[CANCER RESEARCH 47, 5994-5999, November 15, 1987]

Adriamycin Resistance in Human Tumor Cells Associated with Marked Alterationsin the Regulation of the Hexose Monophosphate Shunt and Its Response toOxidant StressGrace Chao Yeh,1-2 Sandra J. Occhipinti, Ken H. Cowan, Bruce A. Chabner, and Charles E. Myers

Clinical Pharmacology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892

ABSTRACT

We found that Adriamycin increased the pentose phosphate shuntactivity in both Adriamycin-sensitive (WT) and Adriamycin-resistant(ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and eumene hydroperoxide markedly stimulated pentose-shuntactivity in ADR" but only moderately increased the activity in WT cells.Furthermore, the altered oxidation-reduction regulation is associated withchanges intrinsic to the key enzymes of the pentose-shunt pathway,glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydro-genase and with glutathione peroxidase. We found the \'„„,values for

glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADR" than WT cellsand the K„of NADP* were 10-fold lower in ADR" than WT. Theactivity of glutathione reductase in ADR" is 42% of that in WT. In spite

of these changes, the response of the cells to both hydrogen peroxide andorganic peroxide is not limited by either the capacity of the pentose shuntor glutathione reductase, but is determined by the activity of glutathioneperoxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase inADR" may, however, seriously limit the activity of cytochrome P-450

reductase, a major enzyme of Adriantycin conversion to a free radical.

INTRODUCTION

Adriamycin, one of the anthracyciines, is widely used inchemotherapy against a wide range of human tumors. AlthoughAdriamycin and daunomycin are two of the most active anti-cancer drugs, there is no clear consensus as to the cytotoxicmechanism of these drugs. The drugs bind to DNA and cellmembranes, and these interactions have been proposed as abasis for cytotoxicity. On the other hand, both drugs undergocomplex oxidation and reduction reactions which yield a rangeof reactive products that are potentially cytotoxic. These includeSuperoxide, hydrogen peroxide, and hydroxyl radical, as well asdrug radicals and the drug quinone methide. Recently, Duro-si«>\v( 1) and Sinha (2, 3) have reported that the formation ofoxygen radicals from the drugs may be directly related to theircytotoxic effect on MCF-7 human tumor cells. Both Doroshowand Sinha (1-3) found that radical scavengers decreased thecytotoxicity of these drugs for MCF-7. These observationssuggest that free radical formation may play an important rolein the antitumor action of anthracyciines. In order to furthertest this hypothesis, we have selected and characterized anAdriamycin-resistant subline (ADRR) from the MCF-7 humanbreast cancer cell line (WT) (4, 5). The resistant cell line ADRRis approximately 200-fold more resistant to Adriamycin thanthe WT, and the resistance has been stable for over a year inthe absence of drug. The resistance is associated with multiplehomogeneously staining regions characteristic of gene amplifi-

Received 11/20/86; revised 5/28/87; accepted 8/13/87.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.

1To whom requests for reprints should be addressed, at Bldg. 10. Km 6N119,National Cancer Institute, NIH. Bethesda, MD 20892.

2Current affiliation: Department of Pharmacology, Uniformed Services, University of the Health Sciences, Bethesda, Maryland 20814.

cation. Over 50 different amplified DNA sequences have beenisolated from this resistant cell line (6) through the use of thein gel Si nuclease digestion technique of Roninson (7). Inaddition to these karyotypic and genetic changes, the ADRR

has exhibited biochemical changes which are consistent withdrug free radical formation as the cytotoxic event. In the WT,Adriamycin exposure results in abundant hydroxyl radical formation. In contrast, ADR" forms no detectable hydroxyl radi

cals, even at drug concentrations 10 times that lethal to thisresistant cell line (2, 3). This decreased hydroxyl radical formation is associated with a marked (45-fold) increase in glutathione transferase activity and a more modest increase in glutathione peroxidase activity. In our laboratory, Batist et al. (5)have purified the dominant glutathione transferase in ADRR to

homogeneity and have shown that it contains organic peroxidase activity. Since every mechanism by which Adriamycincauses hydroxyl radical formation requires the availability ofperoxide, the increased activity of this enzyme as well as thatof glutathione peroxidase may decrease hydroxyl radical formation through enhanced peroxide clearance.

