The proline TP53 variant stimulates likely lymphangiogenesis in an orthotopic mouse model of pancreatic cancer.

BACKGROUND: Pancreatic cancer is a deadly disease characterised by high incidence of TP53 mutations. Restoration of TP53 function is perceived as a highly attractive therapeutic strategy, whose effects are not well characterised. METHODS: The current work adapted an inducible strategy of stage-specific reexpression of wild-type (wt) TP53 in an in vivo orthotopic mouse model of pancreatic cancer. RESULTS: The reconstitution of wt TP53 function in TP53-mutant DanG and MiaPaCa-2 cells caused G1 cell cycle arrest but no evidence of apoptosis induction. Consistent with subcutaneous xenograft models, we found that wt TP53 reduced primary tumour growth. Wt TP53 reexpression during early tumour growth led to significant increase in vascularisation. This correlated with an unexpectedly high rate of micro-metastases in lymph nodes of animals with wt TP53 induction, despite the 90% decrease in median primary tumour weight. Reexpression of wt TP53 later in tumour development did not significantly affect the number of CD31-reactive vessels, but increased lymphatic vessel density. CONCLUSION: The increased number of lymphatic vessels and micro-metastases suggests that wt TP53 induction complexly affected the biology of different tumour constituents of pancreatic cancer. Our observation suggests that combination of the inducible system with an orthotopic model can yield important insights into in vivo pancreatic cancer biology.

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.

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.

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.

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.

Molecular mechanism of interferon alfa-mediated growth inhibition in human neuroendocrine tumor cells.

BACKGROUND & AIMS: Although human neuroendocrine tumors respond to interferon (IFN)-alpha treatment in vivo, the underlying mechanisms of growth inhibition are poorly understood. To characterize the antiproliferative effects at a molecular level, we explored the growth-regulatory action of IFN-alpha in the human neuroendocrine tumor cell lines BON and QGP1. METHODS: IFN-alpha receptor expression and signal transduction were examined by reverse-transcription polymerase chain reaction, immunoblotting, subcellular fractionation, and transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. Expression and activity of cell cycle-regulatory molecules were determined by immunoblotting and histone H1-kinase assays. RESULTS: Both cell lines expressed IFN-alpha receptor mRNA transcripts. Ligand binding initiated phosphorylation of Jak kinases and Stat transcription factors, resulting in Stat activation, nuclear translocation, and transcription from an ISRE-reporter construct. Prolonged IFN-alpha treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-alpha-treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G(1) synchronized QGP1 cells. IFN-alpha-treated cells entered S phase in parallel to control cultures, but their progress into G(2)/M phase was delayed. Both cellular cyclin B levels and CDC 2 activity were substantially reduced. The extent and time course of this reduction corresponded to the observed S-phase accumulation. CONCLUSIONS: IFN-alpha directly inhibits growth of human neuroendocrine tumor cells by specifically delaying progression through S phase and into G(2)/M. These cell cycle changes are associated with inhibition of cyclin B expression, resulting in reduced CDC2 activity.

Stimulation of both CCK-A and CCK-B receptors activates MAP kinases in AR42J and receptor-transfected CHO cells.

It was recently found that cholecystokinin (CCK) activates mitogen-activated protein kinases (MAPK) in isolated rat pancreatic acini. The present study evaluates whether one or both types of CCK receptors are capable of MAPK activation in pancreatic AR42J acinar cells as well as CHO cells transfected with CCK-A or CCK-B receptors. CCK significantly increased p44 MAPK and p42 MAPK activities in AR42J cells. Minimal, half-maximal, and maximal responses were observed at 30 and 500 pM and 10 nM, respectively, after CCK-8 stimulation and at 100 pM and 1.5 and 30 nM, respectively, after gastrin stimulation. Glycine-extended gastrin had no effect at 100 nM and a small but significant effect at 1 microM. The CCK-B receptor antagonist L365,260 almost totally blocked MAPK activation in AR42J cells after stimulation with gastrin and glycine-extended gastrin and substantially reduced the activation of both kinases by CCK-8, while the CCK-A receptor antagonist L364,718 was much less effective. The CCK-A-selective agonist A71376, however, was an effective stimulant of MAPK activity. In an alternative approach, stably transfected CHO cells bearing either CCK-A or CCK-B receptors were stimulated with CCK-8. Each receptor induced a time-dependent increase in activity of both MAPKs by five- to sixfold in CCK-A- and CCK-B-bearing cells. In conclusion, both CCK-A and CCK-B receptors activate MAPK in AR42J cells and in transfected CHO cells.