caused by ferrous iron-dependent formation of OHradicals. Superoxide dismutase, previously purged by dialysis,reduced strand break rates of AA and 6-OHDA (data notshown). Essentially the same results could be observed usingthe fluorescence assay (see legend to Fig. 3ÃŸ).

DNA Strand Breaks in SK-N-SH Cells after Treatment with6-OHDA and H2O2. Treatment of SK-N-SH cells with H2O2 or6-OHDA led to dose-dependent DNA strand break formation(Fig. 4). FeSO4-pretreated SK-N-SH cells were more sensitiveagainst 6-OHDA and H2O2 than DFO-pretreated cells. In

contrast to the cell-free system, AA alone had no effects in thistest system. However, AA enhanced the effects of 6-OHDA aswell as of H2O2 in DFO and in FeSO4-pretreated cells. Themost distinct differences were observed in DFO-pretreated cellsusing intermediate concentrations (100 MM)of H2O2 (P< 0.01)and 6-OHDA (P< 0.05); 3 independent experiments, 2-4-fold.The amount of alkaline stable DNA ("control") was 70 Â±5%in DFO-pretreated cells compared to 61.9 Â±11% in the FeSO4-pretreated group (6 independent experiments, each 2-4-fold; P> 0.05). Strand break rate after incubation with 75 ÃŸMH2O2was greatly reduced in both groups when the incubation wascarried out in the presence of the iron chelator 1,10-phenan-throline (100 UM). It dropped from 37% to 10% in the DFOgroup and from 42% to 5% in the FeSO4 group.

Poly (ADP-Ribose) Polymerase Activity after Treatment with6-OHDA and H2O2. Fig. 5 shows the effects of H2O2 and 6-OHDA on poly(ADP-ribose) polymerase activity in DFO- andFeSO4-pretreated SK-N-SH cells. AA enhanced the effects inboth groups. Similar to DNA strand break experiments, thedifferences between incubations with and without AA weremore pronounced in DFO-pretreated cells. The basal enzymeactivity in FeSO4-pretreated cells was higher than in DFO-pretreated cells (3.34 Â±0.55 versus 2.49 Â±0.93 pmol protein-bound poly(ADP-ribose)/min/106 cells (5 independent experi

ments, 2 fold, P > 0.05).Decrease of NAD* and ATP in SK-N-SH cells after Treatment

with 6-OHDA and H2O2. Fig. 6 shows the decrease of NAD+levels in SK-N-SH cells after incubation with 6-OHDA andH2O2. The effects were more intense in FeSO4-pretreated cells.

- so-

_ 8(

Â°60-

J.40-

25 100 250

Fig. 4. DNA strand breaks in DFO- and FeSO4-pretreated SK-N-SH cells byI!.â€¢().â€¢and 6-OHDA in the absence (â€¢)and presence (â€¢)of 1 mM ascorbic acid(expressed in percentage of controls, set as 100%; n = 5). Number of independentexperiments: 25 MM,n = 2; 100 MM,n = 3; 250 MM,n = 3. Experiments werecarried out 2-fold or 4-fold; SEM (bars), 4.6-12.1%

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250

100!

25 250

100

Fig. 5. Poly(ADP-ribose) polymerase activity in DFO- and FeSO4-pretreatedSK-N-SH cells after treatment with 6-OHDA and H2O2 in the absence (â€¢)orpresence (â€¢)of 1 rtiMascorbate, expressed as percentage of controls (set as 100%;n = 5). Single experiments in duplicate for 25 MMand 100 tÂ¡M;two experimentsin duplicate for 250 ^M (SEM (bars), 2.1-8.3%).

AA clearly enhanced the effects of H2O2 in DFO-pretreatedcells whereas the effects of 6-OHDA were amplified in DFO-as well as in FeSO4-pretreated cells. The basal levels of NAD*of DFO-pretreated cells were 1.53 Â±0.31 nmol/106 cells; thoseof FeSO4-pretreated cells were 1.62 Â±0.16 nmol/106 cells (4

independent experiments; P > 0.05).The decrease of ATP levels in SK-N-SH cells after exposure

to H2O2 and 6-OHDA is shown in Fig. 7. Again, the effectswere more pronounced in FeSO4-pretreated cells. Similarly asshown for NAD-I-, AA enhanced the ATP-depleting effects of6-OHDA more strongly than those of H2O2. The control levelsof DFO-treated cells were 6.96 Â±1.54 nmol/106 cells and ofFeSO4-treated cells 7.90 Â±0.70 nmol/106 cells (4 experiments;

P > 0.05).

