β-七叶皂苷抗白血病作用研究及分子机理探讨
|
摘要
癌症是威胁人类生命恶性疾病之一，全世界因患癌症死亡的人数每年在500万以上，中国每年新发现癌症患者有160多万，死亡130多万。其中白血病是一类临床常见的造血系统恶性肿瘤，俗称“血癌”，占癌症总发病数的5％，在我国各年龄组肿瘤死亡率中，白血病居第6位(男性)和第8位(女性)，在儿童及35岁以下成人中则占第1位。中国以草药治病的历史悠久，药用植物品种多，从这些植物中开发抗癌新药的前景十分看好。三尖杉酯碱、喜树碱(生物碱)、紫杉醇和β-榄香烯(萜类)等就是成功的例子。因为迄今为止，也只有从天然产物中，才能找到如此结构新颖和作用独特的化合物。从植物中探索抗癌新药，已是科学家们热衷的研究方向。尤其白血病治疗方面，常规化疗药阿霉素等给病人所带来的痛苦已家喻户晓，而骨髓移植费用昂贵，一般家庭无法承担。因此，很有必要从天然中草药有效成分中寻找高效、低毒的抗白血病药物。
β-七叶皂苷(Beta-escin，aescin)是从七叶树科中国天师栗(Aesculuswilsonii Rebd)、欧洲七叶树(Aesculus hippocastannum／Horse chestnut)、日本七叶树(Aesculus turbinata Blume)和浙江七叶树(Aesculus Chinensis var.chekiangensis Fang)干燥成熟果实娑罗子(Semen Aesculi)中提取的天然三萜糖苷类化合物，具有抗炎症渗出、增加静脉张力、改善微循环促进肢体功能恢复等作用。β-七叶皂苷目前临床主要用于脑水肿、脑梗死、颅脑外伤及肢体肿胀等疾病的治疗。近年来，有关β-七叶皂苷抗肿瘤活性已有所报道：邹明、郭维等研究发现七叶皂苷具有抑制人鼻咽癌细胞KB、小鼠肝癌细胞H22、小鼠肉瘤细胞S180及大肠癌HCT-8细胞生长的作用，Patlolla JM等报道β-七叶皂苷能够抑制小鼠ACF(结肠癌癌前病变)的形成，以及诱导HT-29人克隆结肠癌细胞凋亡的作用。但有关β-七叶皂苷抗白血病作用的研究目前尚未见报道。本实验旨在从细胞药效、整体动物药效和分子作用机理探讨的角度，多层次、多水平地研究β-七叶皂苷的抗白血病作用，为开发该药成为治疗白血病新药提供理论和实验依据，并为寻找、发现和合成新型抗癌先导化合物提供有用线索。
实验所用β-七叶皂苷冻干粉针，为山东烟台绿叶制药有限公司产品，从七叶树科中国天师栗(Aesculus wilsonii Rebd)干燥成熟果实娑罗子(SemenAesculi)中提取；阿霉素为浙江海正药业股份有限公司产品。人急性髓细胞白血病(human acute myelogenous leukemia)细胞株HL-60、人慢性髓细胞白血病(human chronic mylogenous leukemia)细胞株K562及小鼠急性淋巴细胞白血病(murine lymphoid leukemia)细胞株P388和L1210由中科院上海生化细胞所细胞库提供。DBA／2近交小鼠，体重18-22g，由中科院上海实验动物中心提供。我们采用半固体集落培养法和台盼蓝拒染法观察β-七叶皂苷对HL-60和K562细胞的体外增殖抑制作用，采用MTT法观察β-七叶皂苷对P388细胞的体外增殖抑制作用；并建立P388和L1210小鼠淋巴细胞白血病移植模型观察β-七叶皂苷对体内荷瘤小鼠的生命延长率和小鼠腹水变化；实验进一步以HL-60和K562为靶细胞，采用细胞形态学观察、Annexin V-FITC／PI双标记法、DNA片断(DNA ladder)分析以及流式细胞仪分析DNA含量等方法观察β-七叶皂苷对白血病细胞的诱导凋亡作用，以探讨其分子作用机制。实验结果用SPSS 10．0 for windows统计软件分析处理，两组间差异比较采用Student's t检验，多组资料之间的比较采用单因素方差分析，以P<0．05为差异具有显著性意义。IC_(50)应用GWBASIC(LOGIT)软件非线性回归计算获得。
细胞药效学实验结果表明：30μg/ml的β-七叶皂苷作用于K562细胞24、48和72 h后，CFU-K562集落形成百分率分别为76．5±7．6％，49．5±5．4％和33．2±4．7％，与对照组的100％相比，具有明显的抑制作用(P<0．05)；50μg/ml的β-七叶皂苷作用于K562细胞24、48和72 h，CFU-K562集落形成百分率分别为30．5±5．2％，19．8±4．6％和11．2±3．6％，与对照组的100％相比，具有明显的抑制作用(P<0．01)。高浓度(70μg／ml)的β-七叶皂苷作用于K562细胞24、48和72 h，CFU-K562集落形成百分率接近零及等于零。低浓度(10μg/ml)的β-七叶皂苷对CFU-K562集落的形成没有影响。K562细胞与10，30和5μg/ml的β-七叶皂苷共同孵育24、48和72 h后，细胞活率无明显影响，但高浓度(70μg／ml)的β-七叶皂苷，使K562细胞活率明显下降，从原来的97．5±2．5％降到50％以下，呈现明显的细胞毒活性。
30μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h后，CFU-HL-60集落形成百分率分别为68．5±5．4％，45．2±4．9％and 33．2±5．7％，与对照组的100％相比，具有明显的抑制作用(P<0．05)；50μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h，CFU-HL-60集落形成百分率分别为35．5±4．1％，27．8±3．2％and 16．2±2．9％，与对照组的100％相比，具有明显的抑制作用(P<0．01)。高浓度(70μg/ml)的β-七叶皂苷作用于HL-60细胞24、48和72h，CFU-HL-60集落形成率百分接近零及等于零。低浓度(10μg／ml)的β-七叶皂苷对CFU-HL-60集落的形成没有影响。HL-60细胞与10，30和50μg/ml的β-七叶皂苷共同孵育24、48和72h后，细胞活率无明显影响，但高浓度(70μg／ml)的β-七叶皂苷，使HL-60细胞活率明显下降，从原来的97．8±2．1％降到50％以下，呈现明显的细胞毒活性。β-七叶皂苷对体外培养的P388细胞也具有增殖抑制作用，并呈明显的浓度-效应和时间-效应关系。β-七叶皂苷作用P388细胞72小时IC_(50)为23．15±4．61μg／ml，与阿霉素联合用药具有协同作用。
整体动物药效学实验结果表明：β-七叶皂苷对P388小鼠淋巴细胞白血病移植模型具有延长生命期作用，高(4．5 mg／kg)、中(3．5 mg／kg)、低(2．5mg／kg)剂量组对DBA／2近交小鼠移植性肿瘤的生命延长率分别为23．5％、29．4％和17．6％，其中高、中剂量组与对照组相比具有显著性差异(P<0．01)；阿霉素与β-七叶皂苷低剂量联合用药治疗组，生命延长率为47．1％与阿霉素治疗组(2mg／kg)的29．4％相比具有显著性差异(P<0．01)。β-七叶皂苷对L1210小鼠淋巴细胞白血病移植模型也具有明显的延长生命期作用，高(4．5 mg／kg)、中(3．5 mg／kg)、低(2．5 mg／kg)剂量组对DBA／2近交小鼠移植性肿瘤的生命延长率分别为25．8％、32．3％和12．9％，其中高、中剂量组与对照组相比具有显著性差异(P<0．01)；阿霉素与β-七叶皂苷低剂量联合用药治疗组，生命延长率为45．2％与阿霉素治疗组(2mg／kg)的29．0％相比具有显著性差异(P<0．01)。
分子机理探讨结果显示：30和50μg／ml的β-七叶皂苷作用于K562细胞24 h后，细胞形态呈现明显的凋亡特征；Annexin V-FITC分析结果显示K562细胞经30和50μg／ml的β-七叶皂苷作用24 h后，Annexin V~+和PI~-(早期凋亡)细胞分别占总细胞数的29．61±3．11％和40．71±4．32％，与对照组的1．58±0．97％相比结果具有显著差异(P<0．05)。DNA片断(DNA ladder)分析结果显示30和50μg／ml的β-七叶皂苷作用于K562细胞48和72h后，琼脂糖凝胶电泳显示180bp倍数的DNA ladder；DNA含量分析结果显示K562细胞经30和50μg／mlβ-七叶皂苷作用48h后，流式细胞仪直方图可见明显的细胞凋亡小峰Ap，即Sub-G1期细胞，分别占细胞总数的11．2±1．6％和33.2±3．1％，与对照组的1.3±0．6％相比具有显著性差异(P<0．05)，同时伴随有G1-S期细胞周期阻滞现象。
30μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h后，细胞形态呈现明显的凋亡特征；AnnexinV-FITC分析结果显示HL-60细胞经20、30和50μg／ml的β-七叶皂苷作用24 h后，AnnexinV~+和PI~-(早期凋亡)细胞分别占总细胞数的19．61±3．15％、55．15±5．11％和46．91±6．21％，与对照组的1．15±0．95％相比结果具有显著差异(P<0．05)。DNA片断(DNA ladder)分析结果显示20、30和50μg／ml的β-七叶皂苷作用于HL-60细胞48h后，琼脂糖凝胶电泳显示180bp倍数的DNA ladder；DNA含量分析结果显示HL-60细胞经30和50μg／ml的β-七叶皂苷作用48h后，流式细胞仪直方图可见明显的细胞凋亡小峰Ap，即Sub-G1期细胞，分别占细胞总数的21．8±3．6％和42．4±4．5％，与对照组的1．2±0．2％相比具有显著性差异(P<0．05)，同时伴随有G1-S期细胞周期阻滞现象。
综上所述，β-七叶皂苷(Beta-escin,aescin)，一种从七叶树科中国天师栗(Aesculus wilsonii Rebd)干燥成熟果实娑罗子(Semen Aesculi)中提取的天然三萜皂苷类化合物，对HL-60人急性髓性白血病细胞、K562人慢性髓性白血病细胞和P388小鼠急性淋巴白血病细胞均具有较强的体外增殖抑制作用，作用呈明显的时间和剂量依赖性；高和中剂量的β-七叶皂苷作用于P388和L1210淋巴白血病移植小鼠，可明显延长荷瘤小鼠的生存时间，提高生命延长率。且低剂量的β-七叶皂苷与阿霉素具有协同作用，可以明显提高常规化疗药阿霉素的疗效。另外β-七叶皂苷还可以诱导K562和HL-60细胞凋亡和细胞周期阻滞，提示其抗白血病的作用机制与诱导细胞凋亡的分子途径有关。该研究为开发β-七叶皂苷成为治疗白血病新药提供理论和实验依据，并为寻找、发现和合成新型抗癌先导化合物提供有用线索。
Beta-escin, a natural triterpenoid saponin, isolated from the seed of Aesculus hippocastannum (Europe), Aesculus turbinata Blume (Japane) and Aesculus wilsonii Rebd (China), is known to generate wide variety of biochemical and pharmacological effects. Specially, it has been shown to be effective as an alternative to medical treatment for chronic venous insufficiency. Beta-escin was found to display significant anti-proliferative activity on sarcoma, lung, and hepatic cell lines. and apoptotic activity on human HT-29 colon cancer cell line recently. The aim of the present work was to determine the anti-proliferative and apoptotic activity of beta-escin in various leukemia cells by in vitro and in vivo methods. Another set of experiments were used in order to investigate the mechanisms involved in beta-escin activity. To our knowledge, this is the first report that about beta-escin's effects on leukemia cells. The results present here should be useful in the search for newpotentialantileukemic agents.
