【作者】 王云彬；

【导师】 谢立平；

【作者基本信息】 浙江大学 ， 外科学， 2008， 博士

【摘要】 第一部分二烯丙基三硫化物(DATS)对人膀胱癌T24细胞增殖的影响目的研究大蒜提取物二烯丙基三硫化物(DATS)于体外对人膀胱癌细胞系T24细胞增殖和细胞周期的影响及其可能机制。方法采用MTT法观察不同浓度的DATS(10,20,40,和80μmol/L)作用不同时间(24,48和72h)后对T24细胞增殖的影响。同时应用集落形成试验观察不同浓度的DATS(0,0.5,1,5,10,20和40μmol/L)对T24细胞集落形成的影响。T24细胞经不同浓度DATS(10,20,40,和80μmol/L)作用24小时后,采用流式细胞仪进行细胞周期分析。并应用Western blotting方法检测细胞周期相关蛋白Cdc25C的表达,以观察DATS对其的影响。结果1.DATS对T24细胞的增殖有明显的抑制作用,MTT法显示随着药物浓度升高(2.5～100μmol/L)和作用时间的延长(24-72h)其抑制作用逐渐增强,作用24h后,5-100μmol/L的抑制率范围为13%～81%,而作用48h及72h后抑制率范围则分别升至25%～87%及31%～96%。呈明显剂量和时间依赖性。2.DATS抑制T24细胞集落形成,随着浓度增高集落形成率逐步下降,浓度为20μmol/L时集落形成抑制率已达100%。3.经流式细胞仪检测,不同浓度DATS(10-40μmol/L)作用T24细胞24h后,G2/M期细胞逐渐增多,表明DATS可引起G2/M期停滞,表现为剂量依赖性。4.不同浓度DATS(10-80μmol/L)作用T24细胞24h后,Cdc25C蛋白表达随药物浓度增加逐渐降低。结论DATS能显著抑制T24细胞增殖和集落形成,其抑制作用呈时间和剂量依赖性。DATS能显著抑制T24细胞中Cdc25C蛋白的表达,这可能是诱导T24细胞G2/M期停滞的分子机制之一。第二部分二烯丙基三硫化物(DATS)对人膀胱癌T24细胞凋亡的影响目的研究DATS在体外对人膀胱癌细胞系T24细胞凋亡的诱导作用,并探讨其诱导凋亡的相关信号通路等分子机制。方法研究DATS抗膀胱癌增殖作用是否为诱导细胞凋亡所致。用光学显微镜及荧光染色形态学观察经DATS作用后T24细胞产生的凋亡形态学改变。并应用流式细胞仪(Annexin V/PI双染法)检测细胞凋亡率的变化。采用Western blotting方法检测不同浓度DATS(10,20,40,和80μmol/L)作用24小时后T24细胞中caspase-3、PARP等蛋白在诱导凋亡中的表达,同时进一步研究了诱导细胞凋亡的分子机理,包括细胞中抗凋亡的PI3K/Akt通路及Bcl-2家族蛋白的表达。结果1.DATS作用于T24细胞后明显诱导细胞凋亡并呈显著剂量依赖性。2.光学显微镜显示不同浓度DATS(10,20,40,和80μmol/L)作用于T24细胞24小时后使其细胞密度呈剂量依赖性降低。同时观察到凋亡细胞所具有的典型形态学改变。3.流式细胞仪(Annexin V/PI双染法)检测不同浓度的DATS(10～80μmol/L)对T24细胞作用24h后细胞凋亡呈剂量依赖性增加。细胞发生坏死比例为0.2%～1.3%,而发生凋亡的比例为6.2%～20.0%,较之对照组0.3%明显为高,且呈剂量依赖性。其中早期凋亡细胞比例为4.8%-12.2%,晚期凋亡细胞比例为1.4%～7.8%,均较之对照组为高。4.DATS作用于T24细胞后easpase-3和PARP均被激活,表现为proeaspase-3的减少和裂解的PARP片段(89KD)出现,并呈显著的剂量依赖性。5.DATS作用于T24细胞后p-PDK1,p-Thr308-Akt,和p-Ser473-Akt的表达逐渐降低,呈剂量依赖性,但t-Akt的表达不受影响。6.DATS作用于T24细胞后Bax和BAD表达逐渐增高,Bcl-2和Bcl-xL的表达逐渐降低,呈显著的剂量依赖性。而且Bax/Bcl-2的比例逐渐增高,呈显著的剂量依赖性。结论DATS能诱导T24细胞凋亡,凋亡的诱导作用呈显著剂量依赖性。DATS诱导产生凋亡的机理可能通过抑制T24细胞PI3K/Akt信号通路的激活从而调节Bcl-2家族等蛋白并最终诱导凋亡发生。第三部分二烯丙基三硫化物(DATS)对人膀胱癌T24细胞侵袭转移的影响目的研究DATS于体外对人膀胱癌细胞系T24细胞侵袭转移能力的影响及其可能机制。方法应用MTT法检测DATS对T24细胞粘附纤维连接蛋白(fibronectin)能力的影响,应用Transwell小室进行人工重组基底膜侵袭实验检测DATS对T24细胞侵袭能力的影响,并应用划痕愈合试验检测DATS对T24细胞迁移能力的影响。采用RT-PCR方法检测不同浓度DATS(10,20,40,和80μmol/L)作用24小时后的T24细胞中MMP-9 mRNA和MMP-2 mRNA的表达。采用Western blotting方法检测不同浓度DATS(10,20,40,和80μmol/L)作用24小时后的T24细胞中MMP-9蛋白的表达。结果1.粘附试验表明DATS(10～80μmol/L)作用24小时后,能有效抑制T24细胞与细胞外基质的粘附,且随药物浓度增高,抑制作用增强。2.划痕试验显示DATS作用不同时间(0,12和24小时)对T24细胞迁移力的影响随浓度增加和时间的延长而增强,呈剂量和时间依赖性。3.侵袭试验表明DATS(10～80μmol/L)作用于T24细胞24h后,随着药物浓度的升高,T24细胞的侵袭力逐渐降低,呈剂量依赖性。4.DATS可减少T24细胞中MMP-9 mRNA和蛋白的表达,随药物浓度增高而表达减少,呈剂量依赖性。结论DATS能显著抑制T24细胞的粘附、迁移和侵袭能力。DATS的侵袭转移抑制能力可能与降低MMP-9的表达有关。更多还原

【Abstract】 Part 1 Effects of DATS on proliferation of human bladder carcinoma T24 cellsObjective To investigate the effects of diallyl trisulfide(DATS)on growth inhibition, cell cycle and corresponding molecular mechanism of T24 cells.Methods The cytotoxic effect of DATS on human bladder cancer T24 cells was determined with varying concentration of DATS(10,20,40,and 80μmol/L)treatment for 24,48,and 72 hours by MTT assay.The antiproliferative or anticarcinogenic effect of DATS in T24 cells was determined by anchorage-dependent colony-forming assay.In colony-forming assay,the cells