The Role And Mechanism Of FoxM1 In Myeloid Leukemia

BACKGROUD:Myelocytic leukemia is divided into acute myeloid leukemia(AML)and chronic myeloid leukemia(CML).AML is the most common form of acute leukemia,with the characteristics of rapid disease development and only several months natural course.The patients of AML except acute promyelocyte leukemia(APL)are always offered the combination of standard-dose cytarabine with an anthracycline,but the response to the treatment is poor.As the fact that AML is a kind of highly heterogeneous disease,drugs that target on important molecules in AML progression cannot meet the clinical needs.Further study of mechanism of AML progression and finding new potential targets become more and more important in basic science.CML evolve through three phases,including a chronic phase(CP)at first and then enter an accelerated phase(AP),followed by a blast crisis(BC).Tyrosine kinase inhibitors,a BCR/ABL kinase inhibitor,is effective in chronic phase CML patients,however has poor response to accelerated phase and blast crisis CML patients.Homoharringtonine(HHT)is a natural alkaloid isolated from various cephalotaxus species in China.Study has shown that HHT can through independent BCR/ABL pathway effect on accelerated phase,blast crisis and even tyrosine kinase inhibitor drug-resistant CML patients.Considering the side effects of HHT,such as heart and bone marrow suppression,hyperglycemia and so on,clinical application of HHT is limited.To promote HHT's drug effect,discovering molecules that has synergy sensitization effect with HHT to reduce the clinical effective dosage of HHT is the most important strategy.Forkhead box M1(FoxM1)is a transcription factor,has positive regulation of normal cell cycle execution during G1-S,G2 and M phase transitions to promote cell proliferation and mitosis.Studies have shown that FoxM1 is overexpressed in some solid tumors,inducing abnormal cell proliferation to promote malignant transformation.As FoxM1 does not express in most normal mature tissue cells,it is considered to be a potential target of anti-tumor.The role of FoxM1 in acute leukemia is relatively infrequent reported.B-cell lymphoma-2(BCL2)is one of the most popular oncogene through inhibiting apoptosis to promote the malignant transformation.Whether FoxM1 could regulate BCL2 to have biological effect in AML deserves attention.HHT is a kind of cell cycle specificity drug,while FoxM1 mainly regulates molecular transcription related to cell cycle then participate in tumorigenesis by mediating cell proliferation.The inhibition of FoxM1 can improve chemotherapy drugs sensitivity in solid tumor cells.FoxM1 and HHT can both regulate the cell cycle.There is no report on whether interventing FoxM1 expression can enhance the coordination sensitization of HHT's anti-leukemia effect.OBJECTIVE:1.To investigate the role of FoxM1 and BCL2 in the pathogenesis of acute myeloid leukemia.2.To study inhibition of FoxM1 sensitizes leukemia K562 cells to HHT.METHODS:1.qRT-PCR and immunofluorescence analysis of FoxM1 at mRNA and protein levels in 17 AML-de novo patients,17 AML-CR patients,17 AML-RR patients and 15 healthy controls.HL60 cells and K562 cells were transfected with FoxM1 siRNA,then the cell proliferation was detected by cell proliferation assay and of colony-formation assay on soft agar,and the cell apoptosis was determined by flow cytometry.The expression of FoxM1 and BCL2 at mRNA and protein levels was detected by RT-q PCR and Western blotting.The activity of BCL2 promoter was examined by Luciferase reporter assay with FoxM1 targetting.2.In K562 cells incubated with HHT at different concentrations(0?mol/L,0.015?mol/L,0.03?mol/L,0.045?mol/L)and different times(0h,24 h,48h,72h),FoxM1 mRNA and protein levels were detected by qRT-PCR and Western Blot.FoxM1 siRNA was transfected into K562 cells with 0.015?mol/L HHT after 6 h.After 72 h incubation,cell proliferation was detected by MTT and soft agar assay,and the proportion of apoptotic K562 cells was determined by flow cytometry.The expression of c-myc and Sp1 were detected by qRT-PCR and Western Blot.RESULTS:1.FoxM1 expression level in the AML-de novo patients was significantly higher than that in the healthy controls.As compared with the AML-de novo patients,FoxM1 expression in the AML-CR patients was reduced,the FoxM1 expression level was the highest in the AML-RR patients.FoxM1 expression was inhibited in the HL60 cells and K562 cells transfected with FoxM1 siRNA.Transfection with FoxM1 siRNA in the HL60 cells and K562 cells inhibited the proliferation as compared with NC siRNA transfection,and impaired the colony-formation ability.On the contrary,Transfection with FoxM1 siRNA promoted the cell apoptosis.FoxM1 regulated BCL2 expression positively.2.FoxM1 expression was reduced time-dependently and dose-dependently,suggesting that HHT mediated the downregulation of FoxM1 in K562 cells.In K562 cells treated with FoxM1 siRNA and HHT,cell proliferation was inhibited and apoptosis was promoted much more significantly.Therefore,inhibition of FoxM1 sensitizes leukemia K562 cells to HHT.The expression of c-myc and Sp1 was positively regulated by FoxM1.CONCLUSION:1.FoxM1 promotes the pathogenesis and development of AML by regulating BCL2 expression.Inhibition of FoxM1 expression suppresses cell proliferation and promotes cell apoptosis.FoxM1 is a potential target for AML treatment.2.HHT inhibited FoxM1 expression in K562 cells.FoxM1 inhibition sensitized K562 cells to HHT.