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Cancer Prevention and Control (Y.Z., D.S., G.C., P.L.) and Chemical Carcinogenesis Programs (A.K.S., S.A.), Penn State Cancer Institute, Hershey, Pennsylvania; and Departments of Pharmacology (Y.Z., D.S. A.K.S., S.A., P.L.) and Public Health Sciences (G.C., P.L.), College of Medicine, Pennsylvania State University, Hershey, Pennsylvania
(Received April 12, 2007; Accepted July 3, 2007)
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Abstract |
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; Osborne, 1998; Cuzick et al., 2003; Howell et al., 2003). Adjuvant TAM treatment significantly increases recurrence-free survival and overall survival in patients with estrogen receptor-positive breast cancer (Osborne, 1998; Howell et al., 2003). As a selective estrogen receptor modulator, TAM competes with estrogen for binding to the estrogen receptor and therefore inhibits tumor growth by interfering with the survival and proliferative signals regulated by estrogen. Although TAM is generally well tolerated, there is significant interindividual variability in the clinical efficacy of TAM as well as in the toxicities of TAM. For instance, TAM resistance and relapse have developed in approximately 30% of patients with estrogen receptor-positive breast cancer (Early Breast Cancer Trialists' Collaborative Group, 1998). In addition to its antiestrogenic-related side effects such as hot flashes and vaginal bleeding (Osborne, 1998), TAM may also increase the risk for endometrial cancer (van Leeuwen et al., 1994; Rutqvist et al., 1995). TAM also has partial estrogenic effects that may be linked to reduced risk of ischemic heart disease and osteoporosis (McDonald and Stewart, 1991; Rutqvist and Mattsson, 1993). The mechanisms underlying variability in response to TAM and TAM-related toxicities are not very clear but may be related to the altered patterns of TAM metabolism.
After oral administration, TAM is extensively metabolized by phase I and phase II enzymes into several primary and secondary metabolites (Fig. 1). Two of its hydroxylated metabolites, trans-4-OH-TAM and 4-hydroxy-N-desmethyl-TAM (also known as endoxifen), exhibit high affinity for estrogen receptors, having up to 100 times more antiestrogenic activity in vitro than TAM itself or other TAM metabolites (Crewe et al., 1997; Dehal and Kupfer, 1997; Coller et al., 2002; Crewe et al., 2002; Coller, 2003; Hu et al., 2003; Desta et al., 2004). Because both 4-OH-TAM and endoxifen are abundant in the serum of women treated with TAM, with endoxifen being present at 6- to 12-fold the levels of 4-OH-TAM (Stearns et al., 2003; Desta et al., 2004; Jin et al., 2005), these two TAM metabolites may be the major contributors to the antiestrogenic properties of TAM. Although cis-4-OH-TAM is primarily thought to be a weak estrogen agonist (Furr and Jordan, 1984), studies on individual diastereomeric forms of endoxifen have not been examined previously.
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A major mode of phase II metabolism of TAM is glucuronidation catalyzed by the UDP glucuronosyltransferase (UGT) family of enzymes. TAM is excreted predominantly through the bile, a process facilitated by TAM conjugation to glucuronic acid catalyzed by UGTs (Lien et al., 1989). TAM-glucuronide conjugates have been identified in the urine, serum, and bile of TAM-treated breast cancer patients (Lien et al., 1988, 1989; Poon et al., 1993), and it has been suggested that glucuronidation within target tissues such as adipose tissue of the breast may also be important in terms of TAM metabolism and overall TAM activity (Nowell et al., 2005). Although studies have not yet been performed to examine the antiestrogenic activity of glucuronidated TAM metabolites, previous studies have demonstrated that methylation of the free phenolic group of 4-OH-TAM (producing a methyl ether) significantly decreases the affinity of 4-OH-TAM for the estrogen receptor (Allen et al., 1980).
