BON1 Cell Line Research Articles

Exploring the therapeutic potential of simvastatin in pancreatic neuroendocrine neoplasms: insights into cell cycle regulation and apoptosis.

Pancreatic neuroendocrine neoplasm (pNEN) poses significant challenges in clinical management due to their heterogeneity and limited treatment options. In this study, we investigated the potential of simvastatin (SIM) as an anti-tumor agent in pNEN. We conducted cell culture experiments using QGP-1 and BON-1 cell lines and assessed cell viability, proliferation, migration, and invasion following SIM treatment. To further validate our findings, we performed in vivo experiments using a mouse xenograft model. Additionally, we investigated the underlying molecular mechanisms by analyzing changes in cell cycle progression, apoptosis, and signaling pathways. SIM treatment suppresses pNEN growth both in vitro and in vivo, and led to G1 phase arrest in QGP-1 cells. In contrast, SIM affected both the G1-S and G2-M phase transitions in the BON-1 cell line and induced apoptosis, indicating diverse mechanisms of action. Furthermore, SIM treatment resulted in decreased expression of mutant p53 (mutp53) in BON-1 cells, suggesting a potential therapeutic strategy targeting mutp53. Modulation of the MAPK pathway was also implicated in QGP-1 cells. Our study highlights SIM as a promising candidate for pNEN treatment by inducing cell cycle arrest or apoptosis, potentially through the p53 and MAPK pathways. Further research is warranted to fully elucidate SIM's mechanisms of action and evaluate its therapeutic potential in clinical settings.

THU487 Xenograft of VHL-deficient Pancreatic Neuroendocrine Neoplasm Cells - A Novel Low-grade PNEN In Vivo Model

Abstract Disclosure: A. Telerman: None. Y. Yossef: None. A. Chmelnik: None. A. Tirosh: None. Background: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome caused by a germline mutation in the VHL tumor suppressor gene. Although VHL-related pancreatic neuroendocrine neoplasms (vPNEN) have been studied, their molecular pathogenesis is not fully understood. Aims: To generate a mouse model for studying the mechanisms that promote vPNEN development in vivo. Methods: We induced a frameshift mutation in VHL using the CRISPR/Cas9 technique in the BON1 cell line, originating from high-grade PNEN (FS-BON1). We validated the pseudohypoxic nature of the cells by real-time polymerase chain reaction of VEGF and EPO in FS-BON1 compared to VHL wild-type BON1 (WT-BON1). We compared cell line-derived xenografts (CDXs) growth in athymic Nude-Foxn1nu female mice injected with WT-BON1 (n=9), FS-BON1 (n=9) and media (purified bovine serum, n=5). Tumor diameter and calculated volume and mice weight were recorded weekly. Plasma chromogranin A (CgA) levels were measured in mice sera by ELISA. Results: FS-BON1 showed upregulation of VEGF and EPO compared with WT-BON1, confirming their pseudohypoxic microenvironment. At 14 weeks, 0/9 FS-BON1 CDX reached a volume of 1 cm3 compared with 5/9 of the WT-BON1 group (p=0.03). In time-dependent analysis, FS-BON1-derived xenografts grew significantly slower than WT-BON1 xenografts (p=0.02). No significant differences in CgA serum levels were found between the mice groups. Conclusions: We report a new pseudohypoxic in vivo PNEN model. The pVHL-deficient cells show an unexpected indolent course, suggesting it as a new model for low-grade PNEN. This CDX mouse model can be utilized in further studies to evaluate the mechanism responsible for vPNEN development and for evaluating interventions in low-grade PNEN. Presentation: Thursday, June 15, 2023

Feasibility of the chick chorioallantoic membrane model for preclinical studies on tumor radiofrequency ablation

