Human Carcinoid Tumor Research Articles

Human bronchial carcinoid tumor initiating cells are targeted by the combination of acetazolamide and sulforaphane

BackgroundBronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior.MethodsBronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids.ResultsData demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice.ConclusionsHuman bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.

An autoregulatory network between menin and pri-miR-24-1 is required for the processing of its specific modulator miR-24-1 in BON1 cells.

Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary cancer complex syndrome manifesting a variety of endocrine and non-endocrine neoplasms and lesions. MEN1 is characterized by tumours of the parathyroids, of the neuroendocrine cells of the gastroenteropancreatic tract, and of the anterior pituitary. The MEN1 gene, a tumour suppressor gene, encodes the menin protein. Loss of heterozygosity (LOH) at 11q13 is typical of MEN1 tumours in agreement with Knudson's two-hit hypothesis. We previously showed that the MEN1 parathyroid tumorigenesis is under the control of an "incoherent feedback loop" between miR-24-1 and the menin protein that generates a "Gene Regulatory Network" (GRN) that mimics the second hit of Knudson's hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Here we show, in the BON1 cell line derived from lymphnode metastasis of a human carcinoid tumour of the pancreas, that menin binds specifically to the primary RNA sequence pri-miR-24-1 by promoting the miR-24-1 biogenesis. Network simulation showed a new feed-forward loop between menin, microRNA-24-1 and Musashi-1 proteins. This result shows a novel mechanism whereby menin, a RNA-binding protein, facilitates the processing of its specific miRNA by regulating the dynamics of the menin-miR-24 Gene Regulatory Network at the level of pri-miRNA processing.

Abstract 3050: Human rectal carcinoid tumors highly express DCLK1, a putative cancer stem cell marker

Abstract Background and Aim: Doublecortin-like kinase 1 (Dclk1) is known to regulate neuronal differentiation, migration, and neurogenesis (Shu et al., Neuron 2006). Accumulating evidence suggests that the protein is a putative marker for intestinal and pancreatic stem cells, including cancer stem cells (CSCs) of those organs (Gagliardi et al., Pathol Res 2012, Nakanishi et al., Nat Genet 2013, Bailey et al., Gastroenterology 2013). Since carcinoid tumor is defined as a neuroendocrine tumor, we assessed whether or not the Dclk1 protein, a marker of CSC, was expressed in human rectal carcinoid tumor tissues. Patients and Methods: Eighteen patients (9 male, 9 female; mean age, 51) with rectal carcinoid tumors were included in this study. Informed consent was obtained from all of the patients. The tumors were safely resected by endoscopic mucosal resection (EMR) method between 2003 and 2012 in the Kurume University Hospital. Mean diameter of the tumors was 5.4 mm (range, 2.0-8.0 mm). Immunohistochemical staining was performed to examine the Dclk1 protein expression, accompanied by the staining for the commonly-used neuroendocrine markers synaptophysin, chromogranin A, and CD56. The immune-reactivity was evaluated according to the following criteria: the intensity of staining was scored in each specimen on a scale of 0 to 3, in which 0 = negative staining, 1 = weakly positive staining, 2 = moderately positive staining, and 3 = strongly positive staining. The extent to which positive cells were seen in each specimen (‘‘extent of distribution’’ of positive cells) was estimated on a scale of 0 to 2, in which 0 = positive staining in 0% to 20% of cells; 1, in 21% to 60%; and 2, in 61% to 100%. Sum of the scores was calculated for each specimen. Results: The total score for Dclk1, synaptophysin, CD56, and chromogranin A was 87/90, 86/90, 60/90, and 34/90, respectively. Conclusions: Human rectal caricinoid tumor highly expressed Dclk1, and the high expression rate indicated that the protein expression was a novel feature for the neuroendocrine tumor. It is suggested that Dclk1 is a potential marker of not only CSCs but also non-epithelial tumors, such as carcinoid tumor, regulating crucial tumor cell behaviors. Citation Format: Yu Ikezono, Hironori Koga, Mitsuhiko Abe, Takafumi Yoshida, Toru Nakamura, Jun Akiba, Hirohisa Yano, Osamu Tsuruta, Takuji Torimura, Michio Sata. Human rectal carcinoid tumors highly express DCLK1, a putative cancer stem cell marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3050. doi:10.1158/1538-7445.AM2014-3050