Beta-escin was purchased from Shandong Luye Pharmaceutical Co., Ltd., (Shandong, China), which was isolated from the seed of Aesculus wilsonii Rebd (Semen Aesculi). Doxorbicin (DOX) was provided by Zhejiang Haizheng Pharmaceutical Co. Ltd., The human acute myelogenous leukemia cell line HL-60、human chronic mylogenous leukemia cell line K562 and murine lymphoid leukemia cell line P388 and L1210 were purchased from Shanghai Institutes for Biological Sciences (SIBS, Shanghai China). Cleaning inbred strain DBA/2 mouse (18-22g) male/female, were supplied by Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS, Shanghai China).
The anti-proliferative effects of beta-escin in various leukemia cell lines in vitro were detected by soft agar colony formation assay, MTT assay or/and trypan blue exclusion cell viability assay. In vivo study, mice inoculated with P388 or L1210 murine lymphoid leukemia cells were treated with ten daily doses of beta-escin and each other day of DOX, and were observed for survival. Antileukemic effects were assessed by calculating the increase in lifespan (ILS). The molecular mechnism were expored by the methods of morphology. Annexin V-FITC /PI double staining analysis, DNA fragmentation analysis and flow cytometry DNA content assay using K562 and HL-60 as target cells. The statistic precession was carried out in SPSS 10.0 for windows. Comparison with difference between two groups was performed by student's t-test, the differences between various groups were compared by one-way analysis of variance (ANOVA) and the P value less than 0.05 was considered statistically significant. The IC50 values were obtained by nonlinear regression using the GWBASIC (LOGIT) software.
Results showed that after exposure to 30μg/ml of beta-escin for 24, 48 and 72h. the percentage of CFU-K562 colony formation were 76.5±7.6%, 49.5±5.4% and 33.2±4.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-K562 colony formation were 30.5±5.2%, 19.8±4.6% and 11.2±3.6% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h. a significant suppression (P<0.001) of CFU-K562 were showed by percentage of 93.5%. 100% and 100% respectively. In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24, 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-aescin showed obvious cytotoxic effect in K562 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h.After exposure to 30μg/ml of beta-escin for 24, 48 and 72h, the percentage of CFU-HL-60 colony formation were 68.5±5.4%, 45.2±4.9% and 33.2±5.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-HL-60 colony formation were 35.5±4.1%, 27.8±3.2% and 16.2±2.9% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h, a significant suppression (P<0.001) of CFU-HL-60 were showed by percentage of 92%, 100% and 100% respectively In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24. 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-escin showed obvious cytotoxic effect in HL-60 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h. Beta-escin was also found to be able to inhibit the proliferation of P388 murine leukemia cells by MTT assay. The effects were in dose- and time- dependent manner. The IC_(50) value of beta-escin in P388 cells after 72h was 23.15±4.61μg/ml.
In vivo study showed that after treating the mice inoculated with P388 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 23.5% (p<0.01), 29.4% (p<0.01) and 17.6% respectively. Moreover, we observed 47.1% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg). which is significantly increased compared with 29.4% of ILS when mice were treated with single DOX (P<0.01). After treating the mice inoculated with L1210 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 25.8% (p<0.01), 32.3% (p<0.01) and 12.9% respectively. Moreover, we observed 45.2% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg), which is significantly increased compared with 29.0% of ILS when mice were treated with single DOX (P<0.01).
The molecular mechnism results showed that morphological evidence of apoptosis, including reduction in cell volume, chromatin condensation and vacuolization were observed in K562 cells treated with 30 and 50μg/ml of beta-escin for 24h. Significant increase of population of annexin V~+ and PI~- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (30-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (30-50μg/ml) for 48 and 72h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in K562 cells.
Morphological evidence of apoptosis, including vacuolization, nuclear fragmentation and formation of apoptotic body, were observed in HL-60 cells treated with 30μg/ml of beta-escin for 24, 48 and 72h respectively. Significant increase of population of annexin V~+ and PI~- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (20-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (20-50μg/ml) for 48h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in HL-60 cells.
In summary, beta-escin, a natural triterpenoid saponin from the seed of Aesculus wilsonii Rebd (Semen Aesculi), can effectively inhibit cell proliferation in human acute mylogenous leukemia HL-60 cells, human chronic mylogenous leukemia K562 cells and murine lymphoid leukemia P388 cells in vitro, that were in a dose- and time- dependent manner. Beta-escin can also significant increase the lifespan in mice inoculated with P388 or L1210 murine lymphoid leukemia cells, and low dose of showes coordination effects in combination with DOX by increasing DOX's antileukemic efficacy. Moreover, beta-escin can induce apoptosis and led to an accumulation of sub G1 population in K562 and HL-60 leukemia cells, which indicate that the molecular mechanism of beta-escin's antiproliferate activity is related to its apoptitic ability. The data presented here indicate that beta-escin is a potent natural inhibitor of proliferation and inducer of apoptosis in various leukemia cells and beta-escin has the potential to be a promising candidate for cancer treatment.
