钙调素拮抗剂EBB抑制人乳腺癌细胞生长的实验研究
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摘要
全球恶性肿瘤发病率在今后20年间将增加50%,而在我国,恶性肿瘤已成为威胁国人生命的最大疾病。因此,克服肿瘤是亟待解决的问题。治疗肿瘤药物种类众多,寻找抑制肿瘤生长、转移和复发的特异性药物成为药物研究的一个热点。
中草药是从植物中提取出来的,与传统化疗药物相比,其副作用少,安全性高,逐步成为化疗药物研究的热点。在植物类抗肿瘤药物中,许多中药如紫杉醇类药物、喜树碱类药物都取得了很好的结果。以中药有效成分为先导化合物,经化学结构修饰,得到疗效好,毒副作用小的新型抗肿瘤药物,是当今抗肿瘤药物研究的一个重要方向。中草药小檗胺及其衍生物EBB显示出较强的抗肿瘤活性。
小檗胺是小檗属植物中提取的双苄异喹啉类生物碱,是一种天然钙调素拮抗剂,具有抗肿瘤、促进白细胞增生等作用。其衍生物[O-(4-乙氧基)-丁基]-小檗胺(O-4-Ethoxyl-Butyl-Berbamine,EBB)是国内外已知最强的钙调素拮抗剂之一,具有与小檗胺相似的药理作用,但其拮抗钙调素的能力是小檗胺的一百多倍。
先前的研究表明,EBB在体外抑制多种肿瘤细胞增殖、耐药逆转及实体瘤细胞转移;在体内,还具有抗小鼠肝癌作用。本次研究的目的在于探讨EBB对人乳腺癌细胞的药物作用,探讨其抗肿瘤作用机制。采用四唑盐(MTT)比色法在体外评价不同浓度EBB对乳腺癌细胞的生长抑制作用;用Annexin V-FITC/PI双染色法和流式细胞术检测低于IC_(30)EBB对细胞凋亡的影响;并用流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)水平的改变;在激光共聚焦显微镜下观察EBB作用后肿瘤细胞内线粒体、内质网分布及细胞内游离Ca~(2+)水平变化;同时采用RT-PCR法检测EBB对细胞内凋亡相关基因survivin mRNA表达水平的影响。结果表明MCF-7细胞EBB的IC_(50)值为10.53μmol/L,并且此抑制作用可被超氧化物歧化酶(superoxide dismutase,SOD)所减弱;EBB能诱导MCF-7细胞凋亡;EBB作用能使MCF-7细胞内的ROS呈剂量依赖性升高,并在作用2小时左右达到峰值;EBB能够明显增加细胞内钙离子浓度,并下调凋亡抑制蛋白survivin的基因表达;在激光共聚焦显微镜下可见,EBB作用后线粒体呈药物剂量依赖性减少、破坏,而内质网则出现药物剂量依赖性肿胀。因此我们得出结论,EBB对人乳腺癌MCF-7细胞具有明显抗肿瘤作用,作用机制可能通过氧化应激反应来诱导肿瘤细胞凋亡。
Survivin基因是凋亡抑制蛋白家族(IAP)中分子量最小的成员,是一种肿瘤特异性凋亡抑制因子,主要分布于胚胎和肿瘤组织,正常分化成熟组织几乎检测不到。研究表明,survivin与许多肿瘤的复发和预后密切相关,其主要通过抑制caspase蛋白酶活性而抑制细胞凋亡。
本实验采用PCR法扩增survivin序列,经酶切、连接,克隆至表达载体pAYZ,转化E.coli 16C9,经测序鉴定阳性克隆菌落插入片段为正确编码的survivin基因序列。经低磷培养基AP5诱导表达,用His-tag亲和层析柱纯化,为进一步研究其生物学特性,靶向survivin治疗恶性肿瘤奠定基础。
Malignancy incidence will increase 50%in the next twenty years in the world.At the same time,it has been the most serious sickness that affects our nationality's lives. Therefore,how can we cure malignancy is the key problem in biomedical research. Looking for anti-tumor agents,specifically agents that can inhibit tumor metastasis presents a promising research in drug discovery.Traditional plant-derived medicines are safe and have been used to treat cancer for centuries.We have known that some plant-source medicine just like Paclitaxel or camptothecin,showed good anti-tumor effects.In order to develop more potent pharmacophore structure,charged groups were introduced into the plant-based formulations of chinese traditional medicine that led to discovery of a new synthetic derivatives possessing anti-tumor medicine with less side-effect.In this respect,Chinese herb extraction Berbamine and its derivative 0-4-Ethoxyl-Butyl-Berbamine(EBB) really showed an attractive anti-tumor activity.