Amperometric detection of vesicular exocytosis from BON cells at carbon fiber microelectrodes

Amperometry at ultramicroelectrodes is nowadays a routine analytical technique for quantitative real-time monitoring of vesicular exocytosis at single cell levels. The method performance and ease of application stem from the fact that it simply involves an electrochemical oxidation at a microelectrode surface of neurotransmitters released by a given cell. The exocytotic fluxes are thus converted into a current and appear as a succession of amperometric spikes, whose frequency and shapes are particularly informative about the dynamics of the release process, while their integrals give a direct measure of the quantity of molecules released. In this article, we wish to evaluate and describe the amperometric detection of exocytotic release by BON cells, a cell line derived from a metastatic human carcinoid tumor of the pancreas and recently involved in combined approaches of exocytosis (particularly fluorescence/electrochemistry). The whole exocytotic activity (frequency, spike magnitude) of BON cells is rather weak by comparison with those observed with others cell models like chromaffin cells but remain suitable for amperometric investigations.

Abstract 5212: Akt signaling enhances carcinoid cell secretion, migration, and invasion

Abstract Carcinoid tumors are slow growing neuroendocrine tumors characterized by the secretion of peptides and biogenic amines such as serotonin (5-HT) and TGF-β. Although relatively indolent, carcinoid tumors of the GI tract can metastasize to the liver and release secretory products into the systemic circulation resulting in the carcinoid syndrome. PI3K/Akt signaling is increased in carcinoid tumors and is hypothesized to promote carcinoid progression. The purpose of our current study was to delineate the role of Akt signaling in carcinoid cell secretion, migration, and invasion using the BON cell line, derived from a human pancreatic carcinoid tumor. Methods. i.) BON cells were transfected with either non-targeting control shRNA (BON shControl) or shRNA to PTEN (BON shPTEN), the natural inhibitor of Akt activation. Conditioned media was collected from BON shControl and shPTEN cells and assessed for 5-HT and TGF-β by ELISA. Western blots were performed to assess changes in the expression of 5-HT and TGF-β. ii.) Scratch and Boyden chamber assays were performed to compare the migration and invasion, respectively, of BON shControl and shPTEN cells. The protein levels of the epithelial-to-mesenchymal transition (EMT) markers E-cadherin, a cell adhesion protein, and snail were assessed in both cell groups with western blot. Cells were injected into the pancreas of athymic nude mice and liver metastases were assessed via immunofluorescence. Results. i.) Knockdown of PTEN increased the secretion of 5-HT and TGF-β from BON cells. Compared to control, lysates from BON shPTEN cells displayed increased expression of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, and TGF-β. ii.) Compared to BON shControl cells, BON shPTEN cells exhibited a 3.2- and a 1.6-fold increase in migration at 24 h and 48 h, respectively. At 24 h BON shPTEN cells displayed an 8-fold increase in invasion, while at 48 h a 2.6-fold increase in invasion was noted compared to BON shControl cells. Knockdown of PTEN in BON cells significantly decreased E-cadherin expression, whereas snail expression was increased. BON shPTEN and BON shControl cells formed liver metastases in 60% and 10% of mice, respectively. Conclusions. The Akt pathway enhances carcinoid cell secretion and metastatic potential by increasing expression and synthesis of secretory products and by altering expression of EMT markers, respectively. In addition to promoting metastasis, Akt signaling may also contribute to carcinoid syndrome. These results suggest that targeting Akt signaling may provide a dual strategy to decrease peptide secretion and inhibit carcinoid cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5212.

Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

Overactivation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Human carcinoid BON cells were treated in vitro with LY294002, a PI3-kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for 6 days. The effect of LY294002 or Akt1 siRNA treatment was assessed by Western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (26%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease.

Hydrodynamic Extrusion Of Membrane Nanotubes From Neuroendocrine Bon Cells: Role Of Membrane Trafficking

Dynamics of extrusion of membrane nanotubes from giant vesicles (GUVs) and red blood cells (RBCs) are well understood. In GUVs, extrusion dynamics (tether length as a function of time) are governed by the membrane tension, whereas for RBCs adhesion of the plasma membrane (PM) to the cytoskeleton dominates. In the case of cells with endomembranes extrusion dynamics are complicated by lipid trafficking to and from the PM. Constitutive fusion of vesicles with the PM (exocytosis) continuously adds membrane to the PM while the reverse process of membrane retrieval (endocytosis) helps maintaining a stable steady-state cell surface area. In addition, some cells are capable of calcium-regulated secretion in which specialized secretory vesicles await a trigger signal for undergoing rapid fusion with the PM within seconds. The excess membrane added to the PM is retrieved via compensatory endocytosis on a longer time-scale (minutes). We have been using endocrine BON cells, a cell line derived from a human carcinoid tumor, which are capable of calcium-regulated secretion. Secretion is stimulated, almost instantly during tether extrusion, by UV-uncaging of a photolabile calcium chelator loaded into the cytosol. Thus, a significant transient membrane addition to the PM can be turned on at will and the consequences for extrusion dynamics can be studied.We observe mainly two types of response upon stimulation: a sudden jump in tether length L of ∼10 μm followed by an increased extrusion velocity, or a sudden increase of slope. Both responses are consistent with exocytotic addition of area to the PM. However, only a fraction of such responses are well-correlated with UV-uncaging of intracellular calcium. This suggests exocytosis, or other mechanisms of membrane addition to the PM may independently be triggered by other means, possibly by tether pulling itself.

Carotid Body Tumor: Review of the Literature and Report of a Case With a Rare Sensorineural Symptomatology

lanreotide of patients with advanced neuroendocrine gastrointestinal tumors: Clinical and biological effects. Ann Oncol 8:1041, 1997 31. Oberg K, Eriksson B: The role of interferons in the management of carcinoid tumors. Acta Oncol 30:519, 1991 32. Kaltsas GA, Papadogias D, Makras P, et al: Treatment of advanced neuroendocrine tumours with radiolabeled somatostatin analogues. Endocr Relat Cancer 12:683, 2005 33. Nilsson O, Kolby L, Bernhardt P, et al: GOT1 xenografted to nude mice: A unique model for in vivo studies on SSTR-mediated radiation therapy of carcinoid tumors. Ann N Y Acad Sci 1014:275, 2004 34. Kolby L, Bernhardt P, Johanson V, et al: Successful receptormediated radiation therapy of xenografted human midgut carcinoid tumour. Br J Cancer 93:1144, 2005 35. Kvols LK, Reubi JC, Horisberger U, et al: The presence of somatostatin receptors in malignant neuroendocrine tumor tissue predicts responsiveness to octreotide. Yale J Biol Med 65:505, 1992 36. Chakravarthy A, Abrams RA: Radiation therapy in the management of patients with malignant carcinoid tumors. Cancer 75:1386, 1995 37. Schupak KD, Wallner KE: The role of radiation therapy in the treatment of locally unresectable or metastatic carcinoid tumors. Int J Radiat Oncol Biol Phys 20:489, 1991

A Novel Chromogranin-A Promoter-Driven Oncolytic Adenovirus for Midgut Carcinoid Therapy

