水飞蓟宾膀胱灌注抑制大鼠膀胱癌发生及其诱导膀胱癌细胞凋亡的机制研究
|
摘要
研究背景：
膀胱肿瘤是我国泌尿系统中最常见的恶性肿瘤，且发病率呈不断增高趋势。临床上约70%-80%左右的初发膀胱肿瘤为非肌层浸润性肿瘤，经尿道膀胱肿瘤电切术（Trans-urethral Resection of Bladder Tumor，TUR-BT）则是较常用的手术方法，但术后5年肿瘤复发率高达60%-70%，且有10%-25%的膀胱肿瘤患者经一次或多次复发后，恶性度逐渐增高，并可能发展成为肌层浸润性膀胱肿瘤，严重影响着膀胱肿瘤患者的治疗效果及预后。
目前预防非肌层浸润性膀胱肿瘤术后复发及向恶性进展的可能有效办法之一就是TURBt术后辅以膀胱灌注化疗药物或免疫制剂。但无论是常用化疗药物（吡柔比星、丝裂霉素C等）、生物免疫制剂（卡介苗等）还是两者联合使用，虽然近期均有一定的疗效，但远期仍有22.7%-26.2%的患者存在复发；且两种灌注方法都可能存在不同程度的骨髓抑制、过敏反应及膀胱刺激征等全身或局部毒副作用。鉴于目前膀胱灌注药物的疗效不稳定性及毒副作用，寻找一种更有效且更安全的新型膀胱灌注药物仍是膀胱肿瘤治疗方面研究的重点。
水飞蓟宾（Silibinin）作为一种传统保肝药物在临床上已经被广泛应用于治疗慢性肝炎、肝硬化及中毒性肝损害等疾病。近年来的研究则发现，水飞蓟宾具有广谱抗肿瘤作用。自Agarwal R等1999年在PNAS杂志（美国科学院院报）上首次报道了水飞蓟宾可以发挥抗前列腺癌效果后，先后有多个研究小组均发现了水飞蓟宾可以显著抑制结肠癌、胃癌、肺癌、乳腺癌、肝癌、卵巢癌及皮肤癌等多种肿瘤的生长，提示了水飞蓟宾作为新型抗肿瘤药物的潜在可能性。对于水飞蓟宾抑制多种肿瘤的分子机制，目前认为可能与通过阻滞肿瘤细胞周期，诱导凋亡，抑制血管生成，逆转多药耐药及抑制侵袭转移等途径有关。其中，肿瘤细胞的凋亡诱导是肿瘤化疗中至关重要的机制之一，也是评价化疗药物疗效的重要指标之一，因此，凋亡诱导作用及凋亡相关信号通路（包括外源性细胞膜受体及内源性线粒体凋亡信号通路）的参与在水飞蓟宾的抗肿瘤机制中逐渐受到研究者的关注。
在膀胱肿瘤RT4、T24及TCCSUP细胞中，有研究报道水飞蓟宾可以诱导其发生凋亡并抑制其体内外增殖能力，提示了水飞蓟宾在膀胱肿瘤的药物治疗中的潜在前景。但是，对于外源性及内源性凋亡信号通路在水飞蓟宾所诱导的凋亡过程中的作用未进行充分的探讨及研究，换言之，水飞蓟宾诱导膀胱肿瘤细胞发生凋亡的确切分子机制仍不甚清楚；此外，在以上研究所采用的细胞系中，从细胞的来源及恶性程度来说，膀胱癌RT4细胞代表分化良好的乳头状尿路上皮癌，T24细胞代表浸润性膀胱癌、TCCSUP细胞则代表高级别高浸润性膀胱癌，而临床上膀胱肿瘤的类型以非肌层浸润性膀胱癌最为常见，高复发性是这类肿瘤的特点之一，因此这些细胞均未能很好的代表浅表性（非肌层浸润性）膀胱癌特别是高危浅表性膀胱癌细胞的特征，无法很好的模拟临床；最重要的是，这些研究在体内实验部分均局限在通过口服给药途径给予水飞蓟宾对膀胱肿瘤进行干预，而对于膀胱灌注水飞蓟宾的可能性则未涉及，对于膀胱灌注水飞蓟宾对膀胱肿瘤的影响尚未见任何报道。
研究目的：
1.以膀胱癌5637细胞系（代表高危浅表性膀胱癌）为研究对象，辅以建立裸鼠皮下移植瘤模型，围绕线粒体及线粒体内相关凋亡分子的功能，探讨水飞蓟宾诱导膀胱癌5637细胞凋亡的相关分子机制；
2.以膀胱灌注N-甲基亚硝基脲（MNU）诱导大鼠原位膀胱肿瘤为体内动物研究模型，探讨膀胱灌注水飞蓟宾对于膀胱肿瘤的影响，为进一步将水飞蓟宾作为膀胱肿瘤术后膀胱灌注化疗新的选择方案奠定重要的理论及实验依据。
研究方法：
1.采用MTT法观察水飞蓟宾对膀胱癌5637细胞增殖的影响；应用TUNEL法及Annexin V-FITC/PI标记后应用流式细胞术检测水飞蓟宾对膀胱癌5637细胞凋亡的影响。
2.通过分光光度法检测水飞蓟宾干预后caspase-3及caspase-9的活化程度；使用Western Blot法检测水飞蓟宾干预后cleaved caspase-3、cleaved caspase-8、cleavedcaspase-9、PARP及survivin的表达情况；应用流式细胞术及TUNEL法检测广谱caspase抑制剂z-VAD-fmk预处理对水飞蓟宾所诱导的膀胱癌5637细胞凋亡的影响。
3.采用JC-1标记后应用流式细胞术检测水飞蓟宾对膀胱癌5637细胞线粒体膜电位的影响；提取水飞蓟宾干预后膀胱癌细胞质、线粒体、细胞核蛋白及总蛋白，采用Western Blot检测AIF、Cyto C、Smac/DIABLO、HtrA2/Omi的亚细胞定位情况；应用免疫荧光技术观察水飞蓟宾对膀胱癌细胞AIF核转位的影响。
4.观察口服不同剂量水飞蓟宾对人膀胱癌裸鼠皮下移植瘤生长的影响，记录口服水飞蓟宾对裸鼠体重、瘤重及饮食量的影响；采用TUNEL法检测移植瘤细胞凋亡发生的情况；应用免疫组织化学染色及Western Blot法检测水飞蓟宾对移植瘤细胞内Cleaved Caspase-3、Survivin、AIF等凋亡相关蛋白的影响。
5.通过膀胱灌注MNU诱导大鼠原位膀胱肿瘤，观察早期（第一周）及晚期（第八周）膀胱灌注不同剂量水飞蓟宾对MNU所诱导的大鼠膀胱肿瘤发生及发展的影响；记录及测量膀胱灌注水飞蓟宾对大鼠体重、膀胱总重量及饮食量的影响；采用TUNEL法检测大鼠膀胱癌细胞凋亡发生的情况。
结果：
1. MTT结果显示水飞蓟宾可通过剂量及时间依赖性的方式抑制膀胱癌5637细胞的体外生长；流式细胞术及TUNEL染色结果提示水飞蓟宾还可以诱导膀胱癌5637细胞发生凋亡。
2.水飞蓟宾可显著激活caspase-3、caspase-8、caspase-9及PARP的活化，并下调凋亡抑制因子survivin的表达，但广谱caspase抑制剂z-VAD-fmk不能完全抑制及逆转水飞蓟宾所诱导的膀胱癌细胞凋亡。
3. JC-1标记线粒体后采用流式细胞术检测可发现水飞蓟宾可通过剂量依赖的方式诱导膀胱癌5637细胞线粒体膜电位发生去极化，增加线粒体膜电位崩溃的阳性细胞数；广谱caspase抑制剂z-VAD-fmk不能抑制水飞蓟宾所诱导的线粒体膜电位的下降；Western Blot结果则提示了水飞蓟宾可以显著增加细胞质内Cyto C、AIF以及HtrA2/Omi的表达水平，下调线粒体内Cyto C、AIF以及HtrA2/Omi的表达，但对Smac/DIABLO的亚细胞定位无明显影响，提示水飞蓟宾干预后可引起膀胱癌5637细胞线粒体内Cyto C、AIF以及HtrA2/Omi的释放；此外，水飞蓟宾对膀胱癌5637细胞的总蛋白中Cyto C、AIF、Smac/DIABLO以及HtrA2/Omi的表达均无明显影响。
4. Western Blot结果表明水飞蓟宾可以增加5637细胞核内AIF的表达水平；免疫荧光标记AIF则发现了水飞蓟宾干预后AIF转位至细胞核明显增加，证实了水飞蓟宾广谱caspase抑制剂z-VAD-fmk不能抑制水飞蓟宾所诱导的AIF核转位。
5.口服水飞蓟宾可显著抑制裸鼠膀胱癌皮下移植瘤的生长；口服水飞蓟宾对裸鼠的体重、饮食及一般情况未产生影响；TUNEL染色证明了水飞蓟宾可以引起移植瘤细胞发生凋亡；免疫组织化学染色结果表明水飞蓟宾可以上调移植瘤内CleavedCaspase-3的表达、下调survivin的表达并增加AIF在细胞核内的表达；Western Blot结果也证实了水飞蓟宾可上调Cleaved Caspase-3及AIF的表达并下调survivin的表达。
6.早期（第一周）及晚期（第八周）膀胱灌注水飞蓟宾均可显著降低膀胱灌注MNU所诱导的大鼠发生非肌层浸润性膀胱癌及肌层浸润性膀胱癌的比例；TUNEL染色证实了水飞蓟宾可以引起大鼠膀胱癌细胞发生凋亡；膀胱灌注水飞蓟宾对大鼠的体重、饮食及一般情况均未产生影响，也未影响到正常大鼠的膀胱组织学类型。
结论：
1.水飞蓟宾可通过剂量及时间依赖的方式抑制膀胱癌5637细胞的体外增殖并诱导其发生凋亡；
2.外源性及内源性凋亡信号通路均参与了水飞蓟宾所诱导的膀胱癌细胞凋亡；
3.水飞蓟宾通过激活Cyto C/Omi/caspase依赖及AIF/caspase非依赖途径诱导膀胱癌5637细胞发生凋亡；
4.口服水飞蓟宾可显著抑制膀胱癌裸鼠皮下移植瘤的生长，且该抑制作用也与caspase依赖性及caspase非依赖性凋亡通路的激活有关；
5.早期及晚期膀胱灌注水飞蓟宾均可抑制MNU所诱导的大鼠膀胱癌的发生及发展，且该抑制作用是通过诱导细胞凋亡来实现的。
6.水飞蓟宾可能是一种具有发展前景且相对安全的预防及治疗膀胱肿瘤的新型膀胱灌注药物。
Introduction:
Bladder cancer poses a health problem worldwide. It is the most common urologicmalignancy in China. Of all newly diagnosed cases of bladder cancers, approximately70%-80%present as non-muscle invasive (superficial) tumors. The standard treatment for patientswith superficial bladder cancer is transurethral resection (TUR) of tumors. However,approximately60%-70%of these tumors will recur, with10%-25%showing progression toa higher stage or grade within post-operative5years.
