modulation of cytotoxicity of menadione sodium bisulfite...

[CANCER RESEARCH 45, 5257-5262, November 1985]

Modulation of Cytotoxicity of Menadione Sodium Bisulfite versus LeukemiaL1210 by the Acid-soluble Thiol Pool1

StevenA.Akman,2MarianDietrich,RowanChlebowski,PhyllisLimberg,andJeromeB.Block

Division of Medical Oncology, UCLA School of Medicine, Harbor-UCLA Medical Center, Terranee, California 90509

ABSTRACT

We investigated the mechanism of antitumor activity of thewater-soluble derivative of menadione, menadione sodium bisul

fite (vitamin KJ, versus murine leukemia L1210. Vitamin Ka, inconcentrations >27 Â¿Â¿M,caused time- and concentration-dependent depletion of the acid-soluble thiol (GSH) pool. Maximal GSH

depletion to 15% of control occurred at 45 /Â¿Mvitamin Â«3.VitaminKa-mediated GSH depletion and vitamin Ks-mediated growth

inhibition were abrogated by coincubation with 1 mw cysteine or1 mw reduced glutathione but not by 1 mw ascorbic acid or 180fiM a-tocopherol. Low concentrations of vitamin Ka (9-27 Â¿Â¿M)

elevated both the GSH pool and the total glutathione pool, thelatter to a greater degree. Vitamin Ka also caused an increasedrate of Superoxide aniÃ³ngeneration by L1210, maximal at 45 I*Mvitamin Ka (300% of control), and a concentration-dependent

depletion of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) and total nicotinamide adenine dinucleotide phosphate (NADP) pools. Forty-fifty % depletion of the NADPH pool

occurred after exposure to 27 /Â¿Mvitamin Â«3and 100% occurredat 36 nu vitamin Ka; 27 /tw vitamin Â«3is a nontoxic concentrationof vitamin Ka. Loss of NADPH and total NADP was preventedby coincubation with 1 mw cysteine but not by coincubation withascorbic acid or a-tocopherol. We conclude that tumor cellgrowth inhibition by vitamin Kais modulated by acid-soluble thiols

and may be caused by GSH pool and/or NADPH depletion.Toleration of partial NADPH depletion by L1210 cells may indicate that a threshold level of NADPH loss of >50% is necessaryfor toxicity. NADPH depletion may be a toxic effect common toquinone drugs. Equitoxic concentrations of vitamin K;1, phyllo-

quinone, lapacho!, dichlorolapachol, and doxorubicin causedL1210 NADPH pools to deplete to 30 Â±10 (SD), 60 Â±10, 60 Â±11, and 80 Â± 12% of control, respectively. In contrast, GSHdepletion may not be a common mechanism of toxicity. Of thesequiÃ±ones,only vitamin K3caused significant GSH depletion whenstudied in equitoxic concentrations.

INTRODUCTION

The naphthoquinone derivative menadione (2-methyl-1,4-

naphthoquinone) has been shown to inhibit the growth of tumorcells in vitro (1-6). In the human tumor stem cell soft agar cloning

assay (7), menadione causes inhibition of clonal growth of a widevariety of tumor cell types (3). Its anticancer activity with thisassay is comparable or superior to currently used standardchemotherapeutic agents, such as doxorubicin. Menadione is inuse in early trials in patients with advanced cancer (2) with theantimetabolite 5-fluorouracil. The mechanism of tumor cell

1Supported in part by a grant from the California Institute For Cancer Research.2To whom requests for reprints should be addressed.

Received 9/4/84; revised 7/24/85; accepted 7/26/85.

growth inhibition by menadione is not known. Due to its quinonestructure, menadione participates as a powerful oxidation-reduction agent in oxygen-consuming electron transfer reactions catalyzed by microsomal enzymes (8-12). Menadione semiquinone

free radical is generated during reduction of the quinone (9,10);this radical is short lived, producing Superoxide radicals (9, 10,12, 13). The consequences of menadione reduction have beeninvoked as the cause(s) of menadione toxicity in normal hepa-tocytes (8, 13) and neutrophils (14), and menadione is thoughtto be responsible for: (a) depletion of NAD(P)H with subsequentdepletion of mitochondrial ATP (15); (b) Superoxide and peroxyradical generation, causing peroxidative damage (13, 16, 17);and (c) depletion of cellular GSH3 (13, 14). The data presented

in this paper suggest that menadione potently depletes tumorcell GSH and NADP pools and that acid-soluble thiols are impor

tant modulators of menadione antitumor activity.