β-七叶皂苷(Beta-escin，aescin)是从七叶树科中国天师栗(Aesculuswilsonii Rebd)、欧洲七叶树(Aesculus hippocastannum／Horse chestnut)、日本七叶树(Aesculus turbinata Blume)和浙江七叶树(Aesculus Chinensis var.chekiangensis Fang)干燥成熟果实娑罗子(Semen Aesculi)中提取的天然三萜糖苷类化合物，具有抗炎症渗出、增加静脉张力、改善微循环促进肢体功能恢复等作用。β-七叶皂苷目前临床主要用于脑水肿、脑梗死、颅脑外伤及肢体肿胀等疾病的治疗。近年来，有关β-七叶皂苷抗肿瘤活性已有所报道：邹明、郭维等研究发现七叶皂苷具有抑制人鼻咽癌细胞KB、小鼠肝癌细胞H22、小鼠肉瘤细胞S180及大肠癌HCT-8细胞生长的作用，Patlolla JM等报道β-七叶皂苷能够抑制小鼠ACF(结肠癌癌前病变)的形成，以及诱导HT-29人克隆结肠癌细胞凋亡的作用。但有关β-七叶皂苷抗白血病作用的研究目前尚未见报道。本实验旨在从细胞药效、整体动物药效和分子作用机理探讨的角度，多层次、多水平地研究β-七叶皂苷的抗白血病作用，为开发该药成为治疗白血病新药提供理论和实验依据，并为寻找、发现和合成新型抗癌先导化合物提供有用线索。
实验所用β-七叶皂苷冻干粉针，为山东烟台绿叶制药有限公司产品，从七叶树科中国天师栗(Aesculus wilsonii Rebd)干燥成熟果实娑罗子(SemenAesculi)中提取；阿霉素为浙江海正药业股份有限公司产品。人急性髓细胞白血病(human acute myelogenous leukemia)细胞株HL-60、人慢性髓细胞白血病(human chronic mylogenous leukemia)细胞株K562及小鼠急性淋巴细胞白血病(murine lymphoid leukemia)细胞株P388和L1210由中科院上海生化细胞所细胞库提供。DBA／2近交小鼠，体重18-22g，由中科院上海实验动物中心提供。我们采用半固体集落培养法和台盼蓝拒染法观察β-七叶皂苷对HL-60和K562细胞的体外增殖抑制作用，采用MTT法观察β-七叶皂苷对P388细胞的体外增殖抑制作用；并建立P388和L1210小鼠淋巴细胞白血病移植模型观察β-七叶皂苷对体内荷瘤小鼠的生命延长率和小鼠腹水变化；实验进一步以HL-60和K562为靶细胞，采用细胞形态学观察、Annexin V-FITC／PI双标记法、DNA片断(DNA ladder)分析以及流式细胞仪分析DNA含量等方法观察β-七叶皂苷对白血病细胞的诱导凋亡作用，以探讨其分子作用机制。实验结果用SPSS 10．0 for windows统计软件分析处理，两组间差异比较采用Student's t检验，多组资料之间的比较采用单因素方差分析，以P<0．05为差异具有显著性意义。IC_(50)应用GWBASIC(LOGIT)软件非线性回归计算获得。
细胞药效学实验结果表明：30μg/ml的β-七叶皂苷作用于K562细胞24、48和72 h后，CFU-K562集落形成百分率分别为76．5±7．6％，49．5±5．4％和33．2±4．7％，与对照组的100％相比，具有明显的抑制作用(P<0．05)；50μg/ml的β-七叶皂苷作用于K562细胞24、48和72 h，CFU-K562集落形成百分率分别为30．5±5．2％，19．8±4．6％和11．2±3．6％，与对照组的100％相比，具有明显的抑制作用(P<0．01)。高浓度(70μg／ml)的β-七叶皂苷作用于K562细胞24、48和72 h，CFU-K562集落形成百分率接近零及等于零。低浓度(10μg/ml)的β-七叶皂苷对CFU-K562集落的形成没有影响。K562细胞与10，30和5μg/ml的β-七叶皂苷共同孵育24、48和72 h后，细胞活率无明显影响，但高浓度(70μg／ml)的β-七叶皂苷，使K562细胞活率明显下降，从原来的97．5±2．5％降到50％以下，呈现明显的细胞毒活性。
30μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h后，CFU-HL-60集落形成百分率分别为68．5±5．4％，45．2±4．9％and 33．2±5．7％，与对照组的100％相比，具有明显的抑制作用(P<0．05)；50μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h，CFU-HL-60集落形成百分率分别为35．5±4．1％，27．8±3．2％and 16．2±2．9％，与对照组的100％相比，具有明显的抑制作用(P<0．01)。高浓度(70μg/ml)的β-七叶皂苷作用于HL-60细胞24、48和72h，CFU-HL-60集落形成率百分接近零及等于零。低浓度(10μg／ml)的β-七叶皂苷对CFU-HL-60集落的形成没有影响。HL-60细胞与10，30和50μg/ml的β-七叶皂苷共同孵育24、48和72h后，细胞活率无明显影响，但高浓度(70μg／ml)的β-七叶皂苷，使HL-60细胞活率明显下降，从原来的97．8±2．1％降到50％以下，呈现明显的细胞毒活性。β-七叶皂苷对体外培养的P388细胞也具有增殖抑制作用，并呈明显的浓度-效应和时间-效应关系。β-七叶皂苷作用P388细胞72小时IC_(50)为23．15±4．61μg／ml，与阿霉素联合用药具有协同作用。
整体动物药效学实验结果表明：β-七叶皂苷对P388小鼠淋巴细胞白血病移植模型具有延长生命期作用，高(4．5 mg／kg)、中(3．5 mg／kg)、低(2．5mg／kg)剂量组对DBA／2近交小鼠移植性肿瘤的生命延长率分别为23．5％、29．4％和17．6％，其中高、中剂量组与对照组相比具有显著性差异(P<0．01)；阿霉素与β-七叶皂苷低剂量联合用药治疗组，生命延长率为47．1％与阿霉素治疗组(2mg／kg)的29．4％相比具有显著性差异(P<0．01)。β-七叶皂苷对L1210小鼠淋巴细胞白血病移植模型也具有明显的延长生命期作用，高(4．5 mg／kg)、中(3．5 mg／kg)、低(2．5 mg／kg)剂量组对DBA／2近交小鼠移植性肿瘤的生命延长率分别为25．8％、32．3％和12．9％，其中高、中剂量组与对照组相比具有显著性差异(P<0．01)；阿霉素与β-七叶皂苷低剂量联合用药治疗组，生命延长率为45．2％与阿霉素治疗组(2mg／kg)的29．0％相比具有显著性差异(P<0．01)。
分子机理探讨结果显示：30和50μg／ml的β-七叶皂苷作用于K562细胞24 h后，细胞形态呈现明显的凋亡特征；Annexin V-FITC分析结果显示K562细胞经30和50μg／ml的β-七叶皂苷作用24 h后，Annexin V~+和PI~-(早期凋亡)细胞分别占总细胞数的29．61±3．11％和40．71±4．32％，与对照组的1．58±0．97％相比结果具有显著差异(P<0．05)。DNA片断(DNA ladder)分析结果显示30和50μg／ml的β-七叶皂苷作用于K562细胞48和72h后，琼脂糖凝胶电泳显示180bp倍数的DNA ladder；DNA含量分析结果显示K562细胞经30和50μg／mlβ-七叶皂苷作用48h后，流式细胞仪直方图可见明显的细胞凋亡小峰Ap，即Sub-G1期细胞，分别占细胞总数的11．2±1．6％和33.2±3．1％，与对照组的1.3±0．6％相比具有显著性差异(P<0．05)，同时伴随有G1-S期细胞周期阻滞现象。
30μg／ml的β-七叶皂苷作用于HL-60细胞24、48和72 h后，细胞形态呈现明显的凋亡特征；AnnexinV-FITC分析结果显示HL-60细胞经20、30和50μg／ml的β-七叶皂苷作用24 h后，AnnexinV~+和PI~-(早期凋亡)细胞分别占总细胞数的19．61±3．15％、55．15±5．11％和46．91±6．21％，与对照组的1．15±0．95％相比结果具有显著差异(P<0．05)。DNA片断(DNA ladder)分析结果显示20、30和50μg／ml的β-七叶皂苷作用于HL-60细胞48h后，琼脂糖凝胶电泳显示180bp倍数的DNA ladder；DNA含量分析结果显示HL-60细胞经30和50μg／ml的β-七叶皂苷作用48h后，流式细胞仪直方图可见明显的细胞凋亡小峰Ap，即Sub-G1期细胞，分别占细胞总数的21．8±3．6％和42．4±4．5％，与对照组的1．2±0．2％相比具有显著性差异(P<0．05)，同时伴随有G1-S期细胞周期阻滞现象。
综上所述，β-七叶皂苷(Beta-escin,aescin)，一种从七叶树科中国天师栗(Aesculus wilsonii Rebd)干燥成熟果实娑罗子(Semen Aesculi)中提取的天然三萜皂苷类化合物，对HL-60人急性髓性白血病细胞、K562人慢性髓性白血病细胞和P388小鼠急性淋巴白血病细胞均具有较强的体外增殖抑制作用，作用呈明显的时间和剂量依赖性；高和中剂量的β-七叶皂苷作用于P388和L1210淋巴白血病移植小鼠，可明显延长荷瘤小鼠的生存时间，提高生命延长率。且低剂量的β-七叶皂苷与阿霉素具有协同作用，可以明显提高常规化疗药阿霉素的疗效。另外β-七叶皂苷还可以诱导K562和HL-60细胞凋亡和细胞周期阻滞，提示其抗白血病的作用机制与诱导细胞凋亡的分子途径有关。该研究为开发β-七叶皂苷成为治疗白血病新药提供理论和实验依据，并为寻找、发现和合成新型抗癌先导化合物提供有用线索。
Beta-escin, a natural triterpenoid saponin, isolated from the seed of Aesculus hippocastannum (Europe), Aesculus turbinata Blume (Japane) and Aesculus wilsonii Rebd (China), is known to generate wide variety of biochemical and pharmacological effects. Specially, it has been shown to be effective as an alternative to medical treatment for chronic venous insufficiency. Beta-escin was found to display significant anti-proliferative activity on sarcoma, lung, and hepatic cell lines. and apoptotic activity on human HT-29 colon cancer cell line recently. The aim of the present work was to determine the anti-proliferative and apoptotic activity of beta-escin in various leukemia cells by in vitro and in vivo methods. Another set of experiments were used in order to investigate the mechanisms involved in beta-escin activity. To our knowledge, this is the first report that about beta-escin's effects on leukemia cells. The results present here should be useful in the search for newpotentialantileukemic agents.
Beta-escin was purchased from Shandong Luye Pharmaceutical Co., Ltd., (Shandong, China), which was isolated from the seed of Aesculus wilsonii Rebd (Semen Aesculi). Doxorbicin (DOX) was provided by Zhejiang Haizheng Pharmaceutical Co. Ltd., The human acute myelogenous leukemia cell line HL-60、human chronic mylogenous leukemia cell line K562 and murine lymphoid leukemia cell line P388 and L1210 were purchased from Shanghai Institutes for Biological Sciences (SIBS, Shanghai China). Cleaning inbred strain DBA/2 mouse (18-22g) male/female, were supplied by Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS, Shanghai China).
