Chromogranin A Secretion Research Articles

6365 An Atypical Case Of Large Metastatic Pheochromocytoma With Negative Catecholamine Secretion

Abstract Disclosure: D. Caesario: None. J.K. Fernandes: None. Introduction: Pheochromocytomas (PCCs) are rare tumors arising from chromaffin cells of the adrenal medulla. Diagnosis is suggested by clinical presentations and confirmed by biochemical hypersecretion of metanephrines (MN) and catecholamines (CA). We report an atypical case of large metastatic PCC with normal MN and CA. Case Report: A 61-year-old female presented with uncontrolled HTN despite taking amlodipine, losartan, and carvedilol. No other symptoms reported. She had presented 12 years prior with an 8.7 cm left adrenal mass (AdM) that was incidentally found on CT scan, but she was lost to follow-up. Repeat imaging for her current presentation revealed enlargement of left AdM to 15 cm, centrally necrotic, with absolute washout of 63%. There was extensive local regional involvement, including possible invasion of the pancreas; abutment to left kidney, left renal vein, left hepatic lobe, and stomach; encasement and possible invasion of the splenic vein; and formation of perisplenic collateral vessels. Metastatic lesions were also seen in the liver, bones, and lungs. DOTATATE PET/CT scan showed variable uptake, not a definitive pattern for PCC or neuroendocrine tumor (NET). Labs showed normal levels of plasma fractionated MN and CA, dopamine, renin, aldosterone, cortisol, ACTH, and DHEA-S. Chromogranin A (CgA) was elevated to >8000 ng/dL. Biopsy taken from one of the liver lesions was consistent with metastatic PCC. No family history of NET. Genetic testing was offered, but was deferred. Due to the large tumor burden, she was determined to be nonsurgical and started on systemic therapy with sunitinib. Doxazosin was also added, which better controlled her BP. After 9 months of sunitinib therapy, a repeat CT scan showed overall stable metastatic lesions, but the left AdM had enlarged to 16 cm. Given the slowly worsening disease, patient was switched to temozolomide. She is still actively being treated and monitored today. Discussion: The clinical features of PCC result from excessive CA secretion. HTN is the hallmark of CA release, but the amount, type, and pattern of CA secretion is extremely variable, leading to varied clinical presentations. CgA secretion is also elevated in PCC, but is generally a non-specific marker as its level is also increased in multiple NETs and non-endocrine medical conditions. About 9% of patients with PCC may have normal biochemical workup, with this being even rarer in malignant cases. In general, the amount of CA, MN, and CgA released by the tumor is also positively correlated with the tumor size. The case above is unique in that despite the large tumor burden the patient only presented with HTN and had normal MN and CA levels. Her CgA was significantly elevated, which could correlate with the tumor size, but was interestingly the only marker elevated. It appears that CgA could be a useful adjunct in the diagnosis of PCC, especially in cases where PCC appeared to be non-secretory. Presentation: 6/3/2024

Chromogranin A: a useful biomarker in castration-resistant prostate cancer

PurposeThe natural history of prostate cancer (PC) almost always evolves to castration-resistant prostate cancer (CRPC) status, sometimes comprising pure or mixed neuroendocrine prostate cancers (NEPC) differentiation. In CRPC, monitoring using only prostate-specific antigen (PSA) is not optimal since neuroendocrine differentiated cells do not secrete PSA. Thus, monitoring with PSA and chromogranin A (CgA) may be useful. This review aims to evaluate evidence for the usefulness of CgA assessments during the monitoring of prostate cancer.MethodThis review was based on three recent meta-analysis concerning CgA and prostate cancer. Further data were obtained from PubMed and Embase databases by searches using keywords, including chromogranin A and prostate cancer.ResultsCgA levels remain largely unchanged during the early PC evolution. The development of NEPC is characterised by lower PSA secretion and increased CgA secretion. Data supporting the prognostic value of high CgA baseline levels for survival are contrasting and scarce. However, increasing CgA levels early during treatment of metastatic (m)CRPC suggests resistance to treatment and predicts shorter survival, particularly in men with high baseline levels of CgA levels. In men with mCRPC, the first-line chemotherapy may be more appropriate than other agents when baseline CgA levels are high. Also, increasing CgA levels during treatment may indicate disease progression and may warrant a change of therapy.ConclusionCgA monitoring at baseline and regularly during mCRPC management may be useful for monitoring disease evolution. An increased CgA baseline levels and increasing CgA levels may assist physicians with choosing and modifying therapy.

