Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyoestradiol-bis-sulphamate

The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously shown that the 3-sulphamoylated derivatives of 2-methoxyoestrogens are more potent than the non-sulphamoylated compounds. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth with the IC(50) for 2-MeOE2bisMATE (0.4 microM) being six-fold lower than that for 2-MeOE2 (2.5 microM). Oestrogen sulphamates are potent inhibitors of steroid sulphatase (STS) activity. 2-MeOE2bisMATE was found to retain its STS inhibitory activity and in a placental microsome assay system it was equipotent with oestrone-3-O-sulphamate (EMATE). An in vivo study was also carried out to compare the potency of 2-MeOE2bisMATE with that of EMATE and the non-steroidal STS inhibitor, 667 coumarin sulphamate (667 COUMATE). After a single oral dose (10mg/kg) some recovery of STS activity was detected by day 3 (10%) with activity partially restored (55%) by day 7 after administration of 667 COUMATE. For the other two steroidal compounds, STS activity remained almost completely inactivated for up to 5 days with complete restoration of activity occurring by day 15. The anti-proliferative and STS inhibitory properties of 2-MeOE2bisMATE suggest that it has considerable potential for development as a novel anti-cancer drug.     

Growth inhibition of multi-drug-resistant breast cancer cells by 2-methoxyoestradiol-bis-sulphamate and 2-ethyloestradiol-bis-sulphamate

There is currently considerable interest in the use of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) for the treatment and prevention of breast cancer. We have previously shown that sulphamoylation of 2-MeOE2 and related derivatives greatly enhances their ability to inhibit the proliferation of ER+ and ER- breast cancer cells. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-ethyloestradiol-bis-sulphamate (2-EtE2bisMATE) with that of 2-MeOE2 to inhibit the proliferation of breast cancer cells when grown on three different substrata: plastic, collagen I and Matrigel. The human breast cell line MCF-7 was utilised for these studies together with its doxorubicin resistant variant, MCF-7 DOX40 and mitoxantrone resistant variant, MCF-7 MR, as a longitudinal model of in vitro drug resistance. On a plastic substratum all three cell lines were sensitive to the effects of 2-MeOE2bisMATE and 2-EtE2bisMATE whereas MCF-7 cells and the MCF-MR variant cells were resistant to the effects of 2-MeOE2 at 1 microM. The sensitivity of the cell lines to those compounds also remained significant when grown on more physiological substrata. All of the drugs tested arrested cells in the G2/M phase of the cell cycle. The finding that breast cancer cells that are resistant to conventional chemotherapeutic agents remain sensitive to 2-substituted oestrogen sulphamates offers considerable potential for the treatment of women with drug-resistant breast cancer.     

2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line

In anticancer research where the focus is on finding agents that induces cell death while leaving non-tumorigenic cells less affected, a novel 2-methoxyestradiol derivative has come forth. 2-Methoxyestradiol-bis-sulfamate (2-MeOE2bisMATE) is a 2-methoxyestradiol derivative produced by bis-sulphamoylation, which possesses increased antiproliferative activity and biological availability. Several questions remain regarding the type of cell death mechanisms and possible induction of autophagy by 2-MeOE2bisMATE. The aim of this in vitro study was to investigate the cell death mechanisms exerted by 2-MeOE2bisMATE in an adenocarcinoma cell line (MCF-7) by analyzing its influence on cell growth, morphology, and possible induction of cell death. Spectrophotometry (crystal violet staining), transmission electron microscopy (TEM), light microscopy (hematoxylin and eosin staining), and fluorescent microscopy (Hoechst 33342, propidium iodide and acridine orange) were employed. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 75% in MCF-7 cells after 24 h and to 47% after 48 h of exposure. TEM demonstrated membrane blebbing, nuclear fragmentation, and chromatin condensation indicating the hallmarks of apoptosis. Light microscopy revealed the presence of several cells blocked in metaphase, and apoptotic cells were also observed. Fluorescent microscopy demonstrated increased lysosomal staining; suggesting the induction of autophagy. 2-MeOE2bisMATE shows therapeutic potential, as an, anticancer agent, and the investigation of the cell death mechanisms used by 2-MeOE2bisMATE, thus, warrants further investigation.     