Cytochrome P-450 reductase, a major enzyme in the conversion of Adriamycin to a free radical, is an NADPH-dependentflavin reductase. In addition, both glutathione transferase andglutathione peroxidase also require NADPH to keep glutathione in its reduced, active form. Thus, NADPH is important toboth drug-free radical formation and detoxification of the resultant reactive oxygen species. The formation of NADPH istightly regulated by the activity of the pentose phosphate shunt(Fig. 1), and alteration in the activity of this pathway is acommonly used index of cellular oxidant stress (8, 9). In addition, Henderson and coworkers have shown that Adriamycinfree-radical formation stimulates pentose phosphate shunt activity in RBC (10) and in the heart (11). Rush and Alberts (12)showed that the organic peroxide, terf-butylhydroperoxide,stimulated pentose phosphate shunt activity in freshly isolatedrat hepatocytes. For these reasons, we have examined theregulation of pentose-shunt activity in both Adriamycin-sensitive and -resistant cell lines. The results indicate marked alterations in the regulation of this pathway by exogenous peroxides.The oxidation-reduction response to peroxides was markedlyhigher in ADR" than in Adriamycin-sensitive WT. Furthermore, we found that the key enzyme of the pentose-shuntpathway, glucose-6-phosphate dehydrogenase, was significantlydecreased in ADRR.

MATERIALS AND METHODS

Materials. Hydrogen peroxide, eumene hydroperoxide, glucose-6-phosphate, 6-phosphogluconate, NADP*, NADPH, oxidized glutathi

one, flavin adenine dinucleotide, glutathione reductase, and glucose-6-phosphate dehydrogenase were purchased from Sigma. [l-MC]Glucoseand [6-14C]glucosewere purchased from Amersham Corp., and hyamine

was purchased from New England Nuclear.Cells. Human breast cancer cell line MCF-7 WT and an Adriamycin-

resistant MCF-7 clone, ADR", were plated at an initial concentration

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GLUCOSE

G-6-P

G6PD

•NADP<

site 2

6PGD

6PGD

-NADPH

O,

GSH

GSSG

H202

sitel Y GSH-PX

H20

RIBULOSE-5-P

RIBOSE-5-P

PRPP

Fig. 1. The pemose phosphate-shunt pathway coupled with glutathione cycle.5-P, S-phosphate; PRPP, 5'-phosphoribosyl-I-pyrophosphate; GSH-PX, gluta

thione peroxidase.

of 3 to 4 x IO5cells/25-cm2 plastic tissue culture flask and grown for

3 or 4 days in RPMI 1640 medium (Biofluids) supplemented with 10%fetal bovine serum and 2 m\i L-glutamine at 37*C in a humid atmosphere containing 5% CO... The ADR" cell line was maintained in

continuous exposure to 10 ¿iMAdriamycin for five passages, and thecells were subsequently passaged in drug-free medium at least 4 timesprior to experimental use. For the experiments, ADRR cells in the

fourth to 15th passage without drug were replicate plated. Flaskswithout cells but otherwise identically treated served as blanks.

Pentose Phosphate Activity. Pentose phosphate activity was assessedusing the oxidation of I u( labeled glucose by a method previouslydescribed (13). In brief, 25-cm2-flask monolayer cells were incubatedwith 2 ml of Earle's balanced salt solution. The concentration of glucosewas 2.5 mM, and 2 pCi of [l-MC]gIucose/assay were used. The flasks

were gassed with 95% air and 5% CO2 and sealed with a serum stoppercontaining a plastic center well. Following a I-h incubation, 0.3 ml of6 N sulfuric acid were injected into the incubation mixture to stop thereaction, and 0.3 ml of hyamine were placed within the center well.Evolved "CO.. was trapped in hyamine with an equilibration period of

45 min at room temperature. The radioactivity contained in hyaminewas measured by liquid scintillation spectrometry.