The goal of the present study is to test the hypothesis that glucuronidation of hydroxylated TAM metabolites reduces the antiestrogenic activity of 4-OH-TAM and endoxifen. In this study, the relative antiestrogenic activities of glucuronidated conjugates of the trans- and cis-isomers of 4-OH-TAM and endoxifen compared with their unconjugated counterparts were examined. In addition, this is the first study examining the relative antiestrogenic activities of individual endoxifen isomers.
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Materials and Methods |
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Endoxifen Synthesis. Endoxifen was synthesized as described elsewhere (Sun et al., 2007). Briefly, 4-OH-TAM was refluxed with ethylchloroformate in toluene followed by treatment of the resulting intermediate (4-hydroxy-N-ethoxy-N-methyltamoxifen) with ethylene glycol, hydrazine hydrate, and potassium hydroxide at 140°C. The trans- and cis-endoxifen mixture was characterized and verified by 1H nuclear magnetic resonance.
Purification and Collection of TAM Metabolite Isomers. trans-4-OH-TAM, cis-4-OH-TAM, trans-endoxifen, and cis-endoxifen were separated from the trans-/cis-4-OH-TAM (70%:30% ratio) and trans-/cis-endoxifen stocks by HPLC as described elsewhere (Sun et al., 2007). Briefly, isocratic elution was performed using a Luna C18 analytical column (250 mm x 4.6 mm, 5 µ; Phenomenex, Torrance, CA) in series with a C18 guard column (4.0 mm x 3.0 mm, 5 µ, Phenomenex). trans-4-OH-TAM and cis-4-OH-TAM were purified by separation and elution using 20% buffer A (0.1% TEA, pH 7.4) and 80% acetonitrile, whereas trans-endoxifen and cis-endoxifen were purified with 5% buffer A (0.25% TEA, pH 7.4) and 95% acetonitrile, both at a 1 ml/min flow rate. All isomers underwent a second identical HPLC purification after initial separation to eliminate any possible contamination. Pure trans- and cis-isomers of 4-OH-TAM and endoxifen were collected individually after elution from the HPLC column and stored in DMSO at -20°C until use.
Preparation and Collection of Glucuronide Conjugates. 4-OH-TAM isomers can be glucuronidated at the N-amino position as well as O-glucuronidated at the 4-hydroxyl position, whereas endoxifen isomers are glucuronidated solely at the 4-hydroxyl position (Sun et al., 2007). The O-Gluc products of trans-4-OH-TAM, cis-4-OH-TAM, trans-endoxifen, and cis-endoxifen were prepared by incubation with pig liver microsomes, which were prepared essentially as described previously for human liver microsomes (Fang and Lazarus, 2004; Wiener et al., 2004b). After preincubation with alamethicin (on ice for 15 min), pig liver microsomes (100 µg) were incubated in 50 mM Tris-HCI (pH 7.4), 10 mM MgCI2, 4 mM UDP-glucuronic acid with either the trans-4-OH-TAM/cis-4-OH-TAM (70:30) or trans-endoxifen/cis-endoxifen mixtures (concentration of 
100 µM each) at 37°C for 4 h in a total reaction volume of 200 µl. Reactions were terminated by addition of 200 µl of cold methanol, mixtures were centrifuged at 16,100g at 4°C for 10 min, and 200 µl of supernatant was injected onto the HPLC column (Gemini C18 250 mm x 4.6 mm, 5 µm; Phenomenex). The gradient elution conditions for separation of trans- and cis-4-OH-TAM-O-Gluc were initiated with 65% buffer A and 35% acetonitrile for 15 min, followed by a subsequent linear increasing gradient to 75% acetonitrile (25% buffer A) in 1 min and then maintained at 75% acetonitrile for 10 min. The flow rate was 1 ml/min. For trans- and cis-endoxifen O-Gluc separation, buffer A and acetonitrile started at 67 and 33%, respectively.