The <Emphasis Type="Italic">in vitro</Emphasis> effects of 2-methoxyestradiol-bis-sulphamate on cell numbers,membrane integrity and cell morphology,and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line

2-methoxyestradiol (2ME2) exerts estrogen receptor-independent anti-proliferative, anti-angiogenic and anti-tumor activity in vitro and in vivo. Due to its low bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is a bis-sulphamoylated derivative of 2ME2 with anti-proliferative activity. The aim of this study was to investigate cell signaling events induced by 2-MeOE2bisMATE in a non-tumorigenic cell line (MCF-12A) by analysing its influence on cell number, morphology and membrane integrity, and the possible induction of apoptosis and autophagy. Dose- and time-dependent studies revealed that 48 h exposure to 2-MeOE2bisMATE (0.4 μM) resulted in a decrease in cell numbers to 79%. A slight increase in the level of lactate dehydrogenase production was observed in the 2-MeOE2bisMATE-treated cells. Morphological studies revealed an increase in the number of cells in metaphase. Hallmarks of apoptosis were also found, namely nuclear fragmentation and apoptotic bodies. In addition, increased lysosomal staining was observed via fluorescent microscopy, suggesting the induction of another type of cell death, namely autophagy. Since 2-MeOE2bisMATE is regarded as a potential anti-cancer agent, it is also imperative to investigate the susceptibility of non-tumorigenic cells to its influence. The data generated from this study contributes to the understanding of the action that 2-MeOE2bisMATE exerts on the non-tumorigenic MCF-12A breast epithelial cell line.     

Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates

Sulfamoylated derivatives of the endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2 (7)), such as 2-methoxy-3-O-sulfamoyl estrone (2-MeOEMATE (1)), display greatly enhanced activity against the proliferation of human cancer cells and inhibit steroid sulphatase (STS), another current oncology target. We explore here the effects of steroidal D-ring modification on the activity of such 2-substituted estrogen-3-O-sulfamates in respect of inhibition of tumour cell proliferation and steroid sulphatase. The novel 17-deoxy analogues of 2-MeOEMATE and the related 2-ethyl and 2-methylsulfanyl compounds showed greatly reduced inhibition of MCF-7 proliferation. Introduction of a 17alpha-benzyl substituent to such 2-substituted estrogen sulfamates also proved deleterious to anti-proliferative activity but could, in one case, enhance STS inhibition with respect to the parent substituted estrone sulfamate. In contrast, selected 17-oxime derivatives of 2-MeOEMATE displayed an enhanced anti-proliferative activity. These results illustrate that enhanced in vitro anti-cancer activity can be achieved in the 2-substituted estrogen sulfamate series and highlight, in particular, the importance of potential hydrogen bonding effects around the steroidal D-ring in the activity of these molecules. The SAR parameters established herein will assist the future design of anti-proliferative and anti-endocrine agents as potential therapeutics for both hormone dependent and independent cancers.     

2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases

2-Methoxyoestradiol (2-MeOE2) is an endogenous oestrogen metabolite which inhibits tubulin polymerisation and has anti-tumour and anti-angiogenic activity. 2-MeOE2 induces apoptosis in a wide range of cancer cell types and has recently been demonstrated to cooperate with TRAIL to induce apoptosis in breast cancer cells. 2-Methoxyoestradiol-3,17-bis-O,O-sulphamate (2-MeOE2bisMATE) is a sulfamoylated derivative of 2-MeOE2 with enhanced activity and improved pharmacokinetic properties, and 2-MeOE2bisMATE is a promising candidate for early clinical trials. It is important, therefore, to understand the mechanisms by which 2-MeOE2bisMATE acts, and whether it retains the ability to cooperate with TRAIL. We demonstrate that 2-MeOE2bisMATE-induced apoptosis of CAL51 breast cancer cells was associated with rapid activation of caspase 3 and 9, but not caspase 8 (as measured by BID cleavage) and was completely prevented by the caspase inhibitor zVADfmk. Interfering with Fas- or TRAIL-receptor function did not prevent 2-MeOE2bisMATE-induced apoptosis. Whereas CAL51 cells were resistant to TRAIL-induced apoptosis, 2-MeOE2bisMATE and TRAIL cooperated to induce cell death. This apoptosis was associated with enhanced activation of caspases, but not increased expression of the DR5 TRAIL receptor, previously demonstrated to be induced by 2-MeOE2. Therefore, 2-MeOE2bisMATE-induced apoptosis is dependent on caspases and like 2-MeOE2, 2-MeOE2bisMATE can overcome resistance to TRAIL by stimulating activation of downstream caspases. Our results suggest that 2-MeOE2bisMATE and TRAIL might be a particularly effective combination of anti-cancer agents.     

