Taurolidine Antiadhesive Properties on Interaction with E. coli; Its Transformation in Biological Environment and Interaction with Bacteria Cell Wall

The taurine amino-acid derivative, taurolidine, bis-(1,1-dioxoperhydro-1,2,4-thiabiazinyl–4)methane, shows broad antibacterial action against gram-positive and gram-negative bacteria, mycobacteria and some clinically relevant fungi. It inhibits, in vitro, the adherence of Escherichia coli and Staphylococcus aureus to human epithelial and fibroblast cells. Taurolidine is unstable in aqueous solution and breaks down into derivatives which are thought to be responsible for the biological activity. To understand the taurolidine antibacterial mechanism of action, we provide the experimental single crystal X-ray diffraction results together with theoretical methods to characterize the hydrolysis/decomposition reactions of taurolidine. The crystal structure features two independent molecules linked through intermolecular H-bonds with one of them somewhat positively charged. Taurolidine in a biological environment exists in equilibrium with taurultam derivatives and this is described theoretically as a 2-step process without an energy barrier: formation of cationic taurolidine followed by a nucleophilic attack of O(hydroxyl) on the exocyclic C(methylene). A concerted mechanism describes the further hydrolysis of the taurolidine derivative methylol-taurultam. The interaction of methylol-taurultam with the diaminopimelic NH2 group in the E. coli bacteria cell wall (peptidoglycan) has a negative ΔG value (−38.2 kcal/mol) but a high energy barrier (45.8 kcal/mol) suggesting no reactivity. On the contrary, taurolidine docking into E. coli fimbriae protein, responsible for bacteria adhesion to the bladder epithelium, shows it has higher affinity than mannose (the natural substrate), whereas methylol-taurultam and taurultam are less tightly bound. Since taurolidine is readily available because it is administered in high doses after peritonitis surgery, it may successfully compete with mannose explaining its effectiveness against bacterial infections at laparoscopic lesions

Gene Expression Signature of Normal Cell-of-Origin Predicts Ovarian Tumor Outcomes

The potential role of the cell-of-origin in determining the tumor phenotype has been raised, but not adequately examined. We hypothesized that distinct cells-of-origin may play a role in determining ovarian tumor phenotype and outcome. Here we describe a new cell culture medium for in vitro culture of paired normal human ovarian (OV) and fallopian tube (FT) epithelial cells from donors without cancer. While these cells have been cultured individually for short periods of time, to our knowledge this is the first long-term culture of both cell types from the same donors. Through analysis of the gene expression profiles of the cultured OV/FT cells we identified a normal cell-of-origin gene signature that classified primary ovarian cancers into OV-like and FT-like subgroups; this classification correlated with significant differences in clinical outcomes. The identification of a prognostically significant gene expression signature derived solely from normal untransformed cells is consistent with the hypothesis that the normal cell-of-origin may be a source of ovarian tumor heterogeneity and the associated differences in tumor outcome

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy

NRF2 Modulates Aryl Hydrocarbon Receptor Signaling: Influence on Adipogenesis▿ †

The NF-E2 p45-related factor 2 (NRF2) and the aryl hydrocarbon receptor (AHR) are transcription factors controlling pathways modulating xenobiotic metabolism. AHR has recently been shown to affect Nrf2 expression. Conversely, this study demonstrates that NRF2 regulates expression of Ahr and subsequently modulates several downstream events of the AHR signaling cascade, including (i) transcriptional control of the xenobiotic metabolism genes Cyp1a1 and Cyp1b1 and (ii) inhibition of adipogenesis in mouse embryonic fibroblasts (MEFs). Constitutive expression of AHR was affected by Nrf2 genotype. Moreover, a pharmacological activator of NRF2 signaling, CDDO-IM {1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole}, induced Ahr, Cyp1a1, and Cyp1b1 transcription in Nrf2+/+ MEFs but not in Nrf2−/− MEFs. Reporter analysis and chromatin immunoprecipitation assay revealed that NRF2 directly binds to one antioxidant response element (ARE) found in the −230-bp region of the promoter of Ahr. Since AHR negatively controls adipocyte differentiation, we postulated that NRF2 would inhibit adipogenesis through the interaction with the AHR pathway. Nrf2−/− MEFs showed markedly accelerated adipogenesis upon stimulation, while Keap1−/− MEFs (which exhibit higher NRF2 signaling) differentiated slowly compared to their congenic wild-type MEFs. Ectopic expression of Ahr and dominant-positive Nrf2 in Nrf2−/− MEFs also substantially delayed differentiation. Thus, NRF2 directly modulates AHR signaling, highlighting bidirectional interactions of these pathways

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy

Differentiation of NUT Midline Carcinoma by Epigenomic Reprogramming

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC

OCLER and FNLER tumor histopathology in immunodeficient nude (Nu/Nu) mice.

<p><b>A-B</b>, Hematoxylin and eosin (H&E) stains of representative formalin-fixed paraffin-embedded (FFPE) tumor sections from OCLER (A) and FNLER (B) xenografts revealed focal micropapillary structures. The predominant morphology was diffuse sheets of cells with a poorly differentiated tumor architecture (scale bar  =  20 µM). <b>C-D</b>, PAX8 immunoperoxidase stains of representative FFPE tumor sections from OCLER (C) and FNLER (D) xenografts confirmed that xenografts retained their PAX8 expression (scale bar  =  20 µM).</p

Tumor formation, tumor burden and ascites in the OCLER and FNLER xenograft model.

a<p>Values shown are means ± s.d. across all mice injected with each cell type that had any evaluable tumor mass (the sum of 1× intraperitoneal and 2× subcutaneous sites per mouse).</p>b<p>Formation of ascites was only evaluated among mice that developed tumors.</p>c<p><i>P</i>-values were calculated using the Mann-Whitney test.</p

Culture and characterization of normal ovarian epithelial and fallopian tube epithelial cells.

<p><b>A</b>, Replicate plates of normal fallopian tube epithelial cells were cultured in WIT-fo medium (green and purple lines) or standard medium (red, grey lines, 1:1 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12, supplemented with 0.1% BSA, 5% serum). In WIT-fo medium, normal fallopian tube epithelial cells from two different patients divided continuously for at least 30 days and > 100 days and reached at least 12 and >20 population doublings respectively (green and purple lines). In contrast, matched cells from the same donors growth arrested in the DMEM/Ham’s F12 medium (red, grey lines). Normal fallopian tube epithelial cells were isolated from additional patients 3 and 4. <b>B</b>, Normal ovarian epithelial cells were cultured in WIT-fo medium (purple and blue lines) or standard medium (red line, MCDB 105/Medium 199 (M199) (1∶1 mixture) with 10% FBS and 2 mm l-glutamine). Primary normal ovarian epithelial cells were from patients 2 and 3 (cells cultured in standard medium were from patient 3). Matched cells from patient 3 growth arrested in the MCDB105/M199 medium (red line). <b>C-D</b>, Normal human ovarian tissue; immunoperoxidase staining of formalin-fixed paraffin embedded (FFPE) sections with PAX8 demonstrates that ovarian inclusion cyst epithelium is PAX8⁺ (brown nuclear stain) (<b>C-D</b>) while ovarian surface epithelium is in general PAX8 negative (<b>C</b>), rare presence of rare Pax8 positive cells have been reposted on the ovarian surface. <b>E,</b> Normal human fallopian tube tissue; double immunoperoxidase staining of FFPE sections shows ciliated cells are FOXJ1⁺ (nuclear brown) and non-ciliated cells are PAX8⁺ (nuclear red). <b>F,</b> Normal human fallopian tube tissue; double immunoperoxidase staining of FFPE sections shows that ciliated cells are FOXJ1⁺ (nuclear brown) and non-ciliated cells are CK7⁺ (cytoplasmic red) (scale bar (C-F)  =  20 µM). <b>G,</b> Summary of cell type specific characterization markers.</p