For N-Gluc products, homogenates (50 µg) from human UGT1A4-overexpressing HK293 cells (Wiener et al., 2004a) were used in glucuronidation reactions (performed as described above in a total reaction volume of 100 µl) because pig liver cannot form N-glucuronidated products of TAM or its metabolites (unpublished results). For trans- and cis-4-OH-TAM-N-Gluc, HPLC separation started at 60% buffer A and 40% acetonitrile. As described elsewhere, no N-Gluc products of trans-or cis-endoxifen were detected in glucuronidation reactions using human liver microsomes or homogenates from UGT1A4-overexpressing cell lines (Sun et al., 2007). TAM-N-Gluc was synthesized using UGT1A4-overexpressing cell homogenates as described previously (Sun et al., 2006), and separation was started with 50% buffer A-50% acetonitrile for 16 min, with the gradient increased linearly up to 90% acetonitrile in 4 min and maintained for 10 min. Pure trans- and cis-glucuronidated products were collected after HPLC separation and purification, dissolved in DMSO, and stored at -20°C until use.
Cell Culture and Drug Treatment. MCF-7 human breast cancer cells were routinely cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (maintenance media) at 37°C-5% CO2. Three days before treatment, cells were resuspended and washed twice in phosphate-buffered saline and then plated at 2 x 105 cells/dish in phenol red-free DMEM supplemented with 5% charcoal-stripped bovine calf serum (treatment media) to remove estrogens from the culture medium. For dose-response and time course studies of E2-induced PGR gene expression, MCF-7 cells were treated with vehicle (0.1% DMSO) or various doses of E2 for up to 24 h in treatment media, with cells harvested and pelleted at 0, 1, 3, 6, 12, and 24 h at 37°C-5% CO2 after E2 treatment and stored at -70°C until used. For studies with TAM metabolites, MCF-7 cells in treatment media were treated with E2 (1 x 10-10 M) and TAM or TAM metabolites (1 nM-1 µM) for 12 h at 37°C and 5% CO2. No treatment and vehicle (0.1% DMSO) controls as well as positive controls (1 x 10-10 ME2) were included in all experiments, which were performed three times independently.
Examination of 4-OH-TAM- and Endoxifen-Glucuronides in Culture Media. A 1-ml sample of culture medium was collected before and after a 12-h incubation of MCF-7 cells with 4-OH-TAM- or endoxifen-glucuronides as described above. The 4-OH-TAM- or endoxifen-glucuronides in culture media were determined by using an Acquity UPLC system (Waters, Milford, MA). The sample preparation was as described above with 10 µl of supernatant injected onto an Acquity UPLC column (BEH C18 1.7 µm, 21 mm x 100 mm; Waters). The gradient elution conditions for separation of trans- and cis-4-OH-TAM-O-Gluc and trans- and cis-endoxifen-O-Gluc was initiated with 70% buffer A (0.5 M NH4AOc, pH = 5.0) and 30% acetonitrile for 3.5 min, followed by a subsequent linear increasing gradient to 75% acetonitrile (25% buffer A) over 0.5 min, and then maintained at 75% acetonitrile for 2 min. The flow rate was 0.3 ml/min.
PGR Expression Analysis. Total RNA was extracted from treated 2 x 105 MCF-7 cells using the RNeasy Mini kit and QIAshredder (QIAGEN) as per the manufacturer's protocols and stored at -70°C until use. cDNA was synthesized in a 20-µl reaction including 100 ng of total RNA, 0.5 mmol dNTP mix, 0.5 µg oligo(dT)12-18 primer, and 200 U of SuperScript II RNase reverse transcriptase as per the manufacturer's recommendations and stored at -70°C until use. Real-time PCR was performed using the TaqMan assay with relative quantification (
Ct method) (http://www.biocompare.com/pcr/tutorial/qpcr). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as the endogenous housekeeping gene to normalize for expression for each sample. A 10-µl reaction included TaqMan Universal PCR Master Mix (1x final concentration; Applied Biosystems; Foster City, CA), primer and probe mix of either PGR or glyceraldehyde-3-phosphate dehydrogenase (1x final concentration; Applied Biosystems) and 40 ng of cDNA. Reactions were performed in 384-well plates using the ABI 7900 HT Sequence Detection System (Applied Biosystems). The thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Quantitative values of Ct and relative quantity were calculated by the feature of the Ct study option in SDS2.2.2 software (Applied Biosystems). All of the reagents and reaction conditions were preoptimized and validated by the manufacturer.
