D-ring modified estrone derivatives as novel potent inhibitors of steroid sulfatase

A series of novel D-ring modified derivatives of estrone was synthesized and tested as inhibitors of steroid sulfatase (STS). The steroidal D-ring was cleaved via an iodoform reaction and thermal condensation of the resulting marrianolic acid derivative gave 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives, where a piperidinedione moiety is in place of the D-ring. This synthetic approach was found to give a higher overall yield than the literature method of Beckmann rearrangement. A range of alkyl side chains have been introduced on the nitrogen atom of the imido-ring and the corresponding 3-O-sulfamates synthesized. The new D-ring modified estrone derivatives bearing a propyl (39) and a 1-pyridin-3-ylmethyl (46) moiety had IC(50) values of 1 nM when tested in placental microsomes for the inhibition of STS. These compounds are therefore up to 18-fold more potent than EMATE, the very first highly potent irreversible steroidal STS inhibitor.     

2-phenylindole sulfamates: inhibitors of steroid sulfatase with antiproliferative activity in MCF-7 breast cancer cells

A number of 2-phenylindole sulfamates with lipophilic side chains in 1- or 5-position of the indole were synthesized and evaluated as steroid sulfatase (estrone sulfatase) inhibitors. Most of the new sulfamates inhibited the enzymatic hydrolysis of estrone sulfate in MDA-MB 231 breast cancer cells with IC₅₀ values between 2 nM and 1 μM. A favorable position for a long side chain is the nitrogen of a carbamoyl group at C-5 of the indole when the phenyl ring carries the sulfamate function. These derivatives inhibit gene activation in estrogen receptor (ER)-positive MCF-7 breast cancer cells in submicromolar concentrations and reduce cell proliferation with IC₅₀ values of ca. 1 μM. All of the potent inhibitors were devoid of estrogenic activity and have the potential for in vivo application as steroid sulfatase inhibitors.     

Nonsteroidal compounds designed to mimic potent steroid sulfatase inhibitors

Chemical synthesis and enzyme inhibition results are reported for a series of nonsteroidal sulfatase inhibitors, 1-(p-sulfamoyloxyphenyl)-5-(p-t-butylbenzyl)-5-alkanols and the lower active phenolic analogues. These compounds conserve some structural elements from the previously reported potent steroidal inhibitor 3-O-sulfamate-17alpha-(p-t-butylbenzyl)-17beta-hydroxy-estra-1,3,5(10)-triene, while the C18-methyl group and the hydrocarbon backbone represented by the steroid rings B, C, and D were replaced with a free conformational chain. Using estrone sulfate (100 microM) as substrate and homogenate of transfected HEK-293 cells as source of steroid sulfatase activity, the IC(50) values of the best inhibitors, the undecanol derivatives, were 0.4+/-0.1 and >300 nM, respectively, in the sulfamate and phenolic series. Although these sulfamoylated nonsteroidal inhibitors appear a bit less active than their steroidal analogues, they are however more potent than known inhibitors estrone-3-O-sulfamate and p-(O-sulfamoyl)-N-tetradecanoyl tyramine. The optimal side-chain length for the inhibition of steroid sulfatase activity was found to be six carbons, which corresponds to the number of carbons that mimic the B, C and D steroid rings, between C6 and C17. Furthermore, compounds with only the t-butylbenzyl group or the alkyl chain of six carbons are less potent inhibitors compared to the one that include both of these hydrophobic substituents. Such results suggest that compound from this later category better mimic the steroidal inhibitor.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.     

Development of (p-O-sulfamoyl)-N-alkanoyl-phenylalkyl amines as non-steroidal estrone sulfatase inhibitors

Estrogen levels in breast tumors of postmenopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for treatment of estrogen-dependent breast cancer. Several steroidal compounds have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. However, these compounds and their metabolites may have undesired effects, including estrogenicity. To avoid the problems associated with a potentially active steroid nucleus, we designed and synthesized a series of nonsteroidal estrone sulfatase inhibitors, the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines. The compounds synthesized vary in the length of their alkanoyl chain and in the number of carbons separating the phenyl ring and the carbonyl carbon. The ability of these compounds to inhibit estrone sulfatase activity was tested using human placental microsomes and intact cultured human breast cancer cells. Estrogenicity was also evaluated, using growth of estrogen-dependent human breast cancer cells. All of the test compounds inhibited estrone sulfatase activity of human placental microsomes to some extent, with the most effective compound having an IC50 value of 72 nM. In general, compounds with longer alkanoyl chains (12-14 carbons) were more effective than those with shorter chains. The test compounds also inhibited estrone sulfatase activity in intact cultures of MDA-MB-231 human breast cancer cells. Again, the longer chain compounds were more effective. In both the placental and breast cancer cell sulfatase assays, the optimal distance between the phenyl ring and the carbonyl carbon was 1-2 carbons. The MCF-7 cell proliferation assay revealed that estrone and estrone-3-O-sulfamate were both estrogenic, but the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines were not. Our data indicate the utility of (p-O-sulfamoyl)-N-alkanoyl phenyl alkylamines for inhibition of estrone sulfatase activity. Furthermore, our data support the concept that nonsteroidal estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.     

Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.     

New fluorogenic substrate for the first continuous steroid sulfatase assay

The screening for new inhibitors of steroid sulfatase requires an efficient test system. To overcome the shortcomings of the available discontinuous fluorimetric assay, several coumarin-type compounds were investigated as potential new substrates. 3,4-Benzocoumarin 7-O-sulfate was found to have appropriate substrate properties for the establishment of the first direct continuous assay of steroid sulfatase.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

Synthesis, structure, and screening of estrogenic and antiestrogenic activity of new 3,17-substituted-16,17-seco-estratriene derivatives

The starting compound for synthesis of new 16,17-seco-estratriene derivatives was 3-benzyloxy-17-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile (1b), obtained from estrone in several synthetic steps. 17-Tosyl, -chloro-, bromo-, and -iodo- derivatives 2b, 4b, 5b, and 6b were prepared directly from secocyanoalcohol 1b, while the 17-fluoro-derivative 3b was obtained from tosylate 2b in the reaction with tetrabutyl ammonium fluoride. The corresponding 3-hydroxy derivatives of these compounds were produced by action of hydrogen in presence of Pd/C, except the 3-hydroxy-17-iodo derivative 6a, which was obtained from 3-hydroxy-17-tosyloxy derivative 2a. All the newly synthesized compounds in biological tests on experimental animals exhibited an almost total loss of estrogenic activity, while most of them even prevented the action of endogenous estrogens. On the other hand, most of them, except compounds 3a and 6b, partially hindered the action of estradiol benzoate, behaving as moderate antagonists.     

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase,devoid of residual estrogenic activity

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ.Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.     

Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.     

The difluoromethylene group as a replacement for the labile oxygen in steroid sulfates: a new approach to steroid sulfatase inhibitors

Several estrone sulfate and estradiol sulfate analogues, in which the sulfate group was replaced with an alpha,alpha-difluoromethylenesulfonate group or an alpha,alpha-difluoromethylenetetrazole group, were examined as inhibitors of steroid sulfatase (STS). These compounds were 4.5-10.5 times more potent than their non-fluorinated analogues. Moreover, the presence of the fluorines changed the mode of inhibition from mixed to competitive. The inhibitor bearing the alpha,alpha-difluoromethylenetetrazole group exhibited an affinity for STS approaching that of the natural STS substrate, estrone sulfate. Possible reasons for the enhanced affinity of the fluorinated compounds compared to their non-fluorinated counterparts are discussed.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Δ4-isomer are sulfated at the 17β-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3-OH and 3β-OH Tib can form both 3- and 17-monosulfates as well as disulfates. Only the 3β-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4′-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.     

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.     

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.     

Synthesis,biological evaluation,and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4–15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC₅₀ 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.     

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARalpha and RXR and involves the phosphoinositide 3-kinase and ERK-MAP kinase pathways

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.     

Sulfamoyloxy-substituted 2-phenylindoles: antiestrogen-based inhibitors of the steroid sulfatase in human breast cancer cells

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells following the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC₅₀ values in the submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC₅₀s for sulfatase inhibition were 0.3 and 0.2 μM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells.