No abstract

We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol-and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was steroland Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators. The AEBS binds selective estrogen receptor modulators (SERM) that contain a cationic aminoethoxy side chain, but has no affinity for estrogens and non-cationic antiestrogens, such as Faslodex, 668. 2 A class of derivatives of Tx has been developed that bind to the AEBS with high affinity and selectively. This family of compounds includes as lead compounds tesmilifene (DPPE, N,N 0 -diethylamino-4-(phenylmethylphenoxy)-ethanamine,HCl) 3 and PBPE. 4 These compounds have no affinity for estrogen receptors and did not modulate other known targets of Tx such as acyl-coA:cholesterol acyltransferase and protein kinase C, 5 allowing AEBS-related cellular events to be studied. Despite a lack of a clear understanding of its mechanism of action at that time, tesmilifene was evaluated for the treatment of breast and prostate cancer with some encouraging results in phase II and III clinical trials.6-8 However, a pivotal phase III trial was recently aborted because of the lack of a therapeutic outcome. This strongly reinforces the need to understand better the mechanism of action of drugs to optimize their clinical use.The AEBS has been reported to bind unsaturated fatty acids and oxysterols (7-ketocholesterol, 7-ketocholestanol and 7-hydroxycholesterol), suggesting a link between the AEBS and the metabolism of lipids and sterols. This led us to identify the AEBS as a hetero-oligomeric complex composed of two subunits: the 3b-hydroxysterol-D 7 -reductase (DHCR7) and 3b-hydroxysterol-D 8 -D 7 -isomerase (D8D7I); these two enzymes are invol...