MATERIALS AND METHODS

Chemicals and Reagents. L-Cysteine, vitamin K3, ascorbic acid,NADPH, NADP+, reduced glutathione, Superoxide dismutase (EC

1.15.1.1, from bovine blood), glutathione reductase (EC 1.6.4.2), and allreagents for the assay for pyridine nucleotides were purchased fromSigma Chemical Co. (St. Louis, MO). a-Tocopherol:polyethylene glycol

100:succinate was purchased from Eastman Chemical Co. (Kingsport,TN). This derivative of a-tocopherol is soluble in boiling water at concen

trations Â«50 mW. DTNB was purchased from K and K Laboratories(Hollywood, CA). Lapachol [1,4-naphthoquinone-2-hydroxy-3-(methyl-2-butenyl), NSC 11905] and dichlorolapachol [2-hydroxy-3-(3,3-dichlo-roallylH ,4-naphthoquinone, NSC 125771] were received as gifts from

the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment,National Cancer Institute, Bethesda, MD. Doxorubicin was obtained fromAdria Laboratories (Columbus, OH). Phylloquinone (Aqua Mephyton) wasobtained from Merck, Sharpe, and Dohme (Westpoint, PA). All chemicalswere used as received.

Cell Culture. Leukemia L1210, a murine leukemia maintained in longterm liquid suspension culture, was used in all experiments. L1210 cellswere maintained in 15-ml tissue culture flasks (Falcon Plastics, Oxnard,

CA) at an initial concentration of 100,000 cells/ml using Roswell ParkMemorial Institute Medium 1640 (Grand Island Biological Co., GrandIsland, NY) supplemented with 15% heat-inactivated (56Â°Cfor 30 min)

fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ng/m\),and i -glutamina (2 mw) at 37Â°C in an atmosphere containing 5% CO2

and 95% air. Cultures were fed twice weekly and were in logarithmicgrowth phase at the time of all experimental studies.

Growth inhibition by the drugs reported in these studies were observedby the dose-response method described previously (18). Briefly experiments were carried out in 15-ml tissue culture flasks. Supplemented

Roswell Park Memorial Institute Medium 1640, drug(s), and cells inlogarithmic growth were added to the flasks and incubated at 37Â°C in

'The abbreviations used are: GSH, total-acid soluble thiols, reduced form;

vitamin K3. menadione sodium bisulfite; GSSG, total acid soluble thiols, oxidizedform; DTNB, 5,5'-dithiobis-<2-nitrobenzoic acid); SOD, Superoxide dismutase (EC

1.15.1.1).

CANCER RESEARCH VOL. 45 NOVEMBER 1985

5257

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MODULATION OF REDUCED GLUTATHIONE BY VITAMIN

an atmosphere of 5% CO2 with 95% air. Cells were added at an initialconcentration of 100,000 cells/ml. Drugs were diluted in Dulbecco's

phosphate-buffered saline (Grand Island Biological Co.) (19). Each ex

perimental point was derived from triplicate cultures. After 16 h ofincubation, cells were washed free of drug with two washes of Ros wellPark Memorial Institute Medium 1640 and incubated in fresh supplemented media. Cells were fed with new media on day 4. Cell countswere performed on days 4 and 7 on a Model ZBI Coulter Counter(Coulter Electronics, Hiateah, FL).

Acid-soluble Thiols. Acid-soluble thiol pools were determined for

aliquot s of 10 to 50,000,000 cells by modification of the method ofBeutter ef al. (20). Cells were cooled on tee for 5 min and harvested bycentrifugation at 280 x g at 4Â°C. After removal of aliquots for protein

assay by the method of Bradford (21), the resultant cell pellets werelayered onto 600 n\ of 0.14 M m-phosphoric acid containing 4.6 HIM

EDTA and 3.47 M sodium chloride, overlaid with 500 Â¿ilof silicone fluidDC550 (Dow Chemical, Midland, Ml):light mineral oil, 84:16, in 1.5-ml

Eppendorf centrifuge tubes. Tubes were centrifuged at 15,000 x g for10 s in a microcentrifuge (Fisher Scientific, Tustin, CA), allowing 100%of the cells to migrate into the m-phosphoric acid layer, with less than

