Stefan Rosewicz

Human Galectin-2: Novel Inducer of T Cell Apoptosis with Distinct Profile of Caspase Activation

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a β-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to β1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.

Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the α5β1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G1 cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the α5β1 integrin.

Gastrin and Phorbol 12-Myristate 13-Acetate Regulate the Human Histidine Decarboxylase Promoter through Raf-dependent Activation of Extracellular Signal-regulated Kinase-related Signaling Pathways in Gastric Cancer Cells

Gastrin stimulates transcription of the human histidine decarboxylase (HDC) gene through binding to the G-protein-coupled cholecystokinin-B/gastrin receptor. We have explored the possibility that mitogen-activated protein kinase cascades play a role in mediating the effects of gastrin on transcription in a gastric cancer (AGS-B) cell line. Gastrin and phorbol 12-myristate 13-acetate (PMA) treatment of AGS-B cells was found to increase the phosphorylation of tyrosine residues of extracellular signal-regulated kinases (ERKs) 1 and 2 and increase ERK activity as determined by thein vitro phosphorylation of myelin basic protein. Reporter gene assays also demonstrated that gastrin and PMA stimulated Elk-1- and c-Myc-dependent transactivation, consistent with gastrin- and PMA-induced activation of ERKs. Overexpression of wild type ERK-1 and ERK-2 or activation of endogenous ERKs using activated MEK-1 (mitogen-activated protein kinase kinase or ERK kinase) overexpression stimulated HDC promoter activity in a dose-dependent fashion. Interruption of the ERK-related pathway using expression vectors for kinase-deficient ERKs or an ERK-specific phosphatase (PAC-1) blocked gastrin- and PMA-stimulated HDC promoter activity. In contrast, inhibition of the Jun kinase pathway using an interfering dominant negative SEK-1 (stress-activated protein kinase/ERK-1) mutant did not inhibit HDC promoter activity. Furthermore, whereas gastrin stimulated phosphorylation of Shc proteins and association with Grb2, activation of the HDC promoter was not influenced by expression of dominant negative Ras (N15 or N17) proteins. However, gastrin stimulated Raf-1 kinase activity, and activation of the HDC promoter was blocked by coexpression of a dominant negative Raf-1 construct. Overall, these data demonstrate that gastrin regulates HDC transcription in a Rafdependent, Ras-independent fashion predominantly through activation of the ERK-related pathway.

Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice

Galectins have recently emerged as central regulators of the immune system. We have previously demonstrated that carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Here, we investigate the potential therapeutic effect of galectin-2 in experimental colitis. Galectin-2 expression and its binding profile were determined by immunohistochemistry. Acute and chronic colitis was induced by dextran sodium sulfate administration and in a T-cell transfer colitis model. Apoptosis was assessed by TdT-mediated dUTP-biotin nick-end labeling, and cytokine secretion was determined by cytometric bead array. We show that galectin-2 was constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated. Our study provides evidence that galectin-2 induces apoptosis in vivo and ameliorates acute and chronic murine colitis. Furthermore, galectin-2 has no significant toxicity over a broad dose range, suggesting that it may serve as a new therapeutic agent in the treatment of inflammatory bowel disease.

Oxidative stress activates the human histidine decarboxylase promoter in AGS gastric cancer cells

Oxidant stress is thought to play a role in the pathogenesis of many gastric disorders. We have recently reported that histidine decarboxylase (HDC) promoter activity is stimulated by gastrin through a protein kinase C- and extracellular signal-regulating kinase (ERK)-dependent pathway in gastric cancer (AGS-B) cells, and this transcriptional response is mediated by a downstream cis-acting element, the gastrin response element (GAS-RE). To study the mechanism through which oxidant stress affects gastric cells, we examined the effects of hydrogen peroxide (H2O2) on HDC promoter activity and intracellular signaling in AGS-B cells. H2O2(10 mm) specifically activated the HDC promoter 10–12-fold, and this activation was blocked by both mannitol andN-acetylcysteine. Hydrogen peroxide treatment of AGS-B cells increased the phosphorylation and kinase activity of ERK-1 and ERK-2, but did not affect Jun kinase tyrosine phosphorylation or kinase activity. In addition, treatment of AGS-B cells with H2O2 resulted in increasedc-fos/c-jun mRNA expression and AP-1 activity, and also led to increased phosphorylation of epidermal growth factor receptor (EGFR) and Shc. H2O2-dependent stimulation of HDC promoter activity was completely inhibited by kinase-deficient ERKs, dominant-negative (N17 and N15) Ras, and dominant-negative Raf, and partially blocked by a dominant-negative EGFR mutant. In contrast, protein kinase C blockade did not inhibit H2O2-dependent induction of the HDC promoter. Finally, deletion analysis demonstrated that the H2O2 response element could be mapped to the GAS-RE (nucleotides 2 to 24) of the basal HDC promoter. Overall, these studies suggest that oxidant stress activates the HDC promoter through the GAS-RE, and through an Ras-, Raf-, and ERK-dependent pathway at least partially involving the EGFR.

