In vitro investigation 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 20U/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 20U/mL heparin concentrations (p < 0.005 and p < 0.001, respectively) while no apoptosis was detected in 0U/mL heparin concentration at 0, 1, and 2h. The apoptotic percentages of the lymphoblasts were higher at the first hour than those at 0 and 2h in 10 and 20U/mL heparin levels (p < 0.001). The highest apoptosis was found at first hour in 20U/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 20U/mL heparin concentrations than those in 0U/mL heparin concentration at 0, 1, and 2h (p < 0.001). There were no significant differences between the caspase-3 and -8 activities in 10 and 20U/mL heparin concentrations at 1 and 2h (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.