High-dose folic acid and its effect on early stage diabetic foot ulcer wound healing.

High-dose folic acid (HDFA; vitamin B9)-5 mg, given daily, has not been evaluated as a treatment to improve early stage-diabetic foot ulcer (ES-DFU) wound healing. However, HDFA has been demonstrated to correct: (a) endothelial dysfunction and decreased nitric oxide (NO) bioavailability, associated with type-2 diabetes mellitus (T2DM); and (b) hyperhomocysteinemia (HHcy) that may promote impaired DFU-wound healing. Measures of wound area (cm2 ) reduction (wound closure; WC), over a 4-week period (4 W-WC), greater than 50% of the wound area, have been reported as a robust indicator of the potential for DFU-wound healing. By using this model, we examined the effectiveness of a wound treatment in promoting progressive healing and complete wound closure for the chronic, nonhealing DFU-wound. To investigate this possible relationship between HDFA and ES-DFU wound healing, a retrospective cohort study of medical records, between November 2018 and April 2019, was performed for Veterans with T2DM and ES-DFUs following treatment with HDFA. During the study period 29 (n = 29) Veterans with ES-DFU wounds who received HDFA treatment were identified. Medical record reviews of this retrospective cohort of ES-DFU Veterans receiving HDFA report 90% (26/29) experiencing complete DFU-wound closure during the study period. Of the 29 Veterans with ES-DFUs receiving HDFA, the medical records of nine (30%), with healed wounds, provided documentation suitable for 4 W-WC, pre- and post-HDFA treatment study comparisons. This study documents significant (P < .05) improvements comparing 4 W-WC values for standard treatment for Veterans with poorly progressing, worsening or stagnating ES-DFU-wounds to those for the same subjects following HDFA treatment. These observations suggest that chronic ES-DFUs treated with HDFA may experience significantly improved wound closure and complete healing (re-epithelialization) when compared with standard treatments without HDFA. With validation from RCTs, HDFA may be established as an effective treatment to promote wound healing and closure for nonhealing ES-DFUs.

Nonnuclear Estrogen Receptor Activation Improves Hepatic Steatosis in Female Mice.

Estrogens have the potential to afford atheroprotection, to prevent excess adiposity and its metabolic complications including insulin resistance, and to lessen hepatic steatosis. Cellular responses to estrogens occur through gene regulation by nuclear estrogen receptors (ERs), and through signal initiation by plasma membrane-associated ER. Leveraging the potentially favorable cardiometabolic actions of estrogens has been challenging, because their reproductive tract and cancer-promoting effects adversely impact the risk to benefit ratio of the therapy. In previous works, we discovered that an estrogen dendrimer conjugate (EDC) comprised of ethinyl-estradiol (E2) molecules linked to a poly(amido)amine dendrimer selectively activates nonnuclear ER, and in mice, EDC does not invoke a uterotrophic response or support ER-positive breast cancer growth. In the present investigation, we employed EDC to determine how selective nonnuclear ER activation impacts atherosclerosis, adiposity, glucose homeostasis, and hepatic steatosis in female mice. In contrast to E2, EDC did not blunt atherosclerosis in hypercholesterolemic apoE-/- mice. Also in contrast to E2, EDC did not prevent the increase in adiposity caused by Western diet feeding in wild-type mice, and it did not affect Western diet-induced glucose intolerance. However, E2 and EDC had comparable favorable effect on diet-induced hepatic steatosis, and this was related to down-regulation of fatty acid and triglyceride synthesis genes in the liver. Predictably, only E2 caused a uterotrophic response. Thus, although nonnuclear ER activation does not prevent atherosclerosis or diet-induced obesity or glucose intolerance, it may provide a potential new strategy to combat hepatic steatosis without impacting the female reproductive tract or increasing cancer risk.

Estrogen receptor-α and aryl hydrocarbon receptor involvement in the actions of botanical estrogens in target cells.

Botanical estrogen (BE) dietary supplements are consumed by women as substitutes for loss of endogenous estrogens at menopause. To examine the roles of estrogen receptor α (ERα) and aryl hydrocarbon receptor (AhR) and their crosstalk in the actions of BEs, we studied gene regulation and proliferation responses to four widely used BEs, genistein, daidzein, and S-equol from soy, and liquiritigen from licorice root in breast cancer and liver cells. BEs and estradiol (E2), acting through ERα, stimulated proliferation, ERα chromatin binding and target-gene expression. BEs but not E2, acting through AhR, bound to xenobiotic response element-containing chromatin sites and enhanced AhR target-gene expression (CYP1A1, CYP1B1). While E2 and TCDD acted quite selectively through their respective receptors, BEs acted via both receptors, with their AhR activity moderated by negative crosstalk through ERα. Both ERα and AhR should be considered as mediators of the biology and pharmacology of BEs.

