Selective Estrogen Receptor Modulators. Antonio Cano

Chapter 4. Cellular and Molecular Basis for Acute Nongenomically Mediated Actions of SERMs

• Mario Di'az

• Jorge Marrero-Alonso

• Benito Garcia Marrero

• Raquel Marin

• Tomas Gomez

• Rafael Alonso

4.1

Introduction

Compelling evidence accumulated over the past three decades have demonstrated that, besides their ability to antagonize estrogen binding to their intracellular specific estrogen receptors (ER), selective estrogen receptor modulators (SERMs) can affect a number of biochemical processes in eukaryotic cells. Experimental data from in vivo and in vitro studies have revealed that SERMs and estrogens are surprisingly pleiotropic molecules affecting molecular targets in both estrogen receptor positive (ER+) and negative (ER-) cells. Such “alternative” actions of SERMs and estrogens are typically independent of canonical ERs and do not involve transcriptional or translational events, thereby mediated nongenomically, and usually initiated (and accomplished) within seconds to minutes after presentation of the molecule (Falkestein et al. 2000; Nadal el al. 2001). The spectrum of SERM-induced acute actions includes a wide set of molecular targets, from modulation of ion channels and signaling molecules to alteration of membrane fluidity. In the following sections we review data from different laboratories, including ours, in the context of cellular and molecular evidences for acute nongenomic effects of SERMs observed at pharmacological circulating concentrations. Special emphasis will be placed on actions that might underlie clinically relevant beneficial effects as well as undesirable side effects.

4.2

Cellular and Molecular Targets for Rapid Actions

4.2.1

Interaction with Ion Channels

4.2.1.1

Sodium and Potassium Channels

Electrophysiological studies on primary cultures of hypothalamic neurons and C1300 neuroblastoma cells have shown that the triphenylethylene SERMs tamoxifen and toremifene are able to rapidly inhibit macroscopic voltage-gated tetradotoxin-sensitive Na+ currents (TTX-sensitive INa, IC50 ≈ 1-2 μM) and delayed rectifier K+ currents (IDR) (IC5o ≈ 2-3 μM), while only toremifene exhibits a significant inhibition of transient outward (Ito) currents (IC50 ≈ 3μM) (Hardy et al. 1998). Similar results have been reported in rat cortical glial cells for voltage-gated TTX-sensitive INa and IDR (Smitherman and Sontheimer 2001). Moreover, in isolated cardiac myocytes, tamoxifen inhibits voltage-gated delayed rectifier K+ in a time-, concentration-, and voltage- dependent fashion (Liu et al. 1998) and, more importantly, inhibits the inward rectifier (IK1 and Ito). Inhibition of IK1 and Ito is especially noticeable since it markedly prolongs the action potential duration, decreases the maximal rate of depolarization, and decreases the resting membrane potential in cardiac myocytes (He et al. 2003). The results of these studies suggest that inhibition of Ito, IDr, and IK1 by tamoxifen may contribute to prolonged QT interval of the electrocardiogram observed in some patients receiving tamoxifen treatment, thereby potentially causing untoward life-threatening polymorphic ventricular arrhythmias (De Ponti et al. 2000; Pollack et al. 1997).

Fig. 4.1. Cellular model illustrating cell types in vascular wall involved in vasorelaxation induced by SERMs. Putative targets of SERMs are indicated within cyan tags. SERMs directly affect L-type VDCC, BK β1 subunit in smooth muscle cells, and ER in endothelial cells. L-type VDCC: L-type voltage-dependent calcium channel; BK: calcium-activated large conductance K+ channel; PKG: protein kinase G; eNOS: endothelial nitric oxide synthase; GC: soluble guanylate cyclase; cGMP: cyclic GMP; V: electrochemical membrane potential; ER: estrogen receptor. See text for further details

Tamoxifen also modulates a different set of K+ channels, namely large- conductance calcium-activated potassium channels (BK), by directly interacting with the regulatory β1 subunit of the channel protein (Dick et al. 2001). Interaction of tamoxifen, as well as 4-OH-tamoxifen and the impermeant analog ethylbromide tamoxifen (EBTx), with the β1 subunit dramatically alters the Ca2+/voltage sensitivity of BK, increasing the channel open probability (EC50 = 0.65 - 0.96 μM) and decreasing the unitary conductance of the channel pore (Dick et al. 2001,2002). Interestingly, the stimulatory effect of tamoxifen on β1 is mimicked by 17β-estradiol (Valverde et al. 1999), though tamoxifen is nearly fivefold more effective than 17β-estradiol (Dick et al. 2001). These results are clinically relevant since BK channels play a key role in maintaining the dynamic equilibrium between vasoconstriction and vasodilation in vascular smooth muscle, thereby controlling blood pressure. Activation of BK channels leads to hyperpolarization of the cell membrane, which causes deactivation of voltage-dependent calcium channels and subsequent vasodilation (reviewed in Patterson et al. 2002). Positive modulation of large-conductance potassium channels by tamoxifen and 17β-estradiol likely underlie the acute endothelium-independent relaxing effect of these compounds on vasculature (Patterson et al. 2002; Valverde et al. 1999) (Fig. 4.1).