Salivary Chromogranin A: A Novel Diagnostic Bio-Marker for Alzheimer's Disease

Early diagnosis of Alzheimer's disease will be helpful as no clinical method is available to determine the role of mild cognitive impairment. Chromogranins are soluble glycophosphoproteins which activate microglial cells leading to neurotoxic phenotype. There is need for biomarkers through non-invasive approach to identify incipient Alzheimer’s disease patients with mild cognitive impairment. Chromogranin A present in saliva samples was determined using ELISA. The immunoreactive patterns of Salivary CgA were assayed in dementia and compared to those observed in Alzheimer's disease. Salivary CgA level in Alzheimer’s disease patients was 6.54 pmol/ml and 0.23 pmol/ml in control group. Plasma CgA in dementia patients was 85.76 ng/ml and 60.34 ng/ml in control. Statistical analysis showed significant difference level of P ≤ 0.05. This study showed that salivary CgA levels were reduced at early stages of AD. Chromogranin A (CgA) in saliva exhibited significant reduction in immunoreactivity and to be selectively associated with prion protein deposits, CgA was only found in Amyloid beta plaques. This shows influence of constitutive amyloid protein on chromogranin secretion and role of CgA in AD neurodegenerative process. This study shows that biochemical and psychosocial stress can play major role in CgA and acts as potential biomarker for the diagnosis of AD type dementia.

Pharmacological Characterization of Veldoreotide as a Somatostatin Receptor 4 Agonist.

Veldoreotide, a somatostatin analogue, binds to the somatostatin receptors (SSTR) 2, 4, and 5. The current aim was to assess its pharmacological activity as an SSTR4 agonist. G-protein signaling was assessed using a fluorescence-based membrane potential assay in human embryonic kidney 293 (HEK293) cells stably co-expressing G-protein-coupled inwardly rectifying potassium 2 channels and the individual SSTR2, SSTR4, and SSTR5, and in human BON-1 cells stably expressing these SSTRs. Veldoreotide effects on chromogranin A (CgA) secretion and cell proliferation were examined in BON-1 cells. In HEK293 transfected cells, veldoreotide showed a high efficacy for activating the SSTR4; octreotide and pasireotide had little activity (Emax, 99.5% vs. 27.4% and 52.0%, respectively). Veldoreotide also activated SSTR2 and SSTR5 (Emax, 98.4% and 96.9%, respectively). In BON-1 cells, veldoreotide activated SSTR2, SSTR4, and SSTR5 with high potency and efficacy. CgA secretion was decreased to a greater degree in the BON-1 cells expressing SSTR4 versus the cells expressing SSTR2 and SSTR5 (65.3% vs. 80.3% and 77.6%, respectively). In the BON-1 cells expressing SSTR4, veldoreotide inhibited cell proliferation more than somatostatin SS-14 (71.2% vs. 79.7%) and to a similar extent as the SSTR4 agonist J-2156 in the presence of SSTR2 and SSTR5 antagonists. Veldoreotide is a full agonist of SSTR2, SSTR4, and SSTR5.

A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors

Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Chromogranin A in the early steps of the neurosecretory pathway.

Chromogranin A (CgA) is a soluble glycoprotein stored with hormones and neuropeptides in secretory granules (SG) of most (neuro)endocrine cells and neurons. Since its discovery in 1967, many studies have reported its structural characteristics, biological roles, and mechanisms of action. Indeed, CgA is both a precursor of various biologically active peptides and a granulogenic protein regulating the storage and secretion of hormones and neuropeptides. This review emphasizes the findings and theoretical concepts around the CgA-linked molecular machinery controlling hormone/neuropeptide aggregation and the interaction of CgA-hormone/neuropeptide aggregates with the trans-Golgi membrane to allow hormone/neuropeptide targeting and SG biogenesis. We will also discuss the intriguing alteration of CgA expression and secretion in various neurological disorders, which could provide insights to elucidate the molecular mechanisms underlying these pathophysiological conditions.

Significance of Chromogranin A in Diagnosis and Prognosis of Neuroendocrine Tumors

We present the comparative study of plasma chromogranin A (CgA) levels measured in 232 patients with different types of neuroendocrine tumors (NETs) and 66 healthy individuals using enzyme immunoassay Chromogranin A ELISA kit (Dako). CgA levels were significantly higher in patients with all types of NETs than in healthy subjects. CgA secretion were highly variable. The study demonstrate high diagnostic sensitivity of CgA, which was 80.6 % in overall group of NETs with specificity 98.5 %. We confirmed the prognostic significance of CgA, in this case the high basal levels of CgA (above 100 U/l) were significantly associated with less favorable prognosis of progression free survival after different types of treatment. The data confirms high efficiency of CgA as biomarker, which measurement enhances the accuracy of diagnosis and prognosis of NETs.

