Geldanamycin增强顺铂的抗肿瘤作用

研究抗生素苯醌安莎霉素(Geldanamycin ,GDM)对人肝癌BEL 7402细胞周期的影响和联合应用顺铂 (CDDP)的体外体内抗肿瘤作用。方法 :采用MTT法检测药物的生长抑制作用 ,[3H] TdR掺入法测定DNA合成抑制作用 ,流式细胞术分析细胞周期 ,小鼠移植性肝癌H22模型研究药物单用或联合应用时的体内抗肿瘤作用。结果 :MTT法测得GDM对BEL 7402细胞的生长抑制IC50为0 28μmol/L。GDM处理BEL 7402细胞4h ,抑制DNA合成的IC50为14μmol/L。1 0、10μmol/LGDM处理BEL 7402细胞24h后S期细胞比例明显下降 ,并持续到48h ;0 1、1 0、10μmol/L的GDM引起G2/M期的明显阻断 ,24h时达高峰值 ,48h后开始回复。体外0 1μmol/L和0 2μmol/LGDM均增强CDDP对BEL 7402细胞的细胞毒作用。小鼠移植肝癌H22模型中 ,单用1 5mg/kgCDDP组两批试验的抑瘤率均为73 % ,顺铂联用0 38mg/kgGDM两批试验的抑瘤率分别为89 %和82% ,两者有显著性差异(P<0 05) ,其CDI值分别为0 41和0 72。结论

Geldanamycin产生菌生物合成相关基因的克隆与分析

目的 从 geldanamycin产生菌吸水链霉菌 (S.hygroscopicus)中克隆生物合成基因 ,为阐明生物合成机制及改造其结构奠定基础。方法 以链霉菌 /大肠埃希氏菌穿梭 cosmids p KC5 0 5为载体构建了插入片段为 2 0～ 30 kb的 S.hygroscopicus总 DNA文库。以利福霉素中与 3-氨基 - 5 -羟基苯甲酸 (AHBA)合成相关基因为探针 ,经菌落杂交 ,Southern杂交筛选同源基因片段。测序结果与 Genbank进行同源性比较分析 ,并以attp- 噬菌体载体 KC5 15利用基因阻断实验对克隆的基因片段进行功能鉴定。结果 构建的基因文库含重组基因比率高、基因组覆盖率高 ,稳定性好。经同源基因探针杂交 ,从文库中筛选出 9个阳性 cosmids克隆p CGB2 0、p CGB2 6、p CGB2 8、p CGB2 9、p CGB36、p CGB45、p CGB49、p CGB6 3、p CGB75。测序分析表明 ,cosmidp CGB2 0中 Bam HI- Bam HI 8kb片段两端的不完整 ORF编码蛋白与竹桃霉素 (oleandomycin) PKS Module 5、Module 6 (一致性为 79% )和红霉内酯合成酶 ORF2 (一致性为 75 % )、ORF1(一致性为 73% )、ORF3(一致性为 70 % )高度同源 ;Bam HI- Bam HI3kb片段一端与编码类结核分枝杆菌的磷酸吡哆醛依赖的氨基转移酶家族中半胱氨酸脱硫酶的 DNA有同源性 ,该家族与 AHBA合成酶关系密切。

Geldanamycin对HER2/neu高表达人乳腺癌细胞系SKBr3增殖及迁移能力的影响

目的探讨Hsp90抑制剂苯醌安莎霉素类抗生素(GA)对HER2/neu高表达人乳腺癌细胞系SKBr3增殖及迁移能力的影响。方法用Western蛋白印迹法检测GA介导的HER2/neu酪氨酸激酶的降解;MTT法分析GA对肿瘤细胞存活率的影响;流式细胞仪测定GA对肿瘤细胞周期的影响;RT-PCR和real-time PCR分析GA对cyclin D1表达的影响以及分析GA对肿瘤细胞迁移能力的干预作用。结果GA以剂量/时间依赖性的方式降解了HER2/neu酪氨酸激酶的表达并抑制了SKBr3乳腺癌细胞的增殖,这种增殖抑制效应表现在:GA干预后乳腺癌细胞的存活率明显降低;GA阻断了乳腺癌细胞细胞周期的进展,使细胞周期停滞于G1期,而这两种表现与cyclin D1表达量的减低有着明显的关系。GA的干预也同时降低了肿瘤细胞的迁移能力。结论本研究证实GA能够降解HER2/neu酪氨酸激酶并抑制HER2/neu高表达人乳腺癌细胞系SKBr3的增殖和迁移能力。