The anti-proliferative effects of beta-escin in various leukemia cell lines in vitro were detected by soft agar colony formation assay, MTT assay or/and trypan blue exclusion cell viability assay. In vivo study, mice inoculated with P388 or L1210 murine lymphoid leukemia cells were treated with ten daily doses of beta-escin and each other day of DOX, and were observed for survival. Antileukemic effects were assessed by calculating the increase in lifespan (ILS). The molecular mechnism were expored by the methods of morphology. Annexin V-FITC /PI double staining analysis, DNA fragmentation analysis and flow cytometry DNA content assay using K562 and HL-60 as target cells. The statistic precession was carried out in SPSS 10.0 for windows. Comparison with difference between two groups was performed by student's t-test, the differences between various groups were compared by one-way analysis of variance (ANOVA) and the P value less than 0.05 was considered statistically significant. The IC50 values were obtained by nonlinear regression using the GWBASIC (LOGIT) software.
Results showed that after exposure to 30μg/ml of beta-escin for 24, 48 and 72h. the percentage of CFU-K562 colony formation were 76.5±7.6%, 49.5±5.4% and 33.2±4.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-K562 colony formation were 30.5±5.2%, 19.8±4.6% and 11.2±3.6% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h. a significant suppression (P<0.001) of CFU-K562 were showed by percentage of 93.5%. 100% and 100% respectively. In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24, 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-aescin showed obvious cytotoxic effect in K562 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h.After exposure to 30μg/ml of beta-escin for 24, 48 and 72h, the percentage of CFU-HL-60 colony formation were 68.5±5.4%, 45.2±4.9% and 33.2±5.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-HL-60 colony formation were 35.5±4.1%, 27.8±3.2% and 16.2±2.9% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h, a significant suppression (P<0.001) of CFU-HL-60 were showed by percentage of 92%, 100% and 100% respectively In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24. 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-escin showed obvious cytotoxic effect in HL-60 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h. Beta-escin was also found to be able to inhibit the proliferation of P388 murine leukemia cells by MTT assay. The effects were in dose- and time- dependent manner. The IC_(50) value of beta-escin in P388 cells after 72h was 23.15±4.61μg/ml.
In vivo study showed that after treating the mice inoculated with P388 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 23.5% (p<0.01), 29.4% (p<0.01) and 17.6% respectively. Moreover, we observed 47.1% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg). which is significantly increased compared with 29.4% of ILS when mice were treated with single DOX (P<0.01). After treating the mice inoculated with L1210 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 25.8% (p<0.01), 32.3% (p<0.01) and 12.9% respectively. Moreover, we observed 45.2% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg), which is significantly increased compared with 29.0% of ILS when mice were treated with single DOX (P<0.01).
The molecular mechnism results showed that morphological evidence of apoptosis, including reduction in cell volume, chromatin condensation and vacuolization were observed in K562 cells treated with 30 and 50μg/ml of beta-escin for 24h. Significant increase of population of annexin V~+ and PI~- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (30-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (30-50μg/ml) for 48 and 72h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in K562 cells.
Morphological evidence of apoptosis, including vacuolization, nuclear fragmentation and formation of apoptotic body, were observed in HL-60 cells treated with 30μg/ml of beta-escin for 24, 48 and 72h respectively. Significant increase of population of annexin V~+ and PI~- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (20-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (20-50μg/ml) for 48h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in HL-60 cells.
In summary, beta-escin, a natural triterpenoid saponin from the seed of Aesculus wilsonii Rebd (Semen Aesculi), can effectively inhibit cell proliferation in human acute mylogenous leukemia HL-60 cells, human chronic mylogenous leukemia K562 cells and murine lymphoid leukemia P388 cells in vitro, that were in a dose- and time- dependent manner. Beta-escin can also significant increase the lifespan in mice inoculated with P388 or L1210 murine lymphoid leukemia cells, and low dose of showes coordination effects in combination with DOX by increasing DOX's antileukemic efficacy. Moreover, beta-escin can induce apoptosis and led to an accumulation of sub G1 population in K562 and HL-60 leukemia cells, which indicate that the molecular mechanism of beta-escin's antiproliferate activity is related to its apoptitic ability. The data presented here indicate that beta-escin is a potent natural inhibitor of proliferation and inducer of apoptosis in various leukemia cells and beta-escin has the potential to be a promising candidate for cancer treatment.
引文
[1] Bader P, Niemeyer C, Willasch A, Kreyenberg H, Strahm B, Kremens B, Gruhn B, Dilloo D, Vormoor J, Lang P, Niethammer D, Klingebiel T, Beck JF. Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy. Br J Haematol, 2005, 128(5):649-658.
