Triphenylethylene Antiestrogens Research Articles

Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia—Impact on enzyme activity and response to cytotoxics

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1–10μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

Abstract 4528: Genotoxic and estrogenic properties of new antiestrogens

Abstract Long-term administration of tamoxifen (TAM) to breast cancer patients and women at high risk of developing this disease gives serious side effects, including endometrial cancer. These side-effects are due to the DNA damaging and/or estrogenic action of the drug. Several antiestrogens have been developed in last three decades, but were dropped out from the clinical trials due to their undesirable effects on the uterus. To avoid this serious side-effect, we have synthesized a new triphenylethylene antiestrogen, E-3-{4-[(E)-4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]-phenyl} acrylic acid (SS1020) and a new benzopyran antiestrogen, 2E-3-{4-[(7-hydroxy-2-oxo-3-phenyl-2H-chromen-4-yl)-methyl]-phenyl}-acrylic acid (SS5020). SS1020 and SS5020 had antitumor potential much higher than that of TAM or other antiestrogens at the human equivalent doses (TAM 0.3-1.0 mg/kg/day), against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats or human MCF-7 breast cancer xenograft implanted into athymic nude mice. To evaluate their genotoxic activity, the capability of forming hepatic DNA adducts in rats was determined. When rats were treated orally at a dose molar equivalent to TAM (20 mg/kg/day), a high level (∼1 adduct/105 nucleotides) of DNA adducts was detected in TAM-treated rats whereas no DNA adducts were observed in rats treated with SS1020 or SS5020. To evaluate the estrogenic potential, the uterotrophic activity of our compounds was determined in ovariectomized (OVX) rats at a dose molar equivalent to TAM (10 mg/kg/day), which is 30 times higher than the human equivalent dose of TAM. TAM had high uterotrophic activity while SS1020 and SS5020 did not present significant uterotrophic activity. Therefore, SS1020 and SS5020, lacking estrogenic and genotoxic actions, are expected not to induce reproductive cancers. Interestingly, the antitumor activities of several antiestrogens examined were, rather, increased significantly to the lowering uterotrophic activity evaluated with OVX-rats, but not to their antiproliferative activities against cultured estrogen receptor positive human MCF-7 breast cancer cells. The use of uterotrophic assay, not antiproliferative assay, may be a suitable screening system to exploit antiestrogen alternatives. Non-genotoxic and non-estrogenic SS1020 and SS5020, having strong antitumor potency superior to that of TAM, could be safer alternatives for breast cancer therapy and prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4528. doi:10.1158/1538-7445.AM2011-4528

Anti‐breast cancer potential of SS1020, a novel antiestrogen lacking estrogenic and genotoxic actions

Long-term treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. Several antiestrogens developed in last decades have been discontinued from clinical testing because of their undesirable effects on the uterus. To avoid such serious side-effect while increasing the drug's anti-breast cancer potential, new triphenylethylene antiestrogens, 2E-3-{4-[(E)-4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]-phenyl} acrylic acid (SS1020) and 2E-3-{4-[(Z)-4-chloro-1,2-diphenylbut-1-enyl]phenyl}acrylic acid (SS1010), were designed as safer alternatives. Unlike TAM, SS1020 does not present significant uterotrophic potential in rats; in contrast, SS1010, a compound removing a 4-OH moiety from SS1020, represented weak uterotrophic activity. The structurally related compounds 4-hydroxytamoxifen, toremifene, ospemifene, raloxifene (RAL) and GW5638 all have uterotrophic activity. In addition, SS1020 and SS1010 exhibit no genotoxic activity to damage hepatic DNA in rats. Therefore, SS1020 was selected as a safer antiestrogen candidate and used for evaluating the antitumor potential in animals. At the human equivalent doses of TAM, SS1020 had antitumor potential much higher than that of TAM, RAL and GW5638 against 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. The growth of human MCF-7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS1020. SS1020, lacking estrogenic and genotoxic actions and having strong antitumor potency superior to that of TAM and RAL, could be a safer alternative for breast cancer therapy and prevention.

