地塞米松协同vorinostat促进AML1-ETO蛋白泛素化降解诱导Kasumi-1细胞分化和凋亡

Dexamethasone and vorinostat cooperatively promote differentiation and apoptosis in Kasumi-1 leukemia cells through ubiquitination and degradation of AML1-ETO

目的探讨地塞米松联合组蛋白去乙酰化酶（HDAc）抑制剂vorinostat对Kasumi一1细胞增殖、分化和凋亡的影响及可能的作用机制，为其用于治疗急性髓系白血病提供理论依据。方法以二甲基亚砜为对照组，地塞米松（20nmol／L）、vorinostat（1mmol／L）以及二者联合作用于Kasumi．1细胞24、48h，用MTT法和流式细胞术检测Kasumi-1细胞的生长抑制率、分化抗原的表达和凋亡率。处理48h后用Westernblot法检测AMLl-ETO蛋白水平，用免疫共沉淀-Westemblot法检测其泛素化修饰的变化。结果以地塞米松、vorinostat单独处理以及二者联合处理Kasumi-1细胞48h，发现联合处理组Kasumi-1细胞的增殖抑制率、分化抗原CDl3表达率和凋亡率分别为（42．06±8．20）％、（52．83±8．97）％、（52．92±2．53）％，对照组分别为（6．96＋0．39）％、（21．73±2．03）％、（6．96±0．39）％，地塞米松组分别为（17．30＋3．49）％、（22．53±4．51）％、（19．57±2．17）％，vorinostat组分别为（33．82±9．41）％、（43．93±9．04）％、（42．98±3．01）％，联合处理组较其他3组增高，差异均有统计学意义（P值均〈0．05）。联合处理组与对照组和单药处理组比较，Kasumi-1细胞内AML1-ETO蛋白水平下降，AML1-ETO泛素化修饰水平提高。结论地塞米松和vorinostat通过协同促进AML1-ETO融合蛋白泛素化修饰和泛素蛋白酶体途径降解，抑制Kasumi-1细胞增殖，促进Kasumi-1细胞分化和凋亡。

Objective To probe the effects of dexamethasone （DEX） combined with histone deacetylase （HDAC） inhibitor vorinostat on inhibiting proliferation and inducing differentiation and apoptosis in Kasumi-1 leukemia cells, and its possible mechanisms in order to provide a theoretical basis for the treatment of AML1-ETO positive AML. Methods The cell survival, differentiation and apoptosis rates were tested by MTT or flow cytometry analysis after Kasumi-1 cells were treated by DMSO, DEX （20 nmol/L）, vorinostat （1 gmol/L） or DEX （20 nmol/L） in combination with vorinostat （1μmol/L）. WB and IP-WB were performed to detect AML1-ETO and its ubiquitination. Results Treatment with the combination of DEX and vorinostat for 48 h led to statistically significant differences of inhibited proliferation [ （42.06±8.20）% 1, increased differentiation [ （52.83± 8.97）%] and apoptosis [ （52.92 i 2.53）%1 of Kasumi-1 cells when compared with vorinostat [ （33.82±9.41）%, （43.93±9.04）% and （42.98± 3.01 ）%, respectively], DEX [ （ 17.30±3.49）%, （22.53±4.51 ）% and （19.57±2.17）%, respectivelyl or control [ （6.96±0.39）%, （21.73±2.03）% and （6.96~0.39）%, respectively]. Also significant ubiqutination and decreased AML1-ETO protein in Kasumi-1 cells after the combination treatment over single agent or control were observed. Conclusion The results indicated that DEX and vorinostat could synergistically inhibit the Kasumi-1 cells proliferation, induce Kasumi-I cells differentiation and apoptosis through ubiquitination and degradation of AML 1-ETO.