4.2.1.2

Calcium Channels

The ability of estrogens and nonsteroidal SERMs to modify calcium channel activity in smooth muscle cells was initially inferred from competition binding studies. Thus, it has been shown that tamoxifen and clomiphene compete with dihydropyridine calcium channel antagonists (3H-nitrendipine) in binding in membrane fractions of human and rabbit urinary bladder and myometrium (Batra 1990). On the other hand, functional studies in different preparations of vascular and visceral smooth muscle have revealed that estrogens, xenoe- strogens, and SERMs induce relaxation through ER-independent mechanisms. Thus, in vitro studies on isolated uterine, vascular, detrusor, and intestinal smooth muscles from different species, including humans, have shown that tamoxifen rapidly inhibits spontaneous and agonist-induced contractile activity by interfering with transmembrane calcium influx (Cantabrana and Hidalgo 1992; Di'az 2002; Fernandez et al. 1993; Lipton 1987; Lipton et al. 1984; Ratz et al. 1999; Song et al. 1996). Reported IC50 values for triphenylethylene SERMs in these preparations were in the submicromolar range, i.e., for tamoxifen and ethylbromide tamoxifen in mouse duodenal muscle values were 0.85 and 0.37 μM, respectively (Di'az 2002; J. Marrero-Alonso et al. in press), and for uterine muscles values were 0.70 and 0.58 μM, respectively (J. Marrero-Alonso et al. in press) (Fig. 4.2). In addition, direct electrophysiological evidence has demonstrated that tamoxifen inhibits calcium entry through L-type calcium channels in A7r5 and aortic smooth muscle cells (Song et al. 1996), isolated colonic myocytes (Dick et al. 1999), and other nonmuscle cells, like clonal pituitary cells (Sartor et al. 1988) and PC12 neurosecretory cells (Greenberg et al. 1987). Recently, similar effects on vascular voltage-dependent L-type calcium channels and contractile response of cerebral arteries (Tsang et al. 2004) and pulmonary vessels (Chan et al. 2004) have been reported for the more recent benzotiophene SERM raloxifene.

Obviously, the effects of tamoxifen and derivatives and of raloxifene on L-type calcium channels from aortic and other blood vessels would reduce vascular smooth muscle contractility. This action, in synergy with the aforementioned effect on BK channels, would reduce blood peripheral resistance and blood pressure, which may partially account for the reduction in cardiovascular risk (Da Costa et al. 2004; Trump et al. 1992) (Fig. 4.1).

Fig. 4.2. Effects of triphenylethylene SERMs on spontaneous and depolarization- induced contractions in visceral smooth muscle. Tamoxifen (a) and ethylbromide tamoxifen (EBTx, b) rapidly and reversibly inhibit spontaneous peristaltic activity in duodenal muscle. Both compounds also inhibit depolarization- induced tonic contraction of uterine muscle (c). The inhibition of L-type voltage- dependent calcium channels underlies the relaxing effects illustrated here. Drugs concentrations were 10 μM in all cases. %RA: percent of activity related to maximal activity

On the other hand, we have shown that 4-OH-tamoxifen is as effective as tamoxifen in relaxing duodenal and ileal smooth muscle through inhibition of L-type calcium channels (Di'az 2002). Since tamoxifen is metabolized in the liver to produce the active circulating metabolite 4-OH-tamoxifen, the existence of effects induced by this metabolite provides a critical indication of an in vivo pharmacological action. Indeed, levels of tamoxifen and 4-OH- tamoxifen shown in intestinal preparations to cause a significant reduction of calcium channels are well within the range of tamoxifen concentrations clinically observed in humans and might provide a clue to explaining the occurrence of gastrointestinal disorders in patients receiving high-dose tamoxifen therapies.

4.2.1.3

Chloride Channels

Maxi-Cl- channels are large conductance voltage-dependent anion channels with widespread distribution in eukaryotic cells, yet they have rarely been recorded in intact cells. We have reported that Maxi-Cl- channels are activated in intact neuroblastoma C1300, vascular A7r5 myocytes, and NIH3T3 cells by low micromolar concentrations of the extracellular triphenylethylene SERMs tamoxifen and toremifene, as well as by the nonpermeant analog ethylbromide tamoxifen (Di'az 1999; Di'az et al. 2001; Hardy and Valverde 1994; Valverde et al.

2002), which suggests the involvement of membrane antiestrogen binding sites for these compounds (Fig. 4.3). The fact that Maxi-Cl- channels are generally inactive in whole cells but activate upon membrane-patch excision has suggested the existence of regulatory mechanisms that keep Maxi-Cl- channels closed under nonstimulatory conditions, probably due to either basal phosphorylation of the channel protein or putative regulatory subunits. In this sense, our observations on neuroblastoma C1300 and NIH3T3 fibroblasts revealed that activation of Maxi-Cl- channels by antiestrogens is triggered upon activation of an okadaic acid-sensitive PP2A-like phosphatase in response to tamoxifen and its derivatives, which dephosphorylates Maxi-Cl- channels and switches them to a voltage-sensitive active state (Di'az et al. 2001). Interestingly, activation of Maxi-Cl- channels can be prevented by preincubation with 17β-estradiol via activation of protein kinase A (PKA), which likely keeps the channels in their phosphorylated inactive state (Di'az et al. 2001). The physiological significance of the modulation of Maxi-Cl- channels by estrogens and triphenylethylene SERMs remains unresolved, but a number of studies have emphasized their role in transmembrane electrolyte transport and setting of the membrane potential (Gelband et al. 1996).

