[CANCER RESEARCH57, 1909-1914, May 15. 19971

ABSTRACT

MeIatOIIin, the chief hormone secreted by the pined gland, has beenpreviously shown to inhibit human breast cancer cell growth at the physio.

logical concentration of 1 ni@iin vitro. In this study, using the estrogenreceptor (ER)-positive human breast tumor cell line MCF-7, we have shownthat 10 @iML-buthionine-[S,R]-sulfoximine (L-BSO), an inhibiter of y-glutamylcysteine synthetase (the rate.limiting enzyme in glutathione synthesis),

blocks the oncostatic action of 1 nM melatOnin over a 5-day incubation,indicating that glutathione is required for melatonin action. The result wasrepented with ZR7S-1 cells, suggesting that the glutathione requirement is ageneral phenomenon among ER@breast cancer cells. Addition of exogenousglutathione (1 ,.LM)to L-BSO.treated groups restored the melatonin responsein both cell lines. Further demonstration ofthe importance ofglutathione wasshown using the ER breast tumor cell line HSS78T, which is normallyunresponsive to melatonin. Growth in this cell line was inhibited in thepresence of 1 pt@iethacrynic acid (an inhibitor of glutathione S.transferase)plus 1 nMmelatonin, and this effect was blocked with 10 @ML-BSO. We alsoobserved a steady decrease O1intraCeIIUIarglutathione in MCF-7 cells over a5-day incubation, suggesting that these cells metabolize glutathione differ.ently than do normal cells.

INTRODUCTION

Melatonin, the hormone produced and secreted by the pineal glandduring the night in virtually all mammalian species including man, exertsoncostatic effects on a variety of neoplasms, most notably breast cancer(1—3).Pharmacological levels of melatonin are effective in inhibiting thegrowth of mammary cancers in rodent models of either spontaneous (4,5), transplantable (6, 7), or carcinogen-induced breast cancer (8—14).Theenhancement of chemically induced mammaiy carcinogenesis by eitherpinealectomy or constant light in rats eliminates the nocturnal melatoninsurge (1, 9, 10, 13—16),suggesting that physiological levels of melatoninmay also be important in inhibiting breast cancer growth. Furthermore,the nocturnal amplitude of melatomn secretion is blunted in breast cancerpatients (17), including those with ER3-positive disease (18), suggestingthat a link exists between physiologically relevant nocturnal concentrations ofmelatonin and breast cancer growth. In support ofthis hypothesis,we previously demonstrated that melatonin in the physiological range (10

@Mto 1 nM) inhibits MCF-7 human breast cancer growth in vitro over a

5—7-dayincubation period (19—22).Our initial finding of a direct inhibitoly effect of melatonin, particularly at physiological concentrations, onMCF-7 cell proliferation in vitro has now been replicated in a number oflaboratories (23—31).Two research groups, however, reported a cytostaticor cytotoxic action of melatonin on MCF-7 cell growth only at pharmacological levels (32, 33), whereas one of the groups cited above subsequently failed to find an effect of melatonin on MCF-7 cell growth at anyconcentration over a 12-day culture period (34). These discrepanciescould be attributed to different clonal lines ofMCF-7 cells (35) as well as

Received 11/12/96; accepted 3/24/97.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by the Stephen C. Clark Research Fund.

2 To whom requests for reprints should be addressed, at Bassett Research Institute, 1

Atwell Road, Cooperstown, NY 13326-I 394.3 The abbreviations used are: ER, estrogen receptor; L-BSO, L-buthionine-[S,R]

sulfoximine; TGF, transforming growth factor; FBS, fetal bovine serum.

to different culture conditions, particularly the serum used (36, 37), whichare factors known to alter the responsiveness of these cells to hormonesand chemotherapeutic agents including estrogen and tamoxifen (38).

