2418.Full-Text.Pdf

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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).

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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.

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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 every 2 to 3 days. corresponding to relative fluorescence units immediately Animal Treatment before addition of initiator. The net antioxidant capacity The study protocol was approved by the institutional À [AUC = (AUCinh AUCun) / AUCun] was calculated from Animal Care and Use Committee of University of Illinois the area under the curve of the BODIPY11 fluorescence at Chicago. Female juvenile Sprague-Dawley rats were decay in the presence (AUCinh) and absence (AUCun)of received at 12 days of age from Harlan and 11 pups were an antioxidant (22). housed in a cage with a nursing dam. Animals had Cell Culture free access to standard rat chow (Harlan Teklad) and Hepa 1c1c7 murine hepatoma cells were supplied by Dr. water and were allowed 1week to acclimate. Nineteen- to h J.P. Whitlock, Jr. (Standford University, Stanford, CA) and 21-day-old rats were treated with 17 -estradiol (E2 as the were cultured in a-MEM with 1% penicillin-streptomycin benzoate, 0.1mg/kg/d), DMA (10mg/kg/d), arzoxifene and 10% fetal bovine serum (Atlanta Biologicals). HepG2 (10 mg/kg/d), or F-DMA (10 mg/kg/d) s.c., either alone cells stably transfected with ARE-luciferase reporter were or challenged with E2 (0.1mg/kg/d), for 3 days. On the kindly provided by Dr. A.N. Kong (Rutgers University, fourth day, animals were sacrificed and livers were excised, Piscataway, NJ) and cultured in F-12 medium with 10% rapidly frozen in liquid nitrogen, and stored at À80jC until

Figure 2. Beneficial and detrimental consequences of SERM oxidative metabolism resulting primarily from formation of reactive oxygen species (ROS) and electrophilic quinoid metabolites capable of covalent modification of biomolecules,most importantly by Michael-type additions to thiols.

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NQO1assay. The rats were maintained on a controlled additional 24 h. The luciferase activity was determined as light cycle of 14:10 h (light/dark) at 25jC with water and previously described (27). The data were obtained from food ad libitum. All animal procedures were reviewed and three separate experiments and expressed as fold induction approved by the University of Illinois at Chicago before over control [treated cells/DMSO-treated cells (F SD)]. implementation. Cell Growth Determination NQO1ActivityAssay Cells were plated and treated as described in the NQO1 For rat liver samples, liver was homogenized in assay. The IC50 values were determined as previously 0.25 mol/L sucrose and homogenate was centrifuged at described (27). 15,000 Â g for 20 min at 4jC (24). Supernatant was collected Statistics and one-fifth volume of 0.1mol/L CaCl 2/0.25 mol/L One-way ANOVA analysis with Dunnett’s posttest or sucrose was added, incubated on ice for 30 min, and Tukey’s multiple comparison test was done using Graph- centrifuged as before. Clear cytosolic supernatant was used Pad Prism version 4.00 for Windows, GraphPad Software. immediately or stored at À80jC. Concentrated supernatant was diluted 50 times in PBS and triplicates were assayed as below. Protein concentration was determined by the Results bicinchoninic acid method (Pierce). Net Antioxidant Capacity of X-DMA SERMs For cultured liver cells, Hepa 1c1c7 cells were plated on To test the redox and antioxidant activities of the X-DMA 96-well plates, incubated overnight, and treated with test SERMs, net antioxidant capacity was assayed using the sample, DMSO, or bromoflavone. After 48 h, the NQO1 BODIPY11 fluorescent probe. Decay of fluorescence reflects activity was assayed as previously described (25). Induc- the formation and