Galectin-1 sensitizes carcinoma cells to anoikis via the fibronectin receptor α5ß1-integrin.

Anoikis resistance is a hallmark of transformed epithelial cells. Here, we show that treatment of anoikis-resistant carcinoma cell lines with the endogenous lectin galectin-1 (Gal-1) promoted apoptosis via interaction with the unligated fibronectin receptor α(5)ß(1)-integrin. Gal-1 efficiency correlated with expression of α(5)ß(1)-integrin, and transfection of the α(5)-subunit into deficient cell lines conferred Gal-1 binding and anoikis stimulation. Furthermore, Gal-1 and the α(5)- and ß(1)-integrin subunits co-precipitated in Gal-1-stimulated cells undergoing anoikis. Other members of the galectin family failed to be active. The functional interaction between Gal-1 and α(5)ß(1)-integrin was glycan dependent with α2,6-sialylation representing a switch-off signal. Desialylation of cell surface glycans resulted in increased electrophoretic mobility of α(5)ß(1)-integrin and facilitated Gal-1 binding and anoikis stimulation. On the level of signaling, Gal-1-stimulated anoikis was prevented by filipin, which impaired the internalization of α(5)ß(1)-integrin via cholesterol-enriched microdomains, and by pretreatment with a caspase-8 inhibitor. We propose that Gal-1/α(5)ß(1)-integrin interaction participates in the control of epithelial integrity and integrin sialylation may enable carcinoma cells to evade this Gal-1-dependent control mechanism.

The oral multitarget tumour growth inhibitor, ZK 304709, inhibits growth of pancreatic neuroendocrine tumours in an orthotopic mouse model.

BACKGROUND AND AIMS: Current systemic therapies for neuroendocrine tumours (NETs) do not provide sufficient control of tumour growth. However, efficient evaluation of novel drugs is hindered by the lack of a suitable preclinical animal model. Here an orthotopic mouse model of pancreatic NET is established and used to study the action of ZK 304709, a first in class, oral multitarget tumour growth inhibitor. ZK 304709 is an inhibitor of cyclin-dependent kinases (Cdks) 1, 2, 4, 7 and 9, vascular endothelial growth factor receptor-type kinases (VEGF-RTKs) 1-3 and platelet-derived growth factor receptor-type kinase beta (PDGF-RTKss). METHODS: BON and QGP-1 human NET cells were used to study proliferation, survival and cell cycle distribution in vitro. For induction of orthotopic NETs, BON cells were injected into the pancreas of NMRI(nu/nu) mice. Primary tumour growth and metastatic spread were recorded after 9 weeks, and apoptosis, microvessel density and lymphatic vessel density were determined. RESULTS: ZK 304709 dose-dependently suppressed proliferation and colony formation of NET cells. Direct effects on NET cells were consistent with Cdk inhibition and involved G(2) cell cycle arrest and apoptosis induction, which was associated with reduced expression of MCL1 (myeloid cell leukaemia sequence 1), survivin and hypoxia-inducible factor 1alpha (HIF1alpha). Apoptosis similarly occurred in vivo in ZK 304709-treated orthotopic BON tumours, resulting in a 80% reduction of primary tumour growth. In contrast, treatment with lanreotide or 5-fluorouracil and streptozotocin failed to inhibit tumour gowth. ZK 304709 also reduced tumour microvessel density, implicating antiangiogenic mechanisms. CONCLUSION: BON orthotopic tumours provide an informative model for preclinical drug evaluation in NETs. In this model, ZK 304709 achieved efficacious tumour growth control via induction of apoptosis and inhibition of tumour-induced angiogenesis.

Magnetic resonance cholangiopancreatography: image quality, ductal morphology, and value of additional T2- and T1-weighted sequences for the assessment of suspected pancreatic cancer.

PURPOSE: To assess image quality and duct morphology on magnetic resonance cholangiopancreatography (MRCP) and also the value of additional T2- and T1-weighted sequences for differentiation of benignity and malignancy in patients with suspected pancreatic tumors. MATERIAL AND METHODS: One-hundred-and-fourteen patients received MRCP and unenhanced and contrast material-enhanced MR imaging. MR results were analyzed independently by two blinded readers, and subsequently correlated with the results from surgery, biopsy, and follow-up. Assessment included the evaluation of image quality, duct visualization and morphology, and the differentiation of pancreatic lesion status (benign versus malignant). RESULTS: Overall, 49 patients had benign final diagnoses, while 65 had a malignant diagnosis. Image quality of single-shot thick-slab MRCP was rated significantly better than the MIP images of multisection MRCP. With MRCP alone, the two readers' accuracy in the assessment of pancreatic lesion status was 72% (95% CI, 64% to 83%) and 69% (95% CI, 56% to 77%), respectively; with the addition of T2- and T1-weighted images the accuracy significantly improved to 89% (95% CI, 82% to 95%) and 84% (95% CI, 77% to 92%) for readers 1 and 2, respectively. CONCLUSION: Single-shot thick-slab MRCP and multisection MRCP provide complementary results; however, single-shot MRCP had superior image quality. Moreover, assessment of ductal morphology with MRCP alone facilitated the diagnosis of different pathologic conditions of the pancreatobiliary system in the majority of patients. However, with the addition of T2- and T1-weighted sequences the overall diagnostic accuracy was significantly improved and thus we consider that a comprehensive MR approach should comprise both MRCP techniques and parenchymal sequences.

