NEUROPEPTIDE-Y AND NEUROPEPTIDE-Y 3-36 - ISOLATION FROM HUMAN PANCREATIC ENDOCRINE TUMORS

Using an antiserum raised to the C-terminal region of neuropeptide Y (NPY) which does not cross-react with pancreatic polypeptide (PP), immunoreactivity has been detected in two different endocrine tumours of the human pancreas in concentrations permitting isolation and structural analysis. In a clinically-typical gastrinoma, resected from the head of pancreas, the concentration of NPY immunoreactivity was 3.4 nmol/g. Reverse phase HPLC analysis of extracts of this tumour resolved a single immunoreactive peptide coeluting with synthetic human NPY. The molecular mass of the isolated peptide, determined by mass spectroscopy, was 4270 Da, which was in close agreement with that derived from the deduced primary structure of human tumour NPY (4271.7 Da), obtained by gas-phase sequencing. A somatostatinoma, resected from the region of the ampulla of Vater, contained 3.8 nmol/g of NPY immunoreactivity and isolation of this immunoreactive peptide followed by structural analyses, indicated a molecular structure consistent with NPY 3-36. These data suggest that NPY immunoreactivity detected in human pancreatic endocrine tumours is molecularly heterogenous, a finding which may be of relevance in the symptomatology of such tumours as attenuation of the N-terminus of this peptide generates receptor selectivity.

THE CORRECTED PRIMARY STRUCTURE OF CHICKEN (AVIAN) PANCREATIC-POLYPEPTIDE

Chicken (avian) pancreatic polypeptide was the first member of the pancreatic polypeptide (PP)/neuropeptide Y (NPY) superfamily to be discovered and structurally-characterised. In this 36 amino acid residue, C-terminally amidated peptide, residues 22 and 23 were identified as Asp and Asn, respectively. However, sequencing of chicken PP using modem automated gas-phase sequencing technology has revealed that the original primary structure is incorrect in that residue 22 is Asn and that residue 23 is Asp. After digestion of chicken PP with endoproteinase Asp-N, fragments of chicken PP corresponding in molecular mass to residues 16-22 and 23-36, were unequivocally identified. The corrected primary structure of chicken PP is therefore: Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asn-Asp-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.

RABBIT PANCREATIC-POLYPEPTIDE

1. In almost all studies involving localization or quantitation of regulatory peptides, an essential prerequisite is the generation of specific antisera in rabbits. Despite this almost universal practice, the primary structures of some established regulatory peptides, such as pancreatic polypeptide (PP), of the rabbit, remain unknown.

THE PRIMARY STRUCTURE OF PANCREATIC-POLYPEPTIDE FROM A PRIMITIVE INSECTIVOROUS MAMMAL, THE EUROPEAN HEDGEHOG (ERINACEOUS-EUROPAEUS)

Pancreatic polypeptide (PP) has been isolated from extracts of the pancreas of the European hedgehog (Erinaceous europaeus) which is a representative of the order Insectivora, deemed to be the most primitive group of placental mammals. Pancreatic tissues were extracted in acidified ethanol and the peptide was purified chromatographically using a PP C-terminal hexapeptide amide specific radioimmunoassay to monitor purification. Two major PP-immunoreactive peptides were baseline-resolved following the final analytical reverse phase HPLC fractionation. Each was separately subjected to plasma desorption mass spectroscopy (PDMS) and gas-phase sequencing. The molecular masses of each peptide were similar: (I) 4237.6 +/- 4 Da and (II) 4238.2 +/- 4 Da. The full primary structures of each peptide were deduced and these were identical: VPLEPVYPGDNATPEQMAHYAAELRRYINMLTRPRY. The peptides were deemed to be amidated due to their full molar cross-reactivity with the amide-requiring PP antiserum employed in radioimmunoassay. The molecular mass (4233.8 Da) calculated from the sequence was in close agreemeent with PDMS estimates and the reason for the different retention times of each peptide is unknown at present. Hedgehog PP exhibits only 2 unique amino acid substitutions, at positions 1 (Val) and 19 (His), when compared with other mammalian analogues.

Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum

Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspase-and mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH 2-terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 a-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER. © 2012 Macmillan Publishers Limited All rights reserved.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.