In Vitro and In Vivo Evidence for Lack of Endovascular Remodeling by Third Trimester Trophoblasts

The placental–decidual interaction through invading trophoblasts determines whether a physiological transformation of the uterine spiral arteries is established or not. Trophoblast-orchestrated artery remodeling is central to normal placentation. Dysregulated uteroplacental interaction and vascular remodeling are thought to be associated with the molecular events underlying the pathology of late pregnancy anomalies including preeclampsia. Although the exact gestational age at which trophoblast invasion ceases is not known, it remains unclear whether late pregnancy trophoblasts retain the ability to transform the uterine arteries. Here, we have developed a dual cell, in vitro culture system that mimics the vascular remodeling events during normal pregnancy. We demonstrate that first and third trimester trophoblasts respond differentially to interactive signals from endothelial cells when cultured on matrigel. Term primary trophoblasts or immortalized third trimester extravillous TCL1 trophoblasts not only fail to respond to signals from endothelial cells but also inhibit endothelial cell tube formation. In contrast, HTR8 cells, representing a first trimester trophoblast cell line with invasive properties, undergo spontaneous migration and synchronize with the endothelial cells in a capillary network. This disparity in behavior was confirmed in vivo using a matrigel plug assay. Poor expression of VEGF C and VEGF receptors coupled with high E-cadherin expression by term primary trophoblasts and TCL1 cells contributed to their restricted interactive and migratory properties. We further show that the kinase activity of VEGF R2 is essential for proactive crosstalk by HTR8 cells. This unique behavior of first trimester trophoblasts in the presence of endothelial cells offers a potential approach to study cell–cell interactions and to decipher modulatory components in the serum samples from adverse pregnancy outcomes

The Cranberry Flavonoids PAC DP-9 and Quercetin Aglycone Induce Cytotoxicity and Cell Cycle Arrest and Increase Cisplatin Sensitivity in Ovarian Cancer Cells

Cranberry flavonoids (flavonols and flavan-3-ols), in addition to their antioxidant properties, have been shown to possess potential in vitro activity against several cancers. However, the difficulty of isolating cranberry compounds has largely limited anticancer research to crude fractions without well-defined compound composition. In this study, individual cranberry flavonoids were isolated to the highest purity achieved so far using gravity and high performance column chromatography and LC-MS characterization. MTS assay indicated differential cell viability reduction of SKOV-3 and OVCAR-8 ovarian cancer cells treated with individual cranberry flavonoids. Treatment with quercetin aglycone and PAC DP-9, which exhibited the strongest activity, induced apoptosis, led to caspase-3 activation and PARP deactivation, and increased sensitivity to cisplatin. Furthermore, immunofluorescence microscopy and western blot study revealed reduced expression and activation of epidermal growth factor receptor (EGFR) in PAC DP-9 treated SKOV-3 cells. In addition, quercetin aglycone and PAC DP-9 deactivated MAPK-ERK pathway, induced downregulation of cyclin D1, DNA-PK, phospho-histone H3 and upregulation of p21, and arrested cell cycle progression. Overall, this study demonstrates promising in vitro cytotoxic and anti-proliferative properties of two newly characterized cranberry flavonoids, quercetin aglycone and PAC DP-9, against ovarian cancer cells

Mechanism and Uses of a Membrane Peptide that Targets Tumors and Other Acidic Tissues \u3cem\u3eIn Vivo\u3c/em\u3e

The pH-selective insertion and folding of a membrane peptide, pHLIP [pH (low) insertion peptide], can be used to target acidic tissue in vivo, including acidic foci in tumors, kidneys, and inflammatory sites. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The fluorescence signal is stable for \u3e4 days and is approximately five times higher in tumors than in healthy counterpart tissue. In a rat antigen-induced arthritis model, pHLIP preferentially accumulates in inflammatory foci. pHLIP also maps the renal cortical interstitium; however, kidney accumulation can be reduced significantly by providing mice with bicarbonate-containing drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an α-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP nanotechnology introduces a new method of detecting, targeting, and possibly treating acidic diseased tissue by using the selective insertion and folding of membrane peptides

Inhibition of DUSP6 sensitizes ovarian cancer cells to chemotherapeutic agents via regulation of ERK signaling response genes

Dual specificity phosphatase 6 (DUSP6) is a protein phosphatase that deactivates extracellular-signal-regulated kinase (ERK). Since the ovarian cancer biomarker human epididymis protein 4 (HE4) interacts with the ERK pathway, we sought to determine the relationship between DUSP6 and HE4 and elucidate DUSP6’s role in epithelial ovarian cancer (EOC). Viability assays revealed a significant decrease in cell viability with pharmacological inhibition of DUSP6 using (E/Z)-BCI hydrochloride in ovarian cancer cells treated with carboplatin or paclitaxel, compared to treatment with either agent alone. Quantitative PCR was used to evaluate levels of ERK pathway response genes to BCI in combination with recombinant HE4 (rHE4), carboplatin, and paclitaxel. Expression of EGR1, a promoter of apoptosis, was higher in cells co-treated with BCI and paclitaxel or carboplatin than in cells treated with chemotherapeutic agents alone, while expression of the proto-oncogene c-JUN was decreased with co-treatment. The effect of BCI on the expression of these two genes opposed that of rHE4. Pathway focused quantitative PCR also revealed suppression of ERBB3 in cells co-treated with BCI plus carboplatin or paclitaxel. Finally, expression levels of DUSP6 in EOC tissue were evaluated by immunohistochemistry, revealing significantly increased levels of DUSP6 in serous EOC tissue compared to adjacent normal tissue. A positive correlation between HE4 and DUSP6 levels was determined by Spearman Rank correlation. In conclusion, DUSP6 inhibition sensitizes ovarian cancer cells to chemotherapeutic agents and alters gene expression of ERK response genes, suggesting that DUSP6 could plausibly function as a novel therapeutic target to reduce chemoresistance in EOC

Septin-2 is overexpressed in epithelial ovarian cancer and mediates proliferation via regulation of cellular metabolic proteins

Epithelial Ovarian Cancer (EOC) is associated with dismal survival rates due to the fact that patients are frequently diagnosed at an advanced stage and eventually become resistant to traditional chemotherapeutics. Hence, there is a crucial need for new and innovative therapies. Septin-2, a member of the septin family of GTP binding proteins, has been characterized in EOC for the first time and represents a potential future target. Septin-2 was found to be overexpressed in serous and clear cell human patient tissue compared to benign disease. Stable septin-2 knockdown clones developed in an ovarian cancer cell line exhibited a significant decrease in proliferation rates. Comparative label-free proteomic analysis of septin-2 knockdown cells revealed differential protein expression of pathways associated with the TCA cycle, acetyl CoA, proteasome and spliceosome. Further validation of target proteins indicated that septin-2 plays a predominant role in post-transcriptional and translational modifications as well as cellular metabolism, and suggested the potential novel role of septin-2 in promoting EOC tumorigenesis through these mechanisms

Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

While selective overexpression of serum clinical biomarker Human epididymis secretory protein 4 (HE4) is indicative of ovarian cancer tumorigenesis, much is still known about the mechanistic role of the HE4 gene or gene product. Here, we examine the role of the secretory glycoprotein HE4 in ovarian cancer immune evasion. Through modified subtractive hybridization analyses of human peripheral blood mononuclear cells (PBMCs), we have characterized gene targets of HE4 and established a preliminary mechanism of HE4-mediated immune failure in ovarian tumors. Dual specificity phosphatase 6 (DUSP6) emerged as the most upregulated gene in PBMCs upon in vitro exposure to HE4. DUSP6 was found to be upregulated in CD8+ cells and CD56+ cells. HE4 exposure reduced Erk1/2 phosphorylation specifically in these cell populations and the effect was erased by co-incubation with a DUSP6 inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI). In co-culture with PBMCs, HE4-silenced SKOV3 human ovarian cancer cells exhibited enhanced proliferation upon exposure to external HE4, while this effect was partially attenuated by adding BCI to the culture. Additionally, the reversal effects of BCI were erased in the co-culture with CD8+ / CD56+ cell deprived PBMCs. Taken together, these findings show that HE4 enhances tumorigenesis of ovarian cancer by compromising cytotoxic CD8+ and CD56+ cells through upregulation of self-produced DUSP6

The Use of Simulation Based Learning: With Bachelor of Science Pharmaceutical Science and PharmD Students

Background and purpose: Interprofessional education between bachelor of science pharmaceutical science (BSPS) students and Doctor of Pharmacy (PharmD) students is rare. According to the Association of American Medical Colleges, more than 80% of medical schools incorporate simulation based teaching within all four years of the curriculum. Educational activity and setting: The University of Rhode Island College of Pharmacy healthcare simulation lab has developed integrated educational opportunities for both groups of students by offering independent study opportunities that allow BSPS students to collaborate with PharmD students. A recent example of this model includes BSPS student development of patient cases which are integrated into high-fidelity human patient simulators with faculty assistance. A senior BSPS student researched and designed four clinical patient cases which were presented to P3 pharmacy students. Findings: In all four cases, there was an increase in knowledge and attitudes following the simulation. Qualitative comments from students noted the importance of patient education and an enhanced ability to manage disease and side effects. Summary: The nature of the simulation lab at the University of Rhode Island is a platform that can be modeled by other institutions with both PharmD and BSPS programs

Effects of doxazosin on vascular collagen synthesis, arterial pressure and serum lipids in the spontaneously hypertensive rat

Hypertension in various experimental models, including spontaneously hypertensive rats (SHR), is associated with elevated rates of vascular collagen synthesis. The sympathetic nervous system is an important factor in the etiology of hypertension in SHR. The primary purpose of this study was to determine the effects of the α1adrener-gic receptor antagonist doxazosin on aortic collagen synthesis and on systolic arterial pressure in SHR. Doxazosin was administered either short-term (20 or 200 mg/kg/day by gavage over 5 days) or long-term (0.02 or 0.20 g/L in the drinking water over 8 weeks). Rates of collagen synthesis were determined by incubating aortic segments with 14C-proline in vitro and then measuring either the formation of 14C-hydroxyproline by means of high-performance liquid chromatography, or the amount of radioactivity liberated by collagenase digestion. Systolic arterial pressure was monitored with the standard tail-cuff technique. Both doses of doxazosin depressed aortic collagen synthesis at 8 weeks of treatment, but neither dose had any effect at 4 weeks. In the short-term study only the higher acute dose of doxazosin significantly reduced aortic collagen synthesis; the lower dose had no effect. In the short-term study doxazosin reduced systolic arterial pressure, with a maximum effect at 1-2 days. Tolerance to the depressor effect developed over the remaining 3-4 days, especially with the higher dose. In the 8-week study, the lower doxazosin dose had no effect on systolic arterial pressure, and the higher dose exerted a biphasic effect, moderately but significantly reducing systolic arterial pressure at 1 and 8 weeks of treatment. Assays of serum doxazosin levels suggested that the tolerance to the depressor effect was partially, but not completely, pharmacokinetic. The higher dose of doxazosin significantly reduced serum triglycerides, but none of the other treatment regimens affected serum lipid profiles (very-low-density, low-density or high-density lipoproteins or total cholesterol). The results suggest that the inhibition of vascular collagen synthesis by doxazosin in SHR may be to some extent independent of reductions in arterial pressure. © 1987 Raven Press. New York

Lipopolysaccharide and cecal ligation/puncture differentially affect the subcellular distribution of the pregnane X receptor but consistently cause suppression of its target genes CYP3A

The repressed expression of cytochrome P450 (CYP) enzymes in septic patients contributes significantly to therapeutic failures. Mice treated with sepsis-inducing agent lipopolysaccharide (LPS) sequentially express reduced mRNA levels of the pregnane-X receptor (PXR) and its target genes Cyp3a(s), suggesting that reduction of Cyp expression is associated with the repression of PXR. The present study was undertaken to determine whether septic rats induced by LPS and cecal ligation/puncture (CLP) express reduced levels of rat PXR protein and whether the subcellular distribution of PXR is altered in septic conditions. Rats were treated with LPS (55 vs. 1 mg/kg) or underwent CLP, and the expression of CYP3A and PXR was determined. In LPS-treated rats, the expression of CYP3A enzymes was consistently decreased regardless of the doses used. In contrast, high dose and repeated low dose of LPS caused significant decreases on the nuclear PXR, whereas the opposite was true with the cytosolic PXR. When rats were administered with only a single low dose of LPS, both nuclear and cytosolic PXR levels were significantly increased. In the CLP model, rats undergoing CLP for 30 h expressed significantly lower levels of CYP3A but the PXR levels were not significantly altered. In addition, when rats were treated with dexamethasone, a significant induction of CYP3A was detected. However, such an induction was markedly antagonized by the treatment with LPS. The differential changes on the levels of the nuclear PXR and CYP3A between LPS and CLP models suggest that PXR plays negligible roles in the constitutive expression of CYP3A. The antagonism of LPS against dexamethasone-mediated CYP3A induction suggests that endotoxemia minimizes the inducibility of PXR target genes

Biosynthesis of carotenoids by Phycomyces blakesleeanus mutants in the presence of nitrogenous heterocyclic compounds

Pyridine, imidazole and some of their derivatives stimulate lycopene and γ-carotene synthesis-simultaneously inhibiting β-carotene formation in Phycomyces blakesleeanus Strain C115. Isonicotinoly-hydrazine has a toxic effect on Strains C9 and C115 and 1-methylimidazole on Strain C115 in the concentrations of 1 g/l. Compounds which cause an accumulation of lycopene and γ-carotene usually cause an increase in phytoene synthesis and the disappearance of β-zeacarotene. The effect of succinimide, 4-hydroxypyridine, and isonicotinoylhydrazine on Strain C9 has also been studied. When β-picoline and 2-methylimidazole treated C115 mycelia were washed and resuspended in phosphate buffer at pH 5·6 β-zeacarotene reappeared and β-carotene increased with the simultaneous decrease in lycopene and γ-carotene. The sum of β-carotene, γ-carotene up to 3days of resuspension was almost equal to the total of these at zero time. These results show that the inhibitory action of these compounds is on the enzymes responsible for cyclization of acyclic carotenes. This inhibition varies with the nature of the substituent on the heterocyclic ring and pyridine derivatives having pKa values of 6 ± 1 show the greatest degree of inhibition. © 1973