reaction of the probe with peroxyl tion of NQO1activity was calculated by comparing the radicals in the aqueous acetonitrile medium, formed from NQO1specific activity of sample-treated cells with that of the thermal decay of the azo initiator azobisamidino- solvent-treated cells. CD values represent the concentration propane (22). This assay of peroxyl radical trapping is required to double NQO1induction. Chemopreventive relevant to inhibition of lipid peroxidation, wherein net index (CI = IC50/CD) is calculated from IC50 for inhibition antioxidant activity can be quantified by the area under of cell growth (24). the curve. X-DMA SERMs (10 Amol/L) were incubated For cultured MCF-7 cells, NQO1activity was assayed with BODIPY11 and azobisamidinopropane initiator, the using 2,6-dichlorophenolindophenol as the substrate observed retardation of fluorescence decay in the presence according to Lhoste et al. (26) with minor modifications. of SERMs indicating a modest ability to inhibit BODIPY The final reaction mixture contained 25 mmol/L Tris- oxidation by peroxyl radical trapping (Fig. 3). The X-DMA HCl (pH 7.4), 0.7 mg/mL bovine serum albumin, 0.01% SERMs that are able to act as traditional phenolic anti- A A Tween 20 (w/v), 5 mol/L FAD, 200 mol/L NADH, and oxidants (X = MeO, F, Br, SO2CH3) modestly retarded the 40 Amol/L 2,6-dichlorophenolindophenol, and assays rate of reporter oxidation, with no discernable dependence were carried out in the presence and absence of 20 Amol/L in this assay on the electron-withdrawing nature of the dicumarol. The presence of 20 Amol/L dicumarol com- 4¶-substituent; however, the two SERMs that are able to pletely inhibited enzyme-dependent 2,6-dichlorophenolin- undergo two-electron oxidation to a quinoid (DMA, NH2- dophenol reduction. NQO1activity was described as the DMA) were significantly better antioxidants. dicumarol-inhibitable decrease in absorbance of 2,6-dichlo- Induction of NQO1in Liver Cells rophenolindophenol for 5 min at 600 nm on a microplate Inducible cytoprotective enzymes such as NQO1have reader. NQO1activity was normalized to the protein con- commonly been labeled as phase II enzymes, and this label centration, which was determined by the bicinchoninic is used herein, although the appropriateness of this label is acid assay using bovine serum albumin as a standard. problematic and recognized (28). X-DMA SERMs were Induction of activity was calculated as described above. administrated to Hepa 1c1c7 cells in different concentra- Immunoassay tions and NQO1 activity was measured. The Hepa 1c1c7 Briefly, 10 Ag of tissue or cell lysis protein were separated cell system is widely used to screen chemopreventive by electrophoresis in NuPAGE Novex 4% to 12% Bis-Tris agents because the NQO1enzyme is readily induced to gel (Invitrogen) and transferred onto nitrocellulose mem- measurable levels that allow comparison of agents (29). branes (Bio-Rad). Membranes were blocked with 5% non- Induction of NQO1activity in the presence of SERMs was fat dry milk and incubated with primary polyclonal compared with solvent and to bromoflavone as a positive h antibodies to NQO1(1:500)or -actin (1:500) and then control (Table 1; ref. 24). All SERMs, but not E2, induced with horseradish peroxidase–linked chicken anti-goat NQO1 in Hepa 1c1c7 cells. At the lower concentration immunoglobulin G (1:1,000; all from Santa Cruz Biotech- of the SERMs studied, induction by DMA, Br-DMA, and nology). Western blots were detected with the ECL kit NH2-DMA was significant (P < 0.05), whereas at the higher (Amersham Biosciences). concentration, all seven X-DMA SERMs reached signifi- ARE-Luciferase Reporter Assay cance. Interestingly, BTC was seen to be a potent inducer HepG2-ARE-Luc cells were plated on a six-well plate of NQO1, giving 3-fold induction at 2.5 Amol/L, whereas overnight; treated with DMSO, test samples, or positive DMA only reached 2.2-fold induction at twice the concen- control bromoflavone on the second day; and incubated for tration. The DMA series SERMs as a family seem to have