2% contamination by supernatant (22, 23). Supernatant and oil layerswere removed and cell pellets were dispersed in the m-phosphoric acid

solution by gentle homogenization with a precooled glass rod. Paniculatedebris was removed by centntugation at 15,000 x g for 10 min. Aliquots(500 /il) of supernatant were mixed with 1.5 ml of a reaction mixturecontaining DTNB (Ellman's reagent; 0.05 mg/ml) in 0.25% sodium citrate

and 0.3 M dibasic sodium phosphate. The absorbance of the reactionmix at 412 nm was determined on a Model 25 double beam spectropho-

tometer (Beckman Instruments, Fullerton, CA).Total glutathkxie content of 10 to 20 million log phase growth cells

were determined by a modification of the method of Akerboom and Sies(24). Cell suspensions were placed in Eppendorf centrifuge tubes containing 500 n\ of ice-cold 0.6 N perchloric acid overlaid with 500 Â¡Aof

DC550 silicone fluid:mineral oil (84:16) and centrifuged at 15,000 x g for15 s, after which the oil layers were removed. The acid extracts wereneutralized with 1 N KOH in 0.3 M A/-morpholinopropanesulfonic acidbuffer. KCIO4 precipitates were removed by centrifugation and 50-^1

aliquots of the supernatant s were placed in cuvets containing 1 ml of 0.1M potassium phosphate buffer (pH 7.0), 0.7 UM NADPH, 20 IM DTNB(Ellman's reagent), and 0.2 unit of glutathione reducÃase (Sigma type 3,

from yeast). Loss of NADPH fluorescence in the cuvets (excitationwavelength, 360 nm; emission wavelength, 460 nm) was recorded usingan Aminco-Bowman photofluorometer (Travenol, Inc., Elk Park, IL).

Glutathione content per cuvet was calibrated to the rate of NADPHdisappearance by addition of authentic GSSG standard to each cuvet.Protein content per cell suspension was assayed by the method ofBradford (21 ), using bovine serum albumin type V as standard.

Superoxide Generation. Superoxide generation by L1210 cells wereassayed by the method of Smith et al. (12). One million cells per assaywere suspended in 1.35 ml of Krebs-Henseleit buffer plus or minus SOD(0.02 mg/ml) and warmed at 37Â°C for 2 min. Fifty /J of cytochrome c

(Sigma type VI, from horse heart; 30 mg/ml) plus or minus 150 fit ofvarious concentrations of vitamin Â«3were added and the suspensionswere incubated for an additional 30 min at 37Â°C. After incubation,

suspensions were cooled on tee for 5 min, and 1-ml aliquots were layeredover 500 pi of a mixture of DC550 silicone fluid:mineral oil (84:16) in 1.5-

ml Eppendorf tubes and centrifuged at 15,000 x g for 10 s to remove100% of the cells. Absorbance at 550 nm of the supernatant was assayedimmediately. The absorbance due to Superoxide generation was calculated as the difference between the absorbance in the absence of SODand the absorbance in the presence of SOD. Concentration of Superoxidewas determined using the molar extinction coefficient Am = 21.0 x 10~3M"' cm-1 (25).

NADPH:NADP* Pools. Pyridine nucleotide pools were assayed by a

modification of the cycling spectrophotometric assay of Bemofsky andSwan (26). From 10 to 20 million cells per data point were harvested by

centrifugation at 280 x g for 10 min at 4Â°C. Cells were then washedthree times in ice-cold Dulbecco's phosphate-buffered saline (19) supple

mented with 10 mM glucose. After cells were washed, NADPH poolswere determined on cells which were extracted with 300 n\ of tee-cold0.5 N KOH in 50% ethanol, heated for 5 min at 90Â°C, recooled to 0Â°C,

and neutralized with 0.5 M triethanolamine in 0.4 M dibaste potassiumphosphate. NADPH in the neutralized alkaline extracts was converted toNADP* prior to assay by addition of 10 MM GSSG and 0.2 unit ofglutathione reductase (Sigma type III, from yeast) and incubation at 37Â°C

for 30 min. The extracts were then acidified with ice-cold 2 M m-

phosphoric acid, centrifuged at 15,000 x g for 5 min to remove protein,and reneutralized with 2 N KOH in 0.3 M N-morpholinopropanesulfonicacid. NADP4 pools were determined on cells extracted with 0.2 M m-

