As_2O_3联用华蟾素对K562细胞Bcr-Abl磷酸化的影响

Effects of As_2O_3 in Combination with Cinobufacini on Bcr-Abl Protein Tyrosine Phosphorylation in K562 Cells

目的研究As2O3联用华蟾素对K562细胞Bcr-Abl蛋白酪氨酸磷酸化的影响,探讨其抗白血病的分子机制,为As2O3和华蟾素联合应用治疗CML提供理论依据。方法采用细胞增殖实验检测细胞生长;采用Annexin-V/PI双染实验、DNAPI染色及DNA电泳等方法测定细胞凋亡;运用Westernblot检测K562细胞Bcr-Abl蛋白酪氨酸磷酸化水平。结果在As2O3和华蟾素作用下K562细胞生长受抑伴随活力下降,1.0μmol/LAs2O3、0.125μg/mL华蟾素、0.25μg/mL华蟾素、1.0μmol/LAs2O3+0.125μg/mL华蟾素、1.0μmol/LAs2O3+0.25μg/mL华蟾素作用K562细胞24h和48h后,增殖抑制率分别为(24±1.3)%、(21±1.5)%、(38±3.1)%、(57±2.7)%、(66±3.3)%及(49±2.9)%、(48±2.7)%、(61±2.1)%、(77±3.8)%、(82±4.2)%,细胞凋亡率分别为(4.8±0.5)%、(5.6±0.7)%、(9.8±0.6)%、(11.9±1.2)%和(15.2±1.5)%及(11.0±0.9)%、(12.9±1.1)%、(18.4±1.5)%、(21.0±2.0)%、(28.0±1.9)%,凋亡细胞百分率呈时间剂量依赖关系;DNA电泳出现"梯"状条带;Bcr-Abl蛋白酪氨酸磷酸化水平出现时间剂量依赖性下调。结论 As2O3和华蟾素能诱导K562细胞凋亡和抑制其增殖,两药联用具有协同作用,机制与下调K562细胞Bcr-Abl蛋白酪氨酸磷酸化有关。

Objective To investigate the effects of As203 in combination with cinobufacini on Bcr-Ab] protein tyrosine phosphorylation in K562 cells and their molecular mechanism of antileukcmia to provide theoretical basis for clinical therapy of chronic myelogenous leukemia （ CML ） cases. Methods Cell growth was analyzed by the cell proliferation experiment. Apoptosis was analyzed by Annexin-V/PI staining, DNA-PI staining and DNA gel electrophoresis. Bcr-Abl protein tyrosine phosphorylation was studied by means of Western blot. Results After exposure to As203 and cinobufaeini, the growth of K562 ceils was inhibited and the viability of K562 cells was decreased. After treated with 1.0 ~ mol/L As203, 0.125 p, g/mL cinobufacini, 0.25μ g/mL cinobufacini, 1.0μmol/ L As203＋0. 125μg/L, g/mL cinobufacini, 1.0μ mol/L As203＋0.25μ g/mL cinobufacini for 24 and 48 hours, the proliferation inhibition rates were （ 24 ±l.3 ） %, （21±1.5）%, （38±3.1）%, （57±2.7）%, （66±3.3）%and（49±2.9）%, （48±2.7）%, （61±2.1）%, （ 77± 3.8 ） %, （ 82 ±4.2 ） ~~, and the apoptosis rates of K562 cells were （ 4.8 ± 0.5 ） %, （ 5.6 ± 0.7 ） %, （ 9.8 ±0.6 ） %, （ 11.9 ± 1.2 ） %, （ 15.2 ± 1.5 ） % and （ 11.0± 0.9 ）%, （ 12.9 ± 1.1 ） %, （ 18.4 ± 1.5 ） %, （21.0 ± 2.0）%, （28.0±1.9 ） %, The percentage of apoptotic ceils was a time- and dose-dependent manner. Typical DNA ladder was shown by DNA gel electrophoresis. Tyrosine phosphorylation level of Bcr-Ab] was decreased in a time- and dose-dependent manner. Conclusion AszO3 and cinobufaeini might induce apoptosis of K562 cells and inhibit its proliferation by down-regulating tyrosine phosphorylation of Bcr-Ab] protein. The two drugs have synergistic effect.