丙戊酸钠对Kasumi-1细胞裸鼠移植瘤的生长抑制作用及机制探讨

Inhibitory effect of valproic acid on xenografted Kasumi-1 tumor growth in nude mouse and its mechanism

目的研究组蛋白脱乙酰化酶（HDAC）抑制剂丙戊酸钠（VPA）体内给药对白血病细胞系Kasumi-1细胞裸鼠移植瘤生长的影响，探讨可能的作用机制。方法建立Kasumi-1细胞裸鼠移植瘤模型，分为对照组和VPA治疗组进行实验，观察并记录各组裸鼠移植瘤的生长情况，计算肿瘤体积和肿瘤生长抑制率。HE染色观测肿瘤细胞形态。采用末端脱氧核糖核酸转移酶介导的dUTP原位缺口末端标记（TUNEL）法检测肿瘤细胞凋亡。RT—PCR和Western blot方法分析粒-单核细胞集落刺激因子（GM—CSF）、组蛋白脱乙酰化酶1（HDAC1）、乙酰化组蛋白H3（Ac—H3）、Survivin mRNA或蚩白的表达。染色质免疫共沉淀（ChIP）法检测VPA对GM—CSF基因启动子区域染色质内组蛋白H3乙酰化程度的影响。结果①VPA体内用药明显抑制裸鼠Kasumi—1细胞移植瘤的生长，VPA治疗组肿瘤体积及重量与对照组相比明显缩小或降低，瘤体抑制率（IR）为57．25％。②HE染色可见肿瘤细胞分化及凋亡程度增加，凋亡指数VPA组[（3．66±0．77）％]与对照组[（0．27±0．11）％]相比明显升高。③与对照组比较，VPA治疗组胞核内HDAC1蛋白表达水平明显降低，Ac—H3蛋白表达水平明显升高；GM—CSFmRNA和蛋白表达水平明显升高；GM—CSF启动子区域染色质内组蛋白H3的乙酰化程度明显升高；Survivin的mRNA表达水平明显降低。结论VPA对Kasumi—1细胞裸鼠移植瘤有明显的生长抑制作用，诱导肿瘤细胞分化及凋亡，其机制可能与通过抑制SmMvin基因表达诱导细胞凋亡，和通过抑制细胞核内HDAC表达，增强组蛋白乙酰化程度，影响GM—CSF基因启动子区域染色质内的组蛋白乙酰化修饰，增加AMLI靶基因GM—CSF的转录诱导细胞分化有关。

Objective To investigate in vivo inhibitory effect of histone deacetylase （HDAC） iuhibitor valpruic acid（VPA） on xenografted Kasumi-1 tumor in nude mice and its mechanism. Methods Xeno- grafted Kasumi-1 tumor mouse model was established by subcutaneous inoculation of Kasumi-1 cells. Xenotransplanted nude mice were assigned into control or VPA treatment groups. Volume of the xenografted tmnors was measured and compared between the two groups. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling （TUNEL） was applied to detection of tumor cell apoptosis. The gene expression of （,M-CSF, HDACI ,Ac-H3 and survivin was studied with semi-quantitative RT-PCR and Western blotting. ChIP method was used to assay the effects of VPA on acetylation of histone H3 within GM-CSF promoter region. Results （1）VAP significantly inhibited xenografted Kasumi-1 tumor growth. The calculated inhibition rate was 57.25%. （2）Morphologic study showed that VPA induced differentiation and apoptosis of Kasumi-1 tumor cells. The apoptosis index of VAP treatment group [ （ 3.661 ± 0. 768） % 1 was significantly higher than that of control group [ （0. 267± 0.110）% ]. （3）Compariug to those in control group, the level of nuclear HDACI protein was significantly decreased, the Ac-H3 protein expression level was increased, the mRNA and protein expression levels of GM-CSF and acetylation of histone H3 were remarkably increased, and the gene expression level of survivin significantly decreased in VPA treatment group. Conclusion VAP significantly inhibites xenografted Kasumi-1 tumor growth and induces tumor cell differentiation and apoptosis. The mecha- nism may be decrease of survivin gene expression, inhibition of nuclear expression of HDAC, promotion of histone protein acytelation level and acetylation of histone H3 within GM-CSF promoter region, and increase of GM-CSF transcription.