Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells

Nonsteroidal agent tamoxifen (Tam), a therapeutic/chemopreventive agent for breast cancer, inhibits protein kinase C (PKC), which is considered tea be one of its extra-estrogen receptor sites of action, This drug is required at higher (> 100 mu M) concentrations to inhibit PKC in the test tube, whereas it is required at lower (1-10 mu M) concentrations to induce inhibition of cell growth in estrogen receptor-negative cell types, To identify additional mechanisms of action of Tam on PKC and cell growth, studies with MDA-MB-231, an estrogen receptor-negative breast carcinoma cell type, have been carried out, Upon treatment with 5-20 mu M Tam, a cytosol to membrane translocation of PKC occurred within 30 min, which was then followed by a down-regulation of the enzyme within 2 h. A transient generation of Ca2+/lipid-independent activated form of PKC was observed during this period, Rapidly growing cells require nearly 2-3-fold lower concentrations (2-5 mu M) of Tam than do confluent cells to induce changes in PKC, Furthermore, phorbol ester binding observed with intact cells also decreased in Tam-treated cells only under the conditions PKC was inactivated, Unlike phorbol esters, Tam did not directly support the membrane association of PKC. The release of arachidonic acid correlated with the PKC membrane translocation. Studies carried out with [H-3]Tam revealed that Tam partitioned into the membrane, and there was no appreciable covalent association of [3H]Tam with cellular proteins within this limited time period (2 h). Various antioxidants (vitamin E, vitamin C, beta-carotene, catalase, and superoxide dismutase) inhibited all these cellular effects of Tam. Moreover, vitamin E strikingly blocked Tam-induced growth inhibition. To determine whether oxymetabolites of Tam can affect PKC permanently, OH-Tam was tested with purified PKC. In contrast to Tam, which reversibly inhibited PKC, OH-Tam permanently inactivated the enzyme by modifying the catalytic domain at lower concentrations. The vicinal thiols present within this domain were found to be required to induce this inactivation. This effect was partially blocked by various antioxidants. This is the first report showing the role of oxidative stress in mediating the actions of Tam. Taken together these results suggest that Tam, by initially partitioning into the membranes, induces a generation of transmembrane signals and an oxidative stress to elicit the membrane association of PKC, followed by an irreversible activation, and subsequent down-regulation of this enzyme, which, in part, may lead to cell growth inhibition.