Steroid sulphatase inhibitors for breast cancer therapy

In contrast to aromatase inhibitors, which are now in clinical use, the development of steroid sulphatase (STS) inhibitors for breast cancer therapy is still at an early stage. STS regulates the formation of oestrone from oestrone sulphate (E1S) but also controls the hydrolysis of dehydroepiandrosterone sulphate (DHEA-S). DHEA can be reduced to 5-androstenediol (Adiol), a steroid with potent oestrogenic properties. The active pharmacophore for potent STS inhibitors has now been identified, i.e. a sulphamate ester group linked to an aryl ring. This has led to the development of a number of STS inhibitors, some of which are due to enter Phase I trials in the near future. Such first generation inhibitors include the tricyclic coumarin-based 667 COUMATE. Aryl sulphamates, such as 667 COUMATE, are taken up by red blood cells (rbc), binding to carbonic anhydrase II (CA II), and transit the liver without undergoing first-pass inactivation. 667 COUMATE is also a potent inhibitor of CA II activity with an IC₅₀ of 17 nM. Second generation STS inhibitors, such as 2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE), in addition to inhibiting STS activity, also inhibit the growth of oestrogen receptor negative (ER⁻) tumours in mice and are anti-angiogenic. As the active pharmacaphores for the inhibition of aromatase and STS are now known it may be possible to develop third generation inhibitors that are capable of inhibiting the activities of both enzymes. Whilst exploring the potential of such a strategy it was discovered that 667 COUMATE possessed weak aromatase inhibitory properties with an IC₅₀ of 300 nM in JEG-3 cells. The identification of potent STS inhibitors will allow the therapeutic potential of this new class of drug to be explored in post-menopausal women with hormone-dependent breast cancer. Second generation inhibitors, such as 2-MeOE2bisMATE, which also inhibit the growth of ER⁻ tumours should be active against a wide range of cancers.     

The effects of 2-methoxy oestrogens and their sulphamoylated derivatives in conjunction with TNF-alpha on endothelial and fibroblast cell growth, morphology and apoptosis

2-methoxyoestradiol (2-MeOE2) is a potent anti-angiogenic agent. Its 3- and 17-sulphamoylated derivatives have been demonstrated to induce G2-M cell cycle arrest and apoptosis in breast cancer cells in vitro as well as tumour regression in rats in vivo with greater potency than the parent oestrogen. To determine whether the anti-cancer properties of these derivatives can be synergistically enhanced with low-dose TNF-alpha co-treatment, we investigated the effects of these treatments in adult human fibroblasts and human umbilical vein endothelial cells (HUVECs). Treatment of fibroblasts with 0.1 microM 2-methoxyoestradiol-3,17-bis sulphamate (2-MeOE2bisMATE) but not 2-MeOE2 caused a reversible morphology change and induced G2-M arrest (from 12 to 33%) but not subsequent apoptosis. In contrast, treatment of HUVECs did not induce morphology change or G2-M arrest. Using a nucleosomal ELISA assay, we showed that TNF-alpha (20 ng/ml) combination treatment synergistically increases 0.1 microM 2-MeOE2bisMATE-induced but not 0.1 microM 2-MeOE2-induced apoptosis in HUVECs. These results suggest that TNF-alpha co-treatment may be a beneficial method of increasing the potency of 2-substituted oestrogens as anti-angiogenic agents through synergistic induction of apoptosis in endothelial cells while maintaining low cytotoxicity to fibroblasts.     

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

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.     

Regulation of steroid sulphatase expression and activity in breast cancer

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.     

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

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 (IC(50)>10 microM). 2-MeOE2 did inhibit the growth of the cells (IC(50)<10 microM), but to a lesser extent than any of the sulphamoylated derivatives tested (IC(50)<1.0 microM). Cells treated with the sulphamoylated derivatives became detached and rounded, displaying a characteristic apoptotic appearance. FACS analysis revealed induced G(2)/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.     

Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/mTOR signaling pathway in human breast cancer cells

The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC₅₀ values 0.057 and 0.051 μM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7).     

Synthesis, in vitro and in vivo activity of benzophenone-based inhibitors of steroid sulfatase

Steroid sulfatase (STS) is an important new therapeutic target in oncology. Attempts to design nonsteroidal STS inhibitors, because of the oestrogenicity of the original lead oestrone 3-O-sulfamate in rodents, have led to the discovery of benzophenone-4,4'-O,O-bis-sulfamate (BENZOMATE, 3). The nonfused bicyclic BENZOMATE is a highly potent STS inhibitor in vitro, inhibiting STS activity in intact MCF-7 breast cancer cells by > 70% at 0.1 microM and in placental microsomes by > 98% at 10 microM. When MCF-7 cells were pre-treated with 3 at 1 microM and then washed to remove unbound inhibitor, the initial 94% inhibition was reduced to 89% suggesting that 3, like other sulfamate-based STS inhibitors, inhibits the enzyme irreversibly. This agent also inhibits rat liver STS activity by 84% and 93% respectively 24 h after a single dose of 1 or 10 mg/kg, demonstrating that BENZOMATE possesses similar in vivo potency to the established potent nonsteroidal inhibitor 667COUMATE. Several modifications were made to BENZOMATE structurally and effects on in vitro activity were examined. These structure-activity relationship studies show that its carbonyl and bis-sulfamate groups are pivotal for activity, although conformational flexibility is not required. Two rigid anthraquinone-based sulfamate derivatives however showed inhibitory activity significantly better than BENZOMATE in the MCF-7 cell assay. BENZOMATE and related analogues therefore represent an important class of non-steroidal STS inhibitor and lead compounds for future drug design.     