BackgroundWe evaluated the feasibility of a chick chorioallantoic membrane (CAM) tumor model for preclinical research on tumor radiofrequency ablation (RFA).MethodsFertilized chicken eggs were incubated and divided into five cohorts: RFA for 30 s (n = 5), RFA for 60 s (n = 5), RFA for 120 s (n = 4), sham (n = 8), and controls (n = 6). Xenografting using pancreatic neuroendocrine tumor cells of the BON-1 cell line was performed on embryonic day (ED) 8. The RFA was performed on ED 12. Survival, stereomicroscopic observations, and histological observations using hematoxylin–eosin (H&E) and Ki67 staining were evaluated.ResultsThe survival rates in the 30-s, 60-s, and 120-s, sham and control cohort were 60%, 60%, 0%, 100%, and 50%, respectively. Signs of bleeding and heat damage were common findings in the evaluation of stereomicroscopic observations. Histological examination could be performed in all but one embryo. Heat damage, bleeding, thrombosis, and leukocyte infiltration and hyperemia were regular findings in H&E-stained cuts. A complete absence of Ki67 staining was recorded in 33.3% and 50% of embryos in the 30-s and 60-s cohorts that survived until ED 14, respectively.ConclusionsThe CAM model is a feasible and suiting research model for tumor RFA with many advantages over other animal models. It offers the opportunity to conduct in vivo research under standardized conditions. Further studies are needed to optimize this model for tumor ablations in order to explore promising but unrefined strategies like the combination of RFA and immunotherapy.Relevance statementThe chick chorioallantoic membrane model allows in vivo research on tumor radiofrequency ablation under standardized conditions that may enable enhanced understanding on combined therapies while ensuring animal welfare in concordance with the “Three Rs.”Key points• The chorioallantoic membrane model is feasible and suiting for tumor radiofrequency ablation.• Radiofrequency ablation regularly achieved reduction but not eradication of Ki67 staining.• Histological evaluation showed findings comparable to changes in humans after RFA.• The chorioallantoic membrane model can enable studies on combined therapies after optimization.Graphical

New Therapy Options for Neuroendocrine Carcinoma of the Pancreas-The Emergent Substance GP-2250 and Gemcitabine Prove to Be Highly Effective without the Development of Secondary Resistances In Vitro and In Vivo.

Simple SummaryNeuroendocrine carcinoma of the pancreas is a highly aggressive form of neuroendocrine tumor associated with poor survival and increasing occurrence. GP-2250 is an emergent substance showing antineoplastic properties, especially in combination with Gemcitabine. This study was the first to evaluate the antineoplastic effects of GP-2250 on pancreatic neuroendocrine carcinoma. The combination of GP-2250 and Gemcitabine showed highly synergistic effects in a cell culture model, as well as in mice, without the development of secondary resistances. These findings form the basis for further clinical evaluation of a highly promising combination therapy.Neuroendocrine carcinoma of the pancreas (pNEC) is an aggressive form of neuroendocrine tumor characterized by a rising incidence without an increase in survival rates. GP-2250 is an oxathiazinane derivate possessing antineoplastic effects, especially in combination with Gemcitabine on the pancreatic adenocarcinoma. The cytotoxic effects of the monotherapy of GP-2250 (GP-2250mono) and Gemcitabine (Gemmono), as well as the combination therapy of both, were studied in vitro using an MTT-assay on the QGP-1 and BON-1 cell lines, along with in vivo studies on a murine xenograft model of QGP-1 and a patient-derived xenograft model (PDX) of Bo99. In vitro, Gemmono and GP-2250mono showed a dose-dependent cytotoxicity. The combination of GP-2250 and Gemcitabine exhibited highly synergistic effects. In vivo, the combination therapy obtained a partial response in QGP-1, while GP-2250mono and Gemmono showed progressive disease or stable disease, respectively. In Bo99 PDX, the combination therapy led to a partial response, while the monotherapy resulted in progressive disease. No development of secondary resistances was observed, as opposed to monotherapy. This study was the first to evaluate the effects of the emerging substance GP-2250 on pNEC. The substance showed synergism in combination with Gemcitabine. The combination therapy proved to be effective in vitro and in vivo, without the development of secondary resistances.

Generation and characterization of CRISPR/Cas9-mediated MEN1 knockout BON1 cells: a human pancreatic neuroendocrine cell line

Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g., posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.

