Cell Carcinoid Tumors Research Articles

LYVE-1 immunocytochemical staining for gastrointestinal carcinoids

By immunocytochemical staining for lymphatic vessels using anti-lymphatic vessel hyaluronan receptor (LYVE-1) antibody, pancreatic islets and some pancreatic endocrine tumours (PETs) were positively stained for the cytoplasm in addition to lymphatic vessels. The current study was extended to investigate possible immunostaining of gastrointestinal carcinoids using goat antihuman LYVE-1 antibody. Lymphatic vessels were immunostained by LYVE-1 and blood vessels were immunostained by factor 8 (F-8). Among 27 gastrointestinal carcinoids, six cases (22%) including five primary and one metastatic carcinoids were positive in the cytoplasm for LYVE-1 in addition to lymphatic vessels and there were abundant lymphatic and blood vessels at the margin of the carcinoids. These six LYVE-1 positive cases consisted of three of 10 small intestinal, one of five appendiceal and two of seven large intestinal carcinoids. There was no LYVE-1 positive staining in the adjacent normal gastro-intestinal mucosa. The presence of LYVE-1 immunostaining in some gastrointestinal carcinoids may support a structure-function relationship of lymphatic vessels/hyaluronan receptor for modulating synthesis and secretion of hormones and amines by carcinoid tumour cells.

Pulmonary Carcinoid Tumor Masquerading as a Synovial Sarcoma

A rare case of pulmonary carcinoid tumor exhibiting morphologically as a monophasic synovial sarcoma is reported. The patient is a 37-year-old man who presented with shortness of breath for several weeks. The chest computed tomographic scan showed a 3.5 cm pulmonary mass in the left lower lobe.The lesion was an oval, tan nodule displaying features of monophasic synovial sarcoma. Immunohistochemical studies were performed and included a carcinoid tumor as a main differential diagnosis. Reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization studies were negative for a molecular evidence of synovial sarcoma. This case further emphasizes the capacity of pulmonary carcinoid tumor cells to show various morphologic expressions even toward a mesenchymal differentiation mimicking a synovial sarcoma.

Extensive Expression of Collapsin Response Mediator Protein 5 (CRMP5) is a Specific Marker of High-grade Lung Neuroendocrine Carcinoma

The diagnosis of high-grade neuroendocrine tumors has strong clinical relevance because it identifies patients at higher risk of an unfavorable outcome who should receive multimodal treatment. However, these tumors can be mistaken for poorly differentiated nonsmall cell carcinoma or carcinoid lung tumors. In fact, no immunohistochemical marker can currently distinguish between histologic lung subtypes. Because the collapsin response mediator protein (CRMP) family is involved in an autoimmune disease associated with small cell lung carcinoma, we explored the relationship between CRMP5 expression and lung tumor behavior. Using World Health Organization morphologic criteria, 123 lung neuroendocrine tumors and 41 randomly selected non-neuroendocrine tumors were classified. CRMP5 protein expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining method. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade neuroendocrine lung tumors, including small cell lung carcinoma and large cell lung neuroendocrine carcinoma, but not in any of the squamous cell carcinomas or lung adenocarcinomas in our series. In contrast, the majority of low-grade neuroendocrine lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with 2 different staining patterns of either scattered positive cells or small foci of positive cells. Our findings point at CRMP5 as a novel marker for routine pathologic evaluation of lung tumors surgical samples in distinguishing between highly aggressive neuroendocrine carcinoma and the other lung cancers.

Autoantibodies to epithelial cells in patients on long-term therapy with leucocyte-derived interferon-alpha (IFN-α)

During routine screening for anti-nuclear antibodies, using rat liver tissue as substrate, a reactivity against bile duct epithelium was observed in sera from carcinoid tumour patients given human leucocyte-derived IFN-alpha (HuLe IFN-alpha). In a retrospective study, initiated by this observation, the development of serum antibodies to bile duct epithelium was observed in nine out of 12 patients with carcinoid tumours and in three out of 14 patients with hairy-cell leukaemia during their treatment with HuLe IFN-alpha. However, no bile duct reactivity was observed in sera from carcinoid or hairy-cell leukaemia in patients given recombinant IFN-alpha. When analysing the reactivity of positive sera against a panel of rat and human tissues, a uniform reactivity was observed against simple epithelial cells lining the gastrointestinal tract, pancreatic secretory ducts, fallopian tube, kidney tubuli, mesothelium and also against carcinoid tumour cells. The mechanisms promoting autoreactivity against this simple epithelial cell autoantigen is so far unknown. The cytoplasmic as well as the restricted staining pattern of simple epithelial cells may indicate autoreactivity against certain cytoskeletal intermediate filaments, such as cytokeratin 19, 18 and 8, known to be exclusively present in simple epithelial cells and tumours derived from them.

Endoscopic Decision of Complete Resection Is More Practical Than Pathologic Decision in the Endoscopic Resection for Rectal Carcinoids

Background/Aim: Rectal carcinoids are often described as low-grade malignancy and are usually treated by endoscopic resection. However, reports on the outcomes of endoscopic resection for rectal carcinoids remain limited to several single-institution studies. We reviewed the experiences of endoscopic resection for rectal carcinoids in Korea. Methods: This study retrospectively analyzed the medical records of 409 patients with rectal carcinoid at 14 university hospitals in Korea from January 1999 until June 2007 in the aspects of age, sex, tumor size, complete resection, invasion, recurrence, and complications. Residual disease was determined when carcinoid tumor cells were detected histologically at the resection site in 6 months after endoscopic resection. Results: The mean age at diagnosis was 50 years (range 15-90). Male to female ratio was 1.1: 1. The rectal carcinoids were measured 7.66 ± 4.96 mm in maximal diameter. Three hundred fifty-four patients were treated with endoscopic resection, whereas 55 patients underwent operation. In the endoscopic resection group, complete resection rates were 88.9% based on the endoscopic appearance (CR-E) and 59.3% based on the pathologic evaluation (CR-P). The agreement between CR-E and CR-P was low (κ = 0.188). In 83 of 88 patients (94%) whose disease were defined CR-E but were not achieved CR-P, residual diseases were not found on 6 months follow-up endoscopic biopsy. Complications of endoscopic resection occurred in 13 patients, but were managed endoscopically in all cases. The median follow-up period was 26 months (range 6-147). Local recurrence developed in 2 of 210 patients who had complete resection. One patient was treated with a second endoscopic resection, and the other patient underwent operation. No recurrence has been reported until 26 months follow-up after the second endoscopic resection and operation. The 3-year overall survival rate was 100%. There were 14 patients with metastasis at the time of diagnosis. Independent factors predicting metastasis were age (p = 0.0329), tumor size (p < 0.001), central depression (p < 0.001), ulcer (p < 0.001), lymphatic invasion (p < 0.001), and venous invasion (p < 0.001) in multivariate analysis. Conclusions: Endoscopic resection is an effective therapeutic modality for selected cases of rectal carcinoids (≤10 mm). Discrepancies were observed between CR-E and CR-P, but CR-E better predicted complete resection state probably because endoscopic cauterization might have eradicated minimal tumor cells close to the resected margin. Patients with age, tumor size >10 mm, central depression, ulcer, and lymphatic or vascular invasion have higher metastatic potential.

Germ Cell Origin of Testicular Carcinoid Tumors

Carcinoids are neuroendocrine tumors and most frequently occur within tissues derived from the embryonic gut. These tumors can occur in any organ site but are rare in the testis. The cell type giving rise to testicular carcinoid is unknown. We hypothesized that testicular carcinoid may have a germ cell origin. We describe our analysis of protein and genetic markers of germ cell neoplasia, using immunohistochemistry and fluorescence in situ hybridization, in four testicular carcinoid tumors. All four cases of testicular carcinoid tumor arose in a background of mature teratoma. Isochromosome 12p was identified in carcinoid tumor cells in all four samples. 12p overrepresentation was also observed in three cases. Isochromosome 12p and 12p overrepresentation were present in cells of coexisting mature teratoma in three cases. Carcinoid tumors showed strong immunoreactivity for synaptophysin and chromogranin, but no immunoreactivity for OCT4, CD30, c-kit, TTF-1, and CDX2. Membranous and cytoplasmic staining for beta-catenin was detected in three cases. Our findings suggest that testicular carcinoid represents a phenotypic expression of testicular teratoma and is of germ cell origin.

Antitumor activity of rapamycin and octreotide as single agents or in combination in neuroendocrine tumors

The mammalian target of rapamycin (mTOR) signaling pathway has emerged as a promising target for cancer therapy. Rapamycin inhibits mTOR activity but induces upstream signaling, leading to Akt activation, potentially limiting antitumor activity. Octreotide, a somatostatin analog, decreases phosphatidylinositol-3-kinase/Akt signaling in some models, and thus theoretically may enhance rapamycin's antitumor activity. The aim of this study was to determine the antitumor activity of rapamycin and octreotide as single agents and in combination in neuroendocrine tumors. In carcinoid cell lines BON-1 and NCI-H727, cell proliferation was significantly inhibited by rapamycin in vitro, although rapamycin treatment did lead to Akt phosphorylation. Octreotide had limited antiproliferative effects alone, and did not demonstrate synergistic or additive interactions with rapamycin. Furthermore, octreotide did not overcome rapamycin-induced Akt phosphorylation. In vivo, rapamycin alone caused significant tumor suppression. Octreotide alone did not inhibit in vivo tumor growth and did not enhance rapamycin-mediated growth inhibition. In conclusion, rapamycin causes significant growth inhibition in carcinoid tumor cell lines in vitro and in vivo, thus mTOR is a promising therapeutic target for neuroendocrine tumors. Octreotide does not enhance the efficacy of rapamycin's antiproliferative effects in the models tested, and does not inhibit rapamycin-mediated feedback activation of Akt. Further study is needed in order to determine whether octreotide or other somatostatin analogs enhance the efficacy of mTOR inhibitors in other models.

Physician Recommendations to Patients Taking Chronic Systemic Glucocorticoid Therapy.

Multiple endocrine neoplasia type 1 (MEN 1) is a rare autosomal dominant disorder with variable expressivity. A kindred of four Newfoundland families with this syndrome was identified in the 1970s and this has been called MEN1 Burin. A mutation R460X in the menin gene was identified in 1997. Affected individuals present with primary hyperparathyroidism, pituitary adenomas, pancreatic islet cell tumours, carcinoid tumors and other manifestations. Previous work has shown a higher than expected prevalence of pituitary adenomas and carcinoid tumors and a lower than expected prevalence of pancreatic islet cell tumors compared to the general MEN 1 literature. However, it was suspected from recent clinical experience that the incidence of pancreatic tumors in the MEN 1 Burin kindred may be higher than originally described. Members of the MEN1 Burin kindred were identified thorough previous work by Dr J. Green and the MEN/VHL clinic of the Endocrinology and Medical Genetics departments, and their medical records reviewed. The relative prevalence of the different clinical manifestations of MEN1 including pancreatic islet cell tumors were calculated to define the phenotype of MEN1 Burin kindred living in Newfoundland and any changes in the natural history of MEN1 Burin. To date 121 patients from the MEN 1 Burin kindred have been identified with the mutation R460X. Of the 116 patients with records available, 101 patients are clinically affected. There have been 97 individuals with primary hyperparathyroidism, 38 pituitary adenomas, 31 pancreatic tumors, and 11 carcinoid tumors. Members of the MEN 1 Burin kindred with the R460X mutation have a prevalence of primary hyperparathyroidism of 96%, pituitary adenoma of 38%, pancreatic islet cell tumors of 31%, and carcinoid tumors of 11%. There was a higher prevalence of pancreatic islet cell tumors in the MEN1 Burin kindred than previously reported but still not as high as the prevalence of pancreatic islet cell tumors in the general MEN 1 literature. ABSTRACT #182

CD8+ T cells against multiple tumor-associated antigens in peripheral blood of midgut carcinoid patients.