The anticancer potential of steroidal saponin, dioscin, isolated from wild yam (Dioscorea villosa) root extract in invasive human breast cancer cell line MDA-MB-231 in vitro.

Previously, we observed that wild yam (Dioscorea villosa) root extract (WYRE) was able to activate GATA3 in human breast cancer cells targeting epigenome. This study aimed to find out if dioscin (DS), a bioactive compound of WYRE, can modulate GATA3 functions and cellular invasion in human breast cancer cells. MCF-7 and MDA-MB-231 cells were treated in the absence/presence of various concentrations of DS and subjected to gene analysis by RT-qPCR, immunoblotting, and immunocytochemistry. We determined the ability of MDA-MB-231 cells to migrate into wound area and examined the effects of DS on cellular invasion using invasion assay. DS reduced cell viability of both cell lines in a concentration and time-dependent manner. GATA3 expression was enhanced by DS (5.76 µM) in MDA-MB-231 cells. DS (5.76 µM)-treated MDA-MB-231 cells exhibited the morphological characteristic of epithelial-like cells; mRNA expression of DNMT3A, TET2, TET3, ZFPM2 and E-cad were increased while TET1, VIM and MMP9 were decreased. Cellular invasion of MDA-MB-231 was reduced by 65 ± 5% in the presence of 5.76 µM DS. Our data suggested that DS-mediated pathway could promote GATA3 expression at transcription and translation levels. We propose that DS has potential to be used as an anti-invasive agent in breast cancer.

Dietary licorice root supplementation reduces diet-induced weight gain, lipid deposition, and hepatic steatosis in ovariectomized mice without stimulating reproductive tissues and mammary gland.

SCOPE: We studied the impact of dietary supplementation with licorice root components on diet-induced obesity, fat accumulation, and hepatic steatosis in ovariectomized C57BL/6 mice as a menopause model. MATERIALS AND METHODS: We evaluated the molecular and physiological effects of dietary licorice root administered to ovariectomized C57BL/6 mice as root powder (LRP), extracts (LRE), or isolated isoliquiritigenin (ILQ) on reproductive (uterus and mammary gland) and nonreproductive tissues important in regulating metabolism (liver, perigonadal, perirenal, mesenteric, and subcutaneous fat). Quantitative outcome measures including body weight, fat distribution (magnetic resonance imaging), food consumption, bone density and weight (Dual-energy X-ray absorptiometry), and gene expression were assessed by the degree of restoration to the preovariectomized health state. We characterized histological (H&E and oil red O staining) and molecular properties (expression of certain disease markers) of these tissues, and correlated these with metabolic phenotype as well as blood levels of bioactives. CONCLUSION: Although LRE and ILQ provided some benefit, LRP was the most effective in reducing body weight gain, overall fat deposition, liver steatosis, and expression of hepatic lipid synthesis genes following ovariectomy. Our data demonstrate that licorice root provided improvement of multiple metabolic parameters under conditions of low estrogen and high-fat diets without stimulating reproductive tissues.

Licorice root components in dietary supplements are selective estrogen receptor modulators with a spectrum of estrogenic and anti-estrogenic activities.

Licorice root extracts are often consumed as botanical dietary supplements by menopausal women as a natural alternative to pharmaceutical hormone replacement therapy. In addition to their components liquiritigenin (Liq) and isoliquiritigenin (Iso-Liq), known to have estrogenic activity, licorice root extracts also contain a number of other flavonoids, isoflavonoids, and chalcones. We have investigated the estrogenic activity of 7 of these components, obtained from an extract of Glycyrrhiza glabra powder, namely Glabridin (L1), Calycosin (L2), Methoxychalcone (L3), Vestitol (L4), Glyasperin C (L5), Glycycoumarin (L6), and Glicoricone (L7), and compared them with Liq, Iso-Liq, and estradiol (E2). All components, including Liq and Iso-Liq, have low binding affinity for estrogen receptors (ERs). Their potency and efficacy in stimulating the expression of estrogen-regulated genes reveal that Liq and Iso-Liq and L2, L3, L4, and L6 are estrogen agonists. Interestingly, L3 and L4 have an efficacy nearly equivalent to E2 but with a potency ca. 10,000-fold less. The other components, L1, L5 and L7, acted as partial estrogen antagonists. All agonist activities were reversed by the antiestrogen, ICI 182,780, or by knockdown of ERα with siRNA, indicating that they are ER dependent. In HepG2 hepatoma cells stably expressing ERα, only Liq, Iso-Liq, and L3 stimulated estrogen-regulated gene expression, and in all cases gene stimulation did not occur in HepG2 cells lacking ERα. Collectively, these findings classify the components of licorice root extracts as low potency, mixed ER agonists and antagonists, having a character akin to that of selective estrogen receptor modulators or SERMs.

