The human Ec peptide: the active core of a progression growth factor with species-specific mode of action

Abstract

ObJECTIvE: preferential IgF-1Ec expression has been firmly associated with skeletal muscle repair mechanisms, post-infarction remodeling of the myocardium, the pathophysiology of endometriosis and prostate cancer biology. Therefore, we have studied the possible biological significance of synthetic Ec peptide, a putative cleavage product of IgF-1Ec in pC-3 cells and C2C12 myoblasts. DESIgN: We had previously designed and synthesized commercially peptides corresponding to the human Ec and its mouse igf1 counterpart as well as synthetic peptides that correspond to parts of the hEc. Using proliferation and mitogenic signaling assays, we tested their effect on pC-3 cells and C2C12 myoblasts at different doses and in different culture conditions. RESUlTS: human Ec, hEc, was documented as exerting progression but not competence growth factor actions, activating ERK1/2 without affecting akt phosphorylation in pC-3 cells. a narrow concentration range of hEc (5-50nm) stimulated the growth of pC-3 cells grown in culture media supplemented with 10% FbS. hEc did not stimulate the growth of pC-3 cells cultured with media containing 0.5% FbS or in mouse C2C12 myoblasts under any culture conditions. The activity of hEc was blocked by a neutralizing anti-human IgF-1Ec antibody but not by a neutralizing anti-human IgF-1 receptor antibody. The synthetic mouse Ec was inactive in human pC-3 cells; however, it stimulated significantly the proliferation of mouse C2C12. by analyzing the bioactivity of synthetic hEc fragments, we documented that hEc's active core is located in the last 4aa of its C-terminal end. CONClUSION: The hEc peptide is an important progression factor for human pC-3 prostate cancer cells.

The COOH-terminus of the IGF-1Ec Isoform Enhances the Proliferation and Migration of Human MCF-7 Breast Cancer Cells

“…Interestingly, the exogenous administration of hEc stimulated the growth of the ER + wtMCF-7 cells but not this of the (triple negative) wtMDA-MB-231 cells. Notably the mEc, the counterpart of hEc, a product of the IGF-1Eb transcript of mouse igf1, did not stimulate the wtMCF-7 cells and MDA-MB-231 breast cancer cells, suggesting that the amino acid differences between the hEc and mEc may account for distinct biological activities and species specific actions (17). In addition, hEc stimulated the wtMCF-7 cells within a relatively narrow concentration range (6.25-25 nM), presenting saturating effects at higher concentrations.…”

“…These cell lines were selected based on the minimal mRNA expression of hEc previously documented (16). Recombinant human IGF-1 (rhIGF-1, Chemicon international Inc., Temecula, CA, USA), commercially-synthesized human Ec (hEc; 24 amino acids at the C-termnial end of human IGF-1Ec) peptide, mouse Ec (mEc; 25 amino acids at the C-terminal end of mouse IGF-1b) and scrambled peptide of 24aa (negative control) were used as treatment factors for 48 h. Concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 nm, were selected based on previous knowledge (17). wtMCF-7 cells were plated at approximately 50% confluence one day prior to transfection with pIRES2-EGFP-hEc construct (MCF-7Ec cells) or empty pIRES2-EGFP vector (mock; mMCF-7 cells) according to the manufacturer's instructions (Lipo 2000, Invitrogen, Carlsbad, CA, USA).…”

Role of Alternatively Spliced Messenger RNA (mRNA) Isoforms of the Insulin-Like Growth Factor 1 (IGF1) in Selected Human Tumors

“…This activity was inhibited by sole addition of anti-hEc peptide antibodies, but not using a neutralizing anti-IGF1R antibody. Additionally, it was proven that hEc’s active core is located in the last four aa of its terminal end [ 18 ]. Furthermore, activating influence of interleukin 6 (IL-6) on IGF1Ec isoform up-regulation and secretion of PEc by PC cells was reported.…”

“…Pro-proliferative and anti-apoptotic properties, migration and invasion promotion, as well as EMT, angiogenesis, and metastasis induction mostly concern the IGF1Ec (MGF) isoform, both as the human Ec peptide [ 14 , 18 , 108 , 166 , 170 ] and IGF1Ec pro-forms [ 16 ]. These effects have been proved in hormone-sensitive breast cancer [ 16 , 170 ], prostate cancer [ 18 , 30 , 108 , 166 ], and osteosarcoma cells [ 14 , 15 ]. In prostate cancer cells, hEc exerts progression but not competence growth factor action, activating ERK1/2 without Akt phosphorylation [ 18 , 108 ].…”

“…Biological activity of the mature IGF1 and its isoforms in physiology is mostly dependent on the phosphorylation of type I IGF1 receptor (IGF1R) [ 12 , 13 ]. In turn, in cancer cells, IGF1R-, insulin receptor (INSR)- and/or hybrid IGF1R/INSR-independent activity has also been described [ 14 , 15 , 16 , 17 , 18 ].…”