Enzyme Assays. Glucose-6-phosphate dehydrogenase and 6-phos-phogluconate dehydrogenase activities were assayed by spectrophoto-metric measurement of the rate of appearance of NADPH at 340 nm(14). The glutathione reducíaseactivity was measured with and withoutadded FAD1 by following the rate of NADPH decrease at 340 nm by

the spectrophotometric method (15). Protein determination was measured by the method of Lowry et al. (16).

Computer Simulations. The computer simulations of pentose-shuntresponse to oxidative stress were done using the Stella program (HighPerformance Systems, Inc., Manchester, VT) on a Macintosh Pluscomputer. NADPH was assumed to be a competitive inhibitor ofNADPH* utilization by G6PD. The equation used was that of typical

competitive inhibition.

»= V«,x |NADP*1/AT„x NADPH)/ATil + NADP*

1The abbreviations used are: FAD, flavin adenine dinucleotide; G6PD, glu-cose-6-phosphate dehydrogenase; GSH, reduced glutathione; 6PGD, 6-phospho-gluconate dehydrogenase; G6P, glucose-6-phosphate; GSSG, oxidized glutathione: GR. glutathione reducíase.

0.0

[Adriamycin] mMFig. 2. Effect of Adriamycin on the pentose-shunt activity in intact human

breast cancer cells. Monolayer cells of WT and ADR" at late log stage (Day 4)were incubated as described in "Materials and Methods." Two nCi of |1-UC]-

glucose, 2.5 HIMglucose, and Adriamycin at various concentrations were addedto the medium. The duration of incubation was 60 min. Reaction was stopped byaddition of 0.3 ml of 6 N I I..S(>.,into the flask. Evolved "CO¡was trapped inhyamine. The radioactivity contained in hyamine was measured by liquid scintillation spectrometry. Adriamycin at various concentrations was added to themedium, and the duration of incubation was 60 min. Values for WT (D) andADR* (*) cells are shown. Points, mean of at least three determinations; ears,

SE.

The sum of NADPH plus NADP* was set at 800 JIM,the concentration

measured in the two cell lines. The initial size of the NADPH andNADP* was set at 400 ^M each. The rate of oxidation was set at a

fixed value. The progress of the reaction was simulated through numerical integration using the Runge-Kutta method. When equilibriumwas attained, the concentrations of NADP+ and NADPH were re

corded. This process was repeated for the range of oxidation ratesshown in Fig. 6 with the enzyme kinetic properties set for first the wildtype and then the drug-resistant cell line.

RESULTS

Stimulation of the Pentose Phosphate Shunt by Adriamycin.We found the control levels of pentose-shunt activity in ADR"

and WT cells were 25.31 ±1.51 and 29.93 ±4.1 nmol/h/mgprotein (mean ±SE), respectively. These values for pentose-shunt activity are very high compared to those reported forother mammalian tumor cell lines. Breast tissue is known topossess higher pentose-shunt activity than most normal tissue,

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200

180

[ Hydrogen Peroxide ] mM

300

[Cumene Hydroperoxide] mMFig. 3. .I, effect of hydrogen peroxide on pentose-shunt activity in intact WT

and ADRR cells. The incubation conditions are as described in "Materials andMethods" and in the legend for Fig. 2. Hydrogen peroxide at various concentrations was added to the medium, and the duration of incubation was 60 min.Values for WT (D) and ADR" (*) cells are shown. Points, mean of at least threedeterminations; bars, SE. B, effect of eumene hydroperoxide on pentose-shuntactivity in intact WT and ADR" cells. Details as in A.

presumably to support the synthesis of milk fat (17). The keyenzyme of pentose phosphate shunt, G6PD, may be associatedwith intraductal proliferation. It has also been suggested thatmalignant epithelial cells exhibit a higher level of activity ofG6PD than do normal cells (18-22). The pentose shunt inMCF-7 may therefore merely reflect the properties of its tissueof origin and malignancy. In spite of the already elevatedactivity of the pentose shunt in both cell lines, Adriamycinaddition caused a marked enhancement of "CO, production

(Fig. 2). In addition, the relative responsiveness to Adriamycinin the two cell lines was nearly identical.

