2418 Structural modulation of reactivity/activity in design of improved benzothiophene selective estrogen receptor modulators: induction of chemopreventive mechanisms Bolan Yu, Birgit M. Dietz, Tareisha Dunlap, of ARE-mediated induction of phase II enzymes. The cor- Irida Kastrati, Daniel D. Lantvit, Cassia R. Overk, relation of SERM structure with antioxidant activity and Ping Yao, Zhihui Qin, Judy L. Bolton, NQO1 induction also suggests that oxidative bioactivation and Gregory R.J. Thatcher of SERMs may be modulated to enhance chemopreventive activity. [Mol Cancer Ther 2007;6(9):2418–28] Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois Introduction Selective estrogen receptor modulators (SERM) are clini- Abstract cally important for treatment and prevention of breast The benzothiophene selective estrogen receptor modula- cancer and for therapy of postmenopausal symptoms. The tors (SERM) raloxifene and arzoxifene are in clinical use therapeutic mechanism of action of SERMs is widely and clinical trials for chemoprevention of breast cancer believed to be tissue- and cell-selective agonist or antago- and other indications. These SERMs are ‘‘oxidatively nist activity at the estrogen receptor (ER; ref. 1). The labile’’ and therefore have potential to activate antioxidant optimization of ligand binding to the two ER isoforms, ERa responsive element (ARE) transcription of genes for and ERh, is often used to guide SERM design. A major cytoprotective phase II enzymes such as NAD(P)H- drug discovery goal is the ‘‘ideal SERM’’ that is antiestro- dependent quinone oxidoreductase 1 (NQO1). To study genic in breast and endometrial tissue but proestrogenic in this possible mechanism of cancer chemoprevention, a the vasculature and brain, which would be of use in cancer family of benzothiophene SERMs was developed with chemoprevention and represents an attractive alternative to modulated redox activity, including arzoxifene and its hormone replacement therapy. The majority of SERMs, metabolite desmethylarzoxifene (DMA). The relative anti- because of common chemical structural elements, can be oxidant activity of these SERMs was assayed and cor- oxidatively bioactivated to reactive metabolites. These related with induction of NQO1 in murine and human liver metabolites are not only potentially genotoxic but also cells. DMA was found to induce NQO1 and to activate have the potential to induce cytoprotective phase II ARE more strongly than other SERMs, including raloxifene enzymes such as NAD(P)H-dependent quinone oxidore- and 4-hydroxytamoxifen. Livers from female, juvenile rats ductase (NQO1). However, there has been little attention to treated for 3 days with estradiol and/or with the benzo- structural modifications of SERMs designed to control thiophene SERMs arzoxifene, DMA, and F-DMA showed oxidative bioactivation and via ER-independent mecha- substantial induction of NQO1 by the benzothiophene nisms thereby to enhance chemopreventive activity and to SERMs. No persuasive evidence in this assay or in MCF-7 attenuate toxicity (2). breast cancer cells was obtained of a major role for the The ER accommodates planar polycyclic phenols, and estrogen receptor in induction of NQO1 by the benzothio- hence all SERMs, or their active metabolites, have poly- phene SERMs. These results suggest that arzoxifene aromatic phenolic scaffolds; SERMs are therefore generally might provide chemopreventive benefits over raloxifene redox active and susceptible to oxidative metabolism and other SERMs via metabolism to DMA and stimulation in vivo (3–6). The active metabolite of the archetype SERM tamoxifen (7) is 4-hydroxytamoxifen (HOT), a triphenyl- ethylene phenol formed on oxidative metabolism (Fig. 1A; ref. 8). Further oxidative metabolism of HOT yields quinoid Received 4/12/07; revised 6/28/07; accepted 7/30/07. reactive metabolites: a quinone methide and an o-quinone Grant support: NIH grants CA102590,CA102590-S1,and CA79870. (3). Similarly, other SERMs are metabolized to reactive The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked quinoids, including the diquinone methides formed by advertisement in accordance with 18 U.S.C. Section 1734 solely to the benzothiophene SERMs raloxifene and arzoxifene indicate this fact. (Fig. 1B; refs. 4, 5). These quinoids are able to act as Note: B. Yu and B.M. Dietz contributed equally to this work. electrophiles and oxidants leading to modification of cel- Requests for reprints: Gregory R.J. Thatcher,Department of Medicinal lular biomolecules and thus potentially to cytotoxicity and Chemistry and Pharmacognosy,University of Illinois at Chicago,833 