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四种抗癌物质对微管蛋白体外聚合及热变性的影响

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维普资讯 http://www.cqvip.com 物理化学学报(Wuli Huaxue Xuebao) Novermber Acta Phys.一Chim.Sin.,2002,18(11):1009~1013 Studies of the Effects of Anti-tumor Compounds on the Assembly and Melting DenaturatiOn of Tubulin in vitro Zhou Xiao.Lei Beijing, 100083 J Wang B ao-Huai Li Zhi・-Fen Zeng Hui・-Hui (Institute ofPhysical Chemistry Peking University,Beijing lOO871 School ofPharmaceutical Science,Peking Universiyt Abstract The effects of four kinds of anti—tumor compounds,taxol,carboplatin,ZYZ(the extractive from gleditsi— a),F43(the extractive from squamocin),on the assembly and melting denaturation of tubulin in vitro were studied by urbiditty and differential scanning calorimetry.The experimental results show that taxol and ZYZ enhance the assembly of tubulin,while carboplatin and F43 have no effect on the assembly of ubultn when tihe concentrations of lla four kinds of compounds are 15 I ̄mol。L一1.At the same ime,ttaxol and ZYZ can make the melting points oftubuin risewhile the ,meling pointts of tubuin don’t change in the presence of carboplatin and F43The different interaction mechanisms of .the compounds and tubulin have been discussed according to the above experimental results. Keywords Tubulin,Taxol, Carboplatin, ZYZ, F43,Turbidity,DSC(differential scanning calorimetry) 1 Introduction Microtubules are an important component of the assembly of brain tubulin fn vitro.while there are vari— eties ofresults obtained by different research groups . he experTimental results have indicated that some an- cytoskeleton of most eukaryotic cells.The temporal and spatial control of assembly and disassembly of mi- ti-tumor drugs(e.g.cisplatin)could interact not only with DNA but also with actin and microtubule in the cel1 cytoplasmt8-12】. crotubules is regarded as a key factor in cellar func— tion.Microtubules and their subunits( 一 tubulin het- erodimers,MW l 10,000)maintain a dynamic equi!ib— rium in the cell cytoplasm.In addition,it is postulated hat the labiftity of cytoplasmic microtubules caused by drugs is related to their equilibrium.If he equilitbrium in the cell cytoplasm is disturbed by some drugs,poi— sons or some metal ions entering into the cell,it will cause some serious and unnegligable results.Therefore, investigation of the effects of some anti--umor com-t- pounds on the assembly of tubulin in vitro plays very Taxol,an anti—tumor drug,enhances microtubule assembly and stabilizes microtubules fn vitro[ 。 . Carboplatin is also an anti—tumor dmg,but its effect on he assembly ofttubulin is not clear.ZYZ and F43 have been proved to be anti--umor compounds experimen-t- tally.Their effects on tubulin have not been studied.In this paper,turbidity and differential scanning calorime— try(DSC)were used to explore the effects of the above four agents on the assembly of ubulin itn vitro in order to understand the interaction mechanism between the compounds and tubulin. important role in the generation of cell shape and un- derlying a cell motile function.Calorimetry and ther— ma1 analysis methods have been used to observe the Received:March 28,2002;Revised:June 20,2002. po ̄ed by NSFC(20133030) 2 Experimental Correspondent:Wang Bao—Huai(E—mail:bhwang@pku.edu.ca).‘The Project Sup- 维普资讯 http://www.cqvip.com 1010 Acta P s.一 Sin.(Wuli Huaxue Xuebao),2002 VO1.18 2.1 Materials and melting denaturation of tubulin was fo1lowed by a Taxol was provided by Dr.Qinhong Cao,College Micro DSC llI differentia1 scanning calorimeter of Biological Science,Chinese Agricultural Universi— ty.and dissolved in a dilute DMSO aqueous solu. tion.Carboplatin was from Dezhou Pharmaceutical Factory.Shandong Province.It was dissolved in water nad kept at 4'12 before the experiments.ZYZ and F43 were provided by School of Pharmaceutical Science. Peking University.ZYZ is soluble in water and its molecular weight is 1 000—2 000.The molecular weight for calculating of ZYZ is 1 100.The aqueous solution which contained 2 mmol・L一 of ZYZ was kept at一20℃.F43 is insoluble in water.The solvents ofr F43 are acetone。chloroform,DMSO and so on. In the experiments,F43 was made up 2 mmol・L一 DMSO solution.The molecular weight of F43 is 600~ 700 and its molecular weight for calculating is taken as 650.GTP and ATP(≥85%)were from Mingzhu Dongfeng BiOtechnO1Ogica1 Company,Shanghai.It was kept一20℃in the presence of some drying agents. Proteins of porcine brain microtubule were isolat。 ed and purified by two cycles of assembly and de. assembly with a modified methOd Of Williams[13】.Tl1e concentration of tubulin was determined by the method of Bradford【 .with BSA as a standard.The pure tubu. 1in was diluted to 2.5 g・L。 bv 0.1 mol・L一 PEM buffer(0.1 mo1・L_。PIPES.1 Inn'lo1・L EGTA. 0.5 mmol‘L~MgCh,pH=6.9).All the experi— ments were conducted immediately after purification. nad finished within two days. 2.2 Apparatus and Procedures Microtubule assembly was monitored by the tur. bidity assay of Gaskin[ .Measurements were carried out every 2 s at 350 mn on an UV.3100 UV.VIS.NIR Recording Spectrophotometer(S}{IMADZU,Japan) equipped with a TCL一260 temperature controlled cell holder.Sample of tubulin was kept in ice—bottle.Agent was added into tubulin,mixed.and moved into a cu. veae(2 mm light path) The cuveue was put in the holder at 37℃.and the absorbance of tubulin was recorded immediately. hTe COUrSe of the temperature—induced assembly (Setraam,France).0.4 mL solution of tubulin(2.5 g。L )was added into hte sample cell,while the same volume of 0.1 mol・L PEM buffer was added into hte reference cel1.After a 30 min waiting for thel'inal balance in the calorimeter at 0℃.the temperature was ramped up to 100℃at aheating rate of 1℃・min。1. The effects of agents were observed with the same pro— cedure except that agent was added into the tubulin sample cell before measurement. 3 Results and Discussion The turbidity.time Curves of tubulin assembly are shown in Fig.1.It can be seen that the time depen— dence of absorbance of tubulin takes on a shape of S.The platforms in curves at the beginning was caused by no assembling of tubulin at low temperatures as it was iust tkaen out from the ice bottle.Then the ab. sorbance of tubulin increases with assembly degree rapidly and reaches a constant value in several min. utes.The height of assembly platform indicates the as— sembly degree of tubulin and the slope of turbidity curve shows the speed of utbulin assembly. From Fig.1,the absorbance oftubulin in the pres— ence of taxol is higher than that in the absence of it.The experimental results show that taxol can lead to i 昌 t/s Fig.1 Turbidity curves of tubulin(Tb)assembly at 37℃and the effects of four kinds of anti・tumor compounds(15 p,mol・L ) a)Tb,b)Tb+F43,c)Tb+carboplatin,d)Tb+ZYZ. e)1’b+Taxol 维普资讯 http://www.cqvip.com No.1 1 Wang Bao.Huai et a1.:Effects of Anti.tumor Compounds on the Characteristics of Tubulin 10 1 1 tubulin assemble at 1ow temperatures.Almost no plat. form appears in the turbidity—,ime curve at tthe begin—, ning.Then the absorbance of tubulin increases contin. uously and tends toward stability at the end. Heatflow/mW For other compounds,ZYZ also enhances the as. sembly of tubulin but the enhancing extent is lower han tthat oftaxo1.Carboplatin and F43 show no effect on tubulin assembly under the experimental concentra. tion of compounds. he absorbaTnce oftubulin at the 600 th second af- ter starting the experiments in the presence and in the absence of the compounds are listed in Table 1.Here he absorbance of ttubulin in absence of the compounds is taken as one hundred per cent.From Table 1,the in. teraction extent between the four kinds of anti.tumor compounds and tubulin can be seen clearly. Table 1 The effects of four kinds of anti.tumor Fig.2 20 30 40 50 7'/℃ 60 70 80 DSC curves of he assembly and melting tdenaturation of tubulin and the eftects of four kinds of anti-tumor compounds compounds on the turbidity of tubulin(Tb) assembly at 37℃ at(27.3±0.1)℃and A Hm is about一(1.2±0.2) ×10。J・mo1—1.This is contributed to the assembly of ubultin.The second peak appears at(58.8±0.5)℃ nd A Hm ias about f3.6±0.3)×10。J・mo1’1.This is contributed to the melting denaturation of tubu. 1in.The third peak appears at around 80℃and is The DSC curves of assembly and melting denatu. ration of tubuHn with and without the compounds from 5℃to 95℃are shown in Fig.2.the temperatures and he entthalpy changes of the assembly and melting de— naturation of tubuHn are 1isted in Table 2.The curves — exothermic.This is contributed to the aggregation of protein after melting denaturation of tubulin.The ex. perimental results show that carboplatin and F43 have no effect on the assembly and melting denaturation of ubulin at tthe concen ̄ation of 15 Ixmo1・L一1. From Fig.2,in the presence of taxol,the peak of ubulin assembly disappeatrs.It suggests that tubulin assembly has completed at low temperatures.The in the presence and in the absence of carboplatin and F43 are very similar to the curve of assembly and melting denaturation of tubulin.The first peak appears Table 2 The onset and top temperatures(To and Tf)of melting peaks and he tmelting enthalpies(A 日) oftubulininthepresenceandinthe absenceofthefourkinds ofanti.tumor compounds 维普资讯 http://www.cqvip.com l0l2 Acta尸hys.-Chin ̄Sin.(Wuli Huaxue Xuebao).2002 VO1.18 melting point of tubulin raises 2.5℃and peak widens evidently,and isn’t a single peak.It indicates that as- semblized tubulin is more stable in the presence of tax. O1 than that without taxo1.In the presence of ZYZ.the peak of tubulin assembly disappears,and the melting peak splits into three smaller peaks.The melting point of tubulin raises 4.4℃according to the first small splitten peak.It shows that the results from interaction between the compound and tubulin causes the regional co.ordinations in tubulin molecular change【 .It can be concluded that ZYZ has also strong effect on the as. sembly and melting of tubulin at the experimental con- centration.The results from DSC tell us that both taxol nad ZYZ can change the structure of tubulin and cause hte variations in temperature and peak shape of assem- bly and melting denaturation of utbulin. hTe results of turbidity and DSC experiments, suggest that there is no interaction between carboplatin or F43 with tubulin at the concentration of 15 mO1・ L。 .Taxol and ZYZ have stronger effects on the as. sembly and melting denaturation of tubulin.They can enhance the assembly of tubulin and make the assem. blied tubulin more stable.There is no conclusion about the effects of the four kinds of anti.tumor compounds on the assembly and melting denaturation of tubulin at higher concentration.Further research is in progress. Similar to DNA.tubulin is also the target molecule of some anti.tumor compounds.In the recent years,the studies on anti.tumor small molecular com. pounds targetting tubulin play an important role in molecular design,synthesis and screen of anti.tumor drugs.Thermodynamic investigations have indicated that there are three interaction situations between an. ti-tumor drugs and tubulin,i.e.no interaction(such as carboplatin),enhancing the assembly of tubulin(such as taxo1)and stabilizing it[5-6]and idsturbing hte stable equilibrium of hte assembly and disassembly of tubifin (such as cisplatin).An ideal anti.tumor drug should 11 some cancer cells effectively and shouldn’t hurt hte normal cells in human body.From the thermody. namic point of view,an ideal anti-tumor drug should enhance the assembly of tubulin and stabilize it or doesn t react with tubulin evidently,but shouldn t dis. turb the dynamic equilibrium of the assembly and dis. assembly of tubilin and further make the normal cells in human body be hutted.Thus it can be seen from the experimental results of this paper that both ZYZ and F43.especially ZYZ.should be explored further as ideal anti-tumor drugs.At present.ZYZ and F43 are being further purified and their molecular structures are being determined. Acknowledgments We are grateful to Dr.Qihnong Cao,College of Biological Science,Chinese Agricul- utral University,for providing pure taxol and helping in isolating and purifying brain microtubules. References 1 Sutherland J W H.Sturtevant J M.Proc.Nat1.Acad.Sci 1976,73:3565 2 Hinz H J,GorbunoffM J,Price B.Timasheff S N.Biochem., 1979,18:3084 3 Berkowitz S A.Velicelebi G.Sutherland J W H.Sturtevant J M Proc.Nat1.Acad.Sci.c,趴.1980.77:4425 4 KlumpH,HardR.Thermochira.Acta,1983,65:93 5 HanG S,YenLF,WangBH,ZhangYM.ActaP s.-Chira. Sin.,1997,13:969 【韩公社,阎隆飞,王保怀,张有民.物理 化学学报(m‘ HuaxueXuebao),1997,13:969】 6 rangY,WangBH,LiZF,ZhangYM,HanG S,YenLF Acta Phys.-Chim.Sin.,1999,15:182 【杨勇,王保怀,李芝 芬,张有民,韩公社,阎隆飞.物理化学学报( ‘ffHuaxueXue. bao),1999。15:182】 7 ZhouXL,rangY,WangBH,LiZF,LiuAX,YenLF. Chinese Chemical Letter,2000.1l:243 8 Pe ̄ot V,Briand C,Crevat A,Braguer D,Chauvet—Monges A M, Sad J C.Cancer Treat.Rep.,1983,67(7-8):641 9 Pavlenko V K,Tulub A A.Dok1.Akad.Nauk sssR.1991.319: 498 10 ZengHH,WangBH,ZhangYM,WangK.Thermochim.Acta, 1995,265:3l ll ZengHH,WangK,WangBH,ZhangYM.Biological Macromolecu s.1996。18:16l l2 Zeng H H,Wang B H,Wang K.Thermochim.Acta,2001,373:l 13 Williams R C,Jr Lee J C.Me ̄ods in Enzymology,New York: Academic Press。1982.85(Part B J:376 14 Bradford M M.Ana1.Biochem.,1976。72:248 15 Gaskin F,Cantor C R,Shelanski M L. Mo1.Bio1.,1974。89: 737 16 Stwiver J W,Kama ̄U.Biochemistry,1990。29:2556 维普资讯 http://www.cqvip.com No.11 Wang Bao—Huai et a1.:Effects of Anti—tumor Compounds on the Characteristics of Tubulin 1013 四种抗癌物质对微管蛋白体外聚合及热变性的影响 周晓蕾 王保怀 李芝芬 曾慧慧 (北京大学物理化学研究所,北京 100871;北京大学药学院,北京 100083) 摘要用浊度法和差示扫描量热法研究了四种小分子抗癌物质[紫杉醇、卡铂、猪芽藻提取物(ZYZ)和番荔 枝素提取物(F43)】对微管蛋白体外聚合及热变性的影响。实验结果表明:当四种化合物的浓度均为l5 Ixmol・L 时,紫杉醇和猪芽藻提取物(ZYZ)能促进微管蛋白的聚合并使其热变性温度升高,而卡铂和番荔 枝素提取物(F43)却对微管蛋白的聚合及热变性温度无明显影响。综合上述实验结果,对四种抗癌物质与微 管蛋白相互作用的机理进行了讨论。 关键词: 微管, 紫杉醇, 卡铂, 猪芽藻提取物(ZYZ), 番荔枝素提取物(F43), 浊度, 差示扫描量热法(DSC) 中图分类号:0642 2002—03—28收到初稿,2002—06—20收到修改稿.联系人:王保怀(E.mail:bhwang@pku.edu.cn). 项目 ‘国家自然科学基金(20133030)资助 

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