Intravesical chemotherapy and/or immunotherapy are widely used as adjuvant therapies toprevent recurrence and progression of superficial disease after TUR. Although manychemical agents have shown some evidence of activity, their toxicity and incompleteefficacy have limited their use as common intravesical agents. These factors highlight theurgent need to search for novel adjuvant intravesical agents to reduce the recurrence rate.
Silibinin, a natural flavonoid, is the major bioactive component of silymarin isolated frommilk thistle. Accumulating evidence indicates that silibinin has anticancer activity in varioustumor cells, including cancers of prostate, breast, skin, colon, lung, and kidney. Themanifold inhibitory effects of silibinin against various cancer cells include growth inhibition,anti-inflammation, cell cycle regulation, apoptosis induction, chemosensitization, inhibitionof angiogenesis, reversal of multidrug resistance and inhibition of invasion and metastasis.Induction of apoptosis is believed to be one of the major mechanisms of action for silibininagainst cancer cells, although the details are yet to be elucidated. There is increasing interestin elucidating the mechanisms of action for silibinin as an effective agent for chemopr-evention and chemotherapy against various types of cancers.
Although silibinin-induced apoptosis in bladder transitional cell carcinoma RT4(representinga well-differentiated papillary noninvasive tumor phenotype), T24(representing high-gradetumor) and TCCSUP (representing high-grade invasive tumor) cells were previouslyreported, the details of molecular mechanisms for apoptotic regulation are yet to beelucidated. Additionally, the extensive work mentioned above focused on the effects of oraladministration of silibinin against bladder cancer. However, intravesical administration of chemotherapeutic agent, instead of oral administration, is widely used as adjuvant therapy toprevent recurrence and progression of superficial bladder cancer after TUR. As the standardof care in individual bladder cancer patients with high-risk clinical and pathologic features(Ta, T1, and Tis), intravesical therapy receives more attention by urologists and their patientsthan oral administration. However, so far there was still no report on the effects ofintravesical silibinin against bladder cancer.
Objectives:
1. The first purpose of this study was to determine the effects of intravesical silibinin againstbladder cancer induced by intravesical instillation of MNU.
2. In the present study, using human bladder cancer5637cells (having the same molecularfeatures of high-risk superficial bladder cancer) as the model system, we also explored themolecular mechanisms of silibinin-induced apoptosis in vitro and in vivo, focusing on twomitochondrial cell death pathways, namely the Cyto c/Omi/caspase-dependent and theAIF/caspase-independent pathways.
Methods:
1. Bladder cancer5637cell viability was assessed using a tetrazolium-based assay (MTTassay). Silibinin-induced apoptosis was determined by TUNEL and/or Annexin V-FITC/PIstaining followed by flow cytometry.
2. Caspase-3and caspase-9activity after silibinin treatment was assessed by a colorimetricsystem. Western Blotting was used to examine the activation of caspase-3、caspase-8、caspase-9and PARP. Flow cytometry and TUNEL staining were used to quantitate apoptoticcells after silibinin and/or z-VAD-fmk treatment.
3. Mitochondrial membrane potential collapse was determined using JC-1probes followedby flow cytometry analysis. Mitochondrial, Cytosolic, Nuclear Fractions and total lysateswere prepared. Subcellular localization of AIF、Cyto C、Smac/DIABLO and HtrA2/Omiwere then analysed by Western Blotting. Immunofluorescence staining was carried out onsilibinin-treated cells with antibody against AIF.
4. The tumor volume, tumor weight, body weight and average diet consumption weremeasured to determine the anti-cancer effects of oral silibinin against human bladder cancerxenografts in nude mice. Apoptosis induction was examined in vivo using the TUNEL assay,which labels DNA strand breaks. The expression of Cleaved Caspase-3, Survivin and AIFwas then assessed by immunohistochemistry and Western blotting.
5. Orthotopic rat bladder cancer model was established by intravesical administration ofMNU. Silibinin was instilled intravesically either beginning at week10after four doses instillation of MNU for8weeks or beginning at week1for17weeks before instillation ofMNU. Diet consumption and activity of each rat were observed daily, while Body weightswere recorded weekly throughout the study. All the rats were sacrificed at week18, andhistopathological changes and average bladder weight of each group were investigated.TUNEL staining was used to investigate the apoptotic effects of intravesical silibinin againstbladder cancer.
Results:
1. Silibinin inhibited the growth of bladder caner5637cells in vitro in a dose-and time-dependent manner as determined by MTT assay. Flow cytometry analysis and TUNELstaining demonstrated that100and200μM silibinin treatment exerted strong apoptoticeffects on5637cells in a dose-dependent manner.
2. Silibinin activated caspase-3and caspase-9, as observed by its increased cysteine proteaseactivity for individual substrate, in a dose-dependent manner. Additionally, silibininincreased the cleaved subunits of caspase-3(17and19kDa), caspase-9(35kDa), and PARP(89kDa), indicating that silibinin induced the activation of caspase cascade. However, pancaspase inhibitor z-VAD-fmk did not completely reverse silibinin-induced apoptosis. Thespecificity and efficacy of z-VAD-fmk activity in5637cells was confirmed by caspase3activity assay which clearly demonstrate that pretreatment with z-VAD-fmk decreasedcaspase-3activity.
3. Using a cationic lipophilic dye JC-1as a marker of mitochondrial membrane potential(ΔΨm), flow cytometric studies revealed that silibinin induced a dose-dependent loss of ΔΨm.In addition,80μM of z-VAD-fmk showed no protective effects on the dissipation of ΔΨminthe cells treated with200μM silibinin for48h. As determined by Western Blotting, theprotein levels of mitochondrial cytochrome c, HtrA2/Omi and AIF decreased in silibinin-treated cells. Concomitantly, compared to untreated group, silibinin treatment stronglyincreased the amount of cytochrome c, HtrA2/Omi and AIF in the cytosolic fraction,indicating a release from the mitochondria into the cytoplasm. Surprisingly, no release ofSmac/DIABLO was observed after silibinin treatment. Additionally, silibinin had no effectson the expression of cytochrome c, HtrA2/Omi, Smac/DIABLO and AIF in total lysates.
4. Exposure to silibinin increased the amount of AIF in the nuclear fractions as determinedby western blotting. Results from immunofluorescence staining also supported the findings.Untreated cells had AIF localized to the cytosol and the treatment with200μM of silibininfor48h resulted in remarkable translocation of AIF to the nucleus. Pretreatment with pancaspase inhibitor z-VAD-fmk was unable to prevent AIF localization to the nucleus.
5. The growth of5637tumor xenografts were inhibited significantly following oral silibinintreatment at the dose levels of200and300mg/kg. The average tumor masses in the control rats were2-to3-fold (P <0.05) greater than that of200and300mg/kg silibinin treated mice.The average body weights and daily diet consumption of the control and silibinin-treatedmice showed no difference throughout the experiment. As determined by TUNEL assay, thenumber of apoptotic cells increased from8.5%in the control group to12.5%,21%(P <0.05)and37.5%(P <0.05) in100,200, and300mg/kg silibinin-treated groups, respectively,demonstrating significant apoptotic effects of silibinin in vivo. Furthermore, the microscopicexamination of stained tumor sections showed decreased expression of survivin andincreased expression of cleaved caspase-3and AIF in the silibinin-treated groups.6. Four doses of intravesical MNU to rats resulted in the induction of hyperplasia, papillarydysplasia, atypia, superficial and muscle invasive bladder carcinoma at the end of the17-week study. There were significant differences in histopathological changes among thesilibinin-treated groups and the MNU or MNU+DMSO group (P <0.05). Intravesicalsilibinin beginning either at week1or at week10effectively inhibited carcinogenesis andprogression of bladder cancer in rats by reducing the incidence of superficial and invasivebladder lesions, suggesting its chemopreventive and chemotherapeutic effects againstbladder cancer. No toxicity, locally or systemically, was observed in rats receivingintravesical silibinin alone. In addition, in vivo apoptotic effects of intravesical silibinin onMNU–induced bladder cancer were observed by TUNEL staining.
Conclusions:
1. Silibinin inhibited cell growth and induced apoptosis in dose-and time-dependent mannerin bladder cancer5637cells.
2. Both intrinsic and extrinsic apoptotic signal pathways were involved in silibinin-inducedapoptosis.
3. Silibinin induced-apoptosis in human bladder cancer was mediated by the activation oftwo mitochondrial death pathways, namely the Cyto c/caspase-dependent and the AIF/caspase-independent pathways involving selective translocation of Omi/HtrA2.
4. Silibinin inhibits the growth of human bladder tumor xenograft in athymic nude mice,which was associated with apoptosis induction, increased translocation of AIF, anddownregulation of survivin.