[2]杨建民，王斌,楼敬伟，张妲，闵碧荷，王健民．ST淋巴细胞性白血病1例报 告并文献复习．白血病淋巴瘤，2006，15(1)：52-53．
[3] Kim R, Tanabe K, Uchida Y, Emi M. Inoue H, Toge T. Current status of the molecular mechanisms of anticancer drug -induced apoptosis. Cancer Chemother Pharmacol, 2002, 50(5):343-352.
[4] Reed JC. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med, 2001, 7(7):314-319.
[5] Su IJ, Cheng AL, Tsai TF, Lay JD. Retoic acid-induced apoptosis and regression of a refractory Epsein-barr virus-containing T cell lymophoma expressing multidrug-resistance phenotypes. Br J Haematol, 1993,85(4):826-828.
[6] Manabe A, Coustan-Smith E, Kumagai M, Behm FG, Raimondi SC, Pui CH.Campana D. Interleukin-4 inducesprogrammed cell death (apoptosis) in casesof high-risk acutelymphoblastic leukemia. Blood, 1994, 83(7):1731-1737.
[7] Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon witha wide-ranging implications in tissue kinetica. British Journal of Cancer, 1972,26(4):239-257.
[8] Kerr JF, Winterfold CM, Harmon BV. Apoptosis: Its significance in cancer andcancer therapy. Cancer. 1994, 73(8):2013-2026.
[9] Hickman JA, Potten CS. Merritt AJ, Fisher TC. Apoptosis and cancerchemotherapy. Philos Trans R Soc Lond B Biol Sci, 1994, 345(1313):319-325.
[10] Hockenbery D. Defining apoptosis. Am J Pathol, 1995,146(1):16-19.
[11] Los M, Burek CJ, Stroh C, Benedyk K, Hug H, Mackiewicz A. Anticancerdrugs of tomorrow: apoptotic pathways as targets for drug design. Drug DiscovToday, 2003, 8(2):67-77.
[12] Nicholson DW, Thornberry NA. Apoptosis, Life and death decisions. Science,2003, 299(5604):214-215.
[13] Marx J. Nobel prize in physiology or medicine. Tiny worm takes a star turn.Science, 2002, 298(5593):526-527.
[14]李蒙，于建勋．单味中药诱导肿瘤细胞凋亡的实验研究现状及展望．中 国中西医结合杂志，2001，21(1)：74-76．
[15]牛泱平，Helen T，Chong BH．人参皂苷诱导HL-60细胞凋亡的研究．中 国中西医结合杂志，2002，22(6)：449-453．
[16]牛泱平，钱煦岱．人参三醇对HL-60白血病细胞的抑制增殖及诱导凋亡 作用．中华实用中西医杂志，2004，17(21)：188-189．
[17]骆晓梅．彭涛．王林．七叶皂苷的药学和治疗作用．国外医学药学分册， 2002，29(3)：168-171．
[18] Hu XM, Zeng FD. Inhibitory effect of beta-aescin on inflammatory processfollowing focal cerebral ischemia-reperfusioninrats. Zhongguo Yaolixue YuDulixue Zazhi, 2005, 19 (1):1-6.
[19] Wei F, Ma LY, Jin WT, Ma SC, Han GZ, Khan IA, Lin RC. Antiinflammatorytriterpenoid saponins from the seeds of Aesculus chinensis. Chem Pharm Bull(Tokyo), 2004, 52 (10): 1246-1248.
[20] Sirtori CR. Aescin: pharmacology, pharmacokinetics and therapeutic profile.Pharmacol Res, 2001, 44(3): 183-193.
[21]侯广平．池广明．七叶皂苷的药理作用及其主要临床应用．中国药师， 2004，7(3)：206-208．
[22]韦继政，李素珍，余利平．七叶皂苷治疗急性脑出血53例临床研究．中国??药物与临床，2005，5(8)：622-623．
[23] Guo W. Xu b, Yang XW, Liu Q, Cui JR. Preliminary studies of effect sodium aescinate aginst cancer in vitro and in vivo. Zhongguo Yaoliexue Tong Bao, 2003, 19(3):351-352.
[24]邹明．韩英，纪欣．七叶皂苷抑制大肠癌HCT-8细胞生长及提高癌细胞 对5-FU敏感性的研究．中华消化杂志，2001，21(4)：248-249．
[25] Patlolla JM, Raju J, Swamy MV, Rao CV. Beta-Escin inhibits colonic aberrant crypt foci formationin rats and regulates the cell cycle growth by inducing p21 (waf1/cip1) in colon cancer cells. Mol Cancer Ther, 2006, 5(6):1459-1466.
[26] Mosmann T. Rapid colorimetric assay for cellular growth and surival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): 55-63.
[27] Niu YP, Jin JM, Gao RL, Xie GL. Chen XH. Effects of Ginsenosides Rg1 and Rb1 on Proliferation of Human Marrow Granulocyte-Macrophage Progenitor Cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2001, 9(2): 178-80.
[28] Niu YP. Qian XD, Wang WX. Effect of panaxadiol saponin and panaxtrol saponin on proliferation of human bone marrow hemopoietic progenitor cells. Zhongguo ZhongXi Yi Jie He Za Zhi, 2004, 24(2): 127-129.
[29] Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003.4(12):721-729.
[30] Pec MK, Aguirre A, Moser-Thier K, Fernandez JJ, Souto ML, Dorta J, Diaz-Gonzalez F. Villar J. Induction of apoptosis in estrogen dependent and independent breast cancer cells by the marine terpenoid dehydrothyrsiferol. Biochem Pharmacol,2003, 65(9): 1451-1461.
[31] Vermes I, Haanen C, Steffens-Nakken H. Reutelingsperger C. A novel assay for apoptosis. Flow cytometic detection of phospha-tidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Method, 1995. 184(1):39-51.
[32] Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Research, 1994, 22(24):5506-5507.
[33] Arends MJ, Morris RG, Wyllie AH. Apoptosis: The role of endonuclease. Am J Pathol, 1990, 136(3):593-608.
[34] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods, 1991, 139(2):271-279.
[35] Lacaille-Dubois MA. Wagner H. In Studies in Natural Products Chemistry: Atta-Ur--Rahman, Ed.; Elsevier: Amsterdam, 2000, Vol.21: pp 633-687.
[36] Kikuchi Y, Sasa H, Kita T, Hirata J, Tode T, Nagata I. Inhibition of human ovarian cancer cell proliferation in vitro by ginsenoside Rh2 and adjuvant effects to cisplatin in vivo. Anticancer Drugs, 1991, 2(1):63-67.
[37] Tode T, Kikuchi Y, Kita T, Hirata J, Imaizumi E, Nagata I. Inhibitory effects by oral DOXinistration of ginsenoside Rh2 on the growth of human ovarian cancer cells in nude mice. J Cancer Res Clin Oncol, 1993,120(1-2):24-26.
[38] Lee YN, Lee HY, Chung HY, Kim SI, Lee SK, Park BC, Kim KW. In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells. Eur J Cancer, 1996, 32A(8):1420-1428.
[39] Park JA, Lee KY, Oh YJ, Kim KW, Lee SK. Activation of caspase-3 protease via a Bcl-2-insensitive pathway during the process of ginsenoside Rh2-induced apoptosis. Cancer Lett, 1997, 121(1):73-81.
[40] Yui S, Ubukata K, Hodono K, Kitahara M, Mimaki Y, Kuroda M, Sashida Y. Yamazaki M. Macrophage-oriented cytotoxic activity of novel triterpene saponins extracted from roots of Securidaca inappendiculata. Int Immunopharmacol 2001, 1(11): 1989-2000.
[41] Gaidi G, Miyamoto T, Laurens V, Lacaille-Dubois MA. New acylated triterpene saponins from Silene fortunei that modulate lymphocyte proliferation. J Nat Prod, 2002, 65(11 ):1568-1572.
[42] Haddad M, Laurens V, Lacaille-Dubois MA. Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia. Bioorg Med Chem. 2004, 12(17): 4725-4734.
[43] Mujoo K, Haridas V, Hoffmann JJ, Wachter GA, Hutter LK, Lu Y, Blake ME, Jayatilake GS, Bailey D, Mills GB, Gutterman JU. Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis. Cancer Res, 2001, 61(14):5486-5490.
[44] Haridas V, Higuchi M, Jayatilake GS, Bailey D, Mujoo K, Blake ME, Arntzen CJ, Gutterman JU. Avicins: triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation. Proc Natl Acad Sci USA, 2001, 98(10):5821-5826.