DISCUSSION

The aim of the present study was to investigate the effects ofAA on iron-dependent DNA strand break formation and related

processes in neuroblastoma cells after incubation with H2O2and 6-OHDA. Numerous investigations document the interac

tions and cytotoxic effects of these substances in differentbiological systems (e.g. Refs. 42-47). The biochemical featuresof neuroblastoma cells (high ferritin content, endogenous H2O2production) seem to render them especially vulnerable by AA.

Experiments with 6-OHDA, H2O2, and AA in a cell-freesystem using isolated CGC PM2 DNA showed that 6-OHDA

and AA induced strand breaks, whereas H2O2 was effective onlyin the presence of AA. In agreement with other reports, DNAstrand break formation is apparently caused by iron-dependentformation of OH radicals (48). Analogous processes may occurwithin neuroblastoma cells. These cells contain iron-rich ferritin which is dispersed within the whole cell areas (13). Therefore, iron release by substances such as AA and 6-OHDA shouldbe feasible, in principle, on multiple regions. We supposed thatAA could inflict damage upon these iron-rich cells in thepresence of H2O2, which could be delivered by appropriatedrugs (e.g. 6-OHDA) or formed endogenously in criticalamounts under certain conditions (see below). The ability ofneuroblastoma cells to accumulate AA would be a presupposition for this concept. SK-N-SH cells indeed incorporate iteffectively (Fig. 2). The range of AA in normal serum is about50-150 /Â¿Mbut is often markedly reduced in cancer patients

(49). Uptake of large amounts of AA can enhance serum levels2-3-fold (49). Under cell culture conditions, these amounts leadto an intracellular AA concentration in neuroblastoma cells inthe millimolar range. Calculated from the uptake experimentshown in Fig. 2, the intracellular ascorbate concentration inSK-N-SH cells raised from 0.45 mM after incubation with 50/Â¿MAA to 1.14 mM after 30 min incubation with 200 juMAA.The basal levels in SK-N-SH cells are negligibly low since theculture media commonly used do not contain this vitamin.

The present study showed that 6-OHDA and H2O2 causedsimilar toxic effects in a concentration-dependent manner onDNA and the metabolic pathways related to DNA strand break

25 250

25

25

Fig. 6. Decrease of NAD* concentration in DFO- and FeSCVpretreated SK-N-SH cells by H2O2 and 6-OHDA in the absence (â€¢)and presence (â€¢)of 1 mMascorbic acid, expressed as percentage of controls (set as 100%; n = 4); singleexperiments for 25 ^M and 100 ^M; two experiments for 250 ^M. Bars, SEM.

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ASCORBATE. FERRITIN, AND REACTIVE OXYGEN COMPOUNDS IN NEUROBLASTOMA

25C

Fig. 7. Decrease of ATP concentration in DFO- and FeSCVpretreated SK-N-SH cells by H2O2 and 6-OHDA in the absence (â€¢)and presence (â€¢)of 1 mMascorbic acid, expressed as percentage of controls (set as 100%; n = 4). Singleexperiments for 25 MMand 100 ^M; two experiments for 250 MM.Bars, SEM.

formation [activation of poly(ADP-ribose) polymerase and decline of NAD+ and ATP], Under the experimental conditions

described, cells with high ferritin content proved to be morevulnerable to 6-OHDA and H2O2, especially in the intermediateconcentration range (100 fi\t). These results are in agreementwith published reports indicating that iron also enhanced theDNA strand break rates caused by H2O2 in other cells (31).The enhancing effect of elevated cellular iron content on oxi-dative damaging processes is not restricted on DNA; e.g., therate of lipid peroxidation in different brain regions was foundto correlate closely with their respective ferritin contents (50).