In recent years, significant progress has been made toward elucidatingthe cellular and molecular mechanisms by which physiological melatonininhibits MCF-7 cell growth in culture (3). For example, melatonin notonly delays the progression of MCF-7 cells from GdGI to S-phase of thecell cycle (22, 26), but it also modulates both constitutive and estrogeninduced growth factor activity in these cells (39) and inhibits the mitogenicactionsof estrogen(20),epidermalgrowthfactor(39),andprolactin (40). Results showing that the oncostatic action of melatonin on breastcancer cells seems to be restrictedto ER@cells (20) led to studiesshowing that physiological melatonin suppresses ER expression (41) viaan inhibition of the transcriptional regulation of ER mRNA in MCF-7cells (42). Recent studies in MCF-7 cells also show that melatoninmodulates the steady-state mRNA expression of a variety of other estrogen-regulated proteins such as p52 and the progesterone receptor (43).Furthermore, melatonin also modulates the expression ofTGFs in MCF-7cells such as TGF-a and TGF-j3 as well as some proto-oncogenes such as

c-myc and c-los (43).In spite of these important advances, no definitive signal transduction

pathway has been shown to convey the oncostatic message of melatoninto the intracellular processes controlling the proliferation of MCF-7 orany other cancer cell types. For example, although melatonin (i.e.,

2-['251]iodomelatonin) binding sites have been reported to exist in avariety of tissues, including melanoma (44—46)and carcinogen-inducedrat mammary tumors (47), little to no melatonin binding has been foundin MCF-7 cell membranes (45), suggesting that the recently cloned,membrane-bound, high-affinity melatonin receptors (48) are not involvedin the oncostatic action of melatonin in this cell line.

Increasing attention is now being devoted to the intracellular compartment, particularly the nucleus, as an important site for some of melatoiün'sactions in some cells and tissues (49, 50). This is based on severalconverging lines of evidence including the demonstration of the potentantioxidant and free-radical scavenging capacity of melatonin (51), and

its localization and binding in the nucleus (52) as well as its ability toserve as a ligand with the RZRIROR family of orphan nuclear receptors(53). There is currently no evidence, however, that such pathways mediate the oncostatic effects of melatonin on cancer cells.

In studies relating to its antioxidant properties (51), pharmacological doses of melatonin administered to female rats have been shown

to increase both the intramammary and intrahepatic levels of glutathione and glutathione S-transferase (54). Additionally, melatoninstimulates glutathione peroxidase activity in brain tissue (51). Theseresults suggest that glutathione, a tripeptide molecule that represents

the most prevalent nonprotein thiol synthesized by mammalian cells,may be involved in the mechanisms mediating some of the actions ofmelatonin. Glutathione plays a critical role in a metabolic pathway,using NADPH to provide the intracellular environment with a reducing milieu. Thus, it functions in reductive processes that are essential

for protein metabolism, DNA synthesis, and enzyme regulation aswell as drug and hormone metabolism by forming conjugates withthese compounds either spontaneously or through a mechanism catalyzed by glutathione S-transferase. Via its redox cycling, glutathioneis a potent antioxidant and thereby provides cells with a substantial

1909

Physiological Melatonin Inhibition of Human Breast Cancer Cell Growth in Vitro:

Evidence for a Glutathione-mediated Pathway'

David E. Blask,2 Sean T. Wilson, and Fred Zalatan

Bassett Research Institute, Cooperstown, New York 13326-1394

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GLUTATHIONEREQUIREMENTIN MELATONINONCOSTASIS

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degree of protection against oxidative stress, free radical damage, andother types of toxicitiy (55, 56). Additionally, through its complexmetabolism, glutathione has an important impact on the therapeuticresponsiveness or resistance of cancer cells to anticancer drugs. Forexample, suppression of intracellular glutathione synthesis with LBSO, an irreversible inhibitor of the rate-limiting enzyme -y-glutamylcysteine synthetase, can make cancer cells either more or less sensitive to the cytotoxic effects of antineoplastic agents (57).

On the basis of the evidence that pharmacological levels of melatoninincrease glutathione and glutathione-metabolizing enzymes in vivo (51,54)andthatL-BSO-inducedglutathionedepletionas wellasethacrynicacid inhibition of glutathione S-transferase alters the sensitivity of cancer

cells to anticancer drugs (57—59),we decided to test the hypothesis thatthe antiprohferative effect of physiological melatonin on MCF-7 cellgrowth in vitro is dependent on glutathione. We tested this postulate byexamining the effects of L-BSO-induced glutathione depletion on theresponsiveness of MCF-7 cells to physiological melatonin and supportedthese results by examining another ER@ human breast cancer line,ZR75-l. We also tested whether HS578T human breast cancer cells,which are ER and are not normally affected significantly by melatonin(20), could be made responsive to melatomn when glutathione metabolism is altered by the addition of ethacrynic acid.

