afimoxifene and droloxifene

Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormone-dependent breast cancer. However, tamoxifen has been shown to increase the risk of endometrial cancer which has stimulated research for new effective antiestrogens, such as droloxifene and toremifene. In this study, the potential for these compounds to cause cytotoxic effects was investigated. One potential cytotoxic mechanism could involve metabolism of droloxifene and toremifene to catechols, followed by oxidation to reactive o-quinones. Another cytotoxic pathway could involve the oxidation of 4-hydroxytoremifene to an electrophilic quinone methide. Comparison of the amounts of GSH conjugates formed from 4-hydroxytamoxifen, droloxifene, and 4-hydroxytoremifene suggested that 4-hydroxytoremifene is more effective at formation of a quinone methide. However, all three substrates formed similar amounts of o-quinones. Both the tamoxifen-o-quinone and toremifene-o-quinone reacted with deoxynucleosides to give corresponding adducts. However, the toremifene-o-quinone was shown to be considerably more reactive than the tamoxifen-o-quinone in terms of both kinetic data as well as the yield and type of deoxynucleoside adducts formed. Since thymidine formed the most abundant adducts with the toremifene-o-quinone, sufficient material was obtained for characterization by (1)H NMR, COSY-NMR, DEPT-NMR, and tandem mass spectrometry. Cytotoxicity studies with tamoxifen, droloxifene, 4-hydroxytamoxifen, 4-hydroxytoremifene, and their catechol metabolites were carried out in the human breast cancer cell lines S30 and MDA-MB-231. All of the metabolites tested showed cytotoxic effects that were similar to the parent antiestrogens which suggests that o-quinone formation from tamoxifen, droloxifene, and 4-hydroxytoremifene is unlikely to contribute to their cytotoxicity. However, the fact that the o-quinones formed adducts with deoxynucleosides in vitro implies that the o-quinone pathway might contribute to the genotoxicity of the antiestrogens in vivo.

Topics: Animals; Antineoplastic Agents; Benzoquinones; Breast Neoplasms; Cell Survival; Deoxyribonucleosides; DNA Adducts; Female; Glutathione; Indolequinones; Indoles; Microsomes, Liver; Quinones; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Tamoxifen; Toremifene; Tumor Cells, Cultured

Some studies have shown the potential relevance of the oxidation products of 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative activation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen (3OHTAM), 1-(4-hydroxyphenyl)-1, 2-diphenylethene, and phenol (PhOH), catalyzed by myeloperoxidase (MPx), horseradish peroxidase (HRP), lactoperoxidase, mushroom tyrosinase, and nonenzymatic initiators in vitro under a variety of conditions and in cells. Differences in activation of the phenolics by the enzymes were directly compared using cis-parinaric acid (PnA)-loaded human serum albumin. All phenolics were substrates for the enzymes, but MPx only weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded metabolically with PnA so that effects on phospholipids could be monitored by HPLC with fluorescence detection, PhOH plus H2O2 caused massive oxidation across all phospholipid classes. 4OHTAM dose-dependently protected phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poor substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of those three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trapping of glutathionyl radicals (GS*) with DMPO and quantifying the ESR-silent nitrone form of the GS-DMPO adduct by HPLC showed that neither 3OHTAM plus H2O2 nor 4OHTAM plus H2O2 caused a significant level of GSH oxidation with isolated MPx, nor did the latter in HL60 cells, whereas PhOH plus H2O2 was a potent source of GS* in both systems. Both 4OHTAM and 3OHTAM formed the nitrone adduct under cell-free conditions when activated with HRP. The data show that the substrate specificity of a given (myelo)peroxidase determines if a phenolic exerts pro- (through generation of reactive phenoxyl radicals) or antioxidant (through radical scavenging) properties in intracellular environments.

