BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance.MethodsSeveral established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine.ResultsComparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect.ConclusionsWe developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.

The affinities for the benzodiazepine binding site of the GABA(A) receptor of 21 flavonoids have been studied using [(3)H]flumazenil binding to rat cortical membranes in vitro. We show that flavonoids with high affinity for the benzodiazepine receptor in vitro spanning the whole efficacy range from agonists (1q) to inverse agonists (1l) can be synthesized. The receptor binding properties of the flavonoids studied can successfully be rationalized in terms of a comprehensive pharmacophore model recently developed by Cook and co-workers (Drug Des. Dev. 1995, 12, 193-248), supporting the validity of this model. However, in contrast to the requirement by the model that an interaction with the hydrogen bond-accepting site A2 is necessary for compounds to display inverse agonistic activity, 6-methyl-3'-nitroflavone (1l), which cannot engage in such an interaction, nevertheless displays inverse agonism. The analysis of the binding affinities of 3'- and 4'-substituted flavones in terms of the pharmacophore model has yielded new information for the further development of the pharmacophore model.

Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4␣ (HNF4␣), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1-and HNF4␣-dependent expression of cholesterol 7␣ hydroxylase (CYP7A1) and sterol 12␣ hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4␣, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.cholesterol 7␣ hydroxylase ͉ sterol 12␣ hydroxylase ͉ farnesoid X receptor ͉ small heterodimer partner B ile acids (BAs) are cholesterol derivatives essential for absorption of dietary lipids and fat-soluble vitamins and maintenance of cholesterol BA homeostasis (1, 2). In humans the major BA biosynthetic pathway is initiated by cholesterol 7␣ hydroxylase (CYP7A1) to produce two primary BAs, cholic acid and chenodeoxycholic acid (CDCA). Sterol 12␣ hydroxylase (CYP8B1) catalyzes the synthesis of cholic acid and determines the ratio of cholic acid to CDCA in the bile (1). In addition to emulsification of dietary lipids, cholic acid and CDCA are ligands for farnesoid X receptor (FXR/NR1H4) (3-5). Ligandbound FXR regulates a number of target genes involving BA transport and metabolism (6). BAs also feedback-regulate BA biosynthesis, where activated FXR induces small heterodimer partner (SHP/NR0B2) gene expression, and SHP in turn inhibits liver receptor homolog 1 (LRH-1/NR5A2) or hepatocyte nuclear factor 4␣ (HNF4␣/NR2A1) activities on the BA response elements (BAREs) of CYP7A1 and CYP8B1 promoters (7-10). BAs can also act via FXR-independent pathways that use PKC (11) and JNK signaling (12, 13) to suppress HNF4␣-mediated expression of human CYP8B1 (hCYP8B1) (10,11,14). Although the physiological role of SHP in BA biosynthesis is well documented, mechanistic details of repression by SHP remain unclear. Recent studies indicate that SHP may repress its targets (i) via direct binding and blocking the coactivator interaction interface of its target nuclear receptors (NRs), (ii) by antagonizing CREB binding protein (CBP)/p300-dependent coactivator functions on NRs via recruitment of a coinhibitor protein like EID1, and (iii) by recruiting corepressor complexes that include histone deacetylases (HDAC) 1, 3, and 6, Sin3A...

Human ␥-aminobutyric acid type A (GABA A ) receptors were expressed in the baculovirus/Sf-9 insect cell expression system using recombinant cDNA of ␣ 1 ␤ 2 ␥ 2s subunits. ؊6 M or higher, gradually suppressed the peak amplitude of GABA response. A protein kinase A inhibitor, a protein kinase C inhibitor, and a Ca 2؉ chelator did not modify the effects of DHA on GABA-induced chloride ion current. Six unsaturated fatty acids other than DHA were examined. Arachidonic acid mimicked the effect of DHA while e.g. oleic acid had no effect.The inhibition of the GABA response in the presence of DHA was also observed in cells expressing GABA A receptors of ␣ 1 and ␤ 2 subunit combinations. The data show that the ␥ subunit is essential for DHA and arachidonic acid to potentiate the GABA-induced Cl ؊ channel activity and to affect the desensitization kinetics of the GABA A receptor.Polyunsaturated fatty acids are essential fatty acids that are abundantly bound in the phospholipids of brain membranes. The fatty acids influence the membrane fluidity of the neuronal plasma membrane as well as the functional properties of integral membrane protein (1). Arachidonic acid (AA) 1 and docosahexaenoic acid (DHA) are polyunsaturated fatty acids localized in the 2-position of membrane phospholipids and are liberated by various intracellular enzymes such as phospholipase A 2 and diacylglycerol lipase (2).Recently, various actions of these free fatty acids on the functional properties of neuronal plasma membrane neurotransmitter receptors have been reported, such as N-methyl-Daspartic acid receptor (3, 4), kainate receptor (4), and potassium channel receptor (5, 6). As for the effect of unsaturated fatty acids on ␥-aminobutyric acid type A (GABA A ) receptors, an electrophysiological study demonstrated that AA and DHA potently suppressed the GABA A receptor-mediated current response in the rat substantia nigra neurons (7). Receptor binding studies have shown marked effects of unsaturated fatty acids on brain GABA A receptor complexes in vitro (8 -11), as well as on human recombinant GABA A receptors expressed in Sf-9 insect cells (12). These findings suggest a variety of actions of fatty acids on the GABA A receptor-channel complex. The diversity of effects induced by free fatty acids might be due to ontogenetic, phylogenetic, and regional differences of the GABA A receptor subunit composition in the brain regions studied (13). Furthermore, a recent study on recombinant human GABA A receptors (12) reported that the effect of unsaturated fatty acids was dependent on the subunit composition of the GABA A receptor complexes. In addition, various combinations of GABA A receptor subunits might be expressed in the same neuron, complicating the interpretation of data on the effect of free fatty acids on the GABA A receptor complex obtained at the level of single cells, as, for example, by whole cell patch clamp techniques.The insect cell Sf-9/baculovirus expression system in combination with electrophysiological techniques provide the means for t...

In this review, estrogenic effects in depression, anxiety, and neurodegenerative disorders are summarized. Moreover, preclinical findings from in vitro and animal models are discussed. There is a correlation between decreased estrogen levels (e.g., premenstrually, during the postpartum period, and perimenopausally) and increased anxiety and depressive symptoms. Several studies show beneficial effects of estrogen treatment in women with anxiety and depressive symptoms. Recent data indicate that the estrogen receptor (ER) beta appears to be a major mediator of estrogenic effects in depression and anxiety. Additionally, both preclinical and clinical findings suggest that activation of estrogen receptors have an important role in neuroprotective and neurodegenerative processes in the mammalian central nervous system (CNS).