The Molecular Mechanism Of Anti-angiogenic Activity Induced By ShengMaBieJia Decoction In Acute Myeloid Leukemia Via PI3K/AKT Pathway

Background and purposeAcute Myeloid Leukemia(AML)is a hematological malignancy derived from hematopoietic stem cells(HSCs)that manifests as myeloid hypertrophy and mutations.AML shows the malignant proliferation of leukemia stem cells,resulting in the inhibition of hematopoietic cells,which finally causes a functional lesion of multiple organ.Angiogenesis in hematological tumors has been verified to play a crucial role in stimulating disease procession.The extensive vessels network in bone intima supports the proliferation and further differentiation of hematopoietic stem cells.In pathological situations,leukemia cells and vascular endothelial cells stimulate each other in the bone marrow cavity,resulting in an imbalance of growth factors that stimulates neovascularization,which finally affects the sensitivity of chemotherapeutics and prognosis of the disease,increaseing the rate of drug resistance and the incidence of complication.ShengMaBieJia decoction(SMBJD)is described in the Synopsis of Prescriptions of the Golden Chamber(also called JinGuiYaoLue).It is mostly used in skin diseases,immune diseases and other systems,because of the function of regulating immunity and anti-inflammatory.According to the theory of traditional Chinese medicine,as well as combining with clinical experience,modified ShengMa BieJia Decoction is applied to hematological disease,which can reduce adverse events(such as hematopoietic suppression,immune deficiency,gastrointestinal reactions,etc.),improve the effectiveness of chemotherapy,and better the life of patients.Our pre-research shows that SMBJD has cytotoxic effect on AML cell lines,inducing the apoptosis of AML cells through MAPK pathway.SMBJD also inhibits the growth of tumors in tumor-bearing mice.This study intends to explore whether the anti-leukemia effect of SMBJD is realized via its anti-angiogenesis effect in AML,and the possible molecular mechanism.MethodsFor in vivo,the chicken chorioallantoic membrane(CAM)and mice xenograft models are treated with different dosage of SMBJD.The anti-angiogenic activity is assessed according to microvessel density(MVD)in CAM.and immunohistochemistry(IHC)targeting CD31 and VEGFR2 in HL60 xenograft models.Western blotting and polymerase chain reaction(PCR)are used to detect the expression of protein and mRNA of tumor.For in vitro,high performance liquid chromatography(HPLC)is used for analyze the active ingredients of SMBJD.The activity of proliferation is measured in AML cell lines and HUVECs.The ability of migration and tube formation is used to observe the fuction of HUVECs intervened by SMBJD,conditional media(CM)or VEGF165.Enzyme-linked immunosorbent assay to detect the secretion of VEGF in AML cells.Western blots and polymerase chain reaction(PCR)assays were used to examine the levels of PI3K,AKT,and VEGF.ResultsHPLC analyses revealed the active constituents of SMBJD as Liquiritin,Ferulic acid,Cimifugin,Isoferulic acid,and Glycyrrhizic acid.In vivo,CAM models exhibited a decrease in MVD,and the tissues of xenografted mice possessed reduced CD31 and VEGFR2 expression.WB and PCR showed that the level of relative protein and mRNA was decreased.In vitro,SMBJD treatment decreased cellular migration,chemotaxis,and tube formation at non-cytotoxic concentrations(2,4,8 mg/mL)in a time-and dose-dependent manner.Conditioned media(CM)from AML cells(HL60,NB4 and KG-1 cells)treated with non-cytotoxic doses of SMBJD inhibited chemotactic migration and tube formation in vitro,also.Moreover,the function of HUVECs stimulated by VEGF165 was also inhibit by SMBJD.ELISA displayed that SMBJD reduced the secretion of VEGF.The expression of PI3K,AKT,p-PI3K,p-AKT,VEGF and VEGFR2 was downregulated in Western blotting and PCR experiments.Conclusion1.SMBJD has inhibit the microvessel density(MVD)in CAM.Also,it downregulates the expression of VEGF?VEGFR2 in WB and PCR in AML tumor bearing mice models,also the immunohistochemical of CD31 and VEGFR2.2.SMBJD decreases both the secretion of VEGF in AML cells and the function of HUVECs.3.The protein level of VEGF,VEGFR2,PI3K,AKT and the relevant mRNA are inhibited in HUVECs cultured with CM.This affect is obvious following the increasing dosage of SMBJD in CM.4.The anti-angiogenic activity of SMBJD may be achieved via downregulating the secretion of VEGF in AML cells and PI3K/AKT pathway.