Specific Eradication In Quiescent Acute Myeloid Leukemia Cells Induced By Fenretinide

Resistance to chemotherap y is a major problem in the treatment of acute myeloid leukemia(AML). Since conventional chemotherapies generally target rapidly proliferating leukemic cells, resistance and subsequent relapse may be caused by unresponsiveness of non-proliferating(slowly-proliferating) leukemic cells to these agents. It is therefore crucial that therapies are developed using cell death pathways independent of cell cycle status. Fenretinide(N-(4-hydroxyphenyl) retinamide, 4-HPR) is a reactive oxygen species(ROS) inducer applied in treatment of numerous solid tumor cells, and also exerts a strong cytotoxicity to leukemia stem cell-enriched CD34+C D38- AML and CML(chronic myeloid leukemia) cells, thus showing its potential in eradicating relatively quiescent tumor cells. However, few attempts have been made to compare the sensitivity of ROS inducers between rapidly and slowly proliferating leukemia cells.To this end, we manipulated the cell cycle status of HL-60 cells by serum starvation to further determine the sensitivity of rapidly and slowly proliferating leukemic cells to fenretinide treatment. Flow cytometry experiments and colony formation assay were performed to determine the effects of fenretinide on viability and function of HL-60 cells in different cycle status, and realtime RT-PCR and western blot experiments were carried out to reveal mechanisms involved in fenretinide induced apoptosis.We found in this research that fenretinide shows a stronger apoptosis- inducing and colony formation- inhibiting potential in relatively quiescent HL-60 cells, and has induced more ROS in these cells. Accumulation of intracellular ROS and deregulate of NF-κB signaling pathway were involved in fenretinide-induced apoptosis of these cells. O ur conclusions are as follows: 1. Fenretinide has stronger cytotoxicity and colony formation inhibiting effects on quiescent HL-60 cells than proliferative ones, which may relate to more ROS generated in the former under fenretinide treatment. 2. Fenretinide eradicates quiescent HL-60 cells through the induction of intracellular ROS and inhibition of NF-κB signaling pathway. O ur findings indicates that fenretinide, as a cell cycle independent compounds, could selectively eradicate quiescent AML cells, which shows its potential in overcoming drug-resistance and relapse.Accurate classification of acute myeloid leukemia(AML) based on morphology, immunology, cytogenetics and molecular genetics is an important basis of AML diagnosis, prognosis and chemotherapy. Meanwhile, it also offers critical information for development of new chemotherapy agents based on primary AML regimens. About half of AML patients who bear normal karyotypes are facing different prognosis, which shows the limitation of cytogenetics-based AML classification. Rapid development of the second- generation DNA sequencing technology in recent years has revealed several mutations in genes related to occurrence of AML(FLT3, NPM1, CEBPA, etc.), and some of which have become important basis of AML classification and prognosis. These mutations participate in AML occurrence and progression in different ways, and may involved in signaling pathways used by different chemotherapy agents. Therefore, it is of great importance to clear molecular genetics features of AML specimens used in chemotherapy reagent researches. However, it is not clear that whether fenretinide has different effect on AML samples with distinct gene mutations.To clarify whether AML cells bearing these mutations have different sensitivity to fenretinide, 32 cases of AML samples were collected in this research. Flow cytometry experiments were performed to determine cell viability with or without fenretinide treatment. Gene mutation status(FLT3-ITD/TK D, NPM1, NRAS, IDH1, CEBPA) of these samples were detected with performance of PCR assays and DNA sequencing technology. Two parts of data were then aggregated and analyzed.We found in this research that primary CD34+CD38- AML cells showed varying degrees of viability reduction in different samples under fenretinide treatment. Cells bearing FLT3 mutation had significant lower viabilities than those without FLT3 mutation. However, similar outcomes were not observed in cells with other kinds of mutations. Our conclusions are as follows: 1. Fenretinide induces apoptosis of AML C D34+CD38- cells. 2. AML stem cells bearing FLT3-ITD/TKD mutation are more sensitive to fenretinide treatment than those with wild type FLT3. These conclusions show potential value of fenretinide in improving AML treatment, especially in patients bearing FLT3 mutations.