Zhao Xiao-ying, He Zhi-wen, Wu Dong, Xu Rong-zhen
Department of Hematology, Second Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310009, China.
Chin Med J (Engl). 2007 May 5;120(9):802-6.
Currently, resistance and relapse are still major problems in acute promyelocytic leukemia (APL) cases. Thus, new agents that override the resistance are crucial to the development of curative therapies for APL. In this study, we investigated the effects of berbamine on the proliferation of APL cell line NB4 and its possible mechanisms.
NB4 cells were treated with berbamine at different concentrations (0-64 microg/ml) for 72 hours. MTT assay was used to determine proliferation inhibition of NB4 cells. Cell apoptosis was evaluated by both flow cytometry (FCM) and morphological examination. PML/RAR-alpha and survivin mRNAs were measured by nested-RT-PCR and RT-PCR, respectively. Activated-caspase 3 was determined by FCM.
Berbamine greatly inhibited the proliferation of NB4 cells in dose- and time-dependent manners, and its IC50 value was 3.86 microg/ml at 48 hours. Both morphological observations and FCM results showed that berbamine induced apoptosis of NB4 cells with concomitant increase of activated caspase-3 and decrease of survivin mRNA. After treatment with berbamine at 8 microg/ml for 48 hours, the percentage of apoptotic cells increased from 2.83% to 58.44% (P<0.01), and the percentage of cells with activated-caspase 3 elevated from 2.06% to 70.89% (P<0.01), whereas, level of survivin mRNA was reduced to 38.24% of control (P<0.01). However, no significant change was observed in PML/RAR-alpha mRNA.
Berbamine induces caspase-3-dependent apoptosis of leukemia NB4 cells via survivin-mediated pathway, suggesting that berbamine may be a novel potential agent against APL with a mechanism distinct from that of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).
目前，耐药性和复发仍是急性早幼粒细胞白血病（APL）病例中的主要问题。因此，能克服耐药性的新型药物对于APL根治性疗法的发展至关重要。在本研究中，我们调查了小檗胺对APL细胞系NB4增殖的影响及其可能机制。
用不同浓度（0 - 64微克/毫升）的小檗胺处理NB4细胞72小时。采用MTT法测定NB4细胞的增殖抑制情况。通过流式细胞术（FCM）和形态学检查评估细胞凋亡。分别采用巢式逆转录聚合酶链反应（nested - RT - PCR）和逆转录聚合酶链反应（RT - PCR）检测PML/RAR - α和生存素（survivin）的信使核糖核酸（mRNA）。通过FCM检测活化的半胱天冬酶 - 3（activated - caspase 3）。
小檗胺以剂量和时间依赖性方式显著抑制NB4细胞的增殖，48小时时其半数抑制浓度（IC50）值为3.86微克/毫升。形态学观察和FCM结果均显示，小檗胺诱导NB4细胞凋亡，同时活化的半胱天冬酶 - 3增加，生存素mRNA减少。用8微克/毫升小檗胺处理48小时后，凋亡细胞百分比从2.83%增至58.44%（P < 0.01），活化的半胱天冬酶 - 3阳性细胞百分比从2.06%升至70.89%（P < 0.01），而生存素mRNA水平降至对照的38.24%（P < 0.01）。然而，PML/RAR - α mRNA未观察到显著变化。
小檗胺通过生存素介导途径诱导白血病NB4细胞发生半胱天冬酶 - 3依赖性凋亡，提示小檗胺可能是一种新型潜在抗APL药物，其作用机制不同于全反式维甲酸（ATRA）和三氧化二砷（ATO）。