HSP90的抑制剂Geldanamycin对乳腺癌细胞侵袭、转移能力的影响

目的:探讨H SP90抑制剂Geldanam ycin(GA)对高转移性乳腺癌细胞株M DA-MB-231、MDA-M B-435s侵袭、转移能力的影响。方法:噻唑兰比色(M TT)法检测两株细胞对人工基底膜(Matrigel)的粘附能力变化;TranswellCham ber体外侵袭运动实验研究GA处理前后M DA-M B-231、M DA-M B-435s细胞侵袭、运动能力的改变。结果:GA处理后的M DA-M B-231和M DA-MB-435s细胞的粘附、侵袭、运动能力与未处理组细胞相比均明显下降,差异具有显著性(P<0.05和P<0.01)。结论:H SP90抑制剂Geldanam ycin(GA)能够明显抑制乳腺癌细胞株M DA-MB-231、MDA-M B-435s粘附、侵袭和运动能力。

Docking of Human Heat Shock Protein 90 with Selenoderivatives of Geldanamycin

The interference of human heat shock protein 90 (HSP90) in many signalling networks associated with cancer progression makes it an important drug target. In the present work, we investigated the binding ability of 9 selenoderivatives of geldanamycin (GMDSe) at the N-terminal domain of HSP90 derived from Protein Data Bank (PDB code: 1YET) based on ligand-protein docking. All selenoderivatives interacted positively with HSP90, yet the binding strength decreased when replacing monovalent oxygen in position 1 (GMDSe1) or 9 (GMDSe9). Hydrogen-bonding and lipophilic interactions between selenoderivatives and amino acid residues in the inhibitor site of HSP90 were thermodynamically the main forces driving the binding stability. Molecular electrostatic potential surfaces of the selenoderivatives showed marked non polar areas, which were probably involved in the lipophilic interactions with the hydrophobic residues of amino acids. Interestingly, the amino acid residues forming the hydrogen bonds with GMD were also involved in the hydrogen-bonding interactions with the selenoderivatives. Moreover, HSP90 interacted with the GMDSe6 and GMDSe7 selenoderivatives stronger than with GMD, while maintaining lipophilic interactions and hydrogen bonds with amino acid residues like Asp93, which are catalytically crucial for therapeutic properties of HSP90 inhibitors. This finding should guide further studies of pharmacophore properties of GMD selenoderivatives in order to explore their therapeutic properties. It is noteworthy that selenium has been suggested to reduce the risk of various types of cancers.

Geldanamycin inhibits proliferation and motility of Her2/neu-overexpressing SKBr3 breast cancer cells

Objective Benzoquinone ansamycin antibiotic, geldanamycin (GA), is a new anticancer agent that could inhibit Hsp90 by occupying its NH2-terminal ATP-binding site. This study was to investigate the antitumor efficacy of GA on Her2/neu tyrosine kinase overexpressing human breast cancer cell line SKBr3. Methods The degradation of Her2/neu tyrosine kinase was analyzed by Western blotting, the proliferation index was determined by MTT assay, cell cycle distribution was detected by flow cytometry, Cyclin D1 mRNA transcription was measured by RT-PCR and real-time PCR, and cell motility was evaluated by the cell culture insert model. Results GA induced a dose-and a time-dependent degradation of the Her2/neu tyrosine kinase protein and concurrently, the inhibition of cancer cell proliferation. The antitumor effects mediated by GA included: GA treatment decreased the survival rates of cancer cells, and led to a dose-dependent G1 arrest. Furthermore, this antitumor effect was proved to be related to declined transcription of Cyclin D1. Concurrently, the motility of cancer cells was reduced by GA. Conclusion GA treatment could induce the degradation of Her2/neu tyrosine kinase efficiently, inhibit cancer cell proliferation and reduce motility in Her2/neu tyrosine kinase overexpressed human breast cancer cell line SKBr3.