The aim of the study was to identify immunogenic HLA-A*0201-binding epitopes derived from a number of classical midgut carcinoid-associated proteins. CD8(+) T cells recognizing tumor-associated antigen (TAA) epitopes are of great interest for the establishment of immunotherapy as a novel treatment for this type of malignancy. Midgut carcinoid tumor specimens were microdissected and expression levels of potential TAAs were investigated by quantitative real time PCR. HLA-A*0201-binding motifs were selected using HLA peptide binding prediction algorithms and stabilization of HLA-A*0201 was verified using TAP-deficient T2 cells. Peripheral blood of midgut carcinoid patients was analyzed for peptide epitope recognition and the feasibility of generating peptide-reactive CD8(+) T cells in healthy blood donors was examined by an in vitro stimulation protocol using mature DCs. Activation of patient and healthy donor CD8(+) T cells was analyzed by intracellular flow cytometry staining of interferon gamma. Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), and islet autoantigen 2 (IA-2) are properly expressed by midgut carcinoid tumor cells, with CGA mRNA expressed to greatest level. Midgut carcinoid patients have increased frequencies of peripheral blood CD8(+) T cells recognizing a pool of HLA-A*0201 peptides derived from these proteins compared to healthy age-matched individuals. Activated peptide-specific CD8(+) T cells could also be generated in healthy blood donors by in vitro stimulation. We have identified a number of immunogenic midgut carcinoid-associated peptide epitopes recognized by CD8(+) T cells. We show that midgut carcinoid patients display immune recognition of their tumors. Memory CD8(+) T cells in patient blood are of great interest when pursuing an immunotherapeutic treatment strategy.

The Development and Characterization of a Human Midgut Carcinoid Cell Line

Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents. We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2). Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1 cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets. CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors in mice. Electron microscopy revealed round, pleomorphic, electron-dense neurosecretory granules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small "salt-and-pepper" nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-beta, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors 1 to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations. The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.

Valproic Acid Activates Notch-1 Signaling and Regulates the Neuroendocrine Phenotype in Carcinoid Cancer Cells

Carcinoid tumors are neuroendocrine malignancies that frequently metastasize and secrete hormones that cause debilitating symptoms in patients. In this study we report the effects of valproic acid (VPA), a drug long used for the treatment of epilepsy, on the growth and neuroendocrine phenotype of human carcinoid cancer cells. VPA treatment of gastrointestinal and pulmonary carcinoid cells resulted in a dose-dependent inhibition of cancer cell growth. Western blot analysis revealed degradation of cyclin D1 and an increase in cyclin-dependent kinases p21 and p27 with VPA treatment. Flow cytometry confirmed that the mechanism of VPA-induced growth inhibition is G(1) phase cell cycle arrest. Furthermore, VPA suppressed expression of the neuroendocrine tumor marker chromogranin A. In addition to these effects, VPA also increased levels of full-length Notch-1 and the active Notch-1 intracellular domain. Luciferase reporter assays incorporating the centromere-binding factor 1 (CBF-1) binding site and the achaete-scute complex-like 1 (ASCL-1) promoter confirmed the functional activity of VPA-induced Notch-1. Transfection of Notch-1 small-interfering RNA into carcinoid tumor cells blocked the effects of VPA on Notch-1 activation, ASCL-1 suppression, p21 induction, and cell growth inhibition. VPA also suppressed growth of carcinoid tumors in vivo in a mouse tumor xenograft experiment. These findings confirm the important role of Notch-1 in regulating the growth and neuroendocrine phenotype of carcinoid tumor cells. On the basis of this study, a clinical trial of VPA for patients with advanced carcinoid cancer will be conducted. Disclosure of potential conflicts of interest is found at the end of this article.

