Zhu Guo-Hua, Zhang Qi, Dai Hai-Ping, Jl Ou, Shen Qun
Department of Western Medicine Diagnostics, Nanjing University of Chinese Traditional Medicine, Nanjing, 210046, Jiangsu Province, China; Department of Hematology, The First Hospital Affiliated to Nanjing University of Chinese Traditional Medicine, Nanjing, 210009, Jiangsu Province, China.
Department of Western Medicine Diagnostics, Nanjing University of Chinese Traditional Medicine, Nanjing, 210046, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1423-8. doi: 10.7534/j.issn.1009-2137.2013.06.011.
This study was purposed to explore the effects induced by puerariae radix flavones (PRF) on human acute myeloid leukemia SHI-1 cells, apoptosis induced by PRF in vitro and its molecular mechanism. SHI-1 cells were treated with PRF in various concentration, then the inhibitory effect of cell proliferation were detected by MTT method, the cell cycle was analyzed by flow cytometry, the mRNA expression levels of Caspase-3, Caspase-8, Caspase-9, Bcl-2 and MLL-AF6 were detected by real-time polymerase chain reaction (R-T PCR), the protein expression levels of MAPK, p-MAPK and NF-κB were assayed by Western blot, and the activity of MMP was analyzed by Gelatin zymography. The results indicated that the PRF could inhibit the proliferation of SHI-1 cells in a time-and dose-dependent manner, and the cell cycle was arrested in S phase. When SHI-1 cells were treated with 25, 50 and 75 µg/ml PRF respectively, mRNA levels of Caspase-3, Caspase-8 and Caspase-9 increased in a dose-dependent manner (P < 0.05), Bcl-2 mRNA decreased in a concentration-dependent manner (P > 0.05), and the mRNA level of fusion gene MLL-AF6 did not changed as compared with the control group. Different concentration of PRF was used to treat SHI-1 cells, the expression levels of intracellular JNK, p-JNK, P38 MAPK and p-P38 MAPK increased in the concentration-dependent manner (P < 0.01); the expression of p-ERK1/2 and NF-κB decreased in the concentration-dependent manner, and the activity of MMP-2 and MMP-9 in the cell supernatant did not change in each groups. It is concluded that a certain concentration of PRF can induce the apoptosis of SHI-1 cells in vitro, its molecular mechanism may be related to the activation of Caspase hydrolase, activation of MAPK, downregulation of NF-κB, Bcl-2 and other signal molecules. However, it seemed that all these effects are not relate with the MLL-AF6 fusion gene.
本研究旨在探讨葛根黄酮（PRF）对人急性髓系白血病SHI-1细胞的影响、PRF体外诱导细胞凋亡及其分子机制。用不同浓度的PRF处理SHI-1细胞，采用MTT法检测细胞增殖抑制作用，流式细胞术分析细胞周期，实时聚合酶链反应（R-T PCR）检测Caspase-3、Caspase-8、Caspase-9、Bcl-2和MLL-AF6的mRNA表达水平，Western blot检测MAPK、p-MAPK和NF-κB的蛋白表达水平，明胶酶谱法分析MMP活性。结果表明，PRF能以时间和剂量依赖性方式抑制SHI-1细胞增殖，使细胞周期阻滞于S期。分别用25、50和75μg/ml PRF处理SHI-1细胞时，Caspase-3、Caspase-8及Caspase-9的mRNA水平呈剂量依赖性升高（P<0.05），Bcl-2 mRNA呈浓度依赖性降低（P>0.05），融合基因MLL-AF6的mRNA水平与对照组相比无变化。用不同浓度PRF处理SHI-1细胞，细胞内JNK、p-JNK、P38 MAPK及p-P38 MAPK的表达水平呈浓度依赖性升高（P<0.01）；p-ERK1/2及NF-κB的表达呈浓度依赖性降低，各实验组细胞培养上清中MMP-2和MMP-9活性无变化。结论：一定浓度的PRF可体外诱导SHI-1细胞凋亡，其分子机制可能与Caspase水解酶激活、MAPK激活、NF-κB及Bcl-2等信号分子下调有关。然而，所有这些作用似乎均与MLL-AF6融合基因无关。
