ATPR Induces Acute Myeloid Leukemia Cells Differentiation Via RARα/LCHB/ERK Molecular Axis

Acute myeloid leukemia(AML)is characterized by impaired differentiation of myeloid hematopoietic stem/progenitor cells.The standard treatment for the past 40 years has been "3+7" therapy(anthracyclinesin combination with cytarabine),but there are many side effects.Different from traditional chemotherapy,differentiation therapy is to selectively induce tumor cells to differentiate into normal cells through drugs,without obvious toxic and side effects on normal tissue cells,so as to achieve the goal of curing tumors.Although ATRA,as a classic differentiation drug,has been widely used in the clinical treatment of AML,it is only effective in 10% of AML type M3 acute promyelocytic leukemia(APL),and patients who relapse during treatment often develop resistance to ATRA or retinoic acid syndrome.Therefore,it is necessary to develop new differentiation agents with low toxicity and high efficiency in the treatment of AML.Taking ATRA as the prototype,this research group synthesized a typical new compound 4-amino-2-trifluoromethyl-phenyl retinate(ATPR).It has been proved that it has a strong differentiation induction activity on various hematological malignancy.However,the exact mechanism of ATPR in AML has not been clarified.Abnormal glucose metabolism is one of the main characteristics of tumor cells.In the aerobic environment,tumor cells prefer to use glycolysis pathway rather than oxidative phosphorylation to provide energy.The whole process is accompanied by a large amount of lactic acid accumulation,which leads to abnormal tumor microenvironment and induces metastasis and invasion of tumor cells,which is an important reason for aggravating malignant development of tumor.This abnormal glucose metabolism also contributes to multidrug resistance and significantly increases the recurrence and mortality of cancer.Therefore,targeting tumor cells with the characteristics of abnormal glucose metabolism can be used for more efficient and safe targeted treatment of tumor and improve the prognosis of patients.In recent years,it has been reported that high glycolysis levels exist in a variety of AML cell lines and primary cells of AML patients,and serum glycolysis related molecular lactate dehydrogenase(LDH)levels in AML patients are also closely related to drug resistance and relapse after chemotherapy.These results suggest that targeting AML by regulating the glycolysis pathway may provide a new idea for the future exploration of AML therapy.Proteomic analysis of ATPR and ATRA on NB4 cells showed that,unlike ATRA,ATPR can selectively downregulate lactate dehydrogenase B(LDHB),a key catalytic enzyme in glycolysis,suggesting that LDHB may play a potential role in ATPR induced differentiation of leukemia cells.This study intend to further study the role and molecular mechanism of LDHB gene in ATPR induced AML cell differentiation,so as to lay a theoretical foundation for better application of ATPR in AML induced differentiation therapyObjectives: To elucidate the role and molecular mechanism of LDHB in ATPR induced AML cells differentiation and to clarify the pharmacodynamics of ATPR in AML in vivo experiments.Methods:1.The role of retinoic acid receptor in ATPR-induced AML cell differentiationCCK-8 assay was used to detect the effects of ATPR(10-9-10-5 M,72 h)on the growth activity of AML cell lines(NB4,HL-60,KG-1,U937 and MOLM-13);CCK-8 assay was used to detect the effects of ATPR at different concentrations(10-9-10-5 M)and treatment time(24-72 h)on the growth viability of NB4 and MOLM-13 cells;Western blotting was used to detect the effects of ATPR(10-6 M,24-72 h)at different time on the expression of retinoic acid receptor(RARα,RARβ and RARγ)in AML cell lines(NB4 and MOLM-13)and PML-RARα fusion protein specific in NB4 cells;q RT-PCR was used to detect the effect of ATPR(10-6 M,24-72 h)at different time on the m RNA expression of retinoic acid receptors(RARα,RARβ and RARγ)and downstream target genes(CRABP2 and CYP26A1)in AML cell lines(NB4 and MOLM-13);RARα inhibitor Ro41-5253(10-5 M)was added 1 h before ATPR treatment,and the effect of Ro41-5253 on the cell cycle and differentiation of NB4 