P76: Carcinoid tumor cell growth suppression directly relates to levels of Notch1 expression

Background: Gastrointestinal (GI) carcinoid tumors are characterized by the secretion of neuroendocrine (NE) hormones such as chromogranin A (CgA) and serotonin, which can cause debilitating symptoms. Activation of certain cell signaling pathways has been shown to suppress tumor growth and reduce NE hormone secretion. We have previously established that the activation of the Notch1/hairy enhancer of split 1 (HES-1)/achaete-scute homolog 1 (ASCL1) pathway leads to a reduction in NE tumor cell proliferation.

Extrapulmonary Small Cell Carcinoma of Liver

We report a case of extra pulmonary small cell carcinoma of the liver. Neuroendocrine tumors include all tumors exhibiting neuroendocrine characteristics, such as small cell carcinoma, carcinoid tumor and islet cell tumor of pancreas. Neuroendocrine tumors can involve various organs such as lungs, G.I. tract, pancreas, endometrium and ovaries. Small cell carcinoma has a high incidence of early dissemination due to its aggressive nature. Small cell carcinoma can be of pulmonary and extrapulmonary origin. Extra pulmonary small cell carcinoma mainly affects middle aged or elderly population, with greater than 70% of affected patients being older than age 50. Extra pulmonary small cell carcinoma is an uncommon malignancy that shares many of the histologic features of pulmonary small cell carcinoma. So far, approximately 55 cases of primary hepatic neuroendocrine tumors have been reported in English literature. Chart review and review of literature using Medline and relevant bibliographies. A 68 year old man presented with abdominal pain, nausea, vomiting, jaundice and weight loss. Liver panel was consistent with cholestatic jaundice. Ultrasound of abdomen revealed heterogenous and increased echogenicity of the liver with hepatosplenomegaly. CT scan of the abdomen demonstrated enlarged liver with heterogeneous parenchyma. EGD, ERCP and Colonoscopy were negative. Liver biopsy revealed a focus of malignant cells which were positive for synaptophysin and TTf-1 and negative for chromogranin. Post liver biopsy work up included CT scan of chest which revealed atelectasis with no discrete lesions. Extensive work up did not reveal involvement of any other site. Our patient decided not to undergo chemotherapy and chose to be hospice care. Clinical progression of his disease was rapid and he died within two months of diagnosis. Primary hepatic neuroendocrine tumor is very rare. This diagnosis is considered after an occult primary neruoendocrine malignancy outside the liver has been excluded by intensive search. We report a rare case of extrapulmonary small cell carcinoma of liver presenting as cholestatic jaundice. [figure 1]Figure 1

Effect of pan-inhibitory somatostatin analogues, topotecan and bortezomib on the human enterochromaffin neoplastic cell line KRJ-I

