土贝母皂苷对人低分化上皮样鼻咽癌细胞(CNE-2Z)微管的作用

目的：探讨土贝母皂苷(皂苷)对人低分化上皮样鼻咽癌细胞株(CNE-2Z)微管的影响：方法：采用细胞免疫荧光法观察皂苷处理的CNE-2Z细胞微管的变化，蛋白免疫印迹法分析皂苷处理的CNE-2Z细胞中聚合和未聚合微管蛋白含量的变化。结果：免疫荧光图像结果表明，皂苷作用的CNE-2Z细胞丧失正常的微管网络，蛋白免疫印迹结果证实皂苷使CNE-2Z细胞未聚合的微管蛋白比例增加，而且这一作用与剂量和时间呈正相关。结论：皂苷能抑制CNE-2Z细胞内微管的聚合，提示皂苷是一种有效的抗微管剂、     

山茱萸拮抗线粒体缺陷致神经细胞微管及微管相关蛋白表达异常

目的观察中药山茱萸水提液对线粒体呼吸链复合体IV(即细胞色素C氧化酶)抑制剂叠氮钠(NaN3)致神经细胞骨架系统损伤的拮抗作用,探讨山茱萸在防治阿尔采末病中的机制。方法山茱萸水提液(0.125～8g生药·L-1)分别与SH-SY5Y人神经母细胞瘤细胞及原代培养新生大鼠海马神经元共孵育24～48h,加入64mmol·L-1叠氮钠作用4～6h,MTT、LDH法测定细胞存活率,激光共聚焦显微镜观察细胞微管结构及微管相关蛋白表达的变化。结果1～4g·L-1山茱萸能明显拮抗叠氮钠致神经细胞存活率下降;使损伤的微管结构基本恢复正常,微管相关蛋白表达增加,尤以突起内最为明显。结论中药山茱萸能明显拮抗线粒体缺陷致神经细胞损伤,其机制与保护细胞骨架系统有关,对于防治阿尔采末病等神经退行性疾病有一定应用前景。     

文蛤多肽抑制肿瘤细胞微管蛋白聚合

目的探讨文蛤多肽(Mere15)抑制人肺癌A549细胞增殖作用及其机制。方法 MTT比色法检测细胞增殖抑制率;流式细胞仪分析细胞周期分布的变化;微管蛋白免疫荧光检测技术检测Mere15对微管蛋白聚合的影响;Western blot检测Mere15对p21蛋白表达的影响。结果 Mere15可抑制A549细胞生长,抑制率呈剂量和时间依赖性;随着处理时间的增加,A549细胞G2/M期比例逐渐升高,微管蛋白聚合受到抑制,p21蛋白表达水平逐渐升高。结论 Mere15具有抑制A549细胞增殖的作用,其机制可能与抑制微管蛋白聚合有关。     

具有抗肿瘤活性的嘧啶类化合物研究进展

嘧啶类化合物因其广泛的生物活性在医药、农药等领域备受关注,特别是近年来大量具有抗肿瘤活性的嘧啶类化合物被报道,其中一些化合物已进入临床试验阶段。虽然这些化合物都含有嘧啶结构,但它们的作用机制却表现出多样性。如抑制细胞周期蛋白依赖性激酶、酪氨酸蛋白激酶、碳酸酐酶、二氢叶酸还原酶和干扰微管蛋白聚合等。本文将根据嘧啶类化合物的作用靶点,介绍其在抗肿瘤药物研究中的最新进展。     

去甲斑蝥素对体外微管蛋白聚合的抑制作用

目的探讨去甲斑蝥素(NCTD)对微管蛋白聚合-解聚过程的影响。方法采用蛋白凝胶电泳鉴定从猪脑中提取的微管蛋白,并用Bradford法测定微管蛋白含量。在经过秋水仙碱和紫杉醇验证稳定的37℃和4℃平衡体系内加入NCTD 10,20和30μmol.L-1观察微管蛋白聚合-解聚活性。人卵巢癌SK-OV-3细胞加入NCTD 60μmol.L-1作用8 h,取上清和沉淀,Western印迹法检测微管蛋白含量。结果与空白对照组相比,在37℃聚合反应中,NCTD 10,20和30μmol.L-1组对微管蛋白聚合的抑制率分别为(5.5±2.7)%,(7.1±1.2)%和(27.5±0.4)%,在此浓度范围内NCTD随浓度的升高抑制作用增强(P<0.05),但对微管蛋白在4℃的解聚影响不显著。NCTD 60μmol.L-1作用人卵巢癌细胞后,细胞中游离微管蛋白增加了(91.5±8.5)%,聚合微管蛋白量减少了(51.8±3.8)%,说明NCTD抑制了人卵巢癌细胞SK-OV-3中微管蛋白的聚合。结论 NCTD对体外微管蛋白的聚合具有一定的抑制作用。     

丹酚酸C诱导肝癌HepG2细胞有丝分裂阻滞及凋亡的研究

目的研究丹酚酸C(salvianolic acid C,SalC)的体外抗肿瘤细胞增殖作用及其分子机制。方法 MTT法检测Sal C对人肿瘤细胞的增殖抑制作用,流式细胞术检测细胞周期分布及细胞凋亡,Giemsa染色进行细胞有丝分裂的形态学观察,微管蛋白聚合实验检测Sal C对微管蛋白聚合活性的影响,比色法检测细胞中Caspase-3和Caspase-6的活性。结果丹酚酸C能抑制多种人肿瘤细胞增殖,其中对HepG2细胞的抑制作用较明显。流式细胞术分析发现Sal C使HepG2细胞阻滞于G2/M期并随作用时间延长出现明显的凋亡峰,细胞中Caspase-3和Caspase-6的活性升高。Gi-emsa染色提示HepG2细胞发生有丝分裂阻滞。Sal C能明显抑制微管蛋白的聚合。结论 Sal C具有抗肿瘤细胞增殖活性,通过抑制微管蛋白聚合诱导细胞有丝分裂阻滞从而诱导凋亡。     

文蛤体液蛋白体内外抗肿瘤作用的研究

[摘要]目的：由文蛤体液中获得一种新的抗肿瘤蛋白。方法：采用电镜技术、流式细胞仪技术、微管蛋白α-tubulin的荧光染色技术、电泳技术,研究了MML作用后细胞凋亡、细胞周期及微管蛋白的变化情况;建立裸鼠肝癌皮下移植瘤模型,观察MML的体内抗肿瘤活性。结果：发现MML可显著抑制肿瘤细胞生长, IC50达到52.2μg&#8226;mL-1。作用机制的研究表明MML可以增加肿瘤细胞膜通透性、抑制微管蛋白聚合、使肿瘤细胞停滞在G2/M期;MML对人肝癌BEL-7402移植性肿瘤小鼠具有显著的抗肿瘤作用。结论：文蛤体液蛋白是一种新型的抗肿瘤蛋白,主要通过增加细胞膜的通透性、引起细胞凋亡、抑制微管蛋白聚合产生抗肿瘤作用。     

半边旗提取物5F体外对猪脑微管蛋白聚合的影响

目的　研究半边旗提取物5F对微管蛋白聚合的影响。方法　在离体条件下,将受试化合物加入到微管蛋白聚合解聚反应体系中,于37℃、350nm下检测反应体系吸光值的变化。结果　5F能抑制反应体系吸光值升高,提示它对微管蛋白聚合具有抑制作用并能抑制其解聚。结论　5F能抑制体外微管蛋白聚合,从而可能是影响细胞的细胞周期及诱导凋亡途径之一。     

人参皂苷对蛋白磷酸酶抑制剂冈田酸拟阿尔采末病细胞模型的影响

目的探讨人参皂苷(ginsenoside,GS)对蛋白磷酸酶抑制剂冈田酸(OA)诱导拟Alzheimer病(AD)细胞模型神经细胞tau蛋白的磷酸化、微管、细胞凋亡和凋亡调节因子的影响。方法GS与人神经母细胞瘤细胞系SK-N-SH细胞预孵育24h,弃去培养基,然后用OA10nmol·L-1与SK-N-SH细胞共孵育6h;用倒置显微镜观察细胞形态的变化,激光共聚焦显微镜观察微管变化,Western blot方法观察磷酸化tau蛋白、凋亡因子Bcl-2、Bax和Caspase-3的表达,用TUNEL法观察凋亡细胞的变化。结果正常SK-N-SH神经细胞铺展良好,OA模型组细胞突起断裂,GS显示了细胞保护作用。通过激光共聚焦显微镜观察,正常SK-N-SH细胞微管粗壮、连续,而OA模型组微管断裂、消失;GS能够减少OA引起的微管破坏作用。OA模型组tau蛋白Ser-199/202和Ser-404位点磷酸化水平较正常对照组明显增高,非磷酸化水平较正常对照组明显下降;GS 50mg·L-1和100mg·L-1组使神经细胞tau蛋白Ser-199/202和Ser-404位点磷酸化水平较OA模型组明显下降,GS 50mg·L-1和100mg·L-1组的tau蛋白Ser202非磷酸化水平较模型组则明显升高;正常对照组未见凋亡细胞;OA模型组凋亡细胞明显增多,Bax和Caspase-3表达水平较正常对照组明显增高,Bcl-2水平明显下降;GS能够明显抑制OA诱导的细胞凋亡,减少Bax和Caspase-3表达。结论人参皂苷对蛋白磷酸酶抑制剂OA所致的神经细胞病理变化有明显的保护作用,可能是通过抑制tau蛋白过度磷酸化,防止细胞凋亡来发挥作用的,提示该药在防治AD方面可能具有良好的应用前景。     

