Isolation and Characterization of Pancreatic Cancer (PC) Diabetogenic Factor: A 14 Aminoacids Peptide Corresponding to the N-Terminal Sequence of a S-100 Calcium Binding Protein
Greco E1, Basso D1, Fogar P2, Pucci P4, Flagiello A4, Baldo G2, Giunco S2, Navaglia F1, Zambon CF2, Falda A1, Valerio A3, Pedrazzoli S2, Plebani M1
Departments of 1Laboratory Medicine, 2Medical and Surgical Sciences, 3Clinical and Experimental Medicine, University of Padua. Padua, Italy. 4CEINGE Advanced Biotechnologies and Department of Organic Chemistry and Biochemistry, University of Naples. Naples, Italy
ABSTRACT
Background PC-associated diabetes is consequent to the action of a tumor peptide less than 10,000 Da.
Aim To identify the PC-associated diabetogenic peptide.
Methods Tumor homogenates from PC patients with (n=15) or without (n=8) diabetes, and normal pancreas homogenates (n=6) were subjected to 16.5% SDS-PAGE. After Comassie staining a band of approximately 1,500 Da was evidenced in tumor tissues only from diabetic PC. This band was cutted and sequenced by automatic Edman degradation.
Results The sequence obtained revealed a 14 aa peptide of 1589,88 Da, corresponding to the N-terminal sequence of an S-100 calcium binding protein. This peptide was synthesised and its effects on glucose metabolism were tested in cultured C2C12 myoblasts. These cells were cultured with different amounts (from 1 nmol/L to 2 mmol/L) of the 14 aa peptide. Glucose and lactate were measured in the supernatants after 24, 48 and 72 hours of incubation. In control myoblasts glucose concentration declined from 21.50±0.48 mmol/L (mean±SE) to 6.3±0.56, while lactate increased from 3.20±0.08 mmol/L to 34.50±1.24 after 72 hours of incubation. Fifty nmol/L 14 aa peptide caused a significant reduction in glucose consumption and in lactate production after 72 hours of incubation with respect to control (Student’s t test: t=3.87; P<0.05). At the same concentrations the 14 aa peptide caused also myoblasts phenotypic alterations (accumulation of cells at the periphery of culture wells, lack of differentiation in myotubes and presence of polynucleated cells).
Conclusions the N-terminal 14 aa peptide from an S-100 calcium binding protein is produced by PC causing diabetes mellitus; this peptide impairs glucose catabolism by myoblasts in vitro and this might determine hyperglycemia in vivo; its identification in patients’ biological fluids might be helpful to diagnose PC when a recent onset diabetes mellitus occurs.
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