Effects of Ketoconazole on ACTH-Producing and Non-ACTH-Producing Neuroendocrine Tumor Cells

Prolonged remission of hypercortisolism with steroidogenesis inhibitors has been described in patients with ectopic adrenocorticotropic hormone (ACTH) syndrome. The anti-proliferative and pro-apoptotic effect of ketoconazole in human cancer cells was previously suggested. The aim of this study was to explore the effects of ketoconazole on ACTH-producing and non-ACTH-producing neuroendocrine tumor (NET) cell lines. The effects of ketoconazole alone, and in combination with somatostatin analogs, were evaluated in two human cell lines: DMS-79 (ectopic ACTH-producing small cell lung carcinoma) and BON-1 (human pancreatic NET). Total DNA measurement, apoptosis, cell cycle, chromogranin A (CgA)/proopiomelanocortin (POMC) expression by qRT-PCR, serotonin, CgA, and ACTH secretion assays were performed. In both cell lines, ketoconazole significantly suppressed cell growth and colony formation in a dose and time-dependent manner. The effect in DMS-79 was primarily cytotoxic, while it was more apoptotic in BON-1 cells. Ketoconazole also induced increase in G0/G1 phase in both cell lines and arrest in phase G2/M of BON-1 cells. Ketoconazole did not affect the secretion of serotonin, CgA, ACTH, or the mRNA expression of CgA and POMC. Decreased serotonin secretion was observed after the combination treatment with pasireotide. These results suggest a direct effect of ketoconazole on cell proliferation, apoptosis, and cell cycle in both ACTH- and non-ACTH-producing NET cells.

Effects of novel somatostatin-dopamine chimeric drugs in 2D and 3D cell culture models of neuroendocrine tumors.

Control of symptoms related to hormonal hypersecretion by functioning neuroendocrine tumors (NETs) is challenging. New therapeutic options are required. Since novel in vitro tumor models seem to better mimic the tumor in vivo conditions, we aimed to study the effect of somatostatin and dopamine receptor agonists (octreotide and cabergoline, respectively) and novel somatostatin-dopamine chimeric multi-receptor drugs (BIM-065, BIM-23A760) using 2D (monolayer) and 3D (spheroids) cultures. Dose-response studies in 2D and 3D human pancreatic NET cell cultures (BON-1 and QGP-1) were performed under serum-containing and serum-deprived conditions. Cell proliferation, somatostatin and dopamine receptor expression (SSTs and D2R), apoptosis, lactate dehydrogenase, as well as serotonin and chromogranin A (CgA) release were assessed. The following results were obtained. 3D cultures of BON-1/QGP-1 allowed better cell survival than 2D cultures in serum-deprived conditions. SSTs and D2R mRNA levels were higher in the 3D model vs 2D model. Octreotide/cabergoline/BIM-065/BIM-23A760 treatment did not affect cell growth or spheroid size. In BON-1 2D-cultures, only BIM-23A760 significantly inhibited CgA release -this effect being more pronounced in 3D cultures. In BON-1 2D cultures, cabergoline/BIM-065/BIM-23A760 treatment decreased serotonin release (maximal effect up to 40%), being this effect again more potent in 3D cultures (up to 67% inhibition; with BIM-23A760 having the most potent effects). In QGP-1, cabergoline/BIM-065 treatment decreased serotonin release only in the 3D model. In conclusion, cultures of NET 3D spheroids represent a promising method for evaluating cell proliferation and secretion in NET cell-line models. Compared to 2D models, 3D models grow relatively serum independent. In 3D model, SST-D2R multi-receptor targeting drugs inhibit CgA and serotonin secretion, but not NET cell growth.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 μM) and PACAP (0.1μM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2μM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronicallyincreasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Impaired Processing of Prohormones in Secretogranin III–Null Mice Causes Maladaptation to an Inadequate Diet and Stress

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

Antisecretory Effects of Chimeric Somatostatin/Dopamine Receptor Ligands on Gastroenteropancreatic Neuroendocrine Tumors.

The recent finding that gastroenteropancreatic neuroendocrine tumors expressed the dopaminergic D2 receptor in addition to somatostatin (sst) receptors suggested that multiple targeting approaches might decrease hormone hypersecretion more effectively than sst agonists alone. To test this hypothesis, (i) we measured the expression of sst receptor type 2 (sst2 receptor) and D2 receptor in 11 gastroenteropancreatic neuroendocrine tumors and (ii) we compared the ability of lanreotide, cabergoline, their combination, and sst/D2 chimeric ligands to decrease chromogranin A (CgA), gastrin, or serotonin release in primary cultures derived from these tumors. Moderate to high positivity was observed for sst2 receptor and D2 receptor, the latter being more expressed in pancreatic tumors. Lanreotide decreased CgA secretion in all cultures, but only 3 tumors responded to cabergoline. No additivity was observed in lanreotide. BIM 23A781 decreased CgA release to the same extent as lanreotide, whereas the other chimeric ligands were less efficient. However, BIM 23A781 was 50 times less potent than lanreotide. Similar patterns were found for gastrin or serotonin. No improvement was brought by the sst/D2 combination or chimeric ligands. Factors that underlie these tissue-specific differences remain to be elucidated.

