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中药复方青黛、雄黄、丹参治疗急性早幼粒性白血病分子机制 Wang L, Zhou G B, Liu P, et al. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia[J]. Proceedings of the National Academy of Sciences（PNAS）, 2008, 105(12): 4826-4831. IF:9.674 复方青黛、雄黄、丹参Realgar-Indigo naturalis formula (RIF）由雄黄、青黛、丹参组成，在这个复方中，雄黄的主要成份是四硫化四砷（A），青黛的有效成份是靛玉红（I），丹参的有效成份是丹参酮 IIA（T）。该研究应用小鼠APL（急性早幼粒性白血病疾病）模型，发现ATI联用比任何一个成份单用都有明显增强的疗效；与单个或者2个成份组合相比，3者联用可以显著增加癌蛋白PML-RARα的降解，增强骨髓细胞再分化调节因子的活性，并促进细胞G0/G1阻滞。而且，T和I可以上调水甘油通道蛋白9的基因表达活性，从而促进砷更多的进入恶性早幼粒细胞发挥作用。 文章摘要： To enhance therapeutic efficacy and reduce adverse effects, practitioners of traditional Chinese medicine (TCM) prescribe a combination of plant species/minerals, called formulae, based on clinical experience. Nearly 100,000 formulae have been recorded, but the working mechanisms of most remain unknown. In trying to address the possible beneficial effects of formulae with current biomedical approaches, we use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model. The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza,with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as major active ingredients, respectively. Here, we report that the ATI combination yields synergy in the treatment of a murine APL model in vivo and in the induction of APL cell differentiation in vitro. ATI causes intensified ubiquitination/degradation of promyelocytic leukemia (PML)-retinoic acid receptor (RAR) oncoprotein, stronger reprogramming of myeloid differentiation regulators, and enhanced G1/G0 arrest in APL cells through hitting multiple targets compared with the effects of mono- or biagents. Furthermore, ATI intensifies the expression of Aquaglyceroporin 9 and facilitates the transportation of A into APL cells, which in turn enhances A-mediated PML-RAR degradation and therapeutic efficacy. Our data also indicate A as the principal component of the formula, whereas T and I serve as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae at the molecular, cellular, and organism levels may be a good strategy in exploring the value of traditional medicine. 注： 雄黄【归经】入心、肝、胃经 【功能主治】燥湿，祛风，杀虫，解毒。治疥癣，秃疮，痈疽，走马牙疳，缠腰蛇丹，破伤风，蛇虫蟹伤，腋臭，臁疮，哮喘，喉痹，惊痫，痔瘘 青黛【归经】归肝经。 【功能主治】清热解毒，凉血，定惊。用于温毒发斑，血热吐衄，胸痛咳血，口疮，痄腮，喉痹，小儿惊痫。 丹参【归经】归心、肝经。 【功能主治】祛瘀止痛，活血通经，清心除烦。用于月经不调，经闭痛经，症瘕积聚，胸腹刺痛，热痹疼痛，疮疡肿痛，心烦不眠；肝脾肿大，心绞痛。 急性早幼粒细胞白血病（Acute promyelocytic Leukemia，APL）是一种特殊类型急性白血病，APL的5种染色体易位均累及17号染色体上的RARα基因。t（15；17），即维A酸受体α基因与15号染色体的早幼粒细胞白血病（PML）基因形成PML-RARα融合基因，易位见于绝大多数APL患者。 下面简略看一下研究结果： Fig. 1. 体内研究（A）ATI显著延长荷PML-RAR阳性白血病细胞小鼠的生存时间。(P< 0.001) （B）并且无显著毒性，对小鼠的体重无影响。（C）ATI促进细胞成熟（注：Gr-1阳性细胞为中性粒细胞，Mac阳性细胞为巨噬细胞） In vivo therapeutic efficacies of ATI on an APL murine model. (A) ATI significantly prolongs the life span of mice bearing PML-RAR-positive leukemic cells compared with those treated with mono- or bitreatment of A, T, and I (P< 0.001). (B) ATI does not cause loss of body weight, suggesting that ATI might probably not cause severe toxicity. (C) Treatment with ATI results in cell maturation revealed by an accumulation of Gr-1 and Mac-1-positive cells in BM and peripheral blood (PB). (Dosage of agents used: A, 10 mg/kg; T, 50 mg/kg; I, 50 mg/kg. A is administrated by i.v. injection, whereas T and I are given orally.) Fig. 2. ATI 诱导白血病细胞NB4, NB4-R2, 以及原始白血病细胞终末分化。 （A）APL细胞形态学检测 （B）NBT活性检测，CD11b/CD14表达检测 （C） 原始白血病细胞CD11b表达 （D）A, T, and I 联合增效作用。 