Chemotherapy of Breast Cancer-Additive Anticancerogenic Effects by 2-Methoxyestradiol?

Life Sciences 75 (2004) 1205–1210 www.elsevier.com/locate/lifescie

Chemotherapy of breast cancer-additive anticancerogenic effects by 2-methoxyestradiol?

A.O. Mueck*, H. Seeger, J. Huober

Section of Endocrinology and Menopause, Department of Obstetrics and Gynecology, University of Tuebingen, University Women’s Hospital, Calwerstrasse 7, 72 076 Tuebingen, Germany Received 9 October 2003; accepted 25 February 2004

Abstract

2-Methoxyestradiol (2ME) is an endogenous estradiol metabolite, which acts antiproliferative in various tumor cell lines independent of the hormone receptor status. We investigated whether combinations of 2ME with various chemotherapeutic or endocrine compounds may result in an additive effect on the proliferation of human breast cancer cells. The breast cancer cell lines MCF-7 (receptor-positive), BM (receptor-negative) and a MCF-7 line transfected with the aromatase gene were used. All cell lines were incubated in the concentration range of 0.8 AMto25AM with 2ME alone and in equimolar combinations with the following compounds: epirubicine, daunorubicine, paclitaxel, docetaxel, carboplatin, vinorelbine, 5-fluorouracil, mafosfamide and 4-OH tamoxifen. The effect of letrozole and 2ME alone and in equimolar combinations was tested in the concetration range of 0.6 to 1 AM. Proliferation was measured after 4 days using the ATP-chemosensitivity test. In MCF-7 cells 2ME in combination with 4OH-tamoxifen, epirubicine, docetaxel, 5-fluoprouracil, mafosfamide and carboplatin led to an additive effect. In BM cells only 2ME combined with 4OH-tamoxifen, daunorubicine and mafosfamide showed an additive action. Both letrozole and 2ME were nearly similar effective in inhibition of the aromatase gene. Here no additive effect was found. 2ME displayed antiproliferative actions in various human breast cancer cells. In addition 2ME was able to increase the antiproliferative property of certain antihormones and cytostatic substances. Furthermore 2ME exhibits a similar property as compared to letrozole in inhibiting the aromatase activity. Since 2ME was well tolerated in a recently conducted phase II trial in patients with refractory metastatic breast cancer, the combination of 2ME with chemotherapeutics or antihormones may offer a new clinically relevant treatment regimen. D 2004 Elsevier Inc. All rights reserved.

Keywords: 2-Methoxyestradiol; Chemotherapeutics; Breast cancer cells; Proliferation

* Corresponding author. Tel.: +49-7071-2984801; fax: +49-7071-29-4801. E-mail address: [email protected] (A.O. Mueck).

Introduction

2-Methoxyestradiol (2ME), an endogenous estradiol metabolite, elicits an antiproliferative potency in various tumor cell lines and endothelial cells independent of the hormone receptor status (Fotsis et al., 1994; Lippert et al., 2000, 2003). The mechanism(s) responsible are not fully elucidated, however, different actions such as influence on tubulin polymerization, enhanced expression of p53 consecutively followed by apoptosis and inhibition of the proliferation of vascular endothelial cells have been demonstrated (Zhu and Connery, 1998). In in vitro and in vivo studies an additive effect of 2 ME has been shown at least with ionising radiation (Amorino et al., 2000). In the present study we investigated whether combinations of 2ME with various chemotherapeutic or endocrine compounds may result in an additive effect on the proliferation of human breast cancer cells.

