Study On The Role Of Desferamine-mediated The Wnt/β-catenin And P38MAPK/ERK1/2 Signaling Pathways In Acute Lymphoblastic Leukemia And Mechanism

Acute lymphoblastic leukemia(ALL)is a common hematological malignancy.Patients with ALL account for approximately 78%of patients with pediatric leukemia and about 20%of patients with adult leukemia.The reasons of refractory or relapsed acute leukaemia are not well known.However,refractory or relapsed acute leukaemia likely relates to multiple factors,including patients’age,leukocyte counts,immunophenotypes,genes,time from chemotherapy to CR,minimal residual disease(MRD),etc.Although treatment effects for pediatricALL have been improved considerably in the past decades,treating pediatricpatients with relapsed/refractory ALL is still difficult.The death rate of children with refractory/relapsed ALL is very high,which is associated with chemotherapy resistance,high mortality by reinduction,etc.Therapeutic strategy on relapsed/refractory ALL is always a major therapeutic challenge,which bothers hematology researchers.Iron is essential for the metabolism,survival,and growth of almost all cells and organisms.However,excess iron,which leads to the generation and propagation of oxygen and hydroxyl-free radicals,is potentially toxic in cells.Disturbed iron homeostasis is now recognized as part of the pathophysiology of various disease states including cardiovascular complications,neurodegenerative diseases,aging,infection,and cancer.Several forms of cancers,including lung,pancreas,colon,and breast,have been reported to associate with significant iron overload.With regard to the complex roles of iron in cancer,more and more research indicates that iron chelation therapy can be used to treat cancer patients with iron overload.Iron chelators can affect the total iron content of cells,and are mainly used in clinical treatment of iron overload diseases,such as neuroblastoma,liver cancer,breast cancer,ovarian cancer and leukemia.Experiments have confirmed that iron deficiency can inhibit the proliferation of a variety of tumor cells and induce apoptosis.The use of iron chelators is expected to become a new strategy for treating cancer.Inappropriate activation of the canonical Wnt/β-catenin signaling pathway contributes to the development of a numerous human cancers.Deregulation of the Wnt/β-catenin pathway has been reported in acute myeloid leukemias(AML)where expression of β-catenin is correlated with poor prognosis.In addition,the Wnt/β-catenin signaling has been shown to be required for the development of the highly proliferative leukemia stem cells(LSC)that is thought to maintain leukemias.The widespread deregulation of the Wnt pathway in diverse cancers makes it an attractive therapeutic target.Abnormal activation of ERK1/2 signaling pathway plays an important role in the occurrence and development of cancer.Studies have found that the ERK1/2 and Wnt/β-catenin signaling pathways play a synergistic role in some biological processes.For example,bergamot polyphenol can regulate bone biomarkers by activating the ERK1/2 and Wnt/β-catenin pathways in bone cells,thus affecting bone development and growth.Long non-coding RNA CASC2c inhibits proliferation of liver cancer cells by inhibiting the activity of ERK1/2 and Wnt/β-catenin signaling pathways.However,further experiments are needed to determine whether the suppression of ALL proliferation and invasion,is associated with both the ERK1/2 and Wnt/β-catenin signaling pathways.In this study,we investigated the role and mechanism of deferriamine mediated Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathways in acute lymphoblastic leukemia.Firstly,in order to determine iron overload and reactive oxygen species in serum and cells of ALL patients,the content of ferritin and reactive oxygen species in serum of ALL patients were detected by electrochemiluminescence immunoassay and Fenton method,respectively,and the variable iron pool(LIP)level of ALL cells was determined by fluorescence spectrophotometer.Secondly,in order to study the effect of deferoxamine on the biological of ALL cell lines and mechanism,CCK-8,Transwell and flow cytometry were performed to detect the deferoxamine on ALL cell proliferation,invasion,apoptosis,cell cycle and reactive oxygen species;in addition,the mechanism of DFO function in ALL cell lines was discussed in this paper,and Western blot was carried out to determin the effect of DFO on cell proliferation,invasion,apoptosis-related marked molecules and the Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathways related protein expression.Finally,a mouse model of ALL xenograft was established to further examine the effects of DFO on the growth and reactive oxygen species of ALL xenograft.This study aimed to explore the roles and mechanism of DFO on acute lymphoblastic leukemia,which would provide new treatment target and safe and effective treatment method for ALL.This study is divided into three parts:The first part:Iron overload and reactive oxygen species in serum and cells of ALL patients;The second part:Biological effect of DFO on ALL cell lines and mechanism.The third part:Effect of DFO on tumor growth and serum reactive oxygen species in ALL nude mice.Main Content:The first part:Iron overload and reactive oxygen species in serum and cells of ALL patientsMethods1.Serum ferritin content of ALL patients was detected by electrochemiluminescence immunoassay.2.Reactive oxygen species(ROS)in serum of ALL patients were detected by Fenton method.3.The level of variable iron pool(LIP)in ALL cells was detected by fluorescence spectrophotometer.Results1.Compared with normal serum,serum ferritin content and reactive oxygen species level were significantly increased in ALL patients.2.Variable iron pool(LIP)levels were significantly increased in ALL cells compared with peripheral blood mononuclear cells of healthy volunteers.The second part:Biological effect of DFO on ALL cell lines and mechanismMethods1.CCK-8 assay was used to detect the effects of different concentrations of DFO on the activities of ALL cell lines NALM-6 and Jurkat.2.Fluorescence spectrophotometer was used to detect the effect of DFO on iron pool LIP in ALL cell lines NALM-6 and Jurka.3.The effects of DFO on the Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathway in ALL cell lines NALM-6 and Jurkat were detected by Western blot.4.CCK-8 was performed to determin the effect of DFO on the proliferation of ALL cell lines NALM-6 and Jurkat.5.The effect of DFO on the invasion of ALL cell lines NALM-6 and Jurkat was detected by Transwell assay.6.Flow cytometry was carried out to assess the effect of DFO on apoptosis and cell cycle of ALL cell lines NALM-6 and Jurkat.7.The effects of DFO on the expression of proteins Ki67,Bcl-xl,Bid,cleaved caspase-9,MMP-2 and MMP-7 in ALL cell lines NALM-6 and Jurkat were detected by Western blot.8.The effect of DFO on the activity of Caspase-9 in ALL cell lines NALM-6 and Jurkat was detected by ELISA.9.Flow cytometry assay was performed to detect the effect of DFO on reactive oxygen species(ROS)in ALL cells.Results:1.DFO could reduce the level of LIP by chelating iron ions in ALL cell lines NALM-6 and Jurkat,thereby inhibiting proliferation and invasion ability,promoting apoptosis,blocking cell cycle in G1 and G2 phases,and reducing the proportion of cells in S phase.2.After DFO treatment,the protein expressions of Ki67,Bcl-xl,cleaved caspase-9,MMP-2 and MMP-7 and reactive oxygen levels in NALM-6 and Jurkat cell lines were significantly decreased,the expression level of Bid was significantly increased,and the activity of Caspase-9 was significantly enhanced,further suggesting that DFO can regulate the proliferation,invasion,apoptosis and reactive oxygen levels of ALL cells.3.In NALM-6 and Jurkat cells,DFO could inhibite the expression of β-catenin,c-Myc,cyclinD1,p-p38MAPK and p-ERK1/2 proteins involved in the Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathways.The third part:Effect of DFO on tumor growth and serum reactive oxygen species in ALL nude miceMethods:1.The mouse models of ALL were constructed,and the volume and weight of the mouse ALL xenografts after DFO or/and Dex treatment were certained.2.Western blot was put into determining the expression of the related proteins in the Wnt/β-catenin and ERK1/2 signaling pathways in each group of xenograft tumors.3.The expression of Ki67 protein was determined by immumohistochemical staining in xenograft tumors tissues.4.The level of reactive oxygen species in serum of ALL nude mice was determined by Fenton method.Results:1.Our results showed that DFO could significantly inhibit the growth of ALL xenografts.2.Western blot analyses revealed that DFO can inhibit the expression of c-Myc,cyclinD1,β-catenin,and p-ERK1/2 protiens in the Wnt/β-catenin and ERK1/2 signaling pathways in xenograft tumor tissues.3.Immunohistochemical detection showed that DFO could inhibit the expression of Ki67 protein in xenograft tumor tissues.4.Compared with the control group,DFO could inhibit the level of reactive oxygen species in serum of ALL nude mice,and this inhibition was significantly weakened after Dex treatment.Conclusions:1.Compared with normal serum,serum ferritin content and reactive oxygen species level were significantly increased in ALL patients.The level of variable iron pool(LIP)was significantly increased in ALL cells compared with normal cells.2.DFO could inhibit the activity of Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathway by reducing the level of LIP through chelating iron in ALL cell lines NALM-6 and Jurkat,inhibit proliferation,invasion ability and reactive oxygen species level,promote cell apoptosis,block cell cycle in G1 and G2 phases,and reduce the proportion of cells in S phase.3.In vivo experiments further showed that DFO could inhibit the Wnt/β-catenin and p38MAPK/ERK1/2 signaling pathways and the level of reactive oxygen species in serum of tumor-bearing mice,thereby inhibiting the growth of ALL transplanted tumors.