Leukemia Anti-angiogenic Therapy In Vivo And Vitro

Objective:Resently, a novel proangiogenic growth factor, angiopoietins (Angs), hasbeen found,which is most significant to the formation of vascular tubularstructure. It has been reported that angiopoietins expression is detected inAML bone marrow and peripheral blood, and in AML cell line K-562. Theaim of this study was to detect the expression of angiopoietins in t(8;21) AMLcell line Kasumi-1cell and Kasumi-1xenograft tumor in nude mice, whileevaluated the effects of valproic acid (VPA), as a histone deacetylase inhibitor,on the angiogenesis of AML and the expression of angiopoietins.Method:Kasumi-1cell line–derived from t(8;21) AML was cultured, and treatedwith different concentration VPA for3d. RT-PCR and Western Blot wereapplied to detect the expression change of mRNA or protein level of Ang1andAng2. Kasumi-1cell line model transplanted subcutaneously in nude micewere established, and xenograft transplanted nude mice were assigned intocontrol and VPA treatment groups. CD34was detected byimmunohistochemistry. The expression change of mRNA or protein level ofAng1, Ang2and VEGF were detected by immunohistochemistry, Westernblot and RT-PCR.Result:1VPA inhibited the proliferation of the Kasumi-1cell and tumor growthof xenograft tumor1.1VPA inhibited the proliferation of the Kasumi-1cellThe cell growth was obviously inhibited with different concentration VPAafter3d in dose-dependent manner. The IC50of VPA was2.46mmol/L for3d.1.2VAP inhibited Kasumi-1xenograft tumor growthVAP significantly inhibited Kasumi-1xenograft tumor growth. With the prolongation of time, the growth speed of tumor were inhibited by VPAconspicuously compared the control group. The tumor volume and weight inthe VAP treatment group are statistically lower than that of the control group.The calculated tumor growth inhibition rate is57.25%.2The influence of VPA on MVD of Kasumi-1xenograft tumorImmunohistochemistry was used to detect the expression of CD34invascular endothelial cell. Compared with control group, CD34expression wasdepressed in VPA group with statistical significance (P<0.01).3The influence of VPA on Ang1of Kasumi-1cell and xenograft tumor3.1The influence of VPA on Ang1of Kasumi-1cell3.1.1The influence of VPA on Ang1mRNA of Kasumi-1cellSemi-quantitation RT-PCR was used to detect the expression of Ang1mRNA. Compared with control group, Ang1mRNA expression was depressedin different concentration VPA for3d with dose-dependence and statisticalsignificance(P<0.01,1~3mM VPA group vs control group).3.1.2The influence of VPA on Ang1protein of Kasumi-1cellWestern blot was used to detect the expression of Ang1protein. Comparedwith control group, Ang1protein expression was depressed in differentconcentration VPA for3d with dose-dependence and statisticalsignificance(P<0.01,1~3mM VPA group vs control group).3.2The influence of VPA on Ang1of Kasumi-1Xenograft tumor3.2.1The influence of VPA on Ang1mRNA of Kasumi-1XenografttumorSemi-quantitation RT-PCR was used to detect the expression of Ang1mRNA. Compared with control group, Ang1mRNA expression was depressedin VPA group with statistical significance (P<0.01).3.2.2The influence of VPA on Ang1protein of Kasumi-1XenografttumorWestern blot was used to detect the expression of Ang1protein.Compared with control group, Ang1protein expression was decreased in VPAgroup with statistical significance (P<0.01). Immunohistochemistry was used to detect the expression of Ang1protein.Compared with control group, Ang1protein expression was decreased in VPAgroup.4The influence of VPA on Ang2of Kasumi-1cell and xenograft tumor4.1The influence of VPA on Ang2of Kasumi-1cell4.1.1The influence of VPA on Ang2mRNA of Kasumi-1cellSemi-quantitation RT-PCR was used to detect the expression of Ang2mRNA. Compared with control group, Ang2mRNA expression was depressedin different concentration VPA for3d with dose-dependence and statisticalsignificance(P<0.01,1~3mM VPA group vs control group).4.1.2The influence of VPA on Ang2protein of Kasumi-1cellWestern blot was used to detect the expression of Ang2protein.Compared with control group, Ang2protein expression was depressed indifferent concentration VPA for3d with dose-dependence and statisticalsignificance(P<0.01,1~3mM VPA group vs control group).4.2The influence of VPA on Ang2of Kasumi-1Xenograft tumor4.2.1The influence of VPA on Ang2mRNA of Kasumi-1XenografttumorSemi-quantitation RT-PCR was used to detect the expression of Ang2mRNA. Compared with control group, Ang2mRNA expression was depressedin VPA group with statistical significance (P<0.01).5.2.2The influence of VPA on Ang2protein of Kasumi-1XenografttumorWestern blot was used to detect the expression of Ang2protein.Compared with control group, Ang2protein expression was decreased in VPAgroup with statistical significance (P<0.01).Immunohistochemistry was used to detect the expression of Ang2protein.Compared with control group, Ang2protein expression was decreased in VPAgroup.5The influence of VPA on VEGF of Kasumi-1Xenograft tumor5.1The influence of VPA on VEGF mRNA of Kasumi-1Xenograft tumorSemi-quantitation RT-PCR was used to detect the expression of VEGFmRNA. Compared with control group, VEGF mRNA expression wasdepressed in VPA group with statistical significance (P<0.01).5.2The influence of VPA on VEGF protein of Kasumi-1XenografttumorWestern blot was used to detect the expression of VEGF protein.Compared with control group, VEGF protein expression was decreased inVPA group with statistical significance (P<0.01).Conclusion:1. VPA inhibited the proliferation of the Kasumi-1cell and tumor growthof xenograft tumor.2. The histone deacetylase inhibitor VPA inhibites angiogenesis ofKasumi-1xenograft tumor in nude mice.3. We validated the expression of Ang1and Ang2mRNA in Kasumi-1cell.4. VPA inhibited the expression of proangiogenic growth factor:angiopoietins and VEGF.