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Toxicol. Res.

Vol. 27, No. 2, pp. 85-93 (2011)
Open Access DOI:10.5487/TR.2011.27.2.085

The Effect of Selective Estrogen Receptor Modulators
(SERMs) on the Tamoxifen Resistant Breast Cancer Cells
Bo Yoon Chang, Sae Am Kim, Bindu Malla and Sung Yeon Kim
Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea

(Received May 8, 2011; Revised May 13, 2011; Accepted May 16, 2011)

Selective estrogen receptor modulators (SERMs) are synthetic molecules which bind to estrogen
receptors (ER) and can modulate its transcriptional capabilities in different ways in diverse estrogen
target tissues. Tamoxifen, the prototypical SERM, is extensively used for targeted therapy of ER
positive breast cancers. Unfortunately, the use of tamoxifen is associated with acquired resistance and
some undesirable side effects. This study investigated the availability of the conventional SERMs on the
TAM-resistance breast cancer cells. SERMs showed more effectiveness in MCF-7 cells than tamoxifen
resistant cells, except toremifene and ospemifene. Especially, toremifene was more efficacious in
tamoxifen resistant cells than MCF-7. Ospemifene had similar cytotoxic activity on the two types of
breast cancers. The other SERMs used in this experiment didn’t inhibit efficiently the proliferation of
tamoxifen resistant cells. These results support the possibility to usage of toremifene on tamoxifen
resistant cancer. The effectiveness by toremifene on tamoxifen resistant cells might be different
pathways from the apoptosis and the autophagy. Further study should be needed to elucidate the
underlying mechanism of effect of toremifene on tamox-ifen resistant cancer.
Key words: Selective estrogen receptor modulators (SERMs), Tamoxifen resistant breast cancer,
Toremifene, Apoptosis, Autophagy

INTRODUCTION estrogen receptors and both of those estrogen receptors
are different from uterine estrogen receptors. The selective
Breast cancer is one of the common cancers over the world usage of drugs was made possible by the fact that the ERs
in women, approximately 180,000 new case and 40,000 per of different target tissues vary in chemical structures (Lev-
year in Unites States were reported. The inci-dence of this enson and Jordan, 1999; Osborne et al., 2000; Johnston,
disease in several Asian countries has been dramatically 2001). These drugs are called selective estrogen receptor
increased. Over seventy per cent breast can-cers have modula-tors (SERMs). SERMs are synthetic molecules
estrogen receptors, especially estrogen receptor alpha (ERα) which bind to estrogen receptors ER-α and ER-β and can
and require the hormone to grow. Lowering the estrogen modulate its transcriptional factors in different ways in
levels can slow the growth of the breast can-cer. Breast diverse estrogen target tissues (Jordan, 1988; Lerner and
cancers are treated with drugs that interfere with the Jordan, 1990; Avi-oli, 1999).
estrogens not to bind to the estrogen receptors (ERs). Cells in Tamoxifen, the prototypical SERM, is extensively used for
other tissues in the body, such as bones and the uterus, also targeted therapy of ER positive breast cancers. For almost
have ERs. But each ER has a slightly different structure three decades, this drug has been as a first-line therapy in
depending on the kind of cell it is in. It means that breast cell both early and advanced breast cancer. Unfortunately, a long
estrogen receptors are different from bone cell term use of tamoxifen for the control of tumor growth is
associated with acquired resistance and some undesirable
side effects. Around 50% of advanced breast cancer does not
Correspondence to: Sung Yeon Kim, Institute of Pharmaceutical
have susceptibility to first-line therapy with tamoxifen. Also
Research and Development, College of Pharmacy, Wonkwang Uni-
versity, Iksan 570-749, Korea almost all patients with metastatic disease and approx-imately
E-mail: sungykim@wku.ac.kr 40% of the patients that receive tamoxifen as a adjuvant
therapy experienced tumor relapse and die from their disease
Abbreviations: SERM; selective estrogen receptors, TAM;
tamox-ifen, TOR; toremifene, OSP; ospemifene, IDO; (Normanno et al., 2005). The postulated mech-anism of
idoxifene, RAL; ralox-ifene resistance and/or insensitivity to SERM therapy

