Comparison of Day-Specific Serum LH, Estradiol, and Progesterone with MiraTM Monitor Urinary LH, Estrone-3-glucuronide, and Pregnanediol-3-glucuronide Levels in Ovulatory Cycles.

Background and Objectives: Fertility tracking apps and devices are now currently available, but urinary hormone levels lack accuracy and sensitivity in timing the start of the 6-day fertile window and the precise 24 h interval of transition from ovulation to the luteal phase. We hypothesized the serum hormones estradiol (E2) and progesterone (P) might be better biomarkers for these major ovulatory cycle events, using appropriate mathematical tools. Materials and Methods: Four women provided daily blood samples for serum E2, P, and LH (luteinizing hormone) levels throughout their entire ovulatory cycles, which were indexed to the first day of dominant follicle (DF) collapse (defined as Day 0) determined by transvaginal sonography; therefore, ovulation occurred in the 24 h interval of Day -1 (last day of maximum diameter DF) to Day 0. For comparison, a MiraTM fertility monitor was used to measure daily morning urinary LH (ULH), estrone-3-glucuronide (E3G), and pregnanediol-3-glucuronide (PDG) levels in three of these cycles. Results: There were more fluctuations in the MiraTM hormone levels compared to the serum levels. Previously described methods, the Fertility Indicator Equation (FIE) and Area Under the Curve (AUC) algorithm, were tested for identifying the start of the fertile window and the ovulation/luteal transition point using the day-specific hormone levels. The FIE with E2 levels predicted the start of the 6-day fertile window on Day -7 (two cycles) and Day -5 (two cycles), whereas no identifying signal was found with E3G. However, both pairs of (E2, P) and (E3G, PDG) levels with the AUC algorithm signaled the Day -1 to Day 0 ovulation/luteal transition interval in all cycles. Conclusions: serum E2 and (E2, P) were better biomarkers for signaling the start of the 6-day fertile window, but both MiraTM and serum hormone levels were successful in timing the [Day -1, Day 0] ovulatory/luteal transition interval. These results can presently be applied to urinary hormone monitors for fertility tracking and have implications for the direction of future fertility tracking technology.

Hepatic Gene Expression and Metabolite Profiles of Androstenone and Skatole Relative to Plasma Estrone Sulfate Levels in Boars.

Testicular steroids can alter the activity and expression of enzymes within the liver and may influence the metabolism of skatole and androstenone, which are responsible for boar taint. Plasma levels of estrone sulfate (E1S) are indicative of the steroidogenic capacity of the boar and are variable between animals of similar live weights at slaughter. This study aimed to characterize the relationship between steroidogenic capacity and the metabolism of boar taint compounds by relating plasma E1S levels at slaughter weight to the expression levels of genes regulating the metabolism of androstenone and skatole, along with their respective metabolite profiles. RT-qPCR was used to evaluate gene expression in the liver. Hepatocytes were also isolated and treated with androstenone or skatole, with metabolite levels in the incubation media quantified by high-performance liquid chromatography. Plasma E1S levels ranged from 2.2-108.5 ng/mL and were positively correlated with overall skatole metabolism (p = 0.038), the production of metabolites 3-methyloxindole (p = 0.026) and 3-hydroxy-3-methyloxindole (p = 0.036), and expression levels of key genes involved in skatole metabolism, specifically CYP2C33 (p = 0.0042), CYP2C49 (p = 0.022), and CYB5R1 (p = 0.017). There was no association between androstenone metabolism and plasma E1S concentrations; however, there was evidence of possible co-regulation amongst genes involved in the metabolism of androstenone, skatole, and estrogens. These findings indicate that steroidogenic capacity is related to the rate of skatole, but not androstenone metabolism, in slaughter-weight boars.

Preincubation-dependent inhibition of organic anion transporting polypeptide 2B1.