Anti-Inflammatory Activity of Geldanamycin and Its Derivatives in LPS-Induced RAW 264.7 Cells

Geldanamycin (1) had been isolated as a major compound from Streptomyces zerumbet W14;an endophyte of Zingiber zerumbet (L.) Smith. Two new geldanamycin derivatives;17-(tryptamine)-17-demethoxygeldanamycin (2) and 17-(5’-methoxytryptamine)-17-demethoxygeldanamycin (3) were synthe- sized and their anti-inflammatory activity was evaluated in LPS-induced macrophage RAW 264.7 cells by investigating their effects on the inhibition of production of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10. The data obtained were consistent with the modulation of TNF-α, IL-1β, IL-6, IL-10 production by these derivatives at concentration of 1 to 5 μg/ml. A similar effect was also observed when LPS-induced NO release and PGE2 production were tested. The inhibitory effects were shown in concentration-dependent manners. From the obtained results, it was concluded that two new gelda- namycin derivatives possess anti-inflammatory activity on LPS-induced RAW 264.7 cells. They could be useful for the management of inflammatory diseases.

Geldanamycin Combination with Colcemid Induces Mitotic Arrest through Stabilization of bubR1 Mitotic Kinase in Human Tumor Cells

Mitosis-targeted anti-cancer therapies gained much attention in recent years. However, lack of tumor selectivity poses limitations to the current anti-mitotic drugs to be used as broad-spectrum anti-cancer agents. In this study, we show that combination treatment of colcemid, an inhibitor of microtubule polymerization with geldanamycin, an inhibitor of cancer chaperone, Hsp90 irreversibly targets mitosis through mitotic kinase bubR1 stabilization. When the individual and combination drugs treatments were tested against tumor cells (IMR-32 and HeLa) and non-tumor cells (SRA01), the combination treatment showed significant increase in cytotoxicity only in tumor cells followed by G2/M cell cycle block. The IMR-32 cells showed enhanced cytotoxicity in response to combination treatments, compared to HeLa cells. Further studies revealed that the G2/M arrest in IMR-32 correlates with both increased bubR1 nuclear localization and metaphase arrest. The siRNA knockdown of bubR1 has decreased tumor cell response to geldanamycin suggesting Hsp90-dependent regulation of bubR1. The combination treatment also showed inactivation of non-canonical β-catenin signaling suggesting inhibition of cancer growth. In addition, the combination treatment has significantly affected the distribution and functions of bubR1 downstream mitotic kinases such as aurks and plk1 indicating the combinatorial attack of combination treatment. In conclusion, we demonstrate that colcemid and GA combination treatment compromises the division potential of tumor cells interfering with the mitosis through bubR1 kinase.

Effects of Geldanamycin on Expression of Bcl-2 in Human Cervical Cancer HeLa Cells

OBJECTIVE Geldanamycin,a natural product of Streptomyces geldanus,binds the heat shock protein 90(Hsp90),a cell chaperone protein that interacts with Bcl-2.In this study,we investigated whether geldanamycin(GA)inhibits proliferation of HeLa cells through induction of apoptosis by decreasing the level of Bcl-2 expression. METHODS HeLa cells,a human cervical cell line,were cultured in vitro and treated with different concentrations of GA(0,0.02,0.2, 2,10μmol/L)for 24 h.or were treated for different lengths of time at a GA concentration of 10μmol/L.Proliferation of the cells was analyzed by an MTT assay,and cell apoptosis was determined by staining the cells with annexin V.In addition,cellular mRNA levels for Bcl-2 and Hsp90 were determined by the semiquantitative polymerase chain reaction(PCR),and the levels of Bcl-2 and Hsp90 protein expression were determined by Western blots. RESULTS Treatment of cells with GA was found to inhibit HeLa cell proliferation in a concentration and time-dependent manner.The inhibition was a result of increased cellular apoptotic levels.Further analyses showed that while the mRNA and protein expression levels of Hsp90 were not affected,GA treatment significantly reduced the level of Bcl-2 mRNA and protein expression in a concentration-dependent manner that correlated with the observed inhibition of cell proliferation. CONCLUSION GA can inhibit proliferation and increase apoptosis of HeLa cells by decreasing the transcription and expression of an anti-apoptotic gene bcl-2,probably through interaction and functional inhibition of Hsp90.

