Department of Clinical Pharmacology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
J Cancer Res Clin Oncol. 2012 Dec;138(12):2095-102. doi: 10.1007/s00432-012-1292-1. Epub 2012 Jul 26.
In our previous publication, we have shown that dihydroartemisinin could significantly inhibit the growth of CML K562 cells by its anti-proliferative and inducing apoptotic effects. Given the pivotal effect of Bcr/Abl tyrosine kinase and its downstream signal factors on CML cell proliferation and survival, we extend our study to investigate the effect of DHA on Bcr/Abl and related signal factors to further illuminate the possible mechanisms of the effect of DHA on CML cells.
The expression of Bcr/Abl was analyzed with PCR and Western blotting methods at both mRNA and protein levels. Measurement of protein expression and tyrosine phosphorylation activity of Bcr/Abl, AKT, ERK1/2, NF-κB and cytochrome c were performed with Western blotting and immunoprecipitation methods. Using the activity kits analyzed the activity of caspase 9 and caspase 3.
The treatment with DHA results in a significant suppression on Bcr/Abl expression and leads to a concentration-dependent reduction on the Bcr/Abl tyrosine activity. Moreover, it also results in a strong influence on the downstream signal factors of Bcr/Abl, which includes inhibition of tyrosine kinase activity of AKT and ERK1/2, suppression of NF-κB protein expression, promotion of the cytochrome c release and the consequential activation of caspase 3/9 in CML K562 cells.
Together with our previous report, our data show that the growth inhibitory effect of DHA on CML cells might be due to the influence on Bcr/Abl expression and its downstream signal factors. DHA might be a potential novel anti-CML drug candidate and worthy of further study.
在我们之前的研究中，已经证明青蒿琥酯可以通过抗增殖和诱导凋亡作用显著抑制 CML K562 细胞的生长。鉴于 Bcr/Abl 酪氨酸激酶及其下游信号因子对 CML 细胞增殖和存活的关键作用，我们进一步研究了 DHA 对 Bcr/Abl 及相关信号因子的影响，以进一步阐明 DHA 对 CML 细胞作用的可能机制。
采用 PCR 和 Western blot 法分别从 mRNA 和蛋白水平检测 Bcr/Abl 的表达。采用 Western blot 和免疫沉淀法检测 Bcr/Abl、AKT、ERK1/2、NF-κB 和细胞色素 c 的蛋白表达和酪氨酸磷酸化活性。用活性试剂盒分析 caspase 9 和 caspase 3 的活性。
DHA 处理显著抑制 Bcr/Abl 的表达，并导致 Bcr/Abl 酪氨酸活性呈浓度依赖性降低。此外，它还对 Bcr/Abl 的下游信号因子产生强烈影响，包括抑制 AKT 和 ERK1/2 的酪氨酸激酶活性、抑制 NF-κB 蛋白表达、促进细胞色素 c 释放，并随后激活 CML K562 细胞中的 caspase 3/9。
结合我们之前的报告，我们的数据表明 DHA 对 CML 细胞的生长抑制作用可能是由于对 Bcr/Abl 表达及其下游信号因子的影响。DHA 可能是一种有潜力的新型抗 CML 药物候选物，值得进一步研究。