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greater potential for chemoprevention than raloxifene and NQO1and DMA as a weaker but significant inducer HOT, as determined by the CD measured (Table 1). (Fig. 4A). The observations in the two cell lines indicated CD and chemopreventive index (CI = IC50/CD) have that X-DMA SERMs have the potential to induce NQO1in been used to identify new chemopreventive agents. The the liver. X-DMA SERMs showed IC50 values for inhibition of cell Induction of NQO1 In vivo growth in murine and human liver cells of 4 to 20 Amol/L Three of the X-DMA SERMs were selected for study (Table 1). The best agents were BTC, DMA, and NH2-DMA in vivo: DMA, arzoxifene, and F-DMA. To study the role of with CIs of 16, 1.4, and 1.6, respectively. For comparison, ER signal transduction in the action of the X-DMA SERMs, A the values reported for bromoflavone are CD = 0.01 mol/L E2 was included, alone and in coadministration arms. The and CI = 17,000, whereas for the well-studied chemo- juvenile rat model, commonly used in study of estrogenic preventive agent sulforaphane, CD = 0.43 Amol/L and CI = activity, using 19- to 21-day-old female Sprague-Dawley 26 (24). Although cytotoxicity was observed for benzothio- rats, was selected (8). The organs of rats at this age are fully phene SERMs in liver cancer cells, liver toxicity is not estrogen responsive, but the hormone is not yet being revealed in clinical data on raloxifene and arzoxifene (30). produced in the ovaries, so this rat model can offer a clear The SERMs were also assayed in the human liver cell line hormone background when studying SERM activity (32). HepG2, which is important because of the availability of Eight groups of immature female rats were treated daily HepG2 cells transfected with the ARE-luciferase reporter with E2 (0.1mg/kg) or vehicle control, with or without (27, 31). BTC was confirmed as a strong inducer of X-SERMs (10 mg/kg), for 3 days. Animals were sacrificed on the 4th day and individual NQO1activity was analyzed in the excised liver. The selected dosage compares to 6 or 20 mg/kg arzoxifene daily in a chemoprevention study in mice (33) and 10 to 50 mg/kg arzoxifene daily in phase I human clinical studies, which reported no adverse events compared with placebo (30). The cumulative data showed that induction of NQO1 was observed in all animals treated with X-DMA SERMs: arzoxifene, DMA, and F-DMA induced NQO1activity to about 1.6-, 1.5-, and 1.3-fold that of the control animals, respectively (Fig. 5A and B). Fold induction was significant for all three X-DMA SERMs relative to the E2 treatment group and was significant for arzoxifene relative to the vehicle control group (P < 0.05). Although not reaching significance, the E2 treatment group showed a trend toward reduced levels of hepatic NQO1relative to control. Adult female Sprague-Dawley rats have been reported to show elevated NQO1in response to E 2 treatment (32), but the effects of E2, which are well known to be highly dose sensitive, are expected to be more complex in adult animals endogenously producing E2. A study on NQO1levels in the tissue of ovariectomized mice treated with E2 over 14 days showed high tissue variability but significantly reduced uterine NQO1(34). The observed enzyme activity was substantiated by immunoassay of NQO1protein expression by Western blot (Fig. 5C), confirming that the three X-DMA SERMs induced elevated levels of NQO1protein relative to control in rat liver, in contrast to E2. Finally, in female juvenile rats, the influence of E2 coadministration on the putative chemopreventive actions of the X-DMA SERMs was not significant (Fig. 5D). The simplest explanation for this observation is that NQO1induction by the SERMs in the liver is not ER mediated. InductionofNQO1inBreastCancerCells