Volume-sensitive chloride channels are also sensitive to triphenylethylene SERMs. Thus, tamoxifen, 4-OH-tamoxifen, and toremifene were all found to be high-potency fast blockers ofvolume-sensitive chloride channels in nearly all cell types analyzed so far (Di'az 1996; Valverde et al. 1993; Zhang et al. 1994) by mechanisms independent of estrogen receptor activation that involve direct interaction with the channel protein (Fig. 4.3). Furthermore, the IC50 for tamoxifen-induced inhibition of volume-sensitive channels (≈ 0.3 μM) is, by far, lower than for any other blocker of this type of chloride channels identified to date (Zhang et al. 1994). Interestingly, lens fibers express volume-sensitive chloride channels that play a crucial role in maintaining the hydroelectrolytic equilibrium for normal lens clarity. It is known that one of the side effects of tamoxifen therapies is cataracts (Gerner 1992; Jordan and Murphy 1990). The fact that tamoxifen readily blocks volume-sensitive chloride channels in isolated patches of lens fibers, and that tamoxifen reduces lens transmittance in lens organ cultures at similar concentrations (Zhang et al. 1994), has raised the hypothesis that tamoxifen induces opacity and cataract formation through its effect on channel function.

Fig. 4.3. Modulation of different types of chloride currents by tamoxifen in a single rat aortic A7r5 cell (shown in H). Currents were elicited under voltage- clamp conditions in response to the voltage protocol depicted in G, in symmetrical N- methyl-D-glucamine chloride. The cell was initially recorded under isotonic conditions (A) and afterwards exposed to hypotonicity for different times (B, C). Volume-activated chloride currents activated by hypotonic- ity were completely blocked by tamoxifen (D). Under isotonic conditions, tamoxifen also activates Maxi-Clcurrents in the same cell (E), which were inhibited by niquel chloride (F). VH: holding potential

4.2.1.4

Neurotransmitter Receptors

Tamoxifen can compete with the binding of histamine and antihistaminergic compounds such as DPPE (N,N-Diethyl-2-[(4-phenylmethyl)-phenoxy]ethanamine hydrochloride) in rat brain microsomes and can antagonize histamin-ergic contraction of canine tracheal smooth muscle preparations (Brandes et al. 1987; Kroeger and Brandes 1985). Comparison of relative binding affinities of tamoxifen with those of histamine agonists and antagonists revealed a common binding entity that is neither H1 nor H2 (Kroeger and Brandes 1985). Similarly, tamoxifen competes with high affinity the binding of 3H-domperidone (Kd=0.62nM) to the D2 dopaminergic receptor in membrane preparations of rat brain (Hiemke and Ghraf 1984). The Ki for tamoxifen in this system (≈ 12 M) was one order of magnitude larger than the Ki for dopamine (≈ 1 μM), but much smaller than for 17β estradiol (Hiemke and Ghraf 1984). These interactions of tamoxifen with dopaminergic systems may be clinically relevant since they could explain the emetic effects of antiestrogens, which are among the most common mild side effects of adjuvant therapies (Hiemke and Ghraf 1984).

Other neurotransmitter receptors are equally susceptible to modulation by SERMs. For instance, Ben-Baruch and coworkers (1982) have investigated the possible interaction between the triphenylethylene drug clomiphene citrate and muscarinic receptors in homogenates from various regions of rat brain. Binding analyses and dissociation kinetics studies using the highly specific antagonist 3H-N-methyl-4-piperidyl benzilate (4-NMPB) have shown that clomiphene binds in a positively cooperative pattern to muscarinic receptors (Ben-Baruch et al. 1982). More recently, both tamoxifen and clomiphene have been shown to compete with quinuclidinyl benzilate (QNB) for their binding to muscarinic receptors in membrane fractions of human and rabbit urinary bladder and myometrium, with IC50 values ranging from 5.0 to 13.6 μM (Batra 1990). In addition, electrophysiological studies on adult- type human muscle nicotinic receptors expressed in Xenopus oocytes have shown that tamoxifen and toremifene inhibit inward cationic currents with IC5o values of 1.2 μM (Allen et al. 1998). Interestingly, tamoxifen (and also the impermeant analog ethylbromide tamoxifen) was also able to noncompetitively block another member of the nicotinic receptor family, the ionotropic 5-HT3 receptor channel, in neuroblastoma x glioma NG108-15 hybrid cells with high affinity (IC50 = 0.22 μM for EBTx) (Allen et al. 2000).

4.2.2

Multidrug Resistance and P-glycoprotein

Clinical success in the treatment of tumors with chemotherapy has significantly improved over the past several years, though treatment failure due to drug resistance of cancer cells has remained a major problem. The classical form of multiple drug resistance (MDR) is perhaps the most common type of drug resistance and represents the overexpression of a transmembrane glycoprotein pump (P-170 or Pgp) that mediates an energy-dependent active efflux of a spectrum of structurally and functionally unrelated drugs (reviewed in Mansouri et al. 1992; Ling 1997). Drug transport by P-170 is stoichiometrically coupled to ATPase hydrolysis in a high chemical-potential coupling transition state, where two ATP molecules are bound before the drug is moved to the external side of the membrane (Al-Shawi et al. 2003). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug- stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. A number of studies in cellular models of drug resistance have shown that the triphenylethylene SERMs tamoxifen, its metabolites (4-OH-tamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene all stimulate Pgp ATPase activity and reverse drug resistance (Berman et al. 1994; Chatterjee and Harris 1990; Li et al. 2001; Rao et al. 1994), often equalling the maximal stimulation obtained by verapamil, the best known MDR chemosensitizer (Rao et al. 1994). Interestingly, a single report on adriamycin-resistant MCF-7 cells showed that triphenylethylene SERMs were effective inhibitors of ceramide- induced toxicity, while the raloxifene analog LY117,018 was without influence (Lucci et al. 1999). These results suggest that triphenylethylene SERMs, but not benzotiophene derivatives, reverse the multidrug-resistant phenotype by directly interacting with Pgp, thus interfering with its anticancer-drug-extruding activity (Rao et al. 1994).