The use of replication-selective oncolytic adenoviruses is an emerging therapeutic approach for cancer, which thus far has not been employed for carcinoids. We therefore constructed Ad[CgA-E1A], a novel replication-selective oncolytic adenovirus, where the chromogranin A (CgA) promoter controls expression of the adenoviral E1A gene. The Ad[CgA-E1A] virus was evaluated for E1A protein expression, replication ability, and cytolytic activity in various cell lines. It was also evaluated for treatment of xenografted human carcinoid tumors in nude mice. To use Ad[CgA-E1A] for the treatment of carcinoid liver metastases, it is important that normal hepatocytes do not support virus replication to minimize hepatotoxicity. We therefore evaluated CgA protein expression in normal hepatocytes. We also evaluated CgA gene expression in normal hepatocytes and microdissected tumor cells from carcinoid metastases. We found that Ad[CgA-E1A] replicates similarly to wild-type virus in tumor cells with neuroendocrine features, including the BON carcinoid cell line and the SH-SY-5Y neuroblastoma cell lines, whereas it is attenuated in other cell types. Thus, cells where the CgA promoter is active are selectively killed. We also found that Ad[CgA-E1A] is able to suppress fast-growing human BON carcinoid tumors in nude mice. Furthermore, CgA is highly expressed in microdissected cells from carcinoid metastases, whereas it is not expressed in normal hepatocytes. Ad[CgA-E1A] is an interesting agent for the treatment of carcinoid liver metastases in conjunction with standard therapy for these malignancies.

Carcinoid tumor cell growth suppression by PI3 Kinase-Akt pathway inhibition

Introduction: The PI3 Kinase-Akt signaling pathway is known to promote tumor cell survival by suppressing pro-apoptotic proteins. We have previously shown that human carcinoid tumor cells utilize several signal transduction pathways to modulate cellular growth and neuroendocrine (NE) marker production. Furthermore, human carcinoid cells have endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that the inhibition of the PI3 Kinase-Akt pathway may lead to a reduction of carcinoid tumor cell growth and NE marker production. Methods: Human gastrointestinal (GI) carcinoid BON cells were treated with varying concentrations of the PI3 kinase inhibitor LY294002 (0-100 μM) for a two-day time period. Western analyses were performed for active, phospho-Akt, total Akt, and the NE marker human achaete-scute homolog1 (hASH1). Cellular growth was measured both by MTT assay and cell counts for 8 days. Results: Treatment of BON cells with LY294002 led to a dose-dependent reduction in the levels of the active, phosphorylated Akt, illustrating the successful inhibition of the PI3 Kinase-Akt pathway. Moreover, LY294002 significantly suppressed carcinoid tumor cell growth and production of hASH1. Importantly, these reductions in cellular growth and NE marker production were directly proportional to the degree of Akt inhibition. Conclusion: Human carcinoid tumor cell growth and hASH1 production appears to be dependent upon activation of the PI3 Kinase-Akt signaling pathway. Therefore, inhibition of this important signal transduction pathway could be a potential therapeutic strategy to treat and palliate patients with carcinoid tumors.