Figure 3. Net antioxidant capacity toward peroxyl radicals of X-DMA SERMs (10 Amol/L),assayed by measurement of azobisamidinopropane- To explore the influence of SERMs on NQO1induction in a h À induced fluorescence decay of BODIPY11 reporter in 40% acetonitrile in estrogen-sensitive cells, ER ,ER , and ER( ) MCF-7 breast 10 mmol/L phosphate buffer at 37jC. A, relative net antioxidant capacity cancer cells were studied. Jiang et al. (35) have assessed À as given by AUC [AUC = (AUCinh AUCun) / AUCun]. B, representative phase II enzyme induction in seven cell lines in response to curves for the time course of BODIPY11 oxidation as measured by relative fluorescence units (RFU),normalized relative to 100% relative fluores- sulforaphane and other chemopreventive agents, reporting cence units immediately before addition of initiator. that Hepa 1c1c7 cells were highly inducible, but that

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Table 1. Inhibition of cell growth and relative NQO1 induction by SERMs in Hepa 1c1c7 cells

A A Compounds IC50 ( mol/L), IC50 ( mol/L), Relative induction, Relative induction, CD CI EHOMO Hepa 1c1c7 HepG2 5.0 Amol/L* 2.5 Amol/L* (Amol/L) (eV)

DMSO (0.5%) — — 1.01 F 0.03 1.0 F 0.05 — — — Bromoflavone (0.67 Amol/L) — — — 5.80 F 0.50 — — — BTC 27.1 F 6.0 22.1 F 1.8 3.10 F 0.45c 2.90 F 0.40c 1.7 15.9 — DMA 5.6 F 1.4 13.5 F 1.0 2.21 F 0.44c 1.65 F 0.38c 4.11.4 À5.49 H-DMA 4.7 F 1.1 8.7 F 2.11.90 F 0.57c 1.26 F 0.05 5.9 0.8 À5.69 MeO-DMA (arzoxifene) 6.5 F 2.3 10.6 F 1.4 1.84 F 0.35c 1.36 F 0.10 6.1 1.1 À5.45 Br-DMA 4.3 F 1.4 8.6 F 2.8 1.73 F 0.43c 1.51 F 0.50b 6.5 0.7 À5.77 F-DMA 5.6 F 1.3 9.1 F 2.6 1.68 F 0.37b 1.35 F 0.13 7.3 0.8 À5.72 F F F b F b À NH2-DMA 12.3 5.4 19.2 4.0 1.61 0.20 1.51 0.22 7.6 1.6 5.20 F F F b F b À SO2Me-DMA 8.4 2.2 6.4 0.6 1.63 0.12 1.50 0.10 7.8 1.1 6.01 HOT 7.3 F 1.0 14.5 F 2.2 1.24 F 0.21 1.12 F 0.13 21 0.3 — Raloxifene 10.2 F 3.3 24.7 F 6.3 1.31 F 0.17 1.20 F 0.14 16 0.6 — F F F F E2 22.6 7.129.8 1.7 1.09 0.310.90 0.20 53 0.4 —

NOTE: Data shown are mean F SD for at least triplicate measurements. *Induction of NQO1activity relative to DMSO control. cP < 0.01, treatment significantly different from the control (one-way ANOVA with Dunnett’s posttest). bP < 0.05, treatment significantly different from the control (one-way ANOVA with Dunnett’s posttest). others, including MCF-7 and MDA-MB-231breast cancer noted (37). Nevertheless, the results presented herein are cells, were poorly responsive to chemopreventive agents compatible with an ER-independent mechanism for NQO1 (e.g., sulforophane induced NQO1 2-fold in Hepa 1c1c7 induction by DMA in MCF-7 cells. cells, but <1.2-fold in MCF-7 cells). Background NQO1 Activation of ARE-Luciferase activity was observed to be significantly higher in MCF-7 To investigate the possible mechanism of NQO1induc- than in Hepa 1c1c7 cells (data not shown). Nevertheless, tion by X-DMA SERMs, HepG2-ARE-C8 cells were treated one group has reported NQO1induction by SERMs in with SERMs and luciferase induction was assayed. HepG2- MCF-7 cells, stating that HOT induced NQO1via an ER h- ARE-C8 is a human hepatocarcinomal cell line that was dependent pathway (36). Another group has reported stably transfected with the pARE-T1-luciferase construct. NQO1induction in both ER a- and ERh-transfected cell Luciferase induction in this cell line reflects the ability to lines derived from MCF-7 cells by tamoxifen and HOT (34). activate the endogenous ARE, which regulates expression Thus, we initially studied the MCF-7 cell line stably of many phase II enzymes. A good correlation between transfected with ERh, but observed only very weak ARE-luciferase induction and NQO1induction is antici- induction of NQO1by the X-DMA SERMs raloxifene pated and indeed is shown in Fig. 4B. BTC was observed to and HOT (Supplementary data).1 At concentrations from strongly activate ARE, giving 13-fold luciferase induction 1nmol/L to 