5. Intravesical silibinin effectively inhibited the carcinogenesis and progression of bladdercancer in rats initiated by MNU by reducing the incidence of superficial and invasive bladderlesions without any side effects, which was accompanied with pro-apoptotic effects.
6. Silibinin may prove to be a new form of intravesical chemotherapy in the inhibition ofcarcinogenesis and progression of bladder cancer, providing a basis for future clinical trialsof intravesical silibinin used in patients with bladder cancer.
膀胱肿瘤是我国泌尿系统中最常见的恶性肿瘤，且发病率呈不断增高趋势。临床上约70%-80%左右的初发膀胱肿瘤为非肌层浸润性肿瘤，经尿道膀胱肿瘤电切术（Trans-urethral Resection of Bladder Tumor，TUR-BT）则是较常用的手术方法，但术后5年肿瘤复发率高达60%-70%，且有10%-25%的膀胱肿瘤患者经一次或多次复发后，恶性度逐渐增高，并可能发展成为肌层浸润性膀胱肿瘤，严重影响着膀胱肿瘤患者的治疗效果及预后。
目前预防非肌层浸润性膀胱肿瘤术后复发及向恶性进展的可能有效办法之一就是TURBt术后辅以膀胱灌注化疗药物或免疫制剂。但无论是常用化疗药物（吡柔比星、丝裂霉素C等）、生物免疫制剂（卡介苗等）还是两者联合使用，虽然近期均有一定的疗效，但远期仍有22.7%-26.2%的患者存在复发；且两种灌注方法都可能存在不同程度的骨髓抑制、过敏反应及膀胱刺激征等全身或局部毒副作用。鉴于目前膀胱灌注药物的疗效不稳定性及毒副作用，寻找一种更有效且更安全的新型膀胱灌注药物仍是膀胱肿瘤治疗方面研究的重点。
水飞蓟宾（Silibinin）作为一种传统保肝药物在临床上已经被广泛应用于治疗慢性肝炎、肝硬化及中毒性肝损害等疾病。近年来的研究则发现，水飞蓟宾具有广谱抗肿瘤作用。自Agarwal R等1999年在PNAS杂志（美国科学院院报）上首次报道了水飞蓟宾可以发挥抗前列腺癌效果后，先后有多个研究小组均发现了水飞蓟宾可以显著抑制结肠癌、胃癌、肺癌、乳腺癌、肝癌、卵巢癌及皮肤癌等多种肿瘤的生长，提示了水飞蓟宾作为新型抗肿瘤药物的潜在可能性。对于水飞蓟宾抑制多种肿瘤的分子机制，目前认为可能与通过阻滞肿瘤细胞周期，诱导凋亡，抑制血管生成，逆转多药耐药及抑制侵袭转移等途径有关。其中，肿瘤细胞的凋亡诱导是肿瘤化疗中至关重要的机制之一，也是评价化疗药物疗效的重要指标之一，因此，凋亡诱导作用及凋亡相关信号通路（包括外源性细胞膜受体及内源性线粒体凋亡信号通路）的参与在水飞蓟宾的抗肿瘤机制中逐渐受到研究者的关注。
在膀胱肿瘤RT4、T24及TCCSUP细胞中，有研究报道水飞蓟宾可以诱导其发生凋亡并抑制其体内外增殖能力，提示了水飞蓟宾在膀胱肿瘤的药物治疗中的潜在前景。但是，对于外源性及内源性凋亡信号通路在水飞蓟宾所诱导的凋亡过程中的作用未进行充分的探讨及研究，换言之，水飞蓟宾诱导膀胱肿瘤细胞发生凋亡的确切分子机制仍不甚清楚；此外，在以上研究所采用的细胞系中，从细胞的来源及恶性程度来说，膀胱癌RT4细胞代表分化良好的乳头状尿路上皮癌，T24细胞代表浸润性膀胱癌、TCCSUP细胞则代表高级别高浸润性膀胱癌，而临床上膀胱肿瘤的类型以非肌层浸润性膀胱癌最为常见，高复发性是这类肿瘤的特点之一，因此这些细胞均未能很好的代表浅表性（非肌层浸润性）膀胱癌特别是高危浅表性膀胱癌细胞的特征，无法很好的模拟临床；最重要的是，这些研究在体内实验部分均局限在通过口服给药途径给予水飞蓟宾对膀胱肿瘤进行干预，而对于膀胱灌注水飞蓟宾的可能性则未涉及，对于膀胱灌注水飞蓟宾对膀胱肿瘤的影响尚未见任何报道。
研究目的：
1.以膀胱癌5637细胞系（代表高危浅表性膀胱癌）为研究对象，辅以建立裸鼠皮下移植瘤模型，围绕线粒体及线粒体内相关凋亡分子的功能，探讨水飞蓟宾诱导膀胱癌5637细胞凋亡的相关分子机制；
2.以膀胱灌注N-甲基亚硝基脲（MNU）诱导大鼠原位膀胱肿瘤为体内动物研究模型，探讨膀胱灌注水飞蓟宾对于膀胱肿瘤的影响，为进一步将水飞蓟宾作为膀胱肿瘤术后膀胱灌注化疗新的选择方案奠定重要的理论及实验依据。
研究方法：
1.采用MTT法观察水飞蓟宾对膀胱癌5637细胞增殖的影响；应用TUNEL法及Annexin V-FITC/PI标记后应用流式细胞术检测水飞蓟宾对膀胱癌5637细胞凋亡的影响。
2.通过分光光度法检测水飞蓟宾干预后caspase-3及caspase-9的活化程度；使用Western Blot法检测水飞蓟宾干预后cleaved caspase-3、cleaved caspase-8、cleavedcaspase-9、PARP及survivin的表达情况；应用流式细胞术及TUNEL法检测广谱caspase抑制剂z-VAD-fmk预处理对水飞蓟宾所诱导的膀胱癌5637细胞凋亡的影响。
3.采用JC-1标记后应用流式细胞术检测水飞蓟宾对膀胱癌5637细胞线粒体膜电位的影响；提取水飞蓟宾干预后膀胱癌细胞质、线粒体、细胞核蛋白及总蛋白，采用Western Blot检测AIF、Cyto C、Smac/DIABLO、HtrA2/Omi的亚细胞定位情况；应用免疫荧光技术观察水飞蓟宾对膀胱癌细胞AIF核转位的影响。
4.观察口服不同剂量水飞蓟宾对人膀胱癌裸鼠皮下移植瘤生长的影响，记录口服水飞蓟宾对裸鼠体重、瘤重及饮食量的影响；采用TUNEL法检测移植瘤细胞凋亡发生的情况；应用免疫组织化学染色及Western Blot法检测水飞蓟宾对移植瘤细胞内Cleaved Caspase-3、Survivin、AIF等凋亡相关蛋白的影响。
5.通过膀胱灌注MNU诱导大鼠原位膀胱肿瘤，观察早期（第一周）及晚期（第八周）膀胱灌注不同剂量水飞蓟宾对MNU所诱导的大鼠膀胱肿瘤发生及发展的影响；记录及测量膀胱灌注水飞蓟宾对大鼠体重、膀胱总重量及饮食量的影响；采用TUNEL法检测大鼠膀胱癌细胞凋亡发生的情况。
结果：
1. MTT结果显示水飞蓟宾可通过剂量及时间依赖性的方式抑制膀胱癌5637细胞的体外生长；流式细胞术及TUNEL染色结果提示水飞蓟宾还可以诱导膀胱癌5637细胞发生凋亡。
2.水飞蓟宾可显著激活caspase-3、caspase-8、caspase-9及PARP的活化，并下调凋亡抑制因子survivin的表达，但广谱caspase抑制剂z-VAD-fmk不能完全抑制及逆转水飞蓟宾所诱导的膀胱癌细胞凋亡。
3. JC-1标记线粒体后采用流式细胞术检测可发现水飞蓟宾可通过剂量依赖的方式诱导膀胱癌5637细胞线粒体膜电位发生去极化，增加线粒体膜电位崩溃的阳性细胞数；广谱caspase抑制剂z-VAD-fmk不能抑制水飞蓟宾所诱导的线粒体膜电位的下降；Western Blot结果则提示了水飞蓟宾可以显著增加细胞质内Cyto C、AIF以及HtrA2/Omi的表达水平，下调线粒体内Cyto C、AIF以及HtrA2/Omi的表达，但对Smac/DIABLO的亚细胞定位无明显影响，提示水飞蓟宾干预后可引起膀胱癌5637细胞线粒体内Cyto C、AIF以及HtrA2/Omi的释放；此外，水飞蓟宾对膀胱癌5637细胞的总蛋白中Cyto C、AIF、Smac/DIABLO以及HtrA2/Omi的表达均无明显影响。
4. Western Blot结果表明水飞蓟宾可以增加5637细胞核内AIF的表达水平；免疫荧光标记AIF则发现了水飞蓟宾干预后AIF转位至细胞核明显增加，证实了水飞蓟宾广谱caspase抑制剂z-VAD-fmk不能抑制水飞蓟宾所诱导的AIF核转位。
5.口服水飞蓟宾可显著抑制裸鼠膀胱癌皮下移植瘤的生长；口服水飞蓟宾对裸鼠的体重、饮食及一般情况未产生影响；TUNEL染色证明了水飞蓟宾可以引起移植瘤细胞发生凋亡；免疫组织化学染色结果表明水飞蓟宾可以上调移植瘤内CleavedCaspase-3的表达、下调survivin的表达并增加AIF在细胞核内的表达；Western Blot结果也证实了水飞蓟宾可上调Cleaved Caspase-3及AIF的表达并下调survivin的表达。
6.早期（第一周）及晚期（第八周）膀胱灌注水飞蓟宾均可显著降低膀胱灌注MNU所诱导的大鼠发生非肌层浸润性膀胱癌及肌层浸润性膀胱癌的比例；TUNEL染色证实了水飞蓟宾可以引起大鼠膀胱癌细胞发生凋亡；膀胱灌注水飞蓟宾对大鼠的体重、饮食及一般情况均未产生影响，也未影响到正常大鼠的膀胱组织学类型。
结论：
1.水飞蓟宾可通过剂量及时间依赖的方式抑制膀胱癌5637细胞的体外增殖并诱导其发生凋亡；
2.外源性及内源性凋亡信号通路均参与了水飞蓟宾所诱导的膀胱癌细胞凋亡；
3.水飞蓟宾通过激活Cyto C/Omi/caspase依赖及AIF/caspase非依赖途径诱导膀胱癌5637细胞发生凋亡；
4.口服水飞蓟宾可显著抑制膀胱癌裸鼠皮下移植瘤的生长，且该抑制作用也与caspase依赖性及caspase非依赖性凋亡通路的激活有关；
5.早期及晚期膀胱灌注水飞蓟宾均可抑制MNU所诱导的大鼠膀胱癌的发生及发展，且该抑制作用是通过诱导细胞凋亡来实现的。
6.水飞蓟宾可能是一种具有发展前景且相对安全的预防及治疗膀胱肿瘤的新型膀胱灌注药物。
Introduction:
Bladder cancer poses a health problem worldwide. It is the most common urologicmalignancy in China. Of all newly diagnosed cases of bladder cancers, approximately70%-80%present as non-muscle invasive (superficial) tumors. The standard treatment for patientswith superficial bladder cancer is transurethral resection (TUR) of tumors. However,approximately60%-70%of these tumors will recur, with10%-25%showing progression toa higher stage or grade within post-operative5years.