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A). . For measuring Briefly.A. Medium containing float cells was pharmacy in Chosun University.) to determine of 2 weeks and the concentration of 4-hydroxytamoxifen was autophagy as described Chen et al.. fetal bovine serum (FBS).S.. 2007). The absorbance was read at an optical density 490 nm using 7 cell and TAM resistant cell.A. It must (Promega Corp. PBS two times by centrifugation at 1000 g. U. ER and the growth factor receptor pathways (Normanno et al. All chemicals were of the highest grade commercially available. the cells continuously exposed to 4- inhibition of corepressors and cross talk between the hydroxy tamox-ifen (3 µM). (PI) and RNase A were purchased from Sigma Chemical (St. dimethyl USA) by CellQuest software.) using the Cell Quest software cells were continuously exposed to this treat-ment regimen for (BD Biosciences. UT) and 4-hydroxytamoxifen (0. UT). Pellets were washed twice with cold PBS and bovine serum from Hyclone (Logan. Louis.). 2009. U. MCF. U. after exposure Statistical analysis.A. The data of IC50 was pro- cessed by Graphpad Prism 5. MCF-7 and TAM-R was obtained from American Type Culture Collection (ATCC. methaneth-iosulfonate/phenazine methosulfate analyzed by FACScan flow cytometer (Bec-ton Dickinson solution (MTS) was from Promega Corp. 4. MCF-7 cells were washed with PBS. MO). hydroxytamoxifen. propidium iodode (apoptosis) groups were determined and analyzed (Lin et al. Loss of ER in the tumor. the cell growth rates were reduced. and the culture autophagy. A difference of at (TAM-R) cell line (Choi least p < 0. and toremifene and then favorable professor Kang. U. antibiotics was after which the cells were harvested by centrifuga-tion at 1000 from GIBCO (Gaithersburg. Ospemifene and suspended in PBS containing 50 µg/ml PI. steroid-depleted fetal bovine serum samples were analyzed by flowcytometry (Becton Dickin- (Hyclone.). be valuable to do a closer review of SERMs about the dif. 100 units/ ml times. (U. Acri-dine orange (AO). However. 2007). Logan. 2003). U. Toremifene and raloxifene hydrochloride The cells were then washed twice with cold PBS and re- was purchased from Sigma (St. enhancement of coactivators and resistant cells. Then the cell cycle and sub-G1 sulfoxide (DMSO). CA. Twenty microliters of SERMs.. Dulbecco’s Modified Eagle’s removed and trypsin was added to the cells in plates for 3 min.. Flowcytometric analysis of autophagy. On fixed containing 10% fetal bovine serum (FBS). 2007). 2005. the cell pellet was suspended with 10 µg/ml medium was changed to phenol-red-free DMEM containing acridine orange solution at 37oC for 15~20 min and then 10% charcoal-stripped. medium (DMEM).. Data combined from at least three to the medium for 9 months. Chang et al. the cells were then NewYork). 2007).)..S. MD. U. Sunnyvale. TAM resistance cell was offered by tamoxifen.) as instructed by the manu-facturer.S. To maintain the resistance of TAM- ER mutations. Tamoxifen citrate. and 100 mg/ml streptomycin (Knowlden et al. USA) (Knowlden et MATERIALS AND METHOD al. Louis. Shibutani S (State university of dark room for 30 minutes at 37oC. and toremifene and then incubated for 12 hr. Adamo et al. MCF-7 and TAM-R cells(1×106 cells/well ) cells in 6-well plates were Cell culture.5 hr at 37 oC. San Jose. increased..01.. showing the estab-lishment of a tamoxifen-resistant All values were expressed as mean ± SD. replaced with fresh medium containing various con-centrations 7 cells and TAM-R cells for the development of new of tamoxifen and its derivatives for 24 hr. MO.A. SERMs on the two types of breast cancer cell lines. 6hr after seeding. MD.S. gradually increased to 3 µM over a 9-month period. the rate of cell growth gradually independent experiments were analyzed with Student’s test.A. 2009). Both breast cancer cells were ferences of their estrogen antagonistic properties on MCF.1 µM). et al. The son FACScan. Cell proliferation assay. then fixed by using 70% ethanol (in PBS) at 4 oC overnight.05 was considered statistically significant.Y. cells in 6-well plates (2 × 105 cells/well) were treated with Rochville. The MCF-7 cells were cultured at 37oC in 5% treated with SERMs including tamoxifen. 2003. the cells were harvested by trypsin and rinsed with penicillin. 4-hydroxytamoxifen. steroid-depleted fetal rpm for 5 min. 4-hydroxytamox- CO2/95% air in Dulbecco’s Modified Eagle’s medium (DMEM) ifen. Keon Wook of the department of incubated for 12 hr. Cell proliferation assay was carried out using the the appropriate data which compares the existing several Cell Titer 96 Aqueous One Solution Cell Proliferation Assay SERMs’ potency for MCF-7 cells and TAM-R cells. CA. Materials and reagents.. But it is also true that there isn’t experiment.S. The chemicals and cell culture materials were obtained from following source: MCF-7 cell line Cell cycle and sub-G1 group assays..86 B. a precision microplate reader (Molec-ular Devices Corp. CA. 1 µg/ml RNase in a idoxifene was gift from Dr.S. And the devel-opment of SERMs without any of their harmful stocks of SERMs were kept in a deep freezer for the effects (Peng et al.. Initially. Briefly. was related to followings. researchers are working on the dimethyl sulfoxide (DMSO) to make 3 µM solutions.S.A.. In this study we devised to investigate the methanethiosulfonate/phenazine methosul-fate solution (MTS) estrogen antago-nistic properties of several conventional was added to each well and incubated for 1~1. Choi et al. The SERMs were dissolved in For these reasons.