Preincubation with inhibitor in organic anion transporting polypeptide (OATP) in vitro assays may increase the inhibition potency of inhibitors compared to conventional inhibition assays with only short inhibitor coincubation with substrate. The decrease in IC50 may affect prediction of drug-drug interactions (DDI) involving these transporters and inhibitors. Only few drugs, however, have been assessed for the preincubation-dependent inhibition of the OATP2B1 transporter. Therefore, we studied the effect of preincubation on OATP2B1 inhibition with five known OATP2B1 inhibitors (atorvastatin, erlotinib, ezetimibe, ticagrelor and simeprevir) in HEK293 cells transiently overexpressing OATP2B1. IC50 values were determined with and without inhibitor preincubation for 20 min with three different OATP2B1 substrates (dibromofluorescein, DBF; 5-carboxyfluorescein, 5-CF; estrone sulfate). Atorvastatin, ezetimibe, and simeprevir displayed more than 2-fold lower IC50 values after preincubation with at least one of the tested substrates. Altogether, 4 out of 15 inhibitor/substrate combinations exhibited more than 2-fold potentiation of IC50 after inhibitor preincubation. In addition, preincubation by itself, without inhibitor present with the substrate, resulted in more than 50% inhibition of OATP2B1-mediated uptake of DBF and/or 5-CF by atorvastatin, ticagrelor and simeprevir. Thus, erlotinib was the only inhibitor with no indication of potentiation of inhibition by preincubation with any of the tested substrates. In conclusion, preincubation resulted in inhibitor- and substrate-dependent inhibition of OATP2B1. These results support the conclusion that to reduce the risk of false negative DDI prediction, preincubation should be considered also in OATP2B1 inhibition assays.

Comparison of serum concentrations of pregnancy-associated glycoproteins and estrone sulphate during pregnancy in eutocia and dystocia beef cattle.

Preventing dystocia can stabilise beef cattle management. This study aimed to investigate the relationship between serum pregnancy-associated glycoproteins (PAGs) S-N values and estrone sulphate (E1S) concentrations during pregnancy and the calf birth weight in beef cattle and to evaluate their usefulness as new predictive parameters for dystocia due to foetal overgrowth. Thirty-eight pregnant Japanese Black cattle were used. Blood samples were collected at 40, 70, 100, 150, 200, 250, 280, and 285 days after artificial insemination (AI), and birth weight of the offspring was measured. Serum PAGs S-N values and E1S concentrations were measured, and the area under the curve (AUC) and the ratio of change based on 70 days after AI were calculated, followed by calculation of the correlation coefficient with the birth weight of the offspring and comparison between the eutocia (n = 32) and dystocia (n = 6) groups. The birth weight of the offspring was moderately positively correlated with the AUC of serum PAGs S-N values and E1S concentrations in the second (r = 0.425, P < 0.01) and third (r = 0.595, P < 0.01) trimesters, respectively. The ratio of change in serum E1S concentrations between 70 and 280 days after AI was greater (P < 0.05) in the dystocia group (1276.6 ±â€¯229.1 %) than in the eutocia group (852.6 ±â€¯69.6 %). These results suggest that blood PAGs S-N values at mid-pregnancy (100-199 days after AI) and the ratio of changes in blood E1S concentrations between 70 and 280 days after AI may be new parameters for predicting dystocia.

LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bß, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.

Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn's disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

CD44-fibrinogen binding promotes bleeding in acute promyelocytic leukemia by in situ fibrin(ogen) deposition.

Early hemorrhagic death is still the main obstacle for the successful treatment of acute promyelocytic leukemia (APL). However, the mechanisms underlying hemostatic perturbations in APL have not been fully elucidated. Here, we report that CD44 on the membrane of APL blasts and NB4 cells ligated bound fibrinogen, resulting in in situ deposition of fibrin and abnormal fibrin distribution. Clots formed by leukemic cells in response to CD44 and fibrinogen interaction exhibited low permeability and resistance to fibrinolysis. Using flow cytometry and confocal microscopy, we found that CD44 was also involved in platelet and leukemic cell adhesion. CD44 bound activated platelets but not resting platelets through interaction with P-selectin. APL cell-coated fibrinogen-activated platelets directly induce enhanced procoagulant activity of platelets. In vivo studies revealed that CD44 knockdown shortened bleeding time, increased the level of fibrinogen, and elevated the number of platelets by approximately twofold in an APL mouse model. Moreover, CD44 expression on leukemic cells in an APL mouse model was not only associated with bleeding complications but was also related to the wound-healing process and the survival time of APL mice. Collectively, our results suggest that CD44 may be a potential intervention target for preventing bleeding complications in APL.

ADAPTOR PROTEIN Ruk/CIN85REGULATES REDOX BALANCE IN 4T1MOUSE BREAST CANCER CELLS EXPOSED TO PLASMIN(OGEN).

