Unique SERM-like properties of the novel fluorescent tamoxifen derivative FLTX1.

Tamoxifen is a selective estrogen receptor modulator extensively used on estrogen receptor-positive breast cancer treatment. However, clinical evidences demonstrate the increased incidence of undesirable side effects during chronic therapies, the most life threatening being uterine cancers. Some of these effects are related to tissue-dependent estrogenic actions of tamoxifen, but the exact mechanisms remain poorly understood. We have designed and synthesized a novel fluorescent tamoxifen derivative, FLTX1, and characterized its biological and pharmacological activities. Using confocal microscopy, we demonstrate that FLTX1 colocalizes with estrogen receptor α (ERα). Competition studies showed that FLTX1 binding was totally displaced by unlabeled tamoxifen and partially by estradiol, indicating the existence of non-ER-related triphenylethylene-binding sites. Ligand binding assays showed that FLTX1 exhibits similar affinity for ER than tamoxifen. FLTX1 exhibited antiestrogenic activity comparable to tamoxifen in MCF7 and T47D cells transfected with 3xERE-luciferase reporter. Interestingly, FLTX1 lacked the strong agonistic effect of tamoxifen on ERα-dependent transcriptional activity. Additionally, in vivo assays in mice revealed that unlike tamoxifen, FLTX1 was devoid of estrogenic uterotrophic effects, lacked of hyperplasic and hypertrophic effects, and failed to alter basal proliferating cell nuclear antigen immunoreactivity. In the rat uterine model of estrogenicity/antiestrogenicity, FLTX1 displayed antagonistic activity comparable to tamoxifen at lower doses, and only estrogenic uterotrophy at the highest dose. We conclude that the fluorescent derivative FLTX1 is not only a suitable probe for studies on the molecular pharmacology of tamoxifen, but also a potential therapeutic substitute to tamoxifen, endowed with potent antiestrogenic properties but devoid of uterine estrogenicity.

Estrogen receptors in lipid raft signalling complexes for neuroprotection.

Estrogens exert a plethora of actions conducted to brain preservation and functioning. Some of these actions are initiated in lipid rafts, which are particular microstructures of the plasma membrane. Preservation of lipid raft structure in neurons is essential for signal transduction against different injuries, such as Alzheimer's disease (AD). These membrane structures appear to be disrupted as this neuropathology evolves, and that may largely contribute to dysfunction of raft resident proteins involved in intracellular signalling. This review includes a survey of some protein interactions that are involved in the structural maintenance and signal transduction mechanisms for neuronal survival against AD. Particularly relevant are the rapid mechanisms developed by estrogen to prevent neuronal death, through membrane estrogen receptors (mER) interactions with a voltage-dependent anion channel (VDAC) and other protein markers within neuronal lipid rafts. These interactions may have important consequences in estrogen mechanisms to achieve neuroprotection against amyloid beta (Abeta-induced toxicity).

Estradiol and tamoxifen differentially regulate a plasmalemmal voltage-dependent anion channel involved in amyloid-beta induced neurotoxicity.

There is a wealth of information indicating that estradiol exerts rapid actions involved in neuroprotection and cognitive-enhancing effects. Some of these effects appear to delay onset, or even ameliorate, the neuropathology of Alzheimer's disease (AD), although some controversy exists about the beneficial brain effects of estrogen therapies. Therefore, it is crucial to better understand the mechanisms developed by 17ß-estradiol to signal in the brain. At the neuronal membrane, the hormone can rapidly interact with estrogen receptors (mERs) or activate other receptors, such as G protein-coupled and ionotropic receptors. And the list of membrane signalling molecules modulated by estradiol in neurons is increasing. VDAC is a voltage-dependent anion channel, known as a mitochondrial porin which is also found at the neuronal membrane, where it appears to be involved in redox regulation, extrinsic apoptosis and amyloid beta neurotoxicity. Moreover, VDAC is present in neuronal lipid rafts, where it is associated with estrogen receptor α-like (mER), forming part of a macromolecular complex together with caveolin-1 and other signalling proteins related to neuronal preservation. Interestingly, we have recently found that 17ß-estradiol rapidly promotes VDAC phosphorylation through the activation of protein kinase A (PKA) and Src-kinase, which may be relevant to maintain this channel inactivated. On the contrary, tamoxifen, a selective estrogen receptor modulator (SERM), provokes the dephosphorylation of VDAC, and eventually its opening, by activating a cascade of phosphatases, including protein phosphatase 2 (PP2A). This review will focus on the relevance of these novel findings in the alternative estrogen mechanisms to achieve neuroprotection related to AD.

Membrane-initiated signaling of estrogen related to neuroprotection. "Social networks" are required.

Numerous studies indicate that estrogens are crucial in normal brain functioning and preservation against different injuries. At the neuronal membrane, estrogens, binding to estrogen receptors (ERs) or other surface targets, exert rapid actions involving a plethora of signaling pathways that may converge in neuronal survival. Emerging work reveals that at least part of these actions may require the compartmentalization of ERs in signaling platforms, composed of macromolecular signaling proteins and particular lipid composition integrated in lipid rafts. These particular microstructures may provide the optimal microenvironment to trigger multiple ER interactions that may be crucial for neuroprotection against different brain impairments, such as Alzheimer's disease (AD). In this order of ideas, recent evidence has demonstrated that a membrane ER (mER) physically interacts with a voltage-dependent anion channel (VDAC) in lipid rafts from septal, hippocampal and cortical neurons, and these interactions may have important consequences in the alternative mechanisms developed by estrogens to achieve neuroprotection against amyloid beta (Aß)-induced toxicity. This review includes a survey of some of the rapid mechanisms developed by estrogen to prevent neuronal death, and the ER interactions that are involved in the structural maintenance and signal transduction mechanisms important for neuronal survival against AD neuro-pathology. A special emphasis is put on the biological relevance of neuronal membrane VDAC in Aß-related neurotoxicity, and the potential modulation of this channel as a part of a signaling complex with mER, which may be modified in AD brains.