Steroid conjugates of dichloro(6-aminomethylnicotinate)platinum(II): effects on DNA, sex hormone binding globulin, the estrogen receptor, and various breast cancer cell lines

Esters of 6-aminomethylnicotinic acid with various steroidal alcohols were treated with K(2)PtCl(4) to give the N,N-chelated dichloroplatinum(II) complex conjugates 4. Their interaction with plasmid DNA was monitored by electrophoretic mobility measurements. Their affinities towards sex hormone binding globulin (SHBG) and towards the nuclear estrogen receptor ER(alpha) were assessed by competitive displacement radioassays. The inhibitory effect of 4 on breast tumour cells MCF-7 ER(+)/ER(-) and MDA-MB-231 was investigated in vitro. Conjugates with 3-O-linked estrogens 4 a,b or 17-O-linked androgens 4 g bound strongly to SHBG. The conjugate complex 4 b, featuring a 3-O-linked estradiol, also bound strongly and agonistically to the estrogen receptor. It also elicited distinct growth retardation of MCF-7 (ER(+)) cells, presumably by a mechanism different from that of cisplatin.     

A Role for TGFβ Signaling in Preclinical Osteolytic Estrogen Receptor-Positive Breast Cancer Bone Metastases Progression

While tumoral Smad-mediated transforming growth factor β (TGFβ) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFβ signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E₂)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFβ receptors II and I, only cells with TGFβ-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFβ and E₂ each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFβ neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFβ may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E₂ and TGFβ signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.     

Plasma Sphingosine-1-Phosphate Levels Are Associated with Progression of Estrogen Receptor-Positive Breast Cancer

Although numerous experiments revealed an essential role of a lipid mediator, sphingosine-1-phosphate (S1P), in breast cancer (BC) progression, the clinical significance of S1P remains unclear due to the difficulty of measuring lipids in patients. The aim of this study was to determine the plasma concentration of S1P in estrogen receptor (ER)-positive BC patients, as well as to investigate its clinical significance. We further explored the possibility of a treatment strategy targeting S1P in ER-positive BC patients by examining the effect of FTY720, a functional antagonist of S1P receptors, on hormone therapy-resistant cells. Plasma S1P levels were significantly higher in patients negative for progesterone receptor (PgR) expression than in those positive for expression (p = 0.003). Plasma S1P levels were also significantly higher in patients with larger tumor size (p = 0.012), lymph node metastasis (p = 0.014), and advanced cancer stage (p = 0.003), suggesting that higher levels of plasma S1P are associated with cancer progression. FTY720 suppressed the viability of not only wildtype MCF-7 cells, but also hormone therapy-resistant MCF-7 cells. Targeting S1P signaling in ER-positive BC appears to be a possible new treatment strategy, even for hormone therapy-resistant patients.     

The Impact of Soy Isoflavones on MCF-7 and MDA-MB-231 Breast Cancer Cells Using a Global Metabolomic Approach

Despite substantial research, the understanding of the chemopreventive mechanisms of soy isoflavones remains challenging. Promising tools, such as metabolomics, can provide now a deeper insight into their biochemical mechanisms. The purpose of this study was to offer a comprehensive assessment of the metabolic alterations induced by genistein, daidzein and a soy seed extract on estrogen responsive (MCF-7) and estrogen non-responsive breast cancer cells (MDA-MB-231), using a global metabolomic approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that all test compounds induced a biphasic effect on MCF-7 cells and only a dose-dependent inhibitory effect on MDA-MB-231 cells. Proton nuclear magnetic resonance (¹H-NMR) profiling of extracellular metabolites and gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites confirmed that all test compounds shared similar metabolic mechanisms. Exposing MCF-7 cells to stimulatory concentrations of isoflavones led to increased intracellular levels of 6-phosphogluconate and ribose 5-phosphate, suggesting a possible upregulation of the pentose phosphate pathway. After exposure to inhibitory doses of isoflavones, a significant decrease in glucose uptake was observed, especially for MCF-7 cells. In MDA-MB-231 cells, the glutamine uptake was significantly restricted, leading to alterations in protein biosynthesis. Understanding the metabolomic alterations of isoflavones represents a step forward in considering soy and soy derivates as functional foods in breast cancer chemoprevention.     

Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

1,25-Dihydroxycholecalciferol, the hormonally active vitamin D₃ metabolite, is known to exhibit therapeutic effects against breast cancer, mainly by lowering the expression of estrogen receptors and aromatase activity. Previously, the safety of the vitamin D active metabolite (24R)-1,24-dihydroxycholecalciferol (PRI-2191) and 1,25(OH)₂D₃ analog PRI-2205 was tested, and the in vitro activity of these analogs against different cancer cell lines was studied. We determined the effect of the two vitamin D compounds on anastrozole (An) activity against breast cancer based on antiproliferative activity, ELISA, flow cytometry, enzyme inhibition potency, PCR, and xenograft study. Both the vitamin D active metabolite and synthetic analog regulated the growth of not only estrogen receptor-positive cells (T47D and MCF-7, in vitro and in vivo), but also hormone-independent cancer cells such as SKBR-3 (HER-2-positive) and MDA-MB-231 (triple-negative), despite their relatively low VDR expression. Combined with An, PRI-2191 and PRI-2205 significantly inhibited the tumor growth of MCF-7 cells. Potentiation of the antitumor activity in combined treatment of MCF-7 tumor-bearing mice is related to the reduced activity of aromatase by both An (enzyme inhibition) and vitamin D compounds (switched off/decreased aromatase gene expression, decreased expression of other genes related to estrogen signaling) and by regulation of the expression of the estrogen receptor ERα and VDR.     

A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.     

Cytotoxic Triosmium Carbonyl Clusters: A Structure–Activity Relationship Study

A structure–activity relationship (SAR) study of the triosmium carbonyl cluster Os₃(CO)₁₀(NCCH₃)₂ was carried out with a series of clusters of the general formula Os₃(CO)_12?nL_n, cationic osmium clusters and a hemi‐labile maltolato‐Os cluster. The SAR results showed that good solubility in DMSO and at least one vacant site are required for cytotoxicity. In vitro evaluation of these new compounds showed that some are selectively active against estrogen receptor (ER)‐independent MDA‐MB‐231 breast cancer cell lines relative to ER‐dependent MCF‐7 breast cancer cells, suggesting that the compounds have a different biological target specific to MDA‐MB‐231 cells. In particular, the maltolato cluster exhibits strong antiproliferative activity, with an IC₅₀ value of 3 μM after only 24 h incubation. Additionally, biochemical assays conducted with the cationic cluster show that it induces apoptosis, although a biological target has not yet been identified. Further research to establish the molecular targets of these compounds and to develop improved organometallic clusters as potential breast cancer therapeutics is underway.     

Removal of BPA and NPnEOs from Secondary Effluents of Municipal WWTPs by Means of Ozonation

Removal potential of many EDCs by conventional WWTPs is recognized. Nevertheless, in order to reach very low concentrations, further treatment (e.g., chemical oxidation) might be required. In this work, two estrogen-like substances were considered: nonylphenol (NP) (and its parent compounds) and bisphenol A (BPA). The experimental work was conducted at Verona (Northern Italy) WWTP (370,000 p.e.): the effluent was submitted to ozonation in a pilot scale plant. Beside chemical and microbiological analyses, estrogenic activity measurements were also carried out by means of breast cancer MCF-7 cells transfected with the luminescence luciferase gene. Economic feasibility of tertiary ozonation is finally discussed.     

Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.     

Manipulating Estrogenic/Anti-Estrogenic Activity of Triphenylethylenes towards Development of Novel Anti-Neoplastic SERMs

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI₅₀ = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.     

Development of selective estrogen receptor modulator (SERM)-like activity through an indirect mechanism of estrogen receptor antagonism: defining the binding mode of 7-oxabicyclo[2.2.1]hept-5-ene scaffold core ligands

Previously, we discovered estrogen receptor (ER) ligands with a novel three-dimensional oxabicyclo[2.2.1]heptene core scaffold and good ER binding affinity act as partial agonists via small alkyl ester substitutions on the bicyclic core that indirectly modulate the critical switch helix in the ER ligand binding domain, helix 12, by interactions with helix 11. This contrasts with the mechanism of action of tamoxifen, which directly pushes helix 12 out of the conformation required for gene activation. We now report that a much larger substitution can be tolerated at this position of the bicyclic core scaffold, namely a phenyl sulfonate group, which defines a novel binding epitope for the estrogen receptor. We prepared an array of 14 oxabicycloheptene sulfonates, varying the phenyl sulfonate group. As with the parent compound, 5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester (OBHS), these compounds showed preferential affinity for ERα, and the disposition and size of the phenyl substituents were important determinants of the binding affinity and selectivity of these compounds, with those having ortho substituents giving the highest, and para substituents the lowest affinities for ERα. A few analogues exhibit ERα binding affinities that are comparable to or, in the case of the ortho-chloro analogue, higher than that of OBHS itself. In cell-based studies, we found several compounds with activity profiles comparable to tamoxifen, but acting entirely as indirect antagonists, allosterically interfering with recruitment of coactivator proteins to the receptor. Thus, the OBHS binding epitope represents a novel approach to the development of estrogen receptor antagonists via an indirect mechanism of antagonism.     

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.     

The presence of a ferrocenyl unit on an estrogenic molecule is not always sufficient to generate in vitro cytotoxicity

We recently reported the dual (antihormonal and cytotoxic) functionality of ferrocifens, which are organometallic complexes derived from hydroxytamoxifen, the standard molecule in the treatment of hormone-dependent breast cancers. To test the hypothesis that the presence of a ferrocenyl substituent on molecules with an affinity for the estrogen receptor is sufficient to give them cytotoxic properties in vitro, we prepared complexes derived from estradiol with a ferrocenyl substituent at positions 7alpha and 17alpha. The complexes thus obtained retain a satisfactory level of affinity for the estrogen receptor (RBA values higher than 12 %). At low concentrations (0.1-1 microM) the complexes show an estrogenic effect in vitro equivalent to that of estradiol on hormone-dependent (MCF-7) breast cancer cells, and no cytotoxic effect on hormone-independent (MDA-MB-231) breast cancer cells. At high concentrations (up to 50 microM) the 17alpha-ethynylferrocenyl estradiol and 7alpha-ferrocenylmethylthio estradiol become cytotoxic (IC(50)=13.2 microM and 18.8 microM, respectively) while the 17alpha-ferrocenylestradiol remains non toxic. The low toxicity of these compounds support our hypothesis that electronic communication between the ferrocenyl and phenol moieties in the hydroxyferrocifens series is a key parameter in the generation of cytotoxic effects at submicromolar concentrations.     

Bis‐arylidene Oxindoles as Anti‐Breast‐Cancer Agents Acting via the Estrogen Receptor

We report a new family of bis‐arylidene oxindole derivatives that show highly selective estrogen receptor (ER)‐mediated anticancer activity at low‐nanomolar concentrations in ER‐positive (ER+) breast cancer cells. In terms of cell growth inhibition, IC₅₀ values for these compounds in ER+ breast cancer cells are two to three orders of magnitude lower than in ER‐negative (ER?) breast cancer cells and non‐cancer cells. In comparison with known bis‐arylidene drugs, these compounds are at least three orders of magnitude more toxic than tamoxifen and 1.5–4‐fold more toxic than 4‐hydroxytamoxifen in ER+ MCF‐7 cancer cells. These oxindoles inhibit ER transactivation, and their anticancer activities are inhibited in ER‐depleted MCF‐7 cells. Some of these nonsteroidal molecules also exhibit essential properties of selective ER down‐regulation. From the development of two series of bis‐arylidene oxindole‐based compounds, we report a new series of anticancer agents for estrogen‐responsive breast cancer.     

Elucidating Mechanisms of Toxicity Using Phenotypic Data from Primary Human Cell Systems—A Chemical Biology Approach for Thrombosis-Related Side Effects

Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP^® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.