phosphoric acid. After centrifugation at 1500 x g and neutralization ofthe supematants with 1 N KOH in 0.15 M W-morpholinopropanesulfonicacid, NADP* content was determined by measuring the rate of increase

of absorbance of 570 nm light due to reduction of thiazolyi blue in thecycling system containing 300 //I of the cell extracts, 0.1 N Tris-HCI (pH7.8), 5 mM glucose 6-phosphate, 5 mM MgSO,, 0.8 mM phenazinemethylsulfate, 0.2 mM thiazolyi blue, and 0.3 unit glucose-6-phosphate

dehydrogenase (Sigma type IX, from yeast). Each assay was carried outat 25Â°C in cuvets containing 1.2 ml total volume. NADP* content wasdetermined by addition of authentic NADP* standard to each assay. This

assay was linear in the range 50-2000 pmol. The lower limit of sensitivitywas 10 pmol for acid-extracted samples and 100 pmol for alcoholicalkali-extracted samples.

RESULTS

Incubation with vitamin K3caused a time- and concentration-dependent depletion of the GSH pool (Charts 1 and 2). The GSHpool reached a minimum of 15% of control after 16 h of exposureto 72 fiM vitamin Â«3.The concentration-response relationship forGSH depletion was steep, with 16 h exposure to up to 27 *IMvitamin Â«3producing no depletion and >45 JIMvitamin Â«3producing maximal depletion of 85%. Low concentrations of vitaminKa(<27 /tM) actually enhanced the GSH pool, by as much as 2-fokj.

lo.o-i

Â« 8. OH0

a.o>E 6.0-

4. OH

coo

2.0-

H h8 I6

TIME (hr)

Chart 1. GSH pods were measured by the method of Beutler et al. (20) afterincubation with 72 pM vitamin K3 for the times shown. Proteins were assayed bythe method of Bradford (21). Points, mean (n = 2); oars, SO. The GSH pool ofcontrol cells was 9.9 Â±1.2 Â¿/g/mgprotein.


5258



MODULATION OF REDUCED GLUTATHIONE BY VITAMIN Â«3

0 3.6 9 18 27 36 45

[KJ] (/IMI

Chart 2. GSH pools (â€¢)were measured by the method of Beutler ef al. (20)after 16 h incubation with vitamin Â«3in the concentrations shown. Total glutathionepools (O) were determined by the methods of Akerboom and Sies (24). Proteinswere assayed by the method of Bradford (21). Points, means of the percentage ofcontrol (no vitamin Â«3)values (n = 6); oars, SD. The GSH pool of control cellsvaried from 12 to 20 n9 of GSH per mg protein. The total glutathione pool variedfrom 16 to 30 nmo! of glutathione per mg protein.

Low concentrations of vitamin Â«3also elevate the total glutathione pool (Chart 2). The percentage of elevation of the totalglutathione pool at 9-27 nÂ»vitamin K3 was greater than the

percentage of elevation of the GSH pod. Also the total glutathione pool remained at levels equivalent to those of control cellsafter exposure to higher concentrations of vitamin K3 (>36 /IM),despite depletion of the GSH pool.

L1210 cells readily repleted the GSH pool after a short term(1 h) incubation with vitamin K3. Within 3 h after removal from a1-h exposure to media containing 72 ^M vitamin K3, the GSHpool recovered to 90 Â± 5% (SD) of control (n = 3) from aminimum of 60 Â±4% of control. However, L1210 cells showedonly a limited capacity to replete the GSH pool after a prolonged(16-h) exposure to vitamin Kg. Five h after removal of cells froma 16-h exposure to medium containing 36 UM vitamin Â«3,GSH

levels recovered to only 30 Â±13% of control from 10 Â±2%. Nofurther recovery of GSH was observed beyond 5 h; the viabilityof the cell began to deteriorate at that point.

Vitamin K3 more readily depleted the L1210 GSH pool thandid equitoxic concentrations of some other quiÃ±ones.Exposureto 72 MMfor 1 h vitamin Â«3reduced the GSH pool to 20 Â±18%of control, whereas 53 /*M lapachol (110 Â± 9% of control),dichlorolapachol (90 Â±10% of control), and 37 UM doxorubicin(110 Â±14% of control), all equitoxic at the noted concentrationsto L1210 growing in suspension culture (data not shown), didnot reduce the GSH pool at all. The equitoxic concentration ofphylloquinone (670 MM)actually raised the GSH pool to 150 Â±21% of control. These data suggest that GSH depletion may bea more important component of the antitumor activity of vitaminK3 than are these other quiÃ±ones.

