(CANCER RESEARCH 46, 6274-6278, December 1986] Calmodulin Antagonism and Growth-inhibiting Activity of Triphenylethylene Antiestrogens in MCF-7 Human Breast Cancer Cells1 Alberto Gulino,2 Giuseppina Barrera, Alessandra Vacca, Antonietta Farina, Carlo Ferretti, Isabella Screpanti, Mario U. Dianzani, and Luigi Frati Dipartimento di Medicina Sperimentale, Università "La Sapienza", 324, viale Regina Elena, 00161 Rome [A. G., A. V., A. F., I. S., L. F.J, and Istituto di Patologia Generale [G, B., M. U. D.] and Farmacologia [C. F.], Università di Torino, Torino, Italy ABSTRACT The triphenylethylene antiestrogen tamoxifen has been shown previ ously to inhibit both calmodulin and protein kinase C activities, which are involved in the control of cell proliferation. We have studied the effect of several derivatives of the triphenylethylene antiestrogen family on the inhibition of both calmodulin-dependent cyclic adenosine 3':5'-mono- phosphate-phosphodiesterase activity and proliferation of breast cancer cells cultured with 0.5 MMestradiol in order to prevent interaction of these drugs with the estrogen receptor. We have observed that hydrox- ylation of the triphenylethylene molecule significantly decreases its abil ity to inhibit the calmodulin-dependent phosphodiesterase activity in vitro. Furthermore, the growth-inhibiting activity of several antiestrogens and other calmodulin antagonists [R24571, trifluoperazine, ,V-(6-amino- hexyl)-5-chloronaphthalene-l-sulfonamide, and .V-(6-aminolu>\yl)-l- naphthalenesulfonamide) correlated with their antagonistic effects on calmodulin activity. The level of activity was determined as follows: R24571 > tamoxifen = A'-demethyltamoxifen = nafoxidine > 4-hydrox- ytamoxifen > 3,4-dihydroxytamoxifen = trifluoperazine > \-(6-uniim>- hexyl)-5-chloronaphthalene-l-sulfononamide > metabolite A> ,V-(6- aminohexyl)-l-naphthalenesulfonamide. On the other hand both protein kinase C-activating and -inhibiting drugs (phorboltetradecanoate-13-ace- tate and tamoxifen, respectively) have a synergistic inhibitory effect on the growth of MCF-7 cells. Our data suggest that antiestrogen interac tions with calmodulin and not protein kinase C may play a role in mediating the drug-induced estrogen-independent inhibition of breast cancer cell growth. INTRODUCTION Triphenylethylene antiestrogens have been reported to inhibit mammary cancer cell proliferation in vitro (1) and are widely used in the treatment of human breast cancer (2). Since these compounds are able to compete with estrogen for binding to estrogen receptor (1), it has been suggested that antiestrogens induce these effects by antagonizing the growth-enhancing ac tivity of estradiol at the cellular level. However, the presence of specific ¡n irace-1hihir antiestrogen binding sites distinct from the estrogen receptor (3-5) and the finding that TAM3 inhibits the proliferation of estrogen receptor-negative breast cancer cells (6) suggest that this drug may control estrogen-independ ent processes involved in cell proliferation. Calcium ions have been shown to play an important role in the mechanism by Received 11/26/85; revised 7/24/86; accepted 8/27/86. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Italian National Research Council Special Project "Oncology" Contract 85.02201.44 and by the National Foundation for Cancer Research, Bethesda, MD. 2 To whom requests for reprints should be addressed. 