Biochemical Pharmacology, Vol. 35, No. 24, pp. 4463-4465, 1986. 0006-2952/86 $3.00 + 0.00 Printed in Great Britain. © Pergamon Journals Ltd. INHIBITION OF PROTEIN KINASE C MEDIATED SIGNAL TRANSDUCTION BY TAMOXIFEN IMPORTANCE FOR ANTITUMOUR ACTIVITY KIERAN HORGAN, ERYL COOKE, MAURICE B. HALLETr and ROBERT E. MANSEL Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K. (Received 15 May 1986; accepted 11 July 1986) Abstract--Recent studies have demonstrated tamoxifen inhibition of the enzyme protein kinase C (PKC) in vitro. The aim of this study was to investigate the effects of tamoxifen on PKC function in intact human cells. As PKC activates the neutrophil oxidase mechanism the neutrophil was chosen as an experimental model to assess PKC-tamoxifen interaction in these experiments. Neutrophils from healthy volunteers were separated by centrifugation through Ficoll Hypaque. Two separate parameters of oxidase activation; oxygen consumption and reactive oxygen metabolite production were monitored by a Clark electrode chamber and luminol dependent chemiluminescence respectively. Neutrophil chemiluminescence was markedly stimulated by 4 Phorbol-12 myristate-13 acetate (PMA). This stimu- lation was inhibited by tamoxifen; ICs0 = 6.1 - 1.6 #M (£ -+ S.E.M.) N = 6. Neutrophil oxygen consumption was similarly stimulated by PMA and inhibited by tamoxifen. The tamoxifen inhibition was not due to cell toxicity as assessment of cell integrity by the exclusion of trypan blue and measurement of intracellular concentrations of ATP showed no significant differences before and after treatment. Tamoxifen also inhibited neutrophil chemiluminescence which was stimulated by oleoyl acetyl glycerol and mezerein excluding interaction with PMA as an explanation of its inhibitory effect. These results are consistent with tamoxifen inhibition of PKC function in intact human cells. This may be central to its antitumour action. Tamoxifen is a unique anticancer agent. It inhibits the growth of approximately 30% of human breast cancers [1] with minimal side effects [2]. Knowledge of its mode of action would further an understanding of the biology of breast cancer and also enable the development of other effective non-toxic anti- neoplastic compounds. Although tamoxifen has been used for 20 years its mode of action remains unclear. Its ability to bind to oestrogen receptors on breast cancer cells is well documented [3] but the cellular effects of tamoxifen cannot be explained solely by oestrogenic blockade [4]. Clinical studies have also shown that the growth of approximately 25% of receptor negative breast cancers was inhibited [1]. Recent studies have shown that tamoxifen binds to sites distinct from oestrogen receptors [5] but their significance has yet to be established. An extensive review of the literature led Furr and Jordan to con- clude that "there is still no satisfactory molecular mechanism" to explain its action [6]. However, recent advances in our understanding of cancer cell biochemistry suggest new possibilities. It is now recognised that the enzyme protein kinase C (PKC) plays a crucial role in tumour promotion [7]. Two recent reports have demonstrated tamoxifen inhibition of PKC activity in vitro [8, 9]. It is, there- fore, now necessary to determine whether tamoxifen inhibits PKC-mediated processes in intact human cells. It has been established that the neutrophil is a convenient model for the study of PKC activity in tumour promotion [10]. We have also chosen the neutrophil as a source of human cells to assess PKC mediated signal transduction. MATERIALS AND METHODS Peripheral blood was obtained from healthy vol- unteers and the neutrophils were isolated, as pre- viously described [11], by centrifugation through Neutrophil Isolation Medium (Packard United Technologies). 4~ Phorbol-12 myristate-13 acetate (PMA), oleoyl acetyl glycerol (OAG), mezerein (Mz) and tamoxifen citrate were purchased from Sigma Chemical Company (Dorset, U.K.). A sample of OAG was also kindly donated by Professor Y. Nishizuka (Kobe, Japan). All agents were dissolved in dimethyl sulphoxide (DMSO). The concentration of DMSO in the cell suspension never exceeded 0.3~ (v/v). Measurement of oxidase activity. Oxidase acti- vation was determined by disappearance of the substrate oxygen and appearance of the products, reactive oxygen species. Oxygen consumption was measured using a Clark electrode chamber (Rank Bros, Bottisham, Cambridge). The production of reactive oxygen metabolites from neutrophil sus- pensions (approximately 106 per ml) containing lumi- nol (11/~M) was monitored by measurement of chemiluminescence using a purpose built ther- mostatically controlled luminometer, as previously described [12]. ATP measurement. ATP was measured in the supernatant after neutrophil precipitation by per- chloric acid (1.8 M) according to the method of Wet- termark and Styrene [13]. After neutralisation (potassium carbonate, 3M, potassium phosphate buffer, 1 M) 10/A aliquots of the supernatant were 4463