MATERIALS AND METHODS

Materials. Melatonin (lot I l3Hl083), tamoxifen (lot l0lH0649), glutathione (lot 68F-0474), L-BSO, ethacrynic acid, 5,5'-dithio-bis(2-nitrobenzoicacid), glutathione reductase IV (from baker's yeast; lot 4lH8l954),

@-NADPH,5-sulfosalicytic acid, and human insulin were purchased formSigma Chemical Co. (St. Louis, MO). MCF-7 human breast cancer cells were

a generous gift from Dr. Steven Hill (Tulane University, New Orleans, LA).ZR7S-l and H5578T human breast cancer cells were purchased from American Type Culture Collection (Rockville, MD). DMEM was purchased from

Life Technologies, Inc. (Grand Island, NY). All media were supplementedwith 10% FBS (Tissue Culture Biological, Tulare, CA), 1% penicillin (10,000

units/ml), and streptomycin (10,000 @Wml;Life Technologies, Inc.).Cell Culture. MCF-7andZR75—lhumanbreastcancercells werecultured

and maintained in complete DMEM supplemented with 10% FBS in FalconT-l75 culture flasks (Becton Dickenson, Oxnard, CA) at 37°Cin a humidatmosphere containing 5% CO2 and 95% air as described previously (19, 20,

22). H5578T cells were grown under the same conditions except high-glucose

DMEM was used, and 10 @g/mlinsulin were added. Stock flasks of exponentially growing cells were randomly selected, and the growth medium was

removed followed by treatment with 0.2% EDTA in PBS (pH 7.3). After thecells had sloughed from the bottom of the flasks, 10 ml of complete mediumwere added, and the resulting cell suspension was removed and centrifuged at60 X g for 5 mm. After centrifugation, the supernatant was removed, and theremaining cell pellet was resuspended in 12 ml of complete medium, and a

1-ml sample was taken for hemocytometer counts. After the cell number wasdetermined, the cells were adjusted to 3 X l0@cells/mI with complete medium.One ml of this new suspension was added to each Falcon plastic cell culturedish (60 x 15 mm; Becton Dickenson; 4 dishes/treatment group) along withenough medium (3 ml) to facilitate cellular attachment. Four h after initialplating of the cells, the medium from each dish was removed and replaced by5 ml of fresh complete medium containing hormone and/or drugs at the desiredconcentrations. The cells were returned to the incubator and allowed to growfor various time periods ranging from hours to days.

For the growth studies, cells were harvested on various days of incubationby treatment with I ml of PBSIEDTA and passed 3 times through a 25-gauge

needle to obtain a single cell suspension. From this cellular suspension, a I-mIsample was taken for glutathione analysis (see below), and the remainder of thecells were fixed with 50 @.dof 25% glutaraldehyde for hemocytometer counts.Previous studies from our laboratory showed >95% cell viability with the

trypan blue exclusion test.Unless otherwise indicated, the incubations in most experiments were

carried out for a total of 5 days, without medium changes, in the continuous

presence ofeither physiological melatonin (1 nM),L-BSO (10 ELM),glutathione(1 ELM),or vehicle (0.0001% ethanol). In the time course study, cells wereincubated with either melatonin (1 riM),L-BSO (10 SM),melatonin + L-BSO,or vehicle for either 1, 2, 3, 4, or 5 days, and cell counts and glutathionemeasurements were performed at each of these time points. Tamoxifen was

used at a concentration of 1 @.tM.In the HSS78T experiment, ethacrynic acidwas used at a concentration of 1 @M.

Cellular Preparation and Analysis of Total (Reduced + Oxidized)Glutathione. From each culture dish a 1-ml cell suspension (see above) wasremoved and placed in a 1.5-mI tube. After the samples were centrifuged at

500 rpm for 5 mm, the medium was removed and replaced with 100 p3 of 10mM HC1 and 50 @lof 10% 5-sulfosalicytic acid. The cellular samples were

lysed by repeated freezing in a dry ice 2-propanol bath and thawing at room

temperature. Additional cellular preparation and glutathione analysis wereperformed according to the method of Anderson (60). The intracellular levelsof glutathione are expressed as nr@i/l06cells.

Statistical Analysis. Cell number and glutathione levels are expressed asthe mean ±SE. Differences among the means were determined with aone-way ANOVA followed by Student-Neuman-Keul's post hoc test. Differ

ences among various treatment groups were considered statistically significantat P < 0.05.

