2016-07-12, Kohler, Reto, Anugraham, Merrina, Nunez Lopez, Monica, Xiao, Christina, Schoetzau, Andreas, Hettich, Timm, Schlotterbeck, Götz, Fedier, André, Jacob, Francis, Heinzelmann-Schwarz, Viola

Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting

2016, Jähne, Evelyn A., Eigenmann, Daniela E., Moradi-Afrapoli, Fahimeh, Verjee, Sheela, Butterweck, Veronika, Hebeisen, Simon, Hettich, Timm, Schlotterbeck, Götz, Smiesko, Martin, Hamburger, Matthias, Oufir, Mouhssin

Tryptanthrin and (E,​Z)​-​3-​(4-​hydroxy-​3,​5-​dimethoxybenzylidene)​indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-​inflammatory and antiallergic alkaloids, and shown to inhibit COX-​2, 5-​LOX catalyzed leukotriene synthesis, and mast cell degranulation at low μM to nM concns. To assess their suitability for oral administration, we screened the compds. in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compds., validated UPLC-​MS​/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coeff. > 32.0 × 10-​6 cm​/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coeff. values in the presence of the P-​glycoprotein inhibitor verapamil (50 μM) indicated that tryptanthrin was not involved in P-​glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-​resoln. mass spectrometry pointed to extensive phase II metab. of indolinone (sulfation and glucuronidation)​. Possible cardiotoxic liability of the compds. was assessed in vitro by measurement of an inhibitory effect on human ether-​a-​go-​go-​related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-​a-​go-​go-​related gene inhibition was found for tryptanthrin (IC50 > 10 μM) and indolinone (IC50 of 24.96 μM)​. The anal. of compds. using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-​a-​go-​go-​related gene potassium channel at micromolar concns.