新型磷酸二酯酶抑制剂唑嘧啶

<正> 唑嘧啶(又名乐可安,Trapidil,Rocornal),化学命名7-2乙氨基-5-甲基-5-三唑(1.5—a)嘧啶(5-methyl-7—diethylamino-5 triazolo-[1.5-a]phrimidine)结构如下: 一、药理作用:唑嘧啶为一种新的磷酸二酯酶抑制剂,能够产生剂量依赖性的扩张冠状动脉及外周血管作用,使麦角碱、k~+、PGF_2α、新福林、5-HT引起的冠状动脉收缩作用反转。它并能抑制血小板的聚集作用,同时可抑制血栓素A_2的合成,还能对抗血小板生长的诱导因子及促进前列腺素的合成。可以肯定唑嘧啶具有扩张支气管平滑肌的作用。其机制可能是唑嘧啶竞争性抑制     

Comparison of the effects of colchicine and beta-lumicolchicine on cultured adrenal chromaffin cells: lack of evidence for an action of colchicine on receptor-associated microtubules

The involvement of microtubules in adrenomedullary secretion is presently unclear. Evidence exists for a possible role of microtubules in cholinergic nicotinic receptor-related events. We now describe the actions of the microtubule disrupter, colchicine, on primary cultures of bovine adrenal chromaffin cells and compare these with corresponding actions of beta-lumicolchicine. beta-Lumicolchicine is a structural isomer of colchicine which neither binds microtubular protein (tubulin) nor interferes with microtubule assembly. Both colchicine and beta-lumicolchicine were found to inhibit acetylcholine-induced secretion with similar potencies (half maximal inhibitory concentration 0.2-0.5 mM). The inhibitory actions of both drugs are time-dependent and reversible. However, unlike colchicine which has no inhibitory effects on secretion evoked by depolarization with excess K+, beta-lumicolchicine also inhibits K+-induced secretion. Because colchicine and beta-lumicolchicine have similar effects, the selective inhibitory actions of colchicine on nicotinic receptor-mediated secretion cannot in itself be used as evidence in support of a role of microtubules in receptor-mediated events. However, our data do not preclude such a role. Differences in the effect of colchicine and beta-lumicolchicine on K+-evoked secretion suggests different modes of action of these structural isomers on chromaffin cell function.     

The role of GTP Binding and microtubule-associated proteins in the inhibition of microtubule assembly by carbendazim.

The fungicide carbendazim (CBZ) is known to disrupt microtubular structures in the testis and to cause testicular toxicity in rats. To investigate the mechanism underlying the toxicity of CBZ, tubulin and microtubule-associated proteins (MAPs) were isolated from rat testis and brain using two techniques. The effects of CBZ on MT assembly were compared with the known microtubule (MT) disruptors, colchicine and nocodazole. CBZ (100 microM) had no effect on the assembly of MTs from MAP-containing tubulin isolated with one cycle of glycerol-dependent assembly and disassembly while colchicine (40 microM) and nocodazole (12.5 microM) strongly inhibited the assembly reaction. Similarly, formation of MTs from tubulin prepared with two cycles of glycerol-dependent assembly was strongly inhibited by colchicine and nocodazole but only weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP-free tubulin isolated with glutamate. However, the inhibition by CBZ was reversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled MTs caused immediate depolymerization, whereas CBZ did not directly cause depolymerization. However CBZ was an effective inhibitor of the polymerization of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data suggest that CBZ interferes with initial events of MT polymerization, specifically GTP binding, and that MAPs moderate this effect.     