RESULTS

Changesin GlutathioneLevelsduringGrowthof MCF-7Cells.We measured intracellular glutathione levels in MCF-7 cells in vitroover a 5-day incubation period and found that there was a steadydecrease in glutathione (measured as the total of reduced + oxidizedglutathione) during this time (Fig. 1). From day I to day 3, there wasa 21 % decrease in glutathione concentration as the cells increased innumber by over 3-fold. From day 3 to day 5, glutathione levelsdecreased by another 35%, whereas the cell number almost doubled.The decrease in glutathione was not due to depletion of the media,because replenishing the culture dishes with fresh media during thetime course did not prevent this decrease (data not shown).

aC.)

10 ,E.aC

I

4

3

2

I

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20

15

0 1 2 3 4 5

Daysof Incubation

5

Fig. 1. Cell number and intracellular glutathione levels of MCF-7 cells during a 5-dayincubation period. Cells (initial plating density of 3 X l0@cells/dish) were incubated at3TC in a 5% CO2 atmosphere in 60 X 15-mm culture dishes containing S ml of DMEMand 10% FBS. The cell number plot (•)corresponds to the left y-axis, and glutathioneconcentration plot () corresponds to the right y-axis. Mean values (4 dishes/group) areshown ±SE. Similar results were obtained in two other experiments. a p < 0.05 versusday I and versus day 5.

1910

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BSO, an inhibitor of glutathione synthesis, on MCF-7 cell growth andglutathione concentration during the 5-day incubation period. Fig. 2aclearly demonstrates that the combination of L-BSO (10 p.M) andmelatonin blocked the melatonin effect on growth, whereas treatmentwith L-BSO alone had no effect on growth.

We also measured glutathione levels in these groups (Fig. 2b) todetermine the extent of the decrease in glutathione synthesis causedby this concentration of L-BSO, a concentration that was much lowerthan that used in other studies (56, 58). By day 2, the L-BSO grouphad a 40% decrease in glutathione concentration compared to that ofthe control group, although the growth rate was unchanged. Consistent with Fig. 1, glutathione levels decreased during growth for allgroups. The L-BSO effect on glutathione synthesis persisted throughout the incubation period because the relative difference in the levelsbetween the control group and the L-BSO group was maintainedthrough day 5. Similarly, the combination of L-BSO and melatoninmaintained an approximately 25% decrease in glutathione relative tothat of the control group throughout the incubation period.

Effects of the Inhibition of Glutathione Synthesis on theTamoxifen Response in MCF-7 Cells. To determinewhether thedepletion of glutathione by L-BSO specifically inhibited melatoninaction or inhibited oncostatic mechanisms in general, we assessed theinfluence of L-BSO on the oncostatic action of tamoxifen. Table Ishows that treatment of MCF-7 cells with tamoxifen (1 @.tM)over 5days resulted in a 49% decrease in the cell number. Exposure of cellsto the combination of L-BSO (10 @M)and tamoxifen had no effect onthe tamoxifen response. Intracellular glutathione was measured to

4 5 confirm its depletion by L-BSO. Glutathione levels were approxi

mately 50% lower in the L-BSO-treated groups.Effects of the Inhibition of Glutathione Synthesis on the Mela

tomn Response in ZR75-1 Cells. We decided to test whether theinhibition of glutathione synthesis by L-BSO negates the melatoninresponse in another ER@ human breast cancer cell line, ZR75- 1. Table2 shows the results from exposure of MCF-7 and ZR75-l cells tomelatonin (1 nM) for S days. Melatonin incubation with MCF-7 cellsresulted in a 65% decrease in cell number from controls, whereasmelatonin incubation with ZR7S- 1 cells, which grew at a much slowerrate than MCF-7 cells, resulted in a 3 1% decrease in growth fromcontrols. The treatment of either cell line with L-BSO (10 p.M) alonehad no effect on cell growth. When melatonin and L-BSO werecombined, MCF-7 cell growth, as expected, was equivalent to that ofeither control or L-BSO-treated cells. The L-BSO effect on the melatonin response was reproduced in ZR75- 1 cells, because treatmentwith L-BSO combined with melatonin not only negated the effect of

melatonin, but increased cell growth by 20% as compared to controls.Effects of Exogenous Glutathione on the Melatonin Response in

L-BSO-treated MCF.7 and ZR75-1 Cells. We also tested whetherthe addition of glutathione (1 p.M) to the L-BSO/melatonin groupcould restore the melatonin effect on cell growth. Table 2 shows that

Table I Effects of L-BSO on the zamoxifen response in MCF-7cellsMCF-7

cells (initial plating density of 3 X 10@cells/dish) were incubated for 5daysat37'C in a 5% CO2 atmosphere in 60 X 15-mm dishes containing 5 ml of DMEMand10%

