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This study addresses first the role of human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) in breast cancer (BC) cells. The enzyme has a high estrone-activating activity that is subject to strong substrate inhibition as shown by enzyme kinetics at the molecular level. We used BC cells to verify this phenomenon in living cells: estrone concentration increase did reduce the reaction with 0.025 to 4μM substrate. Moreover, 5α-dihydrotestosterone (DHT) demonstrated some inhibition of estrogen activation at both the molecular and cellular levels. The presence of DHT did not change the tendency toward substrate inhibition for estrone conversion, but shifted the inhibition toward higher substrate concentrations. Moreover, a binding study demonstrated that both DHT and dehydroepiandrosterone (DHEA) can be bound to the enzyme, thereby supporting the multi-specificity of 17β-HSD1. We then followed the concentrations of estradiol and performed q-RT-PCR measurements of reductive 17β-HSDs after 17β-HSD1 inhibition. The estradiol decrease by the 17β-HSD1 inhibition was demonstrated lending support to this observation. Knockdown and inhibition of 17β-HSD1 produced reduction in estradiol levels and the down-regulation of another reductive enzyme 17β-HSD7, thus "amplifying" the reduction of estradiol by the 17β-HSD1 modulation itself. The critical positioning of 17β-HSD7 in sex-hormone-regulation as well as the mutual regulation of steroid enzymes via estradiol in BC, are clearly demonstrated. Our study demonstrates that fundamental enzymological mechanisms are relevant in living cells. Moreover, further enzyme study in cells is merited to advance biological and medical research. We also demonstrated the central role of 17β-HSD7 in sex-hormone conversion and regulation, supporting it as a novel target for estrogen-dependent (ER+) BC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a steroidal enzyme which, in breast cancer cells, mainly synthesizes 17-beta-estradiol (E2), an estrogenic hormone that stimulates breast cancer cell growth. We previously showed that the enzyme increased breast cancer cell proliferation via a dual effect on E2 and 5α-dihydrotestosterone (DHT) levels and impacted gene expression and protein profile of breast cancer cells cultured in E2-contained medium. Here, we used RNA interference technique combined with microarray analyses to investigate the effect of 17β-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition. Our data revealed that knockdown of 17β-HSD1 gene, HSD17B1, modulates the transcript profile of the hormone-dependent breast cancer cell line T47D, with 105 genes regulated 1.5 fold or higher (p < 0.05) in estradiol-independent manner. Using Ingenuity Pathway Analysis (IPA), we additionally assessed functional enrichment analyses, including biological functions and canonical pathways, and found that, in concordance with the role of 17β-HSD1 in cancer cell growth, most regulated genes are cancer-related genes. Genes that primarily involved in the cell cycle progression, such as the cyclin A2 gene, CCNA2, are generally down-regulated whereas genes involved in apoptosis and cell death, including the pro-apoptotic gene XAF1, IFIH1 and FGF12, are on the contrary up-regulated by 17β-HSD1 knockdown, and 21% of the modulated genes belong to this latter functional category. This indicates that 17β-HSD1 may be involved in oncogenesis by favoring anti-apoptosis pathway in breast cancer cells and correborates with its previously shown role in increasing breast cancer cell proliferation. The gene regulation occurring in steroid-deprived conditions showed that 17β-HSD1 can modulate endogenous gene expression in steroid-independent manners. Besides, we tested the ability of estrogen to induce or repress endogenous genes of T47D by microarray analysis. Expression of a total of 130 genes were found to increase or decrease 1.5-fold or higher (p < 0.05) in response to E2 treatment (1 nM for 48 h), revealing a list of potential new estrogen-responsive genes and providing useful information for further studies of estrogen-dependent breast cancer mechanisms. In conclusion, in breast cancer cells, in addition to its implication in the E2-dependent gene transcription, the present study demonstrates that 17β-HSD1 also modulates gene expression via mechanisms independent of steroid actions. Those mechanisms that may include the ligand-independent gene transcription of estrogen receptor alpha (ERα), whose expression is positively correlated with that of the enzyme, and that may implicate 17β-HSD1 in anti-apoptosis pathways, have been discussed., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16β-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17β-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1μM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1μM, not at all at 0.1μM, and showed an interesting inhibitory potency on 17β-HSD1 (90, 59 and 22%, at 10, 1 and 0.1μM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17β-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17β-HSD type 7 (17β-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17β-HSD7 in breast carcinoma. Inhibition of 17β-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17β-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17β-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism., (© The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