5% higher than on TAM-R cells. The inhibitory concentration of SERMs in MCF-7 and TAM-R cells The availability of conventional SERMs on the SERMs IC50 P value TAM-resistance breast cancer cells was investigated. 7. Tormifene as a Candidate Drug for Tamoxifen Resistant Cancer 87 RESULTS Table 1.3 ± 1.6 ± 0. The antagonistic effect of TAM on MCF-7 cells *. of three independent experi-ments (* p < 0. Cells were incubated with various concentrations of SERMs.05 in comparison with control). (D) OSP.01. Effect of SERMs including (A) TAM. Table 1).5 ± 4.0 µM on MCF-7 cells and 27. 0. ns stands for not significance.9 * breast cancer cells. .9 µM on experi-ments.5 *** concentration of SERMs than MCF-7 cells to meet IC 50.0 27.7 ± 1. 1. and 0.0 ± 1. respectively.7 ± 0.0 ± 1.7 ** except for TOR (Fig.05. 1. Fig.D. of three independent were 20.3 ns breast cancers showed that TAM-R cells were need a higher IDO 6.5 ± 4.6 9. The IC 50 of tamoxifen (TAM) Data are expressed as mean ± S. TOR 18.7 ± 0.7 ± 0.6 18.3 ± 0. growth inhibition by SERMs was 4-OH TAM 11.D. *** p < 0.3 12.6 ± 0.2 ns The inhibitory concentrations (IC50) against two types of OSP 12. MCF-7 TAM-R summary To deter-mine the effect on the TAM-resistance TAM 20.1 *** measured by cell prolifer-ation assay.1 13. B) 4-OH TAM. Data are expressed as mean ± S.9 ± 4. **.5 ± 0.3 15. RAL 13. (C) TOR. TAM-R cells. (E) IDO and (F) RAL on the viability in MCF-7 and TAM-R cells.001 in comparison with MCF- was 131.

was all about the same inhibitory con-centration between two TAM. and TOR and then incubated in an incubator for 24 hr. of three independent experiments (*p < 0. Data are expressed as mean ± S. 4-OH TAM. 4-hydroxy tamoxifen (4-OH TAM).02 mg/ml EDTA.01. This was a remark-able result when cells. Tamoxifen is mostly acting in vivo indirectly via hydroxy.2 µM on TAM-R cells. The DNA content of cells was measured by flow cytometry and cell cycle profiles were analyzed using CellQuest software. 4-OH TAM. . The IC50 of toremifene (TOR) was 18. The IC50 of idoxifene (IDO) was the most effective Fig. in comparison with control). and TOR treat group.9 ± 4.3 ± compared to other SERMs.7 ± 1. re-suspended in 75% ethanol. Unlike other SERMs.1 µM on MCF-7 cells and TAM-R cells. TOR inhibited the proliferation of TAM-R cells more effectively tion effect compared with that of tamoxifen itself in MCF-7 than MCF-7 cells. types of breast cancers. **p < 0. Sub-G1 area presented apoptosis. TAM. The IC50 of 4-OH TAM was 11.1 µM on MCF-7 lated metabolite.Y. 4-OH cells and 13. Effects of SERMs-induced apoptosis in MCF-7 and TAM-R cells.1% Triton X-100 and 0. 2.D. TAM showed more than two times powerful growth inhibi. respectively. The IC50 of ospemifene (OSP) 1. and stained with 50 µg/ml PI containing 0.3 ± 0. Chang et al. MCF-7 cells and TAM-R cells were treated with SERMs including TAM. OH TAM compare with MCF-7.05.88 B. (A) Flow cytometeric histogram of control group. (B) Apoptotic ratio was calculated with the control group considered to be 1fold.6 µM and 18.R cells showed resistance more than 60% about 4.

