Flow Cytometric Analysis of 4-HPR-induced Apoptosis and Cell Cycle Arrest in Acute Myelocytic Leukemia Cell Line (NB-4)

In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or programmed cell death (apoptosis) can be induced by variety of agents including: Vitamin analogs, demethylating agents, cyclic AMP analogs and anti-proliferative agents. To the best of our knowledge there has been not any study specifically to analyze apoptotic and anti-proliferative effects of 4-HPR (a vitamin analog) in NB-4 cell line. To test whether this drug has activity in acute myeloid leukemia (AML), we first analyzed the anti-proliferative effect of 4-HPR in one AML cell line (NB-4) using MTT Assay. Next we tested whether this drug induced apoptotic cell death. The ability of this compound to induce apoptosis of cancer cells was examined by Annexin V-FITC Assay using Flow cytometry. We also analyzed the cell cycle progression by PI staining using flow cytometry. Using MTT assay, NB-4 cells exhibited increased inhibition of proliferation at micromolar concentrations of 4-HPR at 24, 48 and 72 hrs post treatment. Flow cytometry analysis indicates that 4-HPR is a potent inducer of in vitro apoptotic cell death, and cell cycle analysis revealed an increase in S phase population. In total, the results indicate that 4-HPR is a strong inhibitor of AML cell proliferation and a potent inducer of in vitro apoptotic cell death. Further studies are required to evaluate the in vitro effects of 4-HPR in AML blasts derived from AML patients.