Rational design, synthesis, and biological evaluation of novel C6-modified geldanamycin derivatives as potent Hsp90 inhibitors and anti-tumor agents

Heat shock protein 90(Hsp90) is an appealing anticancer drug target that provoked a tremendous wave of investigations. Geldanamycin(GA) is the first identified Hsp90 inhibitor that exhibited potent anticancer activity, but the off-target toxicity associated with the benzoquinone moiety hampered its clinical application. Until now, structure optimization of GA is still in need to fully exploit the therapeutic value of Hsp90. Due to the structural complexity and synthetic challenge of this compound family, conventional optimization is bound to be costly but high efficiency is expected to be reachable by combining the art of rational design and total synthesis. Described in this paper is our first attempt at this approach aiming at rational modification of the C6-position of GA. The binding affinities towards Hsp90 of compound 1(C6-ethyl) and 2(C6-methyl) were designed and predicted by using Discovery Studio. These compounds were synthesized and further subjected to a thorough in vitro biological evaluation. We found that compounds1 and 2 bind to Hsp90 protein with the IC_(50) of 34.26 nmol/L and 163.7 nmol/L, respectively. Both compounds showed broad-spectrum antitumor effects. Replacing by ethyl, compound 1 exhibited more potent bioactivity than positive control GA, such as in G2/M cell cycle arrest, cell apoptosis and client proteins degradations. The results firstly indicated that the docking study is able to provide a precise prediction of Hsp90 affinities of GA analogues, and the C6 substituent of GA is not erasable without affecting its biological activity.

一株链霉菌的鉴定及其产格尔德霉素的发酵工艺研究

【目的】对链霉菌Streptomyce sp.FIM18-0592进行菌种鉴定,并对其胞外格尔德霉素产量进行发酵工艺优化,旨在提高产量并降低发酵成本。【方法】通过形态特征、培养特征和生理生化特性,结合16S rDNA序列分析构建系统发育树进行菌种鉴定;采用单因素试验优化培养条件及培养基配方,进一步使用最陡爬坡试验和响应面试验优化培养基配方的含量。【结果】通过对链霉菌FIM18-0592的形态特征、培养特征及生理生化特征进行初步培养观察发现,其在ISP2等培养基上生长较好,气生菌丝旺盛,产黑色素。结合16S rDNA分子鉴定,确定该菌为格尔德霉素链霉菌(Streptomyces geldanamycininus)。通过单因素试验对发酵条件以及培养基配方进行优化,得到最适宜菌株发酵的培养条件为转速140 r/min、装液量12%(体积分数)、接种量7%(体积分数)、培养时间144 h。最佳的碳源、氮源、无机盐分别为葡萄糖、黄豆饼粉和硫酸铵。采用最陡爬坡试验和响应面优化试验确定了其最优的发酵培养基为:葡萄糖10.42%、黄豆饼粉1.68%、硫酸铵0.3%、乳酸0.3%、甘油4%、硫酸镁0.1%、碳酸钙0.4%,在此条件下,格尔德霉素的发酵效价达到2887μg/mL,较原始发酵工艺效价提高了66%。【结论】链霉菌FIM18-0592为格尔德霉素链霉菌,通过对其发酵工艺进行优化显著提高格尔德霉素的产量,为格尔德霉素及其衍生物的开发和利用奠定基础。

微生物来源免疫抑制活性化合物SIPI-100-1的研究

目的:分离吸水链霉菌R-100菌株合成的免疫抑制活性化合物。方法:根据筛选使用的发酵条件,进一步优化最佳摇瓶发酵条件,并进行大规模发酵;发酵液经离心、乙酸乙酯萃取、硅胶柱层析和结晶等方法,从其上清液中分离出活性化合物SIPI-100-1,并测定化合物的图谱数据和生物学活性。结果:通过理化性质、质谱、紫外、红外和核磁共振等图谱数据的分析,确定SIPI-100-1化合物结构;生物学活性研究,发现其具有中等强度的免疫抑制活性。结论:吸水链霉菌R-100合成的SIPI-100-1化合物属于安沙类抗生素,与文献报道的Geldanamycin结构一致,免疫抑制活性属于首次发现。

