Study On Iron Oxide Nanoparticles Regulating ROS And Decreasing LSCs Drug Resistance

Acute myeloid leukemia(AML)is a highly heterogeneous hematological malignant disease with worse clinical outcomes after treatment.The disease is characterized by abnormal primitive medullary system cell proliferation,poor cell differentiation,and infiltration of bone marrow,peripheral blood,or other tissues such as live,spleen and lymph node.The patient with AML also reveal the common hematopoiesis suppression,white blood cell abnormal in quality nature and quantitative,and decrease of erythrocytes and platelet.At present,there are many new chemotherapeutic drugs for AML treatment,which can improve the curative effect and prolong the survival of patients.For example,the backbone of treatment is a combination of cytosine arabinoside(Ara-C)with an anthracycline,and this therapy is still the worldwide standard of care.Two-thirds of AML patients achieve complete remission with progression-free survival;however,most of them ultimately relapse due to occurrence of drug resistance in the patients.Therefore,AML chemotherapy resistance is still a difficult point for clinical treatment.To this end,it is extremely urgent to find a treatment plan that can overcome the problem of AML resistant to chemotherapeutics.Leukemia stem cells(LSCs)are considered as the initiator cells of AML,and have the capability of self-renewal,immortalization and multi-directional differentiation,and play an important role in the development,recurrence and drug resistance of AML.Studies have demonstrated that the LSCs possess a low level of reactive oxygen species(ROS),which may be one of important factors related to AML's drug resistance.Iron Oxide Nanoparticles(IONPs)are a member of the recently discovered nanozyme family.It has the function of horseradish peroxidase(HRP)without any modification on the surface.In acidic condition,IONPs can catalyze the reaction of hydrogen peroxide between and hydroxyl radicals(·OH),which leads to an increase in ROS level.In order to find a new treatment scheme for killing LSCs more efficiently,Fluorescence activated cell sorting(FACS)or magnetic associated cell sorting(MACS)was applied to sort LSCs(CD34+CD38– phenotype cells)from human AML cell line KG1 a.Then,we treated LSCs and AML model mice with IONPs(Fe3O4 were used in the study),and Ara-C to investigate the efficacy and possible mechanisms in vitro and in vivo.Objective: To investigate the effects of IONPs on LSCs in vitro and in vivo and related mechanisms to provide a novel way for improving the AML patient's survival.Methods and Contents: 1.Sorting and identification of LSCs: According to the cell surface molecular markers,the CD34+CD38–phenotype cells(LSCs)were firstly isolated from human AML cell line KG1 a by Fluorescence activated cell sorting,then we verified its stemness via the assays of cell proliferation,chemotherapy resistance,colony formation,soft agar clone,and so on.2.Cell experiments in vitro: The LSCs and Non-LSCs were treated with PBS(Control),Ara-C(0.4 ?M),IONPs(150 ?g/ml)and Ara-C+IONPs respectively.Then we analyzed the levels of apoptosis and reactive oxygen species(ROS),and the expressions of related genes and proteins in a various treated cells by Flow cytometry,RT-q PCR,and Western Blot,respectively.3.Therapeutic experiments in vivo: Each nonobese diabetic/ severe combined immunodeficiency disease(NOD/SCID)mouse was inoculated with 5×105Non-LSCs or LSCs via tail vein.Then,we treated the mice with PBS(Control),Ara-C,IONPs and Ara-C+IONPs,respectively,(Ara-C:50 mg/kg/time/each one,IONPs:10 mg/kg/time/each one)one time per 3 days for 3 weeks.After 20 days of treatment,the therapeutic effects in each group was observed,and the mechanisms was analyzed by cytology and histology in peripheral blood and bone marrow.Results: 1.We identified the purity of cells isolated by FACS with flow cytometry(FCM),and the results showed that CD34+CD38–LSCs accounts for about 32% of KG1 a cells.Compared with Non-LSCs,LSCs can form more clones in soft agar medium,generate stronger drug resistance,and differentiate into CD34+CD38+cells,namely non-LSCs.2.In cell experiments in vitro,we found that compared with the Control,Ara-C,and IONPs treatment groups,the cells treated with IONPs+Ara-C showed a significant increases in levels of intracellular ROS and apoptosis,and that the expressions of intracellular pro-oxidation genes of gp91-phox,p40-phox and OGDH were increased,and the anti-oxidantion gene SOD1 was decreased in the levels of m RNA and protein,and the differences are statistically significant(P<0.05 or P<0.01).In the IONPs+Ara-C treatment group,the IFITM3,which is a high expression gene in LSCs,was down-regulated,and CD38 and DEPTOR,which are a low expression genes in LSCs,were up-regulated,suggesting that elevated ROS level could kill more LSCs.3.The results of therapeutic experiments in vivo showed that the numbers of peripheral blood leukocytes and hemoglobin levels were singnificantly decreased in the LSCs group compared with the Non-LSCs control group,and the difference was statistically significant between the groups(P<0.05 or P<0.01).Compared with control,Ara-C and IONPs groups,the analysis of peripheral blood and bone marrow by the blood and bone marrow slides indicated the above mentioned index was lessened and the signs in the AML bearing mice was alleviated,and the difference are statistically significant between the groups(P<0.05 or P <0.01).Conclusions: 1.CD34+CD38–phenotype cells in the human AML cell line KG1 a have LSCs-like properties,namely LSCs.2.In vitro experiments,IONPs combined with Ara-C could promote the apoptosis of LSCs more strongly than Ara-C,which may be related to the increase of intracellular ROS level by IONPs action.3.The experiment in vivo revealed that IONPs combined with Ara-C have a stronger therapeutic effect on the AML bearing mice,which can kill LSCs more effectively and alleviate leukemia signs,suggesting that IONPs combined with Ara-C is a new effective method for treating of AML.