Stimulation of the Pentose Phosphate Shunt by Peroxides.Since the production of NADPH through the pentose phosphate shunt can be utilized for both activation of Adriamycinto a free radical and reduction of peroxides to less toxic products, we next examined the responsiveness of both cells toperoxide addition. We found that hydrogen peroxide (H2O2)increased the pentose-shunt activity in ADRR to a much greatermagnitude than in WT cells (Fig. 3A). In intact MCF-7 cells,incubated in Earle's balanced salt solution with glucose (2.5

HIM), the addition of H2O2 (1.0 mM) increased pentose-shuntactivity from 25.31 ±1.51 to 122.50 ±8.61 (mean ±SE) inADRR but only from 29.93 ±4.10 to 30.25 ±2.95 nmol/h/mgprotein in WT cells (Table 1). As with H2O2, eumene hydro-peroxide stimulated the shunt activity in ADRR to a greaterdegree than in WT (Fig. 3Ä). In ADRR the pentose shunt

increased from 25.31 ±1.51 to 151.77 ±5.59 nmol/h/mgprotein (mean ±SE) but only from 29.93 ±4.10 to 57.46 ±7.31 nmol/h/mg protein in WT cells (Table 1). The peroxide-mediated effect increased with increasing H2O2 or eumenehydroperoxide concentration (Fig. 3, A and B) and incubationtime (data not shown). When hydrogen peroxide and eumenehydroperoxide were added together at saturating concentrationin the incubation mixture, the shunt activity did not increasefurther in either ADRR or WT cells (Fig. 4). Both hydrogenperoxide and eumene hydroperoxide perturb the oxidation-reduction state of the pentose shunt through a common mechanism, the glutathione cycle, through the activity of glutathionetransferase and glutathione peroxidase. This results in rapidoxidation of intracellular GSH and NADPH and increasedNADP* which can be expected to stimulate the pentose shunt

(Fig. 1). Our results are thus consistent with the increasedactivities of glutathione transferase and glutathione peroxidasein ADRR, but do not rule out increased activity on the part of

the pentose phosphate shunt itself.Glucose-6-phosphate Dehydrogenase, 6-Phosphogluconate

Dehydrogenase, and Glutathione Reductase Activities in WT andADRR. The differential oxidation-reduction regulation of thepentose shunt in WT and ADRR cells prompted us to examine

the activities of the key enzymes in the pathway, G6PD and6PGD. We measured G6PD and 6PGD activities under Vmaxconditions in cell lysates and found that the G6PD and 6PGDactivities in ADRR were both lower than in WT; a 50-folddecrease in G6PD and 2-fold changes in 6PGD were observedin ADRR and WT cells, respectively. The Vmaxactivities ofG6PD in WT and ADRR are 625.89 ±40.66 and 14.8 ±1.86

nmol/min/mg protein (mean ±SE), respectively, and the6PGD Vmaxactivities are 28.24 ±1.021 and 13.1 ±0.116nmol/min/mg protein (mean ±SE) in WT and ADRR, respec

tively. We further examined the kinetic properties of G6PDand found that the Km for substrate G6P was identical (WT =0.074 HIM,ADR" = 0.076 mM). In contrast, ADRR has a lOxlower Km for NADP+ than WT: the Km values for NADP+ inWT and ADRR are 57 and 5.8 /¿M,respectively. The enzyme

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Table 1 Effect of peroxides on pentose-shunt activity in WT and ADR* cellsHuman breast tumor cells, WT and ADR", were incubated as described in the

legend for Fig. 2. The duration of incubation with [l-MC)glucose was 60 min. The

concentration of glucose was 2.5 m\i.

300

Incubated controlHydrogen peroxide (1.0 HIM)Cumene hydroperoxide (1.0 IHM)WT29.93

±4.10°

30.25 ±2.9557.46 ±7.31ADR"25.31

±1.51122.50±8.61151.77±5.59"

Mean ±SE of at least three determinations.

150

100

0.5 mM

0.5 mM HA

HA

-WT- -Adr"-

Fig. 4. Effect of hydrogen peroxide (H2O2) and eumene hydroperoxide onpentose-shunt activity in intact human breast cancer cells. The incubation conditions are as described in "Materials and Methods" and in the legend for Fig. 2.