Berbamine is a bisbenzylisoquinoline alkaloid and a natural calmodulin antagonist. Studies showed that Berbamine has anti-tumor effects and increases the leukocyte count. EBB is a derivative of Berbamine,which is one of the most effective calmodulin antagonists.EBB has similar pharmacology mechanism with berbamine,but the ability of EBB to antagonize calmodulin is 100 times more than berbamine.
Previous study showed that EBB inhibited the cell proliferation,reversed drug resistance and suppressed tumor invasion in several kinds of tumor cell lines in vitro. EBB also showed anti-tumor effects in vivo.In this paper,we investigated the effects of EBB on human breast cancer cell line MCF-7 and explored the possible mechanism. MTT assay was used to analyze the growth inhibition effects of EBB on several tumor cell lines.The effects of EBB on reactive oxygen species(ROS) production and cell apoptosis were detected by flow cytometry analysis.Laser scanning confocal microscope was used to dissect out the changes of the mitochondrion,endoplasmic reticulum and cytosolic Ca~(2+) in the cells.The effects of EBB treatment on survivin gene expression was detected by RT-PCR.
Our results showed that EBB inhibited the proliferation of MCF-7 cells(IC_(50), 10.53μmol/L) and the inhibitory abilities were blocked by adding superoxide dismutase
(SOD).EBB induced apoptosis of MCF-7 cells by increasing ROS production and intracellular Ca~(2+) concentration.EBB also decreased the expression of survivin gene,one of the inhibitor of apoptosis protein.Impairment of the mitochondrion and swelling of endoplasmic reticulum were also observed in a dose-dependent manner after EBB treatment.In conclusion,EBB has the potential ability to inhibit the growth of several tumor cell lines.The possible mechanism is due to the induction of apoptosis induced by oxidative stress and the structural and functional changes of the mitochondrion and endoplasmic reticulum.
Survivin is the smallest member of the inhibitor of apoptosis protein(IAP) family, which is abundantly expressed during human embryonic and fetal development,or in tumor tissues,but undetectable in most normal terminally differentiated adult tissues. Recent study indicates that Survivin protein expression is associated with the relapse and poor prognosis of acute leukemia.Survivin inhibits the apoptosis by inhibiting the caspase activity.On the basis of these findings survivin has been proposed as an attractive new target for new anticancer interventions.
In our research,the full-length human survivin was amplified by RT-PCR,and cloned into pAYZ expression vector.The recombinant pAYZ-survivin was induced to express in E.Colil6C9 efficiently,establishing a foundation for further anti-survivin treatment in leukemia or other malignant tumors.