2 Amol/L, no substantial increase in induction relative to control. X-DMA SERMs raloxifene and HOT was observed for HOT (Supplementary data).1 Selected gave 1.5-fold induction, and again DMA was observed to SERMs were then studied in the MCF-7 ERa cell line, with be an exceptional ARE activator, giving almost 3-fold raloxifene and DMA being observed to induce NQO1by induction of ARE-luciferase activity. These data strongly 1.25-fold over control, but again effects did not reach imply that in liver cells, the observed induction of NQO1is significance (Supplementary data).1 In ER(À) MCF-7 cells, mediated via ARE activation. the induction of NQO1by DMA was significant compared with both control and HOT-treated cells (Supplementary data).1 The relative insensitivity of MCF-7 cells to NQO1 Discussion induction is shown by the weak response to the positive SERMs are polyaromatic phenols susceptible to oxidative control bromoflavone, which gave 1.3-fold induction metabolism to electrophilic quinoid metabolites (Fig. 1), compared with 1.2-fold by DMA. Ansell et al. (34) have which may cause either genotoxicity/cytotoxicity or cyto- emphasized that estrogenic actions mediated through the protection via triggering of cellular defense mechanisms ARE are highly dependent on cellular environment to (Fig. 2). The quinoid metabolites, including o-quinones, account for differences in cell culture observations in the quinone methides, and diquinone methides, have variously literature. The idiosyncrasies of MCF-7 cell sublines, been shown to generate reactive oxygen species and to especially toward estrogens and antiestrogens, have been both oxidize and alkylate biomolecules such as proteins and nucleic acids; this has been argued to contribute to cytotoxicity and genotoxicity (3). However, sulfhydryl 1 Supplementary material for this article is available at Molecular Cancer group oxidation or alkylation by quinoids, which are Therapeutics Online (http://mct.aacrjournals.org/). Michael acceptor electrophiles, can trigger chemopreventive

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mechanisms, in particular via reaction with Keap1and the putative beneficial effects, notably chemoprevention via subsequent Nrf2/ARE activation of induction of cytopro- induction of phase II enzymes, are also lost. A family of tective phase II enzymes such as NQO1 (10, 12, 13, 38). The benzothiophene SERMs has been developed (20) to answer balance between the detrimental and beneficial drug the following questions: Does benzothiophene SERM properties associated with oxidative bioactivation and bioactivation lead to induction? Is this property lost if potential outcomes are depicted in Fig. 2. bioactivation to a quinoid is structurally blocked? The The specific semiquinone and electrophilic quinoid 4¶-substituted desmethoxyarzoxifene (X-DMA) family formed from bioactivation of benzothiophene SERMs are includes arzoxifene (X = MeO) and the arzoxifene depicted in Figs. 1B and 2. When the 4¶-MeO group of metabolite DMA (X = OH). Previous comparison of DMA arzoxifene is replaced by groups such as F, Br, H, and and F-DMA showed that DMA is oxidatively bioactivated SO2Me, oxidation to a quinoid is blocked (Fig. 1B; ref. 39). to a diquinone methide, which is a Michael acceptor The potential detrimental effects of quinoid formation are depleting cellular reduced glutathione (Fig. 2), in contrast hence prevented, but this raises the question about whether to F-DMA, which is also an ER ligand but is not bioactivated to a quinoid and does not deplete cellular reduced glutathione (Fig. 1B; refs. 5, 39). SERMs and Oxidative Bioactivation Tamoxifen is oxidatively metabolized to HOT and other electrophilic reactive intermediates leading to formation of DNA adducts and genotoxicity (40). The increased risk of endometrial cancer in long-term tamoxifen therapy is associated, at least in part, with this chemical carcinogenesis