In VitroInvestigation of the Apoptotic Effect of Heparin on Lymphoblasts by Using Flow Cytometric DNA Analysis and Fluorometric Caspase-3 and -8 Activities

Abstract

The apoptotic effect of heparin on the lymphoblasts obtained from 12 newly diagnosed children with acute lymphoblastic leukemia (ALL) was investigated in vitro. The lymphoblasts were incubated with 0, 10, and 20 U/mL heparin concentrations at 0, 1, and 2 h. The percentages of the apoptotic lymphoblasts were calculated by flow cytometry (FCM), and activities of caspase-3 and -8 were simultaneously measured by fluorometric protease activity method. The apoptotic effect of heparin on the lymphoblasts was determined in 10 and 20 U/mL heparin concentrations (p < 0.005 and p < 0.001, respectively) while no apoptosis was detected in 0 U/mL heparin concentration at 0, 1, and 2 h. The apoptotic percentages of the lymphoblasts were higher at the first hour than those at 0 and 2 h in 10 and 20 U/mL heparin levels (p < 0.001). The highest apoptosis was found at first hour in 20 U/mL heparin concentration. Increased concentrations of heparin had an increasing effect on the percentages of the apoptotic lymphoblasts. Significantly higher caspase-3 and -8 activities were determined in 10 and 20 U/mL heparin concentrations than those in 0 U/mL heparin concentration at 0, 1, and 2 h (p < 0.001). There were no significant differences between the caspase-3 and -8 activities in 10 and 20 U/mL heparin concentrations at 1 and 2 h (p > 0.05), while statistically significant differences were simultaneously detected in the apoptotic rates of the lymphoblasts (p < 0.001). This may be due to that the study included the limited patients, or measurement of the caspase activities is a more sensitive method than the FCM analysis for determination of apoptosis because the activation time of the caspases takes a long time period. It was concluded that the apoptotic effect of heparin in vitro on lymphoblasts developed due to the extrinsic pathway of apoptosis via the caspase-3 and -8 activations 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.…”

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

“…Multicycle analyse program was used to detect of apoptosis. The blast cells ongoing to apoptosis were dyed with PI less than the non-apoptotic cells and showed hypodiploid sub-G1 peak on the DNA histogram [4][5][6][7]12,13].…”

“…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.…”

“…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. We first reported the apoptotic effect of heparin in lymphoblasts and stated that heparin induces apoptosis via the activation of the extrinsic pathway [5,6]. Recently, we claimed that heparin induces intrinsic apoptosis of lymphoblasts by the activation of caspase-9 and Cytochrome C (Cyt C) [8].…”