{"paper_id":"a66d86a9-8473-4b93-b489-b61b19b67d96","body_text":"The human Ec peptide: the active core of a progression \ngrowth factor with species-specific mode of action\nEfstathia Papageorgiou,* Anastassios Philippou,* Athanasios Armakolas,*  \nPanagiotis F. Christopoulos, Andreas Dimakakos, Michael Koutsilieris \nDepartment of Experimental Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece\n*Equal contribution to this manuscript \nAbstrAct \nObJECTIvE: preferential IgF-1Ec expression has been firmly associated with skeletal muscle \nrepair mechanisms, post-infarction remodeling of the myocardium, the pathophysiology of \nendometriosis and prostate cancer biology. Therefore, we have studied the possible biological \nsignificance of synthetic Ec peptide, a putative cleavage product of IgF-1Ec in pC-3 cells and \nC2C12 myoblasts. DESIgN: We had previously designed and synthesized commercially peptides \ncorresponding to the human Ec and its mouse igf1 counterpart as well as synthetic peptides \nthat correspond to parts of the hEc. Using proliferation and mitogenic signaling assays, we \ntested their effect on pC-3 cells and C2C12 myoblasts at different doses and in different cul-\nture conditions. RESUlTS: h uman Ec, hEc, was documented as exerting progression but not \ncompetence growth factor actions, activating ERK1/2 without affecting akt phosphorylation \nin pC-3 cells. a  narrow concentration range of hEc (5-50nm) stimulated the growth of pC-3 \ncells grown in culture media supplemented with 10% FbS. hEc did not stimulate the growth \nof pC-3 cells cultured with media containing 0.5% FbS or in mouse C2C12 myoblasts under \nany culture conditions. The activity of hEc was blocked by a neutralizing anti-human IgF-1Ec \nantibody but not by a neutralizing anti-human IgF-1 receptor antibody. The synthetic mouse \nEc was inactive in human pC-3 cells; however, it stimulated significantly the proliferation of \nmouse C2C12. by analyzing the bioactivity of synthetic hEc fragments, we documented that \nhEc’s active core is located in the last 4aa of its C-terminal end. CONClUSION: The hEc \npeptide is an important progression factor for human pC-3 prostate cancer cells.\nKey words: Active core, C2C12 myoblasts, Ec peptide, IGF-1Ec, PC-3 cells\nHORMONES 2016, 15(3):423-434\nAddress for correspondence:\nMichael Koutsilieris, MD, PhD, Professor & Chairman, \nDepartment of Experimental Physiology, Medical School, \nNational & Kapodistrian University of Athens, 75 Mikras Asias \nStreet, 11527, Goudi-Athens, Greece; Tel.: +30 210 7462507; \nFax: +30 210 7462571; E-mail: mkoutsil@med.uoa.gr\nReceived: 07-04-2016, Accepted: 15-07-2016\nResearch paper\nINTRODUCTION\nInsulin-like growth factor 1 (IGF-1) is a key in-\ntercessor in human physiology and pathophysiology, \nincluding cancer.1 Type I IGF-1 receptor (IGF-1R) \nmediates the effects of IGF-1 by triggering two major \nintracellular signaling cascades: the phosphatidylino-\n\n424 E. PAPAGEORGIOU ET AL\nsitol 3-kinase/AKT kinase (PI3K/AKT) pathway \nand the Raf kinase/mitogen activated protein kinase \n(Raf/MAPK) pathway.2,3 IGF-1R-dependent signal-\ning regulates a wide range of cellular responses, \nincluding cell proliferation. IGF-1 can act both as \na competence growth factor, stimulating the “G0 to \nG1 transition” of quiescent/dormant cells, and as a \nprogression growth factor, stimulating the “G1 to G2 \ntransition” of somatic cells in the cell cycle. 4,5 \nBy alternative splicing of exons 5 and 6, human igf1 \nproduces three transcripts, namely IGF-1Ea, IGF-1Eb \nand IGF-1Ec.3 Since they contain exon 3 and exon \n4, all of them can produce mature IGF-1. However, \ntranslation of these transcripts produces different \nE-domain peptides, namely Ea, Eb, and Ec. It is, \ntherefore conceivable that the preferential expression \nof IGF-1Ec detected in several experimental settings \nafter tissue damage supports the need of the injured \ntissue for an auxiliary to IGF-1, Ec-related bioactiv-\nity.6-9 Indeed, several studies have confirmed that \nsynthetic human Ec (hEc) and mouse E (mE) peptide \n[a product of the E domain of IGF-1Eb transcript of \nmouse igf1] possess mitogenic, angiogenic and migra-\ntory growth factor activity in vitro. In addition, other \nstudies in several experimental settings, including \nprostate cancer models, in vitro and in vivo,6,8,19 have \nsuggested that Ec may act via an IGF-1R-independent \nsignaling pathway.6,15-17 Moreover, using molecular \nengineering, we have recently produced human PC-3 \nprostate cancer transfectans with specific Ec overex-\npression (PC-3hEc cells). These PC-3hEc cells have \nbeen documented, using in vitro and in vivo models,20 \nas possessing an increased oncogenic capacity and \ninvasive/metastatic capability.\nHowever, the biologic importance of synthetic E \ndomain-related peptides has been challenged in studies \nusing various bioassay systems, including mesenchy-\nmal stem cells and mouse C2C12 myoblasts.21 Since \nthere exist significant amino acid (aa) differences \n(Table 1) between hEc and its mouse counterpart mE \n(putative product of the IGF-1Eb of mouse igf1), we \nhave analyzed possible differences in the mode of \naction of synthetic hEc and mE, employing various \nexperimental models (human PC-3 prostate cancer \ncells and mouse C2C12 myoblasts) and various cell \nculture conditions.\n22\nHerein we report that hEc peptide [24 amino acids \nof the carboxy (C)-terminal end of human Ec], is a \nsignificant progression factor but not a competence \ngrowth factor, stimulating the growth of human PC-3 \ncells grown in culture media supplemented with 10% \nfetal bovine serum (10% FBS). Notably, hEc did not \nstimulate significantly the growth of mouse C2C12 \nmyoblasts under any experimental condition. In ad-\ndition, mE stimulated the growth of mouse C2C12 \nmyoblasts but not that of human PC-3 cells under \nany experimental condition. Moreover, hEc and all \nof the synthetic fragments that contained the last 4 \namino acids of hEc’s C-terminal end activated ERK1/2 \nwithout affecting AKT phosphorylation in human \nPC-3 prostate cancer cells.\nma TERIalS aND mEThODS\nSynthetic peptides\nWe had previously designed and synthesized com-\nmercially peptides corresponding to human Ec (last \n24 amino acids of human Ec) and to its mouse igf1  \ncounterpart, a putative product of the IGF-1Eb tran-\nscript (last 25 amino acids of the mouse Eb; mE), as \nwell as various synthetic peptides that correspond \nto parts of the hEc, namely hEc (1-12; N-terminal \nend), hEc (13-24; C-terminal end) and hEc (21-24; \nC-terminal end) (Table 1). Furthermore, we synthe-\nsized a scrambled peptide, which was designed to \ncontain the same amino acids of hEc, in a random \nTable 1. The amino acid (aa) sequence of synthetic human (hEc) \nand mouse (mE) peptides* tested for bioactivity, in vitro\n\nCharacterization of the human Ec peptide 425  \nmanner, avoiding any aa sequence that corresponds \nto hEc or mE (Table 1). The scrambled peptide was \nused as negative control. In addition, the commer -\ncially available mature IGF-1 (rhIGF-1, Chemicon \nInternational Inc., Temecula, CA, USA) was used as \npositive control in our bioassays systems.\nCell Cultures\nHuman PC-3 prostate cancer cells (PC-3 cells) \nand mouse C2C12 myoblasts were obtained from the \nAmerican Type Culture Collection (ATCC, Manas-\nsas, V A, USA) and cultured in Dulbecco’s Modi-\nfied Eagle’s Medium (DMEM) (Gibco, Invitrogen, \nCarlsbad, CA), supplemented with 10% fetal bovine \nserum (FBS) (Gibco, Invitrogen), containing 100 U/\nmL penicillin/streptomycin (Gibco, Invitrogen). The \ncells were grown at 37°C in a humidified atmosphere \nof 5% CO2. The cells were initially cultured to reach \n70-80% confluence. Following this, two types of \nexperiments were performed to search for evidence \nas to whether the synthetic peptides under investiga-\ntion are either competence or progression growth \nfactors. In the first series of experiments, the cells \nwere seeded for 24hrs after plating and the media \ncontinued to be supplemented with 10% FBS during \nthe experimental procedure. We then added various \ndoses of the scrambled peptide (negative control), \nmature IGF-1 peptide (positive control), hEc, mE \nand the synthetic hEc fragments, hEc (1-12), hEc \n(13-24), hEc, 21-24 for 48 hrs. In the second type of \nexperiments, after the cell plating, the culture media \nwere changed to contain 0.5% FBS for 48 hrs. The \nlatter is known to increase the distribution of cells \ninto the G0 phase, thus facilitating the testing of \nputative competence growth factor activity in vitro.  \nA competence growth factor “pushes” cells to enter \nthe cell cycle/G0-G1 transition.\n4,5 In both series of \nexperiments, human PC-3 cells and mouse C2C12 \nmyoblasts were challenged with various concentra-\ntions of synthetic peptides for 48 hrs (0.5nM up to \n200nM; final concentration/well).\nCell proliferation assays\n(a) The rate of proliferation/metabolism of cells was \nmeasured using the 3-(4,5-dimethylthiazol-2-yl)-2.5 \ndiphenyl tetrazolium bromide (MTT assays; Sigma \nLtd). Cells were plated in 96-well plates at a cell den-\nsity of 103cells/well and grown in media (150μl/well) \nsupplemented with either 10% or 0.5% FBS, depend-\ning on the experiment. After treatment with synthetic \npeptides, 15μl of (5mg/ml) MTT was added to each \nwell in a humidified atmosphere (37°C, 5%CO2) for \nup to 4 hr. Then MTT was aspirated and 150 ml of \nDMSO was added to each well. The optical density \n(OD) was measured at 450nm using a microplate \nreader (VersaMax; Molecular Devices, Sunnyvale, \nCA, USA), as described previously.23\n(b) The actual number of alive PC-3 and C2C12 \ncells in cultures was assessed using the trypan blue \nexclusion assays, whereby cells were plated (at a cell \ndensity of 8x104 cells/well) in 12-well plates and ex-\nposed to various doses of the synthetic peptides under \ninvestigation. After 48 hrs, the cells were harvested \nand counted by the trypan blue exclusion method, as \ndescribed previously.23\n(c) The DNA content from identical experiments \nwas extracted by the phenol/chloroform method. The \nDNA content was measured using a spectrophotometer \n(Biospec Nano; Shimadzu Scientific Instruments, Co-\nlumbia, MD, USA). This analysis provided evidence \nfor the rate of DNA synthesis in vitro.24\nCharacterization of hEc & mE activity\nIn order to investigate whether IGF-1R mediates \nthe activity of synthetic hEc in the PC-3 cells, the \ncell cultures were pre-incubated for 1 hr with either \na monoclonal anti-human IGF-1R neutralizing anti-\nbody (R&D Systems; Minneapolis, MN, USA) or a \npolyclonal rabbit anti-human IGF-1Ec antibody. The \nlatter was raised against the 24 amino acids of the \nhEc.22 The IGF-1R neutralizing antibody was used at \na concentration of 10 μg/ml (1:50 dilution), following \nthe manufacturer’s recommendation, and the rabbit \nanti-IGF-1Ec antibody was used at 1:50 dilution, as \npreviously described.20\nCell cycle analysis by flow cytometry\nCells were seeded in 12-well plates at a cell density \nof 8x104cells/well and then challenged by synthetic \npeptides, as described above. After the experimental \nprocedure, adherent and floating cells were combined, \nwashed with PBS and fixed overnight at 4°C in 70% \nethanol in PBS. Fixed cells were then stained with \nCyStain DNA 1step (Partec GmbH; Münster, Ger -\nmany). Cell cycle analysis was performed using a \n\n426 E. PAPAGEORGIOU ET AL\nFACS Calibur CyFlow ML Partec flow cytometer, \nusing the ModFit and Flowmax 3.0 software. This \nanalysis provided evidence for the effects of the \nsynthetic peptides under investigation in the distri-\nbution of PC-3 cells into the various phases of the \ncell cycle (G0/G1, S and G2/M phases), as described \npreviously.15,20,23,24\nWestern analysis\nCells were seeded in 6-well plates grown in culture \nmedia supplemented with 10% FBS and challenged \nwith synthetic peptides under investigation for 5, \n15 and 30 min. Cells were extracted using RIPA \nbuffer (Cell Signaling; Beverly, MA, USA) supple-\nmented with protease and phosphatase inhibitors \n(Cell Signaling; Beverly, MA, USA). After 30 min \nof incubation on ice, the cell lysates were cleared \nby centrifugation (14,000 rpm, for 30 min at 4°C). \nProtein concentration was measured using the BCA \nProtein Assay Kit (Pierce Biotechnology; Rockford, \nIL, USA). An equal amount of protein extracts (20 \nμg) was heated at 95°C for 5 min, electrophoresed \nin 12% SDS-PAGE under denaturing conditions \nand transferred onto a PVDF membrane (BIO-RAD \nLaboratories; Hercules, CA, USA). The blots were \nblocked with TBS-T (20 mmol/L Tris-HCl, pH 7.6, \n137 mmol/L NaCl, and 0.1% Tween 20) contain-\ning 5% nonfat dried milk at room temperature for \n1 hr. The membranes were probed overnight with \nprimary antibodies against phospho-ERK1/2 and \nphospho-AKT (Cell Signaling; Beverly, MA, USA) \nat 1:1,000 dilution in TBS/T containing 5% BSA \n(Santa Cruz Biotechnology; Santa Cruz, CA, USA), \nand with GAPDH (1:2,000 dilution; Santa Cruz \nBiotechnology; Santa Cruz, CA). The blots were \nthen washed and incubated with a secondary goat or \nmouse antibody raised against rabbit IgG conjugated \nto horseradish peroxidase (1:2,000 dilution) (Santa \nCruz Biotechnology; Santa Cruz, CA, USA). The \nbands were visualized by exposing the blots to X-ray \nfilm after incubation with ECL substrate for 5 min \n(SuperSignal; Pierce Biotechnology; Rockford, IL, \nUSA), as described previously.\n15,20,23\nStatistical analysis\nOne-way analysis of variance (ANOV A) was \nemployed to evaluate significant changes in all cell \ntreatment conditions compared to controls, except for \nthe MTT assays where two-way ANOV A was used. \nSpecifically for the MTT assay comparisons, in order \nto reveal if the effect of the various factors used for \ncell treatment is stable for different concentrations or \nbetween different FBS levels, the treatment factors \nwere used as the stable factor (group), while their \nconcentrations or %FBS were the repeated factor \n(group X concentration or FBS interactions; SPSS \nv. 22 statistical package, SPSS Inc. Headquarters; \nChicago, USA). Where significant F ratios were \nfound for main effect or interactions (p <0.05), the \nmeans were compared using Tukey’s post-hoc test, \nwhile Bonferroni corrections for multiple compari-\nsons were performed where appropriate. All data are \npresented as mean ± standard deviation (SD). The \nlevel of significance was set at p <0.05.\nRESUl TS\nCharacterization of hEc and mE activity in vitro\nSynthetic hEc stimulated the proliferation of human \nPC-3 cells when grown in culture media supplemented \nwith 10% FBS, as assessed by MTT (Figure 1: panel A) \nand trypan blue assays (Figure 1: panel C). However, \nhEc did not stimulate significantly the proliferation \nof PC-3 cells in culture media supplemented with \n0.5% FBS (Figure 1: panel B and panel D). These \ndata suggest that hEc is a progression but not a com-\npetence growth factor. In addition, analysis of DNA \ncontent confirmed the ability of hEc to stimulate DNA \nsynthesis in PC-3 cells, acting as progression factor \n(Figure 2: panel B) but not as a competence growth \nfactor (Figure 2: panel E).\nHowever, synthetic hEc did not stimulate (stimula-\ntion 20%-30%) mouse C2C12 myoblasts (Figure 3: \npanel A) and synthetic mE did not stimulate human \nPC-3 cells under any experimental conditions (Fig-\nure 1 & Figure 2: panel C and panel F). Moreover, \nsynthetic mE stimulated the growth of mouse C2C12 \nmyoblasts (Figure 3: panel A & panel B). These data \nsuggest that synthetic hEc and mE exert species spe-\ncific actions in vitro.\nThe mature IGF-1 acted both as a competence and \na progression factor in our bioassay systems, while \nthe scrambled peptide did not stimulate PC-3 cells \nand mouse C2C12 myoblasts under any experimental \nconditions (Figure 1; Figure 2 and Figure 3). Inter -\n\nCharacterization of the human Ec peptide 427  \nFigure 1. Proliferation assays on human PC-3 cells. The effects of synthetic peptides on metabolic activity proliferation and on the \nactual cell number of human PC-3 prostate cancer cells in vitro. The growth of human PC-3 cells was analyzed after 48hrs exposure \nto putative growth substances using various concentrations (dose-dependent effect). The rate of proliferation/metabolism of the human \nPC-3 cells was assessed by MTT assays (A) using cell culture media supplemented with 10% fetal bovine serum (FBS). hEc resulted \nin a dose-dependent stimulation of cell proliferation/metabolism at concentrations of 2.5nM up to 50nM, whereas hEc concentration \n>100nM produced an over-dosing effect (A). hEc had no significant effect on human PC-3 cultures supplemented with 0.5% FBS (B). \nUnlike hEc, IGF-1 stimulated the growth of human PC-3 cells in both experimental conditions tested (0.5% and 10% FBS) (A, B). In \naddition, mE and scrambled peptide did not stimulate the growth of human PC-3 cells (A, B). Moreover, the trypan blue exclusion as-\nsays revealed similar results under identical experimental conditions (C, D). Mature IGF-1 produced significant increases in the actual \nnumber of PC-3 cells grown in both culture conditions (0.5% and 10% FBS) (C, D). Scrambled peptide did not stimulate the growth of \nPC-3 cells in any experimental condition (C, D). The results are expressed as means ± SD (X± SD) of three independent experiments \nperformed in triplicate. *: p<0.05, **: p<0.01, ***: p<0.001 significantly different as compared to controls.\nestingly, synthetic hEc exerted its actions within a \nrelatively narrow range of concentrations (5-50nM), \nthus producing a significant over dosing effect at \n100nM. This effect was not noted by mature IGF-1 \nin vitro (Figure 1: panel A and panel C and Figure \n2: panel A and panel D). A similar pattern of activ-\nity was documented also with mE in mouse C2C12 \nmyoblasts (Figure 3: panel A and panel B).\nAnalysis of the cell cycle revealed that the distri-\nbution of PC-3 cells at G1/G0 phase was increased in \ncultures supplemented with 0.5% FBS as compared to \nthose supplemented with 10% FBS (Figure 4: panel \nA vs panel D). The exogenous administration of \n20nM hEc or 20 nM IGF-1 increased the distribution \nof PC-3 cells into S phase at the expense of G1/G0 \nphase (Figure 4: panel B and panel C, respectively). \nThis data corroborates our results obtained by MTT, \ntrypan blue exclusion and DNA content assays.\nMode of hEc actions\nInvestigating whether hEc activity in PC-3 cells \nis mediated by IGF-1R, we analyzed its effects in \npresence and absence of neutralizing anti-IGF-1R \nantibody and specific anti-human IGF1Ec antibody.22 \nSuch analysis revealed that hEc’s activity can be \nblocked by the anti-IGF1Ec antibody but not by \nthe anti-IGF-1R antibody. The IGF-1 was used as a \npositive control for IGF-1R-mediated specific action \n(Figure 5: panel A; trypan blue assays and Figure 5: \npanel B; DNA content assays).\n\n428 E. PAPAGEORGIOU ET AL\nIn addition, analysis of ERK1/2 and AKT phos-\nphorylation by Western blots has revealed that hEc \ncan activate ERK1/2 without affecting AKT (Figure 5: \npanel D). Again, mature IGF-1 was the positive con-\ntrol, knowing that it activates both ERK1/2 and AKT \n(Figure 5: panel C). The scrambled peptide activated \nneither ERK1/2 nor AKT in PC-3 cells after 5, 15, and \n30 min (Figure 5: panel E). These data suggested that \nhEc activates ERK1/2 via a mechanism that cannot \nbe blocked by neutralizing IGF-1R antibody.\nFurthermore, testing of synthetic hEc fragments \nenabled us to document that the N-terminal fragment \nof hEc [hEc (1-12)] is inactive. However, similarly \nto full length hEc, all the C-terminal fragments of \nhEc were active in PC-3 cells grown in culture media \nFigure 2. DNA content. The effects of the synthetic peptides were tested either in PC-3 cells grown in cell culture media supplemented \nwith 10% or in media containing 0.5% FBS at concentrations of 20nM and 100nM for 48hrs. The 20nM of the hEc peptide increased \nsignificantly the DNA content of PC-3 cells grown with 10% FBS, while hEc at a concentration of 100nM produced an over-dosing \neffect (B). In addition, mE concentration of 20nM and 100nM did not increase the DNA content of human PC-3 cells (C, F). IGF-1 \nincreased the DNA content in PC-3 cells grown under both experimental conditions (0.5% and 10% FBS) (A,D). The results are ex-\npressed as means ± SD (X± SD) of three independent experiments performed in triplicate. *: p<0.05, **: p<0.01, ***: p<0.001 signifi-\ncantly different as compared to controls.\nFigure 3. Proliferation assays for mouse C2C12 cells. The ability of synthetic peptides to stimulate the proliferation of mouse C2C12 \nmyoblasts was assessed in PC-3 cells grown in cell culture media supplemented with 10% FBS (A) and 0.5% FBS (B). IGF-1 and \nmouse mE stimulated the growth of C2C12 myoblasts in both experimental conditions. hEc and scrambled peptide did not stimulate \nthe growth of C2C12 myoblasts (A, B). The results are expressed as means ± SD (X± SD) of three independent experiments performed \nin triplicate. *: p<0.05, **: p<0.01, ***: p<0.001 significantly different as compared to controls, \n# statistically significant compared to \nhEc peptide.\n\n\nCharacterization of the human Ec peptide 429  \nsupplemented with 10% FBS [hEc (13-24) and hEc \n(21-24)] (Figure 6: panel A; trypan blue assays and \nFigure 6: panel B; DNA content assay). The analysis \nof ERK1/2 and AKT phosphorylation revealed that \nall the hEc fragments containing the last 4aa of its \nC-terminal end provoked the activation of ERK1/2 \nwithout affecting AKT [hEc (13-24 and hEc (21-24)] \nand acted as progression factors in human PC-3 cells. \nThe N-terminal hEc [hEc (1-12)] did not affect ERK1/2 \nactivation and growth of PC-3 cells (Figure 6: panel \nC, panel D and panel E). Therefore, we concluded \nthat the active core of hEc is located in the last 4aa \nof its C-terminal end.\nDISCUSSION\nThe time frame between two mitotic divisions is \nknown as the somatic “cell cycle”, while “interphase” \nis the time from the end of one mitosis, or mitotic \n(M) phase, until the start of the next one. After com-\npletion of mitosis, cells may either enter a condition \ncalled G1 phase, during which RNAs and proteins \nare synthesized but there is no DNA replication, \nor withdraw from the cell cycle into the G0 phase \n(quiescence/dormancy). G0 phase cells can reenter \nthe cell cycle (G0 to G1 transition) with the action of \ncompetence growth factors, such as platelet-derived \ngrowth factor (PDGF) and basic fibroblast growth \nfactor (bFGF). 25-27 Competence growth factors can \ninitiate this process via the activation of transcrip-\ntion factors which are the products of the so-called \n“early response genes” (proto-oncogenes), such as \nc-fos and c-myc and ras. The gatekeeper p53, among \nother events, controls the G0 to G1 transition. 28,29 In \nFigure 4. Cell cycle analysis. Analysis of phase distribution of human PC-3 cells grown in cell culture media supplemented with 10% \nFBS and 0.5% fetal bovine serum (FBS), after stimulation with the synthetic peptides under investigation, as assessed by flow cytom-\netry. The human PC-3 cells, which were grown with culture media containing 0.5% FBS, had an increased cell distribution in the G1/\nG0 phase as compared to PC-3 cells grown with 10% FBS (A, D). In addition, stimulation with hEc and IGF-1 increased the distribu-\ntion of PC-3 cells in the S phase at the expense of the G1/G0 phase (A, B). This hEc effect was not evident in PC-3 cells grown with \nculture media containing 0.5% FBS (B, E).\n\n430 E. PAPAGEORGIOU ET AL\nvitro, the supplementation of cell culture media with \nsignificant amounts of serum (>5%) provides the cells \nwith competence and progression factors necessary \nto maintain cell survival and growth in vitro. Serum \ndeprivation (0.5% FBS) is an experimental technique \naiming to synchronize somatic cells in the G0 phase \nin vitro. It is well known that cancer cells and cell \nlines require less serum supplementation to achieve \nefficient survival and growth in vitro.\n25-29 \nIn the late G1 phase the cells reach the restriction \npoint (R); beyond this point cells are committed to DNA \nreplication in the S (synthesis) phase. Other growth \nfactors, such as epidermal growth factor (EGF) and \nIGF-1, are progression growth factors, accelerating \nthe transition of cells already in the cell cycle from \nthe G1 to G2 phase. Notably, the progression from \nG2 phase to M phase is independent of further growth \nfactor stimulation.29 The above is crucial background \nFigure 5. Mitogenic effects of the putative growth substances. Analysis of the mitogenic effects of putative growth substances tested \non human PC-3 cells, using cell culture pre-incubated with neutralizing IGF-1R antibody or IGF-1Ec antibody. The analysis involved \ntrypan blue exclusion assays (A) and DNA content assays (B). Pre-incubation with anti-IGF-1R Ab blocked the effect of IGF-1 but it \ndid not alter the proliferative effects of hEc on human PC-3 cells. However, the anti-IGF-1Ec antibody neutralized the proliferative \neffects of hEc on human PC-3 cells. Furthermore, Western blot analysis revealed that IGF-1 activated both ERK1/2 and AKT in PC-3 \ncells (C). However, synthetic hEc activated ERK1/2 but not AKT (D). The scrambled peptide did not activate ERK1/2 and AKT in \nPC-3 cells (E). The results are expressed as means ± SD (X± SD) of three independent experiments performed in triplicate. *: p<0.05, \n**: p<0.01, ***: p<0.001 significantly different as compared to controls.\n\n\nCharacterization of the human Ec peptide 431  \nknowledge in cell physiology that enables analysis \nof the role of putative growth substances in vitro. \nTherefore, when testing for putative mitogens in \ncell cultures using culture media supplemented with \n0%-0.5% FBS (cells trapped in the G0 phase), the \nprocess is not expected to detect any bioactivity if the \nsubstance under investigation is only a progression \nfactor without competence growth factor activity.\n21 \nAccording to our results this is the case of hEc in vitro. \nMoreover, our data revealed that hEc possesses \nspecies-specific activity, acting as a progression factor \nin human PC-3 cells but not in mouse C2C12 myo-\nblasts. hEc did not stimulate significantly (20%-30%; \np>0.05) the growth of C2C12 myoblasts. All the \nsynthetic hEc fragments that contained the last 4aa of \nthe hEc C-terminal end exerted this species-specific \nactivity. The synthetic hEc fragment that contained \nthe N-terminal end was documented as being inactive \nin all bioassay systems. Similarly, mE, which was \nactive in mouse C2C12 myoblasts, did not stimulate \nsignificantly the growth of human PC-3 cells. Since \nthe last 4 amino acids of the C-terminal end of hE \nand mE differ only in the amino acid residue at po-\nsition 23, the role of rat position 23 in hEc appears \ncrucial for its biological action on human PC-3 cells. \nInterestingly, in the literature the first synthetic hEc \npeptide was initially produced bearing an unexplained \nmodification of its sequence at residue 23 (H instead \nof R), apparently copying the aa sequence of the mE \nC-terminal end.31,32 Conceivably, such a change, which \nwas repeated thereafter by other investigators, has \ncontributed to the confusing data about hEc’s activity \nusing either human or mouse in vitro systems.\nFigure 6. Mitogenic effects of the synthetic fragments of hEc. The synthetic fragments of hEc (a) hEc (1-12), (b) hEc (13-24), (c) hEc \n(21-24) were tested for mitogenic activity in human PC-3 cells using trypan blue exclusion assays (A) and DNA content assays (B). \nThe fragment hEc (1-12) did not affect the growth of human PC-3 cells (A, B). However, all the synthetic fragments that contained the \nlast 4 amino acids (aa) of the hEC’s C-terminal end stimulated the growth of human PC-3 cells. In addition, the synthetic fragments, \nhEc (13-24) and hEc (21-24), activated ERK1/2 (D, E), while the synthetic hEc (1-12) did not affect the phosphorylation of ERK1/2 \nin human PC-3 cells (C).\n\n\n432 E. PAPAGEORGIOU ET AL\nFurthermore, synthetic hEc exerted its action within \na very narrow concentration range, reaching a plateau \nof its dose-dependent effects at 50nM (optimal dose \nfor in vitro testing = 20nM). The testing of hEc action \nat a concentration of 100nM produced no significant \nstimulation of the growth of human PC-3 cells in \nvitro. Therefore, it is conceivable that the contradict-\ning reports on hEc’s actions may be attributable to \nthe variability of experimental settings (human and \nmouse models), the narrow range of activity (tests \nperformed at 100nM) and the absence of competence \nfactor activity (testing in media with 0% FBS).\nhEc action was not blocked by neutralizing the \nanti-IGF-1R antibody, while its effect was blocked \nby the anti-human IGF-1Ec antibody. These data \ncorroborate previous reports on hEc action 15,16 and \nthe recently reported oncogenic role of hEc in PC-3 \nprostate cancer cells and immortalized (SV-40) hu-\nman prostate cancer epithelial cells (HPrEC cells), \nboth molecularly engineered to overexpress specifi-\ncally hEC (PC-3hEc and HPrEc-hEc transfectans), in \nvitro and in vivo.20 Interestingly, HPrECP-hEc cells, \northotopically injected into SCID mice, provoked \nmetastases in these mice. Immortalized HPrEC cells \nwithout expression of Ec do not produce metastases; \nhowever, all the SCID mice injected with HPrECP-\nhEc cells died within 12 weeks, showing a remarkable \nincrease in the mortality rate. 20 Furthermore, hEc \noverexpression produced epithelial to mesenchymal \ntransition (EMT) in PC-3hEc cells and PC-3hEc-\ninduced tumours in SCID mice. Notably, hEc-induced \nEMT was causatively mediated by ERK 1/2 activation \nand ZEB-1 expression, however, by a mechanism that \nwas independent of IGF-1R signalling. 20\nIn addition, we recorded strong evidence supporting \nthe notion that IGF-1Ec overexpression in prostate \ncancer tumours (PC-3hEc tumours in SCID mice) \nis provoked by the host’s immune reaction. Indeed, \nPC-3hEc cells are able to attract and to increase the \ninvasiveness of human mesenchymal cells in vitro,  \nwhile IGF-1Ec expression is enhanced in PC-3 cells \nafter co-culturing with pre-sensitized human mesen-\nchymal cells.20 In cancer biology, bone marrow-derived \nmesenchymal stem cells (MSCs) are locally recruited \nto establish a supportive stroma around the tumour, a \nphenomenon elicited by the release of paracrine signals \nby the tumour. Bearing in mind the above, our data \nsuggesting an overexpression of IGF-1Ec by injured \ntissues, such as by the surrounding tissues in prostate \ncancer, is reminiscent of the preferential expression \nof IGF-1Ec post-skeletal muscle damage and that of \nthe myocardium during the post-infarction period.30-36\nInterestingly, species-specific differences of hEc \nand mE actions may point to a possible different mode \nof actions at the receptor level. It has been reported \nthat mE requires IGF-1R for its bioactivity,\n36 while \nhEc appears to act in an IGF-1R independent man-\nner.15,20,33 Thus, the residue rat position 23 of hEc is \nprobably crucial for hEc receptor recognition.\nTaking into consideration all these data, we con-\nclude that hEc is a progression factor for human \nPC-3 cells but not for mouse C2C12 myoblasts, that \nit increases the growth and metastatic capability of \nPC-3 hEc and HPrECP-hEc transfectans in vitro and \nin vivo and that hEc overexpression can induce EMT \nof PC-3 cells via an IGF-1R-independent [possibly \nvia a hEc receptor (hEc.R)].\nCONClUSION\nhEc may have an important role in human prostate \ncancer biology. Since the preferential overexpression \nof IGF-1Ec in several pathologies produces both IGF-1 \nand hEc, it is conceivable that the biological role of \nhEc may be auxiliary to that of IGF-1 (additive and/\nor synergistic/antagonistic actions) in human tissues \nwhich undergo repair/remodeling and/or in the tumour \nmicroenvironment (host-tumour cell interactions). \nWe therefore conclude that further investigation into \nthe possible biological role of E domain products of \nigf1 is warranted. \nDISClOSURE\nThe authors declare they have no competing inter-\nests that might be perceived to influence the results \nand discussion reported in this paper.\nREFERENCES\n 1. Baker J, Hardy MP, Zhou J, 1996 Effects of an Igf1 gene \nnull mutation on mouse reproduction. Mol Endocrinol \n10: 903-918.\n 2. 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