Synthesis, cytotoxic properties and tubulin polymerization inhibitory activity of novel 2-pyrazoline derivatives

A series of novel 1-(3',4',5'-trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives were synthesized and evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization inhibitory activity. Compounds 6d and 6e exhibited remarkable cytotoxic activity against different cancer cell lines with good tubulin polymerization inhibitory activity. Compound 6d exhibited moderate selectivity toward renal cancer and breast cancer subpanels with selectivity ratios of 3.06 and 5.11, respectively, at the cytostatic activity (TGI) level. Compounds 6e and 6d achieved good tubulin polymerization inhibitory activity with IC(50) values of 17 and 40 μM, respectively. The photomicrographs made for compounds 6d and 6e on cellular microtubules indicated that the cytotoxicity of these compounds can be attributed to their ability to interfere with microtubule assembly. Molecular modeling studies involving compound 6e with the colchicine binding site of α,β-tubulin revealed hydrogen-bonding and hydrophobic interactions with several amino acids in the colchicine binding site of β-tubulin.     

Design and biological evaluation of novel tubulin inhibitors as antimitotic agents using a pharmacophore binding model with tubulin

Although the structure has been elucidated for the binding of colchicine and podophyllotoxin as potent destabilizer for microtubule formation, very little is known about MDL-27048, a competitive inhibitor for colchicine and podophyllotoxin. The structural basis for the interaction of antimitotic agents with tubulin was investigated by molecular modeling, and we propose binding models for MDL-27048 against tubulin. The proposed model was not only consistent with previous competition experiment data between colchicine and MDL-27048, but further suggested an additional binding cavity on tubulin. Based on this finding from the proposed MDL-tubulin complex, we performed molecular design studies to identify new antimitotic agents. These new chalcone derivatives exerted growth inhibitory effects on all four human hepatoma and one renal epithelial cell lines tested and induced strong cell cycle arrest at G2/M phase. Furthermore, these compounds exhibited a strong inhibitory effect on tubulin polymerization in vitro. Therefore, we suggest that the validated MDL-27048 model would serve as a potent platform for designing new molecular entities for anticancer agents targeted to microtubules.     

Effect of diethylstilbestrol on the polymerization and alkylation of tubulin

Diethylstilbestrol is a drug used to treat prostate cancer. It is thought to bind to tubulin, the subunit protein of microtubules, at the colchicine‐binding site. We examined its interaction with tubulin in more detail. Diethylstilbestrol inhibits microtubule assembly, and seems to do so more effectively when tubulin polymerization is catalyzed by MAP2 rather than tau. Diethylstilbestrol also inhibits the intrachain cross‐linking of tubulin by N,N′‐ethylenebis‐(iodoacetamide) in a pattern similar to that shown by colchicine and the drugs which bind to tubulin at the colchicine‐binding site. Unlike most of this category of drugs, however, diethylstilbestrol accelerates, rather than inhibits, the decay of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas. It appears, therefore, that diethylstilbestrol interacts with tubulin in a manner similar to that of the analogs of the A‐ring of colchicine, whose effect on tubulin cross‐linking is similar to that of diethylstilbestrol and which also enhance tubulin decay. Drug Dev. Res. 48:104–112, 1999. © 1999 Wiley‐Liss, Inc.     

HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin

10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone (HMBA), a synthetic compound, has been reported to have a potent antitumor activity. In this study, we found that HMBA depolymerized microtubules in MCF-7 cells and produced aberrant spindles in the MCF-7 cells. It also reduced the distance between the centrosomes and activated the mitotic checkpoint proteins BubR1 and Mad2. Further, HMBA inhibited the progression of MCF-7 cells in mitosis and induced apoptotic cell death involving p53 pathway. In vitro, HMBA bound to purified brain tubulin with a dissociation constant of 4.1 ± 0.9 μM. It inhibited microtubule assembly and increased the GTP hydrolysis rate of microtubule assembly. The compound did not alter the binding of 2′(or 3′)-O-(trinitrophenyl) guanosine 5′-triphosphate (TNP-GTP), a fluorescent analogue of GTP, to tubulin suggesting that it did not inhibit the binding of GTP to tubulin. However, we obtained evidence indicating that HMBA perturbed the conformation of the GTP binding site in tubulin. In addition, an analysis of the modified Dixon plot suggested that HMBA competitively inhibited the binding of colchicine to tubulin. A computational analysis of the binding of HMBA to tubulin supported the finding that HMBA shared its binding site with colchicine in tubulin and indicated that the binding of HMBA to tubulin was primarily stabilized through hydrogen bonding.     

Induction of apoptosis through tubulin inhibition in human cancer cells by new chromene-based chalcones

Context: As microtubules are highly involved in cellular growth, it appears to be a preferential target for cancer treatment. Therefore, many efforts have been performed to discover drugs that affect on microtubule function. Several microtubule inhibitors are in various stages of laboratory evaluations and clinical trials.

