Inhibition of angiogenesis induces chromaffin differentiation and apoptosis in neuroblastoma.
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Inhibition of angiogenesis has been shown to reduce tumor growth, metastasis, and tumor microvascular density in experimental models. To these effects we would now like to add induction of differentiation, based on biological analysis of xenografted human neuroblastoma (SH-SY5Y, WAG rnu/rnu) treated with the angiogenesis inhibitor TNP-470. Treatment with TNP-470 (10 mg/kg s.c., n = 15) reduced the tumor growth by 66% and stereological vascular parameters (Lv, Vv, Sv) by 36-45%. The tumor cell apoptotic fraction increased more than threefold, resulting in a decrease in viable tumor cells by 33%. In contrast, the mean vascular diameter (29 microm) and the mean tumor cell proliferative index (49%) were unaffected. TNP-470-treated tumors exhibited striking chromaffin differentiation of neuroblastoma cells, observed as increased expression of insulin-like growth factor II gene (+88%), tyrosine hydroxylase (+96%), chromogranin A, and cellular processes. Statistical analysis revealed an inverse correlation between differentiation and angiogenesis. It is suggested that by inhibiting angiogenesis, TNP-470 induces metabolic stress, resulting in chromaffin differentiation and apoptosis in neuroblastoma. Such agonal differentiation may be the link between angiostatic therapy and tumor cell apoptosis. (+info)
Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model.
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It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G0-phase-arrested cells. Here we further characterized NE differentiation, androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0%, and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgA-positive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days, respectively. However, no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G0-phase-arrested cells. (+info)
Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation.
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OBJECTIVE: Islet amyloid polypeptide (IAPP), also named amylin, is the predominant protein component of amyloid deposits in human islet beta cell tumours of the pancreas (insulinomas). IAPP is co-produced with insulin by islet beta cells. We investigated IAPP expression in relation to insulin expression and to amyloid formation in eleven insulinomas. DESIGN AND METHODS: RNA and protein extracts were prepared from the same pieces of tumour tissue, and from specimens of two normal human pancreata. IAPP and insulin mRNA and peptide content were quantified using Northern blot analysis and radioimmunoassay (RIA) respectively. Molecular forms of IAPP immunoreactivity were analysed by reversed-phase high-performance liquid chromatography (HPLC). The presence of islet hormones and of amyloid was assessed by (immuno)histochemical staining of paraffin sections. Plasma levels of IAPP and insulin prior to tumour resection were determined by RIA. RESULTS: IAPP and insulin mRNA and peptide content varied widely between the tumour specimens, and there was considerable intratumour heterogeneity of peptide content. HPLC analysis indicated correct proteolytic processing of the IAPP precursor protein. Amyloid deposits were detected only in the three tumours with the highest IAPP content. In contrast to insulin, plasma levels of IAPP were not elevated in the insulinoma patients. CONCLUSIONS: The spectrum of hormone production by insulinomas cannot be inferred from only a few tissue sections due to intratumour heterogeneity. Expression of the IAPP and insulin genes is not coupled in insulinomas, which produce properly processed mature IAPP. In addition to IAPP overproduction, additional factors such as intracellular accumulation of IAPP are involved in amyloidogenesis in insulinomas. (+info)
Library of sequence-specific radioimmunoassays for human chromogranin A.
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BACKGROUND: Human chromogranin A (CgA) is an acidic protein widely expressed in neuroendocrine tissue and tumors. The extensive tissue- and tumor-specific cleavages of CgA at basic cleavage sites produce multiple peptides. METHODS: We have developed a library of RIAs specific for different epitopes, including the NH2 and COOH termini and three sequences adjacent to dibasic sites in the remaining part of CgA. RESULTS: The antisera raised against CgA(210-222) and CgA(340-348) required a free NH2 terminus for binding. All antisera displayed high titers, high indexes of heterogeneity ( approximately 1.0), and high binding affinities (Keff0 approximately 0.1 x 10(12) to 1.0 x 10(12) L/mol), implying that the RIAs were monospecific and sensitive. The concentration of CgA in different tissues varied with the assay used. Hence, in a carcinoid tumor the concentration varied from 0.5 to 34.0 nmol/g tissue depending on the specificity of the CgA assay. The lowest concentration in all tumors was measured with the assay specific for the NH2 terminus of CgA. This is consistent with the relatively low concentrations measured in plasma from carcinoid tumor patients by the N-terminal assay, whereas the assays using antisera raised against CgA(210-222) and CgA(340-348) measured increased concentrations. CONCLUSION: Only some CgA assays appear useful for diagnosis of neuroendocrine tumors, but the entire library is valuable for studies of the expression and processing of human CgA. (+info)
Serum gastrin and chromogranin A during medium- and long-term acid suppressive therapy: a case-control study.
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BACKGROUND: Serum chromogranin A (CgA) is regarded as a reliable marker of neuroendocrine proliferation. We previously described increased serum CgA levels during short-term profound gastric acid inhibition. AIM: To investigate serum gastrin and CgA levels in dyspeptic patients during continuous medium- (6 weeks to 1 year), or long-term (1-8 years) gastric acid suppressive therapy. PATIENTS AND METHODS: 114 consecutive dyspeptic patients referred for upper gastrointestinal endoscopy were enrolled in a cross-sectional, case-control study [62 patients on continuous antisecretory therapy, either with proton pump inhibitors (n = 47) or H2-receptor antagonists (H2RA) (n = 15) for gastro-oesophageal reflux disease with or without Barrett's oesophagus or functional dyspepsia, and 52 age- and sex-matched patients without medical acid inhibition and with normal endoscopic findings (control group)]. Omeprazole doses ranged from 20 mg to 80 mg daily and ranitidine from 150 mg to 450 mg daily. Fasting serum CgA and serum gastrin levels were measured by radioimmunoassay (reference values: serum CgA < 4.0 nmol/L; serum gastrin < 85 ng/L). RESULTS: Fasting serum CgA levels positively correlated with serum gastrin in the entire study population (r = 0. 55, P = 0.0001). Median serum CgA values were higher in patients treated with a proton pump inhibitor than H2RA [2.8 (2.0-5.9) nmol/L vs. 2 (1.9-2.3) nmol/L, P < 0.002] and controls [2.8 (2.0-5.9) nmol/L vs. 1.8 (1.5-2.2) nmol/L, P < 0.0001) and did not differ between patients treated with H2RA or controls. Serum gastrin and CgA levels in patients on proton pump inhibitor therapy positively correlated with the degree and duration of acid inhibition. Patients on long-term proton pump inhibitor therapy had significantly higher fasting serum gastrin and CgA than those on medium-term proton pump inhibitor therapy [127 (73-217) ng/L vs. 49 (29-78) ng/L, P < 0.0001 and 4.8 (2.8-8) ng/L vs. 2.1 (1.9-2.6) ng/L, P < 0.001]. No such relation was found in patients on medium- vs. long-term H2RA. Overall, patients with positive Helicobacter pylori serology had higher serum gastrin and CgA levels than those with negative H. pylori serology [51 (27-119) ng/L vs. 27 (14-79) ng/L, P = 0.01, 2.4 (1.9-3.4) nmol/L vs. 2.0 (1.7-2.5) nmol/L, P = 0.05]. CONCLUSIONS: During long-term continuous proton pump inhibitor treatment, serum gastrin and CgA levels are significantly elevated compared to H2RA treatment and nontreated dyspeptic controls. H. pylori infection seems to affect gastric ECL cell secretory function. Increased serum CgA values during long-term profound gastric acid inhibition could reflect either gastric enterochromaffin-like cell hyperfunction or proliferative changes. (+info)
Immunohistochemical characterization of pancreatic tumors induced by dimethylbenzanthracene in rats.
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Dimethylbenzanthracene (DMBA) induces pancreatic adenocarcinomas in rats 9 months after carcinogen exposure, with precursor lesions (tubular complexes) developing 1 month after initiation of treatment. Because previous studies have suggested an acinar cell of origin for these tumors, we investigated the expression pattern of ductal, acinar, and islet cell markers in these cancers to gain insight into their phenotype and cell of origin. Pancreatic neoplasms were induced in rats by implantation of DMBA into the head of the pancreas. Lesions studied included 10 early tubular complexes (DMBA for 2 weeks), 8 tubular complexes (DMBA for 1 month), and 10 adenocarcinomas (DMBA for 9 months). Normal rat pancreas served as a control. For comparison, 5 human ductal adenocarcinomas were also evaluated. Immunohistochemistry with ductal (keratin, cytokeratin 19, cytokeratin 20), acinar (chymotrypsin), and islet (chromogranin A) cell markers was performed to analyze the tissues. Rat tubular complexes and adenocarcinomas revealed strong expression of keratin, cytokeratin 19, and cytokeratin 20 in the cytoplasm of all neoplastic cells, absence of chymotrypsin, and rare immunoreactivity to chromogranin A. Human adenocarcinomas showed strong expression of keratin and cytokeratin 19 in all neoplastic cells, expression of cytokeratin 20 in 5-20% of cells, and absence of chymotrypsin and chromogranin A. Pancreatic adenocarcinomas induced by DMBA in rats express markers consistent with a ductal phenotype, as observed in human tumors. Ductal marker expression in early tumor stages suggests a ductal cell of origin. (+info)
A case of synchronous double primary lung cancer with neuroendocrine features.
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We report a case of unique double primary lung cancers with neuroendocrine features in a 63-year-old male smoker. The mass in the left lower lobe (LLL) was a small cell/large cell carcinoma with spindle cell sarcomatous areas and organoid structure. The mass in the left upper lobe (LUL) was a tubular adenocarcinoma with neuroendocrine features including organoid nests showing occasional rosette formation, nuclear palisading in the periphery of the nests and positive immunoreaction for CD56, chromogranin A and synaptophysin. The difference in histological structures between the two masses led us to diagnose double primary lung cancer. The combination of small cell lung carcinoma and spindle cell carcinoma is very uncommon. The relationship between LLL and LUL tumors remains unclear. Multiple lung cancers with neuroendocrine features have only rarely been reported in the literature. The patient in our case died of widespread cancer 2 years and 4 months after the surgery without adjuvant chemotherapy, a longer postoperative survival time than in cases of ordinary extensive small cell lung cancer. Multiple lung cancers with neuroendocrine features are extremely rare and similar cases have not been reported in the literature. Neuroendocrine differentiation has attracted widespread attention and, therefore, examining neuroendocrine features in lung cancers is important. (+info)
A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245).
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Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198-245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145-245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation. It has been proved to be efficient in measuring CgA levels in patients with neuroendocrine tumours. (+info)