and MOLM-13 cells under ATPR treatment was detected by flow cytometry;Western blotting was used to detect the effect of Ro41-5253 on cell cycle associated proteins(Cyclin A2,CDK4 and Cyclin D3)and differentiation associated proteins(PU.1)under ATPR treatment.2.The role of LDHB in ATPR-induced AML cell differentiationCCK-8 assay was used to detect the effects of glycolytic inhibitor 2-DG(1 m M-16 m M,72 h)at different concentrations on the growth activity of AML cell lines(HL-60,KG-1,NB4 and MOLM-13);Proteomics was used to detect the difference of protein expression between ATPR and ATRA in NB4 cells;The expression of LDHB in AML patients was analyzed in TCGA database;Western blotting was used to detect the expression of LDHB in peripheral blood of normal subjects,peripheral blood/bone marrow of newly diagnosed AML patients and different AML cell lines(HL-60,KG-1,NB4 and MOLM-13);Western blotting was used to detect LDHB protein expression under ATPR treatment(10-7-10-5 M,24-72 h);RARα inhibitor Ro41-5253(10-5 M)was added 1 h before ATPR(10-6 M,72 h)treatment,and Western blotting was used to detect the effect of Ro41-5253 on LDHB protein expression under ATPR treatment.Lentivirus carrying LDHB sh RNA was used to construct LDHB stably silenced NB4 and MOLM-13 cell models.Immunofluorescence and Western blotting were used to verify the construction of the stably silenced LDHB cell model;CCK-8 and KI67 staining were used to detect the effect of silenced LDHB on the proliferation of NB4 and MOLM-13 cells;Wright-Giemsa staining,NBT staining and flow cytometry(CD11b protein involved in differentiation and CD14)were used to detect the effect of silenced LDHB on the differentiation of NB4 and MOLM-13 cells;Flow cytometry was used to detect the effect of silenced LDHB on the cell cycle of NB4 and MOLM-13 cells;Lentivirus was used to construct NB4 and MOLM-13 cell models of stable silencing LDHB.After successfully constructed cells were screened,subcutaneously injected NB4 and MOLM-13 cells of stable silencing LDHB to construct subcutaneously transplanted tumor mouse model of NCG,and the effect of silencing LDHB on tumor-forming ability of NB4 and MOLM-13 cells was observed;Immunohistochemical staining was used to observe the effect of silencing LDHB on the expression of proliferating protein(KI67)and differentiated protein(CD11b).LDHB overexpression plasmid was transfected to construct an overexpressed LDHB cell model,and immunofluorescence and Western blotting were used to verify the construction of the overexpressed LDHB cell model;After transfection of LDHB overexpression plasmid,ATPR(10-6 M,72 h)was treated at the same time;Flow cytometry(differentiation associated protein CD11b)was used to detect the effect of LDHB overexpression on cell differentiation under ATPR treatment;Immunofluorescence assay(proliferation associated protein KI67 staining)was used to detect the effect of LDHB overexpression on cell proliferation under ATPR treatment.3.The role of Raf/MEK/ERK signaling pathway in ATPR-induced AML cell differentiationWestern blotting was used to detect the expression of Raf,p-MEK/MEK and pERK/ERK proteins in NB4 and MOLM-13 cells treated with ATPR at different concentrations(10-5-10-7 M,72 h);RARα inhibitor Ro41-5253(10-5 M)was added 1 h before ATPR(10-6 M,72 h)treatment,and Western blotting was also used to detect the effects of Ro41-5253 on Raf,p-MEK/MEK and p-ERK/ERK protein expression in NB4 and MOLM-13 cells under ATPR treatment;Western blotting was used to detect the effects of silenced LDHB on Raf,p-MEK/MEK and p-ERK/ERK protein expressions in NB4 and MOLM-13 cells;MEK inhibitor PD98059(10-5 M)was added to ATPR(10-6 M,72 h)pretreatment,and Western blotting was also used to detect the effects of PD98059 on cell cycle associated proteins(Cyclin A2,CDK4 and Cyclin D3)and differentiation associated proteins(PU.1)of NB4 and MOLM-13 cells under ATPR treatment.4.Effect of ATPR on NB4 cell ascites transplantation tumor model in NOD/SCID miceIn vivo,NB4 cells were intraperitoneally injected into NOD/SCID mice to construct the ascites graft tumor model,and ATPR(5 mg/kg,10 mg/kg,20 mg/kg)and doxorubicin(1 mg/kg)were intraperitoneally injected,respectively;The general condition