Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

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Abstract

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC50 below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC50 below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.

Introduction

Estrogens support the growth of hormone-dependent tumours of the breast and endometrium. Enzymes involved in estrogen synthesis in women, such as the aromatase and the 17β-hydroxysteroid dehydrogenase type I, therefore, are obvious targets for therapeutic intervention. An enzyme, which has more recently been proposed as a potential point of attack for anti-tumour therapy, is steroid sulfatase (STS) [1], [2]. The rationale for an important role of STS and its substrates estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) in breast cancer is based on the following arguments:

  • 1.

    Estradiol concentrations in breast tumours of postmenopausal women are 10–40 times higher than serum levels [3], [4], [5], [6], [7]. Furthermore, tissue levels of estradiol in breast tumours of post-menopausal women are nearly equivalent to those in premenopausal patients [7], even though plasma estradiol levels are 5- to 60-fold lower after menopause [8]. These observations are best explained by the local formation of the estrogens by enzymes in the tumour [9], [10]. Biosynthesis of estrogens in tumour tissues may occur via two different routes, either from androstenedione (aromatase pathway) [11], [12] or from estrone sulfate (sulfatase pathway) [13], [14]. Direct evidence has been obtained that in situ formation of estrone in breast tumour tissue via the sulfatase pathway is at least 10-fold higher than by the aromatase route [9], [10], [15]. In another study, the activity of STS in both pre- and postmenopausal patients was found to be 50–200 times higher than that of aromatase [16].

  • 2.

    Estrone sulfate, featuring higher concentration [17] and half-life [18] than estradiol in plasma and high concentration in breast tissue [1], appears to represent a reservoir for the formation of biologically active estrogens in the breast tissue [2].

  • 3.

    In addition to the active estrogens estrone and estradiol, androst-5-ene-3β,17β-diol (ADIOL) as another ligand of the estrogen receptor may be important for growth of breast cancer [19], [20], [21], [22]. About 90% of ADIOL are derived from DHEAS in a pathway involving the sulfatase [23]. DHEAS is found in significant amounts in breast cancer tissue [24].

  • 4.

    Recent epidemiological studies identified STS expression as an independent predictor of recurrence in human breast cancer [25] and revealed an increased risk of postmenopausal breast cancer in women with elevated prediagnostic serum levels of estrone sulfate and DHEAS [26].

Both the formation of active estrogens from estrone sulfate and the formation of ADIOL from DHEAS may be blocked by inhibitors of STS. Such compounds could provide an addition to aromatase blockers, which are in clinical use for the treatment of mammary carcinoma, in order to complete the estrogen blockade. To this end, a considerable number of STS inhibitors have been synthesized (see Refs. [27], [28], [29], [30], [31], [32] for some recent examples). In this study, we describe a novel chemotype, which is a more potent blocker of estrone sulfate-stimulated proliferation of MCF-7 breast cancer cells as compared to previously published reference compounds. Importantly, for one selected non-estrogenic derivative, we also demonstrate inhibition of DHEAS-stimulated MCF-7 cell growth, which is not blocked by specific aromatase inhibitors.

Section snippets

Chemicals and reagents

2,5-Dihydroxyacetophenone, pivaloyl, benzoyl and decanoyl chloride were purchased from the Aldrich Chemical Co. Amidochlorosulfonic acid was prepared according to the method of Appel and Berger [33].

Biochemicals were obtained from Sigma (St. Louis, MO). Materials for protein chromatography were bought from Pharmacia, Uppsala, Sweden. Steroids were checked for purity by HPLC. Commercial batches of estrone sulfate or DHEAS (sodium salts) contained up to 3% of estrone or DHEA, respectively.

Synthesis of non-steroidal STS inhibitors

We designed and synthesized novel non-steroidal aryl sulfamates (compounds 4 and 5, Fig. 1) featuring a 4-chromenone ring system as steroid sulfatase inhibitors. The bicyclic ring system serves as estrone AB-ring mimicry and the alkyl groups fill the space of the steroidal CD-rings. The sulfamate functionality is attached to the benzene ring in diagonally opposite position relative to the alkyl groups in the heteroaromatic ring, which is important for high inhibitory potency.

The synthesis of 4

Discussion

The most active inhibitors of steroid sulfatase reported so far are aryl sulfamates, featuring as aryl moiety either 3-hydroxy steroids [28], [29], [30], [31], 7-hydroxy-coumarins [28], [34], or N-alkanoyl-phenylalkyl amines [27], [32], [35]. We have identified a novel non-steroidal chemotype, exemplified by compounds 4 and 5, which exhibit a chromenone group. The present compounds are irreversible enzyme inhibitors; as such, their IC50 values strongly depend on the assay conditions (incubation

Acknowledgements

We thank Dr Dean Evans for providing us with the MCF-7 cells and a protocol for their culture. We thank Dr Dieter Scholz for synthesis of estrone sulfamate and Libuse Malacek for excellent technical assistence.

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