Interferon-α: Regulatory Effects on Cell Cycle and Angiogenesis

In the current study, we investigated the effects of interferon-α (IFN-α) on proliferation and angiogenesis in neuroendocrine tumor disease. Using a panel of human neuroendocrine tumor cell lines, we confirmed functionally active IFN-α signaling by STAT activation and nuclear translocation as well as transactivation. IFN-α results in anchorage-dependent and -independent growth inhibition due to a delayed progression from S-phase to G2 phase of the cell cycle. This was due to substantial reduction in cellular cyclin B levels resulting in the inhibition of Cdc2 kinase activity. In parallel to growth inhibition, we observed a profound inhibition of VEGF gene transcription by IFN-α in human neuroendocrine tumor cells due to an Sp1/Sp3-dependent inhibition of VEGF promoter activity. Treatment of neuroendocrine tumors with IFN-α in nude mice resulted in growth inhibition and inhibition of angiogenesis. Furthermore, treatment of neuroendocrine tumor patients with IFN-α resulted in decreased VEGF expression as well as tumor angiogenesis in liver metastases. In summary, IFN-α acts via direct antiproliferative effects as well as inhibition of tumor angiogenesis mediated by suppression of VEGF gene expression in neuroendocrine tumor disease.

Retinoids: Effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines

BACKGROUND & AIMS Advanced pancreatic carcinoma has a dismal prognosis despite extensive chemotherapeutic trials. The aim of this study was to evaluate the role of retinoids as an experimental therapeutic approach for pancreatic cancer. METHODS Four ductal and one acinar pancreatic tumor cell lines were investigated. Growth was determined by cell number and a human tumor clonogenic assay. In vivo growth was assessed by xenografts transplanted into nude mice. Differentiation was characterized by immunofluorescence microscopy and carbonic anhydrase II gene expression. Retinoid receptors were characterized by Northern blotting and reverse-transcriptase polymerase chain reaction. RESULTS Retinoid treatment results in a time- and dose-dependent growth inhibition in vitro and in vivo of ductal but not acinar pancreatic tumor cells. Retinoid treatment induces a more differentiated phenotype in ductal tumor cells as shown by morphological criteria and increased expression of carbonic anhydrase II. All pancreatic tumor cell lines expressed a broad panel of cellular retinoid binding proteins and nuclear retinoid receptors. Retinoic acid receptor gamma and cellular retinoic acid binding protein II were found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines. CONCLUSIONS Detailed knowledge of nuclear retinoid receptor expression may provide rational strategies for retinoid treatment of pancreatic cancer.

VEGF-D promotes tumor growth and lymphatic spread in a mouse model of hepatocellular carcinoma.

Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro‐(lymph), angiogenic protein VEGF‐D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF‐D exclusively in the tumor cells. The cognate receptor VEGFR‐3 was detected on blood and lymphatic vessels. By clinicopathological analysis VEGF‐D expression was correlated with pT‐stage of the primary, lymph node metastasis and lymphangiosis carcinomatosa. Three out of 4 human HCC cell lines expressed and secreted VEGF‐D. To approach its biological function, VEGF‐D deficient SKHep‐1 cells were stably transfected with VEGF‐D cDNA and effects on tumor progression were determined in vivo. Compared to mock‐transfected controls, subcutaneous tumors derived from VEGF‐D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF‐D expressing tumors exhibited increased microvessel density and increased abundance of peri‐ and intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE‐1 and/or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR‐3 domain blocked VEGF‐D‐induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF‐D expression resulted in an increased rate of intra‐ and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF‐D is involved in growth and lymphatic spread of HCC. Therefore, VEGF‐D might represent a therapeutic target in HCC.

Combined imaging techniques for pancreatic cancer

Advanced pancreatic cancer has a poor prognosis. Early detection with imaging techniques can, however, improve the outlook for patients who undergo surgical resection, the only potential curative treatment. Individual techniques, however, have poor sensitivity for small masses and cannot easily differentiate tumour tissue from pancreatic masses associated with chronic pancreatitis. We combined biochemical detection on positron emission tomography with the anatomical accuracy of computed tomography and were able to improve accuracy of interpretation of imaging for patients with pancreatic cancer.

Retinoic acid receptor beta regulates growth and differentiation in human pancreatic carcinoma cells

BACKGROUND & AIMS Retinoic acid receptor beta (RAR beta) expression is lost or decreased during malignant transformation in human pancreatic adenocarcinoma. The aim of this study was to evaluate the role of RAR beta expression in the propagation of a malignant phenotype in human pancreatic carcinoma cells. METHODS Overexpression of RAR beta in the human pancreatic carcinoma cell line DAN-G was achieved by selecting stable transfected cell clones. Genomic integration and expression were verified by Southern and Northern blotting and electrophoretic mobility shift assays. Growth was determined by cell number and xenografts transplanted into nude mice. Differentiation was examined by immunohistochemistry. RESULTS Overexpression of RAR beta in DAN-G cells inhibited cellular proliferation in vitro and in vivo. Furthermore, RAR beta overexpression resulted in induction of cellular differentiation in xenografted tumors as evidenced by increased tumor cell expression of duct cell differentiation markers carcinoembryonic antigen (CEA), CA19-9, and cytokeratin 7. CONCLUSIONS Decreased expression of RAR beta plays a key role in the maintenance of a malignant phenotype in human pancreatic adenocarcinoma and therefore represents a novel target for experimental strategies in the treatment of pancreatic cancer patients.