Topics: Animals; Antioxidants; Aorta; Cell Survival; Cells, Cultured; Estrogen Antagonists; Fatty Acids; Glutathione; HL-60 Cells; Humans; Lipid Peroxidation; Muscle, Smooth, Vascular; Oxidants; Oxidation-Reduction; Peroxidase; Peroxidases; Phenol; Phospholipids; Rats; Serum Albumin; Tamoxifen

Earlier study suggested that 3,4-dihydroxytamoxifen (tam catechol), a tamoxifen metabolite, is proximate to the reactive intermediate that binds covalently to proteins and possibly to DNA (). The current study demonstrates that rat and human hepatic cytochrome P-450s (CYPs) catalyze tam catechol formation from tamoxifen (tam), 3-hydroxy-tam (Droloxifene), and 4-hydroxy-tam (4-OH-tam). Higher levels of catechol were formed from 4-OH-tam and 3-hydroxy-tam than from tam. Evidence that human hepatic CYP3A4 and 2D6 catalyze the formation of tam catechol from 4-OH-tam and supportive data that the catechol is proximate to the reactive intermediate, was obtained: 1) There was a good correlation (r = 0.82; p

Topics: Animals; Biotransformation; Catechols; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Estrogen Antagonists; Humans; Hydroxylation; In Vitro Techniques; Liver; Mixed Function Oxygenases; Pentobarbital; Protein Binding; Rats; Rats, Sprague-Dawley; Tamoxifen

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4-hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4-hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4-hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls exce

Topics: Aneuploidy; Animals; Anticarcinogenic Agents; Cell Line; Cell Nucleus; Clomiphene; Female; Humans; Liver; Lymphocytes; Micronucleus Tests; Rats; Rats, Wistar; Tamoxifen; Toremifene; Transfection

Since estrogens play a predominant role in the development and growth of human breast cancer, antiestrogens represent a logical approach to the treatment of this disease. The present study compares the effects of the novel nonsteroidal anti-estrogen EM-800 and related compounds with those of a series of anti-estrogens on basal and 17 beta-estradiol (E2)-induced cell proliferation in human breast cancer cell lines. In the absence of added E2, EM-800 and related compounds failed to change basal cell proliferation, thus showing the absence of intrinsic estrogenic activity in the ER-positive T-47D, ZR-75-1 and MCF-7 cell lines. The stimulation of T-47D cell proliferation induced by 0.1 nM E2 was competitively blocked by a simultaneous incubation with EM-652, EM-800, OH-tamoxifen, OH-toremifene, ICI 182780, ICI 164384, droloxifene, tamoxifen and toremifene at apparent Ki values of 0.015, 0.011-0.017, 0.040-0.054, 0.043, 0.044, 0.243 and 0.735 nM, approx. 10 nM and > 10 nM, respectively. Similar data were obtained in ZR-75-1 and/or MCF-7 cells. Moreover, EM-652 was 6-fold more potent than OH-Tamoxifen in inhibiting the proportion of cycling MCF-7 cells. Our data show that EM-800 and EM-652 are the most potent known antiestrogens in human breast cancer cells in vitro and that they are devoid of the estrogenic activity of OH-tamoxifen and droloxifene suggested by stimulation of cell growth in the absence of estrogens in ZR-75-1 and MCF-7 cells.

Topics: Benzopyrans; Breast Neoplasms; Cell Division; Estradiol; Estrogen Antagonists; Female; Humans; Propionates; Tamoxifen; Tumor Cells, Cultured