Intravesical chemotherapy and/or immunotherapy are widely used as adjuvant therapies toprevent recurrence and progression of superficial disease after TUR. Although manychemical agents have shown some evidence of activity, their toxicity and incompleteefficacy have limited their use as common intravesical agents. These factors highlight theurgent need to search for novel adjuvant intravesical agents to reduce the recurrence rate.
Silibinin, a natural flavonoid, is the major bioactive component of silymarin isolated frommilk thistle. Accumulating evidence indicates that silibinin has anticancer activity in varioustumor cells, including cancers of prostate, breast, skin, colon, lung, and kidney. Themanifold inhibitory effects of silibinin against various cancer cells include growth inhibition,anti-inflammation, cell cycle regulation, apoptosis induction, chemosensitization, inhibitionof angiogenesis, reversal of multidrug resistance and inhibition of invasion and metastasis.Induction of apoptosis is believed to be one of the major mechanisms of action for silibininagainst cancer cells, although the details are yet to be elucidated. There is increasing interestin elucidating the mechanisms of action for silibinin as an effective agent for chemopr-evention and chemotherapy against various types of cancers.
Although silibinin-induced apoptosis in bladder transitional cell carcinoma RT4(representinga well-differentiated papillary noninvasive tumor phenotype), T24(representing high-gradetumor) and TCCSUP (representing high-grade invasive tumor) cells were previouslyreported, the details of molecular mechanisms for apoptotic regulation are yet to beelucidated. Additionally, the extensive work mentioned above focused on the effects of oraladministration of silibinin against bladder cancer. However, intravesical administration of chemotherapeutic agent, instead of oral administration, is widely used as adjuvant therapy toprevent recurrence and progression of superficial bladder cancer after TUR. As the standardof care in individual bladder cancer patients with high-risk clinical and pathologic features(Ta, T1, and Tis), intravesical therapy receives more attention by urologists and their patientsthan oral administration. However, so far there was still no report on the effects ofintravesical silibinin against bladder cancer.
Objectives:
1. The first purpose of this study was to determine the effects of intravesical silibinin againstbladder cancer induced by intravesical instillation of MNU.
2. In the present study, using human bladder cancer5637cells (having the same molecularfeatures of high-risk superficial bladder cancer) as the model system, we also explored themolecular mechanisms of silibinin-induced apoptosis in vitro and in vivo, focusing on twomitochondrial cell death pathways, namely the Cyto c/Omi/caspase-dependent and theAIF/caspase-independent pathways.
Methods:
1. Bladder cancer5637cell viability was assessed using a tetrazolium-based assay (MTTassay). Silibinin-induced apoptosis was determined by TUNEL and/or Annexin V-FITC/PIstaining followed by flow cytometry.
2. Caspase-3and caspase-9activity after silibinin treatment was assessed by a colorimetricsystem. Western Blotting was used to examine the activation of caspase-3、caspase-8、caspase-9and PARP. Flow cytometry and TUNEL staining were used to quantitate apoptoticcells after silibinin and/or z-VAD-fmk treatment.
3. Mitochondrial membrane potential collapse was determined using JC-1probes followedby flow cytometry analysis. Mitochondrial, Cytosolic, Nuclear Fractions and total lysateswere prepared. Subcellular localization of AIF、Cyto C、Smac/DIABLO and HtrA2/Omiwere then analysed by Western Blotting. Immunofluorescence staining was carried out onsilibinin-treated cells with antibody against AIF.
4. The tumor volume, tumor weight, body weight and average diet consumption weremeasured to determine the anti-cancer effects of oral silibinin against human bladder cancerxenografts in nude mice. Apoptosis induction was examined in vivo using the TUNEL assay,which labels DNA strand breaks. The expression of Cleaved Caspase-3, Survivin and AIFwas then assessed by immunohistochemistry and Western blotting.
5. Orthotopic rat bladder cancer model was established by intravesical administration ofMNU. Silibinin was instilled intravesically either beginning at week10after four doses instillation of MNU for8weeks or beginning at week1for17weeks before instillation ofMNU. Diet consumption and activity of each rat were observed daily, while Body weightswere recorded weekly throughout the study. All the rats were sacrificed at week18, andhistopathological changes and average bladder weight of each group were investigated.TUNEL staining was used to investigate the apoptotic effects of intravesical silibinin againstbladder cancer.
Results:
1. Silibinin inhibited the growth of bladder caner5637cells in vitro in a dose-and time-dependent manner as determined by MTT assay. Flow cytometry analysis and TUNELstaining demonstrated that100and200μM silibinin treatment exerted strong apoptoticeffects on5637cells in a dose-dependent manner.
2. Silibinin activated caspase-3and caspase-9, as observed by its increased cysteine proteaseactivity for individual substrate, in a dose-dependent manner. Additionally, silibininincreased the cleaved subunits of caspase-3(17and19kDa), caspase-9(35kDa), and PARP(89kDa), indicating that silibinin induced the activation of caspase cascade. However, pancaspase inhibitor z-VAD-fmk did not completely reverse silibinin-induced apoptosis. Thespecificity and efficacy of z-VAD-fmk activity in5637cells was confirmed by caspase3activity assay which clearly demonstrate that pretreatment with z-VAD-fmk decreasedcaspase-3activity.
3. Using a cationic lipophilic dye JC-1as a marker of mitochondrial membrane potential(ΔΨm), flow cytometric studies revealed that silibinin induced a dose-dependent loss of ΔΨm.In addition,80μM of z-VAD-fmk showed no protective effects on the dissipation of ΔΨminthe cells treated with200μM silibinin for48h. As determined by Western Blotting, theprotein levels of mitochondrial cytochrome c, HtrA2/Omi and AIF decreased in silibinin-treated cells. Concomitantly, compared to untreated group, silibinin treatment stronglyincreased the amount of cytochrome c, HtrA2/Omi and AIF in the cytosolic fraction,indicating a release from the mitochondria into the cytoplasm. Surprisingly, no release ofSmac/DIABLO was observed after silibinin treatment. Additionally, silibinin had no effectson the expression of cytochrome c, HtrA2/Omi, Smac/DIABLO and AIF in total lysates.
4. Exposure to silibinin increased the amount of AIF in the nuclear fractions as determinedby western blotting. Results from immunofluorescence staining also supported the findings.Untreated cells had AIF localized to the cytosol and the treatment with200μM of silibininfor48h resulted in remarkable translocation of AIF to the nucleus. Pretreatment with pancaspase inhibitor z-VAD-fmk was unable to prevent AIF localization to the nucleus.
5. The growth of5637tumor xenografts were inhibited significantly following oral silibinintreatment at the dose levels of200and300mg/kg. The average tumor masses in the control rats were2-to3-fold (P <0.05) greater than that of200and300mg/kg silibinin treated mice.The average body weights and daily diet consumption of the control and silibinin-treatedmice showed no difference throughout the experiment. As determined by TUNEL assay, thenumber of apoptotic cells increased from8.5%in the control group to12.5%,21%(P <0.05)and37.5%(P <0.05) in100,200, and300mg/kg silibinin-treated groups, respectively,demonstrating significant apoptotic effects of silibinin in vivo. Furthermore, the microscopicexamination of stained tumor sections showed decreased expression of survivin andincreased expression of cleaved caspase-3and AIF in the silibinin-treated groups.6. Four doses of intravesical MNU to rats resulted in the induction of hyperplasia, papillarydysplasia, atypia, superficial and muscle invasive bladder carcinoma at the end of the17-week study. There were significant differences in histopathological changes among thesilibinin-treated groups and the MNU or MNU+DMSO group (P <0.05). Intravesicalsilibinin beginning either at week1or at week10effectively inhibited carcinogenesis andprogression of bladder cancer in rats by reducing the incidence of superficial and invasivebladder lesions, suggesting its chemopreventive and chemotherapeutic effects againstbladder cancer. No toxicity, locally or systemically, was observed in rats receivingintravesical silibinin alone. In addition, in vivo apoptotic effects of intravesical silibinin onMNU–induced bladder cancer were observed by TUNEL staining.
Conclusions:
1. Silibinin inhibited cell growth and induced apoptosis in dose-and time-dependent mannerin bladder cancer5637cells.
2. Both intrinsic and extrinsic apoptotic signal pathways were involved in silibinin-inducedapoptosis.
3. Silibinin induced-apoptosis in human bladder cancer was mediated by the activation oftwo mitochondrial death pathways, namely the Cyto c/caspase-dependent and the AIF/caspase-independent pathways involving selective translocation of Omi/HtrA2.