3The abbreviations used are: TAM, tamoxifen; CAM, calmodulin; TPA, 12- O-tetradecanoylphorbol-13-acetate; PDE, phosphodiesterase; TFP, trifluopera zine; W-7, Ar-(6-aminohexyl)-5-chloronaphthalene-l-sulfonamide; W-5, W-(6- aminohexylH-naphthalenesulfonamide; MOHT, 4-hydroxytamoxifen; DOHT, 3,4-dihydroxytamoxifen; Met A, metabolite A [(4-dimethylaminoethoxyphenyl)- l-(hydroxy)-2-phenylbutanej; DMT, /V-demethyltamoxifen; NAF, nafoxidine [1- |2-[p-(3,4-dihydroxy-6-methoxy-2-phenyl-l-naphthyl)phenoxy|ethyl|pyrrolidine hydrochloride); EGTA, ethyleneglycol bis(0-ammoethyl etherHVyvyV'A'-tetra- acetic acid; 1C»,concentration of inhibitor giving 50% inhibition of CAM- dependent PDE activity; EGF, epidermal growth factor. which several growth factors control cell proliferation (7). Cal cium is able to transduce the signals of two main pathways. In the first pathway, calcium interacts with an intracellular calcium receptor, termed CAM, which is an ubiquitous protein that is activated by calcium to regulate several enzymes and physiolog ical cellular processes (8). There is accumulating evidence to suggest that CAM is involved in the control of cell proliferation. Not only has cellular transformation to malignancy been asso ciated with an increase of intracellular CAM (9) but a positive correlation between CAM levels and growth rate has also been demonstrated in cancer cells (10). Moreover, CAM antagonists inhibit tumor cell proliferation in vitro (11) and in vivo (12), while they do not inhibit the growth of variant cells missing a specific CAM-binding protein (13). In the second pathway, calcium ions have been reported to act synergistically with diacylglycerol (a product of membrane phosphatidylinositol breakdown) and with the synthetic ana logue tumor promoter TPA in the activation of protein kinase C, which may act to transduce the growth-promoting signals of some growth factors (14). TAM has been recently reported to antagonize both CAM and protein kinase C activities in vitro (15, 16). In this report we investigated whether the interaction of TAM with CAM or protein kinase C could play a role in the control of breast cancer cell proliferation. We first correlated the in vitro CAM-antag- onistic activity of several triphenylethylene derivatives and other CAM antagonists with their effects on the proliferation of MCF-7 human breast cancer cells in culture. Secondly, to study the role of the protein kinase C activation in the prolif eration of MCF-7 cells, we investigated the effect of TPA on the growth of this breast cancer cell line. We observed that CAM antagonism by several antiestrogen derivatives correlates with their estrogen-independent, growth-inhibiting potencies on MCF-7 cells. On the other hand, the protein kinase C- activating and -inhibiting drugs (TPA and TAM, respectively) combined have a greater inhibitory effect on the growth of MCF-7 cells than either of the drugs alone. Our data suggest that CAM rather than protein kinase C antagonism by anties trogens mediates the estrogen-independent antiproliferative ef fect of these drugs. MATERIALS AND METHODS Chemicals. [3H]EstradioI (specific activity, 100 Ci/mmol), [3H]cAMP (specific activity, 20 Ci/mmol), and [3H]thymidine (specific activity, 2 Ci/mmol) were obtained from New England Nuclear (West Germany). Hog brain purified CAM and calmidazolium (R24571) were purchased from Boehringer Mannheim (Mannheim, West Germany). Activator- deficient bovine brain cAMP-PDE, 5'-nucleotidase, cAMP, TFP, W- 7, W-5, diethylstilbestrol, estradiol, and TPA were obtained from Sigma Chemical Co. (St. Louis, MO). We have used both crude and highly purified PDE preparations from Sigma and we have observed no differences in CAM antagonism by the drugs we have studied. TAM (ICI 46474), MOHT (ICI 79280), DOHT (ICI 77307), Met A (ICI 46929), and DMT (ICI 55548), were kindly provided by ICI Pharma (England). NAF (U 11100 A) was a gift from Upjohn Laboratories (Kalamazoo, MI). DEAE-cellulose, Affi-Gel-CAM, and Dowex AG 6274 Research. on November 26, 2021. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from