The use of triphenylethylene SERMs as Pgp inhibitors for clinical application has been hampered by unacceptable toxicity at doses required to achieve adequate cellular concentration, which is likely due to the involvement of proteins with the ability to bind these compounds. For instance, toremifene is able to reverse MDR and to sensitize human renal cancer cells to vinblastine in vitro. However, in vivo toremifene is tightly bound to serum proteins, in particular a 1-acid glycoprotein (AAG), which may limit its tissue availability (Braybrooke et al. 2000). In agreement with this, Chatterjee and Harris (1990) have shown that tamoxifen and 4-OH-tamoxifen were similarly potent in reversing MDR in Chinese hamster ovary (CHO) cells with acquired resistance to adriamycin. However, the addition of AAG (0.5 to 2 mg/ml, the range found in vivo) to cell cultures decreased the effect of tamoxifen on reversing MDR, and at the highest AAG concentration there was a complete reversal of the effects of both tamoxifen and 4-OH-tamoxifen (Braybrooke et al. 2000). Furthermore, AAG has been found to bind 3H-tamoxifen in a nonsaturable and nonspecific manner, in contrast to the binding of tamoxifen to albumin (Chatterjee and Harris 1990). Thus, the use of tamoxifen as a reversal agent for MDR in vivo might be impaired by high binding to AAG.

4.2.3

Signalling Transducers

4.2.3.1

Calcium Signalling

Tamoxifen also affects Ca2+ signalling independently of conventional ERs. This compound binds calmodulin (CaM) in a Ca2+-dependent manner and thus inhibits the many functions that are activated by this Ca2+-binding protein, including control of cell proliferation (Kahl and Means 2003; Lam 1984; Lopes et al. 1990). Some studies have hypothesized that CaM inhibition might be responsible for the ER-independent tamoxifen cytotoxicity (Borras et al. 1994; Li et al. 2001). In isolated mammalian brain membranes, tamoxifen interacts with two different binding sites of CaM, with an apparent dissociation constant of about 6nM and 9 μM, respectively (Lopes et al. 1990). In the micromolar range, there exists cooperativity for tamoxifen inhibition that is competed for by the CaM antagonist trifluoperazine (Lopes et al. 1990). Interestingly, tamoxifen interacts with CaM in its cation-activated form, which induces exposure of a hydrophobic domain of the C-terminal region of CaM. This domain serves the acceptor site for CaM-modulated enzymes or for CaM-antagonist drugs (La Porte et al. 1980). Molecular modeling of tamoxifen and its derivatives’ interaction with CaM has revealed that the benzene rings ofthe triphenylethy- lene moiety and ethyl group (Fig. 4.4) fit in hydrophobic pockets of the protein, while the aminoethoxy side chain extends toward a region of acidic residues close to the hydrophobic cavity (Hardcastle et al. 1995).

Because of its ability to bind CaM, tamoxifen can increase cyclic AMP surges by inhibiting cyclic AMP hydrolysis by the Ca2+-calmodulin-dependent cyclic nucleotide phosphodiesterase (Fanidi et al. 1989; Rowlands et al. 1990). In bovine brain preparations, tamoxifen appears to act as a competitive inhibitor of calmodulin-activated phosphodiesterase with an IC50 of 2 μM, similar to the value reported for trifluoperazine under the same experimental conditions (Lam 1984).

Nonsteroidal SERMs can also amplify signal-induced Ca2+ surges by inhibiting Ca2+-calmodulin-dependent membrane (Ca2+ + Mg2+)-ATPase. For instance, in synaptic plasma membranes and red cell membrane ghosts, tamoxifen and other triphenylethylene compounds (but not estradiol) have been shown to inhibit (Ca2+ + Mg2+)-ATPase in a calmodulin-dependent fashion that was mimicked by trifluoperazine (Malva et al. 1990). In addition, tamoxifen decreases the calcium affinity of (Ca2+ + Mg2+)-ATPase, as does trifluoperazine in heart sarcolemma (Ca2+ + Mg2+)-ATPase. This reduction of the enzyme sensitivity for calcium is probably due to an impairment of calmodulin-induced transition from a low to a high Ca2+ affinity form (Ca- roni and Carafoli 1981). Moreover, tamoxifen greatly reduces trifluoperazine- insensitive calmodulin-independent microsomal (Ca2+ + Mg2+)-ATPase isolated from brain cortex (Malva et al. 1990), a finding that has been interpreted as the result of a direct interaction of this compound with the enzyme at the endoplasmic reticulum. All these evidences suggest that triphenylethylenes could affect cell proliferation through its ability to modulate CaM, a finding that might explain, at least in part, its cytostatic and cytocidal effects on ER(-) tumors and cell lines (Brandt et al. 2004; Fisher et al. 1983).

