MONCPT对膀胱癌细胞5637的抗肿瘤作用及其机制研究

【作者】 姜海；

【导师】 陈昭典；

【作者基本信息】 浙江大学 ， 外科， 2008， 博士

【摘要】 膀胱癌是我国泌尿系统最常见的恶性肿瘤，90％以上为移行细胞癌。初次就诊的患者中70％～80％为浅表性膀胱癌，其中70％为Ta期，30％为T1期，不论肿瘤分化程度的高低，都未侵及膀胱肌层。经尿道切除术或者膀胱部分切除术可以切除原发肿瘤，但由于膀胱癌的多中心性和多发性特点，术后5年复发率高达三分之二，而术后15年的复发率则高达88％，复发的患者中约有15％会进展为浸润性膀胱癌。因此，浅表性膀胱癌手术后如何预防复发和疾病进展是泌尿外科医生共同努力的目标。自上个世纪60年代起开始的膀胱内灌注治疗有效地延缓或者阻止了肿瘤的复发和进展，因此，膀胱灌注治疗己成为膀胱癌治疗的重要手段之一。目前膀胱灌注治疗的药物主要为两大类，分别为化学药物和免疫制剂，其中喜树碱类衍生物是膀胱灌注化疗的重要药物之一。喜树碱类衍生物的构效关系分析表明在7，9，10位被基团取代的喜树碱类衍生物具有很强的抗肿瘤活性。尽管在10位被羟基和烷基取代后，如10-甲氧基喜树碱有一定的抗肿瘤活性，但是其有较强的毒副作用限制了它的临床应用。相反在9位引入硝基，如9-硝基喜树碱，不仅能保证其治疗效果，同时还具有较低的毒副作用。但要将喜树碱硝化得到9-硝基喜树碱是比较困难的，因为12-硝基喜树碱会是最主要的产物。而对10—羟基喜树碱进行硝化则容易得到10—甲氧基—9—硝基喜树碱（MONCPT），国内华东师范大学合成了该化合物。该药系我国有自主知识产权的喜树碱类抗肿瘤新药，目前正在进行临床前研究。膀胱癌是泌尿系统最常见的肿瘤，本课题就是考察MONCPT对膀胱癌细胞增殖的抑制作用并对其作用机制进行研究。目的：1、研究MONCPT对膀胱癌5637细胞增殖的抑制作用（在体和离体）；2、考察MONCPT对膀胱癌5637细胞周期的影响，若有影响则进一步探讨其作用机制；方法：1)采用MTT法，评价MONCPT体外抑制膀胱癌5637细胞活性；2)采用裸小鼠皮下移植瘤模型，观察MONCPT对荷人膀胱癌5637裸小鼠的实验治疗作用；3)应用流式细胞术检测不同浓度MONCPT和MONCPT在不同作用时间对5637细胞周期的影响；4)采用Western blotting的方法检测MONCPT对5637细胞p53，CDK7，p21，AKT，Mat1，Wee1，Cyclin B1，CDK1，Cyclin H，Cdc25C等蛋白表达水平的影响；结果：1、MONCPT对人膀胱癌细胞株的体外抑制作用：采用MTT法对人膀胱癌5637细胞进行体外IC₅₀测定，结果表明，MONCPT对人膀胱癌5637细胞作用48小时有较强的抑制增殖作用，IC₅₀值为1．31±0．16μM。结果还显示，随浓度的下降，MONCPT在体外对人膀胱癌5637细胞的抑制率迅速降低，呈明显的剂量依赖关系。2、MONCPT对人膀胱癌5637细胞的实验治疗作用：对裸小鼠体内5637肿瘤的实验治疗作用试验结果显示，MONCPT 5、10和20mg／kg组裸小鼠体重无明显影响（P>0．05）。与相应的溶剂对照组比较，Irinotican10mg／kg和MONCPT 10mg／kg组移植瘤体积从第5天开始显著减小（P<0．05—0．01），而MONCPT 20mg／kg组植瘤体积从第3天开始显著减小（P<0．05），MONCPT 5mg／kg组植瘤体积从第9天开始显著减小（P<0．01）。MONCPT 5mg／kg组在给药后第3、5、7、9、11、13和15天的相对肿瘤增殖率（T／C，％）分别为103．2％、84．6％、95．7％、80．2％、62．0％、41．6％和23．8％，从第13天开始相对肿瘤增殖率小于60％；MONCPT 10mg／kg组相应的相对肿瘤增殖率分别为95．3％、67．7％、61．4％、34．2％、21．2％、11．1％和6．2％，从给药后第9天开始相对肿瘤增殖率小于60％；MONCPT 20mg／kg组相应的相对肿瘤增殖率分别为73．2％、51．9％、40．3％、20．9％、11．7％、6．9％和3．3％，从给药后第5天开始相对肿瘤增殖率小于60％；Irinotican 10．0mg／kg组相应的相对肿瘤增殖率分别为88．1％、70．4％、50．5％、31．3％、20．8％、13．8％和7．3％，从给药后第7天开始相对肿瘤增殖率小于60％。给药15天后剖杀裸小鼠取瘤块称重发现，MONCPT 5mg／kg、10mg／kg和20mg／kg组的人膀胱癌5637移植瘤平均重量分别为0．63±0．12g、0．24±0．06g和0．06±0．03g，与阴性对照组的1．83±0．30g比较均有明显下降（P<0．01），三个剂量组相应的肿瘤抑制率分别为64．8％、86．2％和96．5％，肿瘤抑制率均大于40％。Irinotican 10．0mg／kg组的平均瘤重为0．36±0．09g，与溶剂对照组比较也有显著下降（P<0．01），抑瘤率为81．1％，大于40％。3、MONCPT诱导人膀胱癌5637细胞周期阻滞作用：MONCPT10，1，0．1，0．01μM分别对人膀胱癌5637细胞作用24小时后，DNA分析结果表明，溶剂对照组的G2／M期的肿瘤细胞百分率分别为8．56±2．13％；而MONCPT10，1，0．1，0．01μM处理24小时后，处于G2／M期肿瘤细胞百分率分别为19．7±3．6％、46．9±7．3％、76．8±9.2％和18．55±3．5％，0．1μM的MONCPT能达到最大的肿瘤细胞周期阻滞作用。用0．1μM的MONCPT处理5637细胞12h，24h，36h和48h，并设溶剂对照，收集各组细胞，用流式细胞术检测细胞的周期阻滞率。结果显示，溶剂对照组的G2／M期的肿瘤细胞百分率为11．45±2．3％，随着MONCPT作用时间的延长，G2／M期比率分别为22．4±3.85％（12h）、79．18±7．3％（24h）、50．15±6．