(CANCER RESEARCH 50, 7134-7138, November 15, 1990]

Effect of Tamoxifen on DNA Synthesis and Proliferation of Human MalignantGlioma Lines in Vitro1

Ian F. Pollack, Margaret S. Randall, Matthew P. Kristofik, Robert H. Kelly, Robert G. Selker, andFrank T. Vertosick, Jr.2

Departments of Neurosurgery [I. F. P., M. S. R.. M. P. K., R. G. S., F. T. V.] and Pathology [R. H. A'., F. T. V.], University of Pittsburgh School of Medicine, Pittsburgh,

Pennsylvania 15213, and the West Penn Center for ¡Veuro-Oncology,Pittsburgh, Pennsylvania 15224

ABSTRACT

Previous studies in our laboratory have shown that proliferation ofhuman malignant gliomas in vitro depends in part upon the activation ofprotein kinase C (PKC) and, conversely, can be blocked by inhibitors ofPKC. Here, we examined the effect of tamoxifen, a known PKC inhibitor,on DNA synthesis and proliferation of an established human glioma line(U138) and two low passage cultures of explanted human glioblastomas.Tamoxifen produced a profound, dose-dependent inhibition of both | '11|

thymidine incorporation and cell proliferation, with a 50% effective doseof 20 UK/ml under serum-free conditions and 50 to 200 ng/ml in thepresence of 10% serum. These tumors were estrogen receptor negativeand showed no mitogenic response to estradiol. Furthermore, concentrations of estradiol as high as 10 ng/ml had no effect on the tamoxifen-induced inhibition. This suggests that the mechanism of growth inhibitionby tamoxifen in these gliomas did not involve an estrogen receptor-mediated process but may instead result from its inhibition of PKC. Inview of the profound effect of tamoxifen on cultured gliomas at concentrations that can safely be achieved therapeutically, further in vitro andin vivo studies of this agent are warranted.

INTRODUCTION

The prognosis for patients with malignant gliomas treatedwith conventional surgical, radiotherapeutic, and chemothera-peutic modalities remains poor. Further improvements in patient survival may depend upon understanding and manipulating the pathways that regulate aberrant growth in these tumors.In recent years, interest has been focused on the role of PKC1

in controlling normal and abnormal cell proliferation (1, 2).This Ca2+- and phospholipid-dependent protein kinase is activated by phospholipase C-mediated hydrolysis of inositol phos-pholipids. This reaction produces inositol phosphates, whichmobilize intracellular calcium stores, and diacylglycerol, theendogenous ligand for PKC (1,2). PKC is also the receptor fortumor-promoting agents such as 12-0-tetradecanoyl-13-phor-bol-acetate (3-6), which are potent tumor promoters in a varietyof cell types (6-8). In addition, these agents inhibit differentiation of a number of cell lines (9), enhance proto-oncogeneexpression (9-13), and induce many characteristics associatedwith the transformed phenotype (14-18). Furthermore, over-expression of PKC, induced by transfection of PKC cDNA,leads to morphological changes and enhanced tumorigenicityin cultured fibroblasts (19, 20). Taken together, these findingssuggest that uncontrolled activation of PKC may play a role in

Received 5/21/90; accepted 8/20/90.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.

1This work was supported in part by Competitive Medical Research FundGrant 3783 from the Presbyterian-University Hospital of Pittsburgh (I. F. P.)and American Cancer Society Institutional Research Grant 1N-58-28 (F. T. V.).

2To whom requests for reprints should be addressed, at The Center for Neuro-oncology at Western Pennsylvania Hospital. Division of Neurosurgery. 4800Friendship Ave., Pittsburgh. PA 15224.

' The abbreviations used are: PKC. protein kinase C; BBB. blood-brain barrier;DMSO. dimethyl sulfoxide: ED!0, concentration producing 50% inhibition; ER.estrogen receptor.

the development and/or maintenance of malignant transformation.

In previous studies, we have reported that activation of PKCwith TPA produces a profound mitogenic response in severalmalignant glial cell lines grown under serum-free conditions(21). Conversely, inhibition of PKC with the antibiotic poly-myxin B (22-24) produces a dose-dependent inhibition of bothDNA synthesis and cell proliferation. Unfortunately, manyPKC inhibitors such as polymyxin B have unacceptable toxicityi/i vivo or are unable to cross the BBB, precluding their use inclinical trials. However, recent studies have shown that thetriphenylethylene anti-estrogen tamoxifen (25, 26), which hasbeen used extensively in the treatment of breast cancer (27) andpenetrates the BBB (28), inhibits PKC (29-35) by interferingwith the activity of the catalytic subunit of the enzyme (33, 34).Tamoxifen is known to inhibit proliferation of some ER-nega-tive cell lines in vitro (32, 36), possibly by virtue of PKCinhibition. Therefore, we examined the effect of this agent onDNA synthesis and cell proliferation in cultured malignantglioma lines. We report that tamoxifen is a potent inhibitor ofglioma proliferation in all lines tested, acting by a mechanismindependent of ER blockade.