Fig. 4.4. Structure of tamoxifen molecule showing some substructures (modified from De Medina et al. 2004a)

4.2.3.2

Protein Kinase C (PKC)

One of the most relevant targets for tamoxifen is PKC. These enzymes belong to a family of proteins that play crucial roles in signal transduction and cell growth control (Nishizuka 1992), and numerous studies have demonstrated that PKCs are involved in carcinogenesis and malignant transformations (Caponigro et al. 1997). Recently, in mammary carcinoma, PKC activity was found to be more than twice that found in normal breast tissue from the same patients (Boyan et al. 2003). Moreover, a high positive correlation between PKC activity and tumor severity has been demonstrated in breast cancer specimens, with the relationship being even greater in ER(-) tumors (Boyan et al. 2003). In fact, several PKC inhibitors, in combination with cytotoxic drugs, are being used in clinical trials for cancer treatment (Chen et al. 2003), though the precise role of the different PKC isoforms is not fully understood. Tamoxifen, its 4-OH or N-desmethyl metabolites, and clomiphene have been shown to reduce the activity of partially purified PKC and to inhibit the growth of several cell types in culture (O’Brian et al. 1986,1988).

Tamoxifen has also been reported to inhibit classical PKC isoforms (a, 01, 02, and y, which need calcium, phospholipids, and diacylglycerol (DG) for catalytic activity) in different preparations, both in vitro and in vivo (Horgan et al. 1986; O’Brian et al. 1986), and to induce the translocation of different PKC isoforms from the cytosol to the plasma membrane (Ahn et al. 2003; Cabot et al. 1997). The IC50 values for the inhibition of Ca2+- and phospholipid- dependent PKC by 4-OH-tamoxifen and N-desmethyl-tamoxifen, the two main metabolites of tamoxifen, were 2 and 8 μM, respectively (O’Brian et al. 1986), which are well within the range of concentrations detected in plasma. The inhibitory effect of tamoxifen and its derivatives on PKC activity has been demonstrated in both ER(+) MCF-7 cells and ER(-) HCC38 cells, which is an indication that the blocking effect is ER independent. In agreement with this, triphenylethylenes have been shown to inhibit phorbol ester (PDBu) binding and it is competed for by MgATP. This provides strong evidence that triphenylethylenes can inhibit PKC by binding directly to the enzyme, likely to the ATP-binding region of the active site of the enzyme (O’Brian et al. 1986, 1988), though other reports are consistent with modulation of the catalytic site. Indeed, recent structure-activity studies have shown that the loss of a side chain in tamoxifen molecules triggers the ability of compounds to inhibit the catalytic site of PKC, whereas the absence of both the aminoethoxy side chain and the β-ring makes compounds activators of PKC (De Medina et al. 2004a) (Fig. 4.4).

4.2.3.3

Phospholipases and Lipid Signalling

Hyperactivation of phospholipase D (PLD) in certain tumor-derived cell lines have been reported, and recent findings suggest a role for PLD in transformation and metastasis. Elevated levels of PLD have been demonstrated in human breast cancer tissues (Noh et al. 2000) and human gastric carcinoma cells (Uchida et al. 1999). Furthermore, elevated PLD activity, specifically by the isoform PLD2, was reported in human colon adenocarcinoma cells, human breast adenocarcinoma cells (Fiucci et al. 2000), and human renal cancers (Zhao et al. 2000). PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to choline and phosphatidic acid (PA), which has been implicated in signalling cascades that regulate cell growth and metastasis (reviewed in Foster and Xu 2003). Hydrolysis of PA by phospholipase A2 generates the potent mitogen lysophosphatidic acid, whose serum levels have been shown to increase in correlation with the malignancy degree in ovarian cancer patients (Westermann et al. 1998). Interestingly, it has been shown that tamoxifen can stimulate cellular PLD activity through an ER-independent mechanism (Kiss 1994).

In CCD986SK human mammary fibroblasts, incubation with tamoxifen results in dose- and time-dependent increases in the cellular second messengers PA and diacylglycerol (DG) and activates PLD and phospholipase C (PLC) (Cabot et al. 1997). Moreover, the addition of tamoxifen to cultures elicits selective membrane association of PKCε (Cabot et al. 1997), indicating that tamoxifen exerts considerable extranuclear influence at the transmembrane signalling level. The proposed mechanism of this tamoxifen stimulation involves the PLD activator PKC. Using the ER+ mammary epithelial cell line MCF-12A and the ER highly tumorigenic mammary carcinoma cell line MDA-MB-23, it was recently demonstrated that tamoxifen and raloxifene have differential effects on PLD catalytic activity. Thus, tamoxifen stimulates PLD in both ER- positive and ER-negative cells in vivo and in vitro, whereas raloxifene inhibits PLD activity in these same cell types (Eisen and Brown 2002).

Other laboratories have reported that tamoxifen causes ER-independent stimulation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase activities in GH4C1 cells, a rat pituitary adenoma cell line (Friedman 1994). These enzymes are normally product inhibited by the polyphosphoinositides. It has been suggested that tamoxifen binds to polyphosphoinositides, which thereby releases the kinases from product inhibition. Binding of tamoxifen to the polyphosphoinositides also leads to inhibition of phospholipase C (PLC) activity. Tamoxifen causes inhibition of inositol phosphate accumulation and phosphoinositide breakdown in whole GH4C1 cells in culture. No other enzymes of the phosphoinositide breakdown cascade are inhibited by this drug (Friedman 1994). These findings are interesting since increased concentrations of inositol triphosphate (IP3) have been detected in hepatomas and numerous human carcinomas in both clinical samples and tissue culture cells, where the elevated signal transduction activity, as measured by the IP3 concentration, was downregulated in a time- and dose-dependent fashion by tamoxifen (Weber et al. 1999).