13144 Background: Effective therapeutic options to inhibit the growth and limit the spread of carcinoid tumors are limited. Somatostatin (SST) analogs that target specific SST receptors, though often effective at alleviating the symptoms of carcinoid disease, have limited cytostatic effects on carcinoid tumor cells. The anti-proliferative activity of 2 pan-inhibitory SST analogues and lanreotide were evaluated using the human small intestinal carcinoid cell line KRJ-I. The effects of novel cytotoxic agents used in Phase II carcinoid clinical trials, topotecan (topoisomerase-I inhibitor) and bortezomib (proteasomal inhibitor), were also measured. Methods: The SST receptor profile of KRJ-1 cells was determined using hybridization to U133A chips and confirmed by real time PCR with Taqman probes for SSTR1–5. KRJ-1 cells in serum-free medium were treated with 100 pM to 10 μM of the SST pan inhibitory analogs, BIM-23A719 and BIM-23A779, or lanreotide (BIM-23014C) and 1nM to 1 μM of topotecan or bortezomib for 72 hrs. The 3-(4,5-dimethylthiazole-2 yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to evaluate proliferation which was expressed relative to control cells (no agent added). Results: KRJ-I cells co-express mRNA for SSTR2 and SSTR3 (mean levels ± SEM 0.48 ± 0.13 and 0.73 ± 0.19 copy/copy GAPDH respectively). BIM-23A719 inhibited growth maximally (15 ± 2% of control) at ICmax = 10 pM, whereas BIM-23A779 inhibited growth maximally (11 ± 1% of control) at 100 pM. Lanreotide had no significant inhibitory effect on cell growth and resulted in increased MTT conversion at high concentrations (10 μM). Topotecan and bortezomib inhibited growth maximally at 23 ± 1.5%; ICmax = 50 nM and 32 ± 1.6%; ICmax = 50 nM respectively. Conclusions: Cytotoxic agents are more effective but less sensitive than pan-inhibitory somatostatin analogues for the inhibition of proliferation of the small intestinal carcinoid KRJ-I cell line. The specific SSTR2/3/5 analogue, lanreotide, had no inhibitory effect on KRJ-I proliferation. These results suggest that the KRJ-I cell line is more sensitive to analogues targeting a pan-somatostatin receptor profile than cytotoxic agents. The former may have potential as adjuvant agents for treatment of GI carcinoid tumors. [Table: see text]

Activation of MEK1, a Raf-1 effector, results in morphologic transdifferentiation in pancreatic carcinoid tumor cells

Background: We have previously reported that activation of the Raf-1 signal transduction pathway in carcinoid tumor cells results in dramatic changes in cell morphology as well as suppression of neuroendocrine marker and hormone levels. The mechanism by which Raf-1 activation causes these changes has not been determined. We have previously shown that the protein kinase MEK1 is phosphorylated in response to Raf-1 signaling. We have also reported that inhibition of MEK1 blocks the Raf-1-mediated morphology changes and hormone suppression. Therefore, we hypothesized that MEK1 is an important Raf-1 effector, and that activation of MEK1 is sufficient to cause morphologic transdifferentiation in carcinoid cells. Methods: Human pancreatic carcinoid (BON) cells were stably transduced with a doxycycline-inducible MEK1 construct, creating BON-MEK1 cells. BON and BON-MEK1 cells were then treated with either control or doxycycline at various concentrations (0.1 - 2μg/ml). Western blots were used to confirm the expression of MEK1 and phosphorylated-MEK1/2 proteins. Morphologic changes were evaluated with light microscopy. Results: Western blots using antibodies against phosphorylated and unphosphorylated MEK1 confirmed a dose-dependent increase in protein expression in BON-MEK1 cells treated with doxycycline. BON cells treated with control and doxycycline and BON-MEK1 cells treated with control were all identical in appearance. However, BON-MEK1 cells treated with doxycycline underwent dramatic morphologic changes. The treated BON-MEK1 cells became flatter and developed much sharper cellular borders, mimicking cellular differentiation. Conclusions: Activation of MEK1 in human pancreatic carcinoid tumor cells leads to prominent phenotypic changes that resemble differentiation of gastrointestinal carcinoid cells in vitro. MEK1, therefore, is an important effector in the Raf-1signal transduction pathway, and is sufficient to cause morphological transdifferentiation in carcinoid tumor cells.

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.

