Ross V. Weatherman

Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen.

The antiestrogen tamoxifen is extensively metabolized in patients to form a series of compounds with altered affinity for estrogen receptors (ERs), the primary target of this drug. Furthermore, these metabolites exhibit a range of partial agonist and antagonist activities for ER mediated effects that do not depend directly on their absolute affinity for ERs. Thus, clinical response to tamoxifen therapy is likely to depend on the aggregate effect of these different metabolites resulting from their abundance in the patient, their affinity for the receptors, and their agonist/antagonist profile. A recent study has shown that plasma concentrations of the tamoxifen metabolite 4-hydroxy-N-desmethyl tamoxifen (endoxifen), in patents undergoing tamoxifen therapy, are dependent on the cytochrome P450 (CYP) 206 genotype of the patient and that medications commonly prescribed to patients on tamoxifen therapy can also inhibit endoxifen production. In this study we characterized the properties of this metabolite with respect to binding to ERs, ability to inhibit estrogen stimulated breast cancer cell proliferation and the regulation of estrogen responsive genes. We demonstrate that endoxifen has essentially equivalent activity to the potent metabolite 4-hydroxy tamoxifen (4-OH-tam) often described as the active metabolite of this drug. Since plasma levels of endoxifen in patients with functional CYP2D6 frequently exceed the levels of 4-OH-tam, it seems likely that endoxifen is at least as important as 4-OH-tam to the overall activity of this drug and suggests that CYP2D6 status and concomitant administration of drugs that inhibit CYP2D6 activity have the potential to affect response to tamoxifen therapy.

Synthesis and characterization of fluorescent 4-hydroxytamoxifen conjugates with unique antiestrogenic properties.

Membrane receptors for steroid hormones are currently a subject of considerable debate. One approach to selectively target these putative receptors has been to couple ligands to substances that restrict cell permeability. Using this approach, an analogue of the estrogen receptor ligand 4-hydroxytamoxifen was attached to fluorescent dyes with differing degrees of predicted cell permeability. The conjugates bound to estrogen receptor in vitro, but all three conjugates, including one predicted to be cell-impermeable, inhibited estradiol-induced transcriptional activation. Fluorescence microscopy revealed cytoplasmic localization for all three conjugates. We further characterized a 4-hydroxytamoxifen analogue conjugated to a BODIPY fluorophore in breast cancer cell lines. Those experiments suggested a similar, but not identical, mode of action to 4-hydroxytamoxifen, as the fluorescent conjugate was equally effective at inhibiting proliferation of both tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. While these findings point to significant complicating factors in designing steroid hormone mimics targeted to the plasma membrane, the results also reveal a possible new direction for designing estrogen receptor modulators.

Characterization of molecular and structural determinants of selective estrogen receptor downregulators

Antiestrogens used for breast cancer therapy can be categorized into two classes that differ in their effect on estrogen receptor (ER) alpha stability. The selective estrogen receptor modulators (SERMs) stabilize ER alpha and the selective estrogen receptor downregulators (SERDs) cause a decrease in cellular ER alpha levels. A clinically relevant antiestrogen, GW7604, appears to work through a SERD-like mechanism, despite sharing the same molecular scaffold as 4-hydroxytamoxifen, a SERM. In order to investigate potential structural features of GW7604 responsible for SERD activity, GW7604 and two analogs were synthesized using a new, improved synthetic route and tested for their effects on ER alpha function and cell proliferation. The two analogs, which have an acrylamide or a methyl vinyl ketone replacing the acrylic acid group of GW7604, display lower binding affinity for ER alpha than GW7604, but show similar antagonism of estradiol-induced activation of ER alpha-mediated transcription as GW7604 and inhibit estradiol-induced proliferation of the MCF-7 cell line with a similar potency as GW7604. Unlike GW7604, neither analog has a significant effect on cellular ER alpha levels, suggesting that the carboxylate is a key determinant in GW7604 action and, for the first time, showing that this group is responsible for inducing ER alpha degradation in breast cancer cells.

Tamoxifen promotes superoxide production in platelets by activation of PI3-Kinase and NADPH oxidase pathways

BACKGROUND Tamoxifen is a selective estrogen receptor antagonist that is widely used for treatment and prevention of breast cancer. However, tamoxifen use can lead to an increased incidence of thrombotic events. The reason for this adverse event remains unknown. Previous studies showed that tamoxifen and its active metabolite Z-4-hydroxytamoxifen rapidly increased intracellular free calcium ([Ca(2+)](i)) in human platelets by a non-genomic mechanism that involved the activation of phospholipase C. Platelets play a pivotal role in thrombosis and Ca(2+) elevation is a central event in platelet activation. Therefore the mechanism by which tamoxifen activated Ca(2+) entry into platelets was investigated. METHODS [Ca(2+)](i) was measured using the fluorescent indicator fura-2 and reactive oxygen species were measured using lucigenin in isolated human platelets. RESULTS Tamoxifen analogs E-4-hydroxytamoxifen, with weak activity at the nuclear estrogen receptor and Z-4-hydroxytamoxifen, with strong activity at nuclear estrogen receptor, were equally active at increasing [Ca(2+)](i) and synergizing with ADP and thrombin to increase [Ca(2+)](i) in platelets. This result suggests that the effects of tamoxifen and E- and Z-4-hydroxytamoxifen to increase [Ca(2+)](i) are not mediated by the classical genomic estrogen receptor. The effects of tamoxifen to increase [Ca(2+)](i) were strongly inhibited by apocynin and apocynin dimer. This suggests that tamoxifen activates NADPH oxidase which leads to superoxide generation and in turn caused an increase in [Ca(2+)](i). Free radical scavengers TEMPO and TEMPOL also inhibited tamoxifen-induced [Ca(2+)](i) elevation. Inhibition of phosphoinositide-3-kinase (PI3-kinase), an upstream effector of NADPH oxidase with wortmannin and LY-294,002 also caused substantial inhibition of tamoxifen-induced elevation of [Ca(2+)](i). CONCLUSION Tamoxifen increases [Ca(2+)](i) in human platelets by a non-genomic mechanism. Tamoxifen activates phospholipase Cγ as well as PI3-kinase and NADPH oxidase pathway to generate superoxide which causes the release of Ca(2+) from the endoplasmic reticulum, and promotes Ca(2+) influx into the platelets.

Tamoxifen Stimulates Calcium Entry Into Human Platelets

The anti-estrogenic drug tamoxifen, which is used therapeutically for treatment and prevention of breast cancer, can lead to the development of thrombosis. We found that tamoxifen rapidly increased intracellular free calcium [Ca2+]i in human platelets from both male and female donors. Thus 10 μM tamoxifen increased [Ca2+]i above the resting level by 197 ± 19%. Tamoxifen acted synergistically with thrombin, ADP, and vasopressin to increase [Ca2+]i. The anti-estrogen ICI 182780 did not attenuate the effects of tamoxifen to increase [Ca2+]I; however, phospholipase C inhibitor U-73122 blocked this effect. 4-hydroxytamoxifen, a major metabolite of tamoxifen, also increased [Ca2+]i, but other tamoxifen metabolites and synthetic derivatives did not. Three hydroxylated derivatives of triphenylethylene (corresponding to the hydrophobic core of tamoxifen) which are transitional structures between tamoxifen (Ca2+ agonist) and diethylstilbestrol (Ca2+ antagonist) increased [Ca2+]i slightly (6% to 24%) and partially inhibited thrombin-induced [Ca2+]i elevation (68% to 79%). Therefore the dimethylaminoethyl moiety is responsible for tamoxifen being a Ca2+ agonist rather than antagonist. 4-Hydroxytamoxifen and polymer-conjugated derivatives of 4-hydroxytamoxifen increased [Ca2+]i, with similar efficacy. The ability of tamoxifen to increase [Ca2+]i in platelets, leading to platelet activation, and its ability to act synergistically with other platelet agonists may contribute to development of tamoxifen-induced thrombosis.

Chemical approaches to studying transcription

Gene transcription is one of the most important and complex processes in biology but great advances are being made into understanding its molecular mechanisms. Selective modulators of nuclear receptors that can regulate transcription of specific genes allow for the comparative analysis of different states of transcription. Techniques to monitor the binding of proteins to DNA leading up to transcription have also increased our knowledge of the events involved in the initiation of transcription. While still in its infancy, the use of chemical tools to study transcription shows great promise in dissecting a complex molecular process.

Tamoxifen-Based Probes for the Study of Estrogen Receptor-Mediated Transcription

The nuclear receptors are ideal targets to control the expression of specific genes with small molecules. Estrogen receptor can activate or repress transcription though a number of different pathways. As part of an effort to develop reagents that selectively target specific transcriptional regulatory pathways, analogs of 4-hydroxytamoxifen were synthesized with variations in the basic side chain. In vitro binding assays and cell-based luciferase reporter gene assays confirm that all the derivatives have high affinity for the receptor and high potency at repressing direct estrogen receptor-mediated transcription.

Sensing estrogen's many pathways.

Estrogen receptor (ER) is an important drug target, but it has multiple signaling pathways that are difficult to dissect. A new study reports the development of a multicolor bioluminescent probe that can measure a compounds ability to modulate ER-mediated transcription and to promote an interaction between ER and Src, a key protein in a number of different cell signaling cascades. The discovery provides a new tool for quickly obtaining a more complete picture of the potential effects of a compound on estrogen signaling and could lead to more selective ER modulators with fewer side effects.