The effects of 2-substituted oestrogen sulphamates on the growth of prostate and ovarian cancer cells

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Abstract

The human endogenous metabolite 2-methoxyoestradiol (2-MeOE2) has been shown to inhibit the proliferation of breast cancer cells. We have previously shown that sulphamoylation of a series of 2-substituted oestrogens greatly enhances their ability to inhibit breast cancer cell proliferation and induce apoptosis. In this study, we have investigated the ability of a number of 2-substituted oestrogens and their sulphamoylated derivatives to inhibit the proliferation of two prostate cancer cell lines, an ovarian cancer cell line and its drug-resistant derivatives. 2-Methoxyoestrone, 2-ethyloestrone and 2-ethyloestradiol had little effect on the growth of the cell lines tested (IC50>10 μM). 2-MeOE2 did inhibit the growth of the cells (IC50<10 μM), but to a lesser extent than any of the sulphamoylated derivatives tested (IC50<1.0 μM). Cells treated with the sulphamoylated derivatives became detached and rounded, displaying a characteristic apoptotic appearance. FACS analysis revealed induced G2/M cell cycle arrest. Treatment of cells and subsequent drug removal indicated that the effects of the drugs on the cells were irreversible. Immunoblot analysis indicated that apoptosis may be induced by phosphorylation of BCL-2. From these studies, 2-substituted oestrogen sulphamates are emerging as a potent new class of drug that may be effective against AR+/AR− prostate and ovarian tumours, and against tumours that are resistant to conventional chemotherapeutic regimens.

Introduction

Both prostate and ovarian cancers are heterogeneous: the tumours can include many cell types and are often made up of both androgen/oestrogen receptor positive and negative cells [1], [2]. Although both are initially responsive to the treatment regimens used they often recur within a few years. Prostate cancer is the third highest cause of cancer-related death in men. Tumours can develop and go unnoticed over several years and as such usually present at an advanced stage in older men. By this stage they are usually androgen-independent and far more resistant to treatment than early stage tumours, which are usually hormone-dependent. The level of prostate specific antigen (PSA) in serum is used as a marker to test for prostate cancer and its response to treatment [3]. There is little consensus on the best treatment, particularly when diagnosed at the later androgen-independent stages, as treatments available are often ineffective by this time. Treatments achieving some level of success include diethylstilboestrol [4], and mitoxantrone, both alone and in combination with secondary hormonal therapies [3].

Ovarian cancer is the fifth most fatal type of cancer, with a 5-year survival rate of around 30% [2]. As with prostate cancer, this low survival rate is due to late stage detection of most ovarian cancers. For many years the standard treatment against ovarian cancer has been aggressive surgery followed by chemotherapy. Initially alkylating agents were used in the 1970s, but their use was replaced or combined with that of platinum-based compounds, such as cisplatinum and carboplatin, in the 1980s. More recently, microtubule disrupting and other chemotherapeutic agents have been used, either alone or in combination with those already mentioned [5]. Although these advances have lead to higher response rates and prolonged survival times, there has been little change in overall mortality rate. Initial response rates are high (70–80%), but the tumours often recur in a drug-resistant form [6]. To improve survival rates for ovarian, prostate and other cancers, microarray analysis of differentially expressed genes is now being used to identify markers to detect the cancer at an earlier stage [7].

The human endogenous metabolite 2-methoxyoestradiol (2-MeOE2) has been shown to inhibit the proliferation of ER+/ER− breast cancer cells in vitro [8] and the growth of mammary tumours, B16 melanomas and Meth A sarcomas in mice [9], [10]. It causes G2/M cell cycle arrest and apoptosis in prostate cancer cell lines, and inhibits prostate tumour progression in a transgenic mouse model [11]. 2-MeOE2 also has anti-angiogenic properties [10]. We have previously shown that sulphamoylation of a series of 2-substituted oestrogens greatly enhances their ability to inhibit breast cancer cell proliferation and to induce apoptosis in MCF-7 cells [12], [13]. In addition, 2-methoxyoestrone-3-O-sulphamate (2-MeOEMATE) caused regression of NMU-induced mammary tumours in intact rats [14]. In the present study, we have extended our initial investigations to examine the ability of a number of 2-substituted oestrogens and their sulphamoylated derivatives to inhibit the proliferation of prostate and ovarian cancer cells.

The prostate cell lines tested include an androgen receptor positive cell line, LNCaP [15], and an androgen receptor negative cell line, PC3 [16]. The ovarian cell lines include a parent cell line, A2780, and the adriamycin- and cisplatin-resistant cell lines derived from these cells, A2780cis and A2780adr [17], [18]. The initial studies have shown that the sulphamoylated derivatives are also effective in inhibiting the growth of these cells.

Section snippets

Culture of cell lines

Androgen-responsive LNCaP prostate carcinoma cells, androgen-independent PC3 prostate carcinoma cells, the A2780 ovarian carcinoma cell line and its adriamycin- and cisplatin-resistant derivatives, were purchased from the European Collection of Cell Cultures. The LNCaP cells were grown in RPMI supplemented with 10% FBS, 10 mM HEPES, 2 mM l-glutamine and 1 mM sodium pyruvate; the PC3 cell line maintained in Coon’s modified Ham’s F12 supplemented with 10% FBS and 2 mM l-glutamine; and the A2780 cell

Inhibition of cell proliferation

Prostate cancer cell lines, LNCaP and PC3, and ovarian cancer cell lines, A2780, A2780adr and A2780cis, were treated with three concentrations of the 2-substituted oestrogens, 2-MeOE2 and 2-EtE2, and their sulphamoylated derivatives over 4 days. The cells were also treated with three concentrations of known proliferation inhibitors, taxol and colchicine. Inhibition of growth of the various cell lines in the presence of the treatments is shown in Fig. 2.

The proliferation of both the prostate and

Discussion

These initial proliferation studies indicate that of the compounds tested the sulphamoylated derivatives of 2-EtE2 are the most efficient inhibitors of proliferation of both the prostate cancer cell lines, PC3 and LNCaP, and that at 1 μM they are as effective as taxol and colchicine. The ovarian cell line proliferation is inhibited by the sulphamoylated and bis-sulphamoylated derivatives of both 2-MeOE2 and 2-EtE2, again at 1 μM as efficiently as when treated with taxol or colchicine. The

Acknowledgements

The authors would like to thank Mr. D. Bennetto for technical assistance. This research was supported by Sterix Ltd.

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