FBS supplemented with vehicle (control), tamoxifen (I @sM),L-BSO (10 @sM).ortamoxifen+ L-BSO. Cell number and intracellular glutathione levels were then meas

ured. Mean values (4 dishes/group) are shown ±SE. Similar results were obtained infourotherexperiments.Treatment

Cell no. (day 5) Glutathione (nM/106cells)Control

3.17 ±.06 x 106 3.4 ±0.4Tamoxifen1.62 ±.07 X l0&1 5.7 ±0.6―L-BSO2.94 ±.08 x 106 1.7 ±0.2―Tamoxifen

+ L-BSO 1.59 ±.03 X 10@― 2.3 ±0.4―

a

GLUTAThIONE REQUIREMENT IN MELATONIN ONCOSTASIS

0 1 2 3 4 5

Days of incubation

0 1 2 3

Daysofincubation

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Fig. 2. Top, effect of melatonin and L-BSO on MCF-7 cell growth during a 5-dayincubation period. Cells (initial plating density of 3 X l0@cells/dish) were incubated at37°Cin a 5% CO2 atmosphere in 60 X 15-mm culture dishes containing 5 ml of DMEMand 10% FBS supplemented with vehicle (•),I flMmelatonm (), 10 @.&siL-BSO (A), ormelatonin + L-BSO (Y). Bottom, intracellular glutathione levels during the same 5-dayincubation period. Mean values (4 dishes/group) are shown ±SE. Similar results wereobtainedin twootherexperiments.a,p < ØØ5versusvehicleandversusmelatonin+ LBSO. b p .< 0.05 versus vehicle.

Effects of Melatonin on Growth and Glutathione Depletion inMCF-7Cells. We testedwhethertheoncostaticeffectof melatoninon MCF-7 cells slowed the rate of glutathione depletion. Previousstudies (19) have shown that the melatonin effect on cell growth isobserved at an optimal concentration of 1 ni@i(a physiological concentration). Thus a time course was done over a 5-day incubationperiod at this optimal melatonin concentration. Fig. 2a demonstratesthat by day 2 of incubation, a 35% reduction in cell number wasobserved in the melatomn-treated group compared to that of thecontrol group. By day 5, the cell number was 49% lower in themelatonin-treated group. This reduction in growth rate is comparable

to what has been reported previously (19, 20, 22).Differences in glutathione concentration between the control and

melatonin groups were evident at day 1. By day 2, glutathione levels(Fig. 2b) in the melatonin-treated cells were 29% higher than those incontrol cells, whereas at day 5, the levels were 52% higher. Anothertime course done from time 0 to 24 h (day 1) showed no significantincrease in glutathione in the melatonin group until the 24-h timepoint, which was about the time when a difference was observed incell number (data not shown). Although this increase in glutathioneconcentration in the melatonin-treated cells may have been due mostlyto a slower growth rate, it led us to speculate that glutathione metabolism may be important for the effect of melatonin on cell growth.

Effects of the Inhibition of Glutathione Synthesis on the Melatonin Response in MCF.7 Cells. In the experiment cited above (Fig.

2), we also determined the effects of melatonin combined with La p < 0.05 versus control.b@ < o.os versustamoxifen.

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Table 4 Effects of ethacrynic acid on the melatonin response in HS578T cellsHS578T cells (initial plating density of 3 X I0@cells/dish) were incubated for 5 days

at 37'C in a 5% CO2 in 60 x 15-mm culture dishes containing 5 ml of DMEM (highglucose) and 10% FBS and 10 @sg/mlinsulin and supplemented with vehicle (control),melatonin (1 flM),L-BSO (10 @tM),ethacrynic acid (1 gxM),melatonin + ethacrynic acid,L-BSO + ethacrynic acid, or melatonin + L-BSO and ethacrynic acid. Cell number andintracellular glutathione levels were then measured. Mean values (4 dishes/group) areshown ±SE. Similar results were obtained in two otherexperiments.Cell

no. GlutathioneTreatment (day 5) (nM/l06cells)Control

1.17 ±.15 x 106 8.8 ±0.9Melatonin 0.98 ±.04 X 106 111 ±0.7L-BSO 1.14 ±.06 X 106 4.6 ±0.2―Ethacrynic acid 1.18 ±.10 X 106 8.8 ±1.1Melatonin + ethacrynic acid 0.43 ±.04 X l0@― 29.2 ±3.0―L-BSO + ethacrynic acid 1.23 ±.08 X 106 4.5 ±0.2cMelatonin + L-BSO + ethacrynic acid 1.00 ±.05 X 106 7.1 ±0.3a

p < 0.05 versus control.bp < 0.05 versuscontrol, versusmelatonin, versusethacrynic acid, and versus

melatonin + L-BSO + ethacrynic acid.C p < 0.05 versus ethacrynic acid.

