In our research we compared the effect of doxorubicin (DOX) and doxorubicin-transferrin (DOX-TRF) conjugate on the induction of programmed cell death. All experiments were carried out on human leukemia cells: CCRF-CEM, K562 sensitive and resistant to DOX, (K562/DOX), which are the molecular model for the chronic and acute form of hematological malignancies, respectively. At the same time, studies were also performed on normal, peripheral blood mononuclear cells (PBMCs). The first stages of apoptosis, connected with externalization of phosphatidylserine (PS), were evaluated after comparing the viability of tested cell lines treated with DOX-TRF conjugate or free DOX. Morphological changes of nuclei connected with apoptosis were analyzed by double staining Hoechst 33258/propidium iodide. Subsequently, we conducted a more accurate evaluation of DOX-TRF-trigged cell death by using DNA ladder assay, measuring the activation of caspase-3, -8 and -9 and changes in poly-ADP ribose polymerase (PARP) activity. The percentage of apoptotic cells reached its maximum at 24 and 48 h incubation. Prolonged treatment time with DOX-TRF conjugate progressively increased the level of necrotic cells. At 24-48 h time points, we observed a significant increase in the activity of apoptosis-characterized enzymes (caspases -8, -9, -3). This study provided the evidence that DOX-TRF conjugate triggers apoptotic pathway connected with DNA damage mediated by the activation of pro-caspases and PARP cleavage.
Keywords: Cell death; Doxorubicin–transferrin conjugate; Leukemia cells; Multidrug resistance; PARP cleavage.
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