Galectin-4 Expression in Carcinoid Tumors

Galectins (Gal) are an evolutionarily conserved family of 15 carbohydrate-binding proteins (lectins) that are widely distributed in normal and neoplastic cells in a wide range of organisms. They have roles in inflammation, cell adhesion, tumor progression, and metastasis. The function and distribution of Gal-3 and Gal-1 are well characterized, but less information is available about Gal-4. Recent studies have localized Gal-4 in the enterochromaffin cells of the porcine and murine small intestine. We examined the expression of Gal-4 in primary and metastatic human ileal carcinoid tumors as well as in carcinoid tumors of the stomach, lung, and rectum. A total of 44 primary and 42 ileal metastatic carcinoid tumors were examined by immunohistochemistry using tissue microarrays (TMA) with monoclonal antibodies to Gal-4, Gal-3, and Gal-1. Pulmonary (n = 7), rectal (n = 6), and gastric (n = 6) carcinoids were examined with larger tissue sections. A total of 18 pancreatic neuroendocrine tumors were also examined with larger tissue sections. Western blots of three ileal carcinoids were also done. Gal-4 was most highly expressed in the ileal carcinoids and the levels of expression tended to be higher in primary ileal carcinoids compared to the metastatic tumors (p = 0.069). All 18 pancreatic neuroendocrine tumors were negative for Gal-1, Gal-3, and Gal-4. Western blot showed a 32 kDa band for Gal-4 in the ileal carcinoids. Gal-3 and Gal-1 were not detected in the metastatic ileal carcinoids by Western blotting. Gastric carcinoids also expressed Gal-4, but very few pulmonary or rectal carcinoids were positive for Gal-4 (p = 0.002). Lower levels of Gal-1 and Gal-3 expression were present in ileal carcinoids compared to primary pulmonary and rectal tumors. These results show a differential distribution of Gal-4 in carcinoid tumors in different locations of the gastrointestinal tract and the lungs.

Different expression levels of lumican in human carcinoid tumor and neuroendocrine cell carcinoma

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family and participates in the maintenance of tissue structures and tumor growth. Neuroendocrine cell tumors (NETs) including carcinoid tumors and NE cell carcinomas (NECs) possess numerous neuroendocrine (NE) granules, and differences between these tumors are in terms of their biological and metastatic aggressiveness during tumor progression. The purpose of this study was to examine the expression of lumican in NETs, and to determine whether the presence of lumican may be associated with the growth of NETs by comparing its expression between carcinoid tumors and NECs. Immunohistochemically, the positivity rates of lumican expression in the cytoplasm of the tumor cells were 87.5% in carcinoid tumors and 37.5% in NECs. Those of lumican expression in the stroma adjacent to the tumors were 90.1% in carcinoid tumors and 79.2% in NECs. In situ hybridization analysis revealed the lumican mRNA expression in the cytoplasm of carcinoid tumor and NEC cells. Ultrastructurally, the lumican protein was observed in the rough endoplasmic reticulum and NE granules of NETs and interspaces of collagen fibers. Furthermore, RT-PCR analysis revealed the presence of lumican mRNA in NEC cell lines. These results indicate that the higher expression level of cytoplasmic lumican in carcinoid tumors than in NECs may play a role in the slow growth of these tumors.

Identification and characterization of DISP3 gene in silico

Lipid-modified soluble proteins Hedgehog (SHH, DHH and IHH) and WNT (WNT1, WNT2, WNT2B, WNT3, etc.) share distantly related mechanisms for ligand modification as well as for signaling through seven-transmembrane protein with Frizzled domain. Hedgehog and WNT signaling pathways network together during embryogenesis and carcinogenesis. Dispatched 1 (DISP1) and Dispatched 2 (DISP2) are human homologs for Drosophila Dispatched implicated in the release of lipid-anchored Hedgehog from producing cells. Here, we identified and characterized Dispatched 3 (DISP3) gene by using bioinformatics. DISP3 complete coding sequence was determined by assembling BU170953 EST and KIAA1337 uncharacterized cDNA. DISP3 gene at human chromosome 1p36.22 was linked to D1S2667 microsatellite maker and TERE1 gene, whose locus is associated with prostate cancer, bladder cancer, and liver cancer. DISP3 mRNA was expressed in human embryonic stem (ES) cells, brain, testis, lung carcinoid, neuroblastoma, retinoblastoma and brain tumor. DISPH1 domain with five transmembrane regions (codon 452-637 of DISP3) and DISPH2 domain with four transmembrane regions (codon 1116-1319 of DISP3) were identified as novel domains conserved between DISP3 (1392 aa) and DISP1. The region around DISPH1 and DISPH2 domains of DISP3 protein was the Patched homologous region conserved among Patched family members and DISP family members. Because DISP3 and DISP1 are multi-span transmembrane proteins with the Patched homologous region, DISP3 is predicted to be implicated in the release of lipid-anchored secreted proteins. This is the first report on identification and characterization of the DISP3 gene.