The concentrations of hydrogen peroxide and eumene hydroperoxide were 0.5and 1.0 mM as indicated. The duration of incubation was 60 min. CONT, control.

which couples the glutathione cycle to the pentose shunt isglutathione reducíase. We examined the enzyme activity inboth cell lysates and found a moderate decrease in ADRR

compared to WT, 3.48 ±0.13 and 8.93 ±1.3 nmol/min/mgprotein, respectively. We measured glutathione reducíaseactivity with and without FAD, the coenzyme for the enzyme, andfound essentially similar activities in the presence and absenceof FAD in both cells. Thus availability of the flavin cofactordid not limit the activity of their enzyme in either cell.

These results are surprising in that, while ADRR exhibits

diminished G6PD and glutathione reducíaseactivily comparedlo WT, Ihe response of Ihis cell line lo peroxide challenge issignificanlly enhanced. This suggesls lhal Ihe changes seen inaclivily of these two enzymes do noi limit the capacily of ADRR

lo supply electrons for peroxide reduction. In order lo lesi thishypothesis, we have examined the response of both cell lines tométhylèneblue.

Stimulation of the Penlose Phosphate Shunt by MéthylèneBlue. Melhylene blue oxidizes GSH wilhoul Ihe need of per-oxidase (23) and Ihus provides a means of lesting the capacityof each line lo reduce GSSG wilhout the intermediacy ofglutathione peroxidase. Our results showed an equal responseof the pentose shunl lo melhylene blue in bolh cells (Fig. 5).This clearly shows lhal, in ADR", Ihe drop in Vmaxfor G6PD

does noi aller Ihe capacily of Ihis cell line lo respond looxidalive slress. Il is inleresling lo noie lhal, in bolh cell lines,ihe response of Ihe penlose shunl lo melhylene blue is considerably in excess of Ihe response lo eilher eumene hydroperoxideor hydrogen peroxide. Furthermore, while WT exhibils a pen-lose shunl response lo eumene hydroperoxide and hydrogenperoxide of less lhan 80 nmol/h/mg protein, this cell lineexhibited a maximal response to méthylèneblue of 200 nmol/h/mg protein. Thus ihe response of bolh cell lines, bul especially

o.o 0.4 O.5 O.6

[MéthylèneBlue] mMFig. 5. Effect of méthylèneblue on the pentose-shunt activity in intact human

breast cancer cells. The incubation conditions are as described in "Materials andMethods" and in the legend for Fig. 2. Méthylèneblue at various concentrations

was added to the medium, and the duration of incubation was 60 min. Values forWT (D) and ADR" (»)cells are shown. Points, mean of at least three determi

nations; bars, SE.

WT, lo peroxide is limited by ihe peroxidase aclivily, noi ihecapacily lo reduce GSSG.

DISCUSSION

Drug resislance in lumor cells involves Iwo biochemicalmechanisms: (a) decreased accumulalion of the drug (24, 25)and (b) changes in the enzymes which activate or deloxify Ihedrug (26-28). In our laboralory, Sinha et al. (2,3) reported lhalADRR cells have lower drug accumulalion and lower free-

radical formalion lhan WT cells. Batist et al. (5) further demonstrated thai Ihe free-radical deloxificalion enzymes, glutathi-one Iransferase and glulalhione peroxidase, were markedlyincreased al 45x and 12x higher, respeclively, in ADRR lhanWT. Our results suggested lhal ADR" may exhibil al leasl Iwo

independenl biochemical changes which enhance ils resislancelo Ihe cyloloxicily of Adriamycin: increase in glulalhione peroxidase aclivity (Site 1) and an altered responsiveness on Ihepart of Ihe penlose shunl (Site 2).

We found lhal G6PD, 6PGD, and GR acliviiies are decreasedin ADRR compared wilh WT; however, ihe response of Ihe

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pentose shunt to Adriamycin and méthylèneblue were similarin the two cell lines. Our findings suggested that, in both celllines, the capacity of the pentose shunt exceeds the demands ofthe glutathione cycle. There is evidence that strongly suggeststhat the activity of the pentose shunt is in excess in mostmammalian cells, (a) Under most conditions, feedback inhibition by NADPH results in only a small fraction of the totalcapacity of G6PD being utilized. Thus, in mammalian cells, thepentose-shunt activity is regulated by the ratio of [NADPH]/[NADP'J rather than the Vra,, activity of G6PD (24-31). (b) In

tumor cells, the G6PD activity is markedly elevated comparedto normal cells (18-22). (c) Even in G6PD-deficient subjects,the pentose-shunt activity is not altered compared with normalsexcept under severe oxidative stress (32).