MATERIALS AND METHODS

Cell Culture. The human glioblastoma cell line U138 (37, 38) (American Type Culture Collection, Rockville, MD) was initially grown inmodified Eagle's medium supplemented with Earle's salts, trace ele

ments, 10% fetal calf serum (G1BCO, Grand Island, NY), and thefollowing antibiotics: penicillin G (80 units/ml), streptomycin (80 nf>/ml), and fungizone (0.20 Mg/ml). Cultures were established in 75-cm2culture flasks (Costar, Cambridge, MA), maintained at 37°Cin a

humidified atmosphere with 5% CO2 in air, and subcultured every 4 to7 days with 0.25% trypsin in Hanks' balanced salt solution (GIBCO).

Low passage cultures of two human malignant gliomas were derivedfrom freshly obtained surgical specimens. The pathology of both tumorswas glioblastoma multiforme. Tumor tissue was finely minced, passedthrough 100-f/m nylon mesh, and plated in the nutrient medium described above. After reaching confluence (1 to 3 weeks), the cells weresubcultured as described above. Both lines were composed of >98%cells positive for glial fibrillary acidic protein, a marker for astrocyticdifferentiation (39), and were used on their second to fourth passages.

Effect of Tamoxifen on DNA Synthesis. Cells were seeded onto 96-well round-bottomed microtiter trays (Costar) (5 x IO4cells in 200 n\of medium/well) in serum-supplemented medium and, after a 6-hattachment period, were washed 3 times in serum-free MCDB 105medium (Sigma Chemical Co., St. Louis, MO) (40, 41) with antibioticsand grown for 24 h under serum-free conditions. This medium hasbeen previously shown to maintain the viability of malignant glial cellsfor up to 7 days in culture (42). Cells were then incubated for 48 h withvarious concentrations of tamoxifen citrate (Sigma) (1 ng/ml to 100Mg/ml), with or without platelet-derived growth factor (Amgen Biolog-icals. Thousand Oaks, CA) at 50 ng/ml (a concentration found toproduce a profound mitogenic response in cultured malignant glia)(42), 10% fetal calf serum, or 17j3-estradiol (Sigma) (1 ng/ml to 10 /ig/ml). The selection of this incubation period was based on the known

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EFFECT OF TAMOXIFEN ON HUMAN MALIGNANT GLIOMAS

cell cycle time of these lines in culture (50-96 h). Stock solutions oftamoxifen were made up in DMSO (1%, w/v) and stored at -20°Cin

the dark. Control cells were incubated with various concentrations ofDMSO instead of tamoxifen. All studies were performed in triplicate.Cells were then washed and incubated for 4 h in serum-free mediumwith [3H]thymidine (1 /uCi/ml) (Amersham Corporation, Arlington

Heights, IL). After extensive washing to remove unbound radioactivity,cells were detached by a 30-min incubation at room temperature in2.5% trypsin/0.85% NaCl (GIBCO) and were centrifuged for 20 minat 2000 x g. The pellet was suspended in 6% trichloroacetic acid with0.03% unlabeled thymidine. Following a 30-min incubation period, theacid precipitate was centrifuged. The pellet was then suspended andincubated in fresh trichloroacetic acid solution and centrifuged again.The precipitate was then solubilized in 200 n\ of 0.5 N NaOH with0.1 % bovine serum albumin, transferred to scintillation vials, and mixedwith 5 ml of Ecolite scintillation cocktail (ICN Biomedicals, Cleveland,OH). Incorporated radioactivity was measured in a liquid scintillationcounter.

Effect of Tamoxifen on Cell Proliferation. Cells were seeded into 35-mm Petri plates (Costar) (5 x IO4 cells/plate) in serum-supplementedmedium and, after a 6-h attachment period, were grown under serum-free conditions for 24 h. Tamoxifen with or without serum was thenadded, and the cells were grown for an additional 96 h. Control cellsreceived no tamoxifen. All studies were performed in triplicate. Cellswere then detached with 0.25% trypsin in Hanks' balanced salt solution

and counted in a hemacytometer.Estrogen Receptor Assay. Receptor studies on the E. S. and L. C.