BACKGROUND: Cell surface plasmin is involved in tumor growth and metastatic dissemination by regulating cancer cells adhesion, migration and invasion. Plasmin-induced cell detachment is accompanied by an increased rate of reactive oxygen species (ROS) generation and cell death. However, cancer cells acquire the ability to develop adaptive mechanisms to resist ROS-mediated apoptosis. AIM: To establish the role of adaptor protein Ruk/CIN85 in the control of viability and redox balance in breast adenocarcinoma cells exposed to plasmin(ogen). MATERIALS AND METHODS: Mouse 4T1 cells with the stable overexpression of adaptor protein Ruk/CIN85 (RukUp subline) and corresponding control (Mock subline) were treated with Glu-plasminogen (1-100 nM). Plasminogen to plasmin conversion was monitored spectrophotometrically by cleavage of the specific chromogenic substrate S2251. Specific uPA inhibitor BC11 was used to verify the uPA-mediated mechanism of plasminogen pericellular activation by 4T1 cells. Cell survival rate was assessed by MTT-test and cell proliferation was estimated by colony formation assay. Enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase, as well as hydrogen peroxide (H2O2) levels were measured by spectrophotomertric and fluorometric assays. The intracellular ROS generation was monitored by flow cytometry using H2DCF-DA fluorescent probe. RESULTS: Plasminogen was shown to be converted into an active proteinase plasmin on the surface of carcinoma cells in uPA-dependent manner. Plasmin(ogen) suppressed proliferation and affected survival of both studied 4T1 sublines. However, RukUp cells displayed higher resistance to plasmin(ogen)-induced cytotoxicity than Mock cells. Plasmin(ogen) promoted significant elevation in ROS generation rate in cells with the basal level of Ruk/CIN85 expression. In contrast, RukUp cells appear to be more effective in counteracting prooxidant changes due to the activation of some enzymes of the glutathione system, in particular glutathione peroxidase, and a concomitant decrease of H2O2 accumulation. CONCLUSION: Adaptor protein Ruk/CIN85 is involved in the regulation of redox homeostasis in cancer cells to maintain levels of ROS, thus promoting redox adaptation in cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85 may represent one of the relevant targets in order to diminish the resistance of cancer cells to ROS-mediated apoptosis.

Algorithms with Area under the Curve for Daily Urinary Estrone-3-Glucuronide and Pregnanediol-3-Glucuronide to Signal the Transition to the Luteal Phase.

Background and Objectives: Home fertility assessment methods (FAMs) for natural family planning (NFP) have technically evolved with the objective metrics of urinary luteinizing hormone (LH), estrone-3-glucuronide (E3G) and pregnanediol-3-glucuronide (PDG). Practical and reliable algorithms for timing the phase of cycle based upon E3G and PDG levels are mostly unpublished and still lacking. Materials and Methods: A novel formulation to signal the transition to the luteal phase was discovered, tested, and developed with a data set of daily E3G and PDG levels from 25 women, 78 cycles, indexed to putative ovulation (day after the urinary LH surge), Day 0. The algorithm is based upon a daily relative progressive change in the ratio, E3G-AUC/PDG-AUC, where E3G-AUC and PDG-AUC are the area under the curve for E3G and PDG, respectively. To improve accuracy the algorithm incorporated a three-fold cycle-specific increase of PDG. Results: An extended negative change in E3G-AUC/PDG-AUC of at least nine consecutive days provided a strong signal for timing the luteal phase. The algorithm correctly identified the luteal transition interval in 78/78 cycles and predicted the start day of the safe period as: Day + 2 in 10/78 cycles, Day + 3 in 21/78 cycles, Day + 4 in 28/78 cycles, Day + 5 in 15/78 cycles, and Day + 6 in 4/78 cycles. The mean number of safe luteal days with this algorithm was 10.3 ± 1.3 (SD). Conclusions: An algorithm based upon the ratio of the area under the curve for daily E3G and PDG levels along with a relative PDG increase offers another approach to time the phase of cycle. This may have applications for NFP/FAMs and clinical evaluation of ovarian function.

The Story of the Fibrin(ogen) αC-Domains: Evolution of Our View on Their Structure and Interactions.