Tamoxifen also releases arachidonic acid (AA) and stimulates prostacyclin (PGF1a) production from rat liver cells at micromolar concentrations (Levine 2003a). This ability of tamoxifen to release AA is rapid and not affected by preincubation with either actinomycin or the estrogen antagonist ICI182780, indicating its nongenomic nature (Levine 2003b). Since AA and tamoxifen have been associated with the induction of apoptosis (even in ER-negative cells), the induction of AA release by tamoxifen suggests a mechanism for cancer chemoprevention that does not require metabolism by cyclooxygenase (Levine 2003a).

4.2.3.4 Nitric Oxide

In the vascular system, nitric oxide (NO) is synthesized in the endothelium from L-arginine by endothelial nitric oxide synthase (eNOS) (Palmer et al. 1988). NO can diffuse rapidly to smooth muscles, causing relaxation via stimulation of soluble guanylate cyclase, followed by an increase in cyclic GMP (Rapoport et al. 1983) (Fig. 4.1). Subsequent activation of protein kinase G leads to phosphorylation of BK channels, which increases BK open probability and causes vasorelaxation (Patterson et al. 2002). A number of in vitro studies have demonstrated a direct relationship between SERMs (and estrogens) and acute activation of endothelial NO production (Kim et al. 1999; Simoncini and Genazzani 2000). Tamoxifen induces significant endothelium- dependent rapid relaxation in precontracted rabbit coronary arteries (Fig. 4.1). This relaxation is inhibited by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) and also by the estrogen receptor antagonist ICI 182,780 (Figtree et al. 2000). Similar L-NAME-sensitive acute effects caused by toremifene on thoracic aorta (Gonzalez-Pérez and Crespo 2003) and by the novel tryphenylethylene SERM idoxifene have been observed in aortic and mesenteric tromboxane A2-precontracted vessels (Christopher et al. 2002).

Benzothiphene SERMs have also been demonstrated to have an endothelium- dependent relaxing effect. In cultured human umbilical vein endothelial cells, clinically effective concentrations of raloxifene triggered a rapid and dose- dependent release of NO from endothelial cells (Simoncini and Genazzani 2000). Interestingly, raloxifene-induced NO production was abolished by the pure ER antagonist ICI 182,780, though it was not associated with changes in eNOS messenger RNA (Simoncini and Genazzani 2000). Indeed, raloxifene- induced NO production is due to an ER-dependent acute stimulation of eNOS enzymatic activity via a phosphatidylinositol 3-kinase (PI3K) pathway (Simoncini et al. 2002a). Similar ER- and NO-dependent vasodilation has been observed for raloxifene in coronary arteries (Figtree et al. 1999). These studies strongly argue in favor of raloxifene exerting a potentially important direct vasculoprotective effect by stimulating endothelial NO production.

Recently, it was found that EM-652 (acolbifene), a fourth-generation SERM exerting complete antiestrogenic effects on the breast and uterus, potently stimulates endothelial NO production in vitro and in vivo (Simoncini et al. 2002b). EM-652 triggers NO release by human umbilical vein endothelial cells through nongenomic mechanisms, rapidly activating eNOS via an ER- dependent sequential activation of MAPKs and PI3K/Akt pathways independently from gene transcription or protein synthesis. Moreover, EM-652 increases eNOS protein levels during prolonged treatment. Upon pharmacological comparison, EM-652 has been demonstrated to be markedly more potent than the SERMs raloxifene and tamoxifen in increasing NO synthesis from endothelial cells (Simoncini et al. 2002b).

4.2.4

Lipids, Membrane Lipids, and Fluidity

Triphenylethylene SERMs, like most anticancer drugs, are amphiphilic molecules of highly lipophilic character and likely to accumulate in membrane lipids and protein moieties. Experimental studies performed on artificial and biological membranes show that tamoxifen is enriched in lipid bilayers and affects both physical properties and composition (Custodio et al. 1993; Engelke et al. 2002; Wiseman et al. 1993). Thus, tamoxifen, 4-OH-tamoxifen, droloxifene (3-OH-tamoxifen), and other related compounds (such as 17-β-estradiol and cholesterol) inhibit metal-ion-dependent lipid peroxidation in liver micro- somes and brain liposomes in vitro (Wiseman et al. 1992b). Nonetheless, the chemical structure of tamoxifen indicates that it is unlikely to act as a chainbreaking antioxidant because it does not possess easily donatable hydrogen atoms (Wiseman et al. 1992a). Instead, the beneficial antioxidant action of tamoxifen seems to be related to its role as membrane stabilizer against lipid peroxidation, via decreased membrane fluidity. Direct evidence exists that tamoxifen decreases membrane fluidity and increases the physical order of pure phospholipid liposomes, human cancer breast cells, and retinal epithelium (Custodio et al. 1993; Engelke et al. 2002; Wiseman et al. 1993). In addition, a good correlation has been found between decreased membrane fluidity and the antioxidant ability of tamoxifen (Wiseman et al. 1993). Computer molecular modeling indicates that this property of tamoxifen is shared by cholesterol, which stabilizes membranes via interactions between the rigid hydrophobic structure of cholesterol and the saturated, monounsaturated, and polyunsaturated fatty acid chain of phospholipids (Wiseman et al. 1992a).