ATI induces terminal differentiation of NB4, NB4-R2, and primary leukemic cells harvested from APL patients. (A) Morphological examination of APL cells treated with ATI. (B) Detection of NBT reduction activity and CD11b/CD14 expression of cells treated with ATI. (C) Expression of CD11b in primary leukemic cells treated with ATI in vitro. Concentration of agents used: A, 0.375 uM; T, 0.75 uM; I, 0.75 uM. (D) Combination of A, T, and I exerts synergic effects on NB4 and NB4-R2 cells, as reflected by the median-effect method of Chou and Talalay (23). 注： CD11b不成熟的自然杀伤细胞 CD14单核细胞 NBT还原实验为中性粒细胞功能实验。这两个检测的目的是判断诱导分化结果。 NBT还原实验：取500ul新鲜配制含佛波酯（TPA,100ug/ml）10ul的0.2%NBT溶液，分别与等体积细胞悬液混合，37℃温育30min后，光镜下观察，胞浆含蓝灰色颗粒的细胞为阳性细胞，计数200个细胞，得出阳性细胞百分率。或取细胞3-5×105，离心后加入二甲基亚砜100μl混匀,酶标仪570nm波长下测定细胞的A值。 Fig. 3. ATI下调PML-RAR癌蛋白，重新表达分化调节因子。 ATI triggers degradation of PML-RAR oncoprotein and reprogramming of differentiation regulators. (A) Enhanced degradation of PML-RAR is seen in NB4 (Left) and NB4-R2 (Right) cells treated with the ATI combination compared with those treated with mono- or bitreatment of A, T, and I. (B) TI intensifies matrix transfer from the nucleoplasm and ubiquitination of PML-RAR caused by A. Cells are pretreated with MG-132 for 1 h and then with the protocols indicated for 6 h. Western blot is performed as described (24). (C) Effects of ATI on differentiation regulators of NB4 cells at the protein level. (D) Effects of the ATI combination on differentiation regulators at the mRNA level, assessed by semiquantitative RT-PCR. (E) ATI treatment up-regulates the expression of RARβ2 at the mRNA level; this might be due to the dissociation of RARβ2 from HDAC1 upon ATI treatment.
Fig. 4. ATI 对细胞周期调节作用 Effects of ATI on cell cycle progression and cell cycle modulators. (A) ATI treatment leads to the G1/G0 arrest of NB4 and NB4-R2 cells. (B) Effects of the ATI combination on important cell cycle regulators in NB4 (Left) and NB4-R2 (Right) cells. (C) Down-regulation of CDK2 by ATI treatment on NB4 (Upper) and NB4-R2 (Lower) cells results in decrease of phosphorylated H1. Cells are treated with A, T, I, and the combined drugs for 24 h. CDK2 is immunoprecipitated (IP) from cell lysates and assayed for kinase activity using Histone H1 as a substrate. The efficiency of IP is confirmed and quantitated by Western blot analysis, and CDK2 at each treatment group is adjusted to the same level. Fig. 5. T and I 调节通道蛋白 AQP9 T and I facilitate arsenic uptake by malignant promyelocytes via up-regulation of AQP9. (A) Combinatory use of T and/or I increases intracellular arsenic concentration ([As]i) in NB4 cells (*, P= 0.002; #, P= 0.004; **, P=0.002). (B and C) T and/or I in combination with A up-regulate AQP9 expression at both the mRNA (B) and protein (C) levels (Coom, stained with Coomassie blue). (D) Immunofluorescence analysis of AQP9 expression in NB4 cells treated with the ATI combination. (E) AQP9-specific siRNA down-regulates AQP9 expression by approximately one-half in NB4 cells (NB4-AQP9-Si) compared with cells treated with nonsilencing siRNA control (NC). (F) Treatment with AQP9-specific siRNA reduces [As]i in NB4-AQP9-Si cells upon ATI compared with NB4-NC cells (*, P= 0.018; #, P = 0.037; **, P = 0.009). (G) Treatment with AQP9-specific siRNA inhibits differentiation of NB4 cells on ATI, revealed by analysis of CD11b expression. |
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