were tested for their proliferation potential and formation of an individual colony.The cell cycle arrest effect of DATS on human bladder cancer T24 cells was determined with varying concentration of DATS treatment for 24 hours by flow cytometry.The cell cycle modulating protein Cdc25C was determined by immunoblotting.Results1.Using the T24 human bladder cancer cell line,we found that DATS treatment resulted in dose-dependent(2.5/100μmol/L)and time-dependent(24～72 hours) inhibition of cellular proliferation and cell viability.2.Reduction in cell viability with DATS treatment at concentrations of 5～100μmol/L after 24 hours ranged from 13%to 81%,whereas after 48 and 72 hours ranged from 25%to 87%,31%to 96%,respectively.3.There was a drastic decrease in the ability of the T24 cells to form colonies with increasing doses of DATS(0.5～20μmol/L).DATS at dosages of 20μmol/L completely inhibited the proliferation of cells with no colonies formed by the end of 7 days.4.As shown by flow cytometry,DATS treatment(10,20,40,and 80μmol/L for 24h) of the T24 cells resulted in significant G2/M phase cell cycle arrest.5.As shown by immunoblot analysis,DATS treatment(10,20,40,and 80μmol/L for 24 h)of the T24 cells resulted in significant dose-dependent downmodulation of the protein expression of Cdc25C.Conclusions Our data suggested DATS treatment resulted in a significant dose- and time-dependent inhibition in the growth and colonogenic survival of T24 Cells.Our study also suggests that DATS causes a decrease in the protein levels of the Cdc25C, thereby causing a G2/M-phase arrest of the cell cycle.Part 2 Effects of DATS on apoptosis of human bladder carcinoma T24 cellsObjective To investigate the effects of DATS on apoptosis of human bladder carcinoma T24 cells and to identify the altered signaling pathway underlying the response to EGCG exposure.Methods We determined whether DATS -mediated loss of T-24 cell viability was the result of the induction of apoptosis.The induction of apoptosis by DATS (10～80μmol/L)was measured by acridine orange/ethidium bromide assays,and the proapoptotic effect of DATS was also confirmed by analyzing cell morphology and by PI staining and the annexin V method.The extent of apoptosis was quantified by flow-cytometric analysis of DATS-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for Akt proteins,indicative of activation.The role of caspase 3,PARP-1,Bcl-2 family in apoptosis was analyzed by Western blotting.Results1.Using the T24 human bladder cancer cell line,we found that DATS treatment resulted in induction of apoptosis in dose-dependent manner(10～80μmol/L).2.Phase-contrast photomicrographs were taken 24 h after DATS treatment revealed a dose-dependent decrease in cell density.Changes in cell morphology and cell membrane blebbing,which are characteristics of apoptosis,were also detected.3.As shown by PI staining and the annexinⅤmethod,we found that DATS caused a dosage dependent increase in T24 cell apoptosis.It was observed that treatment of T24 cells with 10 and 80μmol/L of DATS for 24 hours increased the