QGP-1 Cells Research Articles

Src Inhibitors: a Synergic Help for Pancreatic Neuroendocrine Tumors Treatment

Context Pancreatic neuroendocrine tumors (pNETs) are becoming high in prevalence and have started showing a more aggressive behavior. Current therapies for advanced pNETs are inadequate as many patients develop primary or secondary resistance to mTOR (mammalian target of rapamicin) inhibitors, in part due to the activation of escape routes such as the PI3K/AKT pathway. SFKs (Src Family of Kinases) are overexpressed in pNETs and have a central role in controlling cell growth, adhesion and migration, also by regulating the mTOR pathway and the activation of the epithelial growth factor receptor in pNETs. SFK inhibitors are already safely used in patients with other tumors but have never been tested on pNETs. Objective To evaluate the use of SFK inhibitors on pNETs both alone and in combination with mTOR inhibitors. Methods Different dosages of dasatinib and bosutinib were tested in two human pNET cell lines, a pancreatic carcinoid (BON-1) and a somatostatinoma (QGP-1), either alone or in combination with the mTOR inhibitor RAD001 (everolimus). Src activation was assessed by monitoring Y416 phosphorylation. mTOR activation was assessed by testing rp-S6 and 4E-BP1 phosphorylation. Escape pathways were investigated through Akt and eIF4E phosphorylation. MTT and colony formation assays were used to evaluate cell proliferation. Results Dasatinib and bosutinib are effective at inhibiting Src activation and, when administrated together with RAD001, escape pathways are not activated. Extremely low dosages of RAD001 reduce cell proliferation when administered in combination with dasatinib or bosutinib. Colony formation assays show a strong reduction in colony number. BON-1 cells respond better than QGP-1 cells to treatments. Conclusions SFK inhibitors are promising new drug options for pNETs, as they reduce tumor cell proliferation when used in combination with RAD001. They allow to reduce RAD001 dosage, which may lower the risk of developing adverse effects in patients. Additional studies on animal models should be pursued to better assess their potential use in clinical therapies.

Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor

Foregut neuroendocrine tumors [NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor (EGFR) by growth factors, gastrointestinal (GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid,BON, the somatostatinoma QGP-1 and the rat islet tumor,Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr1068 EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs.

QGP-1 cells release 5-HT via TRPA1 activation; a model of human enterochromaffin cells

Recently, we discovered that transient receptor potential ankyrin1 channel (TRPA1) is highly expressed in human and rat enterochromaffin (EC) cells, and those TRPA1 agonists such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA) enhance the release of serotonin (5-hydroxytryptamine; 5-HT) from EC cells in vitro. In this study, QGP-1 cells, a human pancreatic endocrine cell line, were found to highly express TRPA1 and EC cell marker genes, such as tryptophan hydroxylase 1 (TPH1), chromogranin A (CgA), synaptophysin, ATP-dependent vesicular monoamine transporter 1 (VMAT1), metabotropic glutamate receptor 4 (mGluR4), beta1-adrenergic receptor (ADB1), muscarinic 4 acetylcholine receptor (ACM4), substance P, serotonin transporter (SERT), and guanylin. Furthermore, the TRPA1 agonists AITC, CA, and acrolein concentration dependently evoked an increase in intracellular Ca(2+) influx and the release of 5-HT in QGP-1 cells. The effects of these TRPA1 agonists were inhibited by ruthenium red, a TRPA1 antagonist, and TRPA1-specific siRNA. These results indicate that the Ca(2+) influx increase and 5-HT release induced by AITC, CA and acrolein in QGP-1 cells were mediated by TRPA1, and that the QGP-1 cell line could be a new model for the investigation of TRPA1 function in the human EC cell.

Homeobox Gene CDX2 Inhibits Human Pancreatic Cancer Cell Proliferation by Down-Regulating Cyclin D1 Transcriptional Activity

Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity. CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

Src KINASES FAMILY REGULATE THE mTOR PATHWAY IN PANCREATIC ENDOCRINE TUMOUR CELLS

Background: The mTOR pathway is frequently deregulated in cancer. During cell adhesion to the ECM, an increase in cap-dependent mRNA translation occurs. Aim: Given the important role played by Src kinases family in pancreatic endocrine tumour (PET) cell adhesion and spreading, we set out to investigate if these kinases modulate the activation of mTOR, during PET cell adhesion. Methods: We have used, as model system, the pancreatic endocrine tumour cell line QGP-1, derived by somatostatinoma. To avoid interference by serum, we performed all the adhesion experiments in absence of FBS. Results: We observed a reduction of the rate of protein synthesis during adhesion of QGP-1 cells on FBS-pre-coated dishes in the presence of the Src kinases inhibitor, PP2. Monitoring the phosphorylation status of the ribosomal protein S6 and 4EBP1 we demonstrated that PP2 treatment strongly reduced mTOR pathway activation during QGP-1 adhesion. Inhibition of Src kinases also reduced the formation of the mRNA translation initiation complex in QGP-1 cells. On these bases, we carried out a microarray analysis on mRNAs purified from polysomal fraction from QGP-1 treated or not with PP2, evidencing a group of genes translationally regulated by Src activity. Conclusions: These results point out a novel mechanism of regulation of translation and suggest a new approach for PETs therapies with mTOR inhibitors.

Aurora kinases as novel drug targets in gastroenteropancreatic neuroendocrine tumor disease: Antiproliferative and pro-apoptotic effects of ZM 447439, a new aurora kinase inhibitor, in BON and QGP-1 cells

22023 Background: The serine/threonine protein kinases aurora A and B, key regulators of mitosis, are emerging as novel drug targets for cancer treatment. Aurora B overexpression has been previously documented by immunohistochemistry in several types of human tumors. In gastroenteropancreatic neuroendocrine tumors (GEP-NETs) the therapeutic possibilities are still not satisfactory. Therefore, we assessed aurora B expression in a series of well-differentiated GEP-NETs and studied the effects of a novel aurora kinase A and B inhibitor (ZM 447439) in our well-differentiated human GEP-NET cell lines BON and QGP-1. Materials and Methods: Tumor specimens from 102 patients (40 foregut, 55 midgut, 7 hindgut) were studied immunohistochemically for aurora kinase expression. The cell lines BON and QGP-1 were treated with increasing concentrations of ZM 447439 (Tocris) with or without coincubation with Streptozocin, Doxorubicin, Octreotide and SOM-230. Growth inhibition was measured with crystal violet assays, apoptosis induction with caspase 3/7 activation and DNA fragmentation. Cell cycle analysis was evaluated by performing FACS analysis. Results: The immunohistochemical analysis of aurora B in our patient collective demonstrated an overexpression in a significant portion of tumors, compared with normal epithelium. Moreover, we demonstrate that ZM 447439 inhibited dose-dependently proliferation of BON and QGP-1 cells. Treatment with ZM 447439 induced apoptosis and dose-dependently led to a cell cycle arrest. In addition, combined treatment with the somatostatin analogues Octreotide and SOM-230 in BON cells and with Streptozocin and Doxorubicin in QGP-1 cells, ZM 447439 augmented significantly the antiproliferative effects of Octreotide, Streptozocin and Doxorubicin, but not in combination with SOM-230. Conclusion: Our results suggest that aurora B may represent a valid target in well differentiated GEP- NETs, since aurora B is overexpressed in these tumors, and aurora kinase inhibition by ZM 447439 seems to be a promising new therapeutic approach, which should be evaluated in further clinical trials. No significant financial relationships to disclose.

Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1

We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O 2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene ( DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.

Src family kinase activity regulates adhesion, spreading and migration of pancreatic endocrine tumour cells

Pancreatic endocrine tumours (PETs) are rare and 'indolent' neoplasms that usually develop metastatic lesions and exhibit poor response to standard medical treatments. Few studies have investigated pathways responsible for PET cell growth and invasion and no alternative therapeutic strategies have been proposed. In a recent microarray analysis for genes up-regulated in PETs, we have described the up-regulation of soluble Src family tyrosine kinases in this neoplasia, which may represent potentially promising candidates for therapy. Herein, we have investigated the expression and function of Src family kinases in PETS and PET cell lines. Western blot analysis indicated that Src is highly abundant in the PET cell lines CM and QGP-1. Immunohistochemistry and Western blot analyses showed that Src is up-regulated also in human PET lesions. Pharmacological inhibition of Src family kinases by the specific inhibitor PP2 strongly interfered with adhesion, spreading and migration of PET cell lines. Accordingly, the actin cytoskeleton was profoundly altered after inhibition of Src kinases, whereas even prolonged incubation with PP2 exerted no effect on cell cycle progression and/or apoptosis of PET cells. A transient increase in tyrosine phosphorylation of a subset of proteins was observed in QGP-1 cells adhering to the plate, with a peak at 75 min after seeding, when approximately 80% of cells were attached. Inhibition of Src kinases caused a dramatic reduction in the phosphorylation of proteins with different molecular weight that were isolated from the cell extracts by anti-phosphotyrosine immunoprecipitation or pull-down with the SH2 domain of Src. Among them, the docking protein p130Cas interacted with Src and is a major substrate of the Src kinases in QGP-1 cells undergoing adhesion. Our results suggest that Src kinases play a specific role during adhesion, spreading and migration of PET cells and may indicate therapeutical approaches directed to limiting the metastatic potential of these cells.

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription‐starting exons and the involvement of Sp1 in its transcriptional regulation

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

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.

Molecular mechanism of interferon alfa–Mediated growth inhibition in human neuroendocrine tumor cells

Background & Aims: Although human neuroendocrine tumors respond to interferon (IFN)-α 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-α in the human neuroendocrine tumor cell lines BON and QGP1. Methods: IFN-α 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-α 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-α treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-α–treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G 1 synchronized QGP1 cells. IFN-α–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-α 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. GASTROENTEROLOGY 2000;118:735-748

Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen.

We have investigated the post-translational modification of carcinoembryonic antigen (CEA) for membrane-anchoring in QGP-1 cells derived from a human pancreatic carcinoma. Pulse-chase experiments with [3H]leucine demonstrated that CEA was initially synthesized as a precursor form with Mr 150,000 having N-linked high-mannose-type oligosaccharides, which was then converted to a mature form with Mr 200,000 containing the complex type sugar chains. The mature protein thus labeled was found to be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that CEA is a phosphatidylinositol-linked membrane protein. This was confirmed by metabolic incorporation into CEA of 3H-labeled compounds such as ethanolamine, myo-inositol, palmitic acid, and stearic acid. The 3H-labeled fatty acids incorporated were specifically removed from the protein by nitrous acid deamination as well as by phosphatidylinositol-specific phospholipase C treatment. Since the available cDNA sequence predicts that CEA contains a single methionine residue only in its carboxyl-terminal hydrophobic domain, processing of the carboxyl terminus was examined by pulse-chase experiments with [35S]methionine. It was found that CEA with Mr 150,000 was initially labeled with [35S]methionine but its radioactivity was immediately lost with chase. Taken together, these results suggest that CEA is anchored to the membrane by simultaneously occurring proteolysis of the carboxyl terminus and replacement by the glycophospholipid immediately after the synthesis.

Coproduction of Carcinoembryonic Antigen and Nonspecific Cross-Reacting Antigen by a Continuous Cell Line From a Human Pancreatic Tumor<xref ref-type="fn" rid="fn2">2</xref>

A simultaneous production of nonspecific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) by the same individual cells of an established human pancreatic cell line (QGP-1) was demonstrated by the immunoperoxidase method. Kinetics of cell proliferation and production of CEA and NCA were analyzed, and active synthesis of both antigens was found to be accompanied with the active proliferation of cultured cells. Both antigens in culture medium were purified by immunoadsorption and gel filtration. Immunochemical studies confirmed that CEA and NCA produced by the QGP-1 cells had properties identical to those of authentic CEA derived from metastatic colorectal carcinoma and to those of NCA from normal lungs, respectively.