Autophagy was determine whether SERMs-decreased via apopto-sis.6 µM against cells was characterized by the treatment of SERM. Apoptosis occurred after treatment with were 13. The IC50 values of raloxifene (RAL) tration of 10 µM. 2. G1 phase from MCF-7 and TAM-R cells were mea-sured to and TOR and then incubated for 12 hr. 6. The AVOs (acidic vesicular organelles) of cells were measured by flow cytometry and cell cycle profiles were analyzed using CellQuest software. Effects of SERMs-induced autophagy in MCF-7 and TAM-R cells. respec-tively. MCF-7 and TAM-R cells were treated with SERMs including TAM. In this experimental condition. As shown in Fig. However the Fig. Tormifene as a Candidate Drug for Tamoxifen Resistant Cancer 89 SERM used in the experiments.3 µM and 15.7 µM on MCF-7 all SERMs of 20 µM applied to TAM-R cells. the percentage of cells with AVOs. (B) Autophagic ratio was calculated with the control group considered to be 1fold. 4-OH TAM. 3). MCF- The effects of SERMs on the cell cycle distribution and sub. (A) This shown isotype untreated (green) and treated (pink). Data are expressed as mean ± S. TAM. of three independent experiments. Apoptotic cell death in TAM-R therapy and survival in breast cancer patients. To determine the role of autophagy by SERMs. The other SERM didn’t show any effect in this experimental SERMs block ER activation and have affected on the condition.7 ± 0. The percentage values represent M1.7 ± 0. number of cells examined. the number of subG1 phase profiles for MCF-7 cells was only clearly DISCUSSION increased after the treatment with 20 mM 4-OH TAM in MCF- 7. there or G2/M phase of the cell cycle. . but the other SERMs had no effect in concen- in both cells. The number of cells in subG1 was not found any evidence related autophagy by of the cell cycle was expressed as a percent-age of the total SERMs in both MCF-7 and TAM-R cells (Fig. Cells were then harvested and stained with acridine orange.5 ± 0. cells and TAM-R cells. 3.6 ± 0. Treat- MCF-7 cells and 9. ment of 10 µM TOR induced significantly subG1 group IDO showed the similar pattern of antagonic effects (apoptosis).5 µM against TAM-R cells. Cells ana-lyzed after stained with acridine orange using with DNA content were designated as being in the G0/G1.D. 7 and TAM-R cells were treated with 4-OH TAM. S flowcytome-try. and TOR and then incubated in an incubator for 24 hr.

2008). conventional SERMs’ possibility for the treatment on TOR was made by the chlorination of TAM. Fig. and position and a pyrrolidino ethoxy side chain replacing the fulvestrant (Shelly et al. TAM. TAM binds antagonism of calmoduline dependent processes (McCague et to cytoplasm estrogen receptors in breast. therefore it has For these reasons.. Kim et al.. and deamino- triphenylethylene and benzothiophene derivatives. resulting in There are various types of conventional SERMs (Fig. based on OH TOR (Berthou et al. 1988) as well as in breast effects (Peng et al. researchers have been working on the greater potency of inhibiting cell multiplication in nor-mal devel-opment of SERMs without any of their unexpected human breast cells (Malet et al. enhanced ER binding and antitumor effect of TAM. TAM acts as an advantages than TAM for the treatment of breast cancer. bazedoxifene. 1994. and idoxifene. including benzothiophene derivatives (arzoxifene). (Table 2) The eliminate free radicals in vitro. 4. It is metabo- tamoxifen resis-tant cancer patients was investigated. IDO has a lot of 2D6 and CYP 3A4 (Stearns et al. IDO is a TAM derivative with an iodine atom in the 4- lasofoxifene. TOR produced two orders of mag-nitude lower DNA types of SERMs have entered clinical development more adducts in rat liver and did not promote hepatocarcinoma in recently. Table 2.. several cancer cell lines in culture (Coezy et al.. metabolized into the major . The TAM is a prodrug that is metabolized to active metabo-lites iodinated TAM placed on the 4-position enhances ER bind-ing 4-OH TAM and endoxifen by cytochrome P450 iso-forms. toremifene. levormeloxifene. rats (Shibutani et al. metabolites for binding to estrogen receptors. 1989).Y. 2001). 2003). pipendoxifene. 4). Raloxifene is a SERM and causes carcinoma on uterus (Labrie et al.. the formation of N-desmethyl TOR. acquired drug resistance. It improved stimulated VEGF production in breast tumor cells. ante-rior pituitary al.. The target of SERMs Site Pure estrogen SERMs Pure 1st generation 2nd generation antiestrogen Bone Agonist Agonist Agonist Antagonist Cholesterol Agonist Agonist Agonist - Uterus Agonist Partial agonist Antagonist Antagonist Mammary Agonist Antagonist Antagonist Antagonist Prototype 17-estradiol Tamoxifen Raloxifene ICI-164384 success of tamoxifen therapy is limited by intrinsic and lite of the antiestrogen. The structure of SERMs. Each of them has its own unique known to inhibit chain reactions of lipoperoxida-tion and to responses to different types of body tissues. are a serious clinical therapeutic problem. 2009). 2003). CYP affinity and inhibits metabolic 4-hydroxylation.. and EM-800). benzopyrans (ormeloxifene. 1999). Unlike with a benzothiophene structure (Jirecek et al. TOR also reduces the side SERMs with triphenylethylene structures are tamoxifen. in humans and other mammals. 4-OH TAM is a metabo. Other TAM. diethylaminoethoxy group in the parent compound. lized by cytochrome P450 1A and 3A4 enzymes. Resistance to tamoxifen as well 4-OH TAM has a higher affinity than TAM and its other as side effects. 4-OH TOR. 2003).90 B. effect of TAM which generates an intrinsic estrogenic effect droloxifene... Chang et al. In this study.. OSP is a novel triphenyl-ethylene compound and is and prostate tissues. TOR was their chemical structures. HMR-3339. IDO estrogen antagonist in mammary gland and blocks estradiol. 1982)..

endometrial carcinoma in breast (Purdie and Beardsworth. 1040-1052. SERM drugs for the prevention of SERMs-decreased via apoptosis.. Azad.. and Gibson. 2010). Can-cer cells evade apoptotic signal. which was a major adverse autophagy is a key mechanism of progression ER a-positive effect of TAM. 10. 1883-1895. The effects of strategies in metastatic breast cancer (MBC) for treatment of SERMs on the cell cycle distribution and sub-G1 phase from tamoxifen-resistant patients. 2005.. Also the researchers Founda-tion Grant funded by the Korean will be able to utilize the data of this study and design Government (KRF-2006-331-E00431). it appears to function lism and estrogen-antagonistic effects on uterine as a protective mechanism against cellu-lar stress and yet the endometrium and breast tissue (Morishima et al. N.. Unlike the other SERMs. Y. although there isn’t a stark con-trast further study should be needed.. (2009). Samaddar et al.B. Food and 2008). In normal cells and in some cancer cells. Metab.. Zanghì.. P. Chen. Cell Death Differ. 317-319. improved SERMs for TAM-resistant cancer... E. induction of autophagy is associated with cell death in some The side effect was less than 1st generation SERMs. it appears to atrophy (Rodriguez et al. uterine can-cer but has anti-osteoporosis effects. (1994). Autophagy plays different role depending on the trials in post-menopausal osteoporosis and urogenital cell type.. Methods for . therefore distribution of the Adamo. RAL tried to be treated as 2nd line effect on TAM-R cells than MCF-7 cells. Recently. 2009... TOR had more anti-estrogenic 1999). in addition to inhibiting the growth of TAM-R cells compared to MCF-7. human breast cancer MCF-7 cells and DMBA-induced Autophagy is an intracellular self eating process involv-ing mammary tumors. RAL has an evidence related autophagy by SERMs in both MCF-7 and antiestrogenic action to inhibit the growth of mammary or TAM-R cells.B. Sci- apoptotic pathway has important effects on the clinical out. Treatment of 10 µM TOR induced signif. OSP binds to estro. Targeting the prodeath and prosurvival func-tions of addition. Gonzalez-Malerva et al.. entering mitosis. but the other SERMs had Pharmacol. between two types of cells. FDA announced approval of RAL breast cancer cells to get anti-estrogen resistance (Schoenlein for reducing the high risk of invasive breast cancer in et al.. The U.. apoptosis and autophagy. Berthou. The effect of TOR in TAM-R cells was not inefficacious in TAM-R cells compared with MCF-7 cells extensively examined. Tormifene as a Candidate Drug for Tamoxifen Resistant Cancer 91 metabolite TOR (deamino-OH-TOR). Belloc. 2011.. Endocrinol. Montalto. Biochem. To elucidate the main mechanisms. (1999). F. 2007). The action mechanism of used 2nd and 3rd line therapies (Miller et al. OSP had similar cytotoxic activity on the two types of breast cancers. L. Cummings et al. 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