Isolation and characterization of pancreatic cancer diabetogenic factor: A 14 aminoacids peptide corresponding to the n-terminal sequence of an s-100 calcium binding protein

Pancreatic cancer (PC)-associated diabetes is consequent to the action of a tumor peptide less than 10,000 Da, although unknown in its biochemical nature. Our aim was to identify and biochemically characterize the PC-associated diabetogenic peptide. Tumor homogenates from PC patients with (n15) or without (n8) diabetes, in comparison with normal pancreas homogenates (n6) were subjected to 16.5% SDS-PAGE. After Comassie staining a band of approximately 1500 Da was evidenced in PC from diabetic, not from non diabetic PC patients or from normal pancreas. The SDS-PAGE separated proteins from a tumor sample obtained from a diabetic patient were transferred to a PVDF membrane, and the 1500 MW band cutted and sequenced by automatic Edman degradation. 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. The identified peptide was then synthesised and its effects on glucose metabolism was tested in cultured C2C12 myoblasts. 60,000 cells were seeded in 24 wells culture plates and cultured with high glucose DMEM added with different concentrations (from 1 nmol/L to 2 mmol/L) of the 14 aa peptide. Glucose and lactate, the end product of glycolysis, 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 mmol/L, while lactate increased from 3.20 0.08 mmol/ L to 34.501.24 mmol/L after 72 hours of incubation. The 14 aa peptide at the concentration of 50 nmol/L caused a significant reduction in glucose consumption and in lactate production (13.55 0.85 mmol/L and 24.25 2.25 mmol/L after 72 hours of incubation) with respect to control (Student’s t test: t3.87, p0.05). At the same concentrations the 14 aa peptide caused also myoblasts phenotypic alterations, which were represented by the accumulation of cells at the periphery of culture wells, by the lack of differentiation in myotubes and by the presence of polynucleated cells. In conclusion 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.