25％（36h）和29．7±3.85％（48h）。4、MONCPT对5637细胞周期相关蛋白表达量的影响：用0．1μM MONCPT分别处理5637细胞12，24，36和48h，并设对照，收集各组细胞，提取蛋白，用Western印迹法检测CDK1、CyclinB1、Cyclin H、MAT1、AKT、Wee1、CDK7，Cdc25C，p21和p53蛋白表达量的变化。结果显示，随给药时间的延长，MONCPT可使人膀胱癌5637细胞p53，p21的表达先增加后下降，MAT1、CyclinB1的表达先下降后增加，CDK1、CDK7、Cdc25C、Cyclin H、AKT、Wee1的表达量下降。讨论和结论：MONCPT是新合成的喜树碱类衍生物。体内外的抗肿瘤实验证实MONCPT对人膀胱癌5637细胞具有很强的抑制增殖作用，同时我们还证实这种作用是可以通过对细胞周期G2／M期阻滞实现的。近几年来，植物来源的抗肿瘤药物受到了广泛的关注。当哺乳动物细胞遇到DNA损伤时，细胞周期会停滞直到错误被纠正，细胞周期G2／M期被阻滞后细胞将停滞生长。相比较G1／S，人们对G2／M了解的并不多，然而这个周期检验点对于肿瘤发生却是非常重要的。G2／M的转变是受到Cdc家族蛋白和cyclin调控的，Cdc2可以被四个转录靶点p53，Gadd45，p21和14—3—3σ所抑制。p53可作为转录因子，诱导另外几种蛋白的产生，如p21，从而达到调控细胞周期的作用。在哺乳动物细胞中，cyclin的结合和CAK对Cdc2的Thr161位磷酸化对于Cdc2-cyclin B复合物的活性都是非常重要的。若没有Cdc25C对Cdc2的Thr14和Tyr15位去磷酸化，且Plk1磷酸化cyclin B时，细胞周期将被停滞。当用0．1μM MONCPT分别处理5637细胞12，24，36和48h，Western印迹法检测结果显示，随给药时间的延长，5637细胞的p53，p21表达先增加后下降，MAT1、CyclinB1表达先下降后增加，CDK1、CDK7、Cdc25C、Cyclin H、AKT、Wee1表达量下降。因此，我们推测，MONCPT使人膀胱癌5637细胞终止在G2／M期的作用可能是其通过诱导细胞p53、p21表达上调和CyclinB1、CDK1、CDK7、AKT、Wee1、Cyclin H、Mat1和Cdc25C表达下调，从而使细胞终止在G2／M期，达到抑制肿瘤细胞增殖的作用。综上所述，我们实验证实，MONCPT对人膀胱癌5637细胞的增殖具有明显的抑制作用，其作用机制可能与p53通路、CDK7、Cdc25C及cyclin B1-CDK1复合物有密切关系，通过阻滞膀胱癌细胞周期而发挥抗肿瘤活性。在体和离体实验证实，MONCPT具有良好的抗膀胱癌实验治疗作用，值得在临床进一步实验以确证疗效。更多还原

【Abstract】 Bladder cancer is the most common urologic cancer in our country. Morethan 90% of bladder cancer is transitional cell carcinoma （TCC）. About 70-80% ofcases are superficial at the time of diagnosis, of which 70% are stage Ta and 30%are stage T1. Low-grade non-invasive tumors may be treated with resection andfulguration. However, despite complete tumor resection, two thirds of patients willdevelop tumor recurrence in five years and by 15 years 88% of patients willdevelop a recurrence. Progression from superficial bladder cancer to deepmuscle invasion occurs in 15% of patients. The high rate of tumor recurrence andpotential progression provides an opportunity to institute chemoprevention orprophylactic therapy. Now intravesical therapy is the important modality forbladder cancer treatment.There are two main categories of intravesical therapy,that is, chemotherapy and immunotherapy. Camptothecin analogs is one of theimportant chemotherapeutic drugs for intravesical therapy.The structure-activity relationship （SAR） analysis of the CPTs hasdemonstrated that substitutions at the 7-, 9-, or 10-positions of most camptothecinanalogs can enhance their antitumor activity. Although the induction of hydroxylor alkoxy at position 10, such as, 10-methoxycamptothecin, has antitumor activity,its low therapy index limited further research. In contrast, the induction of a nitroat position 9, such as 9-nitrocamptothecin, has shown satisfactory activity with lowtoxicity. 