Table2 Effectsof L-BSOand exogenousglutathioneon themelazoninresponseinZR7S-1and MCF-7cellsZR75-

I and MCF-7 cells (initial plating density of 3 X l0@cells/dish) wereincubatedfor5 days at 37'C in a 5% CO2 atmosphere in 60 X 15-mm culture dishes containing5mlof DMEM and 10% FBS supplemented with vehicle (control), melatonin (InM),L-BSO

(10 @LM),glutathione (I sM), melatonin + L-BSO, or melatonin + L-BSOandglutathione.Cell number was then measured. Mean values (4 dishes/group)areshown

±SE. For MCF-7 cells, similar results were obtained in two other experiments.

ZR75-lcellno.Treatment(day 5)

Table 3 Effects of L-BSO and exogenous glutathione on intracellularglutathioneconcentrationin MCF-7cellsMCF-7

cells (initial plating density of 3 X l0@ cells/dish) were incubated for 5daysat

37'C in a 5% CO2 atmosphere in 60 X 15-mm culture dishes containing 5 ml ofDMEMand10% FBS supplemented with vehicle (control), melatonin (1 nM), L-BSO (10@xM),glutathione

(1 i@―O'melatonin + L-BSO, or melatonin + L-BSO andglutathione.Intracellularglutathione levels were then measured. Mean values (4 dishes/group)areshown

±SE. Similar results were obtained in one otherexperiment.GlutathioneTreatment

(nM/l06 cells)

13.1 ±1.945.8±7.4―1.7

±0.7―4.6±1.2―1.0±0.6―16.0±2.lc

GLUTATHIONE REQUIREMENT IN MELATONIN ONCOSTASIS

the exposure of cells to glutathione alone had no effect on the growthof either cell line. In contrast, when MCF-7 cells were coincubatedwith a combination of melatonin, L-BSO, and glutathione, cell growth

was reduced to a rate similar to that of the melatonin-treated cells. Thesame treatment on ZR75- 1 cells resulted in a 20% decrease in cell

number compared to that of the control group and a 29% decreasecompared to that of the L-BSO/melatonin group.

To further investigate the restoration of the melatonin response byglutathione in the presence of L-BSO, glutathione concentrations weremeasured in MCF-7 cells at day 5. Table 3 shows that treatment withglutathione alone resulted in intracellular glutathione levels that were65% lower than controls. Interestingly, in the group treated with glutathione combined with melatonin and L-BSO, a treatment that restored themelatonin response on growth, the level of glutathione was only restoredto that of the control group and was significantly lower than that of themelatonin group. Noteworthy, however, was the relative difference inglutathioneconcentrationin the glutathionelL-BSO/melatoningroupcompared to that ofthe L-BSO/melatonin group; this difference was evengreater than that seen between the control group and the melatonin group.

Effects of Ethacrynic Acid on the MelatOnin Response in HSS78TCells. Because the above evidence suggested that glutathione wasfundamentalto the oncostaticactionof melatonin,we decidedto testwhether cells that are normally much less responsive to melatonincould be made responsive if glutathione metabolism is altered. TheER breast tumor line HS578T was tested in a growth experimentusing ethacrynic acid (I @.LM),which inhibits glutathione S-transferase,an enzyme that conjugates glutathione. Treatment with melatonin (1nM) resulted in only a 16% decrease in cell number compared to the

control group (Table 4). The combination of melatonin and ethacrynic

acid, however, resulted in a decrease of 63% from the control group,whereas ethacrynic acid alone had no effect on cell growth.

We also tested whether L-BSO could negate the effect of ethacrynicacid plus melatonin. When L-BSO treatment (10 p.M) was combinedwith melatonin and ethacrynic acid, cell growth was restored to thelevel of melatonin treatment alone. Treatment with L-BSO alone orwith L-BSO plus ethacrynic acid had no effect on growth compared tothe control group.