pathway (3, 9). Subsequent generations of SERMs in clinical use and preclinical development including the benzothiophene SERMs raloxifene and arzoxifene are based on polyaromatic phenolic scaffolds and have been observed to form quinoids in vitro and in vivo (3–6, 39). Many of these quinoid metabolites have been shown to act as electrophiles and/or to generate reactive oxygen species, although, as yet, there is no evidence that the newer- generation SERMs are carcinogenic. The chemical character of oxidative metabolites formed from individual SERMs is varied, and this is likely to strongly influence the balance between the potential detrimental and beneficial effects. Induction of the phase II enzymes aryl sulfotransferase and hydroxysteroid sulfotransferase has been reported in rat liver and intestine, in both male and female Sprague- Dawley rats, after tamoxifen treatment for 1to 2 weeks (41). In separate studies, based largely on work in MCF-7 cells, HOT and raloxifene have been reported to induce NQO1 via an ERh-dependent mechanism (36). In further studies, NQO1induction by tamoxifen and HOT was reported in both ERa- and ERh-positive cells derived from the MCF-7 line (34). Thus, in the present study, HOT and raloxifene were included with the X-DMA SERMs for comparison. NQO1 Induction via AREActivation Measurement of NQO1activity in vitro and in vivo provides an efficient assay for discovery of chemopreventive agents (29), and NQO1is a biomarker for cancer chemoprevention (42). NQO1, as other inducible phase II enzymes such as glutathione S-transferases and UDP- glucuronyl transferase, provides cytoprotection by detoxification of small molecules; however, NQO1also binds and regulates the stability of the tumor suppressor protein Figure 4. A, incubation of SERMs (2.5 Amol/L) with HepG2 cells for p53, inhibiting p53 degradation (43). Epidemiologic evi- 24 h showing fold induction of NQO1 relative to control as measured by dence indicates that genetically deficient NQO1is a risk enzyme activity. The measured IC50 values in this cell line are annotated to factor for development of malignancies, and, regardless of the plot for reference. B, incubation of SERMs (2.5 Amol/L) with HepG2 cells for 24 h showing fold induction of ARE-luciferase reporter activity the exact mechanism of chemoprevention, NQO1has been relative to vehicle control. proved as a useful biomarker for discovery of agents such

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Figure 5. Induction of liver NQO1 activity in juvenile female rats treated for 3 d (10 mg/kg qd SERMs or 0.1 mg/kg qd E2 or vehicle control). A, distribution of individual rats in treatment groups receiving arzoxifene (Arz),DMA,F-DMA,or E 2. B, cumulative data showing statistical significance of NQO1 induction by arzoxifene relative to control and significant induction by arzoxifene (P < 0.001),DMA ( P < 0.005),and F-DMA ( P < 0.05) relative to the E2-treated group. C, Western blot showing different levels of NQO1 expression in rat liver tissue of different groups. D, cumulative data showing the effects of 10 mg/kg qd X-DMA SERMs in antagonizing and reversing the effects of 0.1 mg/kg qd E2 on cotreatment. Data were analyzed by one-way ANOVA using Tukey’s post hoc analysis. as sulforaphane that have shown efficacy in chemopreven- nisole showed that the observed chemopreventive actions tion of breast and other cancers (44). Sulforaphane, Michael correlated with induction of NQO1, levels of which were acceptors, and other classes of chemopreventive agents elevated in nearly all tissues (47). More recent work on 2,3- have been shown to chemically modify the cysteine t-butyl-4-hydroxyanisole implicated activation of kinase residues of the redox sensor protein Keap1, leading to cascades converging on Nrf2 in NQO1induction (31).The activation of ARE by Nrf2 (16). Ex vivo experiments using X-DMA family members that are blocked from diquinone