AA constantly enhanced the effects of 6-OHDA and H2O:on DNA strand breaks and the related processes in iron- aswell as in DFO-pretreated cells. These effects are of specialinterest due to their potential therapeutic perspective. In thefollowing model an interpretation of the ways AA may interactwith iron and H2O2 in neuroblastoma cells is presented. Inthese cells, endogeneous formation of H2O2 is feasible by different pathways. First, H2O2 is produced during the autoxida-tion of AA and this process is thought to be one reason for itscytotoxicity (17). However, other effects being specific forneuroblastoma cells may play a more important role; AA enhances the biosynthesis of catecholamines in normal sympathetic cells (51, 52) as well as in neuroblastoma cells (53). Innormal sympathetic cells catecholamines are stored in vesicleswhich protect them from oxidation. The important role ofstorage vesicles for preventing H2O2 formation by catecholamines was recently shown. The production of H2O2 was sub-

stanially increased in striata! synaptosomes incubated with do-pamine when this reaction was performed in the presence ofreserpine, an inhibitor of catecholamine uptake into storagevesicles (54). It is therefore most important to point to the factthat neuroblastoma cells contain only a very low number ofvesicles. Consequently, enhanced formation of H2O2 by themonoamine oxidase reaction and by catecholamine autoxida-tion processes occurring in the cytoplasm can be expected.

The cytotoxic effects of H2O2 may be facilitated in neuroblastoma cells by several mechanisms. First, like many othertumor cells, neuroblastoma cells are characterized by low levelsof defense enzymes against H2O2 [catatase, GSH peroxidase(55)], thus decelerating its destruction. These data are in agreement with the observed long half-life time of H2O2 in SK-N-SH cells (8-9 min when 250 MMH2O2 was added to 1.5 x 10"

cells). Additional factors may further impair the effects of bothenzymes. Most neuroblastoma cells lack cystathionase whichnormally converts cystathionine to homoserine and cysteine.Elevated amounts of cystathionine will be found in urine inabout 50-80% of neuroblastoma patients (56). Consequently,only a reduced intracellular cysteine production occurs andcould limit GSH supply necessary for peroxidase activity andother pathways of the cellular defense system (57). Furthermore, AA is known being a catatase inhibitor in vitro (58). Thismight also be true in vivo. Finally, an important aspect ofascorbic acid toxicity is probably its capacity to release ironfrom ferritin as shown in the cell-free system (27). Althoughnot yet proved experimentally, this reaction is assumed to occuralso intracellularely and should facilitate the formation of OHradicals by the Fenton reaction. Thus, in neuroblastoma severalbiochemical processes influenced by AA could act in concertand be responsible for the H2O2-mediated destructive processesdescribed.

In order to support this interpretation, it is worth mentioningthat a very similar biochemical pattern is believed to be responsible for tissue destruction in Parkinson disease. According tothe "endogenous toxin hypothesis" (59, 60), an elevated dopa-

mine production occurs in some areas of the substantia nigrawhich may compensate for loss of dopamine production inalready destroyed tissue compartments. This leads to an enhanced intracellular H2O: formation due to an enhanced dopamine turnover by monoamine oxidase. Since the substantianigra in this disease is characterized by an enhanced iron level(61) and reduced GSH, catatase, and GSH peroxidase activity(62), oxidative damaging processes are favored. Indeed, lipidperoxidation products can be detected in the substantia nigraof Parkinson's patients (63). Similar pathways may play a role

in neuroblastoma cells in the presence of ascorbic acid.Neuroblastoma cells, like other tumor cells, are in a some

what unstable metabolic condition. Permanent high dose application of AA may continually irritate neuroblastoma cells byforcing the described biochemical processes and finally allow abreak in this fragile metabolic balance in those cells that hadsurvived a previous therapy. Therefore, application of highdosages of AA for an unlimited period of time after the end ofthe conventional therapy may be a mild, beneficial, and easilyperformable attempt to prevent relapses in neuroblastoma.

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ASCORBATE. FERRITIN, AND REACTIVE OXYGEN COMPOUNDS IN NEUROBLASTOMA

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1991;51:6066-6072. Cancer Res Gernot Bruchelt, Ingrid U. Schraufstätter, Dietrich Niethammer, et al. Processes in the Neuroblastoma Cell Line SK-N-SH

on Iron-dependent DNA Strand Breaks and Related2O2HAscorbic Acid Enhances the Effects of 6-Hydroxydopamine and

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