We found the response of the pentose shunt to Adriamycinwas similar in the two cell lines. In spite of this similarity, theutilization of the NADPH produced by the pentose shunt mustbe quite different in the two cell lines. In the WT MCF-7 cells,the abundant production of hydroxyl radicals, the low glutathione peroxidase activity, and the lack of response of the pentoseshunt to peroxide addition all suggest that the pentose-shuntstimulation seen in this cell line following Adriamycin additionreflects utilization of NADPH for Adriamycin free-radical formation. In ADRR, the activity of those enzymes shown to reduceAdriamycin to a free radical, xanthine oxidase, cytochrome P-450 reducíase,and cytochrome ¿>5reducíase,is similar to thaiseen in ihe WT cells (4, 5). In this contexl, the absence ofdeteclable hydroxyl radical formation, increased glutathioneperoxidase activity, and enhanced responsiveness of the pentoseshunl lo peroxide addilion all suggest a significant proportionof ihe increase in pentose shunl following Adriamycin addilionand reflecl utili/ut ion of NADPH for peroxide detoxification.

Adriamycin free-radical formation in MCF-7 has been shownto be NADPH dependent (2,3). The major NADPH-dependentenzyme known lo reduce Adriamycin is cylochrome P-450reducíase.This enzyme and cylochrome P-450 ilself are appar-enlly extraordinarily sensitive to inhibilion by NADP+ (33-35).

Thus, il is theoretically possible for a cell to modulate cytochrome P-450 reducíaseactivity by increasing NADP+. There

is evidence thai this may indeed be significant. The activationof the carcinogen benzo(a)pyrene, a cytochrome P-450-media-

600

500

400

300

200

100

20 40 60 100

NADPH Oxidation, MM,min

Fig. 6. Computer simulation of the impact of the altered kinetic properties ofG6PD seen in ADR" on NADP* pool size at various rates of oxidation. See"Materials and Methods" for details of the computer simulation. Values for WT(•)and ADP" (D) cells are shown.

ted event, is much lower in G6PD-deficient erylhrocyles andlymphocytes compared lo normals (36). This activation is apparently not limited by NADPH availability, but rather theinhibition by NADP+ (34, 35). The total NADPH plus NADP+

pool in mosl cells is in excess of 500 MM,while for cytochromeP-450 reducíase,Ihe K, for NADP+ and the Km for NADPHare both below 10 MM. Because the G6PD in ADRR exhibitsboth an altered Vmaxand Km for NADP+, it is difficult to seeintuitiv elv how ihese changes would aller ihe NADPH/NADP*

ralio in a cell. In order lo further clarify ihis point, we havedeveloped a computer simulation of the pentose phosphateshunl using ihe kinetic properties of Ihe G6PD in each cell line.Fig. 6 shows Ihe effecl of a range of oxidalion rates on theNADP+ concentration for enzymes with the kinetic properties

of G6PD from the iwo cell lines. Il is apparenl lhat, as the rateof NADPH oxidalion increased, NADP+ would accumulale loa much greater extent in ADR". These results have led us topropose thai Ihe altered G6PD seen in ADR" is of advantagelo ADRR because, during Adriamycin-induced oxidalive stress,the accumulation of NAD!' leads to inactivation of cytochrome P-450 reducíase.We are currently studying the role ofNADP* as a regulator of Adriamycin free-radical formalion in

Ihis cell line.

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1987;47:5994-5999. Cancer Res   Grace Chao Yeh, Sandra J. Occhipinti, Ken H. Cowan, et al.   Monophosphate Shunt and Its Response to Oxidant StressMarked Alterations in the Regulation of the Hexose Adriamycin Resistance in Human Tumor Cells Associated with

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