Keap1, Keap1/Nrf2, and Nrf2 knockouts showed ablated methide formation (X = H, F, Br, OMe, SO2Me) were induction of NQO1by sulforaphane and other agents, observed in the 1,1-diphenyl-2-picrylhydrazyl radical scav- emphasizing the importance of the Keap1/Nrf2/ARE enging assay to act as phenolic antioxidants (20). Mea- pathway for chemoprevention (45). However, multiple surement herein of the classic antioxidant activity of the kinase cascades converge on Nrf2 phosphorylation and X-DMA SERMs in peroxyl radical trapping showed that both actin modification and gene transactivation may also the superior antioxidants were those able to undergo two- contribute to overall ARE-mediated induction (31, 46). electron oxidation to quinoids (DMA, NH2-DMA; Table 1; Expectations from X-DMA Redox Activity Fig. 3); however, this assay did not distinguish between In comparing the properties of the X-DMA SERMs, the the remaining members of the family, which were poorer simplistic prediction would be that only DMA and possibly antioxidants. In the 1,1-diphenyl-2-picrylhydrazyl assay, NH2-DMA, which are susceptible to bioactivation to antioxidant activity correlated linearly with calculated quinoids, will be capable of oxidation and electrophilic EHOMO values for the phenolic antioxidant X-DMA SERMs modification of Keap1leading to NQO1induction via (Table 1; ref. 20). Zoete et al. (48) have previously correlated Nrf2/ARE. If confirmed by experiment, this would indicate CD values for a family of NQO1inducers with EHOMO that (a) oxidative bioactivation of SERMs is a contributor to values. Thus, the X-DMA redox activity data predict the their chemopreventive activity, and (b) new SERMs should relative induction of NQO1: DMA >> NH2-DMA >> incorporate oxidative lability as a design parameter. arzoxifene > H-DMA > F-DMA > Br-DMA > SO2Me- Not all chemopreventive agents are electrophiles. Early DMA. This prediction was first tested in vitro in liver work on the phenolic antioxidant 2,3-t-butyl-4-hydroxya- cell lines.

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In vitro X-DMA Activity relative to E2 treatment; furthermore, comparison of each In Hepa 1c1c7 cells, DMA was indeed observed to be an SERM treatment group with the relevant SERM + E2 inducer of NQO1and was observed to be the strongest of treatment group showed no significant difference (Fig. 5D). the X-DMA SERMs, and several other X-DMA SERMs Hence, the induction of hepatic NQO1 in vivo by the reached significance versus control (Table 1). At higher X-DMA SERMs does not show estrogenic or antiestrogenic concentration, all SERMs except raloxifene and HOT character and is therefore unlikely to be ER mediated. In reached significance (P < 0.05); E2 had no effect on NQO1 accord with this assessment, the observed induction of activity. CD values also confirmed the prediction of DMA NQO1by X-DMA SERMs in MCF-7 cell lines stably as the strongest NQO1inducer; however, other X-DMA transfected with ERa or ERh was very modest and was SERMs were also inducers and not readily differentiated. not ER dependent. For example, SO2Me-DMA, which is a a h It was postulated above that after bioactivation to a ligand for ER (IC50 = 27 nmol/L) but not for ER (20), diquinone methide, the 2-phenyl-benzothiophene core of gave 1.2-fold induction of NQO1 in both ERa- and ERh- the X-DMA SERMs would provide the reactivity toward positive MCF-7 cells. In ER(À) MCF-7 cells, DMA showed the Keap1sensor required to induce NQO1.Therefore, significant induction of NQO1, which is compatible with BTC, constituting the core of the benzothiophene SERMs, its ability to induce NQO1via ER-independent activation was studied for comparison. In Hepa 1c1c7 cells, BTC was of the ARE. The chemopreventive properties of arzo- indeed observed to be a potent inducer of NQO1, with a CI xifene toward ER(À) breast cancer have previously been value comparable to the chemopreventive agent sulfora- reported