Neurotrophin system expression in human pulmonary carcinoid tumors

Neurotrophin (NT) and NT receptor expression was assessed in 12 typical (TC) and 8 atypical (AC) human pulmonary carcinoids by Western blot and immunohistochemistry. TC and AC carcinoid express to different extent NT and NT receptor proteins. Nerve growth factor (NGF) was expressed by 83% of the TC but not by the AC carcinoids. Brain derived neurotrophic factor (BDNF) was expressed by 33 and 100% of TC and AC carcinoids, respectively. NT-3 was expressed by 58% of the TC and 38% of AC carcinoids. TC carcinoids express high affinity NT receptors while 50% of the AC carcinoids express the TrkB receptor. Our results demonstrate that NGF/TrkA and BDNF/TrkB signaling need to be considered as regulatory pathways that may address survival, differentiation and/or aggressiveness of human pulmonary carcinoids. Contrarily to the BDNF/TrkB, expression of the NGF/TrkA signaling may overcome aggressiveness of carcinoid cells. NTs may be useful as markers in the clinic.

Insulin-like growth factor 1 signaling in human gastrointestinal carcinoid tumor cells

Background Insulin-like growth factor 1 (IGF-1) is an autocrine regulator of carcinoid tumors. Blockade of IGF-1 signaling has been proposed as a therapeutic target in the treatment of patients with carcinoid syndrome. We hypothesized that the induction of parallel raf-1/MEK1 pathways will block IGF-1-mediated chromogranin A (CgA) maintenance. Methods Human gastrointestinal carcinoid tumor cells (BON) were treated with IGF-1 (0-500 ng/mL). Raf-1/MEK1 activation was achieved with an estrogen-inducible raf-1 vector that was transduced into BON cells. Activation of IGF-1/raf-1 pathways was determined by phosphorylation of downstream targets p70s6 and ERK1/2. The secreted and intercellular levels of CgA were measured in conditioned media and whole cell extracts by Western and enzyme-linked immunosorbent assay analysis. Results IGF-1 and raf-1 pathways were activated successfully in BON cells, as shown by high levels of phosphorylated p70s6 and phosphorylated ERK1/2, respectively. Treatment of BON cells with IGF-1 stimulated the release of CgA, while high intracellular CgA levels were maintained. The activation of raf-1/MEK1 reversed the effect of IGF-1 treatment by the depletion of intracellular CgA. Conclusions The induction of the raf-1/MEK1 pathway blocks IGF-1-mediated intracellular neuroendocrine hormone regulation. Therefore, raf-1/MEK1 activation may be a viable method to block IGF-1-mediated cellular effects and serve as a therapeutic target in gastrointestinal carcinoid tumors.

Biokinetics of 111In-DTPA-D-Phe1-octreotide in nude mice transplanted with a human carcinoid tumor

The long time biokinetics of the radiolabeled somatostatin analogues 111In-DTPA-D-Phe1-octreotide was studied in nude mice transplanted with the human carcinoid tumor, GOT1. The results were compared with those from the patient with the original tumor. This patient has been diagnosed and later treated with 111In-DTPA-D-Phe1-octreotide. The animals received about 2 MBq 111In-DTPA-D-Phe1-octreotide (0.1 μg) by injection into a tail vein. The animals were killed 0.5 h-14 d after injection of the radiopharmaceutical. Tumor tissue and normal tissues were collected and weighed and measured for 111In activity. The 111In uptake in the tumor was higher than in all normal tissues except the kidneys. The tumor-to-normal-tissue activity concentration, TNC, increased with time for all normal tissues studied. These data were similar to those observed for the original tumor in the patient. The similar biokinetics for 111In-DTPA-D-Phe1-octreotide in the tumor-bearing mice and the patient makes this animal model suitable as a model for evaluation of therapy of somatostatin receptor (sstr) expressing tumors with radiolabeled somatostatin analogues. Furthermore, the increase with time of TNC both in mice and the patient indicates that long-lived radionuclides are preferred for therapy with radiolabeled somatostatin analogues.