Multiple modes of cell death in neuroendocrine tumors induced by artesunate

BackgroundThe paucity of effective treatment in neuroendocrine tumors (NETs) encouraged us to investigate the therapeutic value of artesunate (ART) promised by its inhibitory effect against various tumors and broad safety profile. MethodsWe evaluated the impact of ART on three NET cell lines, BON-1, QGP-1 and NCI-H727 on cellular and molecular levels. ResultsOur results showed that ART induced endoplasmic reticulum (ER) stress through phosphorylation of eIF2α, which further gave rise to autophagy in all three NET cell lines. Specifically, apoptosis and ferroptosis were also observed in BON-1 cells, which made BON-1 cell line more vulnerable upon ART treatment. The different sensitivities presented on the three cell lines also associated with a differential regulation of p21 on the long run. Co-treatment with p21 inhibitor UC2288 showed an additive effect on QGP-1 and NCI-H727 cell lines indicating p21 upregulation in these two cell lines might confer resistance towards ART treatment. ConclusionsIt is possible to include ART in the treatment of NETs in the future.

Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line.

Pancreatic Neuroendocrine Neoplasms (pNEN) are rare tumors which treatment still represent an important clinical problem, due to the paucity of medical treatments. Due to tumor complexity, techniques as 3D cultures are important to study drug activity in a more realistic model. This study aims to compare three different 3D culture methods in order to understand which one can be considered the best option in terms of experimental easiness and reproducibility in studying the efficacy of a target drug on pNEN. The BON1 cell line was used as a pNEN model and the well-known Receptor Tyrosine Kinase inhibitor Sunitinib was used in order to better investigate the different features of each method. The investigated methods are: (1) 96-well hanging drop plates (HD plates), (2) 24-well plates with a cell-repellent surface, and (3) ultra-low attachment 96-well plates with clear round bottom (ULA plates). The evaluated parameters during the study were: cell seeding, easiness in spheroids formation, morphology, culture maintenance, medium change, spheroids monitoring, picture quality, spheroid perimeter measurement reproducibility error, possibility to perform assays into the seeding plate, overall time of the experiment. Moreover, we investigated how culture methods can influence experimental outcomes evaluating perimeter changes, cell viability and immunohistochemistry of spheroids treated with different Sunitinib concentrations. Results showed that each method has weak and strong points but, considering the easiness of spheroids maintenance and reproducibility results, ULA plates method appears to be the best approach to culture BON1 spheroids and, therefore, to study pNEN.

04:03 PM Abstract No. 225 CRISPR-mediated loss of DAXX protein expression in BON1 cell lines results in ischemia resistance and ischemia-induced epithelial-mesenchymal transition

04:03 PM Abstract No. 225 CRISPR-mediated loss of DAXX protein expression in BON1 cell lines results in ischemia resistance and ischemia-induced epithelial-mesenchymal transition

Abstract 3519: TGF-β pathway alteration in pancreatic neuroendocrine tumors: characterization of a novel SMAD3 translocation

Abstract Pancreatic neuroendocrine tumors (PanNETs) are the second most common malignancy of the pancreas, and the only curative option is represented by surgery. PanNETs can be classified as well- or poorly- differentiated, based on histology. Poorly-differentiated neuroendocrine carcinomas (NECs) represent less than 10% of cases, but they are highly malignant and associated with poor prognosis, and, most importantly, the inconsistency in their clinical management is due to the insufficient data on NEC genetic features. In order to widen our knowledge of PanNET genetic alterations, we performed RNASeq analysis (using Illumina NextSeq platform) of a group of 18 PanNETs and 2 NECs. TopHat-Fusion algorithm identified, among others, a fusion involving SMAD3 transcription factor and CCDC149; the alteration of SMAD3 in a poorly differentiated NEC was particularly interesting since SMAD3 is involved in TGF-b signaling, which has an oncogenic role in malignant cells. The SMAD3-CCDC149 fusion was confirmed in the index case by RT-PCR using primers spanning the breakpoint, followed by Sanger sequencing. The fusion was also detected by FISH analysis performed on both FFPE and frozen samples. By both FISH analysis and Sanger sequencing we identified two other poorly-differentiated NEC samples characterized by the presence of the SMAD3-CCDC149 translocation. Immunohistochemical (IHC) staining revealed that the 3 tumors, positive for the SMAD3-CCDC149 fusion, were characterized by nuclear localization of SMAD3. In order to test the hypothesis that the fusion generates an active form of SMAD3 that localizes exclusively within the nucleus, we transfected BON-1 cell line with plasmids containing either SMAD3 full length or SMAD3 truncated form (the latter mimics the fusion product) showing that only the truncated form of SMAD3 had an exclusively nuclear localization. Interestingly, FISH analysis performed on 3 well-differentiated PanNET samples did not detected the presence of the SMAD3-CDC149 translocation and IHC analysis showed cytoplasmic localization of SMAD3. Moreover, IHC analysis performed on a cohort of 40 cases of non-pancreatic neuroendocrine tumors (NETs) revealed that only poor-differentiated tumors showed a significant percentage of samples with nuclear localization of SMAD3. These data support the hypothesis that the translocation is a typical phenomenon of poorly-differentiated tumors and suggests that it could be involved also in non-pancreatic NETs. In this study we identified a previous unknown SMAD3 translocation in poor-differentiated NECs, which generates an active SMAD3 form. Deepening the knowledge of the fusion product will shed light on PanNET genetic alterations, opening the way to new therapeutic strategies. Citation Format: Emanuela Brunetto, Greta Grassini, Valeria De Pascali, Anna Talarico, Francesca Invernizzi, Michaela Bowden, Massimo Loda, Luca Albarello, Massimo Falconi, Lorenza Pecciarini, Maria G. Cangi, Claudio Doglioni. TGF-β pathway alteration in pancreatic neuroendocrine tumors: characterization of a novel SMAD3 translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2017-3519