and survival period of the mice were recorded,and pathological changes of the main organs(liver,spleen and kidney)were detected by HE staining.Results:1.The role of retinoic acid receptor in ATPR-induced AML cell differentiation3/5 AML cell lines(HL-60,KG-1,NB4,MOLM-13 and U937)were all sensitive to ATPR,and NB4 and MOLM-13 cell lines were the most sensitive to ATPR,and the optimal concentration was 10-6 M,and the optimal action time was 72 h;Compared with the control group,ATPR increased the m RNA and protein expressions of RARα and RARβ receptors in a time dependent manner in NB4 and MOLM-13 cells,decreased the expression of PML-RARα fusion protein specific to NB4 cells,but had little effect on the m RNA and protein expressions of RARγ receptor.ATPR also increased the m RNA expression of downstream target genes(CRABP2 and CYP26A1);Compared with the control group,early addition of RARα receptor inhibitor Ro41-5253 could block ATPR induced differentiation and G0/G1 phase cycle arrest of NB4 and MOLM-13 cells.2.The role of LDHB in ATPR-induced AML cell differentiationCompared with the control group,the addition of glycolytic inhibitor 2-DG inhibited the growth activity of AML cell lines(HL-60,KG-1,NB4 and MOLM-13)in a dosedependent manner,and NB4 cells were most sensitive to 2-DG;Compared with ATRA,ATPR can specifically down-regulate the protein expression of LDHB gene in NB4 cells;TCGA database showed that LDHB gene was highly expressed in various AML types;LDHB expression was increased in peripheral blood/bone marrow blood of AML patients and in different AML cell lines(HL-60,NB4,KG-1 and MOLM-13)compared with normal peripheral blood;ATPR inhibited LDHB gene expression in a concentration-and time-dependent manner in NB4 and MOLM-13 cells;Compared with the control group,Ro41-5253 could reverse the inhibitory effect of ATPR on LDHB gene.In vitro,silencing LDHB induced differentiation and proliferation inhibition of NB4 and MOLM-13 cells compared with control cells;In vivo,silencing LDHB inhibited the growth of NB4 and MOLM-13 xenograft tumors by inducing differentiation and proliferation inhibition,compared with control cells;Compared with the control group,overexpression of LDHB reversed the differentiation of NB4 and MOLM-13 cells induced by ATPR treatment.3.The role of Raf/MEK/ERK signaling pathway in ATPR-induced AML cell differentiationCompared with the control group,ATPR could activate the expression of Raf/MEK/ERK signaling pathway related proteins in a concentration-dependent manner in NB4 and MOLM-13 cells;Compared with the control group,early addition of RARα receptor inhibitor Ro41-5253 blocked the activation of Raf/MEK/ERK signaling pathway related protein expressions in NB4 and MOLM-13 cells by ATPR treatment;Compared with the control group,silencing LDHB could activate the expression of Raf/MEK/ERK signaling pathway related proteins expressions in NB4 and MOLM-13 cells;The addition of MEK inhibitor(PD98059)could reverse the differentiation and G0/G1 phase cycle arrest of NB4 and MOLM-13 cells induced by ATPR.4.Effect of ATPR on NB4 cell ascites transplantation tumor model in NOD/SCID miceIntraperitoneal injection of cyclophosphamide 150 mg/kg,100 mg/kg and 50 mg/kg for two consecutive days inhibited the immune capacity of NOD/SCID mice;After intraperitoneal injection of 150 mg/kg for two consecutive days in NOD/SCID mice,intraperitoneal injection of NB4 cells could form ascites and solid tumors in the abdominal cavity,infiltrate into liver,spleen,kidney and other organs,and damage the normal tissue structure;ATPR alleviated infiltration and destruction of NB4 cells to tissues and organs,and induce differentiation and maturation of ascites and solid tumor cells;ATPR(10 mg/kg,20 mg/kg)significantly prolonged the survival of mice.Conclusion:1.ATPR induced AML cell differentiation via the RARα/LDHB/ERK molecular axis.2.ATPR(10 mg/kg and 20 mg/kg)promoted the differentiation and maturation of abdominal solid tumor cells in NOD/SCID mouse ascites transplanted tumor models,inhibited the infiltration of leukemia cells to the tissues and organs of NOD/SCID mice,and significantly prolonged the survival period of NOD/SCID mouse ascites transplanted tumor models.