Material and methods

2-Methoxyestradiol (2ME), androstenedione and 4-hydroxytamoxifen (4OH-Tam), an active metabolite of tamoxifen were purchased from Steraloids and Sigma Chemical, USA, respectively. Letrozole (Let) was kindly provided by Novartis Pharma GmbH, Nuremberg, Germany. The cytostatica used were epirubicine (Epi, Pharmacia), daunorubicine (Dau, Pharmacia), paclitaxel (Pac, Bristol-Myers Squibb), docetaxel (Doc, Aventis), carboplatin (Car, Bristol-Myers Squibb), vinorelbine (Vin, Pierre Fabre Pharma), 5-fluorouracil (FU, Hexal) and mafosfamide (Maf, Baxter). The compounds were dissolved in ethanol or PBS where appropriate and diluted by an ethanol/PBS mixture to yield a final ethanol concentration of 0.1% per well or by PBS alone. MCF-7, a human estrogen and progestin receptor-positive breast cancer cell line, was purchased from ECACC, UK. BM, a receptor-negative human breast cancer cell line, was established in our own laboratory and characterized by immunohistochemical methods for the expression of estrogen receptor-a and progestin receptors. In addition a MCF-7 cell line transfected with the aromatase gene, kindly provided by Novartis, Switzerland, was used. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% (v/ v) fetal calf serum supplemented with 0.3 mg/ml glutamine, 5 ng/ml bovine insulin and 100 U/ml penicillin plus 100 Ag/ml streptomycin. Ninety six well plates were seeded with approximately 1000 cells per well in assay kit medium. Subsequently, the agents were added to the wells in the concentrations of 0.8 to 25 AM alone and in equimolar combinations. After incubation for 4 days, cell proliferation was measured by an ATP-chemosensitivity test (Andreotti et al., 1991). In brief, proliferation is quantified by measuring light which is emitted during the bioluminescence reaction of luciferine in the presence of ATP and luciferase. For the MCF-7 cells transfected with the aromatase gene, the test concentration range was 0.06 Mto1AM for both 2ME and letrozole. Proliferation was stimulated by the addition of 0.1 pM androstenedione. A.O. Mueck et al. / Life Sciences 75 (2004) 1205–1210 1207

Statistical analysis was done by ANOVA with the logarithmated values followed by Dunnett’s procedure from quadruplicates of two independent experiments. The overall alpha level was set at 0.05.

Results

In MCF-7 cells 2ME alone had an inhibitory effect on the proliferation in the range of 5– 35% as compared to controls. 4OH-Tam showed an antiproliferative effect in the range of 6 to 40% was observed. For epirubicine, daunorubicine, paclitaxel, docetaxel and vinorelbine the reduction of cell proliferation was in the range of 40 to nearly 100 %. For carboplatin, 5- fluorouracil, gemitacine and mafosfamide the values were in the range of 5 to 40%. As seen in Fig. 1, in combination with 2ME an additive effect was found for 4OH-Tam, Epi, Doc, FU, Maf and Car. This additive effect was more pronounced in the lower concentrations in the case of Epi and Doc. In contrast, for Car an additive effect was only seen at the highest concentration of 25 AM. For 5-FU and Maf an additive antiproliferative effect was seen over the entire tested concentration range. In BM cells 2ME elicited an antiproliferative effect with values in the range of 15 to 70%. For 4OH-Tam the corresponding figures were between 1.6 and 98%. Among the cytostatic substances Epi, Dau, Doc and Pac were more effective than Vin, Car, FU, Gem and Maf. As depicted in Fig. 2 in combination with 2ME only 4OH-Tam, Dau and Maf showed an additive antiproliferative effect, which was more pronounced in the lower concentration ranges.

Fig. 1. Changes in proliferation of MCF-7 cells after addition of 2-methoxyestradiol (2-ME), epirubicine (Epi), carboplatin (Car), docetaxel (Doc), 5-fluorouracil (FU), mafosfamide (Maf) and 4-hydroxytamoxifen (4OH-Tam) alone and in equimolar combinations. (means F SD, each concentration in quadruplicates from two independent experiments, *p < 0.05, **p < 0.01 comparing combination vs. monosubstances). 1208 A.O. Mueck et al. / Life Sciences 75 (2004) 1205–1210

Fig. 2. Changes in proliferation of BM cells after addition of 2-methoxyestradiol (2-ME), daunorubicine (Dau), mafosfamide (Maf) and 4-hydroxytamoxifen (4OH-Tam) alone or in equimolar combinations. (means F SD, each concentration in quadruplicates from two independent experiments, *p < 0.05, **p < 0.01 comparing combination vs. monosubstances).

Fig. 3 shows the results of the effect of 2ME and Let on the proliferation of MCF-7 cells transfected with the aromatase gene. Letrozole significantly inhibited the androstenedione-induced proliferation between 20 and 70%. 2ME significantly reduced cell proliferation in the range of 17 to 60%. For 2ME the net proliferative effect of about 15% at a concentration of 1 AM in the absence of androstenedione was considered. The equimolar combination did not show any additive effect.