BIOCHEMICAL MARKERS IN SERUM AND URINE IN THE WORKUP OF PATIENTS WITH NEUROENDOCRINE TUMORS

This review summarizes current data on neuroendocrine tumors (NET), which, unlike other neoplasms, are able to produce biologically active substances (hormones, vasoactive peptides, amines). It is exactly their main characteristic that allows to unify this heterogeneous group and that may determine their clinical course. We present integrated recommendations for biochemical diagnosis and confirmation of over-secretion syndromes based on a panel assessment of NET biochemical markers. Data from the literature are reviewed on evaluation of clinical significance of generic and specific NET markers, as well as the results of the studies performed by the authors themselves. Three hundred and thirty patients were examined with NETs of various localization (pancreas, stomach, small intestine and large intestine, lungs) and with metastatic NET disease with unknown primary location, who were treated in the N.N. Blokhin Russian Cancer Research Center. The control group included 115 healthy individuals. Before and during the treatment, plasma and serum chromogranin A (CgA) and serotonin levels, as well as 5-hydroxyindoleacetic acid (5-HIAA) in a 24-hour urine sample were measured with standardized immunoenzyme plate-based assays (“Chromogranin A ELISA kit”, Dako A/S; “Serotonin ELISA and 5-HIAA ELISA”, IBL International GMBH). We evaluated clinical importance of CgA as a generic NET marker, as well as that of serotonin and its metabolite 5-HIAA as specific markers of the carcinoid syndrome. CgA was shown to be the most efficient biochemical marker for diagnosis, assessment of prevalence and monitoring of NETs. CgA has a high diagnostic sensitivity (63.4 to 88.9%) in various NETs. An association between CgA secretion and prevalence and biological activity of the tumor was confirmed. CgA measurement is particularly important in functionally inactive tumors, where serotonin and 5-HIAA have lower sensitivity, being specific markers of the carcinoid syndrome.

CLINICAL VALUE OF CHROMOGRANIN A IN GASTROENTEROPANCREATIC NEUROENDOCRINE TUMORS

Background: Neuroendocrine tumors (NET) is a heterogeneous group of neoplasms characterized by hypersecretion of biologically active sub- stances that manifests by specific syndromes and determines the clinical course of the disease. The most common NET types are those of gastrointestinal tract. The obligatory biochemical marker used in the examination of NET patients is chromogranin A (CgA). Aim: Evaluation of the CgA value for diagnostics and monitoring of gastrointestinal NETs. Materials and methods: A comparative study of plasma CgA levels was performed in 146 patients with gastroenteropancreatic neuroendocrine tu- mors and 66 healthy individuals using the enzyme immunoassay “Chromogranin A ELISA kit” (Dako A/S, Denmark). Results: CgA levels were significantly higher in patients with NETs of all localizations, such as pancreas, stomach, gut, small and large bowel, than in the healthy subjects (р < 0.000001). In NET patients, CgA secretion was highly variable, with the highest value in the group of patients with gastric NETs (102000 U/l). The highest CgA medians were detected in patients with small intestinal (183.9 U/l), colon (148.4 U/l) and pancreatic (135.9 U/l) NETs. There was an association between CgA secretion and extension or activity of NETs, with the highest median values in patients with hepatic metastases (395 U/l) and those with carcinoid syndrome (352 U/l). The clinical significance of CgA as a NET marker was assessed using the cut-off value of 33 U/l, calculated according to the results in the control group. Overall diagnostic sensitivity of CgA in NET patients was high (85.8%) with a specificity of 98.5%. Conclusion: The results obtained confirm a high sensitivity of CgA as a NET marker whose determination helps to improve accuracy of diagnostics and to assess NET prevalence.

Pasireotide and octreotide antiproliferative effects and sst2 trafficking in human pancreatic neuroendocrine tumor cultures.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) raise difficult therapeutic problems despite the emergence of targeted therapies. Somatostatin analogs (SSA) remain pivotal therapeutic drugs. However, the tachyphylaxis and the limited antitumoral effects observed with the classical somatostatin 2 (sst2) agonists (octreotide and lanreotide) led to the development of new SSA, such as the pan sst receptor agonist pasireotide. Our aim was to compare the effects of pasireotide and octreotide on cell survival, chromogranin A (CgA) secretion, and sst2 phosphorylation/trafficking in pancreatic NET (pNET) primary cells from 15 tumors. We established and characterized the primary cultures of human pancreatic tumors (pNETs) as powerful preclinical models for understanding the biological effects of SSA. At clinically relevant concentrations (1-10 nM), pasireotide was at least as efficient as octreotide in inhibiting CgA secretion and cell viability through caspase-dependent apoptosis during short treatments, irrespective of the expression levels of the different sst receptors or the WHO grade of the parental tumor. Interestingly, unlike octreotide, which induces a rapid and persistent partial internalization of sst2 associated with its phosphorylation on Ser341/343, pasireotide did not phosphorylate sst2 and induced a rapid and transient internalization of the receptor followed by a persistent recycling at the cell surface. These results provide the first evidence, to our knowledge, of striking differences in the dynamics of sst2 trafficking in pNET cells treated with the two SSAs, but with similar efficiency in the control of CgA secretion and cell viability.

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.

Salivary stress markers and psychological stress in simulated microgravity: 21 days in 6° head-down tilt

Spaceflight occurs in an environment of temperature extremes, microgravity, solar and galactic cosmic radiation, lack of atmospheric pressure, and high-speed micrometeorites. Exposure to microgravity and the space environment during space missions of short and long duration has important medical and health implications for astronauts. Psychological well-being is of increasing importance in planned spaceflights and interplanetary missions of long duration. The 6° head-down tilt (HDT) is an established method of mimicking low gravity on earth. The aim of the present study was to determine the effects of 21 days of HDT on psychological stress in 12 healthy male volunteers. Psychological state was assessed by the current stress test, and chromogranin-A (CgA), cortisol, alpha-amylase, and beta-endorphin were measured in saliva. After one week of HDT, all volunteers developed psychological stress, and secretion of CgA, cortisol, alpha-amylase, and beta-endorphin were all significantly higher. Thus, 6° HDT appears to be a valid model to induce psychological stress changes in the immune system, changes that might also be encountered by astronauts and cosmonauts during both a short stay in space, such as that required while orbiting a space station, and in longer spaceflights.

Chromogranin A and the tumor microenvironment.

Chromogranin A (CgA) is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of the adrenal medulla and of many other neuroendocrine cells and neurons. This protein is frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors. Circulating CgA is also increased in patients with other diseases, including subpopulations of patients with non-neuroendocrine tumors, with important prognostic implications. A growing body of evidence suggests that CgA is more than a diagnostic/prognostic marker for cancer patients. Indeed, results of in vitro experiments and in vivo studies in animal models suggest that this protein and its fragments can affect several elements of the tumor microenvironment, including fibroblasts and endothelial cells. In this article, recent findings implicating CgA as a modulator of the tumor microenvironment and suggesting that abnormal secretion of CgA could play important roles in tumor progression and response to therapy in cancer patients are reviewed and discussed.

Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids

Bronchial carcinoids (BCs) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. It has been previously demonstrated that everolimus, or RAD001, an mTOR inhibitor, has potent antiproliferative effects in human endocrine tumors. Our aim was to evaluate the possible antiproliferative effects of everolimus in human BCs in primary culture. We collected 24 BCs that were dispersed in primary cultures, treated without or with 1 nM-1 muM everolimus, 10 nM SOM230 (pasireotide, a somatostatin receptor multiligand), and/or 50 nM IGF1. Cell viability was evaluated after 48 h, and chromogranin A (CgA) as well as vascular endothelial growth factor (VEGF) secretion was assessed after 8 h incubation. Somatostatin receptors, mTOR, and AKT expression were investigated by quantitative PCR. We found that in 15 cultures (67.5%), everolimus significantly reduced cell viability (by approximately 30%; P<0.05 versus control), inhibited p70S6K activity (-30%), and blocked IGF1 proliferative effects. Everolimus also significantly reduced CgA (by approximately 20%) and VEGF (by approximately 15%) secretion. Cotreatment with SOM230 did not exert additive effects on cell viability and secretory activity. AKT expression was similar in responder and nonresponder tissues, while mTOR expression was significantly higher in the responder group, which was characterized by higher CgA plasma levels and bigger tumors with higher mitotic index and angiogenesis. Our data demonstrate that everolimus reduces VEGF secretion and cell viability in BCs with a mechanism likely involving IGF1 signaling, suggesting that it might represent a possible medical treatment for BCs.