Effects Of VPA Combining With Arsenic Trioxide On The Expression Of Angiogenesis Related Factors In NB₄Cell Line

Objective:Arsenic trioxide （As₂O₃） is a highly effective and safe treatment forpatients with refractory/relapsed acute promyelocytic leukemia （APL）.Complete response （CR） rate is high. Rapid degradation of PML/RARA hasbeen reported as a molecular mechanism for the effectiveness of As₂O₃. It canachieve the purpose of the cure by inducing differentiation and promotingcancer cells apoptosis. But clinically white blood cell can growth spurt byusing As₂O₃in the early differentiation stage. Leukemia cells infiltratingincreases the risk of patients. Some scholars think that leukemia cellinfiltration phenomenon is related to vascular endothelial growth factor. VEGFis the most important regulating angiogenesis cytokine. The combination ofVEGF and its receptor（Flt-1）can stimulate vascular endothelial cells to formneovascularization. It can influence the growth,transfer and prognosis ofmultiple tumors. Matrix metal proteinases is a series of proteolytic enzymes. Itplays an important role in the process of tumor invasion and recurrence.MMP-9facilitates the extracellular matrix remodeling through degradation ofcollagen type Ⅳ enzymes and break the basement membrane in order topromote to form blood vessels. MMP-9can increase the expression of VEGF.Survivin is a new member of the inhibitor of apoptosis protein family, It playsan important role in tumor occurrence and development process. Valproic acidsodium （valproic acid, VPA） is a spectrum of antiepileptic drug widely appliedto the clinic. VPA has the function of blocking tumor cell cycle, inducingtumor cell apoptosis and promoting tumor cell autophagy. It has been foundrecently that it could block angiogenesis in cancer treatment. This studyinvestigates the effect of the expression of angiogenesis related factors int（15,17）APL cell line NB4induced by the combination of VPA with different concentration As₂O₃.Methods:NB4cells line was cultured and treated with As₂O₃alone （with dose of0、0.1、0.5、1.0、2.0μmol/L） or in combination with VPA.Cytotoxic reaction wasassessed by MTT assay. RT-PCR was applied to detect the expression changeof mRNA level of VEGF/Flt-1、MMP-9and Survivin.Western Blot wasapplied to detect the expression change of protein level of VEGF andSurvivin.Results:1The influence of survival with different treatment group of NB4cellMTT results showed that different concentrations of As₂O₃alone have acertain degree of inhibition in a dose-dependent manner to the NB4cell andstatistical significance （P<0.05）.The survival rate was depressed in As₂O₃andVPA compared with As₂O₃alone and statistical significance （P<0.05）.Compared between groups of different concentrations As₂O₃and VPA nostatistical significance （P>0.05）.2The influence of different concentrations of As₂O₃on VEGF of NB4cell2.1Semi-quantitation RT-PCR was used to detect the expression ofVEGF mRNA. Compared with control group, VEGF mRNA was increased indifferent concentrations As₂O₃（with dose of0.1、0.5、1.0μmol/L） andstatistical significance （P<0.05）. VEGF mRNA was depressed in2.0μmol/LAs₂O₃and statistical significance （P<0.05）.2.2Western Blot was used to detect the expression of VEGF protein.Compared with control group, VEGF protein was increased in differentconcentration As₂O₃（with dose of0.1、0.5、1.0μmol/L） and statisticalsignificance （P<0.05）. VEGF protein was depressed in2.0μmol/L As₂O₃andstatistical significance （P<0.05）.3The influence of different concentrations of As₂O₃on Flt-1of NB4cellSemi-quantitation RT-PCR was used to detect the expression of Flt-1mRNA. Compared with control group, Flt-1mRNA was increased in different concentrations As₂O₃（with dose of0.1、0.5、1.0μmol/L） andstatistical significance （P<0.05）. Flt-1mRNA was depressed in2.0μmol/LAs₂O₃and statistical significance （P<0.05）.4The influence of different concentrations of As₂O₃on MMP-9of NB4cellSemi-quantitation RT-PCR was used to detect the expression of MMP-9mRNA. Compared with control group, MMP-9mRNA was increased indifferent concentrations As₂O₃（with dose of0.1、0.5、1.0μmol/L） andstatistical significance （P<0.05）. MMP-9mRNA was depressed in2.0μmol/LAs₂O₃and statistical significance （P<0.05）.5The influence of VPA combining with different concentration As₂O₃and As₂O₃alone on VEGF of NB4cell5.1Semi-quantitation RT-PCR was used to detect the expression ofVEGF mRNA. Compared with As₂O₃alone, VEGF mRNA was depressed inVPA combining with As₂O₃and statistical significance （P<0.05）.5.2Western Blot was used to detect the expression of VEGF protein.Compared with As₂O₃alone, VEGF protein was depressed in VPA combiningwith As₂O₃and statistical significance （P<0.05）.6The influence of VPA combining with different concentration As₂O₃and As₂O₃alone on Flt-1of NB4cellSemi-quantitation RT-PCR was used to detect the expression of Flt-1mRNA. Compared with As₂O₃alone, Flt-1mRNA was depressed in VPAcombining with As₂O₃and statistical significance （P<0.05）.7The influence of VPA combining with different concentration As₂O₃and As₂O₃alone on MMP-9of NB4cellSemi-quantitation RT-PCR was used to detect the expression of MMP-9mRNA. Compared with As₂O₃alone, MMP-9mRNA was depressed in VPAcombining with As₂O₃and statistical significance （P<0.05）.8The influence of different concentrations of As₂O₃on Survivin of NB4cell8.1Semi-quantitation RT-PCR was used to detect the expression of Survivin mRNA. Compared with control group, Survivin mRNA wasdepressed in different concentrations As₂O₃（with dose of0.1、0.5、1.0、2.0μmol/L） and statistical significance （P<0.05）.8.2Western Blot was used to detect the expression of Survivin protein.Compared with control group, Survivin protein was depressed in differentconcentrations As₂O₃（with dose of0.1、0.5、1.0、2.0μmol/L） and statisticalsignificance （P<0.05）.9The influence of VPA combining with different concentration As₂O₃and As₂O₃alone on Survivin of NB4cell9.1Semi-quantitation RT-PCR was used to detect the expression ofSurvivin mRNA. Compared with As₂O₃alone, Survivin mRNA was depressedin VPA combining with As₂O₃and statistical significance （P<0.05）.9.2Western Blot was used to detect the expression of Survivin protein.Compared with As₂O₃alone, Survivin protein was depressed in VPAcombining with As₂O₃and statistical significance （P<0.05）.Conclusions:1VPA could enhance the survival rate decline of NB4cells induced byAs₂O₃alone.2Small dose of As₂O₃can induce the expression of angiogenesis relatedfactors VEGF/Flt-1and MMP-9.3The expression of VEGF/Flt-1and MMP-9cultured with VPA+As₂O₃compared with As₂O₃alone both decreased.4Small dose of As₂O₃can induce the expression of Survivin mRNA andprotein depressed. The expression of Survivin decreased cultured with VPA+As₂O₃compared with As₂O₃alone.