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[CANCER RESEARCH 45, 5257-5262, November 1985]
Modulation of Cytotoxicity of Menadione Sodium Bisulfite versus LeukemiaL1210 by the Acid-soluble Thiol Pool1
Division of Medical Oncology, UCLA School of Medicine, Harbor-UCLA Medical Center, Terranee, California 90509
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 M,caused time- and concentration-dependent depletion of the acid-soluble thiol (GSH) pool. Maximal GSH
depletion to 15% of control occurred at 45 /Mvitamin 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 M)
elevated both the GSH pool and the total glutathione pool, thelatter to a greater degree. Vitamin Ka also caused an increasedrate of Superoxide aningeneration 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 /Mvitamin 3and 100% occurredat 36 nu vitamin Ka; 27 /tw vitamin 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 10 (SD), 60 10, 60 11, and 80 12% of control, respectively. In contrast, GSHdepletion may not be a common mechanism of toxicity. Of thesequiones,only vitamin K3caused significant GSH depletion whenstudied in equitoxic concentrations.
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
18.104.22.168, from bovine blood), glutathione reductase (EC 22.214.171.124), 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 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 (56Cfor 30 min)
fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ng/m\),and i -glutamina (2 mw) at 37C 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 37C 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-
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 4C. 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 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 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 reduc-ase (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 c