Xiao Liang, Yin Xiao-Cheng, Cao Qiang-Qiang
Department of Pediatrics, First Affilliated Hospital of Nanhua Medical University, Hengyang, Hunan 421001, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2016 Oct;18(10):1050-1054. doi: 10.7499/j.issn.1008-8830.2016.10.026.
To study the role of the PI3K/AKT signaling pathway in the diallyl disulfide (DADS)-induced apoptosis of K562 cells.
K562 cells in the logarithmic growth phase were treated with 10, 20, 40, or 80 mg/L DADS for 48 hours, then fixed and stained with acridine orange/ethidium bromide (AO/EB), and examined for cellular morphological changes under an inverted microscope. Annexin V-FITC/PI staining was used for determining the apoptotic rates, and Western blot for measuring the expression of AKT, p-AKT, and Caspase-3. Two control groups, blank and solvent, were used as references.
K562 cells treated with DADS for 48 hours exhibited the characteristic morphological features of apoptosis including cell shrinkage, irregular cell shape, and membrane blebbing. AO/EB staining results demonstrated that the number of apoptotic cells with cell shrinkage, pyknotic or bead-like nuclei, chromatin condensation, and orange staining increased with the increasing DADS concentration, and 40 mg/L DADS had the most significant effect. The apoptotic rates of cells treated with 10, 20, 40, and 80 mg/L DADS were all significantly higher than those in the control groups (P<0.05). There were no significant differences in AKT protein expression between the K562 cells treated with different concentrations of DADS; the p-AKT protein expression decreased with the increasing DADS concentration, while the Caspases-3 protein expression increased with the increasing DADS concentration (P<0.05).
DADS induces the apoptosis of K562 cells, probably through inhibiting the protein expression in the PI3K/AKT signaling pathway.
研究磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/AKT)信号通路在二烯丙基二硫化物(DADS)诱导K562细胞凋亡中的作用。
对数生长期的K562细胞分别用10、20、40或80 mg/L DADS处理48小时,然后固定并用吖啶橙/溴化乙锭(AO/EB)染色,在倒置显微镜下观察细胞形态变化。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)染色法测定凋亡率,蛋白质免疫印迹法检测AKT、磷酸化AKT(p-AKT)和半胱天冬酶-3(Caspase-3)的表达。设置空白对照组和溶剂对照组作为参考。
用DADS处理48小时的K562细胞呈现出凋亡的特征性形态学特征,包括细胞皱缩、细胞形状不规则和细胞膜起泡。AO/EB染色结果表明,随着DADS浓度增加,出现细胞皱缩、核固缩或念珠状核、染色质凝聚及橙色染色的凋亡细胞数量增加,40 mg/L DADS的作用最为显著。10、20、40和80 mg/L DADS处理组细胞的凋亡率均显著高于对照组(P<0.05)。不同浓度DADS处理的K562细胞中AKT蛋白表达无显著差异;p-AKT蛋白表达随DADS浓度增加而降低,而Caspases-3蛋白表达随DADS浓度增加而升高(P<0.05)。
DADS可能通过抑制PI3K/AKT信号通路中的蛋白表达来诱导K562细胞凋亡。