The Notch signaling effector Hes1 in gastrointestinal carcinoid tumor cells oncology

The Notch signaling effector Hes1 in gastrointestinal carcinoid tumor cells oncology

Hairy Enhancer of Split-1 (HES-1), a Notch1 effector, inhibits the growth of carcinoid tumor cells

The Notch1-signaling pathway has been shown to regulate the differentiation and growth of carcinoid tumor cells. However, the molecules that mediate Notch1 signaling, as well as their potential roles in regulating the growth of carcinoid tumors, have not been characterized. We and others have shown previously that the transcription factor Hairy Enhancer of Split-1 (HES-1) is upregulated in response to Notch1 signaling, demonstrating that it is a Notch1 effector. We hypothesized that HES-1 may be the essential downstream factor in Notch1-mediated growth regulation of carcinoid tumors. H727 carcinoid tumor cells were transduced stably with a doxycycline-inducible HES-1 construct, creating H727-HES-1 cells. H727-TRE (vector-only control) and H727-HES-1 cells were then treated with varying concentrations of doxycycline to achieve increasing levels of HES-1 protein expression. Cell proliferation was determined with the use of a cell viability assay. Treatment of H727-HES-1 cells with increasing dosages of doxycycline resulted in dose-dependent increases in HES-1 protein by Western blot analysis. Importantly, induction of HES-1 in carcinoid tumor cells led to suppression of tumor cellular proliferation. Moreover, the degree of carcinoid growth inhibition appeared to be proportional to the level of HES-1 induction. HES-1 alone can regulate the growth of carcinoid tumor cells. Furthermore, these results suggest that HES-1 may be the critical downstream effector in the Notch1-signaling pathway.

Conservation of the Notch1 signaling pathway in gastrointestinal carcinoid cells

Gastrointestinal (GI) carcinoid cells secrete multiple neuroendocrine (NE) markers and hormones including 5-hydroxytryptamine and chromogranin A. We were interested in determining whether activation of the Notch1 signal transduction pathway in carcinoid cells could modulate production of NE markers and hormones. Human pancreatic carcinoid cells (BON cells) were stably transduced with an estrogen-inducible Notch1 construct, creating BON-NIER cells. In the present study, we found that Notch1 is not detectable in human GI carcinoid tumor cells. The induction of Notch1 in human BON carcinoid cells led to high levels of functional Notch1, as measured by CBF-1 binding studies, resulting in activation of the Notch1 pathway. Similar to its developmental role in the GI tract, Notch1 pathway activation led to an increase in hairy enhancer of split 1 (HES-1) protein and a concomitant silencing of human Notch1/HES-1/achaete-scute homolog 1. Furthermore, Notch1 activation led to a significant reduction in NE markers. Most interestingly, activation of the Notch1 pathway caused a significant reduction in 5-hydroxytryptamine, an important bioactive hormone in carcinoid syndrome. In addition, persistent activation of the Notch1 pathway in BON cells led to a notable reduction in cellular proliferation. These results demonstrate that the Notch1 pathway, which plays a critical role in the differentiation of enteroendocrine cells, is highly conserved in the gut. Therefore, manipulation of the Notch1 signaling pathway may be useful for expanding the targets for therapeutic and palliative treatment of patients with carcinoid tumors.

ZM336372, a Raf-1 activator, suppresses growth and neuroendocrine hormone levels in carcinoid tumor cells

Neuroendocrine tumors, such as carcinoids, are highly metastatic neoplasms that secrete bioactive hormones resulting in carcinoid syndrome. Few curative treatments exist outside of surgical resection. We have previously shown that activation of the Raf-1 signaling pathway can suppress hormone production in carcinoid tumor cells. In this study, we investigated a novel treatment for carcinoid tumor cell growth based on pharmacologic Raf-1 activation using the compound ZM336372. Treatment of carcinoid tumor cells with ZM336372 resulted in progressive phosphorylation of Raf-1, mitogen-activated protein kinase 1/2, and extracellular signal-regulated kinase 1/2. Importantly, exposure to ZM336372 resulted in a significant reduction of bioactive hormone levels as well as the transcription factor, human achaete-scute homologue-1 in carcinoid tumor cells. Furthermore, treatment with ZM336372 led to a marked suppression of cellular proliferation and induction of the cell cycle inhibitors p21 and p18. In summary, ZM336372 targets both proliferation and palliative issues associated with carcinoid tumor cells, and therefore, warrants further investigation as a possible therapeutic strategy for patients with carcinoid tumors.