Coincubation with 1 rriM cysteine restored the vitamin K3-mediated depletion of the L1210 GSH pool (Table 1); coincuba-

tion with 1 DIM reduced glutathione partially restored the GSHpool. However, reduced glutathione was somewhat unstable inaerobic tissue culture medium with up to 43% oxidation ofglutathione occurring in 30 min in medium in the absence of cells,with or without the presence of vitamin Kg. Coincubation with

Table 1Effect of reducing agents on vitamin Ka-mediateddepletion of the LI 210

acid-soluble thtol poolGSH pools were measured after a 16-h exposure to vitamin K3 Â±reducing

agents by the method of Beutler er al. (20). Proteins were assayed by the methodof Bradford (21). Values shown are representative of at least three experiments.The interexperimentcoefficient of variation is <12% for the first five and the ninthand tenth data points listed and <36% for the sixth through the eighth data points.

Vitamin KaReducingagent (45Â»IM)NoneAscorbic

acid (1.2rriM)a-Tocopherol(0.2 mm)-Reduced

glutathione (1 HIM)-Cysteine(1mu)None

+Ascorbicacid+o-Tocopherol

+Cysteine+Reduced

glutathione +GSH

poolGig/mgprotein)811(1

OOf13(120)13(120)10(90)20(180)4(30)3(20)5(50)23

(200)8(70)

* Units are calibrated to Â¿igof a reduced glutathione standard which produces

an equivalent reduction of DTNB." Numbers in parentheses,percentageof control.

- IOO-

80-

ou

60-

40-

20-

0 3.6

[K.]

Chart 3. Growth curves were generated by placing cells in fresh medium aftera 16-h exposure to the appropriate vitamin K3concentrations (see "Materials andMethods"). Points, mean of the percentageof control (no vitamin K3)cell counts at96 h (n = 2); oars, SD.

the nonthiol reducing agents ascorbic acid and a-tocopherol didnot prevent vitamin K3-mediated GSH depletion (Table 1).

Growth Inhibition. The effect of 16 h exposure to vitamin Â«3on L1210 growth in liquid suspension culture was evaluated.Exposure for 16 h was chosen to assure maximal depletion ofthe GSH pool. Vitamin Â«3inhibited L1210 growth in concentrations >36 nu (Chart 3). The dose concentration-response rela

tionship was quite steep with concentrations <36 A/Mhaving noeffect.

Coincubation of L1210 cells with vitamin K3plus 1 muÃcysteineor 1 HIM reduced glutathione abrogated vitamin remediatedgrowth inhibition (Table 2). However, nontoxic concentrations ofascorbic acid and a-tocopherol did not prevent vitamin Ka-medi

ated growth inhibition.Superoxide Generation. Ohnishi ef al. have shown that mi-

crosomes plus NAD(P)H may reduce vitamin K3 to a semiqui-

none, which has a short lifetime and may produce Superoxide(9,10). In some cells, Superoxide generation has been correlatedwith GSH depletion (27,28,29). In L1210 cells, vitamin Kgcausedalmost a 400% stimulation of Superoxide detected extracellularly


5259



MODULATION OF REDUCED GLUTATHIONE BY VITAMIN

Table 2Effect of reducing agents on vitamin K3-mediatedinhibition of L1210 growth

L1210 cells were grown in liquid suspensionculture in the continuous presenceof 36 MMvitamin K3Â±reducing agent (see "Materials and Methods'). Shown are

the means Â±SD of the percentage of control (i.e., no vitamin Ks exposure) cellcount at 96 h of a representativeexperiment. None of these reducing agents aloneaffected L1210 growth at all at the concentrations shown.

Reducing agent % of normal growth8

NoneAscorbic acid (0.7 mw)a-Tocopherol (0.2 mm)Cysteine (1 RIM)Glutathione(1 DIM)

10Â±110 Â±0.820 Â±2

110Â±10100Â±3

* Cell count of control cells at 96 h was 4.2 Â±0.4 million.

.Â£ I4.0-Q>

O

o.

e I2.0H

4(C

u.O

8.0H

6. OH

4. OH

Â« 2.0-<r

H 1 // 1 1 1 1 t-0 3.6 18 27 36 45 54 72

Chart 4. Superoxide generation by 1 to 5 millionL1210 cells in the presence ofvarying concentrations of vitamin K3(O) or vitamin K3+ 0.2 mw ,,-tocopherol (A)was assayed by the reduction of cytochrome c in the presence and absence ofSOD, by the method of Smith eÃal. (12). Points, means (n = 3); bars, SD.