4. Silibinin inhibits the growth of human bladder tumor xenograft in athymic nude mice,which was associated with apoptosis induction, increased translocation of AIF, anddownregulation of survivin.
5. Intravesical silibinin effectively inhibited the carcinogenesis and progression of bladdercancer in rats initiated by MNU by reducing the incidence of superficial and invasive bladderlesions without any side effects, which was accompanied with pro-apoptotic effects.
6. Silibinin may prove to be a new form of intravesical chemotherapy in the inhibition ofcarcinogenesis and progression of bladder cancer, providing a basis for future clinical trialsof intravesical silibinin used in patients with bladder cancer.
引文
[1] Orntoft T F. Personalised treatment for bladder cancer[J]. Lancet Oncol,2011,12(2):111-112.
[2] Weizer A Z, Tallman C, Montgomery J S. Long-term outcomes of intravesical therapy for non-muscle invasive bladder cancer[J]. World J Urol,2011,29(1):59-71.
[3] Williams S K, Hoenig D M, Ghavamian R, et al. Intravesical therapy for bladder cancer[J]. ExpertOpin Pharmacother,2010,11(6):947-958.
[4] Greenlee H, Abascal K, Yarnell E, et al. Clinical applications of Silybum marianum in oncology.[J].Integr Cancer Ther,2007,6(2):158-165.
[5] Zi X, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancerintervention.[J]. Proc Natl Acad Sci U S A,1999,96(13):7490-7495.
[6] Li L, Zeng J, Gao Y, et al. Targeting silibinin in the antiproliferative pathway.[J]. Expert OpinInvestig Drugs,2010,19(2):243-255.
[7] Martin K R. Targeting apoptosis with dietary bioactive agents.[J]. Exp Biol Med (Maywood),2006,231(2):117-129.
[8] Tyagi A, Agarwal C, Harrison G, et al. Silibinin causes cell cycle arrest and apoptosis in humanbladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase3and PARP cleavages.[J]. Carcinogenesis,2004,25(9):1711-1720.
[9] Singh R P, Tyagi A, Sharma G, et al. Oral silibinin inhibits in vivo human bladder tumor xenograftgrowth involving down-regulation of survivin.[J]. Clin Cancer Res,2008,14(1):300-308.
[10] Jacobs B L, Lee C T, Montie J E. Bladder cancer in2010: how far have we come?[J]. CA Cancer JClin,2010,60(4):244-272.
[11] Jemal A, Bray F, Center M M, et al. Global cancer statistics[J]. CA Cancer J Clin,2011,61(2):69-90.
[12] Jemal A, Siegel R, Xu J, et al. Cancer statistics,2010.[J]. CA Cancer J Clin,2010,60(5):277-300.
[13] Yang L, Parkin D M, Ferlay J, et al. Estimates of cancer incidence in China for2000andprojections for2005[J]. Cancer Epidemiol Biomarkers Prev,2005,14(1):243-250.
[14] Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008.[J]. CA Cancer J Clin,2008,58(2):71-96.
[15] Mungan N A, Kiemeney L A, van Dijck J A, et al. Gender differences in stage distribution ofbladder cancer[J]. Urology,2000,55(3):368-371.
[16] Mungan N A, Aben K K, Schoenberg M P, et al. Gender differences in stage-adjusted bladdercancer survival[J]. Urology,2000,55(6):876-880.
[17] Shariat S F, Sfakianos J P, Droller M J, et al. The effect of age and gender on bladder cancer: acritical review of the literature[J]. BJU Int,2010,105(3):300-308.
[18] Campisi J, Yaswen P. Aging and cancer cell biology,2009[J]. Aging Cell,2009,8(3):221-225.
[19] Schultzel M, Saltzstein S L, Downs T M, et al. Late age (85years or older) peak incidence ofbladder cancer[J]. J Urol,2008,179(4):1302-1306.
[20] Wu X R. Urothelial tumorigenesis: a tale of divergent pathways[J]. Nat Rev Cancer,2005,5(9):713-725.
[21] Jankovic S, Radosavljevic V. Risk factors for bladder cancer[J]. Tumori,2007,93(1):4-12.
[22] Kirkali Z, Chan T, Manoharan M, et al. Bladder cancer: epidemiology, staging and grading, anddiagnosis[J]. Urology,2005,66(6Suppl1):4-34.
[23]凌云.膀胱癌的流行病学和预防[J].国外医学(卫生学分册),2002,29(1):31-35.
[24]韩瑞发，潘建刚.中国人群膀胱癌发病危险因素的Meta分析[J].中华泌尿外科杂志,2006,27(4):243-246
[25] Scelo G, Brennan P. The epidemiology of bladder and kidney cancer[J]. Nat Clin Pract Urol,2007,4(4):205-217.
[26] Montironi R, Mazzucchelli R, Scarpelli M, et al. Morphological diagnosis of urothelial neoplasms[J]. J Clin Pathol,2008,61(1):3-10.
[27] Seitz M, Zaak D, Knuchel-Clarke R, et al.[Urinary bladder tumours. The new2004WHOclassification][J]. Urologe A,2005,44(9):1073-1086.
[28] Puppo P, Conti G, Francesca F, et al. New Italian guidelines on bladder cancer, based on the WorldHealth Organization2004classification[J]. BJU Int,2010,106(2):168-179.
[29] Sexton W J, Wiegand L R, Correa J J, et al. Bladder cancer: a review of non-muscle invasivedisease[J]. Cancer Control,2010,17(4):256-268.
[30] Bischoff C J, Clark P E. Bladder cancer.[J]. Curr Opin Oncol,2009,21(3):272-277.
[31] Kaufman D S, Shipley W U, Feldman A S. Bladder cancer.[J]. Lancet,2009,374(9685):239-249.
[32] Sonpavde G, Sternberg C N, Rosenberg J E, et al. Second-line systemic therapy and emergingdrugs for metastatic transitional-cell carcinoma of the urothelium[J]. Lancet Oncol,2010,11(9):861-870.
[33] Stein J P, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladdercancer: long-term results in1,054patients[J]. J Clin Oncol,2001,19(3):666-675.
[34] Babjuk M, Oosterlinck W, Sylvester R, et al. EAU Guidelines on Non-Muscle-Invasive UrothelialCarcinoma of the Bladder, the2011Update[J]. Eur Urol,2011,59(6):997-1008.
[35] Veenema R J. The role of thiotepa instillations in bladder cancer[J]. JAMA,1968,206(12):2725-2726.
[36] Serretta V, Morgia G, Altieri V, et al. A1-year maintenance after early adjuvant intravesicalchemotherapy has a limited efficacy in preventing recurrence of intermediate risk non-muscle-invasive bladder cancer[J]. BJU Int,2010,106(2):212-217.
[37] Cheng C W, Chan S F, Chan L W, et al. Twelve-year follow up of a randomized prospective trialcomparing bacillus Calmette-Guerin and epirubicin as adjuvant therapy in superficial bladdercancer[J]. Int J Urol,2005,12(5):449-455.
[38] Okamura K, Ono Y, Kinukawa T, et al. Randomized study of single early instillation of (2"R)-4'-O-tetrahydropyranyl-doxorubicin for a single superficial bladder carcinoma[J]. Cancer,2002,94(9):2363-2368.
[39] Nilsson S, Ragnhammar P, Glimelius B, et al. A systematic overview of chemotherapy effects inurothelial bladder cancer[J]. Acta Oncol,2001,40(2-3):371-390.
[40] Lamm D L, Riggs D R, Traynelis C L, et al. Apparent failure of current intravesical chemotherapyprophylaxis to influence the long-term course of superficial transitional cell carcinoma of thebladder[J]. J Urol,1995,153(5):1444-1450.
[41] Bohle A, Jocham D, Bock P R. Intravesical bacillus Calmette-Guerin versus mitomycin C forsuperficial bladder cancer: a formal meta-analysis of comparative studies on recurrence and toxicity[J]. J Urol,2003,169(1):90-95.
[42] Sylvester R J, van der Meijden A P, Lamm D L. Intravesical bacillus Calmette-Guerin reduces therisk of progression in patients with superficial bladder cancer: a meta-analysis of the publishedresults of randomized clinical trials[J]. J Urol,2002,168(5):1964-1970.
[43] Malmstrom P U, Sylvester R J, Crawford D E, et al. An individual patient data meta-analysis of thelong-term outcome of randomised studies comparing intravesical mitomycin C versus bacillusCalmette-Guerin for non-muscle-invasive bladder cancer[J]. Eur Urol,2009,56(2):247-256.
[44] Jain A, Phillips R M, Scally A J, et al. Response of multiple recurrent TaT1bladder cancer tointravesical apaziquone (EO9): comparative analysis of tumor recurrence rates[J]. Urology,2009,73(5):1083-1086.
[45] Hendricksen K, van der Heijden A G, Cornel E B, et al. Two-year follow-up of the phase II markerlesion study of intravesical apaziquone for patients with non-muscle invasive bladder cancer[J].World J Urol,2009,27(3):337-342.
[46] Mckiernan J M, Masson P, Murphy A M, et al. Phase I trial of intravesical docetaxel in themanagement of superficial bladder cancer refractory to standard intravesical therapy[J]. J ClinOncol,2006,24(19):3075-3080.
[47] Dalbagni G, Russo P, Bochner B, et al. Phase II trial of intravesical gemcitabine in bacilleCalmette-Guerin-refractory transitional cell carcinoma of the bladder[J]. J Clin Oncol,2006,24(18):2729-2734.
[48] De Berardinis E, Antonini G, Peters G J, et al. Intravesical administration of gemcitabine insuperficial bladder cancer: a phase I study with pharmacodynamic evaluation[J]. BJU Int,2004,93(4):491-494.
[49] Alexandroff A B, Nicholson S, Patel P M, et al. Recent advances in bacillus Calmette-Guerinimmunotherapy in bladder cancer[J]. Immunotherapy,2010,2(4):551-560.