Abstract 4617: Novel antiestrogens, SS1020 and SS5020, for breast cancer therapy and prevention

Abstract Tamoxifen (TAM) is widely used as a first-line endocrine therapy for breast cancer patients and as a prophylactic agent for women at high risk of developing this disease. Long-term administration of TAM has serious side effects, including endometrial cancer. These side-effects are due to the DNA damaging and/or estrogenic action of the drug. Raloxifene (RAL) approved as a chemopreventive agent for postmenopausal women at high risk for invasive breast cancer retains an estrogenic action. The poor bioavailability of RAL requires high doses to obtain an efficacy equivalent to that achieved with TAM. Several other antiestrogens developed were dropped out from the clinical trials due to their undesirable effects on the uterus. Therefore, development of new antiestrogen alternatives, but free of genotoxic and estrogenic potential, having significant antitumor potential beyond TAM and RAL is urgently required. SS1020 and SS5020 were developed as a new triphenylethylene antiestrogen and benzopyran antiestrogen, respectively, in our laboratory. Although TAM is a hepatic carcinogen and produces a high level of DNA damage in rats, both SS1020 and SS5020 did not produce such DNA damage. Unlike TAM and RAL, SS1020 and SS5020 had no detectable uterotrophic activity in OVX-rats treated even with a high dose (10 mg/kg). These indicate that SS1020 and SS5020 are free of genotoxic and estrogenic activities. The antitumor potential of SS1020 or SS5020 against 7,12-dimethyl-benz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer xenograft in athymic nude mice was superior to that of TAM and RAL. SS1020 has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL. In addition, the development of estrogen-induced mammary tumor in rats was completed inhibited by dietary intake of SS1020, indicating that this compound has preventive potential. Taken together, SS1020 and SS5020 lacking genotoxic and estrogenic actions have anti-breast cancer activity superior to that of TAM and RAL. Both SS1020 and SS5020 are safer and more effective antiestrogen candidates for breast cancer therapy and prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4617.

A New Safer and More Effective Triphenylethylene Antiestrogen (SS1020) for the Treatment and Prevention of Breast Cancer.

Abstract Background: Tamoxifen (TAM) is widely used as a first-line endocrine therapy for breast cancer patients and as a prophylactic agent for women at high risk of developing this disease. Besides the significant benefit, long-term administration of TAM has serious side effects, including endometrial cancer. These side-effects are due to the DNA damaging and/or estrogenic action of the drug. Raloxifene (RAL) approved as a chemopreventive agent for postmenopausal women at high risk for invasive breast cancer retains an estrogenic action. RAL has a poor bioavailability. This makes it difficult to achieve adequate bioavailability by oral administration in humans. High doses are required to obtain an efficacy quivalent to that achieved with TAM. Several other antiestrogens were dropped out from the clinical trials due to their undesirable effects on the uterus. Therefore, development of new antiestrogen alternatives, but free of genotoxic and estrogenic potential, having significant antitumor potential beyond TAM and RAL is urgently required for breast cancer therapy and prevention.Results: SS1020 was developed as a new triphenylethylene antiestrogen in our laboratory. Although TAM is a hepatic carcinogen and produces a high level of DNA damage in rats, SS1020 did not produce such DNA damage. Unlike TAM and RAL, SS1020 had no detectable uterotrophic activity in OVX-rats even treated with a high dose (10 mg/kg). These indicate that SS1020 is free of genotoxic and estrogenic activities. The antitumor potential of SS1020 against 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary carcinoma in rats and human MCF-7 breast cancer xenograft in athymic nude mice was superior to that of TAM and RAL. In addition, the development of estrogen-induced mammary tumor in rats was completed inhibited by dietary intake of SS1020, indicating that this compound has preventive potential. The bioavailability of SS1020 was approximately 35 times higher than that of RAL. The maximum blood concentration (493 ng/ml serum) of SS1020 was observed at 2 hour after the oral treatment while that of RAL (15.6 ng/ml) was at 6 hours, indicating that SS1020 is absorbed rapidly into the body. SS1020 has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL.Conclusion: Taken together, SS1020 lacking genotoxic and estrogenic actions has anti-breast cancer activity superior to that of TAM and RAL and has a preventive potential. SS1020 is a safer and more effective antiestrogen candidate for breast cancer therapy and prevention. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4085.

Identification of covalent attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain

Affinity labeling of human estrogen receptor α (ERα) by high affinity and antiestrogenic estradiol (E 2) 11β-derivatives, 11β-bromoacetamidoethoxyphenylE 2 (11BAEOPE 2) and 11β-bromoacetamidopentoxyphenylE 2 (11BAPOPE 2) was studied using glutathione- S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERα. To identify and quantify the electrophile covalent attachment sites on LBD, [ 14C]11BAEOPE 2- and [ 14C]11BAPOPE 2-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE 2 and 11BAPOPE 2, respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys 530Lys 531 LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the covalent attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD–ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys 530 could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common attachment to Cys 530 of estrogenic E 2 17α-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor α, J. Steroid Biochem. Mol. Biol. 98 (4–5), in press] and antiestrogenic E 2 11β-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.

Endometrial K-ras mutations in postmenopausal breast cancer patients treated with adjuvant tamoxifen or toremifene

Long-term use of tamoxifen is associated with a two- to threefold increased risk of endometrial cancer in postmenopausal women. Toremifene is another triphenylethylene antiestrogen, which is as effective as tamoxifen in postmenopausal breast cancer. Thus far, its use has not been associated with an increased risk of endometrial cancer. K-ras codon 12 mutations seem to be important in endometrial carcinogenesis, and these mutations have been found in endometrial samples of patients on tamoxifen. The present study was undertaken to investigate if there is any difference in the frequency of endometrial K-ras mutations among patients treated with tamoxifen or toremifene. Endometrial samples were taken from 23 postmenopausal breast cancer patients (tamoxifen, n = 11; toremifene, n = 12) before and after 36 months of treatment. DNA was isolated from formalin-fixed paraffin-embedded samples using a routine proteinase K digestion protocol. K-ras mutations in codon 12 were screened using real-time PCR and melting curve analysis in LightCycler equipment. Wild-type PNA oligomer was used to increase the sensitivity of the assay. All baseline samples contained wild-type K-ras, while 10/23 (43%) of the follow-up samples carried a codon 12 mutation. Mutations were identified in 3 of the 11 in the tamoxifen group and in 7 of the 12 in the toremifene group. Seven were transitions (G-->A), and three were transversions (two G-->T, one G-->C). One of the mutations in the toremifene group was associated with a polypoid endometrium. All the other mutations were found in an atrophic (n = 6) or proliferative (n = 3) endometrium. Both tamoxifen and toremifene induce endometrial K-ras codon 12 mutations. The significance of this finding to endometrial carcinogenesis remains to be elucidated.

Effects of tamoxifen on ischemia-induced angiogenesis in the mouse lung.

Obstruction of pulmonary blood flow in the mouse lung causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 5-6 days [Mitzner et al. Am J Pathol 2000; 157(1): 93-101]. Tamoxifen, a triphenylethylene antiestrogen, has been shown to be effective in limiting tumor growth, possibly because of inhibition of angiogenesis. We investigated the effects of tamoxifen on blood vessel development after left pulmonary artery ligation (LPAL). Timed-release pellets of either tamoxifen (free base/15 mg over 21 days) or placebo carrier were implanted subcutaneously in male C57BL/6J mice 6-8 weeks of age. Two days after pellet implantation, the left pulmonary artery was permanently obstructed by suture ligation. New systemic vessel growth was assessed after left ventricular injection of fluorescence labeled microspheres. Tamoxifen slowed the formation of functional blood vessels seven days after LPAL. By 14 days, however, no difference was observed between tamoxifen and placebo treated mice with systemic perfusion to the left lung reaching a maximum of 3.8% and 4.7% of cardiac output respectively. No change in VEGF mRNA expression was observed until 14 days after LPAL when a small increase (2-fold) was observed in both placebo and tamoxifen treated lungs. However, VEGF protein was elevated in both tamoxifen and placebo lungs 24 h after LPAL (approximately 4-fold). These changes in VEGF protein may be due to the presence of trapped inflammatory cells observed in lung sections at this early time point. Although tamoxifen appeared to slow the progression of blood vessel formation, it did not affect VEGF mRNA, therefore likely acting through an estrogen receptor-independent mechanism.