9-nitrocamptothecin was difficultly prepared by nitration of camptothecinbecause the 12-nitrocamptothecin was the main product,and the 10-methoxy-9-nitrocamptothecin could easily be prepared from10-hydroxycamptothecin.The present study was conducted to estimate theanti-cancer activity of MONCPT against bladder cancer both in vitro and in vivo,and the molecular mechanisms of anti-tumor effect.Purposes: To determine the anticancer activity of MONCPT both in vitro and invivo against bladder cancer cell 5637; estimate the effect on cell cycle of bladdercancer cell 5637 and elucidate its mechanism.Methods:1) Cancer cell killing ability of MONCPT was measured by MTT method;2) Tumor growth inhibition activity of MONCPT was measured by murine bearinghuman xenografted tumor models in vivo;3) After MONCPT treating,cell cycle distribution was detected by flow cytometry;4) Protein （p53, CDK7, p21, AKT, Mat1, Wee1, Cyclin B1, CDK1, Cyclin H,Cdc25c） expression was detected by Western blotting;Results:1. Cytotoxicity AssayUsing the MTT method, we determined the cytotoxic activity of MONCPTagainst bladder cancer cell lines （5637 cells）. All the cells exhibiteddose-dependent sensitivity to 48-hours exposure with MONCPT and the IC₅₀value was 1.31±0.16μM. 2. Effect of MONCPT on Tumor Growth in vivo in 5637 Xenografted Models.On the basis of the encouraging in vitro data, the antiproliferation activity ofMONCPT was evaluated in human tumor models xenografted in athymic mice.The study showed that MONCPT possessed of significant effect on tumorweight（P<0.01） compared with the negative control group , but not on athymicmice body weight （P > 0.05）. The tumor weights were 0.63±0.12g （MONCPT5mg/kg）, 0.24±0.06g （MONCPT10mg/kg） and 0.06±0.03g （MONCPT20 mg/kg）.In the negative control group the tumor weight was 1.83±0.30g. Compared withthe control group, the tumor volumes were significantly inhibited from day 5 in thegroups treated with MONCPT （10.0 mg/kg） and Irinotican （10.0 mg/kg） （P<0.05-0.01） and from day 3 in the groups treated with MONCPT （20.0 mg/kg） （P<0.05） , from day 9 in the groups treated with MONCPT （5.0 mg/kg） （P <0.01）.The relative tumor proliferation rates （T/C, %） of 5 mg/kg MONCPT were 103.2%,84.6%,95.7%,80.2%,62.0%,41.6% and 23.8% at treatment day 3, 5, 7, 9,11,13, and 15 respectively, while these of 10 mg/kg MONCPT were 95.3%,67.7%,61.4%,34.2%, 21.2%,11.1% and 6.2%. The T/Cs （%） of 20 mg/kg MONCPTwere 73.2%、51.9%、40.3%、20.9%、11.7%、6.9% and 3.3%, from day 5the T/Cs （%） < 60% . The T/Cs （%） of 10 mg/kg Irinotican were 88.1%、70.4%、50.5%、31.3%、20.8%、13.8% and 7.3%,from day 7 the T/Cs（%） <60%. 