Construction of three-dimensional culture model of human neuroendocrine tumor BON-1 and H727 cell lines

Objective To provide new methods for the study of human neuroendocrine tumor cells in vitro by establishing three-dimensional culture models. Methods Agarose gel, alginate/polyacrylamide copolymer (ALG/PAAm) hydrogel and polyethylene glycol (PEG) crosslinked poly methyl vinyl ether maleic acid alt (PMVE-alt-MA) hydrogel were prepared. Three-dimensional culture models of human neuroendocrine tumor H727 and BON-1 cell lines based on these three materials were constructed, and the morphological changes of the models were observed by a microscope every 24 h. Results The shapes of H727 and BON-1 cells with three-dimensional culture were different from those of two-dimensional culture. After cultured for one week, H727 cells formed different multicellular spheroids with sphere diameter of 350-450, 150-250 and 50-100 μm on agarose gel, ALG/PAAm hydrogel and in PEG crosslinking PMVE-alt-MA hydrogel respectively. And BON-1 cells grew up into multicellular spheroids with sphere diameter of 150-200 μm in PEG crosslinking PMVE-alt-MA hydrogel, but they just formed irregularly shaped multicellular aggregates on the other two culture materials. Conclusion The three materials could be applied for constructing three-dimensional culture models of BON-1 and H727 cell lines. Due to their advantages and disadvantages, the appropriate culture material would be selected according to specific experimental requirements. Key words: Neuroendocrine tumor cell; Three-dimensional culture model; Culture material

Development of an everolimus-resistant BON1 cell line with altered cell cycle and c-Met activity

Development of an everolimus-resistant BON1 cell line with altered cell cycle and c-Met activity

Effects of Somatostatin Analogs and Dopamine Agonists on Insulin-Like Growth Factor 2-Induced Insulin Receptor Isoform A Activation by Gastroenteropancreatic Neuroendocrine Tumor Cells