中草药是从植物中提取出来的,与传统化疗药物相比,其副作用少,安全性高,逐步成为化疗药物研究的热点。在植物类抗肿瘤药物中,许多中药如紫杉醇类药物、喜树碱类药物都取得了很好的结果。以中药有效成分为先导化合物,经化学结构修饰,得到疗效好,毒副作用小的新型抗肿瘤药物,是当今抗肿瘤药物研究的一个重要方向。中草药小檗胺及其衍生物EBB显示出较强的抗肿瘤活性。
小檗胺是小檗属植物中提取的双苄异喹啉类生物碱,是一种天然钙调素拮抗剂,具有抗肿瘤、促进白细胞增生等作用。其衍生物[O-(4-乙氧基)-丁基]-小檗胺(O-4-Ethoxyl-Butyl-Berbamine,EBB)是国内外已知最强的钙调素拮抗剂之一,具有与小檗胺相似的药理作用,但其拮抗钙调素的能力是小檗胺的一百多倍。
先前的研究表明,EBB在体外抑制多种肿瘤细胞增殖、耐药逆转及实体瘤细胞转移;在体内,还具有抗小鼠肝癌作用。本次研究的目的在于探讨EBB对人乳腺癌细胞的药物作用,探讨其抗肿瘤作用机制。采用四唑盐(MTT)比色法在体外评价不同浓度EBB对乳腺癌细胞的生长抑制作用;用Annexin V-FITC/PI双染色法和流式细胞术检测低于IC_(30)EBB对细胞凋亡的影响;并用流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)水平的改变;在激光共聚焦显微镜下观察EBB作用后肿瘤细胞内线粒体、内质网分布及细胞内游离Ca~(2+)水平变化;同时采用RT-PCR法检测EBB对细胞内凋亡相关基因survivin mRNA表达水平的影响。结果表明MCF-7细胞EBB的IC_(50)值为10.53μmol/L,并且此抑制作用可被超氧化物歧化酶(superoxide dismutase,SOD)所减弱;EBB能诱导MCF-7细胞凋亡;EBB作用能使MCF-7细胞内的ROS呈剂量依赖性升高,并在作用2小时左右达到峰值;EBB能够明显增加细胞内钙离子浓度,并下调凋亡抑制蛋白survivin的基因表达;在激光共聚焦显微镜下可见,EBB作用后线粒体呈药物剂量依赖性减少、破坏,而内质网则出现药物剂量依赖性肿胀。因此我们得出结论,EBB对人乳腺癌MCF-7细胞具有明显抗肿瘤作用,作用机制可能通过氧化应激反应来诱导肿瘤细胞凋亡。
Survivin基因是凋亡抑制蛋白家族(IAP)中分子量最小的成员,是一种肿瘤特异性凋亡抑制因子,主要分布于胚胎和肿瘤组织,正常分化成熟组织几乎检测不到。研究表明,survivin与许多肿瘤的复发和预后密切相关,其主要通过抑制caspase蛋白酶活性而抑制细胞凋亡。
本实验采用PCR法扩增survivin序列,经酶切、连接,克隆至表达载体pAYZ,转化E.coli 16C9,经测序鉴定阳性克隆菌落插入片段为正确编码的survivin基因序列。经低磷培养基AP5诱导表达,用His-tag亲和层析柱纯化,为进一步研究其生物学特性,靶向survivin治疗恶性肿瘤奠定基础。
Malignancy incidence will increase 50%in the next twenty years in the world.At the same time,it has been the most serious sickness that affects our nationality's lives. Therefore,how can we cure malignancy is the key problem in biomedical research. Looking for anti-tumor agents,specifically agents that can inhibit tumor metastasis presents a promising research in drug discovery.Traditional plant-derived medicines are safe and have been used to treat cancer for centuries.We have known that some plant-source medicine just like Paclitaxel or camptothecin,showed good anti-tumor effects.In order to develop more potent pharmacophore structure,charged groups were introduced into the plant-based formulations of chinese traditional medicine that led to discovery of a new synthetic derivatives possessing anti-tumor medicine with less side-effect.In this respect,Chinese herb extraction Berbamine and its derivative 0-4-Ethoxyl-Butyl-Berbamine(EBB) really showed an attractive anti-tumor activity.