3. Cell cycle Assay of MONCPT against 5637.5637cells were respectively treated with 10,1, 0.1, 0.01μM MONCPT for 24hours. Collect the treated cells and the control cells, and then use the flowcytometry to detect the cell cycle distribution. It was showed that G2/M distributionwere 19.7±3.6%、46.9±7.3%、76.8±9.2%and 18.55±3.5%respectively,and 8.56±2.13% in control group . 0.1μM MONCPT has the greatest effectto arrest cell cycle in G2/M phase. 5637 cells were treated with 0.1μM MONCPTfor 12, 24, 36 and 48 hours, collectting the treated cells and the control cells, andthen used the flow cytometry to detect the cell cycle distribution. It was indicatedthat the increase of the cell cycle G2/M distribution was detected in 5637 cells.The percentages of cell cycle G2/M in 5637 cells induced by 0.1μM MONCPT for0, 12, 24, 36 and 48 hour were 11.45±2.3%, 22.4±3.85%（12h）, 79.18±7.3%（24h）, 50.15±6.25%（36h） and 29.7±3.85% （48h） respectively.4. Determination of the Expression of Cell cycle G2/M phase-relatedProteins.Incubate the 5637 cells with 0.1μM MONCPT for 12, 24, 36 and 48 hours,collectting the treated cells and the cells without exposure to MONCPT, followedby extracting proteins from these cells. It was showed that MAT1、CyclinB1 wereobviously down-regulated from 12 to 24h, and then up-regulated from 36 to 48hby MONCPT （0.1 pM）. CDK1、CD7、Cdc25C、Cyclin H、AKT、Wee1 weredown-regulated within 48 hours by MONCPT （0.1μM）. The high-level expression of p21 and p53 proteins at 24 h after treatment of MONCPT （0.1μM ） in 5637cells indicated that MONCPT exert its potency through affecting the expression ofproteins related with cell cycle.Discuss and Conclusion:MONCPT is a new synthetic derivative of Camptothecin. Our results showedthat MONCPT exerted potent antiproliferative action on human bladder cancercells in vitro and in vivo. We also identified that cell cycle G2/M phase waseffectively arrest by MONCPT in human bladder cancer 5637 cells, whichindicated that its mechanism of anti-bladder cancer effect was through cell cyclepathway.In previous study, it was reported that MONCPT induced cell cycle G2/Mphase arrest by several pathways simultaneously, including p53 pathway, theMAPK pathway and the activation of Akt. Cell cycle regulation and itsmodulation by various plant-derived agents are gaining