Background: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) express insulin-like growth factor (IGF)-related factors [IGF1, IGF2; insulin receptor (IR)-A, IR-B; IGF-binding protein (IGFBP) 1-3] as well as somatostatin (SSTRs) and dopamine D₂ receptors (D2Rs). Objectives: To (1) compare mRNA expression of IGF-related factors in human pancreatic NET (panNET) cell lines with that in human GEP-NETs to evaluate the usefulness of these cells as a model for studying the IGF system in GEP-NETs, (2) determine whether panNET cells produce growth factors that activate IR-A, and (3) investigate whether somatostatin analogs (SSAs) and/or dopamine agonists (DAs) influence the production of these growth factors. Methods: In panNET cells (BON-1 and QGP-1) and GEP-NETs, mRNA expression of IGF-related factors was measured by quantitative real-time PCR. Effects of the SSAs octreotide and pasireotide (PAS), the DA cabergoline (CAB), and the dopastatin BIM-23A760 (all 100 n<smlcap>M</smlcap>) were evaluated at the IGF2 mRNA and protein level (by ELISA) and regarding IR-A bioactivity (by kinase receptor activation assay) in panNET cells. Results: panNET cells and GEP-NETs had comparable expression profiles of IGF-related factors. Especially in BON-1 cells, IGF2 and IR-A were most highly expressed. PAS + CAB inhibited IGF2 (-29.5 ± 4.9%, p < 0.01) and IGFBP3 (-20.0 ± 4.0%, p < 0.01) mRNA expression in BON-1 cells. In BON-1 cells, IGF2 protein secretion was significantly inhibited with BIM-23A760 (-23.7 ± 3.8%). BON-1- but not QGP-1- conditioned medium stimulated IR-A bioactivity. In BON-1 cells, IR-A bioactivity was inhibited by BIM-23A760 and PAS + CAB (-37.8 ± 2.1% and -30.9 ± 4.1%, respectively, p < 0.0001). Conclusions: (1) The BON-1 cell line is a representative model for studying the IGF system in GEP-NETs, (2) BON-1 cells produce growth factors (IGF2) activating IR-A, and (3) combined SSTR and D2R targeting with PAS + CAB and BIM-23A760 suppresses IGF2-induced IR-A activation.

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 3462: Proteomic profiling reveals novel targets for combination treatment with lanreotide in neuroendocrine tumors

Abstract Somatostatin analogues (SSAs) are well established in the treatment of neuroendocrine tumors (NETs), including small intestinal NET (SI-NET) due to their tumor growth arrest and symptom relieving characteristics. Here we determined the direct effects of lanreotide treatment on NET cell lines and a primary SI-NET culture. The cell lines HC45 and H727 were treated with a pharmacological concentration of 10 nM lanreotide for different time periods and the proteome, analyzed by in-depth HiRIEF LC-MS/MS, was compared with the proteome of non-treated cells. We quantified 6 451 and 7 801 proteins in HC45 and H727. Differential expression of the candidate proteins APC, BIRC5/survivin, BMPER, C14orf142, FYN, INSM1 and SPAG16 was confirmed by Western blotting. The possible direct anti-proliferative effect of lanreotide was evaluated in the primary SI-NET culture and the HC45, H727 and BON1 cell lines, which showed only a slight inhibition of proliferation. However, based on proteomics driven pathway analysis a significant synergy in anti-proliferative effect of lanreotide was observed when the small molecule IGF1R inhibitor, NVP-AEW541, was combined with lanreotide .This effective combination also represents an indirect model of suppressed IGF1 activity in the NET patients treated with SSA. Targeting of survivin with the small molecule YM155 dramatically reduced the proliferation of HC45. We also studied the effect of lanreotide alone or in combination with IGF-1R inhibitor on signaling pathways PI3K/Akt, Erk1/2 and P38. P38, Erk1/2 and GSK3β phosphorylation was equally increased by lanreotide and/or NVP-AEW541, however, lanreotide-induced AktS473 dephosphorylation was reversed after6 hours, unlike consistent AktS473 dephosphorylation after treating with NVP-AEW451 alone or in combination. In conclusion, we report growth inhibitory and potential feedback pro-growth signaling of the SSA lanreotide on NET cells, highlighting the role of SSAs in modulation of IGF1 in NET cell growth arrest and propose combination treatment candidate targets for improved efficacy of this agent. Citation Format: Omid Fotouhi, Hanna Kjellin, Jamileh Hashemi, Ming Lu, Christofer Juhlin, Anders Höög, Jan Zedenius, Catharina Larsson, Janne Lehtiö, Magnus Kjellman. Proteomic profiling reveals novel targets for combination treatment with lanreotide in neuroendocrine tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2015-3462

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca2+ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca2+ ([Ca2+]i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La3+ (100μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca2+]i in NET cells. CAP (20μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10μM). La3+ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca2+, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.

Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1-10 μM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48-72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 μM by inducing caspase-mediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3β (GSK3β) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3β signaling, and inhibits secretory activity in PNN in vitro models, suggesting that Enzastaurin might represent a possible medical treatment of human PNN.

Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co-localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co-localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co-localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post-translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON-1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down-regulated by post-translational modulation and shows potential antiproliferative properties in vitro.