Tamoxifen and its metabolite 4-hydroxytamoxifen can both exist as geometrical isomers. Trans-tamoxifen is an oestrogen receptor antagonist and is used for the treatment of breast cancer. Trans-4-hydroxytamoxifen is 100 times more anti-oestrogenic than trans-tamoxifen. The cis isomers of tamoxifen and 4-hydroxytamoxifen are oestrogenic and weakly anti-oestrogenic or oestrogenic respectively. Both isomers of 4-hydroxytamoxifen have been detected in breast tumours of patients treated with trans-tamoxifen and it has been proposed that enzymatic isomerization of 4-hydroxytamoxifen occurs in vivo, resulting in resistance to tamoxifen therapy. We have investigated the isomerization of 4-hydroxytamoxifen by human liver microsomes and whether it is mediated by cytochromes P450. Microsomes from five of the 12 livers examined catalysed the interconversion of trans- and cis-4-hydroxytamoxifen (0.52 microM) when incubated for 40 min with an NADPH-generating system. Between 51 and 64% conversion of trans-4-hydroxytamoxifen was observed. Cis-4-hydroxytamoxifen was also converted to trans-4-hydroxytamoxifen (range 22-27%). Incubations with control, heat-treated microsomes resulted in approximately 1% isomerization of trans-4-hydroxytamoxifen. The extent of isomerization of trans- to cis-4-hydroxytamoxifen observed in microsomes from the other seven livers (range 2-8%) did not greatly exceed that seen in heat-inactivated microsomes. Enzymatic isomerization required NADPH and was inhibited by SKF 525A and ketoconazole, indicating the involvement of cytochromes P450. Enzymatic isomerization of trans-tamoxifen and trans-droloxifene (the 3-hydroxy synthetic analogue of tamoxifen) was not observed. These findings may have implications for the safe and effective use of tamoxifen.

Topics: Adult; Biotransformation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Resistance; Female; Genetic Variation; Humans; Isomerism; Ketoconazole; Male; Microsomes, Liver; Middle Aged; NADP; Proadifen; Tamoxifen

The effects of tamoxifen, three of its in vivo metabolites and 3-hydroxytamoxifen on cellular proliferation and the induction of four oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-25 and cathepsin D) have been measured in MCF-7 breast cancer cells in phenol red-free culture medium. Tamoxifen and 3-hydroxytamoxifen acted as partial oestrogens to stimulate cell growth and the levels of the pNR-2 and pNR-25 RNAs. They were full oestrogens for the induction of cathepsin D RNA and induced the pNR-1 RNA above the level found in oestrogen-treated cells. N-Desmethyltamoxifen and 4-hydroxytamoxifen behaved like tamoxifen except that N-desmethyltamoxifen did not induce the pNR-2 RNA and was only a partial oestrogen for the induction of cathepsin D RNA, and 4-hydroxytamoxifen did not induce the pNR-2 or pNR-25 RNAs. In the presence of oestradiol, the four anti-oestrogens prevented the stimulation of growth and reduced (pNR-2 and pNR-25) or increased (pNR-1) the RNA levels to those present in MCF-7 cells treated with the anti-oestrogen alone. In contrast, for cathepsin D RNA levels there was a synergistic effect of the anti-oestrogens and oestradiol. The concentration at which each anti-oestrogen was effective was related to its affinity for the oestrogen receptor. Metabolite E was a full oestrogen for the induction of cell proliferation and the oestrogen-regulated RNAs. pNR-25 and pNR-2 RNA levels correlated most closely with effects on cell proliferation. These RNAs are therefore potentially the most useful for predicting the response of breast cancer patients to tamoxifen therapy.

Topics: Breast Neoplasms; Cell Division; Estradiol; Estrogen Antagonists; Gene Expression Regulation; Humans; RNA, Neoplasm; Tamoxifen; Tumor Cells, Cultured

Topics: Animals; Body Weight; Humans; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Organ Size; Receptors, Estrogen; Tamoxifen

