IN VITRO DETERMINATION OF THE APOPTOTIC EFFECT OF HEPARIN ON LYMPHOBLASTS USING DNA ANALYSIS AND MEASUREMENTS OF Fas AND Bcl-2 PROTEINS BY FLOW CYTOMETRY

Abstract

Heparin has an apoptotic effect beside its anticoagulant, anti-inflammatory, antihypertensive, and antiproliferative effects. In this study, the authors detected the percentages of apoptotic lymphoblasts and the expressions of apoptotic Fas protein and antiapoptotic Bcl-2 protein with flow cytometry in vitro after the incubation of lymphoblasts with heparin. Eleven newly diagnosed acute lymphoblastic leukemia (ALL) children were included in the study. Lymphoblasts were incubated in all different levels of heparin concentrations (0, 10, and 20 U/mL) and the percentages of apoptotic lymphoblasts and the percentages of Fas protein and Bcl-2 proteins were simultaneously measured by flow cytometry at 0, 1, and 2 h. At 0, 1, and 2 h, apoptosis was determined when heparin was added in 10- and 20-U/mL concentrations (p <.05). The apoptotic effect of heparin on lymphoblasts was higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.01). The highest apoptosis was detected in the 20-U/mL heparin concentration at the first hour. The expression levels of Fas protein on lymphoblasts were higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.001). The highest expression of Fas protein was observed in the 20-U/mL heparin concentration at the first hour. The expression levels of Bcl-2 protein on lymphoblasts were lower at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.001). The lowest expression of Bcl-2 protein was detected in the 20-U/mL heparin concentration at the first hour. Increased concentrations of heparin had an increasing effect on the percentages of apoptotic lymphoblasts. The expression percentages of Fas protein on lymphoblasts also increased, whereas the expression percentages of Bcl-2 protein on lymphoblasts decreased (p <.05). These results suggest that low-dose heparin may cause significant apoptosis of lymphoblasts in newly diagnosed ALL patients.

The effect of heparin on the cell cycle in human B-lymphoblasts: An in vitro study

“…We demonstrated in this study that the greatest proliferation effect of heparin and the highest SPF ratio were determined in 20 U/ml concentration at 1 h. Recent studies from Erduran et al [30] determined that the greatest apoptotic effect of heparin on the lymphoblasts was detected in 20 U/ml concentration at 1 h. The results of some recent studies were found to be concordant [28][29][30]. Apoptosis and proliferation are important regulators of normal development and homeostasis in the bone marrow.…”

“…On the other hand, heparin causes apoptosis in human peripheral blood neutrophils, lymphoblasts and mononuclear cells, and indicates its apoptotic effect on lymphoblasts via extrinsic or intrinsic pathways [27][28][29]. We demonstrated in this study that the greatest proliferation effect of heparin and the highest SPF ratio were determined in 20 U/ml concentration at 1 h. Recent studies from Erduran et al [30] determined that the greatest apoptotic effect of heparin on the lymphoblasts was detected in 20 U/ml concentration at 1 h. The results of some recent studies were found to be concordant [28][29][30].…”

Roles of vimentin and 14-3-3 zeta/delta in the inhibitory effects of heparin on PC-3M cell proliferation and B16-F10-luc-G5 cells metastasis

“…In our evaluation of the influence of heparin on tumor cell proliferation, cell cycle and apoptosis, we found that it could inhibit proliferation of PC-3M tumor cells and arrest them in the G 0 /G 1 phase. Heparin has been reported to induce apoptosis in tumor cells mediated by caspase-3, caspase-8, Fas, and Bcl-2 [15] . In this study, we did not find a significant influence of heparin on tumor cell apoptosis, although there was a slight increase in number of apoptotic PC-3M cells treated with heparin (125 μg/mL).…”

Apoptotic Effect of Heparin on the Lymphoblasts Using DNA Analysis and Measurements of Intracellular Calcium

“…Beside its anticoagulant, anti-inflammatory, antihypertensive and antiproliferative effects [1][2][3], heparin has the apoptotic effect in lymphoblasts, in vitro [4][5][6]. Manaster et al [7] claimed that 50, 100 and 200 U/ml concentrations of heparin induce apoptosis in human peripheral blood neutrophils, in vitro.…”

“…Manaster et al [7] claimed that 50, 100 and 200 U/ml concentrations of heparin induce apoptosis in human peripheral blood neutrophils, in vitro. In our studies, lymphoblasts were incubated with 0, 5, 10 and 20 U/ml concentrations of heparin at 0, 1 and 2 hours and the highest apoptosis was determined in 20 U/ml heparin concentration at first hour [4][5][6]. The expression of fas, the activities of caspase-3 and -8 increased and the expression of bcl-2 decreased when lymphoblasts were incubated with heparin in 10 and 20 U/ml concentrations at 1 and 2 hours.…”