These effects on cell membrane physicochemical properties could also explain some side effects of tamoxifen. For instance, there is clear evidence that tamoxifen, prescribed for long-term low-dose therapy of breast cancer, induces retinopathy (Pavlidis et al. 1992), although the underlying mechanisms are largely unknown. Recently, studies performed on human retinal pigment epithelial cell line D407 have provided evidence for the involvement of cellular membranes in the cytotoxic action mechanism (Engelke et al. 2002). Tamoxifen increases the physical order of the lipid bilayer in D407 cells, which is accompanied by a compensatory decrease in the cholesterol content of the plasma membrane. In intracellular membranes, phosphatidylcholine content is reduced to 50% of the controls, and this reduction may be related to the sustained activation of protein kinase C via the phospholipase C pathway. Since increased plasma membrane fluidity, as well as sustained activation of protein kinase C, influences the rod outer segment binding and/or ingestion by retinal pigment epithelial cells, these membrane-mediated pathways might contribute to the tamoxifen-induced retinopathy (Engelke et al. 2002).

The compensatory effect of cholesterol observed in D407 cells have also been demonstrated in other cell lines (Cho et al. 1998; Holleran et al. 1998) and may well be a consequence of tamoxifen-induced severe inhibition of lanosterol (to cholesterol)-converting enzymes. In rat liver preparations and CHO cells, sterol ∆8-isomerase (IC50 ≈ 0.21-0.15 μM) was the most sensitive lanosterol-converting enzymes to inhibition (which is noncompetitive) by tamoxifen. In these cells, inhibition of ∆8-isomerase activity was paralleled by a decreased rate of [14C]-mevalonate incorporation into cholesterol (Cho et al. 1998). These findings might explain the fact that administration of tamoxifen to either humans or laboratory animals results in both a marked accumulation of sterol metabolites in serum and a drastic reduction in cholesterol. Clearly, these results provide important insights into the underlying mechanism(s) of tamoxifen’s cardioprotective role by interfering with cholesterol biosynthesis by lanosterol in mammals.

Paralleling the acute effect of tamoxifen and metabolites on cholesterol biosynthesis, triphenylethylenes have been reported to protect against the progression of coronary artery diseases in human and different atherosclerosis animal models by blocking the appearance of the atheromatous plaque, though the precise molecular mechanisms of cardioprotective remain unknown. Recently, evidence for Acyl-CoA:cholesterol acyl transferase (ACAT) being a putative target for tamoxifen has been provided. ACAT catalyzes the biosynthesis of cholesteryl esters, which are the major lipids found in atheromatous plaque, using both long-chain coenzyme-A-activated fatty acids and cholesterol as substrates (Chang et al. 1997). Tamoxifen inhibits ACAT in a concentration- dependent manner on rat liver microsomal extract (De Medina et al. 2004b). More importantly, tamoxifen is able to inhibit ACAT on intact macrophages stimulated with acetylated low-density lipoproteins and block the formation of foam cells, a step that precedes the formation of the atheromatous plaque (De Medina et al. 2004b). Molecular modeling reveals that tamoxifen displays threedimensional structural homology with Sah 58-035, a prototypical inhibitor of ACAT, and that the major structural element of tamoxifen responsible for this effect is the stilbene moiety present in the triphenylethylene backbone (De Medina et al. 2004a) (Fig. 4.4). This work constitutes the first evidence that tamoxifen is an inhibitor of ACAT and foam cell formation at therapeutic doses, and that this may account for its atheroprotective action.

The antioxidant effect of tamoxifen has also been postulated to underlie some beneficial cardiovascular effect of this and other SERMs. Oxidative damage of LDL plays an important role in the development of atherosclerosis, and it has been postulated that these highly lipophilic molecules stabilize LDL against lipid peroxidation by interaction between its hydrophobic rings and the polyunsaturated residues of the phospholipid layer of LDL (Resch et al. 2004; Wiseman 1994). In fact, tamoxifen (and more potently 4-OH-tamoxifen) and raloxifene can protect human LDL against Cu2+-dependent lipid peroxidation (Resch et al. 2004; Wiseman 1994). Recent studies have demonstrated that the in vitro antioxidant activity of raloxifene on LDL in postmenopausal women is substantially more potent than that of tamoxifen or 17-β-estradiol (Arteaga et al. 2003).