Interconversion of trans- and cis-Isomers of 4-OH-TAM or Endoxifen in Cell Culture Medium. One microliter of trans-or cis 4-OH-TAM or endoxifen (10 mM) was added to 1 ml of treatment media preadjusted to various pH values (6.0-8.5) and incubated at 37°C-5% CO2. Aliquots of 50 µl were removed at various time points (up to 24 h) and mixed with 50 µl of acetonitrile and then centrifuged at 16,000g at 4°C for 10 min. Seventy microliters of supernatant was injected onto HPLC immediately after centrifugation, and relative amounts of trans-versus cis-isomers of 4-OH-TAM or endoxifen were determined. Identical experiments were also performed for glucuronidated conjugates of each of the 4-OH-TAM and endoxifen isomers. Possible pH variability of treatment media without 4-OH-TAM was measured after incubation under the same conditions. All experiments were performed three times independently.
Statistical Analysis. Student's t test (two-sided) was used to compare the differences of induction of PGR mRNA expression between different concentrations of test compounds. The statistical significant level was set at 0.05.
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Results |
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). To examine the isomer interconversion in our experimental model, the percentage interconversion between isomers of 4-OH-TAM and endoxifen was measured in treatment media under various pH conditions for up to 24 h. As shown in Fig. 2A, approximately 2% of trans-4-OH-TAM was converted to cis-4-OH-TAM after 12 h of incubation in standard cell culture conditions at pH 
7.5. Less than 5% interconversion was observed after a 24-h incubation at these pH values. Similarly, <2% of cis-4-OH-TAM was converted to trans-4-OH-TAM after a 24-h incubation at pH 7.5 (Fig. 2B). This observation is important because the biological effect of 4-OH-TAM has been suggested to be dependent upon its isomeric state: the trans-isomer is a potent antiestrogen whereas the cis-isomer has been suggested to be a weak estrogen agonist (Furr and Jordan, 1984; Stearns et al., 2003; Desta et al., 2004; Jin et al., 2005). The percentage interconversion of the trans- and cis-isomers of endoxifen were virtually identical to that observed for 4-OH-TAM (results not shown). No interconversion was found for any of the glucuronidated trans-or cis-isomers of TAM, 4-OH-TAM, or endoxifen (results not shown). The pH values of the treatment media used in the cell culture experiments were 8.5 before incubation and 8.0 and 7.7 after 12 and 24 h of incubation, respectively. These results suggested that under the cell culture conditions used in this study, there was minimal interconversion of trans- and cis-isomers of TAM metabolites or their glucuronide conjugates.
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1 x 10-11 M, with maximum induction (20-fold) being observed at 1 x 10-7 ME2 after a 24-h incubation (Fig. 3). The estimated EC50 of E2-induced PGR expression was 1.2 x 10-11 M with a 12-fold induction of PGR mRNA observed after a 12-h treatment (Fig. 3). These results are similar to that observed previously for E2 induction of PGR expression in MCF-7 cells (Lim et al., 2005).
Effects of 4-OH-TAM and Endoxifen Isomers and Their Glucuronide Conjugates on PGR mRNA Expression in the Presence of E2. To minimize the potential confounding effects of isomeric interconversion of 4-OH-TAM and endoxifen on E2 induction of PGR expression, a 12-h time point was used for induction experiments. As shown in Fig. 4, trans-4-OH-TAM-O-glucuronide (Fig. 4A) and trans-endoxifen-O-glucuronide (Fig. 4B) were stable after a 12-h incubation in media with MCF-7 cells. MCF-7 cells were incubated with 1 x 10-10 ME2 and independently treated with each of the isomeric forms of 4-OH-TAM or endoxifen or their O-or N-glucuronide conjugates. As shown previously (Lim et al., 2005), a dose-dependent inhibition of E2-induced PGR gene expression was found for both the trans- and cis-isomers of 4-OH-TAM, with maximal inhibition (complete blockage of E2 induction of PGR gene expression) being attained at 1 x 10-6 M for both isomers (Fig. 5A). An identical pattern of inhibition was observed for both the trans- and cis-isomers of endoxifen (Fig. 5B). These data indicated that each of the isomers of both primary metabolites of TAM exhibited equipotent antiestrogenic effects on PGR gene expression in MCF-7 cells. Interestingly, similar to the effect described previously (Lim et al., 2005), TAM itself exhibited no significant antiestrogenic effect at the same dose range as that used for 4-OH-TAM or endoxifen isomers (Fig. 5A). No effect on PGR gene expression was observed in E2-treated MCF-7 cells for any of the TAM, 4-OH-TAM, or endoxifen glucuronide conjugates examined in this study (Fig. 5, A and B).
