Establishment and Molecular Characterization of Two Patient-Derived Pancreatic Ductal Adenocarcinoma Cell Lines as Preclinical Models for Treatment Response.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is exceedingly poor. Although surgical resection is the only curative treatment option, multimodal treatment is of the utmost importance, as only about 20% of tumors are primarily resectable at the time of diagnosis. The choice of chemotherapeutic treatment regimens involving gemcitabine and FOLFIRINOX is currently solely based on the patient's performance status, but, ideally, it should be based on the tumors' individual biology. We established two novel patient-derived primary cell lines from surgical PDAC specimens. LuPanc-1 and LuPanc-2 were derived from a pT3, pN1, G2 and a pT3, pN2, G3 tumor, respectively, and the clinical follow-up was fully annotated. STR-genotyping revealed a unique profile for both cell lines. The population doubling time of LuPanc-2 was substantially longer than that of LuPanc-1 (84 vs. 44 h). Both cell lines exhibited a typical epithelial morphology and expressed moderate levels of CK7 and E-cadherin. LuPanc-1, but not LuPanc-2, co-expressed E-cadherin and vimentin at the single-cell level, suggesting a mixed epithelial-mesenchymal differentiation. LuPanc-1 had a missense mutation (p.R282W) and LuPanc-2 had a frameshift deletion (p.P89X) in TP53. BRCA2 was nonsense-mutated (p.Q780*) and CREBBP was missense-mutated (p.P279R) in LuPanc-1. CDKN2A was missense-mutated (p.H83Y) in LuPanc-2. Notably, only LuPanc-2 harbored a partial or complete deletion of DPC4. LuPanc-1 cells exhibited high basal and transforming growth factor (TGF)-ß1-induced migratory activity in real-time cell migration assays, while LuPanc-2 was refractory. Both LuPanc-1 and LuPanc-2 cells responded to treatment with TGF-ß1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-ß1 target genes, which is consistent with the absence of DPC4 in LuPanc-2 cells. Both cell lines were able to form spheres in a semi-solid medium and in cell viability assays, LuPanc-1 cells were more sensitive than LuPanc-2 cells to treatment with gemcitabine and FOLFIRINOX. In summary, both patient-derived cell lines show distinct molecular phenotypes reflecting their individual tumor biology, with a unique clinical annotation of the respective patients. These preclinical ex vivo models can be further explored for potential new treatment strategies and might help in developing personalized (targeted) therapy regimens.

Linking Complement C3 and B Cells in Nasal Polyposis.

Nasal polyposis often is characterized by a persistent inflammation of the sinonasal mucosa, disease recurrence after medical or surgical intervention, and asthma comorbidity. Dysregulated complement activation may contribute to immunologic alterations and disease. To date, there is only scattered knowledge on the source and regulation of the central complement factors in the pathogenesis of nasal polyps. Here, we aim to study complement signatures, especially the C3-C3aR axis, and focus on cellular sources and targets in nasal polyps. Expression of complement factors, including C3, C5, and the anaphylatoxin receptors, was analyzed in nasal polyp tissue samples, the corresponding inferior turbinates, and healthy controls using transcriptomic methods and protein measurements. Distinct patterns of complement expression were found in nasal polyps compared to controls, characterized by an increased C3 activation and an increase in C3aR-bearing cells. In contrast, no difference was shown for epithelial-dependent C3 production. Besides low intracellular C3-expression levels for lymphocytes in general, we could identify an enlarged B lymphocyte population in nasal polyps displaying high amounts of intracellular C3. Our data suggest a prominent role for the C3-C3aR-axis in nasal polyps and, for the first time, describe a B cell population containing high levels of intracellular C3, suggesting a new role of B cells in the maintenance of the inflammation by complement.

1,8-cineol inhibits the Wnt/ß-catenin signaling pathway through GSK-3 dephosphorylation in nasal polyps of chronic rhinosinusitis patients.

Chronic rhinosinusitis with nasal polyps (CRSwNP) represents a benign neoplasm of the nasal mucosa, which leads to a decreased breathing capacity and reduced olfaction. The pathogenesis and the molecular mechanisms driving nasal polyps are not very well known. GSK-3 is involved in the regulation of various biosynthetic pathways and various kinases are able to regulate the GSK-3. Therefore, we investigated the effect of the monoterpene oxide 1,8-cineol on the regulation of the Wnt/ß-catenin signaling pathway with its central regulator protein GSK-3 in vitro. We determined GSK-3 expression and phosphorylation as well as the expression of negative regulators (Akt and SGK) and downstream activation of ß-catenin in nasal polyps of patients with CRSwNP by immunohistochemistry and Western blot experiments. In this study we demonstrated for the first time, that 1,8-cineol acts as a potential inhibitor of the Wnt/ß-catenin signaling pathway, by affecting the inhibitory phosphorylation of GSK-3, which is the key regulator of the ß-catenin activity. Our data provide novel insights in the regulatory networks responsible for the progression of CRSwNP and furthermore represent a new mechanism of 1,8-cineol activity, which may lead to novel treatment approaches to this natural drug.

Inhibition of GSK3α/ß impairs the progression of HNSCC.

BACKGROUND: Head and neck squamous cell cancer (HNSCC) is one of the most common tumors worldwide and there is an enormous need for innovative therapy approaches. Several recent studies suggest tumor entity specific roles of glycogen synthase kinase 3 (GSK3) in different human cancers, acting as tumor suppressor or as tumor promoter. Here we describe the role of GSK3 with respect to different parameters within HNSCC progression. METHODS: Base line expression and activity profiles of p-GSK3α/ß (Ser21/9) and p-GSK3α/ß (Tyr279/216) were analyzed by immunohistochemistry and western blotting. Four different permanent HNSCC cell lines were exposed to the potent GSK3α/ß inhibitor SB 216763. Cell viability was controlled via the MTT test. Cell migration was quantified with the Real Time Cell Analyzer (RCTA) xCELLigence. Regulation of the epithelial-mesenchymal transition (EMT) was measured with the Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler™ PCR Array and scratch assays. Taqman probes were used to detect the specific gene expression profiles of inflammatory cytokines Interleukin IL1ß, IL6, IL8, IL10, TNFα and IFNß. RESULTS: Exposure of permanent HNSCC cell lines to the specific GSK3α/ß inhibitor SB 216763 leads to significant growth inhibition, inhibition of migration and decreased levels of active GSK3α/ß in a dose dependent manner.Exposure of HNSCC lines to SB 216763 also resulted in a markable shift of EMT markers and functional EMT dysregulation. Functionally GSK3 differentially mediates the expression of TLR4- and TLR3-induced inflammatory cytokines in HNSCC, whereas no effect of SB 216763 on the NFkB activity was noticed. CONCLUSION: GSK3α/ß plays a crucial role in a variety of regulatory networks for HNSCC cancer progression as it drives proliferation or migration and thus GSK3 could serve as an interesting target for clinical drug development.

Inhibitory Effect of 1,8-Cineol on ß-Catenin Regulation, WNT11 Expression, and Cellular Progression in HNSCC.

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumors worldwide. The high mortality rates have not changed during the last three decades, and thus there is an enormous need for innovative therapy approaches. Several recent studies suggest an important role of the Wnt/ß-catenin signaling pathway in the tumorigenesis of HNSCC. We analyzed the effect of the monoterpene oxide 1,8-cineol on the regulation of the Wnt/ß-catenin signaling pathway and the cellular progression of different HNSCC cell lines. METHODS: Permanent HNSCC cell lines were exposed to varying concentrations and times of 1,8-cineol. Regulation and activity profiles of the Wnt/ß-catenin signaling cascade were analyzed using Western hybridization experiments, MTT assays, real-time PCR-based epithelial to mesenchymal transition array, and immunohistochemistry. RESULTS: Exposure of different cell lines to 1,8-cineol treatment resulted in a dose-dependent inhibition of proliferation and a decreased activity of the WNT/ß-catenin pathway. We can show the inhibition of glycogen synthase kinase 3 (GSK-3)α/ß (Ser-9/21) as well as a corresponding decreased endolysosomal localization, leading to a decreased ß-catenin activity. Furthermore, we can show that exposure to cineol functionally results in a reduced expression of WNT11. CONCLUSION: In this work, we demonstrate for the first time that 1,8-cineol acts as an inhibitor of the Wnt/ß-catenin activity in HNSCC via a decreased inhibition of GSK-3, which lead to reduced levels of WNT11 and a dose-dependent decrease of the cellular progression. Our data represent a new mechanism of 1,8-cineol activity, which may lead to novel molecular targets and treatment approaches of this natural drug.

Melanocortin crosstalk with adipose functions: ACTH directly induces insulin resistance, promotes a pro-inflammatory adipokine profile and stimulates UCP-1 in adipocytes.

The melanocortin (MC) system is a pivotal component of the hypothalamo-pituitary-adrenal (HPA) stress axis and plays an important role in the pathogenesis of obesity and the metabolic syndrome. Adipose dysfunction is implicated in the pathogenesis of these disorders. We investigated direct ACTH effects on adipose functions in immortalised murine white and brown adipocytes. MC receptor types 2 and 5 were expressed at the mRNA and protein levels and were strongly up-regulated during differentiation. Chronic ACTH stimulation did not affect adipogenesis. Insulin-induced glucose uptake in white adipocytes was acutely and transiently reduced by 45% upon ACTH treatment. Visfatin and adiponectin gene expression was reduced by about 50% in response to ACTH, while interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were acutely up-regulated by 2100 and 60% respectively. Moreover, IL-6 secretion was increased by 1450% within 4 h of ACTH treatment. In brown adipocytes, stimulation with ACTH caused a 690% increase in uncoupling protein (UCP)-1 mRNA levels within 8 h, followed by a 470% increase in UCP-1 protein concentrations after 24 h. Consistently, p38 mitogen-activated protein kinase (MAPK) phosphorylation was acutely increased by 1800% in response to ACTH stimulation, and selective inhibition of p38 MAPK abolished the ACTH-mediated UCP-1 protein increase. Taken together, ACTH acutely promotes an insulin-resistant, pro-inflammatory state and transiently enhances energy combustion. In conditions characterised by a dysregulation of the HPA stress axis such as the metabolic syndrome, direct MC interaction with adipocytes may contribute to dysregulated energy balance, insulin resistance and cardiometabolic complications.

Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling.

Calpains are a family of non-lysosomal cytoplasmatic cysteine proteases. Since calpain 10 (CAPN10), a member of the calpain family of proteases, has been found to represent a putative diabetes susceptibility gene, it was argued that calpains may be involved in the development of type 2 diabetes. The functional role of calpains in insulin signaling and/or insulin action is, however, not clear. We investigated the effects of the calpains 1 and 2 inhibitor PD151746 on insulin signaling and insulin action in human hepatoma G2 cells (HepG2). HepG2 cells were incubated without (-PD) or with (+PD) 5.33 micromol/l PD151746 for different times and then stimulated with 100 nmol/l insulin for 0 (t(0)), 5 (t(5)), 15 (t(15)), 30 (t(30)), 45 (t(45)), and 60 (t(60)) min. After solubilization of the cells, insulin receptor kinase activity, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol-3 kinase (PI3-kinase), PI3-kinase activity, Thr(308) phosphorlyation of Akt, amount of protein tyrosine phosphatase-epsilon (PTPepsilon), and glycogen synthase activity were determined. Incubation with PD151746 resulted in a significant reduction of insulin-stimulated glycogen synthesis compared with cells not pre-incubated with the calpain inhibitor (-PD: t(0), 4.90 +/- 1.20%; t(5), 5.90 +/- 1.02%; t(15), 5.29 +/- 0.95%; t(30), 5.60 +/- 1.10%; t(45), 5.52 +/- 0.90%; t(60), 5.67 +/- 0.97%;+PD: t(0), 4.56 +/- 1.10%; t(5), 6.16 +/- 1.05%; t(15), 7.52 +/- 1.09%; t(30), 7.68 +/- 1.10%; t(45), 8.28 +/- 0.89%; t(60), 7.69 +/- 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPepsilon in the cells after 12 h (-PD: t(1), 0.85 +/- 0.18 RU (Relative unit); t(8), 0.87 +/- 0.18 RU; t(12), 0.9 +/- 0.13 RU; +PD: t(1), 0.92 +/- 0.21 RU; t(8), 1.1 +/- 0.15 RU; t(12), 1.34 +/- 0.16 RU; P < 0.05). Calpain inhibition with PD151746 had no effect on the insulin stimulation of the investigated insulin signaling parameters. These results in HepG2 cells suggest that calpains play a role in the hepatic regulation of insulin-stimulated glycogen synthesis independent of the PI3-kinase/Akt signaling pathway.

Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes.

BACKGROUND/AIMS: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. METHODS: Adult rat hepatocytes were cultured+/-dexamethasone for 48 h; insulin actions were studied subsequently. RESULTS: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. CONCLUSIONS: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes.