Díaz-Carballo D, Malak S, Freistühler M, Elmaagacli A, Bardenheuer W, Reusch H P
Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, Germany.
Int J Clin Pharmacol Ther. 2008 Aug;46(8):428-39. doi: 10.5414/cpp46428.
This work is aimed at characterizing nemorosone, isolated from Clusia rosea, as a potential antileukemic agent. In addition, we analyzed its influence on hematopoiesis in a mouse model.
The isolation of nemorosone was carried out employing the RP-HPLC (reversed phase high-performance liquid chromatography) technique. Cytotoxicity was assessed in human leukemia cell lines including parental and chemotherapy-refractory sublines based on the MTT compound. Its effects on the cell cycle were analyzed using FACS (fluorescence-activated cell sorting) and Western blot techniques. Studies on the drug-induced early apoptotic process were carried out by means of fluorescence microscopy. Major signal transducers and the enzymatic inhibition of immunoprecipitated Akt/PKB were detected by Western blot. Hematopoiesis was analyzed in NMRI nu/nu mice after chronic nemorosone treatment, measuring hematological parameters by conventional laboratory techniques.
Nemorosone proved cytotoxic in both parental and chemoresistant leukemia cell lines with IC50 values between 2.10 and 3.10 mg/ml. No cross-resistances could be detected. Cell cycle studies showed apoptosis induction accompanied by an increase in the G0/G1 population in both cell lines studied, whereas a significant decrease in the S-phase was found in Jurkat cells. Nemorosone induced a down-regulation of cyclins A, B1, D1, and E as well as a dephosphorylation of cdc2. Major signal transduction elements such as ERK1/2 and p38 MAPK, as well as important oncoproteins such as c-Myb and BCR/ABL were also found down-regulated. The enzymatic activity of immunoprecipitated Akt/PKB was substantially inhibited in vitro. Moreover, subchronic nemorosone treatment induced reversible monocytosis and thrombocytosis in the mouse model examined.
Here, we demonstrate for the first time that nemorosone exerts cytotoxicity in leukemia cells, partly by targeting the Akt/PKB signal transducer, affecting protein levels and cell cycle progression. Finally, in vivo studies suggest that nemorosone significantly affects hematopoiesis in mice.
本研究旨在鉴定从玫瑰克鲁希亚中分离出的降香萜酮作为一种潜在抗白血病药物的特性。此外，我们还分析了其对小鼠模型造血功能的影响。
采用反相高效液相色谱（RP-HPLC）技术分离降香萜酮。基于MTT化合物评估其对包括亲代和化疗难治性子代在内的人白血病细胞系的细胞毒性。使用荧光激活细胞分选（FACS）和蛋白质印迹技术分析其对细胞周期的影响。通过荧光显微镜研究药物诱导的早期凋亡过程。通过蛋白质印迹检测主要信号转导分子以及免疫沉淀的Akt/PKB的酶活性抑制情况。在慢性给予降香萜酮治疗后的NMRI裸鼠中分析造血功能，通过传统实验室技术测量血液学参数。
降香萜酮在亲代和化疗耐药白血病细胞系中均显示出细胞毒性，IC50值在2.10至3.10 mg/ml之间。未检测到交叉耐药性。细胞周期研究表明，在所研究的两种细胞系中均诱导了凋亡，同时G0/G1期细胞群体增加，而在Jurkat细胞中S期显著减少。降香萜酮诱导细胞周期蛋白A、B1、D1和E下调以及cdc2去磷酸化。还发现主要信号转导元件如ERK１/２和p38丝裂原活化蛋白激酶以及重要的癌蛋白如c-Myb和BCR/ABL下调。免疫沉淀的Akt/PKB的酶活性在体外被显著抑制。此外,在受试小鼠模型中，亚慢性降香萜酮治疗诱导了可逆性单核细胞增多和血小板增多。
在此，我们首次证明降香萜酮对白血病细胞具有细胞毒性，部分是通过靶向Akt/PKB信号转导分子，影响蛋白质水平和细胞周期进程。最后，体内研究表明降香萜酮显著影响小鼠的造血功能。