number of early apoptotic cells(LR)from 4.8%to 12.2%respectively,in a dose-dependent manner compared to 0.1%in untreated control cells.The number of late apoptotic cells(UR)increased from 1.7%to 7.8%compared with 0.2%in non-DATS treated cells.4.Western blot analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent activation of caspase-3 and PARP proteins 24 hours after DATS treatment.5.DATS treatment resulted in an appreciable down-regulation of protein expression of phospho-PDK1,phospho-Thr308-Akt,and phospho-Ser473-Akt without an effect on total Akt expression in T24 cells.6.DATS treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and a concomitant increase in proapoptotic Bax proteins.7.The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with DATS treatment.8.DATS caused an increase in Bad and a decrease in Bcl-xL protein levels.Conclusions Our data suggested that DATS causes an inhibition of phosphatidylinositol 3’-kinase/Akt activation that,in turn,results in modulations in Bcl-2 family proteins in such a way that the apoptosis of T24 cells is promoted.Based on these studies,we suggest that DATS could be developed as an agent for the management of bladder cancer.Part 3 Effects of DATS on invasion and metastasis of human bladder carcinoma T24 cellsObjective The present study was undertaken to examine the effects of DATS on invasion and metastasis of human bladder carcinoma T24 cells and to identify the possible mechanisms.Methods To evaluate the antimetastatic activity of DATS,we first assessed the inhibitive effect of DATS on the adhesion of T24 cells to fibronectin by MTT assay. Wound healing assay and the in vitro invasion assay were used to investigate the antimetastatic activities of DATS against T24 cells.The effect of DATS on the gene expression of MMPs was examined by treating human bladder carcinoma T24 cells with DATS(10～80μmol/L).The transcription levels of MMP-2 and -9 were assessed by reverse transcription-polymerase chain reaction(RT-PCR).The role of MMP-9 in antimetastatic activities was analyzed by Western blotting.Results1.Using the T24 human bladder cancer cell line,we found that DATS treatment resulted in dose-dependent(10～80μmol/L)inhibition of the adhesion activities.2.The cellular motility was evidently inhibited in dose-dependent(10-80μmol/L) and time-dependent(6～24 hours)manner by DATS.3.Similarly,the results of the in vitro invasion assay also displayed that DATS was able to inhibit invasion ability of T24 cells dose-dependently.4.RT-PCR analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent decrease in MMP-9 mRNA levels.5.Western blot analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent decrease in MMP-9 protein levels.Conclusions DATS elicited a significant inhibition of in vitro cell adhesion, migration,and invasion in human bladder carcinoma T24 cells.The inhibition of invasion ability of T24 ceils by DATS was shown to may be attributed to decreases of the expression of MMP-9.Thus,clinical application of DATS may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis.更多还原