吸水链霉菌17997格尔德霉素部分生物合成基因簇的克隆和分析

吸水链霉菌17997(Streptomyceshy groscopicus17997)是我所从中国云南土壤中分离到的格尔德霉素(geldanamycin,GDM)产生菌,GDM具有良好的抗肿瘤和抗病毒活性,但其肝毒性和水溶性差的缺点限制了其在临床上的应用。为了实现对GDM结构的生物学改造,首先要获得GDM的生物合成基因。根据GDM后修饰基因——氨甲酰基转移酶基因(gdmN)的保守序列筛选S.hygroscopicus17997的柯斯质粒基因组文库,共获得6个阳性克隆,选择CT-4阳性柯斯质粒进行亚克隆和测序,又通过PCR延伸的方法获得了与CT4连锁的将近5kb的外源序列,共获得28.356kb的外源DNA序列,其中包含了13个可能阅读框架,通过同源比较证实该序列与S.hygroscopicusNRRL3602中的GDM生物合成基因有很高的同源性。为进一步研究GDM生物合成基因的功能,并通过组合生物学的方法改造GDM的结构奠定了基础。

作用于基质金属蛋白酶的抗生素

基质金属蛋白酶 (matrix metalloproteinases,MMPs)是在与组织重塑有关的生理过程和以细胞外基质过度破坏为主要特征的病理过程中起重要作用的一类锌离子依赖性内肽酶。近些年来 ,MMPs在恶性肿瘤侵袭、生长和转移过程中的作用日益受到重视。我们以在肿瘤细胞破坏和穿过基底膜发生侵袭和转移过程中起重要作用的 MMPs家族成员 IV型胶原酶为研究对象 ,利用抗 IV型胶原酶单克隆抗体 3D6为工具 ,采用间接 EL ISA方法研究了四种抗生素对 IV型胶原酶或能够分泌 IV型胶原酶的 PG细胞的作用。结果表明 ,geldanamycin和博安霉素均能竞争性地抑制 3D6与 IV型胶原酶或 PG细胞的结合 ,说明其作用于 IV型胶原酶并可能对其活性产生抑制作用 ;而米诺环素在实验浓度下仅对 3D6和 PG细胞的结合表现出抑制作用。另外 ,我们发现地红霉素亦能够抑制单抗 3D6与 IV型胶原酶或 PG细胞的结合。结果表明 ,这四种抗生素可能作用于基质金属蛋白酶。

格尔德霉素生物合成的调控基因

从吸水链霉菌17997中克隆了格尔德霉素(Geldanamycin,Gdm)生物合成酶基因簇,通过生物信息学分析发现两个LAL(Large ATP-bindingregulators of the LuxR family)家族的调控基因gdmRI和gdmRII,基因阻断和基因回复实验证实这两个基因产物都正调控Gdm的生物合成。

格尔德霉素滴眼液在离体兔眼角膜的吸收与扩散

目的制备格尔德霉素 ( geldanamycin)滴眼液 ,进行离体兔眼角膜实验 ,检测角膜及人工房水中药物量。方法采用二室渗透池法 ,在不同时间点取人工房水及角膜 ,分别测定药物在角膜吸收量及角膜中药物扩散进入人工房水量 ,对累积数据进行方程模拟。结果药物易被角膜吸收 ,但不易渗透进入房水 ,药物在角膜中不同时间的吸收累积曲线符合Higuchi方程 ,吸收速率常数为10 2 1 9ng/h。角膜中的药物扩散进入人工房水符合零级方程 ,药物在角膜和人工房水中达到平衡的时间分别为 2h和 4h ,药物从角膜扩散进入人工房水有 2h时滞。结论格尔德霉素滴眼液易被兔眼角膜吸收 ,且在角膜中达到较高浓度 。

HSP90在乳腺癌细胞株ZR-75-30中的表达及对粘附侵袭能力的影响

目的:研究热休克蛋白(HeatShockProtein,HSP)90在乳腺癌细胞株ZR-75-30中的表达及对该细胞粘附侵袭能力的影响,并探讨相关机制及意义。方法:体外培养ZR-75-30乳腺癌细胞株,以HSP90抑制剂Geldanamycin(GA)作用处理;免疫印迹技术(Western-blot)检测GA处理前后HSP90在乳腺癌细胞株ZR-75-30中的表达情况;噻唑兰比色(MTT)法检测GA对ZR-75-30细胞的增殖抑制作用;噻唑兰比色(MTT)法检测细胞对人工基底膜(Matrigel)的粘附能力变化;TranswellCham-ber法观察癌细胞侵袭、迁移能力的改变。结果:HSP90抑制剂GA对ZR-75-30细胞具有增殖抑制作用,呈时间剂量依赖关系;GA作用后HSP90在乳腺癌细胞株ZR-75-30中表达明显下降;HSP90抑制剂GA处理的乳腺癌细胞ZR-75-30的粘附率较未加GA的对照组下降明显(P<0.01);在GA未处理对照组侵袭、迁移实验穿膜细胞数分别为95.4±5.2,103±15.4与GA处理实验组穿膜细胞数46.2±4.9,52.4±8.4相比具有显著性差异(P<0.01)。结论:通过抑制HSP90在乳腺癌细胞株ZR-75-30中的表达,GA能明显减弱乳腺癌细胞株ZR-75-30增殖粘附侵袭能力,HSP90与乳腺癌细胞增殖侵袭转移能力相关。

固相萃取-HPLC法研究格尔德霉素在Beagle犬药代动力学

目的:建立格尔德霉素在 Beagle 犬血浆中的同相萃取(SPE)-HPLC 测定方法,并研究其在犬体内的药代动力学。方法:18只 Beagle 犬分为0.25,0.5,1.0 mg·kg^(-1)低、中、高3个剂量组,单次静脉注射格尔德霉素,于给药后不同时间点采集血样。以 N-苯基-1-萘胺作为内标,Oasis HLB 同相萃取柱优化提取富集,HPLC 法测定其浓度;用药代动力学计算程序3P97拟合药物浓度-时间曲线,求算药动学参数。结果:格尔德霉素在0.01-1.0μg·mL^(-1)线性范围内,低、中、高3种不同浓度质控样品(0.01,0.1,1.0μg·mL^(-1))日内 RSD 分别为5.6%,4.6%,1.9%(n=5);日间 RSD 分别为6.9%,3.2%,6.7%(n=5)。绝对回收率分别为85.0%±3.0%,91.1%±2.2%,94.0%±0.7%(n=5);低、中、高剂量组的 T_(1/2β)分别为89.2,116.6,101.6 min;AUC 分别为6.9,12.7,24.9 min·μg·mL^(-1);Vc 分别为858.0,964.4,981.3 mL·kg^(-1);CL 分别为35.9,39.4,40.1mL·min^(-1)·kg^(-1)。结论:固相萃取-HPLC 法测定格尔德霉素准确,快速,回收率高。格尔德霉素在 Beagle 犬体内药代动力学符合二室静脉注射模型。这对以后的临床应用指导具有十分重要的意义。

格尔德霉素生物合成基因功能的验证

格尔德霉素(Geldanamycin,Gdm)作为热休克蛋白90的特异性抑制剂,是非常有前景的抗肿瘤和抗病毒的药物,我们已从吸水链霉菌17997(Streptomyces hygroscopicus17997)的基因文库中获得了Gdm大部分生物合成基因。为了研究主要基因的功能,选择了聚酮合酶基因(Polyketide synthase gene,pks)的第六模块、单加氧酶基因(Mono-oxygenase gene,gdmM)和氨甲酰基转移酶基因(Carbamoyltransferase gene,gdmN)3个基因作为靶点分别进行基因阻断,获得了基因同源双交换的阻断变株△pks、△gdmM和△gdmN。经HPLC检测证实这些基因的阻断变株均不产生Gdm,基因回复实验排除了基因阻断所可能造成的极性效应对其它基因表达的影响,说明所克隆的pks、gdmM和gdmN基因确实是Gdm生物合成所必需的基因。