Triphenylethylene antiestrogen-induced acute relaxation of mouse duodenal muscle. Possible involvement of Ca 2+ channels

The nonsteroidal antiestrogens tamoxifen, 4-OH-tamoxifen and toremifene rapidly inhibited spontaneous contractile activity and reduced basal tone in isolated mouse duodenal muscle. Inhibition was rapid in onset (∼2 min) and was not mimicked by the pure steroidal antiestrogen 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17β-diol (ICI182,780) indicating the involvement of non-genomic mechanisms. Inhibition by tamoxifen and 4-OH-tamoxifen were observed at concentrations comparable to those reached in antiestrogen adjuvant therapy. Antiestrogen-relaxed tissues showed no response to KCl depolarisation or K + channel blockade but displayed clear transient responses to acethylcholine or to the muscarinic receptor agonist carbachol. Frequency analysis showed that spontaneous activity could be readily restored in antiestrogen-relaxed tissues by the exposure to the L-type Ca 2+ channel agonist 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-pyridine-3-carboxilic acid methyl ester (BAY K8644). Our experiments suggest that triphenylethylene antiestrogens relax duodenal intestinal muscle via a mechanism that involves inhibition of L-type Ca 2+ channels but not activation of K + channels.

Activation of Maxi Cl(-) channels by antiestrogens and phenothiazines in NIH3T3 fibroblasts.

The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.

Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells.

Previously we demonstrated that multidrug-resistant (MDR) cancer cells have elevated levels of a glycosylated form of ceramide, glucosylceramide. Here we compared ceramide metabolism and ceramide toxicity in MCF-7 and in adriamycin-resistant (MCF-7-AdrR) human breast cancer cells. MCF-7-AdrR cells were resistant to C6-ceramide (1-10 microM); however, in MCF-7 cells treated with C6-ceramide, viability dropped sharply. Ceramide, when supplemented, was not metabolized by MCF-7 cells. In contrast, ceramide was efficiently converted to glucosylceramide by MCF-7-AdrR cells. Analysis of extracellular [3H]ceramide in radiolabeled cells showed that MCF-7-AdrR cells do not have an enhanced capacity to efflux ceramide compared with MCF-7 cells. Triphenylethylene anti-estrogens, known modulators of drug resistance, were effective inhibitors of ceramide conversion to glucosylceramide, suggesting that blocking ceramide metabolism plays a role in chemosensitization. The anti-progestine, RU486, also blocked glucosylceramide synthesis in cells; however, LY117018, a raloxifene analog, was without influence. We propose that an enhanced capacity to glycosylate ceramide as evidenced in MCF-7-AdrR cells, is a molecular determinant of drug resistance, particularly as regards resistance to ceramide-enhancing agents such as anthracyclines, ionizing radiation, and tumor necrosis factor-alpha.

Structure–Activity Relationships for Triphenylethylene Antiestrogens on Hepatic Phase-I and Phase-II Enzyme Expression