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), TAM glucuronides have been detected in the serum of TAM-treated patients (Lien et al., 1988, 1989). Although glucuronidation pathways are largely hepatic, local glucuronidation activity within target tissues including the breast may impact the local pharmacological effect of TAM (Nowell et al., 2005). Therefore, it is clearly important to understand the potential impact of glucuronidation as a potential deactivation step on the pharmacological effects of TAM.
As described in previous studies, 4-OH-TAM and endoxifen exhibit up to 100 times the level of antiestrogenic activity compared with TAM itself as measured by inhibition of E2-induced increases in cell growth, stimulation of plasminogen activator activity, or induction of PGR gene expression (Jordan et al., 1977; Furr and Jordan, 1984; Katzenellenbogen et al., 1984; Murphy et al., 1990; Stearns et al., 2003; Johnson et al., 2004; Lim et al., 2005). Previous studies also showed that the antiestrogenic activities of 4-OH-TAM and endoxifen were approximately equivalent (Jordan et al., 1977; Stearns et al., 2003; Johnson et al., 2004; Lim et al., 2005). In this report, no effect on E2 induction of PGR expression was observed in cells treated with glucuronide conjugates of active TAM metabolites across the same range of TAM metabolite doses that inhibit E2 induction in vitro and that mimic the TAM concentrations (20 mg/day) administered to women in vivo (Lee et al., 2003; Stearns et al., 2003). This pattern was observed for trans- and cis-isomers of both endoxifen- and 4-OH-TAM-glucuronides. These data strongly suggest that the glucuronide conjugates of both 4-OH-TAM and endoxifen render these metabolites inactive at relevant doses. This is particularly important with respect to residual circulating liver-initiated TAM-glucuronides that were not excreted into bile as well as adipose-dependent glucuronidation effects, because alterations in glucuronidation capacity could therefore significantly impact the local pharmacology within target tissues.
Previous studies examining the antiestrogenic properties of TAM metabolite isomers have been hindered by the interconversion between the cis- and trans-isomers of TAM and TAM metabolites shown to occur in previous experimental systems (Jordan et al., 1981; Lieberman et al., 1983; Katzenellenbogen et al., 1984). The present study is the first to examine the antiestrogenic properties of relatively pure isomers of both 4-OH-TAM and endoxifen. This was made possible by the fact that results from the present study demonstrated that the interconversion properties of TAM metabolite isomers are highly pH-dependent, with decreasing interconversion occurring at higher pH values (7.8-8.4). Although significant interconversion (up to 14%) was observed between trans- and cis-isomers at lower pH values, there was <3% conversion between trans- and cis-isomers for both 4-OH-TAM and endoxifen after a 12-h incubation in the cell culture media used in this study. This level of interconversion remained relatively constant over the time course examined with or without the presence of E2 and/or TAM metabolites. This contrasts with the up to 18% isomer interconversion observed over a similar incubation time in previous study (Katzenellenbogen et al., 1984). Therefore, unlike previous in vitro cell culture systems where the pH may not have been optimal to prevent interconversion between isomers, the antiestrogenic properties of relatively pure isomers of TAM metabolites were examined in this study.
