BMC Cancer 2004, 4:49 http://www.biomedcentral.com/1471-2407/4/49 (EFafilfgleuactre1o6f7tµaMmo),xoifnen6,-kLeYt1o1-7P0G1F81αanpdrotdaumcotixoinfen plus LY118018 Effect of tamoxifen, LY117018 and tamoxifen plus LY118018 (all at 16 µM), on 6-keto-PGF1α production. The rat liver cells were incubated 6-h. The analyses were performed with quintuplicate dishes. Each bar gives the mean value and the brackets the SEM. * Statistically significant vs tamoxifen. pathways leading to apoptosis and COX induction. The intracellularly released AA also can serve as substrate for oxygenases. AA has intrinsic biologic activities that may also affect signaling pathways. Such changes would depend on the lipophilic properties of the compound and the composition of a particular membrane [22] and would vary from cell type to cell type, organelle to organelle and with the growth phase of the cell. The AA release by tamoxifen and other reagents studied in my laboratory occurs with µM concentrations [4,5,19,23,24]. These experiments were carried out in the presence of BSA (1.0 mg/ml), and therefore do not differ- entiate between the protein-bound and free reagent. Thus, they are likely to be overestimated values. Nevertheless, the possibility that general necrotic cell death may cause AA release, must be considered. Tamoxifen, was found not to be toxic at concentrations of 10 to 20 µM for A549 human lung adenocarcinoma (ER-negative) cells [25]. Nor was 10 µM tamoxifen toxic when tested on rat glial cells and breast cancer MCF-7 cells [26]. Even when cell viability of three different breast cell lines (ER-positive MCF-7; ER-negative MDA-MB-239 and ER-negative BT-20 cells) was measured after incubation with 25 µM tamoxifen for 24-h, the loss in viability was due to LFAYiAg1u1rre7el0e1a88se(4fr8omµMr)atanlidvetramceollxsifbeyntianmporxeisfenc(e12ofµLMY)1,17018 AA release from rat liver cells by tamoxifen (12 µM), LY117018 (48 µM) and tamoxifen in presence of LY117018. Cells were incubated for 6-h. The analyses were performed with triplicate dishes. Each bar gives the mean value and the brackets the SEM. * = Statistically significant vs MEM/BSA control. ** = Statistically significant vs tamoxifen. apoptosis [12] and was not the result of necrotic cell death. Concentrations of tamoxifen used in this report are comparable to those found to induce apoptosis, not necrotic cell death. The median concentration of tamoxifen and its metabolites for clinical effectiveness in the treatment of breast cancer varies from 0.8 µM to 2.4 µM, depending on the age of the woman [27]. When measured after a 6-h incubation, 10 µM tamoxifen stimulates deesterification of membrane phospholipids as measured by extracellular AA release, (Fig. 1 and 2). The 6-h incubation may not be optimum for apoptosis to be observed, e.g. after a 6-h incubation of 5 µM tamoxifen with breast cancer cells, apoptosis was induced in about 10% of the cells compared to 8% in control cells [12]. After 24- and 48-h incubation, apoptosis in the tamoxifen treated cells increased to 40 and 70%, respectively. Apoptosis, induced by this membrane perturbation after 6-h incubation, increased 4 to 7 fold after longer Page 6 of 8 (page number not for citation purposes)