(Chart 4). Superoxide production at 30 min was maximally stimulated by 45 UMvitamin K:tand did not further increase at highervitamin Ks concentrations (Chart 4). a-Tocopherol inhibited detectable Superoxide generated in the presence of 36 #jMvitaminKs by 43% (Chart 4). Meaningful measurements of Superoxideproduction in the presence of ascorbic acid or cysteine wereprecluded by the rapid reduction of ferricytochrome c by thesecompounds.

NADP+:NADPHPools. Incubation of L1210 cells with vitaminKs for 16 h caused a concentration-dependent depletion ofNADPH pool with significant depletion of NADPH and total NADPobserved after exposure to vitamin KSin concentrations as lowas 27 //M(Table3). From 40 to 50% depletion of NADPHoccurredin the presence of 27 UMvitamin K,, the concentration belowwhich growth inhibition and GSH depletion occurred (Charts 2and 3). Coincubation with 1 HIMcysteine prevented the depletion

Table 3Effect of vitamin K,-reducing agents on NADPH/NADP+pools in L1210 cells

From 10 to 20 millionL1210 cells per data point were incubated with the agentsshown for 16 h at 37Â°Cin a 5% CO2:95%air atmosphere, after which pyridine

nucleotides were measured by the thiazolyl blue cycling assay of Bemofsky andSwan (26). Proteins were assayed by the method of Bradford (21). Shown are themeans Â±SE of the combined data from 4-7 separate experiments.

nmol/mg protein

AgentNone

Vitamin K3VitaminK.JVitamin

K;,Vitamin K,Vitamin K3:cysteineVitamin K3:ascorbicacidVitamin K3:,,-tocopherolConcentration9

MM18MM27

MM36MM27/iM:l

mu27 I/M:1 rriM27 MM:0.2rriMNADP*0.2

Â±0.040.2 Â±0.040.2 Â±0.070.1 Â±0.04s

<0.010.2 Â±0.07

0.02 Â±0.01<0.01NADPH1

Â±0.21 Â±0.31 Â±0.4

0.6 Â±0.3*

<0.11 Â±0.2<0.1<0.1

' Significantlydifferent than control (P < 0.05 by paired r test).

of NADPH and total NADP caused by 27 MMvitamin KS;coincubation with 0.2 rriMa-tocopherol or 1 mw ascorbic acid did not(Table 3).

The ability of vitamin KSto deplete the L1210 cellular NADPHpool was compared to that of several other quiÃ±ones.Exposurefor 1 h to equitoxic concentrations of the naphthoquinonesvitamin Â«3(72 MM),lapacho! (53 MM),dichlorolapachol (53 MM),and phylloquinone (670 MM)diminished the NADPH pool to 30 Â±10, 60 Â±10, 60 Â±11, and 40 Â±9% of control (n = 3),respectively. The equitoxic concentration of the benzanthroqui-none doxorubicin (37 MM)lowered the NADPH pool to 80 Â±12%of control.

DISCUSSION

The GSH pool is an important determinant of tumor cell viability(30-32). Exposure of leukemia L1210 cells by vitamin Kscausesmarked alterations in the GSH pool; the concentration effectcurve has a Diphasicshape. Exposure to low concentrations (9-27 MM)of vitamin Ks is associated with up to 2-fold elevation ofthe GSH pool, while higher concentrations of vitamin KS(>36MM)cause marked depletion of the GSH pool. Total glutathioneis also increased by a low concentration of vitamin KSto a furtherdegree than is GSH and is unaffected by higher vitamin Ksconcentrations. These data suggest that vitamin Ks has twoeffects on thiol metabolism in L1210 cells: (a) to stimulate newglutathione synthesis; and (b) cause a shift in the GSH:GSSGratio favoring GSSG. At low concentrations of vitamin KS,newthiol synthesis predominates; thus both GSH and GSSG poolsare raised. At higher concentrations, thiol synthesis cannot keepup with rapid oxidation to GSSG; thus the GSH pool is depleted.