South Wood Street,Chicago,IL 60612. Phone: 312-355-5282. genotoxicity (3). Tamoxifen remains an effective clinical E-mail: [email protected] therapeutic for breast cancer, but long-term treatment is Copyright C 2007 American Association for Cancer Research. known to be associated with increased risk of endometrial doi:10.1158/1535-7163.MCT-07-0268 cancer (9). Mol Cancer Ther 2007;6(9). September 2007 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 2419 Figure 1. A, structures of SERMs and other compounds used in this study. B, single-electron oxidation of benzothiophene SERMs and BTC leads to a semiquinone or phenoxy radical,but a second one-electron oxidation to yield a quinoid is blocked for all derivatives except 4 ¶-OH (DMA),NH 2-DMA, and BTC. Cellular defense mechanisms that respond to damage electrophilic reagents, proteolysis is inhibited and dissoci- from oxidative stress and electrophiles, the key causes of ated Nrf2 translocates to the nucleus, binds to ARE, and malignant transformation, represent a target for chemo- promotes phase II enzyme gene expression (17). preventive agents (10). It has been known for some time Reactive metabolites formed from SERMs may cause cell that compounds that possess weak carcinogenic activity stress, alternatively initiating genotoxic/cytotoxic path- have the potential to protect against carcinogenesis (11). ways or contributing to cytoprotection via the Keap1/ The balance between the deleterious effects and chemo- Nrf2 pathway. The opportunity therefore exists to modu- preventive potential of compounds that are able to form late metabolic bioactivation toward cytoprotection by electrophilic and oxidative metabolites is determined by structural modification. Benzothiophene SERMs were their metabolic bioactivation and ultimately by their selected because of the clinical importance of raloxifene in chemical structure (Fig. 2). women’s health (18) and the potential importance of Induction of cytoprotective phase II enzymes such as arzoxifene, an improved next-generation SERM targeted NQO1is mediated largely at the antioxidant/electrophile at chemoprevention (19). A homologous series of arzox- responsive element (ARE) that promotes transcriptional ifene analogues has been developed (Fig. 1) and studied activation of phase II genes (12). Prochaska et al. in vitro and in vivo, showing that ER-independent chemo- (13, 14) suggested that the ‘‘oxidative lability’’ of com- prevention is a realistic target for SERM therapeutics. pounds created the inducer signal, and work on induction by quinones led to the search for a sulfhydryl-rich sensor protein able to transduce modification of sulfhydryl groups Materials and Methods to give ARE activation. Current thinking holds that the Reagents cytosolic regulatory protein Kelch-like ECH-associated BTC {the ‘‘benzothiophene core’’ 2-(4-hydroxyphenyl)- protein 1(Keap1)is the sulfhydryl-rich sensor that benzo[b]thiophen-6-ol} was synthesized following litera- responds to modification by oxidants/electrophiles. Under ture procedures and the X-DMA SERMs using our basal conditions, Keap1associates with nuclear factor improved methods (20, 21). Other reagents were purchased erythroid 2–related factor 2 (Nrf2) and targets it for from Sigma unless stated otherwise. Cell culture media degradation by the ubiquitin-proteasome proteolysis sys- and supplements, gels, and buffers were obtained from tem (15, 16). When Keap1 is modified by oxidative/ Invitrogen. Mol Cancer Ther 2007;6(9). September 2007 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. 2420 Chemopreventive Mechanisms by Benzothiophene SERMs Peroxyl Radical Trapping fetal bovine serum, 1% penicillin-streptomycin, 1% essen- Net antioxidant capacity was assayed by the BODIPY11 tial amino acids, and 0.2 mg/mL insulin (23). ERa-positive, fluorescence decay method. X-DMA (10 Amol/L) and ERh-positive, and ER-negative MCF-7 cells were gene- BODIPY11 stock solutions prepared in 40% acetonitrile/ rously supplied by Drs. D.B. Lubahn and W.V. Welshons 60% 10 mmol/L PBS were added to a 96-well plate and the (University of Missouri, Columbia, MO) and were cul- reaction was initiated by addition of small volumes (10 AL) tured in MEM plus 2.5 mol/L HEPES, 6 Ag/mL insulin, of azobisamidinopropane stock solution in PBS. The 2 mmol/L L-glutamine, 5% stripped calf serum, and 2% fluorescence decay of the probe was monitored to penicillin-streptomycin with or without geneticin. All cells j completion. The emission intensity in relative fluorescence were incubated in 5% CO2 at 37 C and medium was units was measured with time and normalized; with 100% changed