Although much has been established concerning the overall structure and function of fibrinogen, much less has been known about its two αC regions, each consisting of an αC-connector and an αC-domain, but new information has been accumulating. This review summarizes the state of our current knowledge of the structure and interactions of fibrinogen's αC regions. A series of studies with isolated αC regions and their fragments demonstrated that the αC-domain forms compact ordered structures consisting of N- and C-terminal subdomains including ß sheets and suggested that the αC-connector has a poly(L-proline) type II structure. Functionally, the αC-domains interact intramolecularly with each other and with the central region of the molecule, first demonstrated by electron microscopy and then quantified by optical trap force spectroscopy. Upon conversion of fibrinogen into fibrin, the αC-domains switch from intra- to intermolecular interactions to form ordered αC polymers. The formation of αC polymers occurs mainly through the homophilic interaction between the N-terminal subdomains; interaction between the C-terminal subdomains and the αC-connectors also contributes to this process. Considerable evidence supports the idea that the αC-regions accelerate fibrin polymerization and affect the final structure of fibrin clots. The interactions between αC-regions are important for the mechanical properties of clots, increasing their stiffness and extensibility. Conversion of fibrinogen into fibrin results in exposure of multiple binding sites in its αC regions, providing interaction of fibrin with different proteins and cell types during hemostasis and wound healing. This heretofore mysterious part of the fibrinogen molecule is finally giving up its secrets.

Migration and abiotic transformation of estrone (E1) and estrone-3-sulfate (E1-3S) during soil column transport.

Steroid estrogens have received worldwide attention and given rise to great challenges of aquatic ecosystems security, posing potential adverse effects on aquatic organisms and human health even at low levels (ng/L). The present study focused on understanding the mobility and abiotic transformation of estrone (E1) and estrone-3-sulfate (E1-3S) over spatial and time scales during soil transport. Column transport experiments showed that the migration capacity of E1-3S was far stronger than E1 in soil. The calculated groundwater ubiquity score and leachability index values also indicated the high leaching mobility of E1-3S. The hydrolysis of E1-3S and abiotic transformation into estradiol and estriol was observed in the sterilized soil. Furthermore, possible transformation products (e.g., SE239, E2378, E1 dimer538, E1-E2 dimer541) of E1 and E1-3S in soil were analyzed and identified after the column transport experiments. The estrogenic activity was estimated by 17ß-estradiol equivalency values during the transport process in aqueous and soil phases. Additionally, the potential leaching transport to groundwater of E1-3S requires further critical concern.

The Role of Fibrin(ogen) in Wound Healing and Infection Control.

Fibrinogen, one of the most abundant plasma proteins playing a key role in hemostasis, is an important modulator of wound healing and host defense against microbes. In the current review, we address the role of fibrin(ogen) throughout the process of wound healing and subsequent tissue repair. Initially fibrin(ogen) acts as a provisional matrix supporting incoming leukocytes and acting as reservoir for growth factors. It later goes on to support re-epithelialization, angiogenesis, and fibroplasia. Importantly, removal of fibrin(ogen) from the wound is essential for wound healing to progress. We also discuss how fibrin(ogen) functions through several mechanisms to protect the host against bacterial infection by providing a physical barrier, entrapment of bacteria in fibrin(ogen) networks, and by directing immune cell function. The central role of fibrin(ogen) in defense against bacterial infection has made it a target of bacterial proteins, evolved to interact with fibrin(ogen) to manipulate clot formation and degradation for the purpose of promoting microbial virulence and survival. Further understanding of the dual roles of fibrin(ogen) in wound healing and infection could provide novel means of therapy to improve recovery from surgical or chronic wounds and help to prevent infection from highly virulent bacterial strains, including those resistant to antibiotics.

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Synthesis and evaluation of AKR1C inhibitory properties of A-ring halogenated oestrone derivatives.

Enzymes AKR1C regulate the action of oestrogens, androgens, and progesterone at the pre-receptor level and are also associated with chemo-resistance. The activities of these oestrone halides were investigated on recombinant AKR1C enzymes. The oestrone halides with halogen atoms at both C-2 and C-4 positions (13ß-, 13α-methyl-17-keto halogen derivatives) were the most potent inhibitors of AKR1C1. The lowest IC50 values were for the 13α-epimers 2_2I,4Br and 2_2I,4Cl (IC50, 0.7 µM, 0.8 µM, respectively), both of which selectively inhibited the AKR1C1 isoform. The 13α-methyl-17-keto halogen derivatives 2_2Br and 2_4Cl were the most potent inhibitors of AKR1C2 (IC50, 1.5 µM, 1.8 µM, respectively), with high selectivity for the AKR1C2 isoform. Compound 1_2Cl,4Cl showed the best AKR1C3 inhibition, and it also inhibited AKR1C1 (Ki: AKR1C1, 0.69 µM; AKR1C3, 1.43 µM). These data show that halogenated derivatives of oestrone represent a new class of potent and selective AKR1C inhibitors as lead compounds for further optimisations.