widespread attention inrecent years. Mammalian cells respond to DNA-damaging agents by activatingcell cycle checkpoints, acting by delaying cell cycle progression until errors havebeen corrected. Usually these control mechanisms determine a temporaryarrest at a specific stage of the cell cycle to allow the cell to correct possibledefects. The arrest at G2/M prevented the cells from completing the cell cycleand proliferating. In contrast to the G1/S checkpoint, the mammalian G2/Mcheckpoint is poorly understood.This checkpoint prevents the improper segregation of chromosomes, which is likely to be important in humantumorigenesis. G2/M transition provides an effective checkpoint in cell cycleprogression that is regulated by the sequential activation and deactivation of Cdcfamily proteins and cyclin complexes. The Cdc2-cyclin B kinase is pivotal inregulating this transition. Cdc2 is inhibited simultaneously by four transcriptionaltargets of p53, Gadd45, p21, and 14-3-3σ. p53 is a transcription factor thatup-regulates a number of important cell cycle-modulating genes such as p21.Part of the mechanism by which p53 blocks cells at the G2 checkpoint involvesinhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Inmammalian cells, both cyclin binding and phosphorylation by CAK are required forthe activation of Cdc2-cyclin B complex, Thr161 on Cdc2 of the complex isphosphorylated by CAK, a complex containing cyclin H, MAT1 and CDK7. In theabsence of Cdc2 dephosphorylation by Cdc25C, and also the directphosphorylation of cyclin B by Plk1, the accumulation of cyclin B-Cdc2 in thenucleus is prevented and entry into mitosis is stalled. In addition, Plk1 also hasbeen shown to function via the Cdc25C and Cdc2/cyclin B1 positive feedbackloop at the onset of mitosis. Our results were showed that MAT1、CyclinB1 wereobviously down-regulated from 12 to 24h, and then up-regulated from 36 to 48hby MONCPT （0.1μM ）. CDK1、CDK7、Cdc25C、Cyclin H、AKT、Wee1 weredown-regulated within 48 hours by MONCPT （0.1μM ） . The high-levelexpression of p21 and p53 proteins at 24 h after treatment of MONCPT （0.1μM）in 5637 cells indicates that MONCPT exert its potency through affecting theexpression of proteins related with cell cycle. In conclusion, MONCPT exhibited high anti-proliferation activity in humanbladder cancer 5637 cell line both in vivo and in wfro.lt was shown that MONCPTcould induce cell cycle G2/M phase arrest in human bladder cancer 5637 cellsand its mechanism may be associated with cyclinB1-CDK1 complex, p53, CDK7and Cdc25c.更多还原