[50] Gontero P, Bohle A, Malmstrom P U, et al. The role of bacillus Calmette-Guerin in the treatment ofnon-muscle-invasive bladder cancer[J]. Eur Urol,2010,57(3):410-429.
[51] Colombel M, Saint F, Chopin D, et al. The effect of ofloxacin on bacillus calmette-guerin inducedtoxicity in patients with superficial bladder cancer: results of a randomized, prospective, double-blind, placebo controlled, multicenter study[J]. J Urol,2006,176(3):935-939.
[52] Agrawal M S, Agrawal M, Bansal S, et al. The safety and efficacy of different doses of bacillusCalmette Guerin in superficial bladder transitional cell carcinoma[J]. Urology,2007,70(6):1075-1078.
[53] Gee J R, Jarrard D F, Bruskewitz R C, et al. Reduced bladder cancer recurrence rate withcardioprotective aspirin after intravesical bacille Calmette-Guerin[J]. BJU Int,2009,103(6):736-739.
[54] Pan C W, Shen Z J, Ding G Q. The effect of intravesical instillation of antifibrinolytic agents onbacillus Calmette-Guerin treatment of superficial bladder cancer: a pilot study[J]. J Urol,2008,179(4):1307-1312.
[55] Jimenez-Cruz J F, Vera-Donoso C D, Leiva O, et al. Intravesical immunoprophylaxis in recurrentsuperficial bladder cancer (Stage T1): multicenter trial comparing bacille Calmette-Guerin andinterferon-alpha[J]. Urology,1997,50(4):529-535.
[56] Rajala P, Kaasinen E, Raitanen M, et al. Perioperative single dose instillation of epirubicin orinterferon-alpha after transurethral resection for the prophylaxis of primary superficial bladdercancer recurrence: a prospective randomized multicenter study--FinnBladder III long-termresults[J]. J Urol,2002,168(3):981-985.
[57] Joudi F N, Smith B J, O'Donnell M A. Final results from a national multicenter phase II trial ofcombination bacillus Calmette-Guerin plus interferon alpha-2B for reducing recurrence ofsuperficial bladder cancer[J]. Urol Oncol,2006,24(4):344-348.
[58] Gelabert-Mas A, Arango T O, Bielsa G O, et al.[A prospective and randomized study of thecomplete response, index of recurrences and progression in superficial bladder carcinoma treatedwith mitomycin C alone versus mitomycin C and BCG alternatively][J]. Arch Esp Urol,1993,46(5):379-382.
[59] Witjes J A, Caris C T, Mungan N A, et al. Results of a randomized phase III trial of sequentialintravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C alone inpatients with superficial bladder cancer[J]. J Urol,1998,160(5):1668-1672.
[60] Solsona E, Iborra I, Ricos J V, et al. Clinical panurothelial disease in patients with superficialbladder tumors: therapeutic implications[J]. J Urol,2002,167(5):2007-2011.
[61] Flora K, Hahn M, Rosen H, et al. Milk thistle (Silybum marianum) for the therapy of liverdisease.[J]. Am J Gastroenterol,1998,93(2):139-143.
[62] Jacobs B P, Dennehy C, Ramirez G, et al. Milk thistle for the treatment of liver disease: asystematic review and meta-analysis.[J]. Am J Med,2002,113(6):506-515.
[63] Deep G, Agarwal R. Antimetastatic efficacy of silibinin: molecular mechanisms and therapeuticpotential against cancer[J]. Cancer Metastasis Rev,2010,29(3):447-463.
[64] Ramasamy K, Agarwal R. Multitargeted therapy of cancer by silymarin.[J]. Cancer Lett,2008,269(2):352-362.
[65] Kaur M, Agarwal R. Silymarin and epithelial cancer chemoprevention: how close we are tobedside?[J]. Toxicol Appl Pharmacol,2007,224(3):350-359.
[66] Gazak R, Walterova D, Kren V. Silybin and silymarin--new and emerging applications inmedicine[J]. Curr Med Chem,2007,14(3):315-338.
[67] Wang H J, Tashiro S, Onodera S, et al. Inhibition of insulin-like growth factor1receptor signalingenhanced silibinin-induced activation of death receptor and mitochondrial apoptotic pathways inhuman breast cancer MCF-7cells.[J]. J Pharmacol Sci,2008,107(3):260-269.
[68] Son Y G, Kim E H, Kim J Y, et al. Silibinin sensitizes human glioma cells to TRAIL-mediatedapoptosis via DR5up-regulation and down-regulation of c-FLIP and survivin.[J]. CancerRes,2007,67(17):8274-8284.
[69] Yoo H G, Jung S N, Hwang Y S, et al. Involvement of NF-kappaB and caspases in silibinin-induced apoptosis of endothelial cells.[J]. Int J Mol Med,2004,13(1):81-86.
[70] Cui W, Gu F, Hu K Q. Effects and mechanisms of silibinin on human hepatocellular carcinomaxenografts in nude mice.[J]. World J Gastroenterol,2009,15(16):1943-1950.
[71] Bang C I, Paik S Y, Sun D I, et al. Cell growth inhibition and down-regulation of survivin bysilibinin in a laryngeal squamous cell carcinoma cell line.[J]. Ann Otol Rhinol Laryngol,2008,117(10):781-785.
[72] Li L, Gao Y, Zhang L, et al. Silibinin inhibits cell growth and induces apoptosis by caspaseactivation, down-regulating survivin and blocking EGFR-ERK activation in renal cell carcinoma.[J]. Cancer Lett,2008,272(1):61-69.
[73] Hubbard S R, Miller W T. Receptor tyrosine kinases: mechanisms of activation and signaling.[J].Curr Opin Cell Biol,2007,19(2):117-123.
[74] Gschwind A, Fischer O M, Ullrich A. The discovery of receptor tyrosine kinases: targets for cancertherapy.[J]. Nat Rev Cancer,2004,4(5):361-370.
[75] Sharma Y, Agarwal C, Singh A K, et al. Inhibitory effect of silibinin on ligand binding to erbB1and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostatecarcinoma cells.[J]. Mol Carcinog,2001,30(4):224-236.
[76] Tyagi A, Sharma Y, Agarwal C, et al. Silibinin impairs constitutively active TGFalpha-EGFRautocrine loop in advanced human prostate carcinoma cells.[J]. Pharm Res,2008,25(9):2143-2150.
[77] Qi L, Singh R P, Lu Y, et al. Epidermal growth factor receptor mediates silibinin-inducedcytotoxicity in a rat glioma cell line.[J]. Cancer Biol Ther,2003,2(5):526-531.
[78] Pollak M. Insulin and insulin-like growth factor signalling in neoplasia.[J]. Nat Rev Cancer,2008,8(12):915-928.
[79] Singh R P, Sharma G, Dhanalakshmi S, et al. Suppression of advanced human prostate tumorgrowth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increasedapoptosis, and inhibition of angiogenesis.[J]. Cancer Epidemiol Biomarkers Prev,2003,12(9):933-939.
[80] Raina K, Blouin M J, Singh R P, et al. Dietary feeding of silibinin inhibits prostate tumor growthand progression in transgenic adenocarcinoma of the mouse prostate model.[J]. CancerRes,2007,67(22):11083-11091.
[81] Cheung C W, Taylor P J, Kirkpatrick C M, et al. Therapeutic value of orally administered silibininin renal cell carcinoma: manipulation of insulin-like growth factor binding protein-3levels.[J]. BJUInt,2007,100(2):438-444.
[82] Klampfer L. Signal transducers and activators of transcription (STATs): Novel targets ofchemopreventive and chemotherapeutic drugs.[J]. Curr Cancer Drug Targets,2006,6(2):107-121.
[83] Turkson J. STAT proteins as novel targets for cancer drug discovery.[J]. Expert Opin TherTargets,2004,8(5):409-422.
[84] Agarwal C, Tyagi A, Kaur M, et al. Silibinin inhibits constitutive activation of Stat3, and causescaspase activation and apoptotic death of human prostate carcinoma DU145cells.[J].Carcinogenesis,2007,28(7):1463-1470.
[85] Singh R P, Raina K, Deep G, et al. Silibinin suppresses growth of human prostate carcinoma PC-3orthotopic xenograft via activation of extracellular signal-regulated kinase1/2and inhibition ofsignal transducers and activators of transcription signaling.[J]. Clin Cancer Res,2009,15(2):613-621.
[86] Tyagi A, Singh R P, Ramasamy K, et al. Growth inhibition and regression of lung tumors bysilibinin: modulation of angiogenesis by macrophage-associated cytokines and nuclear factor-kappaB and signal transducers and activators of transcription3.[J]. Cancer Prev Res (PhilaPa),2009,2(1):74-83.
[87] Annunziata C M, Davis R E, Demchenko Y, et al. Frequent engagement of the classical andalternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma.[J]. CancerCell,2007,12(2):115-130.
[88] Naugler W E, Karin M. NF-kappaB and cancer-identifying targets and mechanisms.[J]. Curr OpinGenet Dev,2008,18(1):19-26.
[89] Van Waes C. Nuclear factor-kappaB in development, prevention, and therapy of cancer.[J]. ClinCancer Res,2007,13(4):1076-1082.
[90] Dhanalakshmi S, Singh R P, Agarwal C, et al. Silibinin inhibits constitutive and TNFalpha-inducedactivation of NF-kappaB and sensitizes human prostate carcinoma DU145cells to TNFalpha-induced apoptosis.[J]. Oncogene,2002,21(11):1759-1767.
[91] Tyagi A, Raina K, Singh R P, et al. Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder carcinogenesis in male ICR mice.[J]. MolCancer Ther,2007,6(12):3248-3255.
[92] Singh R P, Dhanalakshmi S, Agarwal C, et al. Silibinin strongly inhibits growth and survival ofhuman endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-kappaB:implications for angioprevention and antiangiogenic therapy.[J]. Oncogene,2005,24(7):1188-1202.