tumor lines (derived from a male patient and a female patient, respectively) were performed using the dextran-coated charcoal technique.Briefly, 3 x 10* cells, maintained overnight under serum-free condi

tions, were homogenized by sonication in IO ITIMTris (pH 7.4), 1.5mM EDTA, 20 mM sucrose, and 20 mM sodium molybdate at 4°C,and

centrifuged at 100,000 x g for 1 h. The supernatant was collected andthe protein concentration was determined in an aliquot, using thetechnique of Lowry et al. (43), and was adjusted to between 0.5 and 1ng/ml. To determine the presence or absence of estrogen receptors, 125iil of cytosol were incubated with 3 nM 17/3-[3H]estradiol (100 Ci/mmol) (Amersham), with or without 300 nM unlabeled diethylstilbes-terol (Sigma) (total reaction volume, 150 p\), for 18 h at 4°C.Dextran-

coated charcoal (400 ß\)was then added and the mixture was centrifuged for 30 min at 2000 x g at 4°C.The supernatant was then counted

in a liquid scintillation counter. All studies were performed in duplicate.

1 10 100 1000TAMOXIFEN (ng/ml)

Fig. 1. Effect of varying concentrations of tamoxifen on baseline [JH]thymi-

dine incorporation in three human glioma lines. Bars, SD.

ml, had no effect on tritiated thymidine incorporation. Whenadministered in the presence of tamoxifen, estradiol producedno attenuation of the inhibition induced by tamoxifen. Furthermore, receptor binding studies found no detectable estrogenreceptors in either of the two tumors (E. S. and L. C.) studied.

RESULTS

In each of the tumors tested, tamoxifen produced a dose-dependent inhibition of both baseline (Fig. 1) and mitogen-stimulated (Fig. 2) tritiated thymidine incorporation. The ED50for this effect ranged from 20 ng/ml for cells maintained underserum-free conditions to 50-200 ng/ml in the presence of 10%serum. At concentrations of tamoxifen greater than 200 ng/ml,tritiated thymidine incorporation was less than 5% of controllevels. Addition of the vehicle (DMSO) to control cells atconcentrations up to 0.1% (the amount added to the culturemedium for administration of 10 ng/m\ tamoxifen) had noeffect on thymidine incorporation (data not shown).

Baseline and serum-stimulated cell proliferation were alsonhibited by tamoxifen in a dose-dependent fashion (Fig. 3). At200 ng/ml, tamoxifen produced nearly complete inhibition ofproliferation in each of the tumors. The effect achieved by thisconcentration was cytostatic rather than cytotoxic becausegreater than 95% of cells excluded trypan blue (44). However,concentrations of tamoxifen greater than 5 /¿g/mlproduced adirect toxic effect on the cells, manifested by detachment fromthe culture dish. DMSO alone had no effect on proliferation atconcentrations up to 0.1%.

Estradiol, at concentrations ranging from 1 ng/ml to 10 /ug/

DISCUSSION

The anti-estrogen tamoxifen has long been used in the treatment of postmenopausal women with ER-positive breast carcinoma (25-27) and is a potent inhibitor of the proliferation ofER-positive tumors in vitro (45, 46). Until recently, it wasgenerally acknowledged that the effect of this agent and itsmetabolites on such tumors was largely mediated by competitiveinhibition of estrogen binding to its receptor. However, severallines of evidence suggest that tamoxifen and related triphenyl-ethylenes may have a variety of other antiproliferative effects.First, 13% of patients with ER-negative tumors have beenreported to respond to tamoxifen (47). This agent also inhibitsgrowth of some ER-negative cell lines in vitro (34, 36). Second,tamoxifen has been shown to inhibit proliferation of ER-positive breast cancer lines maintained under serum-free conditions(i.e., in the absence of exogenous estrogen) (48, 49). Third,although the inhibitory effects of low (nanomolar) concentrations of tamoxifen in ER-positive cell lines are reversible byaddition of exogenous estrogens such as estradiol, inhibitioncan no longer be attenuated at higher concentrations of tamoxifen (49, 50). In such tumors, these irreversible effects areunrelated to the affinity of the anti-estrogen for the ER (32,50). Taken together, these studies indicate that these agents

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1 10 100 1000TAMOXIFEN (ng/ml)

Fig. 2. Effect of varying concentrations of tamoxifen on platelet-derivedgrowth factor-stimulated ( ) and serum-stimulated ( ) [3H]thymidine in

corporation in three human glioma lines. Bars, SD.

can inhibit cell proliferation by pathways independent of theER.

The basis for the ER-independent antiproliferative effects ofthe triphenylethylenes has only recently been elucidated. Theseagents have been reported to inhibit the ouabain-sensitive Na+/K+ ATPase and the Mg2+ ATPase (51). Moreover, they interact

with and inhibit binding of cholinergic and dopaminergic receptors (reviewed in Ref. 52) and of a novel subclass of histamin-ergic receptors (53), as well as inhibiting the calmodulin/Ca2"1"-dependent protein kinase (30, 54). O'Brian et al. (29, 32, 34)

and Su et al. (30) have also shown that the triphenylethylenesare potent inhibitors of PKC, a finding that has been confirmedby several other groups (31, 33, 35). PKC is known to play arole in the signal-induced cascade involved in regulating proliferation in many cell types (1,2), including cultured glioma lines(21 ). Conversely, inhibition of PKC by the antibiotic polymyxinB produced profound blockade of both DNA synthesis and cellproliferation in human and rat glioma cell lines (21).

