二烯丙基二硫对人白血病KG1-α细胞增殖的影响

Proliferation inhibition of human leukemia KG1-α cells induced by diallyl disulfide

目的 ：探讨二烯丙基二硫（diallyl disuli de,DADS）对人白血病细胞系KG1-α增殖的抑制作用及其对丙酮酸激酶M2（pyruvate kinase M2,PKM2）的影响。方法 ：首先采用蛋白质印迹法筛选PKM2高表达的白血病细胞株;CCK-8（cell counting kit-8）法检测不同浓度的DADS（50～400μmol/L）作用24、48和72 h后,对高表达PKM2的白血病细胞株KG1-α增殖的影响;FCM法检测对KG1-α细胞周期的影响,Hochest 33258染法色观察对KG1-α细胞凋亡形态学的影响;蛋白质印迹法检测PKM2、PIN1、细胞周期蛋白D1以及p21蛋白的表达;免疫荧光法检测PKM2在细胞质及细胞核中的分布情况。结果 ：PKM2在KG1-α细胞中高表达;CCK-8结果显示,DADS在50～400μmol/L浓度范围内对KG1-α细胞均有明显的抑制作用;FCM法检测结果显示,DADS（50和100μmol/L）作用48 h后,药物组中G0/G1期细胞所占的比例明显增加;Hochest 33258染色结果显示,DADS诱导后KG1-α细胞呈现典型的凋亡形态学改变;DADS作用8 h后,蛋白质印迹法和免疫荧光检测结果均显示,PKM2蛋白在细胞核中的表达水平呈下调趋势;PIN1蛋白的表达水平下调;DADS作用48 h后,KG1-α细胞中细胞周期蛋白D1的表达水平下调,而p21蛋白的表达水平上调（P均〈0.05）。结论 ：DADS对KG1-α细胞增殖抑制的机制可能与下调PKM2的表达,并使其细胞周期阻滞在G0/G1期有关。

Objective： To investigate the inhibition effect of diallyl disulfide （DADS） on proliferation of human leukemia KG1 -α cells and pyruvate kinase M2 （PKM2）. Methods： The expression levels of PKM2 protein in different human leukemia cell lines were detected by Western blotting, in order to find out the leukemia cell line with high expression of PKM2. The growth inhibition of KG1-α cells with high expression level of PKM2 treated with different concentrations of DADS （50-400 μmol/L） for 24, 48 and 72 h was measured by cell counting kit-8 （CCK-8） assay. The effect of DADS on cell cycle was detected by flow cytometry （FCM）. Hochest 33258 staining was used to examine DADS-induced apoptosis in KG1-α cells. The expression levels of PKM2, PIN1, cyclin D1 and p21 proteins were detected by Western blotting. The distribution of PKM2 in cells after treatment with DADS was detected by immunofluoresence. Results： PKM2 was highly expressed in KG1-α cells. DADS （50-400 μmol/L） had the most efficient inhibitory effect on KG1-α cells, and it could significantly arrest the cell cycle at G0/G1 phase after 48 h. Hoechst 33258 staining indicated that the typical morphology of apoptosis could be found in KG1 -α cells induced by DADS. The expression of PKM2 in nucleus was decreased and the expression of PIN 1 was also declined significantly in KG1 -α cells after treatment with DADS for 8 h. The expression of cyclin D1 was decreased and p21 was increased in KG1 -α cells after treatment with DADS for 48 h （both P 〈 0.05）. Condusion：DADS can significantly inhibit the proliferation of KG1-α cells. This mechanism may be related to down-regulation of the expression of PKM2 and the arrest of cell cycle at phase G0/G1.