Objective: A series of chromene-based chalcones with chlorine, methoxy, fluorine, tetrahydropyranyloxy and cyanide substituents were prepared and evaluated for cytotoxic effects against K562 and SK-N-MC cell lines, and the inhibitory effect on tubulin polymerization was studied as well.

Materials and methods: MTT, tubulin polymerization assays and binding measurements were evaluated by using related spectroscopy. Immunocytochemical study, morphological observations and apoptosis assay were examined using a fluorescence microscope and a flow cytometer.

Results: (E)-3-(6-Chloro-2H-chromen-3-yl)-1-(3,4,5-trimethoxyphenyl) prop-2-en-1-one (compound 14) proved to be the most active in this series as an inhibitor of tubulin assembly [IC₅₀, 19.6 µM] and cytotoxic agent on K562 cells [IC₅₀, 38.7 µM]. Furthermore, these compounds exhibited a strong inhibitory effect on tubulin polymerization and reduced the in vitro assembly and bundling of proto-filaments. Also, compound 14 bound to the tubulin with a dissociation constant of 9.4 ± 0.7 µM and induced conformational changes in this protein.

Discussion and conclusion: This study suggests that the compound 14 could be a good antitumor agent because of its biological functions. Compound 14 appears to bind directly to tubulin and thereby perturbs microtubule stability and the function of the spindle apparatus, which causes cancer cells to arrest and undergo apoptosis.     

Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-benzoxazepines

We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.     

Cytotoxic activity and cytostatic mechanism of novel 2-arylbenzo[b]furans

The aims of this study were to screen cytotoxic compounds from 14 newly-synthesized 2-arylbenzo[b]furans and explore their mechanisms of action. Cytotoxicity was determined by the MTT method. Cell-cycle distribution was detected by flow cytometry. Wright-Giemsa staining was performed to demonstrate the morphological features of cells in mitotic phase. Polymerization of tubulin was detected by tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical study. Among the 14 compounds screened, 4-formyl-2-(4-hydroxy-3-methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo[b]furan (ERJT-12) showed significant cytotoxicity. Our results demonstrated that ERJT-12 exhibited anti-cancer activity in a variety of tumour cell lines with an IC50 value (concentration resulting in 50% inhibition of cell growth) of 5.75 approximately 17.29 microM. Cell cycle analysis showed a concentration-dependent accumulation of tumour cells in G2/M phase after treatment with ERJT-12. Further investigation indicated that ERJT-12 blocked the cell cycle in M phase, with separation and dispersion of chromosomes. ERJT-12 inhibited tubulin polymerization in-vitro. Changes of the cellular microtubule network caused by ERJT-12 were also detected, which were similar to the changes caused by colchicine. These results suggested that the anti-cancer activity of ERJT-12 is worth further investigation.     

Tubulin-perturbing naphthoquinone spiroketals

Several natural and synthetic naphthoquinone spiroketals are potent inhibitors of the thioredoxin-thioredoxin reductase redox system. Based on the antimitotic and weak antitubulin actions noted for SR-7 ([8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2'-naphtho[1',8'-de][1',3'][dioxin]), a library of related compounds was screened for tubulin-perturbing properties. Two compounds, TH-169 (5'-hydroxy-4'H-spiro[1,3-dioxolane-2,1'-naphthalen]-4'-one) and TH-223 (5'-methoxy-4'H-spiro[1,3-dioxane-2,1'-naphthalen]-4'-one), had substantial effects on tubulin assembly and were antiproliferative at low micromolar concentrations. TH-169 was the most potent at blocking GTP-dependent polymerization of 10 mum tubulin in vitro with a remarkable 50% inhibitory concentration of ca. 400 nm. It had no effect on paclitaxel-induced microtubule assembly and did not cause microtubule hypernucleation. TH-169 failed to compete with colchicine for binding to beta-tubulin. The 50% antiproliferative concentration of TH-169 against human cancer cells was at or slightly below 1 mum. Flow cytometry showed that 1 mum TH-169 caused an increase in G(2)/M and hypodiploid cells. TH-169 eliminated the PC-3 cells' polyploid population and increased their expression of p21(WAF1) and Hsp70 in a concentration-dependent manner. The antiproliferative effect of TH-169 was irreversible and independent of changes in caspases, actin, tubulin, glyceraldehyde phosphate dehydrogenase or Bcl-x(S/L). This structurally simple naphthoquinone spiroketal represents a small molecule, tubulin-interactive agent with a novel apoptotic pathway and attractive biological function.