In L1210 leukemia, vitamin Ks depletes the GSH pool to asignificantly greater degree than do equitoxic concentrations ofits analogues phylloquinone, lapacho!, and dichlorolapachol orthe benzanthroquinone doxorubicin. Our data strongly suggestthat vitamin K3-mediateddepletion of the GSH pool is intimatelyassociated with its tumor cell growth-inhibitory activity: (a) theconcentrations of vitamin KS required to inhibit L1210 growthand to deplete the L1210 GSH pool are similar; (b) normal L1210growth in the presence of vitamin Â«3can be completely restoredby coincubation with thiols, such as cysteine or glutathione. It isunclear yet whether cysteine protects L1210 cells by serving asa substitute nucleophile or by stimulating new glutathione synthesis. However, other nucleophiles such as ascorbic acid and


5260



MODULATION OF REDUCED GLUTATHIONE BY VITAMIN K3

a-tocopherol neither prevented vitamin Ka-mediated GSH deple

tion nor protected L1210 cells from vitamin K3 toxicity.It is of interest that reduced glutathione protected the cells

from vitamin K3 toxicity in that, unlike cysteine, the tripeptideglutathione does not stimulate the L1210 GSH pool (Table 1).This suggests the possibility that vitamin Ka-mediated cytotox-

icity may occur at or near the cell surface, in proximity toextracellular glutathione. Morrison eÃal. have made a similarsuggestion in hepatocytes (33). Although we cannot entirelyexclude the possibility that glutathione inhibits entry of vitaminKa into the cell by covalently forming vitamin K3:glutathionethioether adducts, the fact that intracellular GSH pool depletionoccurs in the presence of the vitamin K3-reduced glutathione

combination (Table 1) suggests that the cell is subject to somevitamin Â«3effect, despite the presence of reduced glutathione.Further work on the site of vitamin Katoxicity is clearly necessary.

There are several mechanisms by which cellular metabolismof vitamin Â«3may cause a shift in the GSH:GSSG ratio favoringGSSG: (a) vitamin K:( semiquinone radical may reduce molecularoxygen, causing Superoxide and hydrogen peroxide formation(10, 12, 13, 16). Peroxide generation could consume reducedglutathione by the glutathione peroxidase reaction; (Ã³)reductionof vitamin KS utilizes NAD(P)H cofactor (15, 27), causing adepletion of the NAD(P)H pool. NADPH is also a cofactor forreduction of oxidized glutathione (28, 34); loss of NADPH couldimpair glutathione reduction; (c) vitamin Â«3may bind reducedglutathione covalently at position 3 via thio ether formation (16).Since the concentration of GSH in L1210 is much greater than100 /tw and we noted GSH depletion at vitamin Kaconcentrationsof 36 to 54 /Â¿M,it is stoichiometrically unlikely that covalentbinding is a major cause of vitamin K3-rnediated GSH depletion.

In L1210 leukemia, vitamin Ka causes rapid Superoxide generation, probably due to reaction of molecular oxygen with thevitamin K, semiquinone radical. The rate of detectable superox-

ide generation is maximal at 45 /UMvitamin Ka and does notincrease further at higher vitamin Ka concentrations, suggestingthat 45 fÃMvitamin K3 causes the reductase(s) of L1210 responsible for the reduction of vitamin Kato its semiquinone to catalyzeat Vâ„¢,.Therefore a rough estimate for KMof vitamin Ka-mediated

Superoxide generation by L1210 cells is 22 ^M, considerablyhigher than the reported KM value of vitamin KS of 2 pM forNADPH oxidation by liver microsomes (11). However, since only3% of Superoxide generated intracellularly is detected by reduction of cytochrome c extracellulariy (29), only limited inferencespertaining to the rate of Superoxide generation can be made.

The vitamin K3 concentration dependence of stimulation ofSuperoxide generation did not correlate well with the vitamin Kaconcentration dependence of GSH depletion, especially in thecrucial concentration range 18-45 ftw. The good correlation of

growth inhibition with GSH depletion and lack of correlation ofgrowth inhibition with Superoxide generation suggest that growthinhibition was related to the former rather than to the latter.However, the vitamin KS concentration dependence of GSSGgeneration, as estimated from the difference between the totalglutathione pool (expressed as percentage of control) and theGSH pool (also expressed as percentage of control; see Chart2), correlated well with the vitamin Ka concentration dependenceof the Superoxide generation rate. This suggests that oxidationof GSH was related to Superoxide production. GSH depletionwas not observed at the lower concentrations of vitamin Ka

which stimulated Superoxide generation (18-27 /Â¿M)because

enhanced new glutathione synthesis more than compensated fordepletion of GSH by oxidation. The ability of a-tocopherol toinhibit vitamin Ka-mediated intracellular Superoxide generation

without affecting GSH depletion is not consistent with this hypothesis and is as yet unexplained. The caution, however, thatthe extracellular assay system detects only a small fraction ofSuperoxide generated and that a number of transformation stepsoccur before intracellular Superoxide is detected extracellulariymust be reemphasized with respect to overinterpretation of thequantitative effect of a-tocopherol on Superoxide generation.