[93] Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.[J]. Nat RevCancer,2009,9(3):153-166.
[94] Lapenna S, Giordano A. Cell cycle kinases as therapeutic targets for cancer.[J]. Nat Rev DrugDiscov,2009,8(7):547-566.
[95] Meeran S M, Katiyar S K. Cell cycle control as a basis for cancer chemoprevention through dietaryagents.[J]. Front Biosci,2008,13:2191-2202.
[96] Agarwal C, Singh R P, Dhanalakshmi S, et al. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinomaHT-29cells.[J]. Oncogene,2003,22(51):8271-8282.
[97] Kaur M, Velmurugan B, Tyagi A, et al. Silibinin suppresses growth and induces apoptotic death ofhuman colorectal carcinoma LoVo cells in culture and tumor xenograft.[J]. Mol Cancer Ther,2009,8(8):2366–2374
[98] Yang S H, Lin J K, Huang C J, et al. Silibinin inhibits angiogenesis via Flt-1, but not KDR,receptor up-regulation[J]. J Surg Res,2005,128(1):140-146.
[99] Gallo D, Giacomelli S, Ferlini C, et al. Antitumour activity of the silybin-phosphatidylcholinecomplex, IdB1016, against human ovarian cancer[J]. Eur J Cancer,2003,39(16):2403-2410.
[100] Singh R P, Raina K, Sharma G, et al. Silibinin inhibits established prostate tumor growth,progression, invasion, and metastasis and suppresses tumor angiogenesis and epithelial-mesenchymal transition in transgenic adenocarcinoma of the mouse prostate model mice.[J]. ClinCancer Res,2008,14(23):7773-7780.
[101] Singh R P, Deep G, Chittezhath M, et al. Effect of silibinin on the growth and progression ofprimary lung tumors in mice.[J]. J Natl Cancer Inst,2006,98(12):846-855.
[102] Velmurugan B, Gangar S C, Kaur M, et al. Silibinin exerts sustained growth suppressive effectagainst human colon carcinoma SW480xenograft by targeting multiple signaling molecules[J].Pharm Res,2010,27(10):2085-2097.
[103] Thiery J P, Acloque H, Huang R Y, et al. Epithelial-mesenchymal transitions in development anddisease.[J]. Cell,2009,139(5):871-890.
[104] Gupta S C, Kim J H, Prasad S, et al. Regulation of survival, proliferation, invasion, angiogenesis,and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals[J].Cancer Metastasis Rev,2010,29(3):405-434.
[105] Raina K, Rajamanickam S, Singh R P, et al. Stage-specific inhibitory effects and associatedmechanisms of silibinin on tumor progression and metastasis in transgenic adenocarcinoma of themouse prostate model[J]. Cancer Res,2008,68(16):6822-6830.
[106] Mokhtari M J, Motamed N, Shokrgozar M A. Evaluation of silibinin on the viability, migration andadhesion of the human prostate adenocarcinoma (PC-3) cell line[J]. Cell Biol Int,2008,32(8):888-892.
[107] Wu K, Zeng J, Li L, et al. Silibinin reverses epithelial-to-mesenchymal transition in metastaticprostate cancer cells by targeting transcription factors.[J]. Oncol Rep,2010,23(6):1545-1552.
[108] Wu K J, Zeng J, Zhu G D, et al. Silibinin inhibits prostate cancer invasion, motility and migrationby suppressing vimentin and MMP-2expression.[J]. Acta Pharmacol Sin,2009,30(8):1162-1168.
[109] Chen P N, Hsieh Y S, Chiou H L, et al. Silibinin inhibits cell invasion through inactivation of bothPI3K-Akt and MAPK signaling pathways[J]. Chem Biol Interact,2005,156(2-3):141-150.
[110] Lee S O, Jeong Y J, Im H G, et al. Silibinin suppresses PMA-induced MMP-9expression byblocking the AP-1activation via MAPK signaling pathways in MCF-7human breast carcinomacells.[J]. Biochem Biophys Res Commun,2007,354(1):165-171.
[111] Hsieh Y S, Chu S C, Yang S F, et al. Silibinin suppresses human osteosarcoma MG-63cellinvasion by inhibiting the ERK-dependent c-Jun/AP-1induction of MMP-2.[J]. Carcinogenesis,2007,28(5):977-987.
[112] Baguley B C. Multiple drug resistance mechanisms in cancer[J]. Mol Biotechnol,2010,46(3):308-316.
[113] Higgins C F. Multiple molecular mechanisms for multidrug resistance transporters[J]. Nature,2007,446(7137):749-757.
[114] Tyagi A K, Agarwal C, Chan D C, et al. Synergistic anti-cancer effects of silibinin withconventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breastcarcinoma MCF-7and MDA-MB468cells[J]. Oncol Rep,2004,11(2):493-499.
[115] Dhanalakshmi S, Agarwal P, Glode L M, et al. Silibinin sensitizes human prostate carcinomaDU145cells to cisplatin-and carboplatin-induced growth inhibition and apoptotic death[J]. Int JCancer,2003,106(5):699-705.
[116] Rho J K, Choi Y J, Jeon B S, et al. Combined treatment with silibinin and epidermal growth factorreceptor tyrosine kinase inhibitors overcomes drug resistance caused by T790M mutation[J]. MolCancer Ther,2010,9(12):3233-3243.
[117] Agarwal R, Agarwal C, Ichikawa H, et al. Anticancer potential of silymarin: from bench to bedside.[J]. Anticancer Res,2006,26(6B):4457-4498.
[118] Tyagi A, Singh R P, Agarwal C, et al. Silibinin activates p53-caspase2pathway and causescaspase-mediated cleavage of Cip1/p21in apoptosis induction in bladder transitional-cell papillomaRT4cells: evidence for a regulatory loop between p53and caspase2.[J]. Carcinogenesis,2006,27(11):2269-2280.
[119] Santiago A R, Cristovao A J, Santos P F, et al. High glucose induces caspase-independent celldeath in retinal neural cells.[J]. Neurobiol Dis,2007,25(3):464-472.
[120] Thornberry N A, Lazebnik Y. Caspases: enemies within.[J]. Science,1998,281(5381):1312-1316.
[121] Shankar S L, Mani S, O G K, et al. Survivin inhibition induces human neural tumor cell deaththrough caspase-independent and-dependent pathways.[J]. J Neurochem,2001,79(2):426-436.
[122] Joza N, Susin S A, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor inprogrammed cell death.[J]. Nature,2001,410(6828):549-554.
[123] Jr Hail N. Mitochondria: A novel target for the chemoprevention of cancer.[J]. Apoptosis,2005,10(4):687-705.
[124] Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase8mediates the mitochondrial damage in theFas pathway of apoptosis.[J]. Cell,1998,94(4):491-501.
[125] Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl2interacting protein, mediates cytochrome c releasefrom mitochondria in response to activation of cell surface death receptors.[J]. Cell,1998,94(4):481-490.
[126] Wang C, Youle R J. The role of mitochondria in apoptosis.[J]. Annu Rev Genet,2009,43:95-118.
[127] Kroemer G, Reed J C. Mitochondrial control of cell death.[J]. Nat Med,2000,6(5):513-519.
[128] Lorenzo H K, Susin S A. Therapeutic potential of AIF-mediated caspase-independent programmedcell death.[J]. Drug Resist Updat,2007,10(6):235-255.
[129] Kuranaga E. Caspase signaling in animal development[J]. Dev Growth Differ,2011,53(2):137-148.
[130] Ghobrial I M, Witzig T E, Adjei A A. Targeting apoptosis pathways in cancer therapy.[J]. CACancer J Clin,2005,55(3):178-194.
[131] Crawford E D, Wells J A. Caspase Substrates and Cellular Remodeling[J]. Annu Rev Biochem,2011,80:1055-1087.
[132] Ledgerwood E C, Morison I M. Targeting the apoptosome for cancer therapy.[J]. Clin Cancer Res,2009,15(2):420-424.
[133] Olsson M, Zhivotovsky B. Caspases and cancer[J]. Cell Death Differ,2011,18(9):1441-1449.
[134] van Loo G, van Gurp M, Depuydt B, et al. The serine protease Omi/HtrA2is released frommitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhancedcaspase activity.[J]. Cell Death Differ,2002,9(1):20-26.
[135] Daugas E, Nochy D, Ravagnan L, et al. Apoptosis-inducing factor (AIF): a ubiquitousmitochondrial oxidoreductase involved in apoptosis.[J]. FEBS Lett,2000,476(3):118-123.
[136] Susin S A, Lorenzo H K, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor.[J]. Nature,1999,397(6718):441-446.
[137] Kanwar J R, Kamalapuram S K, Kanwar R K. Targeting survivin in cancer: the cell-signallingperspective[J]. Drug Discov Today,2011,16(11-12):485-94
[138] Carrasco R A, Stamm N B, Marcusson E, et al. Antisense inhibition of survivin expression as acancer therapeutic[J]. Mol Cancer Ther,2011,10(2):221-232.
[139] Li F, Ambrosini G, Chu E Y, et al. Control of apoptosis and mitotic spindle checkpoint bysurvivin[J]. Nature,1998,396(6711):580-584.
[140] Margulis V, Lotan Y, Shariat S F. Survivin: a promising biomarker for detection and prognosis ofbladder cancer.[J]. World J Urol,2008,26(1):59-65.
[141] Altieri D C. Survivin, cancer networks and pathway-directed drug discovery[J]. Nat RevCancer,2008,8(1):61-70.
[142] Cui L, Shi Y, Dai G, et al. Modification of N-Methyl-N-Nitrosourea initiated bladdercarcinogenesis in Wistar rats by terephthalic acid.[J]. Toxicol Appl Pharmacol,2006,210(1-2):24-31.
[143]钱立新，刘训良，丁鸿，等. N-甲基亚硝基脲诱导大鼠膀胱肿瘤作用的动态观察[J].中华实验外科杂志,2004,21(5):563-564.