Effects of tamoxifen (TAM), nafoxidine (NFA), 4-hydroxytamoxifen (4-OH-TAM), 3-hydroxytamoxifen (3-OH-TAM), and medroxyprogesterone acetate (MPA) on the clonogenic growth of a hormone-responsive human breast cancer cell line (MCF-7) and its tamoxifen-resistant variant (R-27) were studied. TAM, (10(-6)M) showed an inhibitory effect on the colony formation in a plastic dish of MCF-7 cells only in medium containing DDC-treated FCS (E2(-) medium). With the presence of E2 (10(-8)M) in the medium (E2(+) medium), TAM did not show any effect on cell growth. Irrespective of the presence or absence of E2 in the medium, there was no inhibitory effect of TAM on the clonogenic growth of R-27 cells. The ID50 values, expressed as the suppression of plating efficiencies, obtained by adding NFA, 4-OH-TAM, 3-OH-TAM, and MPA to the MCF-7 cells were shown to be 2 X 10(-7)M, less than 10(-8)M, 1 X 10(-7)M, and 4 X 10(-7)M, respectively in the E2 (-) medium, and 2 X 10(-6)M, 2 X 10(-7)M, 2 X 10(-6)M, and 4 X 10(-8)M respectively in the E2 (+) medium. For the R-27 cells, ID50 values obtained by adding NAF, 4-OH-TAM, 3-OH-TAM, and MPA were 7 X 10(-7)M, 5 X 10(-8)M, 4 X 10(-7)M, and 6 X 10(-8)M, respectively in the E2(-) medium, and 2 X 10(-6)M, 2 X 10(-6)M, greater than 5 X 10(-6)M, and 1 X 10(-8)M, respectively in the E2(+) medium. These results suggest that the antiestrogens used produce their suppressive effects on cell growth depending on the E2 concentration in the medium in these estrogen-responsive cell lines, and that the monohydroxytamoxifens are more potent than TAM in suppressing cell growth. The effects of MPA are shown to be different from the antiestrogen used in that MPA inhibited the growth of both TAM-sensitive and-resistant cells, independent of the presence or absence of E2 in the medium. The R-27 cell line, a variant of the MCF-7 cell line, appears to be a good model for studying antiestrogen resistance and for evaluating the effectiveness of agents against endocrine-resistant breast cancer cells.

Topics: Breast Neoplasms; Cell Division; Cell Line; Cells, Cultured; Drug Resistance; Estrogen Antagonists; Female; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Nafoxidine; Tamoxifen

This study demonstrates in vivo and in vitro properties of the non-steroidal antiestrogens tamoxifen (TAM), 4-OH-tamoxifen (4-OH-TAM) and 3-OH-tamoxifen (K 060 E). In immature rabbit uteri 4-OH-TAM and K 060 E bound to the respective estrogen receptors with a ten-fold higher affinity than TAM. Furthermore, K 060 E exhibited less agonistic (estrogenic) but higher antagonistic (antiestrogenic) activity in the immature rat uterus than TAM and 4-OH-TAM (change of uterine weight). The ratio of agonistic vs antagonistic effect of K 060 E was distinctly lower than in TAM and 4-OH-TAM. In addition, K 060 E reduced by approximately 45% the growth of the transplantable Fisher rat mammary tumor (R 3230 AC) as compared with TAM (33%). We assume that, due to the higher antitumor activity, K 060 E (3-OH-TAM) is a better antiestrogen than TAM.

Topics: Adenocarcinoma; Animals; Binding, Competitive; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Female; Mammary Neoplasms, Experimental; Organ Size; Rabbits; Rats; Rats, Inbred Strains; Receptors, Estrogen; Tamoxifen; Uterus

The antiestrogenic action of 3-hydroxytamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1-(3-hydroxyphenyl)-2 -phenylbut-1-ene] was characterized in vitro and compared with that of tamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. The relative binding affinities of 3-hydroxytamoxifen to estrogen receptor were 3.3% in cytosol of MCF-7 cells and 1.5% in human mammary carcinoma cytosol compared to values of 0.2 and 0.3% for tamoxifen (the affinity of 17 beta-estradiol considered to be 100%). The concentration of 3-hydroxytamoxifen necessary to suppress the 17 beta-estradiol-induced growth stimulation of MCF-7 cells was about tenfold lower than that for tamoxifen. The induction of progesterone receptor in MCF-7 cells by 17 beta-estradiol was inhibited by 3-hydroxytamoxifen. In the absence of 17 beta-estradiol, 3-hydroxytamoxifen gave rise to a moderate increase in the progesterone receptor levels, which demonstrates the partially estrogenic character of hydroxytamoxifen.

Topics: Binding, Competitive; Breast Neoplasms; Cell Division; Cell Line; Cytosol; Dose-Response Relationship, Drug; Estradiol; Female; Humans; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen