Eva Fiala-Beer

Role of Pancreatic Stellate Cells in Pancreatic Cancer Metastasis

Pancreatic stellate cells (PSCs) produce the stromal reaction in pancreatic cancer (PC), and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using both in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approaches. A sex mismatch study (injection of male hPSCs plus female PC cells into the pancreas of female mice) was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the Y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) quantifying human microvascular endothelial cell (HMEC-1) tube formation in vitro on exposure to conditioned media from hPSCs. Transendothelial migration was assessed in vitro by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs plus female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; and iii) exhibited transendothelial migration that was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis, and are able to intravasate/extravasate to and from blood vessels.

Purification and characterisation of proteins with cardiac stimulatory and haemolytic activity from the anemone Actinia tenebrosa.

Three new proteins with cardiac stimulatory and haemolytic activity, designated tenebrosins-A, -B and -C, have been purified from the Australian sea anemone Actinia tenebrosa. These proteins are basic (pI greater than or equal to 9.4), have mol. wt of about 20,000, and have very similar amino acid compositions and N-terminal amino acid sequences. None of the proteins contains cysteine or cystine residues. On isolated, spontaneously beating guinea pig atria they exhibit at 1-2 nM strong positive inotropic and slight to moderate chronotropic effects. In some cases a transient negative inotropic effect occurs prior to onset of the positive inotropic response. The proteins are also haemolytic, producing 50% haemolysis of guinea pig erythrocytes at concentrations similar to those showing positive inotropic effects.

Isolation of Quiescent Human Pancreatic Stellate Cells: A Promising in vitro Tool for Studies of Human Pancreatic Stellate Cell Biology

Background: Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. To date, human PSC biology has been studied using cancer- or inflammation-associated (pre-activated) PSCs, but an in vitro model of quiescent normal human PSCs (NhPSCs) has been lacking. Aims: To (i) isolate and characterize quiescent NhPSCs, and (ii) evaluate the response of culture-activated NhPSCs to cytokines and LPS. Methods: Quiescent NhPSCs were isolated from normal pancreatic tissue using density gradient centrifugation. PSC markers, glial fibrillary acidic protein (GFAP), desmin, α-smooth muscle actin (αSMA) and the lipopolysaccharide (LPS) receptors TLR4 and CD14 were identified by immunoblotting and immunocytochemistry. The effect of platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ) and LPS on NhPSC activation was also assessed. Results: Freshly isolated NhPSCs displayed a polygonal appearance with refringent cytoplasmic lipid droplets. Culture-activated NhPSCs expressed GFAP, desmin, αSMA, TLR4 and CD14, and were responsive to PDGF, TGFβ and LPS. Conclusion: Isolated NhPSCs expressed the same markers as rat PSCs and human cancer-associated PSCs and responded to PDGF and TGFβ similarly to rat PSCs. NhPSC preparations provide a useful in vitro tool to study the biology of PSCs in their physiological, non-activated state.

Dextran structural details from high-field proton NMR spectroscopy

Abstract Proton nuclear magnetic resonance at 500 MHz resolves anomeric proton resonances of dextrans well enough to furnish useful structural information. Minor resonances which would take inordinately long accumulation times to detect by carbon-13 spectroscopy are readily accessible. Enzymic hydrolysis of the dextrans to homologous series of oligosaccharides was useful in assigning anomeric resonances to specific structural features in the polysaccharides. Neighbouring group effects are also discussed.

Upregulation of cytochromes P450 2B in rat liver by orphenadrine

The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)‐inducible CYP2B in rat liver. CYP2B protein was increased to ∼14‐fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40‐fold. Corresponding increases in the activity of CYP2B‐dependent androstenedione 16β‐hydroxylation were measured in microsomes from ORPH and PB‐induced rats. Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH‐induced rat liver. Consistent with this finding, ORPH was found to activate a PB‐responsive enhancer module in constitutive androstane receptor (CAR)‐transfected Hep G2 cells. Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post‐translational component. In PB‐pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post‐translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH.

Phospho-STAT5 accumulation in nuclear fractions from vitamin A-deficient rat liver

The growth hormone (GH)‐responsive cytochrome P450 (CYP) 2C11 is down‐regulated in vitamin A‐deficient (VAD) rat liver. This study assessed the impact of a VAD diet on the hepatic Janus kinase‐Signal Transducers and Activators of Transcription (JAK‐STAT) system that mediates GH signalling. Nuclear tyrosine‐ and serine‐phosphorylated STAT5 accumulated in VAD liver, whereas nuclear JAK2 tyrosine kinase and SHP‐1 phosphatase were decreased. Tyrosine‐phosphorylated SHP‐1 was decreased to 36 ± 14% of control (P < 0.01), indicating its impaired activation in VAD liver. Episodic GH pulses increased nuclear phospho‐STAT5, especially in control liver, but nuclear phospho‐JAK2 and phospho‐SHP‐1 were not restored. CYP2C11 protein and testosterone 16α‐hydroxylation were decreased in VAD liver to 67 ± 16% and 76 ± 19% of control, and were further decreased by GH to 32 ± 8% and 30 ± 14% of control. Thus, hypo‐responsiveness of JAK‐STAT in VAD liver is associated with impaired nuclear phospho‐STAT dephosphorylation.

Structures of water-soluble α-d-glucans synthesized from sucrose by glucosyltransferases isolated from Streptococcus sobrinus culture filtrates

Abstract From three strains of Streptococcus sobrinus -K1-R, -6715-13-201 and -6715-13-27, grown in continuous culture under a variety of defined conditions, three different glucosyltransferases (GTF): GTF-S1, GTF-S3 and GTF-S4, were isolated. The enzymes were used to synthesize, from sucrose, soluble α- d -glucans S1, S3 and S4 respectively, of quite different structure. Methylation analysis, NMR spectroscopy and enzymic hydrolysis were used to determine the structural features of the S1, S3 and S4 polysaccharides. The GTF-S1-type enzymes synthesized highly branched (up to ≈ 34%) glucans having single -(1 → 3)-α-linked d -glucosyl residues attached to approximately one in two of the -(1 → 3)-α-linked d -glucosyl units of the main chain. The GTF-S3 enzymes produced low molecular weight linear -(1 → 3)-α-linked glucans. The GTF-S4 enzymes, released from strains K1-R and 6715-13-201 only in the presence of the surfactant Tween 80, form S4 glucans with branching ranging from ≈ 8 to 11%. Enzymic hydrolysis with an endo-(1 → 3)-α- d -glucanase was used to show that side chains consisting of a sequence of -(1 → 3)-α-linked- d -glucosyl residues are attached to -(1 → 6)-α-linked- d -glucosyl residues of the backbone, in S4 glucans from strains K1-R and 6715-13-201. Side chains of single -(1 → 3)-α-linked glucosyl residues are also present in these S4 glucans, as shown by NMR studies. Strain 6715-13-27 released GTF-S4 enzymes in the absence of Tween 80. The S4 glucans formed by these enzymes have the single side chains almost exclusively, and undergo very limited hydrolysis with the -(1 → 3)-α-glucanase.

Influence of the culture medium on the synthesis of α-D-glucans by Streptococcus cricetus AHT

Abstract Three different α- d -glucosyltransferases (GTFs) were separated from culture filtrates of Streptococcus cricetus strain AHT grown in a complex. standard medium in batch culture or under defined conditions of growth in the chemostat. Two of the enzymes (GTF-s1) converted sucrose into branched soluble dextrans, and the third (GTF-I) produced a relatively linear, water-insoluble, predominantly (1→3)-linked α- d -glucan. When the organism was grown in comlex medium by the removal of the fraction of high molecular weigth, only GTF-S1 and GTF-S2 were released, and GTF-I was detected. The water-insoluble glucan fraction obtained by incubating the cell-free filtrate with sucrose contained from 17 to 25% of (1→3)-glucosidic linkages, and accounted for up to 78 and 4% of the total glucans derived from growth in standard and modified medium, respectively. The soluble glucans produced in the same reaction were fractionated with ethanol to give, from both media, two distinct dextrans comprising (1) a highly branched dextran similar to the S1-dextran product of GTF-S1 and ([it2) a dextran containing fewer branch linkages and up to 86% of (1→6)-α- d -glucosidic linkages. A GTF responsible for the synthesis of the latter dextran was not separated. The structures of the glucan fractions and the products of the separated GTF were examined by enzymic degradation and methylation analysis.

Abstract LB-395: Hepatocyte growth factor: a potential therapeutic target in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic stellate cells (PSCs, which produce the desmoplastic reaction of pancreatic cancer) interact with pancreatic cancer (PC) cells to potentiate PC progression. A candidate factor that may mediate this interaction is the hepatocyte growth factor (HGF). High serum HGF levels in PC patients correlate with poor outcome. However, there is limited knowledge about the role of HGF in PC. Aims : To i) determine whether human PSCs and PC cell lines express HGF; and ii) assess the effects of HGF inhibition on PC progression in an orthotopic mouse model. Methods : i) HGF expression in PSCs and PC cells assessed by RT-PCR, immunoblotting/immunocytochemistry. ii) Orthotopic model: AsPC-1 (a human PC cell line) ± human PSCs implanted into the pancreas mice. One week later, mice divided into groups (n=8 mice/group) and treated with: HGF antibody AMG102 [(Amgen Inc.), 300 or 600μg IP biweekly] or isotype IgG (600 μg IP biweekly). Seven weeks later, tumour size and metastasis assessed. Results : i) PSCs express HGF at both mRNA and protein levels. In contrast, PC cells (MiaPaCa2, Panc-1, AsPC-1) express negligible HGF mRNA. ii) Orthotopic model: a) IgG treated AsPC-1+PSC mice showed larger tumours than mice injected with AsPC-1 alone ([Table][1]); b) 300 and 600μg AMG102 inhibited tumour growth in AsPC-1+PSC mice compared to the IgG treated group. However, AMG102 did not reduce tumour size in mice injected with AsPC-1 alone ([Table][1]). c) 600μg AMG102 inhibited metastasis (liver, diaphragm, mediastinum) in AsPC-1+PSC mice (p<0.05) compared to IgG treated mice. Conclusions : We have shown for the first time that i)human PSCs synthesise HGF; ii)HGF inhibition reduces tumour growth and metastasis of tumours representative of human PC i.e., exhibiting both tumour elements and a stromal reaction, but not in the clinically non-representative cancers formed by PC cells alone. Implication : Targeting the stromal reaction with relevant specific inhibitors may represent a novel therapeutic approach in PC. ![Figure][2] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-395. doi:10.1158/1538-7445.AM2011-LB-395 [1]: #F1 [2]: pending:yes