4.2.5

Specific Antiestrogen-Binding Sites (AEBS)

The existence of specific antiestrogen binding sites was initially reported by Sutherland and coworkers in 1980 in the microsomal fraction of human mammary and endometrial carcinomas (Sutherland et al. 1980). Since then, AEBS have been found in microsomes from most normal and tumori- genic tissues and cells investigated (Jordan and Murphy 1990; Lazier and Bapat 1988) including ER(-) cells (Mehta and DasGupta 1987). Such AEBS correspond to high-affinity (Kd 1 nM for tamoxifen in rat uterus), saturable binding sites that do not bind estradiol or ICI182,780 or ICI164,384 but compounds that retain both a basic aminoether side chain and a di- or tricyclic aromatic ring structure (De Medina et al. 2004a; Watts and Sutherland 1987) (Fig. 4.4). This shows that the important feature for tamoxifen binding to the AEBS is the presence of a dimethylmethano moiety linked to the aminoethoxy side chain (De Medina et al. 2004a) (Fig. 4.4). Such structure-activity relationship outcomes have prompted the development of selective AEBS ligands such as 1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene HCl (PBPE) or 4-(2-morpholinoethoxy) benzophenone (MBoPE), which has been used to demonstrate that these binding sites are membranous multi- proteic complexes that require phospholipids to bind tamoxifen (Kedjouar et al. 2004; Mesange et al. 2002). These studies provide strong evidence that AEBSs are hetero-oligomeric complexes including, among others, car- boxylesterase ES-10, liver fatty acid binding protein (FABP), epoxide hydrolase mEH, 3β-hydroxysterol-∆8-∆7-isomerase, and the 3β-hydroxysterol-∆7- reductase as subunits (reviewed in De Medina et al. 2004a). The latter two proteins are necessary and sufficient for tamoxifen binding in mammary cells (Kedjouar et al. 2004). Altogether, these data indicate that AEBSs are enzymatically linked to cholesterol metabolism at a postlanosterol step under acute regulation by triphenylethylene antiestrogens. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism and tumor growth control (De Medina et al. 2004b).

4.3

Final Considerations

Since the introduction in the early 1980s of tamoxifen, the first SERM used in clinical therapeutics, evidence has steadily grown that this compound is able to affect biochemical processes other than interacting, either in an agonistic or antagonistic manner, with intracellular ERs. Similarly, tamoxifen metabolites (4-OH-tamoxifen and N-desmethyltamoxifen), as well as other triphenylethylene derivatives, share many of the pleiotropic actions of tamoxifen. It seems now that benzothiphene derivatives led by raloxifene or LY156,758, as representatives of another class chemically different from SERMs, can also trigger acute effects in different molecular models. In general, these acute actions are non-ER-mediated and nongenomically transduced and take place with a short delay after exposure to SERMs. More importantly, we have emphasized the fact that these nongenomic actions take place at concentrations that fall within the low micromolar and submicromolar ranges, as indicated by the EC50 or IC50 values reported by different laboratories.

Continual administration of therapeutic doses of tamoxifen (about 40 mg daily as adjuvant for breast cancer) gives serum concentrations that increase linearly with tamoxifen intake, averaging 4-6 μM at the higher dose levels (Trump et al. 1992). Moreover, tamoxifen is a lipophilic compound, meaning its concentration in plasma membranes may be even higher than in serum. In fact, tissue concentration of tamoxifen is approximately 10- to 60-fold higher than in serum (Lien et al. 1991). These observations strengthen the notion that acute nongenomic effects of triphenylethylene and benzothiphene SERMS reviewed here are therapeutically and clinically relevant. Indeed, as we have discussed, some of these demonstrated effects account satisfactorily for both beneficial (i.e., vasorelaxation) and undesirable side effects (i.e., ocular toxicity) reported in individuals receiving different pharmacological therapies based on SERMs. Undoubtedly, these experimental and epidemiological observations will support the rationale for the design and development of new function- and tissue-specific SERMs.

In this sense, we have observed that, unlike tamoxifen, the quaternary derivative ethylbromide tamoxifen fails to block volume-sensitive chloride channels (as those found in lens fibers) in HeLa and C1300 neuroblastoma cells (unpublished data). Likewise, ethylbromide tamoxifen is totally ineffective on delayed rectifier K+ channels in NG108-15 cells, while tamoxifen is a potent reversible blocker (Allen et al. 2000). From this point of view, nonpermeant SERM derivatives are useful pharmacological tools for investigating whether binding sites in membrane targets are located in the extracellular domains of membrane proteins or, because they can partition into the membrane, interact at some level within the lipid bilayers.

As a final remark, though the spectrum of acute actions induced by SERMs from in vitro assays is considerable, it is widely assumed, and has been clinically proven, that tamoxifen toxicity is generally low. Several reasons can be advanced to account for the apparent discrepancy between in vivo and in vitro data. The concentrations of an unbound drug are likely to be different, and insufficient, in tissues and cells that, like nerve cells, have limited access to circulating plasma molecules. This observation could explain the low incidence of neurological disorders in patients under tamoxifen treatments, in spite of proven inhibitory in vitro effects found in critical voltage-dependent Na+ and K+ channels as well as neurotransmitter receptor cationic channels (see above). Furthermore, SERMs are protein bound in serum, and even in vitro the presence of serum proteins (like albumin and AAG) reduces the effectiveness of SERMs. Nevertheless, accumulation will occur after time, especially in long-term therapies, and therefore appropriate concentrations are likely to be reached.

Acknowledgements

Work from the authors’ laboratories has been supported by Grants SAF2001- 3614-C03-01, SAF2001-3614-C03-02, and SAF2004-08316-C02-01 (Ministerio de Ciencia y Tecnologi'a, Spain), PI2003/098 and PI2003/153 (Gobierno de Canarias, Spain), PI042640 (Instituto de Salud Carlos III, Spain), and from the Spanish Network of Neurological Research (CIEN, ISCIII-C03/06). R.M. is a fellow of the “Ramon y Cajal” Programme (Ministerio de Ciencia y Tecnologi'a, Spain).

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