Berbamine is a bisbenzylisoquinoline alkaloid and a natural calmodulin antagonist. Studies showed that Berbamine has anti-tumor effects and increases the leukocyte count. EBB is a derivative of Berbamine,which is one of the most effective calmodulin antagonists.EBB has similar pharmacology mechanism with berbamine,but the ability of EBB to antagonize calmodulin is 100 times more than berbamine.
Previous study showed that EBB inhibited the cell proliferation,reversed drug resistance and suppressed tumor invasion in several kinds of tumor cell lines in vitro. EBB also showed anti-tumor effects in vivo.In this paper,we investigated the effects of EBB on human breast cancer cell line MCF-7 and explored the possible mechanism. MTT assay was used to analyze the growth inhibition effects of EBB on several tumor cell lines.The effects of EBB on reactive oxygen species(ROS) production and cell apoptosis were detected by flow cytometry analysis.Laser scanning confocal microscope was used to dissect out the changes of the mitochondrion,endoplasmic reticulum and cytosolic Ca~(2+) in the cells.The effects of EBB treatment on survivin gene expression was detected by RT-PCR.
Our results showed that EBB inhibited the proliferation of MCF-7 cells(IC_(50), 10.53μmol/L) and the inhibitory abilities were blocked by adding superoxide dismutase
(SOD).EBB induced apoptosis of MCF-7 cells by increasing ROS production and intracellular Ca~(2+) concentration.EBB also decreased the expression of survivin gene,one of the inhibitor of apoptosis protein.Impairment of the mitochondrion and swelling of endoplasmic reticulum were also observed in a dose-dependent manner after EBB treatment.In conclusion,EBB has the potential ability to inhibit the growth of several tumor cell lines.The possible mechanism is due to the induction of apoptosis induced by oxidative stress and the structural and functional changes of the mitochondrion and endoplasmic reticulum.
Survivin is the smallest member of the inhibitor of apoptosis protein(IAP) family, which is abundantly expressed during human embryonic and fetal development,or in tumor tissues,but undetectable in most normal terminally differentiated adult tissues. Recent study indicates that Survivin protein expression is associated with the relapse and poor prognosis of acute leukemia.Survivin inhibits the apoptosis by inhibiting the caspase activity.On the basis of these findings survivin has been proposed as an attractive new target for new anticancer interventions.
In our research,the full-length human survivin was amplified by RT-PCR,and cloned into pAYZ expression vector.The recombinant pAYZ-survivin was induced to express in E.Colil6C9 efficiently,establishing a foundation for further anti-survivin treatment in leukemia or other malignant tumors.
引文
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18.Nair S,Samy TS,Krishan A. Calcium calmodulin,and protein content of adriamycin-resistant and -sensitive murine leukemic cells.Cancer Res. 1986, 46:229-232.
19.Debajit, K B.; Prem Reddy, V.; Mickael, P.; Benu, M.; Nichole, P.;Auther, P.B, J.Biol. Chem., 1998,273(50), 33817.
20.Veigal ML,Vanaman TC,Sedwick WD. Calcium and calmodulin in cell growth and transformation.Biochim Biophys Acta, 1984,738(1-2):21-48.
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24.WangX. The expanding role of mitochondfia in apoptosis. Genes Dev, 200 1, 15(22): 2922—2933.
25.Desagher S,Maninou JC.Mitochondfia as the central controlpoint of apoptosis.Trends Cell Biol,2000,10(9):369-377.
26.A shkenazi A,Dixit VM.Death receptors:signaling and modulation.Science, 1998,281 (5381):1305-1308.
27.Sun XM,MacFarlane M,Zhuang J,Wolf BB,Green DR,Cohen GM.Distinct caspase cascades are initiated in receptor-mediated and chemical—induced apoptosis.Joural of Biological Chemistry ,1999,274(8):5053～5060.
28.Ji ZN, Ye WC, Lju GQ, et al. Inhibition of telomerase activity and bcl-2 expression in berbamine-induced apoptosis in HL-60 cells. PlantaMed, 2002, 68: 596-600.
29.Paul S.Brookes,Yisang Yoon.Calcium,ATP,and ROS:a mitochondrial love-hate triangle.Am J Physiol Cell Physiol 2004,287:817-833.
30.徐磊,赵小英,徐荣臻,等.钙调素拮抗剂小檗胺诱导K562细胞凋亡的研究,中华血液杂志,2003,24:261-262.
31.Malgorzata NS,Wasik MA,Slupianek A,et al.signal transducer and activator of transcription(SIAT)5 activation by BCR/ABL is dependent On intact Src homology SH3 and SH2 domains Of BCR/ABL and is required for leukemogenesis[J].J Exp Med,1999,189(8):1229-1242.
32.Nimman apalli R,O Bryan E,Bhalla K.Geldan amycin an d its an alogue 17-allylamino-17-demethoxygeldan arnycin lowers Bcr—Abl levels and induces apoptosis and differentiation of Bcr.Abl.positive human leukemic blasts.Cancer Res,2001.61:1799.180L4.
33.Guo F.Rocha K.Bali P.et al.Abrogation ofheat shock protein 70 induction as a strateg Y to increase antileukemia activity of heat shock protein 90 inhibitor 17-allylamino-demethoxy geldan amycin.Cancer Res,2005.65:10536-10544
34.林茂芳,孟小莉,蔡真,等.抗凋亡基因survivin在急性白血病细胞表达及其临床意义.中华血液学杂志,2002,23:251-253.
35.Xu R,Dong Q,Yu Y,et al.Berbamine:a novel inhibitor of bcr/abl fusion gene with potent anti.leukemia activity.Leuk Res.2006.30:17-23.
36.何智文,赵小英,徐荣臻,吴东.小檗胺诱导白血病细胞株NB4细胞凋亡及其机制的研究.浙江大学学报(医学版),2006,35,209-214.
37.Ahn EY,Pan G,Oh JH,etal.The combination of calmodulinantagunists and interferon-gamma induces apoptosis through caspase-dependent and -independent pathways in eholangiocarcinoma cells.Am J Pathol,2003,163(5):2053-2063
38.吴东,林茂芳,赵小英,等.小檗胺对JURKAT细胞体内外作用的实验研究.中国实验血液学,2005.13(3):373-378.
39.Rodriguez-Mora OG,LaHar MM,McCubrey JA,et al.Calcium/calmodulin-dependent kinase I and ealcium/calmodulin-dependent kinase kinase participate in the control of cell cycle progression in MCF-7 human breast cancer cells.Cancer Res,2005,65(12):5408-5416.
40.闫美荣,侯金凤,苏秀兰,闫晓红,等.人胚胎组织低分子活性肽协同中药有效成分的抗白血病效应的研究.内蒙古医学院学报,2000,22:81-83
41.王晨光,葛日萍,等.钙调素拮抗剂EBB逆转HL-60/HAR细胞对抗肿瘤药的抗性.中国病理生理杂志,1997,11(7);496-499.
42.Ram Ganapathi,Dale Grabowski,William Rouse,and Francis Riegler.Differential Effect of the Calmodulin Inhibitor Trifluoperazine on Cellular Accumulation,Retention,and Cytotoxicity of Anthracyclines in Doxorubicin (Adriamycin)-resistant P388 Mouse Leukemia Cells.Cancer Research,1984,44:5056-5061
43.Y.C.Mayurl,S.Jagadeesh and K.N.Thimmaiah.Targeting Calmodulin in Reversing Multi Drug Resistance in Cancer Cells.Mini-Reviews in Medicinal Chemistry,2006,6,1383-1389
44.J.L.Jiang,H.C.Chan,Q.Zhou,HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains.CMLS,Cell.Mol.Life Sci.61(2004)2083-2091
45.潘兵,周圆,齐静等.钙调素拮抗剂EBB对HT1080细胞系侵袭能力的影响.中国医学科学院学报,2005,27(3):311-314