The antiestrogen tamoxifen is widely used for endocrine therapy of breast cancer; however, the mechanisms of estrogen receptor-independent interactions of tamoxifen remain ill defined. Here we examine the effect of tamoxifen on the initial steps of cell signal transduction. To this end, phospholipid metabolism and protein kinase C (PKC) translocation were assessed in CCD986SK human mammary fibroblasts treated with tamoxifen. The addition of tamoxifen resulted in dose-dependent and time-dependent increases in the cellular second messengers phosphatidate (PA) and diacylglycerol (DG). On addition of ethanol to the medium, tamoxifen induced the formation of phosphatidylethanol, demonstrating that tamoxifen activates phospholipase D (PLD). Cellular DG also increased in the presence of ethanol, showing that tamoxifen also activates phospholipase C (PLC). In cells prelabeled with choline and ethanolamine, tamoxifen caused increases in choline, phosphorylcholine, ethanolamine and phosphorylethanolamine. Structure-activity relationship studies for activation of PLD revealed that tamoxifen was the most effective, whereas 4-hydroxy tamoxifen was nearly devoid of activity. Phorbol diesters also activated PLD, but estrogen had no influence. Pretreatment of cells with phorbol dibutyrate (PKC down-regulation protocol) blocked phorbol diester- and tamoxifen-induced PLD activity. Exposure of cells to the PKC inhibitor GF 109203X diminished tamoxifen-induced PLD activity. Addition of tamoxifen to cultures elicited selective membrane association of PKC epsilon. We conclude that tamoxifen exerts considerable extra-nuclear influence at the transmembrane signaling level. These events may contribute to effects beyond the scope of estrogen receptor-dependent actions.

We have previously shown that multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H., Bursten, S. L., Giuliano, A. E., and Cabot, M. C. (1996) J. Biol. Chem. 271, 19530-19536). In this study we used the multidrug-resistant human breast cancer cell line MCF-7-Adriamycin-resistant (AdrR), which exhibits marked accumulation of glucosylceramide compared with the parental MCF-7 wild type (drug-sensitive) cell line, to define the relationship between glycolipids and multidrug resistance (MDR). Herein it is shown that clinically relevant concentrations of tamoxifen, verapamil, and cyclosporin A, all circumventors of MDR, markedly decrease glucosylceramide levels in MCF-7-AdrR cells (IC50 values, 1. 0, 0.8, and 2.3 microM, respectively). In intact cells, tamoxifen inhibited glycosphingolipid synthesis at the step of ceramide glycosylation. In cell-free assays for glucosylceramide synthase, tamoxifen (1:10 molar ratio with ceramide) inhibited glucosylceramide formation by nearly 50%. In cell cultures, inhibition of glucosylceramide synthesis by tamoxifen is correlated with its ability to sensitize MCF-7-AdrR cells to Adriamycin toxicity. Moreover, treatment of cells with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis, likewise sensitized MCF-7-AdrR cells to Adriamycin. It is concluded that high cellular levels of glucosylceramide are correlated with MDR, and that glycolipids are a target for the action of MDR-reversing agents such as tamoxifen. The data entertain the notion that drug resistance phenomena are aligned with cell capacity to metabolize ceramide.

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor-independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF-7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme-specific membrane association of PKC-epsilon, which was time-dependent (as early as 5 min post-treatment) and dose-dependent (5.0-20 microM). Tamoxifen did not influence translocation of alpha, beta, gamma, delta or zeta PKC isoforms. Structure-activity relationship studies demonstrated chemical requirements for PKC-epsilon translocation, with tamoxifen, 3-OH-tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N-dimethyl-2-[(4-phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent-induced PKC-epsilon translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC-epsilon has been associated with cell differentiation and cellular growth-related processes, the antiproliferative influence of tamoxifen on MCF-7 cells may be related to the interaction with PKC-epsilon.