The cytotoxic effects of estradiol-17 beta, catecholestradiols and methoxyestradiols on dividing MCF-7 and HeLa cells

In this study the cytotoxic effects of high concentrations (greater than or equal to 1 x 10(-6) M) of estradiol-17 beta (E2), 2-/4-hydroxyestradiol-17 beta (2-/4-OHE2) and 2-/3-/4-methoxyestradiol-17 beta (2-/3-/4-MeOE2) were determined on dividing MCF-7 and HeLa cells. The 2-MeOE2 metabolite followed by 2-OHE2 and E2 (in this order) proved to be extremely toxic to dividing MCF-7 and HeLa cells. The cytotoxic effect on these cells comprised uneven chromosome distribution. Indirect immunofluorescent studies, in which monoclonal anti-alpha-tubulin antibodies were used, showed that these compounds (2-MeOE2 greater than 2-OHE2 greater than E2) at high concentrations caused abnormal and fragmented polar formations as well as disorientated microtubule arrangement in the dividing MCF-7 and HeLa cells. The 4-OHE2 and 3-/4-MeOE2 metabolites had little or no cytotoxic effects on dividing cells. The large number of abnormal metaphases seen in HeLa cells exposed to 2-MeOE2 suggested that this metabolite may be the ultimate cytotoxic compound. The reduction in the number of HeLa cells with abnormal metaphase configurations after exposure to 2-OHE2 plus quinalizarin (an inhibitor of catechol-O-methyltransferase) indicated that the production of 2-MeOE2 is necessary for the formation of abnormal spindles in metaphase. Quinalizarin treatment in the presence of 2-MeOE2 had no effect on the large number of abnormal metaphases. We therefore conclude that neither E2 nor 2-OHE2, but a high concentration of 2-MeOE2 is responsible for abnormal spindle formation. In additional experiments the number of normal and abnormal dividing HeLa cells were greatly reduced when simultaneously exposed to E2 and 2-/4-hydroxylase-inhibitor alpha-naphthoflavone.     

Novel pyrazole derivatives: Synthesis and evaluation of anti-angiogenic activity

The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.     

The nature of inhibition of steroid sulphatase activity by tibolone and its metabolites

Tibolone is used for hormone replacement therapy and acts in a tissue-specific manner being oestrogenic on CNS and bone but not on breast tissues or endometrium. The ability of tibolone and its metabolites to inhibit steroid sulphatase (STS) activity has a crucial role in regulating its tissue-specific effects. In this study, we have examined the ability of tibolone and its non-sulphated and sulphated metabolites to inhibit STS activity in different enzyme preparations and in intact cells. For this, we have used an 'extracellular' method, which measures the amount of product released into culture medium, and an 'intracellular' method, which assesses the extent of product formation within cells. In addition, the nature by which tibolone and some of its metabolites inhibit STS activity was investigated using intact cells and an enzyme kinetic method. In MCF-7 and T47D breast cancer cells and JEG-3 choriocarcinoma cells, which have high STS activity, tibolone and its metabolites were relatively potent inhibitors of STS activity (33-57% inhibition at 10 microM) using the extracellular assay method. In HOS-TE-85 osteoblast-like cells, tibolone and its Delta-4 metabolite were relatively inactive whereas the 3alpha/3beta-hydroxy metabolites and their sulphated conjugates inhibited activity by 39-55%. When STS activity was assessed in HOS-TE-85 cells using an 'intracellular' method tibolone and its 3beta-hydroxy metabolite were inactive. Pre-treatment of breast cancer cells and JEG-3 cells, and removal of drugs prior to assaying for STS activity, revealed that in these cells tibolone and its metabolites were acting mainly as reversible inhibitors. This finding was confirmed in an enzyme kinetic study to measure concentration-dependent STS inhibition. In HOS-TE-85 cells, pre-treatment of cells and removal of compounds before assaying for remaining STS activity indicated that some tibolone metabolites appeared to stimulate STS activity. Possible mechanisms by which this might occur are discussed but, if confirmed, this could contribute to the positive oestrogenic effects that tibolone has on bone.     

Tibolone and its delta-4, 7alpha-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3alpha- and 3beta-hydroxy derivatives and the delta-4, 7alpha-methylnorethisterone (7alpha-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) activities but also stimulate steroid sulphotransferase and 17beta-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7alpha-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7alpha-MeNET, but not the 3alpha- or 3beta-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10muM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7alpha-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17beta-HSD1, tibolone and 7alpha-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.     

Evaluation of synthetic isoflavones on cell proliferation, estrogen receptor binding affinity, and apoptosis in human breast cancer cells

Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.