Evaluation of wild yam (Dioscorea villosa) root extract as a potential epigenetic agent in breast cancer cells.

The present study was designed to evaluate the efficacy of wild yam root extract (WYRE) as a potential demethylating agent using two breast cancer cell lines, MCF-7 (estrogen receptor positive; ER(+)) and MDA-MB-231 (Estrogen receptor negative; ER(-)), and a methylated gene, GATA3, as a potential marker of breast cancer development. The cells were treated with WYRE (0-50 µg/mL) for 72 h and used for viability, mRNA, and methylation analyses. WYRE significantly reduced viability of both cell lines and enhanced mRNA content of GATA3 in a concentration-dependent manner; however, DNMT mRNAs (DNMT1, 3A, 3B) were found to increase significantly only in MDA-MB-231 cells. Global DNA methylation, analyzed as 5'-methyl-2'-deoxycytidine (5-mC) and 5-hydroxymethylcytosine (5-hmC), showed a concentration-dependent enhancement of 5-mC with no alteration in 5-hmC level in MCF-7 cells; however, in MDA-MB-231 cells, in contrast to MCF-7 cells, 5-mC remained unaltered but 5-hmC reduced significantly in all WYRE concentrations (10-50 µg/mL) used in this study. Since 5-hmC is generated from 5-mC by ten-eleven-translocation (TET) enzymes, analysis of TET mRNAs (TET1, TET2, and TET3) in MDA-MB-231 cells indicated a concentration-dependent reduction in TET1 and induction of TET3; however, TET2 remained unaltered. No alterations in any of the TET mRNAs were found in MCF-7 cells. Methylation analysis of GATA3 promoter at specific locus indicates probable demethylating activity of WYRE in MDA-MB-231 cells. We conclude that activation of GATA3 gene in ER(-) MDA-MB-231 cells may occur by altering DNA methylation pattern on the promoter region which may be different from the mechanisms operated in ER(+) MCF-7 cells.

Transcriptomic analysis identifies gene networks regulated by estrogen receptor α (ERα) and ERß that control distinct effects of different botanical estrogens.

The estrogen receptors (ERs) ERα and ERß mediate the actions of endogenous estrogens as well as those of botanical estrogens (BEs) present in plants. BEs are ingested in the diet and also widely consumed by postmenopausal women as dietary supplements, often as a substitute for the loss of endogenous estrogens at menopause. However, their activities and efficacies, and similarities and differences in gene expression programs with respect to endogenous estrogens such as estradiol (E2) are not fully understood. Because gene expression patterns underlie and control the broad physiological effects of estrogens, we have investigated and compared the gene networks that are regulated by different BEs and by E2. Our aim was to determine if the soy and licorice BEs control similar or different gene expression programs and to compare their gene regulations with that of E2. Gene expression was examined by RNA-Seq in human breast cancer (MCF7) cells treated with control vehicle, BE or E2. These cells contained three different complements of ERs, ERα only, ERα+ERß, or ERß only, reflecting the different ratios of these two receptors in different human breast cancers and in different estrogen target cells. Using principal component, hierarchical clustering, and gene ontology and interactome analyses, we found that BEs regulated many of the same genes as did E2. The genes regulated by each BE, however, were somewhat different from one another, with some genes being regulated uniquely by each compound. The overlap with E2 in regulated genes was greatest for the soy isoflavones genistein and S-equol, while the greatest difference from E2 in gene expression pattern was observed for the licorice root BE liquiritigenin. The gene expression pattern of each ligand depended greatly on the cell background of ERs present. Despite similarities in gene expression pattern with E2, the BEs were generally less stimulatory of genes promoting proliferation and were more pro-apoptotic in their gene regulations than E2. The distinctive patterns of gene regulation by the individual BEs and E2 may underlie differences in the activities of these soy and licorice-derived BEs in estrogen target cells containing different levels of the two ERs.

Mechanisms enforcing the estrogen receptor ß selectivity of botanical estrogens.

Because little is known about the actions of botanical estrogens (BEs), widely consumed by menopausal women, we investigated the mechanistic and cellular activities of some major BEs. We examined the interactions of genistein, daidzein, equol, and liquiritigenin with estrogen receptors ERα and ERß, with key coregulators (SRC3 and RIP140) and chromatin binding sites, and the regulation of gene expression and proliferation in MCF-7 breast cancer cells containing ERα and/or ERß. Unlike the endogenous estrogen, estradiol (E2), BEs preferentially bind to ERß, but their ERß-potency selectivity in gene stimulation (340- to 830-fold vs. E2) is enhanced at several levels (coregulator recruitment, chromatin binding); nevertheless, at high (0.1 or 1 µM) concentrations, BEs also fully activate ERα. Because ERα drives breast cancer cell proliferation and ERß dampens this, the relative levels of these two ERs in target cells and the BE dose greatly affect gene expression and proliferative response and will be crucial determinants of the potential benefits vs. risks of BEs. Our findings reveal key and novel mechanistic differences in the estrogenic activities of BEs vs. E2, with BEs displaying patterns of activity distinctly different from those seen with E2 and provide valuable information to inform future studies.

Activation of ERα is necessary for estradiol's anorexigenic effect in female rats.

While there is considerable evidence that the ovarian hormone estradiol reduces food intake in female rats, it is unclear which estrogen receptor (ER) subtype, ERα or ERß, mediates this effect. While several studies have demonstrated that activation of ERα, but not ERß, is sufficient to reduce food intake in ovariectomized (OVX) rats, there are limited data regarding which receptor subtype is necessary. Here we used the selective ERα and ERß antagonists, MPrP and PHTPP, respectively, to investigate this question. We found that antagonism of ERα, but not ERß, prevented the decrease in food intake following acute administration of estradiol in OVX rats. In addition, antagonism of ERα prevented the estrous-related, phasic reduction in food intake that occurs in response to the rise in circulating levels of estradiol in cycling rats. We conclude that activation of ERα is necessary for the anorexigenic effects of exogenous and endogenous estradiol in female rats.

Influence of oil droplet surface charge on the performance of antibody--emulsion conjugates.

Targeted drug delivery requires binding of (and subsequent uptake by) the carrier to target cells. The purpose of our present study is to compare the binding and uptake of emulsions with different electric surface properties to SK-BR3 cell line, which over-expresses the HER2 receptor. Cationic emulsion was prepared by incorporating 0.25% w/w of the cationic lipid, stearylamine in the formulation while the anionic emulsion formulation was identical but lacking stearylamine. Immunoemulsions were prepared by conjugating the 2-iminothiolane derivative of the monoclonal antibody trastuzumab (Herceptin) through the reactive maleimide group of the octadecyl-4-(maleimidomethyl)cyclohexane-carboxylic amide linker which was incorporated in the oil phase of the anionic and cationic emulsions. Cationic emulsion exhibited a droplet size of approximately 130 nm and a zeta potential of +50 mV compared to anionic emulsion with a droplet size of approximately 140 nm and a zeta potential of -30 mV which decreased to -5 mV following antibody coupling. There was no significant difference in the coupling efficiency of trastuzumab to anionic and cationic emulsions which was in the range of 60-70%. The cationic emulsion and immunoemulsion appeared to be physically stable over a long period of time, as indicated by particle-size measurements while the droplets of the anionic immunoemulsion coalesced with time resulting in phase separation within 20 days storage at 4 degrees C. The results of binding and uptake to cells showed that both cationic and anionic immunoemulsions bind and internalized to cells much more than the respective blank emulsions. The enhanced penetration of the probe coumarin-6 with both immunoemulsions clearly indicated that the internalization process was mainly controlled by a cell-receptor endocytosis mechanism mediated by the binding affinity of trastuzumab to the cell surface receptor since the uptake of the cationic immunoemulsion was not significantly different from the uptake of the anionic immunoemulsion. However, only the cationic immunoemulsion might be considered for further investigation in view of its long standing physical stability.

Isocoumarins as estrogen receptor beta selective ligands: Isomers of isoflavone phytoestrogens and their metabolites.

In a search for new ligands selective for the estrogen receptor beta (ERbeta), we prepared a series of non-steroidal compounds having an isocoumarin core structure. An interesting feature of these derivatives is that they bear the same functionalities as the well-known ERbeta -selective, isoflavone phytoestrogens daidzein and genistein, but in an isomeric arrangement. These compounds could be prepared efficiently by electrophilic cyclization of acetylenic ester precursors, followed by simple manipulations to introduce additional substituents. Through a reduction of some of the isocoumarins, we also obtained isomeric analogs of the isoflavone metabolites equol and dehydroequol. The compounds we prepared were evaluated in ER binding assays, and selected compounds were studied further in cell-based gene transcription assays. Several of the isocoumarins and their analogs are high-affinity ligands that show considerable selectivity for ERbeta in terms of binding affinity, and strikingly high ERbeta selectivity in terms of potency in gene transcription assays. Two of the best compounds, which combine high transcriptional potency with an ERbeta selectivity greater than 1000, should prove to be excellent probes of ERbeta function in vivo.

Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta.

Equol is a metabolite produced in vivo from the soy phytoestrogen daidzein by the action of gut microflora. It is known to be estrogenic, so human exposure to equol could have significant biological effects. Equol is a chiral molecule that can exist as the enantiomers R-equol and S-equol. To study the biological activity of racemic (+/-)-equol, as well as that of its pure enantiomers, we developed an efficient and convenient method to prepare (+/-)-equol from available isoflavanoid precursors. Furthermore, we optimized a method to separate the enantiomers of equol by chiral HPLC, and we studied for the first time, the activities of the enantiomers on the two estrogen receptors, ERalpha and ERbeta. In binding assays, S-equol has a high binding affinity, preferential for ERbeta (K(i)[ERbeta]=16 nM; beta/alpha=13 fold), that is comparable to that of genistein (K(i)[ERbeta]=6.7 nM; beta/alpha=16), whereas R-equol binds more weakly and with a preference for ERalpha (K(i)[ERalpha]=50 nM; beta/alpha=0.29). All equol isomers have higher affinity for both ERs than does the biosynthetic precursor daidzein. The availability and the in vitro characterization of the equol enantiomers should enable their biological effects to be studied in detail.

Characterization of the biological roles of the estrogen receptors, ERalpha and ERbeta, in estrogen target tissues in vivo through the use of an ERalpha-selective ligand.

Estrogens elicit many biomedically important responses in different target tissues, and the respective roles of the two estrogen receptors, ERalpha and ERbeta, in mediating these bioactivities is incompletely understood. In this study, we investigated the activity of an ERalpha-selective agonist ligand, propyl pyrazole triol (PPT), in several rat animal models to define the involvement of ERalpha in these biological responses. In a short-term (4 d) uterotrophic assay, PPT was found to be as efficacious as 17alpha-ethinyl-17beta-estradiol in stimulating uterine weight gain and up-regulating complement 3 gene expression. In a 6-wk chronic model, PPT completely prevented the ovariectomy-induced body weight increase and loss of bone mineral density. It also increased uterine weight and markedly reduced plasma cholesterol levels in these mature animals. PPT was also effective in the brain. It increased progesterone receptor mRNA in the arcuate and ventromedial nuclei of the hypothalamus and prevented experimentally induced hot flushes. Our findings indicate that several physiologically relevant estrogen-induced tissue responses can be effectively evoked via ERalpha alone. By providing an approach that is complementary to that of analyzing the phenotype and response of ER knockout animals, our findings also demonstrate that ER subtype-selective ligands can play a valuable role in enhancing our understanding of how estrogens work through the two ER subtypes.

Efficacy evaluation of a novel submicron amphotericin B emulsion in murine candidiasis.

A submicron amphotericin B (AmB) sterile emulsion with a mean droplet size of approximately 100 nm was prepared. The emulsion was stable at 4 degrees C over a period of 4 months. The acute toxicity results showed that the maximum tolerated dose of the AmB emulsion was 2.0 mg kg-1 as compared to 0.5 mg kg-1 for a commercial deoxycholate suspension, Fungizone. Efficacy evaluations of submicron AmB emulsion compared to Fungizone were performed in a murine candidiasis model using either a single or a multiple daily dose administration of 0.4 mg kg-1. Survival (100%) was observed up to 15-18 days post-infection in mice treated with AmB emulsion or Fungizone, while all control non-treated animals had died after 9 days in both single and multi-treatment experiments. Survival (50%) was obtained after 35 days in the single treatment, and 40 days in the multi-treatment, experiment with AmB emulsion, while treatment with Fungizone gave 50% survival after 15 and 20 days, respectively. The overall results indicate that in murine candidiasis, treatment with AmB submicron emulsion was more effective than Fungizone, suggesting a potential therapeutic application.

Towards a new galenic dosage form, the liposomes?

Liposomes used first as models for the study of cellular membrane permeability have been proposed about ten years ago as intracellular carriers of therapeutic substances. Since then a large number of fields of application have been explored. The difficulties encountered are important enough for having hampered any therapeutical application. Nevertheless, the first results are not negligible and provided that the efforts keep their multidisciplinary character, the concept of intracellular carrier should meet some success.