The reductive 17β-hydroxysteroid dehydrogenases which catalyze the last step in estrogen activation for estrogen dependent breast cancer cells were studied. Their biological function and the effects of their knockdown for cancer cell proliferation were demonstrated. The multidisciplinary study involves enzyme catalysis, sex-hormone and cell cycle regulation, as well as cell proliferation in breast cancer cells. Reductive 17β-HSD1, -7 and -12 were studied in the main breast cancer epithelial cells MCF-7 and T47D. Modification of estradiol and 5α-dihydrotestosterone concentrations was monitored by ELISA assay while corresponding cell viability measured by MTT assay. Cell cycle was determined by flow cytometry. Dual activity of estradiol activation and 5α-dihydrotestosterone reduction by 17β-HSD1 and -7 was critical for breast cancer cell (T47D and MCF-7) viability. Cell viability was decreased by 35.8% ± 1.6% in T47D cells after simultaneously knocking down 17β-HSD1 and -7. MCF-7 cell viability was decreased by 29.3% ± 4.2% using a combination of siRNAs and inhibitors. By knocking down 17β-HSD7, we have provided the first demonstration of the significant role of this enzyme in the stimulation of breast cancer cell viability as a result of its high activity on androgen reduction with positive feedback on estradiol production. A further decrease in cell viability was not observed with additional knockdown of 17β-HSD12 after 17β-HSD1 and 7. Breast cancer cell cycle progression was impeded to enter the S phase from G0-G1 after knocking down 17β-HSD1 and -7. In summary, this is the first demonstration that the dual activity in estrone activation and 5α-dihydrotestosterone reduction are the functional basis of reductive 17β-HSDs in breast cancer cells. 17β-HSD1 and -7 are principal reductive 17β-HSDs and major players in the viability of estrogen-dependent breast cancer cells. Combined targeting of these enzymes may be potential for molecular therapy of such cancer., (Copyright © 2015. Published by Elsevier Ltd.)

Estrone and estradiol are both estrogens with estrone being the less potent form and estradiol being the most potent estrogen. The binding of the latter to cellular regulatory elements stimulates the proliferation of breast cancer cells. A high ratio of estradiol/estrone is related to increased cell proliferation, and is of great importance to understanding of breast cancer mechanisms. 17beta-hydroxysteroid dehydrogenase type 1 and type 2 play important roles in the activation of estrone and inactivation of estradiol. Breast cancer cells T47D, MCF-7, BT 20, and JEG 3 as control cells, were chosen to evaluate the contribution of these two enzymes to the ratio. Twenty four hours after addition of different concentrations of estrone and estradiol, the ratio stabilized to around 9/1 in breast cancer cell lines with high expression of type 1 (T47D, BT 20, and JEG 3), whereas it approached 1/5 in cells with low expression of type 1 (MCF-7). The estradiol/estrone concentration ratio was modified to 9/1 in MCF-7 and HEK-293 cells over-expressing type 1. In T47D and BT 20, this ratio was decreased from 9/1 to nearly 1/5 (19/81 and 17/83 respectively) after type 1 knockdown by specific siRNAs. Type 2 is mainly involved in the conversion of estradiol into estrone. This ratio was decreased from 9/1 to 7/3 after over-expression of type 2 in MCF-7 cells already over-expressing type 1. The ratio was further decreased by the addition of the oxidative cofactor, NAD, to the cell culture to facilitate the estradiol to estrone conversion catalyzed by type 2. These results demonstrate that the estradiol/estrone ratio is controlled by both type 1 and type 2 with an additional contribution by NAD, although type 1 is the first determining factor in the cellular environment compared with type 2 and cofactors. Moreover, kinetic studies were carried out in intact cells as a new approach, using HEK-293 cells over-expressing type 1 and T47D breast cancer cells.

The active estrogen estradiol (E2) stimulates breast cancer cell (BCC) growth, whereas the androgen dihydrotestosterone (DHT) has shown an antiproliferative effect. The principal product synthesized by the 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is E2, although we have demonstrated that the purified enzyme also inactivates DHT. However, the direct roles of 17beta-HSD1 in sex-hormone regulation and BCC proliferation have not been completely established. Here, we show that 17beta-HSD1 inhibition suppresses DHT catabolism by 19%, whereas knockdown of the gene expression increases the concentration of DHT by 41% in the T47D BCC line. The 17beta-HSD1/DHT complex crystal structure reveals that DHT binds in both normal and reverse modes, but the latter mode leading to O3 reduction is preferred with stronger interactions. Using RNA interference and an inhibitor of 17beta-HSD1, we demonstrate that 17beta-HSD1 expression is negatively correlated to DHT levels in BCC but positively correlated to estrone reduction, E2 levels, and cell proliferation. 17beta-HSD1 inhibition reduces DHT inactivation, increasing the antiproliferative effect by DHT in T47D cells after 8 d treatment. Thus, 17beta-HSD1 up-regulates BCC growth by a dual action on estradiol synthesis and DHT inactivation. We have further demonstrated that 17beta-HSD1 can enhance the E2-induced expression of the endogenous estrogen-responsive gene pS2, providing an important information regarding the modulation of the estrogen responsiveness by 17beta-HSD1 that may also contribute to BCC growth. These results strongly support the rationale for inhibiting 17beta-HSD1 in breast cancer therapy to eliminate estrogen activation via the sulfatase pathway while avoiding the deprivation of DHT.

Oestradiol is a well-characterized sex hormone that stimulates breast cancer and other oestrogen-related diseases. 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the last step in the synthesis of oestradiol and androstenediol in breast tumour tissue. The enzyme's high expression and activity after simultaneous blockade of oestrogen receptors and inhibition of aromatase in the tumour shows the necessity for its inhibition as a requirement for breast cancer therapy. In the present paper, we report structures of the binary and ternary complexes of 17beta-HSD1 with a new inhibitor E2B {3-[3',17'beta-dihydroxyestra-1',3',5'(10')-trien-16'beta-methyl]benzamide}, and the enzyme inhibition by the later. The IC50 value for E2B was determined to be 42 nM in T47D cells. Multiple interactions between E2B and the enzyme include hydrogen bonds and hydrophobic interactions, as well as pi-pi interactions. A kinetic study demonstrated that E2B inhibits the enzyme's reduction forming oestradiol from oestrone, with a Ki of 0.9+/-0.15 nM. Such strong inhibition is in agreement with its extensive interaction with the enzyme, suggesting its potential as a lead compound for breast cancer therapy. In fact, this possibility is enhanced by its capacity for cell penetration similar to natural steroids. Such inhibitors that block oestrogen synthesis to suppress the sulfatase pathway producing oestradiol can be used in adjuvant therapies with oestrogen receptor blockade, opening a new orientation of breast cancer treatment.

Three series of steroid derivatives, enones 1, enols 2 and saturated alcohols 3, were easily synthesized from estrone according to a sequence of three reactions: an aldol condensation with an aromatic aldehyde (R(a-g)CHO) to afford 1, the carbonyl reduction of 1 to obtain the enol 2, and the double bond reduction of 2 to give 3 with the R(a-g) group 16beta-oriented. All compounds were tested as inhibitors of type 1 17beta-HSD. The inhibitory potency increases in the following order 1<2<3, suggesting that the presence of a flexible 16beta-methylene group allows a better positioning of the aryl moiety. With an IC50 of 0.8 microM, the 16beta-benzyl-E2 (3a) is the best inhibitor in this series.

The steroidogenic enzyme type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is involved in the synthesis of estradiol (E(2)), a hormone well-known to stimulate the growth of estrogen-sensitive tumors. To obtain compounds able to control E(2) formation, two moieties were linked with a methylene side chain: an adenosine moiety for interacting with the cofactor-binding site and an E(2) moiety for interacting with the substrate-binding site. When tested as inhibitors of type 1 17beta-HSD, the hybrid compounds inhibited the reductive activity (E(1) into E(2)) with IC(50) values ranging from 52 to 1,000 nM. The optimal side-chain length was determined to be eight methylene groups for a 16 beta-orientation. The presence of two components (E(2) and adenosine) is essential for good inhibition, since 16 beta-nonyl-E(2) and 5-nonanoyl-O-adenosine, two compounds having only one of the components, did not inhibit the enzyme. Moreover, the 3D-structure analysis of EM-1,745 complexed with type 1 17beta-HSD showed key interactions with both substrate- and cofactor-binding sites.

Crystallographic studies of ligand-protein complexes reveal most preferable ligand binding modes, but do not show less populated modes that may contribute to measurable biochemical and biophysical characteristics of the complexes. In some cases, a ligand may bind a protein in essentially different modes. An example is 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), a steroidogenic enzyme that catalyzes reduction of estrone to estradiol in gonadal and peripheral tissues. The enzyme exhibits a high specificity for estrogens which bind with their C17 atom in the proximity of the NADP(H) cofactor. 17Beta-HSD1 can also bind androgens, but in a reverse binding mode, in which the steroid C3 atom is the closest carbon atom to the cofactor. Here we map the interaction energy of estradiol and dihydrotestosterone binding to 17beta-HSD1. Positions and orientations of the steroids in the ligand-binding tunnel were sampled systematically, and at each combination of these generalized coordinates, the energy was Monte Carlo minimized. The computed maps show energy minima corresponding to the X-ray structures and predict alternative binding modes, in particular, an upside-down orientation in which steroidal face alpha is exposed to protein residues that normally interact with face beta. The methodology can be used for mapping ligand-receptor interactions in various systems, for example, in ion channels and G-protein-coupled receptors that bind elongated ligands in confined space between transmembrane helices.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.

17 beta-Estradiol (E2) is a potent stimulator of certain forms of breast cancer. The final step of E2 biosynthesis is catalyzed by the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), which is an important target for anti-cancer drugs. X-ray crystallography indicated that the binding site for the steroids has a tunnel-like shape. We have used a Monte Carlo-Minimization (MCM) protocol to explore possibilities of interactions of E2 with the binding site tunnel of 17 beta-HSD1. The enzyme was represented by flexible residues having at least one atom within 6 A from either E2 or NADP (as seen in a crystal ternary complex) and by rigid residues having at least one atom within 10 A from E2 or NADP. Special constraints were used to pull the substrate 10 A along the tunnel with 1 A step; the complex was MCM-optimized at each position of the steroid. The optimal binding mode of E2 in 17 beta-HSD agrees with the crystallographic data; however, wide and flat minima of the MCM profile suggest alternative modes of the steroid binding. The advance of the steroid along the tunnel is accompanied by essential conformational rearrangements of the enzyme side chains, noticeable rotation of the substrate along its longitudinal axis, and certain conformational deformations of the substrate. The contributions of the enzyme residues and of the steroid atoms to the intermolecular energy were estimated.

Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. The K(m) of estrone is more than 1000-fold lower than that of DHEA and the K(m) of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17beta-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 --> Val variant showed a significantly decreased K(m) for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a beta-oriented form, which approaches the transition-state conformation for DHEA reduction.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17Beta-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17beta-HSD1-EM519-NADP+ and 17beta-HSD1-EM553-NADP+ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a = 155.59 A, b = 42.82 A, c = 121.15 A, beta = 128.5 degrees for 17beta-HSD1-EM519-NADP+, and a = 124.01 A, b = 45.16 A, c = 61.40 A, beta = 99.2 degrees for 17beta-HSD1-EM553-NADP+, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.