Discussion

Estrogens are known to be a major factor in the etiology of human breast cancer. Therefore, one of the main strategies in the treatment of breast cancer is to prevent the binding of estrogens to its receptor or to reduce systemic or local synthesis of estrogens (Bentrem and Jordan, 2002). Tamoxifen, an antiestrogen, is widely used for endocrine treatment of human breast cancer. Drug resistance, however, remains a significant problem in breast cancer treatment. This is emphasized by the finding that about 15–20% of patients who eventually progress after an initial response to primary treatment are known to respond to second-line therapy with

Fig. 3. Changes in proliferation of MCF-7 cells transfevted with the aromatase gene after addition of 2-methoxyestradiol (2- ME) and letrolzole (Let) alone or in equimolar combinations. (means F SD, each concentration in quadruplicates from two independent experiments, *p < 0.05, **p < 0.01 compared to the effect of androstenedione (And)). A.O. Mueck et al. / Life Sciences 75 (2004) 1205–1210 1209 another endocrine agent (Leonard et al., 1994). Breast cancer cells seem to adapt to tamoxifen treatment by switching to growth factor signalling pathways (Santen, 2003). Recent clinical trials suggest that aromatase inhibitors may be as good as or even better than tamoxifen in neoadjuvant as well as adjuvant therapy of breast cancer, since they can nearly block estrogen synthesis (Santen, 2003). Furthermore aromatase inhibitors may be superior to tamoxifen in the prevention of breast cancer due to the elimination of possible toxic estradiol metabolites (Santen, 2003). Recent research has gained evidence that 2-methoxyestradiol (2-ME), an endogenous estradiol metabolite, may be a candidate for treatment of several cancers because of its anti-carcinogenic and anti-proliferative properties (Zhu and Connery, 1998). As yet little in vitro data are available investigating the combination of 2ME with other compounds currently used for breast cancer treatment. Our results using the anti-hormonal substances tamoxifen and letrozole as well as various cytostatic substances demonstrated that 2ME may be able to enhance the antiproliferative property of certain therapeutic compounds. This was true for tamoxifen, epirubicine, docetaxel, carboplatin, 5-fluorouracil and mafosfamide in the receptor-positve cell line MCF-7. In the receptor-negative cell line BM, again the combination of 2ME with tamoxifen showed an additive antiproliferative effect. Furthermore, 2ME plus daunorubicine and mafosfamide acted additively. These data emphasize that different mechanism(s) by 2ME and by certain therapeutic substances are working and thus may enhance the effectivity of breast cancer treatment. A possible positive influence of 2ME on treatment resistance e.g. to tamoxifen is conceivable, but has to be explored in further experiments and clinical studies. For the first time we compared the efficacy of 2ME as aromatase inhibitor with that of letrozole, a well-known potent inhibitor, demonstrating that 2ME is nearly equipotent to letrozole. However, no additive effect was observed indicating a similar mechanism by 2ME and letrozole. Purohit et al. already demonstrated that 2ME is able to inhibit the basal and TNF-a stimulated conversion of androstenedione into estrone (Purohit et al., 1999). The mechanism by which 2ME blocks the activity of the aromatase gene and thus reduces the formation of estrogens is currently unknown. Enhancing the efficacy of conventional breast cancer therapies such as chemo- or hormonal treatment by new compounds are promising ways in the gynecological oncology. Animal experiments and the preliminary data of a clinical phase II study indicate that 2ME has a very low toxicity and is well tolerated even in high dosages (Fotsis et al., 1994; Miller et al., 2001). First results of a phase I study of the combination of 2ME with docetaxel revealed a good tolerability (EntreMed, data on file). 2ME did not significantly alter the pharmacokinetics of docetaxel and vice versa. Serum levels of 2ME achieved after treatment with a dosage of 1mg were in the range of 100 to 600 nM. Preliminary experiments with a new 2ME formulation demonstrated plasma concentrations up to 10 AM (EntreMed, data on file), thus approximating the effective concentrations in our experiment. Thus the combination of 2ME with different chemotherapeutics or antihormones such as tamoxifen or aromatase inhibitors may provide new clinical options in the treatment of breast cancer. The efficacy of such combinations can be monitored using the chemosensitivity test, which is established in our laboratory for chemotherapeutics since several years and shows a good reproducibility with low standard errors. 1210 A.O. Mueck et al. / Life Sciences 75 (2004) 1205–1210

In vitro data indicate that 2ME may beneficially influence the cardiovascular system by positively modulating the production of vasoactive substances (Seeger et al., 1997, 1999; Dubey and Jackson, 2001). However, these results await confirmation in clinical studies as well as the effect of 2ME on menopausal symptoms such as hot flushes.

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