Fatty Acids Derived from Royal Jelly Are Modulators of Estrogen Receptor Functions

Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E2), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E2 FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E2-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E2-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E2, FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E2, FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E2, induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells

AbstractThe Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells

Molecular basis of estrogen receptor antagonism

Estrogen Receptors (ERs), ERa and ERb, are responsible for mediating the physiological effects of the steroid hormone 17b-estradiol and structurally related compounds. ER signaling is a complex process where the combined effects of accessory proteins, known as coregulators, promoter characteristics and influence from other signaling systems, determine the cell-specific transcriptional response in a given situation. Central to the regulatory mechanism is the ability of ERs to adopt distinct conformations in response to ligands and thereby determine the relative affinity of coregulators. Dysregulation of ER signaling leads to increased cell proliferation and reproductive diseases. Synthetic anti-estrogens can antagonize inappropriate ER signaling constituting the rationale behind their clinical use in cancer therapy. The mechanisms that govern anti-estrogen action are poorly understood with several of the currently applied drugs having unwanted side effects in non-target tissues, and after prolonged treatment tumor cells become resistant to anti-estrogen therapy. Recent findings extend the traditional view of ER antagonists as compounds that solely block estrogen effects to include “active antagonist receptor signaling”. The aim of this thesis was to better understand the molecular details involved in mediating the effects of antagonist-bound ER complexes. To this end we have investigated coregulator interaction surfaces on ERs using peptides that mimic coregulator interactions. A major finding of our studies is that the ER-ligand-binding domains harbor surfaces capable of recruiting coregulators in the presence of antagonists, which may represent bona fide control surfaces involved in mediating antagonist activity. We identified peptide interactions that can serve as models for coregulator mediated subtype and antagonist specific effects (Paper I) and a peptide interaction (Paper II) that serve as a model for coregulator mediated agonist and antagonist action. In addition, a peptide interaction-mechanism overlapping with a known nuclear receptor (NR) corepressor surface was characterized with detailed structural analysis (Paper III). Moreover, the peptide-receptor interaction system turned out to be a very useful system for monitoring receptor conformation after binding of uncharacterized ligands. Phytoestrogens, such as genistein, are being extensively investigated in search of new therapeutic agents for estroge n-related pathologies. 8-prenylnaringenin is a potent plantderived estrogenic compound, whereas naringenin lacking a prenyl-group is not. We therefore specifically investigated the impact of alkyl chain length and degree of branching at C(8) of the naringenin skeleton on estrogenic effects (Paper IV). Taken together, the in vitro bioassays of the 10 novel naringenin derivatives, accessed by a regioselective alkylation scheme at C(8), showed a very interesting relationship between the nature of the alkyl substituent and the established bioactivity. They span an activity spectrum ranging from full agonists to partial agonists to antagonists. Our systematic approach to investigate the impact of the nature of the substituent provides further evidence that subtle modifications to ligands greatly influence their pharmacological properties. Delineation of the biological activity elicited by compounds with various chemical properties together with characterization of the complex ER antagonist signaling is expected to increase our understanding of how to target estrogen-dependent tumors in a more optimal manner

Maximum Tolerated Dose and Pharmacokinetics of Paclitaxel Micellar in Patients with Recurrent Malignant Solid Tumours : A Dose-Escalation Study

Introduction: A water-soluble Cremophor EL-free formulation of paclitaxel, in which retinoic acid derivates solubilize paclitaxel by forming micelles (paclitaxel micellar), was studied for the first time in man to establish the maximum tolerated dose (MTD) and to characterize the pharmacokinetics (PK). Methods: This was an open-label, one-arm, dose-escalating study in patients with advanced solid malignant tumours, for which no standard therapy was available or had failed. Paclitaxel micellar was given as 1-h intravenous infusion every 21 days for 3 cycles, mainly without premedication. Plasma samples were collected during 24 h at the first cycle and paclitaxel concentrations were assayed by high-performance liquid chromatography. PK was evaluated using a two-compartment model. Results: Thirty-four patients received paclitaxel micellar at doses ranging between 90 and 275 mg/m2. MTD was established as 250 mg/m2. Fatigue and neuropathy were the most frequent dose-limiting toxicities. No hypersensitivity reactions were observed. PK of paclitaxel was evaluated in 25 data sets. Paclitaxel micellar had a rapid initial distribution phase, mean half-life 0.55 h, estimated to be completed 3 h after dosing and a mean terminal half-life of 8.8 h. Mean clearance was 13.4 L/h/m2 with fivefold interindividual variability. The residual areas after 10 h and 24 h were 15.7 ± 8.6% and 5.7 ± 3.9% of the area under the plasma concentration–time curve to infinite time (AUCinf), respectively. Conclusion: No new side effects unknown for paclitaxel were observed. Maximum plasma concentration (Cmax) and AUCinf showed a tendency to increase linearly with dose within the 150–275 mg/m2dose range. The possibility to administer paclitaxel micellar without steroid premedication makes it an attractive candidate for further studies in combination with immunotherapy. Trial Registration: EudraCT no: 2004-001821-54

Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells?

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals