Comparison of neuroprotective and erythropoietic effects of erythropoietin and carbamylated-erythropoietin in different cell systems.

CHAMORRO MARÍA EUGENIA; VITTORI DANIELA; VOTA DAIANA; WENKER SHIRLEY; NESSE ALCIRA

Barcelona, España

Congreso; 15th Congress of the European Hematology Association; 2010

Background: In addition to its well-known hematopoietic effects, erythropoietin (Epo) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for Epo-like compounds with more selective neuroprotective action. One such compound extensively assayed is the chemically modified Epo-derivative carbamylated erythropoietin (cEpo), which in experimental assays cEpo has demonstrated non-hematopoietic tissue protection without significant erythropoietic effect. However, less is known about cellular mechanisms of action. Aims: To compare the neuroprotective action between Epo and cEpo and to distinguish their effects upon erythropoietic cells. Methods: Cell viability/proliferation was analyzed by the MTT assay and apoptosis by fluorescent microscopy after Hoechst staining. Models: Cells of neuronal origin: SH-SY5Y cells were cultured in E-MEM:Ham-FBS. Cells of ability to erythroid differentiation: a) Epo-dependent UT-7 cells were cultured in IMDM-FBS with Epo, b) Epo-independent K562 cells were grown in RPMI-1640-FBS and differentiated to erythroid lineage with hemin, and c) physiological colony forming units-erythroid (CFU-E) from Balb/c mice were evaluated by bone marrow cell cultures in methylcellulose-medium. After 48 h, 2,7-diaminofluorene reaction was performed and hemoglobinized colonies were counted. Results: Epo and cEpo acted in similar way (no significant differences) to prevent apoptosis induced by either staurosporine (STP) or TNF-alpha in SH-SY5Y cells (Control 8±1.5%; STP 44±5.2%; Epo-STP 12±2.2%; cEpo-STP 12±1.7%; TNF 32±1.7%; Epo-TNF 9±0.9%; cEpo-TNF 10±2.9%; n=4). Moreover, assays in the presence of inhibitors showed that cell activation by Epo and cEpo involves similar signaling pathways mediated by PI3K and Jak2. Instead, Epo but not cEpo overcame apoptosis induced by TNF-alpha in erythroid differentiated K562 cells (Control 8±2.3%; TNF 17±2.7%; Epo-TNF 8±1.0%; cEpo-TNF 18±1.5%, Epo-TNF vs. Epo P