in a mouse model (33). phane (Table 1). A potential explanation for the weaker induction by DMA and raloxifene, both of which contain the BTC sub-structure, is that for the quinoid-forming Conclusions compounds, direct interaction of the quinoid with the This is the first report that the benzothiophene SERM sensor protein is required for activation and induction; the arzoxifene can induce NQO1 in vivo. Given the interest steric hindrance due to the SERM side arm would inhibit in stimulation of the Nrf2/ARE signaling pathway and interaction with the sensor protein relative to BTC. induction of NQO1in cancer prevention, this result is The observation of significant activation of ARE by both potentially significant for the clinical use of arzoxifene as a BTC and DMA and induction of NQO1in HepG2 cells chemopreventive agent. Comparison of the in vitro and (Fig. 4) strongly supports the concept that appropriate in vivo data suggests that the arzoxifene metabolite DMA benzothiophene SERMs can act as chemopreventive agents is responsible for the observed NQO1induction via Nrf2/ via the Keap1/Nrf2/ARE pathway. It should be noted that ARE activation, suggesting that arzoxifene might provide the character of the quinoid metabolite is expected to be chemopreventive benefit over other SERMs via metabo- of importance: DMA and raloxifene are bioactivated to lism to DMA. In all assays done, the arzoxifene metabolite diquinone methides, which have different chemical char- DMA was superior to raloxifene. The high activities of acteristics to other quinoids. The results confirm that DMA and BTC support the hypothesis that oxidative engineering of the SERM structure to modulate redox bioactivation of benzothiophene SERMs to a quinoid activity affects biological activity of potential therapeutic metabolite can stimulate Nrf2/ARE signaling, likely via significance. direct interaction with the Keap1sensor. However, induc- In vivo X-DMA Activity tion of NQO1, although weaker, was also observed for On the basis of the in vitro data collected in murine and X-DMA SERMs, such as F-DMA, which cannot form qui- human hepatocellular culture, DMA would be predicted to noids but which are phenolic antioxidants. Although the act in vivo as a SERM with chemopreventive activity, in evidence supports the stimulation of Nrf2/ARE signaling addition to any ER-dependent properties, derived from in induction of NQO1by the X-DMA SERMs, further phase II enzyme induction via the Keap1/Nrf2/ARE research is needed to differentiate the exact mechanisms pathway. DMA, arzoxifene, and F-DMA were selected for invoked by quinoid-forming versus phenolic antioxidant comparison of induction of hepatic NQO1in the juvenile SERMs, including the roles of mitogen-activated protein rat model, which is commonly used in assessing estrogenic kinase and protein kinase C pathways. Induction of cyto- and antiestrogenic activity of new SERMs (8, 49). It was protective defenses in tissues to which SERMs are tradi- found that all three X-DMA SERMs at 10 mg/kg/d induced tionally directed as cancer chemopreventive agents also NQO1after only 3 days of treatment, with arzoxifene being warrants further study. the strongest inducer, elevating NQO1activity 1.6-foldover vehicle control (Fig. 5B). Arzoxifene was originally devel- Acknowledgments oped as an improved SERM with increased bioavailability We thank Rezene T. Asghodom for assistance in the animal study. over raloxifene, and because DMA is known to be an arzoxifene metabolite, it is not surprising that arzoxifene References and DMA induced NQO1 in vivo. In the same model, 1. Katzenellenbogen BS,Katzenellenbogen JA. Biomedicine. Defining the treatment with E2 showed a trend toward decreased NQO1 ‘‘S’’ in SERMs. Science 2002;295:2380 – 1. activity relative to vehicle. Increased NQO1induction in 2. Evans DC,Watt AP,Nicoll-Griffith DA,Baillie TA. 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