[Pancreatic carcinoma]. / Pankreaskarzinom.

Despite considerable progress in the areas of epidemiology and molecular genetics, pancreatic cancer is still characterized by a dismal prognosis. The current overview attempts a critical analysis of the published data resulting in recommendations for diagnostic and therapeutic algorithms. The diagnostic work-up of suspected pancreatic cancer patients includes an initial abdominal ultrasound which in case of ambiguous results or the absence of distant metastases is followed by "one-stop-MRI" and explorative laparotomy. Locally confined tumour disease should proceed to resective surgery which however results in cure only in a minority of resected patients even in specialized centers. Adjuvant chemotherapy might be of some benefit in terms of survival. Locally advanced but irresectable disease can be considered for combined radiochemotherapy. Systemic chemotherapy with Gemcitabine -results in marginal benefit of overall survival and may offer clinical benefit to a subgroup of patients. Best supportive care still remains the mainstay of irresectable disease. This summary highlights the urgent need for the development of innovative diagnostic and therapeutic strategies.

Autocrine growth inhibition by transforming growth factor beta-1 (TGFbeta-1) in human neuroendocrine tumour cells.

BACKGROUND AND AIM: The role of transforming growth factor beta-1 (TGFbeta-1) in neuroendocrine tumour biology is currently unknown. We therefore examined the expression and biological significance of TGFbeta signalling components in neuroendocrine tumours (NETs) of the gastroenteropancreatic (GEP) tract. METHODS: Expression of TGFbeta-1 and its receptors, Smads and Smad regulated proteins, was examined in surgically resected NET specimens and human NET cell lines by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting, and ELISA. Activation of TGFbeta-1 dependent promoters was tested by transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. The role of endogenous TGFbeta was assessed by a TGFbeta neutralising antibody and stable transfection of a dominant negative TGFbetaR II receptor construct. RESULTS: Coexpression of TGFbeta-1 and its receptors TGFbetaR I and TGFbetaR II was detected in 67% of human NETs and in all three NET cell lines examined. NET cell lines expressed the TGFbeta signal transducers Smad 2, 3, and 4. In two of the three cell lines, TGFbeta-1 treatment resulted in transactivation of a TGFbeta responsive reporter construct as well as inhibition of c-myc and induction of p21((WAF1)) expression. TGFbeta-1 inhibited anchorage dependent and independent growth in a time and dose dependent manner in TGFbeta-1 responsive cell lines. TGFbeta-1 mediated growth inhibition was due to G1 arrest without evidence of induction of apoptosis. Functional inactivation of endogenous TGFbeta revealed the existence of an autocrine antiproliferative loop in NET cells. CONCLUSIONS: Neuroendocrine tumour cells of the gastroenteropancreatic tract are subject to paracrine and autocrine growth inhibition by TGFbeta-1, which may account in part for the low proliferative index of this tumour entity.

Interferon-gamma inhibits growth of human neuroendocrine carcinoma cells via induction of apoptosis.

Although biotherapy of gastroenteropancreatic neuroendocrine tumors (NET) provides excellent control for the hypersecretion syndrome, tumor regression is rarely observed, implying the need for novel antiproliferative strategies. Here, we demonstrate that human pancreatic QGP-1 NET cells express functionally intact interferon-gamma (IFN-gamma) receptors and downstream effectors, including the putative tumor suppressor interferon regulatory factor-1 (IRF-1). IFN-gamma treatment profoundly inhibited anchorage-dependent and anchorage-independent growth of QGP-1 cells. Concomitant with the onset of growth inhibition, apoptotic cells were detected in cell cycle analyses of IFN-gamma treated cultures. Apoptosis was confirmed by evaluation of DNA fragmentation and PARP cleavage. Immunoblots of IFN-gamma treated QGP-1 cells revealed a substantial upregulation of caspase-1, followed by the appearance of active proteolytic fragments of caspase-3, suggesting that autocatalytic activation of caspase-1 might initiate the caspase cascade. Apoptosis induction by IFN-gamma was also observed in two of four primary cultures established from tumors of patients with for- and midgut NETs, respectively. Taken together our results characterize IFN-gamma as a potent proapoptotic stimulus in a subset of gastrointestinal NETs and suggest an IRF-1 mediated induction of caspase-1 as a relevant underlying mechanism. Based on these results, the potential of IFN-gamma in experimental biotherapeutic treatment of NETs can be further explored.

Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice.

BACKGROUND AND AIMS: Chromogranin A (CgA) is a multifunctional acidic protein specifically expressed in neuroendocrine cells. In the stomach, CgA is found predominantly in enterochromaffin-like (ECL) cells, where it is regulated by gastrin. We investigated the ability of a promoter fragment comprising 4.8 kb of 5'-flanking DNA of the mouse CgA (mCgA) gene to direct cell-specific expression as well as gastrin responsiveness in the gastroenteropancreatic neuroendocrine system. METHODS: Two independent lines of mCgA 4.8 kb-luc transgenic mice were created. Transgene expression was assessed by determination of luciferase activity and reverse-transcription polymerase chain reaction analysis of luciferase messenger RNA. Cell specificity of transgene expression was investigated by immunohistochemical analysis. The influence of hypergastrinemia on transgene expression was determined after repeated omeprazole injections. RESULTS: In both transgenic lines, mCgA 4.8 kb-luc expression paralleled the expression pattern of the endogenous CgA gene. ECL cells were identified as the major gastric cell population expressing the transgene. Omeprazole treatment stimulated expression of the transgene and the endogenous CgA gene selectivity in the gastric corpus (3-4-fold). CONCLUSIONS: mCgA 5'-flanking DNA (4.8 kb) contain the major cis-regulatory element(s) required for cell-specific CgA expression in the neuroendocrine system and gastrin-responsiveness in the gastric corpus. Further analysis of the CgA promoter in transgenic studies may elucidate the general molecular mechanisms underlying cell-specific gene expression in the gastroenteropancreatic neuroendocrine system.

Interferon gamma inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis.

BACKGROUND AND AIMS: The poor prognosis of pancreatic cancer is partly due to resistance to a broad spectrum of apoptotic stimuli. To identify intact proapoptotic pathways of potential clinical relevance, we characterised the effects of interferon gamma (IFN-gamma) on growth and survival in human pancreatic cancer cells. METHODS: IFN-gamma receptor expression and signal transduction were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation, western blot analysis, and transactivation assays. Effects on cell growth and survival were evaluated in terms of cell numbers, colony formation, cell cycle analysis, DNA fragmentation, and poly(ADP ribose) polymerase (PARP) cleavage. RESULTS: All four pancreatic cancer cell lines examined expressed functional IFN-gamma receptors and downstream effectors, including the putative tumour suppressor interferon regulatory factor 1 (IRF-1). IFN-gamma treatment profoundly inhibited anchorage dependent and independent growth of pancreatic cancer cells. Cell cycle analyses revealed subdiploid cells suggesting apoptosis, which was confirmed by demonstration of DNA fragmentation and PARP cleavage. Time and dose dependency of apoptosis induction and growth inhibition correlated closely, identifying apoptosis as the main, if not exclusive, mechanism responsible for growth inhibition. Apoptosis was preceded by upregulation of procaspase-1 and accompanied by proteolytic activation. Furthermore, the caspase inhibitor z-vad-fmk completely prevented IFN-gamma mediated apoptosis. CONCLUSIONS: These results identify an intact proapoptotic pathway in pancreatic cancer cells and suggest that IRF-1 and/or procaspase-1 may represent potential therapeutic targets to be further explored.

Interferon-alpha delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinases.

The potential antiproliferative effects of interferon-alpha (IFN-alpha) in the treatment of hepatocellular carcinoma (HCC) are controversial, and the growth inhibitory mechanisms remain poorly understood. Therefore, the current study was designed to delineate the molecular mechanisms responsible for direct antiproliferative actions of IFN-alpha in HCC cells. IFN-alpha receptor expression and signal transduction were examined by RT-PCR, immunoprecipitation, Western analysis, and transient transactivation assays. Effects of IFN-alpha on cell growth and cell-cycle distribution were evaluated based on cell numbers and flow cytometry. Composition and activity of cyclin-dependent kinase complexes were determined by immunoblotting and histone-H1-kinase assays. Expression of IFN-alpha receptors was found in all 3 HCC cell lines. IFN-alpha binding initiated phosphorylation of Jak1 and Tyk2 kinases leading to Stat1/Stat2 activation, nuclear translocation, and transactivation of an ISRE-luciferase reporter gene construct. IFN-alpha treatment resulted in a time- and dose-dependent reduction of proliferation. Cell cycle analysis of G1-synchronized, IFN-alpha-treated HCC cells revealed a substantial delay in S-phase progression but no alteration of G1/S-phase transition or evidence of apoptotic cell death. Reflecting the time course of S-phase accumulation, cell cycle-dependent induction of Cyclin A and Cyclin B was impaired, resulting in reduced activity of Cdk2 and Cdc2 kinases. Furthermore, Cdc25C was selectively down-regulated. IFN-alpha treatment inhibits growth of HCC cells by specifically delaying S-phase progression, most likely because of inhibition of Cyclin A induction, resulting in decreased activity of the associated Cdk2 and Cdc2 kinases.

Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia. METHODS: VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transient transfection assays. RESULTS: We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5' flanking region -2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions which was significantly decreased following deletion of the region -1286 and -789 suggesting a substantial contribution of the -975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activation of the VEGF gene. CONCLUSION: These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of VEGF gene transcription and an increase in VEGF mRNA stability.

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.

De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop.

BACKGROUND & AIMS: The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. METHODS: VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. RESULTS: Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. CONCLUSIONS: These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.

A novel function for the tumor suppressor p16(INK4a): induction of anoikis via upregulation of the alpha(5)beta(1) fibronectin receptor.

The tumor suppressor gene p16(INK4a) inhibits the kinase activity of the cyclin-dependent kinase 4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G1 phase of the cell cycle. Recent studies suggested that control of the G1/S boundary might not be the sole biological function of p16(INK4a). We hypothesized that p16(INK4a) might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage dependence. Here we provide evidence that stable transfection of p16(INK4a) restitutes apoptosis induction upon loss of anchorage (anoikis) in a variety of human cancer cells. Anoikis in p16(INK4a)-transfected cells was evidenced by DNA fragmentation and poly(ADP-ribose) polymerase cleavage upon cultivation on polyhydroxyethylmethacrylate-coated dishes and was associated with suppression of anchorage-independent growth as well as complete loss of tumorigenicity. p16(INK4a)-mediated anoikis was due to selective transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin receptor as detected by FACS((R)) analysis, immunoprecipitation, Northern blotting, and nuclear run-on assays. Addition of soluble fibronectin and inhibitory alpha(5) antibodies to nonadherent cells completely abolished p16(INK4a)-mediated anoikis, whereas laminin was ineffective. Furthermore, antisense-induced downregulation of the alpha(5) integrin chain in p16(INK4a)-transfected cells restored resistance to anoikis. These data suggest a novel functional interference between a cell cycle-regulating tumor suppressor gene and membrane-bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.

Activation of protein kinase Calpha inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest.

We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)alpha. PKCalpha subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKC(&agr;) activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCalpha, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKC(&agr;) activation arrested cells in the G(1) phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21(cip1) cyclin-dependent kinase inhibitors. Levels of p21(cip1) remained elevated and CDK2 activity repressed in spite of PKCalpha downregulation, indicating that downstream effectors of PKCalpha are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCalpha-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCalpha results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.

Differential expression of matrix metalloproteinases and their tissue inhibitors in colon mucosa of patients with inflammatory bowel disease.

BACKGROUND/AIMS: Alterations in synthesis and breakdown of extracellular matrix components are known to play a crucial role in tissue remodelling during inflammation and wound healing. Degradation of collagens is highly regulated by a cascade of matrix metalloproteinases (MMPs). The current study was therefore designed to determine gene expression patterns of MMPs and their tissue inhibitors (TIMPs) in single endoscopic biopsies of patients with inflammatory bowel disease (IBD). PATIENTS/METHODS: mRNA expression was measured by quantitative competitive polymerase chain reaction (PCR) in biopsies from patients with ulcerative colitis (n=21) and Crohn's disease (n=21). Protein expression was analysed by western blotting and immunohistochemistry. RESULTS: MMP-2, MMP-14, and TIMP-1 mRNAs were marginally increased in inflamed, but 9-12-fold increased in ulcerated colonic mucosa in IBD whereas TIMP-2 mRNA expression remained unchanged. MMP-1 and MMP-3 mRNA expression correlated well with the histological degree of acute inflammation, resulting in more than 15-fold increased MMP-1 and MMP-3 mRNA levels in inflamed versus normal colon samples from patients with ulcerative colitis and Crohn's disease. CONCLUSION: Profound overexpression of MMP-1 and MMP-3 mRNA transcripts suggests an important role for these enzymes in the process of tissue remodelling and destruction in inflammatory bowel disease.