To better understand the mechanism(s) by which tamoxifen induces rat hepatic CYPIIB2 and suppresses GSTA1, structure–activity studies were performed. Compounds employed in these studies included: tamoxifen, fixed-ring tamoxifen, ethylated fixed-ring tamoxifen, pyrrolidino-tamoxifen, 4-iodotamoxifen, idoxifene, and toremifene. With respect to GSTA1 suppression, tamoxifen, fixed-ring tamoxifen, 4-iodotamoxifen, idoxifene, and toremifene were all potent suppressors of GSTA1, while ethylated fixed-ring tamoxifen and pyrrolidino-tamoxifen were completely without activity. The results suggest that the aminoethoxy side chain plays a crucial role in GSTA1 suppression, and that 4-iodination may potentiate this activity. With respect to induction of CYPIIB2, tamoxifen, fixed-ring tamoxifen, and ethylated fixed-ring tamoxifen were inducers of this enzyme, while toremifene and 4-iodotamoxifen were inactive, suggesting that the aminoethoxy side chain is not a structural determinant of CYPIIB2 induction. Because ethylated fixed-ring tamoxifen, toremifene, and 4-iodotamoxifen had differential activities in the two assays, we conclude that CYPIIB2 induction and GSTA1 suppression by triphenylethylenes are the result of two separate and distinct mechanistic pathways. Structure–activity relationships for GSTA1 suppression and CYPIIB2 induction were compared with previously published relationships for triphenylethylene: 1) estrogen receptor relative binding affinity; 2) calmodulin antagonism; 3) antiuterotrophic activity; and 4) antagonism of MCF-7 cell growth. No clear correlation was observed between the effects on CYPIIB2 and these other four activities, suggesting no relationship between the mechanisms responsible for these effects. Similarly, no precise correlation was observed between GSTA1 suppression and these other activities, although rough similarities were observed for relative binding affinity and antiuterotrophic activity. This suggests that the mechanisms responsible for CYPIIB2 induction and GSTA1 suppression are not related to the mechanisms of action for these other documented activities, and may represent different mechanistic pathways.

Genotoxicity Studies with the Antiestrogen Toremifene

ABSTRACTToremifene, a second-generation triphenylethylene antiestrogen used clinically in the chemotherapy of breast cancer and some other cancers, differs in its nonclinical toxicology from its first-generation congener tamoxifen. Tamoxifen produces DNA adducts and tumors in rat liver, whereas assays for DNA adduct formation with toremifene have been negative to weakly positive, and toremifene does not produce liver tumors in rats. to evaluate further toremifene for possible genotoxicity, it was tested in three standard, in vitro assay—reversion of bacterial point mutations, unscheduled DNA synthesis in cultured hepatocytes from two rat strains, and cytogenetics of human lymphocytes in primary cultur—and in one in vivo assay, the mouse, erythrocyte micronucleus assay. the three in vitro assays were conducted with toremifene at up to the limit of cytotoxicity (100 to 250 ng/ml, depending on the system). the bacterial mutagenicity and lymphocyte chromosome aberration assays were performed both in the presence and absence of metabolic activation by Araclor-induced, rat liver S-9, while the hepatocyte unscheduled DNA synthesis assay provides intrinsic bioactivation. to test for chromosome damage in vivo, mice were administered up to 2g/kg toremifene once by gavage, and bone marrow was harvested daily, for three days. Normochromatic and polychromatic bone marrow erythrocytes were examined for micronuclei. Toremifene lacked genotoxicity or myelotoxicity detectable by any of the above assays. These findings, together with the reported absence of DNA binding in rat liver, provide evidence that toremifene is not genotoxic.

Triphenylethylene antiestrogens induce uterine vascular endothelial growth factor expression via their partial estrogen agonist activity

Estradiol induces vascular endothelial growth factor (VEGF) expression in the rat uterus and this may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue. Triphenylethylene antiestrogens such as tamoxifen have mixed agonist/antagonist activity and their specific effects are tissue and gene specific. These drugs exhibit primarily antiestrogenic actions in mammary tissue and are thus used for the treatment of breast cancer. These drugs are also suggested to be inhibitors of angiogenesis. However, uterine side effects of tamoxifen are thought to stem largely from the agonist activity of the drug in this tissue. Since side effects of tamoxifen such as uterine bleeding and endometrial cancer seem likely to have an angiogenic component, we have examined the effects of this drug, its metabolite, 4-hydroxy-tamoxifen and two additional triphenylethylene antiestrogens, nafoxidine and clomiphene, on the expression of VEGF and another estrogen regulated gene, c- fos, using the rat uterus as an experimental system. All four compounds increase uterine VEGF and c- fos mRNA levels indicating that the triphenylethylene class of antiestrogens are predominantly agonists for the induction of these genes in the uterus.

Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11 beta-n-alkyl derivatives bearing a terminal electrophilic group: antiestrogenic and cytotoxic properties.

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11 beta-ethyl (C2), 11 beta-butyl (C4), or 11 beta-decyl (C10) derivatives of estradiol bearing an 11 beta-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4), bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the order of increasing affinity (i) relative to the 11 beta-alkyl arm was ethyl < butyl and (ii) relative to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy. Regardless of the conditions used, including prolonged exposure of the receptor to various pH levels (7-9) and temperatures (0-25 degrees C), the extent of receptor affinity labeling by the 11 beta-ethyl and 11 beta-butyl compounds, if any, was under 10%. This was in sharp contrast to results obtained using 11 beta-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor hormone-binding sites with an EC50 of approximately 10 nM. Estrogenic and antiestrogenic activities of the compounds were determined using the MVLN cell line, which was established from the estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant estrogen-responsive luciferase gene. The two 11 beta-ethyl compounds were mainly estrogenic, whereas the three 11 beta-butyl and the 11 beta-decyl compounds essentially showed antiestrogenic activity. The fact that the chemical reactivities of 11 beta-ethyl and 11 beta-butyl compounds were not compromised by interaction with the estrogen receptor made the synthesized high-affinity compounds potential cytotoxic agents which might be able to exert either (i) a specific action on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All electrophiles were able to irreversibly suppress expression of the luciferase gene; the antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than 4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than 4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11 beta-ethyl and 11 beta-butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues, by coupling them with a toxic moiety.

Toremifene. A review of its pharmacological properties and clinical efficacy in the management of advanced breast cancer.

The triphenylethylene antiestrogen toremifene is a chlorinated derivative of the antiestrogen tamoxifen, an agent which has been widely and successfully used in the treatment of breast cancer. Clinical trials investigating the efficacy of toremifene as first-line endocrine therapy in postmenopausal women with advanced breast cancer (estrogen receptor status positive or unknown) have shown this drug to have similar antitumour activity to that of tamoxifen. In multicentre comparative trials, objective responses (complete and partial) occurred in 20 to 29% of patients treated with toremifene (60 to 240 mg/day) and in 19 to 37.5% of tamoxifen (20 or 40 mg/day) recipients. The duration of response, time to disease progression and median overall survival time were generally similar in both treatment groups. Toremifene is well tolerated. Most drug-related adverse effects are mild or moderate in severity and rarely necessitate discontinuation of therapy. The tolerability profile of toremifene is similar to that reported for tamoxifen, the most common adverse effects being hot flushes, sweating, nausea and/or vomiting, dizziness, oedema, and vaginal discharge and/or bleeding. Thus, toremifene provides an equally effective and well tolerated alternative to tamoxifen for the first-line endocrine therapy of postmenopausal advanced breast cancer. Preclinical studies showing toremifene to have a lower carcinogenic potential than tamoxifen indicate that toremifene may be a preferable agent for long term treatment regimens; however, these findings require confirmation in the clinical setting.

Clomiphene prevents cancellous bone loss from tibia of ovariectomized rats.

Estrogen inhibits postmenopausal bone loss and decreases fracture risk. Unfortunately, estrogen replacement therapy has many undesirable side effects, the majority of which are due to stimulation of reproductive tissues. Tissue specific estrogen agonists provide a promising new alternative to natural estrogens for hormone replacement. Clomiphene (CLO) is a substituted triphenylethylene antiestrogen based on its ability to antagonize estrogen-mediated uterine growth in rodents. CLO is used clinically for the treatment of disorders of ovulation in patients wishing to become pregnant. In order to determine whether CLO has tissue selective actions, we performed a dose-response study in adult (6-month-old) ovariectomized (OVX'd) rats. The rats received daily (gavage) doses of either 17 alpha-ethynyl estradiol (E) (0.1 mg/kg) or CLO (0.01-10 mg/kg) daily for 5 weeks. Long-term loss of ovarian function had no effect on serum cholesterol, greatly decreased uterine weight, cancellous bone area and trabecular number, and increased bone formation rate (BFR) and osteoblast and osteoclast perimeters. E treatment of OVX'd rats prevented uterine atrophy, greatly lowered cholesterol, and prevented many of the bone changes. CLO was a very weak estrogen agonist in supporting uterine weight, a partial agonist in reducing serum cholesterol, and an excellent agonist in maintaining normal bone mass and indices of bone turnover. We conclude from these studies that CLO exhibits pronounced tissue selective estrogen agonism in the rat. Specifically, CLO is effective in preventing cancellous bone loss in the OVX'd rats and has minimal uterotrophic activity.

Effects of toremifene on neonatal rat uterine growth and differentiation.

In the developing rodent uterus, the estrogen agonist activity of triphenylethylene antiestrogens such as tamoxifen alters uterine luminal epithelium morphology and inhibits uterine gland genesis. We examined uterine growth and differentiation in female offspring from date-mated Sprague-Dawley rats given the structurally related antiestrogen, toremifene, by s.c. injection in 10 microl of sesame oil on postnatal days (PND) 1-5, 10-14, or 20-24. Toremifene given on PND 10-14, a period of rapid uterine gland differentiation, caused a dose-related increase in uterine weight, tripled luminal epithelium cell height, and completely inhibited uterine gland development on PND 14 at doses of 10 microg or higher. Based on this dose-response analysis, a 10-microg dose of toremifene was chosen to assess uterine development after neonatal exposure (PND 1-5). Uterine weights and luminal epithelium cell heights were significantly increased by toremifene on PND 5 but returned to control levels by PND 26. Uterine gland numbers were reduced to 50% those of controls on PND 26. Dose-related uterine weight and luminal epithelium cell height increases were also observed in rats given toremifene on PND 20-24. This estrogen agonist activity of toremifene, revealed primarily in the uterine luminal epithelium, indicates that toremifene is developmentally toxic.

Both estradiol and tamoxifen decrease proliferation and invasiveness of cancer cells transfected with a mutated estrogen receptor.

Previous studies have shown that, after wild-type estrogen receptor (ER) transfection in ER-negative breast cancer cells, estradiol but not tamoxifen prevents growth, invasiveness and metastasis of these cells in mice. Because an ER mutation at position 400 converts the triphenylethylene antiestrogen, OH-tamoxifen into a full estrogen agonist, we transfected this mutated form of human ER in an ER-negative rat cancer cell line. This was aimed at inducing an inhibitory, estrogen-like response of tamoxifen in these cells. In two stable ER-positive transfectants, OH-tamoxifen inhibited cell growth and invasiveness in vitro as efficiently as estradiol. The pure antiestrogen, ICI 164,384, was not agonistic alone and antagonized estrogen action. In contrast, the three compounds were ineffective in control mock-transfected cells. When injected into ovariectomized nude mice, ER-negative mock-transfected cells formed tumours which were significantly stimulated by estradiol and inhibited by tamoxifen treatment. This indicates that estradiol and tamoxifen altered the growth of ER-negative tumours via a general effect on the host response. Surprisingly, the hormone responsiveness of ER-positive tumours developed from ER-transfected cells did not significantly differ from that of ER-negative (mock-transfected) tumours. We conclude that transfection of a mutated human estrogen receptor inhibited, through an estrogenic activity of tamoxifen, the growth and invasiveness of these cancer cells in vitro. However, the low expression of ER did not allowed us to obtain the same effect of tamoxifen in vivo.

Antagonism of estrogen receptor and calmodulin association by antiestrogens is not dependent on an interaction with calmodulin

Previously, two antiestrogenic estradiol derivatives (3 and 4) bearing the basic side chain of tamoxifen were shown to impede the binding of the estrogen receptor (ER) to Calmodulin (CaM)-Sepharose. In this study, the interaction of these and related compounds with calmodulin was examined using the cyclic AMP phosphodiesterase (cAMP-PDE) assay. Neither of the steroids gave any significant inhibition of the calmodulin dependent cAMP-PDE activity up to a final concentration of 20 μM. For comparison, tamoxifen and nafoxidine produced ic 50 values of 6.7 μM ±1.0 and 7.4 μM ±1.1, respectively. In addition, a comparison was made of the activity of some triphenylethylene derivatives against CaM dependent cAMP-PDE and the ER-CaM Sepharose assays, but no relationship was observed. Overall, these results demonstrate that inhibition of the ER-CaM association by various steroidal and triphenylethylene antiestrogens does not relate to antagonism of calmodulin function or their binding affinity for the estrogen receptor.