[45] Yang XW, Zhao J, Cui JR, Guo W. Studies on the biotransformation of escin Ia by human intestinal bacteria and the anti-tumor activities of desacylescin I. Beijing Da Xue Xue Bao, 2004, 36(1): 31-35.
[46] Muraki K, Imaizumi Y, Watanabe M. Ca-dependent K channels in smooth muscle cells permeabilized by Beta-escin recorded using the cell-attached patch-clamp technique. Pflugers Arch, 1992, 420(5-6):461-469.
[47] Pearson PJ, Vanhoutte PM. Vasodilator and vasoconstrictor substance produced by the endothelium. Rev Physiol Biochem Pharmacol, 1993, 122:1-67.
[48] Berti F, Omini C, Longiave D. The mode of action of aescin and the release of prostaglandins. Prostaglandins, 1977, 14(2):241-249.
[49] Matsuda H, Li Y, Yoshikawa M. Possible involvement of 5-HT and 5-HT2 receptors in acceleration of gastrointestinal transit by escin Ib in mice. Life Sci, 2000, 66(23):2233-2238. [50] Facino RM, Carini M, Stefani R, Aldini G, Saibene L. Anti-elastase and anti-hyaluronidase activities of saponins and sapogenins from Hedera helix, Aesculus hippocastanum, and Ruscus aculeatus: factors contributing to their efficacy in the treatment of venous insufficiency. Arch Pharm (Weinheim), 1995, 328(10):720-724.
[51] Tutton PJ, Barkla DH. Influence of prostaglandin analogues on epithelial cell proliferation and xenograft growth. Br J Cancer, 1980, 41(1): 47-51.
[52] Stone OJ. Cancer resistance, carcinogenesis and ground substance viscosity. Med Hypotheses, 1986, 20(1): 117-124.
[1] Warner JK, Wang JC, Hope KJ, Jin L, Dick JE. Concepts of human leukemic development. Oncogene, 2004, 23(43):7164-7177.
[2] Wang ZH, Lin q. Effect of berberine hydrochloride on differentiation of HL-60 cells and study of it's mechanism. Journal of China Pharmaceutical University, 2006, 37(2):165-168.
[3] Seki T, Tsuji K, Hayato Y. Moritomo T, Ariga T. Garlic and onion oils inhibit proliferation and induce differentiation of HL-60 cells. Cancer Letters, 2000, 160(1): 29-35.
[4] Luo YY, Zhang TL. Differentiation of HL-60 cells induced by realgar nano-particles. China Journal of Chinese Materia Medica, 2006, 31(16): 1343-1346.
[5] Liu ZG, Yang YM, et al. Differentiation and Apoptosis of NB4 Cells Synergistically Induced by Tan shinone II A and All-trans Retinoic Acid. Journal of Sichuan Universicy, 2004, 35 (6): 788-791.
[6] Wang Z J, Wu Y L, Lin Q, et al. Effect of panaxynol on differentiation of HL-60 cells line in vitro induced. Chnese Traditional and Herbal Drugs, 2003, 34 (8): 736-738.
[7] Liu YQ, Gao J, Gu B, et al. Observation of the differentiation induction effect of Jinglong capsule on tumor cells. Journal of Chinese cancer clinic, 2004, 31(7):380-382.
[8] Zhu ZT, Jin B, Liu YP, et al. Enhancement of all-trans retinoic acid-induced differentiation by bufalin in primary culture of acute promyelocytic leukemia cells. Chinese journal of intern medicine, 2006, 45(4): 315-318.
[9] Zhang CM. Yin XJ, Feng, ZC. Accumulation of γ globin mRNA and induction of erythroid differentiation after treatment of chronic myelocytic leukemia cell line K562 with matrine. Journal of Applied Clinical Pediatrics, 2007, 22(3): 218-221.
[10] Suzuki K, Adachi R, Hirayama A, et al. Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells. British Journal of Haematology, 2005, 130(5): 681-690.
[11] Zuo MX, Chen XG. The status and future of cyclin-dependent kinase inhibitors research[j]. Chin J Oncol 2007, 29(5): 321-325.
[12] Aggarwal BB, Banerjee S, Bharadwaj U, et al. Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines. Biochem Pharmacol, 2007, 73(7):1024-1032.
[13] Liu MJ, Wang Z, Ju Y, et al. Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis. Cancer Chemother Pharmacol, 2005, 55(1):79-90.
[14] Marko D, Schatzle S, Friedel A, et al. Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells. Br J Cancer, 2001, 84(2):283-289.
[15] Edwin B, Go van D, Bart JK, et al. Targeted induction of apoptosis for cancer therapy: current progress and prospects. TRENDS in Molecular Medicine, 2006, 12(8):382-393. [16] Liu JJ, Huang RW, Lin DJ, et al. Antiproliferation effects of ponicidin on human myeloid leukemia cells in vitro[J]. Oncol Rep, 2005, 13(4):653-657.
[17] Tan TW, Tsai HR, Lu HF, et al. Curcumin-induced cell cycle arrest and apoptosis in human acute promyelocytic leukemia HL-60 cells via MMP changes and caspase-3 activation. Anticancer Res, 2006, 26(6B):4361-4371.
[18] Cecchinato V, Chiaramonte R, Nizzardo M, et al Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells. Biochem Pharmacol, 2007, 74(11): 1568-1574.
[19] Wang Y, Li J M, Yang C M, et al. Study on apoptosis of NB4 cells induced by cinabufagin and its mechanism. Tumour, 2005, 25(6): 534-537.
[20] Liu X, Li Y. Effects of Artesunate on K562 Cell Apoptosis and Fas/FasL Expression of Chronic Granulocytic Leukemia. Shanghai Journal of TCM, 2006, 40(10): 63-65
[21] Wang JQ, Zhang SM, Zheng ZJ. Apoptosis of K562 cells induce d by proteins extracted from Nidus Vespae in vitro and its mechanism. Shandong medicine. 2007, 47(17):13-15.
[22] Quiney C, Billard C, Mirshahi P, Fourneron JD, Kolb JP. Hyperforin inhibits MMP-9 secretion by B-CLL cells and microtubule formation by endothelial cells. Leukemia, 2006, 20(4):583-589.
[23] Chen FY, Wang HR, Zhong L, et al. Effects of quercetin on morphology and VEGF secretion of leukemia cells NB4 in vitro. Chin J Oncol, 2006, 28(1): 25-27.
[24] Huang YL, Zhou YX, Zhou D, et al. Celastrol in the inhibition of neovascularization. Chin J Oncol, 2003, 25(5): 429-432.
[25] Cai TG, Cai Y, Chen B. Reversal effect on multidrug resistance and effect on intracellular on Ca~(2+) concentration of HL-60 cells by psoralen. Chinese Traditional and Herbal Drugs, 2006, 37(6): 881-885.
[26] Chang HY, Pan KL, Ma FC, et al. The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin. Chin J Hematol, 2006, 27(4): 254-258.
[27] Xiao XB, Xie ZX. Effects of neferine on expression of glutathione S-transferase-pi in K562/A02 cells in vitro. Journal of Xian Jiaotong University, 2005, 26(5):428-430.
[28] Chen XF, Gu ZL, Yang HH, et al. Induction of apoptosis and inhibition of??telomerase activity by extract of Fagopynum cymosum (Fr4) in human leukemia HL-60 cells. Chinese Pharmacological Bulletin, 2006, 22 (7):836-840.
[29] Chakraborty S, Ghosh U, Bhattacharyya NP, et al. Inhibition of telomeraseactivity and induction of apoptosis by curcumin in K-562 cells. Mutat Res,2006, 596(1-2):81-90.
[30] Mukherjee Nee Chakraborty S, Ghosh U, Bhattacharyya NP, et al.Curcumin-induced apoptosis in human leukemia cell HL-60 is associated withinhibition of telomerase activity. Mol Cell Biochem, 2007 , 297(1-2): 31-39.
[31] Ji ZN, Ye WC, Liu GQ, Huang Y. Inhibition of telomerase activity and bcl-2expression in berbamine-induced apoptosis in HL-60 cells. Planta Med, 2002Jul, 68(7):596-600.
[2]杨建民，王斌,楼敬伟，张妲，闵碧荷，王健民．ST淋巴细胞性白血病1例报 告并文献复习．白血病淋巴瘤，2006，15(1)：52-53．
[3] Kim R, Tanabe K, Uchida Y, Emi M. Inoue H, Toge T. Current status of the molecular mechanisms of anticancer drug -induced apoptosis. Cancer Chemother Pharmacol, 2002, 50(5):343-352.
[4] Reed JC. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med, 2001, 7(7):314-319.
[5] Su IJ, Cheng AL, Tsai TF, Lay JD. Retoic acid-induced apoptosis and regression of a refractory Epsein-barr virus-containing T cell lymophoma expressing multidrug-resistance phenotypes. Br J Haematol, 1993,85(4):826-828.
[6] Manabe A, Coustan-Smith E, Kumagai M, Behm FG, Raimondi SC, Pui CH.Campana D. Interleukin-4 inducesprogrammed cell death (apoptosis) in casesof high-risk acutelymphoblastic leukemia. Blood, 1994, 83(7):1731-1737.
[7] Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon witha wide-ranging implications in tissue kinetica. British Journal of Cancer, 1972,26(4):239-257.
[8] Kerr JF, Winterfold CM, Harmon BV. Apoptosis: Its significance in cancer andcancer therapy. Cancer. 1994, 73(8):2013-2026.
[9] Hickman JA, Potten CS. Merritt AJ, Fisher TC. Apoptosis and cancerchemotherapy. Philos Trans R Soc Lond B Biol Sci, 1994, 345(1313):319-325.
[10] Hockenbery D. Defining apoptosis. Am J Pathol, 1995,146(1):16-19.
[11] Los M, Burek CJ, Stroh C, Benedyk K, Hug H, Mackiewicz A. Anticancerdrugs of tomorrow: apoptotic pathways as targets for drug design. Drug DiscovToday, 2003, 8(2):67-77.
[12] Nicholson DW, Thornberry NA. Apoptosis, Life and death decisions. Science,2003, 299(5604):214-215.
[13] Marx J. Nobel prize in physiology or medicine. Tiny worm takes a star turn.Science, 2002, 298(5593):526-527.
[14]李蒙，于建勋．单味中药诱导肿瘤细胞凋亡的实验研究现状及展望．中 国中西医结合杂志，2001，21(1)：74-76．
[15]牛泱平，Helen T，Chong BH．人参皂苷诱导HL-60细胞凋亡的研究．中 国中西医结合杂志，2002，22(6)：449-453．
[16]牛泱平，钱煦岱．人参三醇对HL-60白血病细胞的抑制增殖及诱导凋亡 作用．中华实用中西医杂志，2004，17(21)：188-189．
[17]骆晓梅．彭涛．王林．七叶皂苷的药学和治疗作用．国外医学药学分册， 2002，29(3)：168-171．
[18] Hu XM, Zeng FD. Inhibitory effect of beta-aescin on inflammatory processfollowing focal cerebral ischemia-reperfusioninrats. Zhongguo Yaolixue YuDulixue Zazhi, 2005, 19 (1):1-6.
[19] Wei F, Ma LY, Jin WT, Ma SC, Han GZ, Khan IA, Lin RC. Antiinflammatorytriterpenoid saponins from the seeds of Aesculus chinensis. Chem Pharm Bull(Tokyo), 2004, 52 (10): 1246-1248.
[20] Sirtori CR. Aescin: pharmacology, pharmacokinetics and therapeutic profile.Pharmacol Res, 2001, 44(3): 183-193.
[21]侯广平．池广明．七叶皂苷的药理作用及其主要临床应用．中国药师， 2004，7(3)：206-208．
[22]韦继政，李素珍，余利平．七叶皂苷治疗急性脑出血53例临床研究．中国??药物与临床，2005，5(8)：622-623．
[23] Guo W. Xu b, Yang XW, Liu Q, Cui JR. Preliminary studies of effect sodium aescinate aginst cancer in vitro and in vivo. Zhongguo Yaoliexue Tong Bao, 2003, 19(3):351-352.
[24]邹明．韩英，纪欣．七叶皂苷抑制大肠癌HCT-8细胞生长及提高癌细胞 对5-FU敏感性的研究．中华消化杂志，2001，21(4)：248-249．
[25] Patlolla JM, Raju J, Swamy MV, Rao CV. Beta-Escin inhibits colonic aberrant crypt foci formationin rats and regulates the cell cycle growth by inducing p21 (waf1/cip1) in colon cancer cells. Mol Cancer Ther, 2006, 5(6):1459-1466.
[26] Mosmann T. Rapid colorimetric assay for cellular growth and surival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): 55-63.
[27] Niu YP, Jin JM, Gao RL, Xie GL. Chen XH. Effects of Ginsenosides Rg1 and Rb1 on Proliferation of Human Marrow Granulocyte-Macrophage Progenitor Cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2001, 9(2): 178-80.
[28] Niu YP. Qian XD, Wang WX. Effect of panaxadiol saponin and panaxtrol saponin on proliferation of human bone marrow hemopoietic progenitor cells. Zhongguo ZhongXi Yi Jie He Za Zhi, 2004, 24(2): 127-129.
[29] Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003.4(12):721-729.
[30] Pec MK, Aguirre A, Moser-Thier K, Fernandez JJ, Souto ML, Dorta J, Diaz-Gonzalez F. Villar J. Induction of apoptosis in estrogen dependent and independent breast cancer cells by the marine terpenoid dehydrothyrsiferol. Biochem Pharmacol,2003, 65(9): 1451-1461.
[31] Vermes I, Haanen C, Steffens-Nakken H. Reutelingsperger C. A novel assay for apoptosis. Flow cytometic detection of phospha-tidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Method, 1995. 184(1):39-51.
[32] Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Research, 1994, 22(24):5506-5507.
[33] Arends MJ, Morris RG, Wyllie AH. Apoptosis: The role of endonuclease. Am J Pathol, 1990, 136(3):593-608.
[34] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods, 1991, 139(2):271-279.
[35] Lacaille-Dubois MA. Wagner H. In Studies in Natural Products Chemistry: Atta-Ur--Rahman, Ed.; Elsevier: Amsterdam, 2000, Vol.21: pp 633-687.
[36] Kikuchi Y, Sasa H, Kita T, Hirata J, Tode T, Nagata I. Inhibition of human ovarian cancer cell proliferation in vitro by ginsenoside Rh2 and adjuvant effects to cisplatin in vivo. Anticancer Drugs, 1991, 2(1):63-67.
[37] Tode T, Kikuchi Y, Kita T, Hirata J, Imaizumi E, Nagata I. Inhibitory effects by oral DOXinistration of ginsenoside Rh2 on the growth of human ovarian cancer cells in nude mice. J Cancer Res Clin Oncol, 1993,120(1-2):24-26.
[38] Lee YN, Lee HY, Chung HY, Kim SI, Lee SK, Park BC, Kim KW. In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells. Eur J Cancer, 1996, 32A(8):1420-1428.
[39] Park JA, Lee KY, Oh YJ, Kim KW, Lee SK. Activation of caspase-3 protease via a Bcl-2-insensitive pathway during the process of ginsenoside Rh2-induced apoptosis. Cancer Lett, 1997, 121(1):73-81.
[40] Yui S, Ubukata K, Hodono K, Kitahara M, Mimaki Y, Kuroda M, Sashida Y. Yamazaki M. Macrophage-oriented cytotoxic activity of novel triterpene saponins extracted from roots of Securidaca inappendiculata. Int Immunopharmacol 2001, 1(11): 1989-2000.
[41] Gaidi G, Miyamoto T, Laurens V, Lacaille-Dubois MA. New acylated triterpene saponins from Silene fortunei that modulate lymphocyte proliferation. J Nat Prod, 2002, 65(11 ):1568-1572.
[42] Haddad M, Laurens V, Lacaille-Dubois MA. Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia. Bioorg Med Chem. 2004, 12(17): 4725-4734.
[43] Mujoo K, Haridas V, Hoffmann JJ, Wachter GA, Hutter LK, Lu Y, Blake ME, Jayatilake GS, Bailey D, Mills GB, Gutterman JU. Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis. Cancer Res, 2001, 61(14):5486-5490.
