Inhibitory Effect Of VPA On Kasumi-1Xenograft Tumor Growth In Nude Mice

Objective:It is the pivotal pathogenesis that t(8;21)AML produces fusion protein AML1-ETO by chromosome translocation to recuit histone deacetylase (HDAC) which represses transcription of AML1target genes. To investigate in vivo tumor suppression effect of HDAC inhibitor Valproic acid in the treatment of Kasumi-1xenograft tumor in nude mice and its mechanism.Method:Kasumi-1xenograft tumor model was established by nude mice subcutaneous inoculation with Kasumi-1cell. Xenograft transplanted nude mice were assigned into control and VPA treatment groups. Growth of xenograft tumor of the two groups of nude mice was measured and recorded. Tumor volume and inhibition rate was calculated and compared between the two different groups. Tumor morphology study was performed with HE (hematoxylin and eosin) staining. TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) was applied to detect tumor cell apoptosis. The gene expression and translation of GM-CSF、HDAC1、 Ac-H3、Survivin were studied with semi-quantitative RT-PCR and Western blotting. ChIP (Chromatin Immunoprecipitation) method was used to assay the effects of VPA on acetylation of histone H3within GM-CSF promoter region.Result:1. The Characteristics of Kasumi-1Xenograft Tumor modelWe established Kasumi-1cell line tumor xenografts. The tumor formation rate of mice is100%. With the prolongation of time, the tumor volume became large markedly and cell growth curve was generated. Semi-quantitation RT-PCR was used to validated the expression of AML1/ETO mRNA. 2. VAP inhibited Kasumi-1xenograft tumor growthVAP significantly inhibited Kasumi-1xenograft tumor growth. With the prolongation of time, the growth speed of tumor were inhibited by VPA conspicuously compared the control group. The tumor volume and weight in the VAP treatment group are statistically lower than that of the control group. The calculated tumor growth inhibition rate is57.25%.3. The influence of VPA on morphocytology of Kasumi-1xenograft tumorThe result showed that, in the control group, the sizes of Kasumi-1xenograft tumor cells were similar and sizes of cell nuclei were also similar; Meanwhile, in the VPA group, the tumor cells were irregular and in disorder, and the size of the cell nuclei was different from each other, and the rate of cytoplasm and nucleus was decreased, pyknosis, karyorrhexis, karyolysis were increased.4. The influence of VPA on cells apoptosis of Kasumi-1Xenograft TumorTUNEL was applied to detect tumor cell apoptosis, and calculated apoptosis index (AI%)。Expressions of apoptotic cells were observed in two groups, the apoptosis index of the VAP treatment group (3.66±0.76)%is higher than that of the control group (0.26±0.11)%, with statistical significance(P<0.05).5. The influence of VPA on expression of Survivin mRNA in Kasumi-1Xenograft tumorSemi-quantitation RT-PCR was used to detect the expression of Survivin mRNA. Compared with control group, Survivin mRNA expression was depressed in VPA group with statistical significance (P<0.01).6. The influence of VPA on expression of GM-CSF in Kasumi-1Xenograft tumorSemi-quantitation RT-PCR was used to detect the expression of GM-CSF mRNA. Compared with control group, GM-CSF mRNA expression was increased in VPA group with statistical significance (P<0.01).Western blot was used to detect the expression of GM-CSF protein. Compared with control group, GM-CSF protein expression was increased in VPA group with statistical significance (P<0.01).SP immunohistochemical staining was used to observe the expression of GM-CSF protein in Kasumi-1xenograft tumor. Compared with control group, GM-CSF protein expression was increased in VPA group.7. The influence of VPA on expression of HDAC1protein in Kasumi-1Xenograft tumorWestern blot was used to detect the expression of HDAC1protein. Compared with control group, HDAC1protein expression was depressed in VPA group with statistical significance (P<0.01).SP immunohistochemical staining was used to observe the expression of HDAC1protein in Kasumi-1xenograft tumor. Compared with control group, HDAC1protein expression was depressed in VPA group.8. The influence of VPA on expression of AC-H3protein in Kasumi-1Xenograft tumorWestern blot was used to detect the expression of AC-H3protein. Compared with control group, AC-H3protein expression was increased in VPA group with statistical significance (P<0.01).SP immunohistochemical staining was used to observe the expression of AC-H3protein in Kasumi-1xenograft tumor. Compared with control group, AC-H3protein expression was increased in VPA group.9. The effects of VPA on acetylation of histone H3within GM-CSF promoter regionChIP method was used to assay the effects of VPA on acetylation of histone H3within GM-CSF promoter region. Compared with control group, acetylation of histone H3within GM-CSF promoter region was increased remarkably in the VPA group.Conclusion:VAP significantly inhibited Kasumi-1xenograft tumor growth, induced tumor cell apoptosis and differentiation. The mechanism underlying tumor inhibition may be due to inhibition of HDAC nuclear expression, enhancing histone protein acytelation level and level of acetylation of histone H3within GM-CSF promoter region, and increasing AML1target gene GM-CSF transcription. Our reasearch can offer theory evidence of target theropy to acute leukemia in vivo.