In the present study, we have shown that another PKCinhibitor, tamoxifen, also was a potent inhibitor of baseline aswell as growth factor- and serum-stimulated DNA synthesisand cell proliferation in human malignant glioma lines. Chouvetet al. (36) also reported a strong inhibition with 4-hydroxyta-moxifen in an ER-negative, progesterone receptor-negativebreast carcinoma line. As in the present study, dose-dependentinhibition of proliferation was achieved with antiestrogen concentrations in the range of 10~8 to IO"6 M, and this effect was

partially reversible by the addition of serum to the culturemedium. In the glioma lines, the ED50 for inhibition by tamoxifen was increased from 5-20 ng/ml under serum-free condi-

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lions to 50-200 ng/ml in the presence of 10% serum. This ismost likely due to binding of tamoxifen by serum proteins (36,49, 55). Alternatively, serum may contain endogenous factorsthat compete for tamoxifen binding sites.

The mechanism of the tamoxifen-mediated inhibition of ourglioma lines remains speculative but appears not to involve anER-mediated effect since, as in the cell line examined by Chouvet et al. (36), the response to tamoxifen was not attenuated byincreasing concentrations of estradiol and no ER were detectedin a receptor binding assay. The antiproliferative effect may bea result of PKC inhibition, particularly in view of the aforementioned role for PKC in glioma proliferation. However, the ED5(Ifor the effect of tamoxifen in our lines was somewhat lowerthan the 50% inhibitory concentration for PKC inhibition notedby O'Brian et al. (100 //M) (29), Su et al. (14 to 30 MM)(30),

and Morgan et al. (6.1 MM)(31 ) in direct assays of PKC activity.Since there are at least seven subclasses of PKC that aredifferentially distributed in various cell types and that havedistinct substrate specificities (2), it may be that the PKCsubclass expressed in malignant gliomas is particularly sensitiveto inhibition by tamoxifen. Clearly, further studies that directlyexamine the effect of tamoxifen on PKC activity in these tumorsare needed before this mechanism of growth inhibition can beaccepted.

Alternatively, the profound growth-inhibitory properties oftamoxifen may stem from one or more of the other effects ofthis agent. We purposely exposed the cell lines to tamoxifenfor prolonged periods, to stimulate the use of tamoxifen in vivo.Such prolonged exposures, however, make a pure anti-PKC

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50 no/ml 200 THX TMXng/ml 50 200

INHIBITOR

Fig. .V Effect of tamoxifen (TMX) on baseline (CONTROL) and fetal calfserum-stimulated (F('S) cell proliferation in three human glioma lines. Bars, SD.*. Significant difference from control at P < 0.05. unpaired Student's / test; **.

P < 0.01.

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EFFECT OF TAMOXIFEN ON HUMAN MALIGNANT GL1OMAS

effect less likely. Nevertheless, the inhibition achieved by ta-

moxifen in these cell lines was demonstrated at concentrationsthat are achieved therapeutically in sera of patients with breastcancer (100 to 2000 ng/ml) (56-59), thus indicating a potentialrole for this agent in the treatment of malignant glial tumors.Although cerebrospinal fluid and brain concentrations of ta-moxifen have been examined in only a handful of studies (28,58), these indicate that there is at least some penetration of theBBB. In addition, this agent has been successfully used to treatcerebral métastasesfrom breast cancer (60, 61) and to inhibitER-mediated effects in various cell populations within thecentral nervous system that lie behind the BBB (62, 63), supporting the notion that tamoxifen administered systemicallycan reach substantial concentrations within the brain. Moreover, the known BBB disruption seen in the vicinity of malignant gliomas in situ would further favor this drug reaching thetumor.

In conclusion, tamoxifen has proven to be a potent inhibitorof human glioma proliferation in vitro, acting by a mechanismindependent of estrogen receptor binding at concentrations thatmay potentially be achievable in vivo with systemic administration. While it is possible that the suppression of proliferationobserved in our cell lines was due to inhibition of PKC, giventhe sensitivity of these lines to PKC inhibition (21), the mechanism by which tamoxifen inhibits these tumors remains unknown. Nevertheless, in view of these encouraging initial resultswith tamoxifen, further in vitro and in vivo studies of this agentin malignant glial tumors are indicated.

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in VitroHuman Malignant Glioma Lines Effect of Tamoxifen on DNA Synthesis and Proliferation of

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