Enhancement of hemoglobin and F-cell production by targeting growth inhibition and differentiation of K562 cells with ribonucleotide reductase inhibitors (didox and trimidox) in combination with streptozotocin

Abstract

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F-cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation. Of the RRIs currently in use, hydroxyurea is the most effective agent for Hb F induction. We have examined the capacity of two novel RRIs, didox (DI) and trimidox (TRI), in combination with streptozotocin (STZ), to induce hemoglobin and F-cell production. The K562 cells were cultured with different concentrations of didox-STZ or trimidox-STZ at a fixed molar ratio of 3:1 and 1:5 for 96 hr, respectively. At pre-determined time intervals, aliquots of cells were obtained and total hemoglobin (benzidine positive) levels, number of F-cells, and Hb F were determined by the differential staining technique, fetal hemoglobin assay kit, and fluorescence cytometry respectively. The effect of combined drug treatment on the growth of K562 cells was examined by isobologram analysis. Our results indicate that a synergistic growth-inhibitory differentiation effect occurred when didox or trimidox was used in combination with STZ on K562 cells. There was an increase in the number of both benzidine-positive normoblasts and F-cells, accompanied by morphologic appearances typical of erythroid maturation. On day 4, the number of benzidine-positive cells showed a 6-9-fold increase and the number of F-cells was between 2.5- and 5.7-fold higher than the respective controls. Based upon these results, treatment with a ribonucleotide reductase inhibitor, such as didox or trimidox, in combination with STZ, might offer an additional promising option in sickle cell disease therapy.