[44] Haridas V, Higuchi M, Jayatilake GS, Bailey D, Mujoo K, Blake ME, Arntzen CJ, Gutterman JU. Avicins: triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation. Proc Natl Acad Sci USA, 2001, 98(10):5821-5826.
[45] Yang XW, Zhao J, Cui JR, Guo W. Studies on the biotransformation of escin Ia by human intestinal bacteria and the anti-tumor activities of desacylescin I. Beijing Da Xue Xue Bao, 2004, 36(1): 31-35.
[46] Muraki K, Imaizumi Y, Watanabe M. Ca-dependent K channels in smooth muscle cells permeabilized by Beta-escin recorded using the cell-attached patch-clamp technique. Pflugers Arch, 1992, 420(5-6):461-469.
[47] Pearson PJ, Vanhoutte PM. Vasodilator and vasoconstrictor substance produced by the endothelium. Rev Physiol Biochem Pharmacol, 1993, 122:1-67.
[48] Berti F, Omini C, Longiave D. The mode of action of aescin and the release of prostaglandins. Prostaglandins, 1977, 14(2):241-249.
[49] Matsuda H, Li Y, Yoshikawa M. Possible involvement of 5-HT and 5-HT2 receptors in acceleration of gastrointestinal transit by escin Ib in mice. Life Sci, 2000, 66(23):2233-2238. [50] Facino RM, Carini M, Stefani R, Aldini G, Saibene L. Anti-elastase and anti-hyaluronidase activities of saponins and sapogenins from Hedera helix, Aesculus hippocastanum, and Ruscus aculeatus: factors contributing to their efficacy in the treatment of venous insufficiency. Arch Pharm (Weinheim), 1995, 328(10):720-724.
[51] Tutton PJ, Barkla DH. Influence of prostaglandin analogues on epithelial cell proliferation and xenograft growth. Br J Cancer, 1980, 41(1): 47-51.
[52] Stone OJ. Cancer resistance, carcinogenesis and ground substance viscosity. Med Hypotheses, 1986, 20(1): 117-124.
[1] Warner JK, Wang JC, Hope KJ, Jin L, Dick JE. Concepts of human leukemic development. Oncogene, 2004, 23(43):7164-7177.
[2] Wang ZH, Lin q. Effect of berberine hydrochloride on differentiation of HL-60 cells and study of it's mechanism. Journal of China Pharmaceutical University, 2006, 37(2):165-168.
[3] Seki T, Tsuji K, Hayato Y. Moritomo T, Ariga T. Garlic and onion oils inhibit proliferation and induce differentiation of HL-60 cells. Cancer Letters, 2000, 160(1): 29-35.
[4] Luo YY, Zhang TL. Differentiation of HL-60 cells induced by realgar nano-particles. China Journal of Chinese Materia Medica, 2006, 31(16): 1343-1346.
[5] Liu ZG, Yang YM, et al. Differentiation and Apoptosis of NB4 Cells Synergistically Induced by Tan shinone II A and All-trans Retinoic Acid. Journal of Sichuan Universicy, 2004, 35 (6): 788-791.
[6] Wang Z J, Wu Y L, Lin Q, et al. Effect of panaxynol on differentiation of HL-60 cells line in vitro induced. Chnese Traditional and Herbal Drugs, 2003, 34 (8): 736-738.
[7] Liu YQ, Gao J, Gu B, et al. Observation of the differentiation induction effect of Jinglong capsule on tumor cells. Journal of Chinese cancer clinic, 2004, 31(7):380-382.
[8] Zhu ZT, Jin B, Liu YP, et al. Enhancement of all-trans retinoic acid-induced differentiation by bufalin in primary culture of acute promyelocytic leukemia cells. Chinese journal of intern medicine, 2006, 45(4): 315-318.
[9] Zhang CM. Yin XJ, Feng, ZC. Accumulation of γ globin mRNA and induction of erythroid differentiation after treatment of chronic myelocytic leukemia cell line K562 with matrine. Journal of Applied Clinical Pediatrics, 2007, 22(3): 218-221.
[10] Suzuki K, Adachi R, Hirayama A, et al. Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells. British Journal of Haematology, 2005, 130(5): 681-690.
[11] Zuo MX, Chen XG. The status and future of cyclin-dependent kinase inhibitors research[j]. Chin J Oncol 2007, 29(5): 321-325.
[12] Aggarwal BB, Banerjee S, Bharadwaj U, et al. Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines. Biochem Pharmacol, 2007, 73(7):1024-1032.
[13] Liu MJ, Wang Z, Ju Y, et al. Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis. Cancer Chemother Pharmacol, 2005, 55(1):79-90.
[14] Marko D, Schatzle S, Friedel A, et al. Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells. Br J Cancer, 2001, 84(2):283-289.
[15] Edwin B, Go van D, Bart JK, et al. Targeted induction of apoptosis for cancer therapy: current progress and prospects. TRENDS in Molecular Medicine, 2006, 12(8):382-393. [16] Liu JJ, Huang RW, Lin DJ, et al. Antiproliferation effects of ponicidin on human myeloid leukemia cells in vitro[J]. Oncol Rep, 2005, 13(4):653-657.
[17] Tan TW, Tsai HR, Lu HF, et al. Curcumin-induced cell cycle arrest and apoptosis in human acute promyelocytic leukemia HL-60 cells via MMP changes and caspase-3 activation. Anticancer Res, 2006, 26(6B):4361-4371.
[18] Cecchinato V, Chiaramonte R, Nizzardo M, et al Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells. Biochem Pharmacol, 2007, 74(11): 1568-1574.
[19] Wang Y, Li J M, Yang C M, et al. Study on apoptosis of NB4 cells induced by cinabufagin and its mechanism. Tumour, 2005, 25(6): 534-537.
[20] Liu X, Li Y. Effects of Artesunate on K562 Cell Apoptosis and Fas/FasL Expression of Chronic Granulocytic Leukemia. Shanghai Journal of TCM, 2006, 40(10): 63-65
[21] Wang JQ, Zhang SM, Zheng ZJ. Apoptosis of K562 cells induce d by proteins extracted from Nidus Vespae in vitro and its mechanism. Shandong medicine. 2007, 47(17):13-15.
[22] Quiney C, Billard C, Mirshahi P, Fourneron JD, Kolb JP. Hyperforin inhibits MMP-9 secretion by B-CLL cells and microtubule formation by endothelial cells. Leukemia, 2006, 20(4):583-589.
[23] Chen FY, Wang HR, Zhong L, et al. Effects of quercetin on morphology and VEGF secretion of leukemia cells NB4 in vitro. Chin J Oncol, 2006, 28(1): 25-27.
[24] Huang YL, Zhou YX, Zhou D, et al. Celastrol in the inhibition of neovascularization. Chin J Oncol, 2003, 25(5): 429-432.
[25] Cai TG, Cai Y, Chen B. Reversal effect on multidrug resistance and effect on intracellular on Ca~(2+) concentration of HL-60 cells by psoralen. Chinese Traditional and Herbal Drugs, 2006, 37(6): 881-885.
[26] Chang HY, Pan KL, Ma FC, et al. The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin. Chin J Hematol, 2006, 27(4): 254-258.
[27] Xiao XB, Xie ZX. Effects of neferine on expression of glutathione S-transferase-pi in K562/A02 cells in vitro. Journal of Xian Jiaotong University, 2005, 26(5):428-430.
[28] Chen XF, Gu ZL, Yang HH, et al. Induction of apoptosis and inhibition of??telomerase activity by extract of Fagopynum cymosum (Fr4) in human leukemia HL-60 cells. Chinese Pharmacological Bulletin, 2006, 22 (7):836-840.
[29] Chakraborty S, Ghosh U, Bhattacharyya NP, et al. Inhibition of telomeraseactivity and induction of apoptosis by curcumin in K-562 cells. Mutat Res,2006, 596(1-2):81-90.
[30] Mukherjee Nee Chakraborty S, Ghosh U, Bhattacharyya NP, et al.Curcumin-induced apoptosis in human leukemia cell HL-60 is associated withinhibition of telomerase activity. Mol Cell Biochem, 2007 , 297(1-2): 31-39.
[31] Ji ZN, Ye WC, Liu GQ, Huang Y. Inhibition of telomerase activity and bcl-2expression in berbamine-induced apoptosis in HL-60 cells. Planta Med, 2002Jul, 68(7):596-600.