Previous studies have shown that the cis- and trans-isomers of 4-OH-TAM exhibit similar patterns of antiestrogenic activities. Interpretation of results from previous studies was difficult because of the possible interconversion between isomers. It was originally suggested that because cis-TAM is estrogenic, the antiestrogenic properties of cis-4-OH-TAM in a rat uterine weight test model may be due to interconversion to its trans-form (Jordan et al., 1981). A later study using a prolactin synthesis model demonstrated a stronger antiestrogenic effect for trans-4-OH-TAM compared with cis-4-OH-TAM and that neither isomer exhibited estrogenic properties, but these data were also confounded by the possibility of interconversion between isomers (Lieberman et al., 1983). Similarly, suppression of MCF-7 cell growth and E2-induced stimulation of plasminogen activator activity by the cis-isomer of 4-OH-TAM was suggested to be due to isomer interconversion (Katzenellenbogen et al., 1984). This suggestion was confirmed in studies of fixed-ring trans- and cis-isomers of TAM and 4-OH-TAM, which prevented isomer interconversion (Murphy et al., 1990). The inhibition of E2-mediated induction of PGR gene expression was roughly equivalent for the trans-versus cis-isomers of 4-OH-TAM in previous studies (Lim et al., 2005), a pattern similar to that observed in the present study for both 4-OH-TAM and endoxifen. These data suggest that the trans- and cis-isomers of these active TAM metabolites exhibit similar properties in terms of inhibition of E2-mediated induction of PGR gene expression and other E2-mediated cellular properties.
This is the first study to examine the antiestrogenic effects of individual isomers of endoxifen. Results from the present study demonstrate that both the trans- and cis-isomers of endoxifen exhibit antiestrogenic activity. Similar levels of reduction in E2-induced PGR gene expression were observed for the two endoxifen isomers at all TAM metabolite doses examined in this study. Interestingly, the same level of inhibition of E2-induced expression of PGR was observed for endoxifen isomers as for isomers of 4-OH-TAM. This is similar to the antiestrogenic properties observed for the two TAM metabolites in previous studies (Stearns et al., 2003; Johnson et al., 2004; Lim et al., 2005). Also similar to that observed in previous studies (Katzenellenbogen et al., 1984) is the fact that TAM exhibited no effect on E2-induced PGR expression in MCF-7 cells at all TAM concentrations tested. This result further supports the fact that the TAM metabolites, 4-OH-TAM and endoxifen, and not TAM itself, are the active antiestrogens in women treated with TAM.
Consistent with the antiestrogenic properties of the trans-isomer of 4-OH-TAM and endoxifen, PGR gene expression was slightly inhibited when MCF-7 cells were incubated in the absence of E2. In contrast, there was no effect on PGR gene expression by the corresponding cis-isomers. This is consistent with a decreased antiestrogenic potential for the cis-isomers of these TAM metabolites.
The results from this study suggest that glucuronidation is an important detoxifying and deactivating metabolic pathway for TAM. Because TAM is an important antiestrogen for the treatment and prevention of estrogen-dependent breast cancer, individuals with higher glucuronidation capacity may therefore deactivate and detoxify TAM at higher rates, potentially increasing risk of breast cancer occurrence and/or recurrence due to lowered drug efficacy. It is also possible that such variations in glucuronidation capacity may alter the risk for TAM-related toxicities. Studies examining the role of UGT pharmacogenetics in different individuals could provide us with important insight into the role of glucuronidation in patient response to TAM.
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ABBREVIATIONS: TAM, tamoxifen, 1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1-(Z)-ene; 4-OH-TAM, 4-hydroxy-TAM; UGT, UDP-glucuronosyltransferase; E2,17ß-estradiol; HPLC, high-performance liquid chromatography; TEA, triethylamine; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle medium; PGR, progesterone receptor; UPLC, ultra-performance liquid chromatography; PCR, polymerase chain reaction; RT, reverse transcriptase.
Address correspondence to: Dr. Philip Lazarus, Division of Population Sciences and Cancer Prevention, Penn State Cancer Institute, Department of Pharmacology, MC-H069, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033. E-mail: plazarus{at}psu.edu
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