Flow cytometric analysis of 4-HPR-induced apoptosis and cell cycle arrest in acute myelocytic leukemia cell line [NB-4]

ABSTRACT

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 had 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 on EAML cell line [NB-4] using MTT Assay. Next we tested whether this drug induced apoptotic cell death. The abilty 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 71 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 5 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-HDR in AML blasts derived from AML patients