Biokinetics of 111In-DTPA-D-Phe1-octreotide in nude mice transplanted with the human carcinoid tumour cell line GOT1

s: Abstracts of an International Conference on Peptide Radiopharmaceuticals in Diagnosis and Therapy Rome, Italy, 25-28 May 2000: Poster Presentations

Differential expression of Jun protooncogenes in human intestinal carcinoid tumors

Differential expression of Jun protooncogenes in human intestinal carcinoid tumors

A novel cytotoxic agent for human carcinoid tumors

Background: Conventional adjuvant therapy for advanced carcinoid tumors remains disappointing; novel therapeutic agents are needed. We have shown previously that inhibiting polyamine biosynthesis with αdifluoromethylornithine (DFMO) slows the growth of carcinoid tumors. However, the clinical utility of DFMO has been limited by its cytostatic property. Synthetic polyamine analogs such as 1,19-bis(ethylamino)-5,10,15-triaztanonadecane (BE-4-4-4-4) appear to be cytotoxic against several human tumors. The purpose of our study was to determine whether BE-4-4-4-4 is a more effective antiproliferative and cytotoxic agent than DFMO on human carcinoid (BON) cells in vitro. Methods: BON cells were treated with either 5 mmol/L DFMO, 0.5 to 10 μmol/L BE-4-4-4-4, or vehicle (control). Ornithine decarboxylase activity was determined by the rate of 14CO 2 production, and intracellular polyamine levels were determined by chromatography. Cell number and viability were determined by Coulter counter and trypan blue exclusion, respectively. Results: BE-4-4-4-4 inhibited ornithine decarboxylase activity and depleted all 3 polyamines. BE-4-4-4-4 decreased cell numbers by 81% compared with control and 27% compared with DFMO. BE-4-4-4-4 also induced a 2-fold increase in cell death compared with control or DFMO. Conclusions: BE-4-4-4-4 is cytotoxic and more effective than DFMO in inhibiting growth of BON cells. Polyamine analogs such as BE-4-4-4-4 may be effective adjuvant therapeutic agents for advanced carcinoid tumors. (Surgery 1998;124:1071-6.)

Interferon-mediated activation of the STAT signaling pathway in a human carcinoid tumor.

Growth inhibition of human cancers (e.g., endocrine tumors) by interferons (IFNs) has been demonstrated, but the exact cellular mechanisms remain largely undefined. IFNs and other cytokines can activate novel Stat (Signal transducers and activators of transcription) proteins, which translocate to the nucleus to activate target genes. The purpose of this study was to determine the effect of IFN-alpha and IFN-gamma on the Stat pathway using a unique human pancreatic carcinoid tumor, BON, established in our laboratory. BON cells were treated with IFN-alpha (500 U/mL) or IFN-gamma (500 U/mL); nuclear protein was extracted at selected intervals. Steady state levels of Stat proteins 1, 3, and 5 were measured by Western blot; protein binding was assessed by electrophoretic mobility shift assay (EMSA) using probes containing either the Stat1/Stat3 or Stat5 binding sites. Treatment with IFN-alpha increased predominantly Stat3 and Stat5 protein levels and binding activities. IFN-gamma increased Stat1, 3, and 5 protein levels, with maximal elevations occurring at 24 to 48 hours after addition; Stat3 and 5 binding activities were also increased. We have shown that both IFN-alpha and IFN-gamma can induce Stat protein binding (particularly Stat3 and Stat5) to their cognate DNA consensus sites and increase Stat protein steady state levels in BON cells. Delineating the signaling pathways altered by IFN treatment will provide a better understanding of downstream gene targets and mechanisms for IFN-mediated growth inhibition of endocrine tumors.