We then measured glutathione levels at day 5, shown in Table 4.Interestingly, ethacrynic acid treatment alone had no effect on theselevels. Treatment with L-BSO alone or in combination withethacrynic acid reduced glutathione by 48% compared to the controlgroup. This indicates that L-BSO did indeed block glutathione synthesis in these cells.

DISCUSSION

Previous studies from our group as well as other investigators havedemonstrated that both physiological and pharmacological concentrations of melatonin inhibit the growth of ER@ human breast cancer celllines in either monolayer or suspension culture (19—31). Althoughphysiological levels of melatonin have been shown to regulate ER andprogesterone receptor expression as well as the expression of a number of estrogen-regulated oncogenes, proteins and growth factors inMCF-7 cells (41—43),the precise cellular and molecular mechanismof the oncostatic effect of melatonin on ER@ breast cancer cells hasremained elusive. The results of the present investigation show thatdepletion of glutathione with L-BSO in MCF-7 cells completelyblocks the antiproliferative effect of a physiological concentration ofmelatonin in vitro. The same result was observed with ZR75-1 cells,indicating that the glutathione requirement is not restricted only toMCF-7 cells but may be a more generalized phenomenon among ER@human breast cancer cell lines that are sensitive to melatonin.

The role of glutathione in melatonin oncostasis was further demonstrated when the L-BSO-induced blockade of melatonin action wasprevented by the addition of exogenous glutathione to both MCF-7 andZR75-l cultures. Although glutathione does not readily reenter most cells

due to its extracellular breakdown by ‘y-glutamyltranspeptidase(55, 56),our observation with exogenous glutathione suggests that enough glutathione escaped extracellular degradation and entered the cells to restorethe melatonin response. Alternatively, ‘y-glutamylcysteinesynthetasemay not have been completely inhibited by the low dose of L-BSO used,and thus enough enzyme activity may have persisted to resynthesizeglutathione from its constituent amino acids after its extracellular degra

MCF-7cellno.(day 5)

2.98 ±.07 x 1061.06 ±.03 x 106a

2.85±.06x 1062.92±.10x 1062.87±.09x 10661.40±.12x lO&

Control 0.80 ±.04 X 106Melatonin 0.55 ±.03 XL-BSO 0.79 ±.06 x 106Glutathione 0.86 ±.04 x 106Melatonin + L-BSO 0.96 ±.05 X l0@―Melatonin + L-BSO + glutathione 0.64 ±.04 X l0@―

ap

GLUTATHIONE REQUIREMENT IN MELATONIN ONCOSTASIS

dation. Interestingly, the addition ofexogenous glutathione alone resultedin intracellular glutathione levels that were significantly lower than thoseof the controls, possibly due to a negative feedback on glutathionesynthesis (55, 56). This result (Table 3), along with the measurement ofintracellular glutathione levels in a melatonin/L-BSO/glutathione-treatedgroup (a measurement that was not higher than that of the correspondingcontrol group), indicates that the absolute amount of glutathionerequiredfor the melatonin response may not be important. With the dynamic stateof glutathione synthesis observed in our studies, it is not surprising thatabsolute glutathione concentrations are not a good indication of whethera certain cellular growth response will be observed.

The requirement of glutathione in the melatonin response prompted usto examine whether this was a specific effect of melatonin or rather aneffect that was secondary to an inhibition of cell growth. Our findingssupport a specific requirement because L-BSO failed to block the antiproliferative effect of tamoxifen. This observation is consistent with thenotion that melatonin and tamoxifen inhibit MCF-7 cell proliferation byfundamentally different mechanisms, in spite of the fact that they sharesome of the same antiestrogemc capabilities (20, 22).

The decrease in glutathione concentration over time, independent of any treatment with L-BSO or melatonin, was a significantdiscovery. Given the earlier finding that glutathione levels increasesharply during the first 4 days of growth of 3T3 fibroblast cells invitro (61), it is quite likely that glutathione metabolism in breastcancer cells is much different from that in normal cells. Thedecrease in glutathione observed in our system is most likely dueto its conjugation to other compounds, possibly tied to a glutathione S-conjugate export pump (62). The dynamics of glutathionemetabolism reported in our study may have implications in cancercell resistance in vivo over a specific time period. In many cases,high glutathione levels are associated with cellular resistance totoxic agents (57). Conversely, high glutathione levels are sometimes associated with increased toxicity (57), which is consistentwith the glutathione requirement observed in our melatonin studies. Because glutathione levels may be changing during tumor

growth and metabolism in vivo, the timing of any glutathionerelated treatment could prove to be important. For example, previous in vitro studies from our laboratory have shown that theoncostatic action of melatonin is markedly reduced if MCF-7 cellsare not exposed to melatonin within 12 h after plating.4

The decrease of glutathione during growth also accounts for theelevated glutathione levels in the melatonin-treated cells. For example,the amount of glutathione in melatonin-treated cells during days 3—5ofincubation corresponds to the levels observed in control cells at the samedensity earlier in the growth curve. An initially elevated glutathionecontent in melatonin-treated cells on day 1 of culture, when cell densitywas identical to that of the control group, may have had a bearing on theapparent elevation of glutathione in melatonin-treated cells at the end ofthe culture period, when cell density was lower than that of the controls.Thus, melatonin-induced slowing of the growth rate seemed to blunt thenormal decline in glutathione to create the impression that melatoninitself had actually raised glutathione levels by the end of the culture

@od,when in fact, the actual rates of the glutathione decline are verysimilar between control and melatonin-treated cells. We have also ohserved similar elevated levels of glutathione in MCF-7 cells treated withother growth inhibitors at the end of S days of incubation.4

A striking result was obtained with HSS78T cells. These cells,which are ER, do not normally respond significantly to melatonin, butexposure to ethacrynic acid plus melatonin caused cell growth to bereduced to an extent similar to that typically observed in ER@ cells. This

finding again demonstrates that glutathione metabolism is somehow tiedto the oncostatic action of melatonin, because altering an enzyme activity(glutathione 5-transferase) central to this metabolism made these cellsresponsive to melatomn. The role of glutathione in this ER system wasfurther indicated by the blockage of the melatonin/ethaciynic acid effectwith L-BSO-induced glutathione depletion. These results may have potential clinical implications because ER breast tumors are usually resistant to standard treatments such as tamoxifen (63). Our results suggestthat breast cancer cells could be made more sensitive to toxic agents ifcellular ability to conjugate glutathione is hampered. These results mayalso change the prevailing ideas on how melatonin works in our in vitrosystem. Strong evidence supports the idea that melatonin action is linkedto the presence ofthe ER in MCF-7 cells (20, 22, 41, 42). Our results withHS578T cells are not necessarily inconsistent with this idea because themechanism of melatonin action may be operating through several different intracellular pathways. It is, however, inconsistent with an absoluterequirement that the ER be present in order for melatonin to inhibitgrowth, particularly because melatonin inhibits the growth of other nonbreast cancer cell lines devoid of the ER (1—3).

Depletion of glutathione may affect other cellular thiols, resulting intheir oxidation to disulfides or the formation of thioesters. Glutathionedepletion has been shown to cause a decrease in microtubule polymerization in cells that may relate to the oxidation of sulthydryl groups (64).In contrast, physiological concentrations of melatonin are known tostabilize microtubules by inhibiting Ca2―/calmodulindepolymerization,which is itself a mitogenic signal transduction mechanism (65). Thus,adequate levels of glutathione may be required to maintain sulThydrylgroups of microtubule-associated proteins in a reduced state in order formelatonin to suppress Ca2@/calmodulin-mediated depolymerization ofthe cytoskeleton and thus cell proliferation. Indeed, other Ca2@/cahnodulin antagonists have been demonstrated to inhibit MCF-7 cell proliferation (66). Furthermore, a recent report suggests that melatonin enhancesthe antioxidant activity ofglutathione (67). Regardless ofthe exact natureof the interaction between melatonin and glutathione, it is clear thatglutathione is involved in some fundamental way in the mechanism ofmelatonin action in human breast cancer cells.

As mentioned earlier, the activity of a number of cytotoxic drugs,including bleomycin, mephalan, and doxorubicin, is enhanced by LBSO-induced glutathione depletion, whereas other cytotoxic agents suchas neocarzinostatin and Taxol actually require reduced thiols for theirantineoplastic activity (57, 58). It is possible that melatonin belongs tothis latter category and requires a reducing intracellular milieu to exert itsoncostatic action at physiological levels in human breast cancer cells.

ACKNOWLEDGMENTS

We thank Maria DeLima for assistance with figure designs.

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1997;57:1909-1914. Cancer Res David E. Blask, Sean T. Wilson and Fred Zalatan

: Evidence for a Glutathione-mediated Pathwayin VitroGrowth Physiological Melatonin Inhibition of Human Breast Cancer Cell

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