We observed that vitamin Ka caused partial depletion of theL1210 cellular NADPH and total NADP pools at concentrationsslightly lower than those which disturbed cell growth and theGSH pool. Depletion of total NADP suggests that exposure tovitamin Ka is associated with either inhibition of NADP synthesisor enhanced degradation, possibly as a consequence of vitaminKa-mediated damage to macromolecules (33). Reversal of vitamin Ka-mediated total NADP pool depletion by cysteine, which

also prevents vitamin K3 cytotoxicity, but not by other reducingagents which do not prevent vitamin Ka cytotoxicity is consistentwith this hypothesis. It must be emphasized that depletion of theL1210 cellular NADPH pool by vitamin K3>unlike the situation inhepatocytes (13), is not simply due to oxidation of NADPH in thepresence of vitamin Ka; altered NADP turnover must be involved,since both NADPH and NADP+ are depleted.

It is difficult to ascertain from these data whether vitamin Katoxicity versus leukemia L1210 was related to GSH depletion, orboth, since the vitamin KSconcentration dependence of growthinhibition, GSH depletion, and NADPH depletion were similar andall of the vitamin Â«3effects were abrogated by cysteine. If vitaminKa toxicity was related to NADPH depletion, there may be athreshold effect, since some depletion of NADPH was observedat a nontoxic vitamin KSconcentration (27 ^M).

The observation that equitoxic concentrations of every qui-

none studied caused significant depletion of the NADPH pool inL1210 cells, albeit to varying degrees, suggests that NADPHdepletion may be a common cytotoxic effect of the quinonedrugs. The relative contribution of NADPH depletion to doxorub-icin-mediated cytotoxicity is probably less than for the naphtho-

quinones, however, since a toxic concentration of doxorubicincaused only minimal NADPH pool depletion.

In contrast, only vitamin Â«3caused significant GSH pool depletion. It has been suggested that GSH depletion may be animportant mechanism of toxicity of other antitumor quiÃ±ones,such as doxorubicin (35-37). Doroshow ef al. (38) noted that

doxorubicin administration in vivo to mice moderately depletedhepatic and cardiac GSH pools. However, our data and those ofothers suggest that GSH depletion is not a general mechanismof toxicity of all antitumor quiÃ±ones.Patterson ef al. (39) observedGSH depletion in rat heart due to some antitumor benzanthro-quinones (e.g., daunorubicin), but not others (e.g., 1,4-bisÂ¡2-[(2-hydroxyethyl)amino]ethylamino|-9,10-anthracenedione). We also

noted a disparity among quiÃ±ones in their effect on the GSHpool; vitamin Ka caused GSH pool depletion, whereas lapacho!,dichlorolapachol, and phylloquinone did not. Also unlike its effecton mouse heart and liver, a toxic concentration of doxorubicindid not affect the GSH pool in cultured L1210 cells.

Since the GSH pool may be an important determinant ofresistance to some chemotherapeutic agents, e.g., certain alkyl-


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MODULATION OF REDUCED GLUTATHIONE BY VITAMIN Â«3

ating agents (40), as well as a mediator of antitumor activity, theidentification of agents which selectively deplete GSH has become an important research goal. Vitamin Ka, which is amongthe most potent GSH-depleting agents of the antitumor quiÃ±ones,may be a useful template for modeling of GSH-depleting

agents with improved therapeutic:toxic ratios.

ACKNOWLEDGMENTS

We would like to thank Dr. James L. Doroshow, City of Hope Medical Center,for helpful discussions and Christie Seltzer and Barbara Young for secretarialassistance.

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1985;45:5257-5262. Cancer Res Steven A. Akman, Marian Dietrich, Rowan Chlebowski, et al.

Leukemia L1210 by the Acid-soluble Thiol PoolversusModulation of Cytotoxicity of Menadione Sodium Bisulfite

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