[144]刘红耀，李晓东，米振国，等. N-甲基亚硝基脲的制备及其诱发膀胱肿瘤的作用[J].中华实验外科杂志,2000,17(6):548-549
[145] Steinberg G D, Brendler C B, Squire R A, et al. Experimental intravesical therapy for superficialtransitional cell carcinoma in a rat bladder tumor model.[J]. J Urol,1991,145(3):647-653.
[146] Steinberg G D, Brendler C B, Ichikawa T, et al. Characterization of an N-methyl-N-nitrosourea-induced autochthonous rat bladder cancer model.[J]. Cancer Res,1990,50(20):6668-6674.
[147] Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases.[J].Drugs,2001,61(14):2035-2063.
[148] Crallan R A, Georgopoulos N T, Southgate J. Experimental models of human bladdercarcinogenesis[J]. Carcinogenesis,2006,27(3):374-381.
[149]颜汝平，王剑松，徐鸿毅.膀胱癌的动物实验模型[J].国外医学.泌尿系统分册,2005,25(4):446-449.
[150]刘俊国，韩瑞发.动物膀胱癌原位模型建立和治疗实验研究进展[J].国际泌尿系统杂志,2006,26(6):748-752.
[151] Schalken J A, van Moorselaar R J, Bringuier P P, et al. Critical review of the models to study thebiologic progression of bladder cancer[J]. Semin Surg Oncol,1992,8(5):274-278.
[152]赵献，颜汝平，李翀，等. OH-BBN诱导不同鼠类膀胱肿瘤的研究现状[J].国际泌尿系统杂志,2010,30(5):649-652
[153] Herman C J, Vegt P D, Debruyne F M, et al. Squamous and transitional elements in rat bladdercarcinomas induced by N-butyl-N-4-hydroxybutyl-nitrosamine (BBN). A study of cytokeratinexpression[J]. Am J Pathol,1985,120(3):419-426.
[154]李云，许传亮，李志华，等.两种构建大鼠膀胱癌模型方法的比较研究[J].临床泌尿外科杂志,2008,23(10):783-784.
[155] Arentsen H C, Hendricksen K, Oosterwijk E, et al. Experimental rat bladder urothelial cellcarcinoma models[J]. World J Urol,2009,27(3):313-317.
[156] Oliveira P A, Colaco A, De la Cruz P L, et al. Experimental bladder carcinogenesis-rodentmodels[J]. Exp Oncol,2006,28(1):2-11.
[157] Drago J R. The Noble rat bladder cancer model. FANFT-induced tumors[J]. Cancer,1984,53(5):1093-1099.
[158] Hicks R M, Wakefield J S. Rapid induction of bladder cancer in rats with N-methyl-N-nitrosourea.I. Histology[J]. Chem Biol Interact,1972,5(2):139-152.
[159] Hamamoto Y, Hamamoto N, Nakajima T, et al. Morphological changes of epithelial rests ofMalassez in rat molars induced by local administration of N-methylnitrosourea.[J]. Arch OralBiol,1998,43(11):899-906.
[160] Knowles M A, Jani H, Hicks R M, et al. N-methyl-N-nitrosourea induces dysplasia and cell surfacemarkers of neoplasia in long-term rat bladder organ cultures.[J]. Carcinogenesis,1985,6(7):1047-1054.
[161] Irving C C, Cox R, Murphy W M. Influence of dose of N-methyl-N-nitrosourea on the induction ofurinary bladder cancer in rats[J]. Cancer Lett,1979,8(1):3-7.
[162]刘红耀，庞东梓，李梦强，等. MNU诱导的近交系大鼠膀胱肿瘤模型[J].肿瘤研究与临床,2005,17(2):89-90.
[163]罗春丽，胡宏波，蔡晓钟，等. MNU诱发SD大鼠膀胱肿瘤原位模型[J].重庆医学,2007,36(14):1352-1354.
[164] Severs N J, Barnes S H, Wright R, et al. Induction of bladder cancer in rats by fractionatedintravesicular doses of N-methyl-N-nitrosourea[J]. Br J Cancer,1982,45(3):337-351.
[165] Chen D, Song D, Wientjes M G, et al. Effect of dimethyl sulfoxide on bladder tissue penetration ofintravesical paclitaxel[J]. Clin Cancer Res,2003,9(1):363-369.
[166] Schoenfeld R H, Belville W D, Jacob W H, et al. The effect of dimethyl sulfoxide on the uptake ofcisplatin from the urinary bladder of the dog: a pilot study[J]. J Am Osteopath Assoc,1983,82(8):570-573.
[167] Hashimoto H, Tokunaka S, Sasaki M, et al. Dimethylsulfoxide enhances the absorption ofchemotherapeutic drug instilled into the bladder[J]. Urol Res,1992,20(3):233-236.
[168] See W A, Xia Q. Regional chemotherapy for bladder neoplasms using continuous intravesicalinfusion of doxorubicin: impact of concomitant administration of dimethyl sulfoxide on drugabsorption and antitumor activity[J]. J Natl Cancer Inst,1992,84(7):510-515.
[169] Hanno P M, Burks D A, Clemens J Q, et al. AUA Guideline for the Diagnosis and Treatment ofInterstitial Cystitis/Bladder Pain Syndrome[J]. J Urol,2011,185(6):2162-2170
[170] Fall M, Baranowski A P, Elneil S, et al. EAU guidelines on chronic pelvic pain[J]. Eur Urol,2010,57(1):35-48.
[171] Michaud D S. Chronic inflammation and bladder cancer[J]. Urol Oncol,2007,25(3):260-268.
[172] Kantari C, Walczak H. Caspase-8and bid: caught in the act between death receptors andmitochondria[J]. Biochim Biophys Acta,2011,1813(4):558-563.
[173] Beere H M, Wolf B B, Cain K, et al. Heat-shock protein70inhibits apoptosis by preventingrecruitment of procaspase-9to the Apaf-1apoptosome[J]. Nat Cell Biol,2000,2(8):469-475.
[174] Saleh A, Srinivasula S M, Balkir L, et al. Negative regulation of the Apaf-1apoptosome byHsp70[J]. Nat Cell Biol,2000,2(8):476-483.
[175] Gurbuxani S, Schmitt E, Cande C, et al. Heat shock protein70binding inhibits the nuclear importof apoptosis-inducing factor[J]. Oncogene,2003,22(43):6669-6678.
[176] Ravagnan L, Gurbuxani S, Susin S A, et al. Heat-shock protein70antagonizes apoptosis-inducingfactor[J]. Nat Cell Biol,2001,3(9):839-843.
[177] Raina K, Agarwal R. Combinatorial strategies for cancer eradication by silibinin and cytotoxicagents: efficacy and mechanisms.[J]. Acta Pharmacol Sin,2007,28(9):1466-1475.
[2] Weizer A Z, Tallman C, Montgomery J S. Long-term outcomes of intravesical therapy for non-muscle invasive bladder cancer[J]. World J Urol,2011,29(1):59-71.
[3] Williams S K, Hoenig D M, Ghavamian R, et al. Intravesical therapy for bladder cancer[J]. ExpertOpin Pharmacother,2010,11(6):947-958.
[4] Greenlee H, Abascal K, Yarnell E, et al. Clinical applications of Silybum marianum in oncology.[J].Integr Cancer Ther,2007,6(2):158-165.
[5] Zi X, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancerintervention.[J]. Proc Natl Acad Sci U S A,1999,96(13):7490-7495.
[6] Li L, Zeng J, Gao Y, et al. Targeting silibinin in the antiproliferative pathway.[J]. Expert OpinInvestig Drugs,2010,19(2):243-255.
[7] Martin K R. Targeting apoptosis with dietary bioactive agents.[J]. Exp Biol Med (Maywood),2006,231(2):117-129.
[8] Tyagi A, Agarwal C, Harrison G, et al. Silibinin causes cell cycle arrest and apoptosis in humanbladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase3and PARP cleavages.[J]. Carcinogenesis,2004,25(9):1711-1720.
[9] Singh R P, Tyagi A, Sharma G, et al. Oral silibinin inhibits in vivo human bladder tumor xenograftgrowth involving down-regulation of survivin.[J]. Clin Cancer Res,2008,14(1):300-308.
[10] Jacobs B L, Lee C T, Montie J E. Bladder cancer in2010: how far have we come?[J]. CA Cancer JClin,2010,60(4):244-272.
[11] Jemal A, Bray F, Center M M, et al. Global cancer statistics[J]. CA Cancer J Clin,2011,61(2):69-90.
[12] Jemal A, Siegel R, Xu J, et al. Cancer statistics,2010.[J]. CA Cancer J Clin,2010,60(5):277-300.
[13] Yang L, Parkin D M, Ferlay J, et al. Estimates of cancer incidence in China for2000andprojections for2005[J]. Cancer Epidemiol Biomarkers Prev,2005,14(1):243-250.
[14] Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008.[J]. CA Cancer J Clin,2008,58(2):71-96.
[15] Mungan N A, Kiemeney L A, van Dijck J A, et al. Gender differences in stage distribution ofbladder cancer[J]. Urology,2000,55(3):368-371.
[16] Mungan N A, Aben K K, Schoenberg M P, et al. Gender differences in stage-adjusted bladdercancer survival[J]. Urology,2000,55(6):876-880.
[17] Shariat S F, Sfakianos J P, Droller M J, et al. The effect of age and gender on bladder cancer: acritical review of the literature[J]. BJU Int,2010,105(3):300-308.
[18] Campisi J, Yaswen P. Aging and cancer cell biology,2009[J]. Aging Cell,2009,8(3):221-225.
[19] Schultzel M, Saltzstein S L, Downs T M, et al. Late age (85years or older) peak incidence ofbladder cancer[J]. J Urol,2008,179(4):1302-1306.
[20] Wu X R. Urothelial tumorigenesis: a tale of divergent pathways[J]. Nat Rev Cancer,2005,5(9):713-725.
[21] Jankovic S, Radosavljevic V. Risk factors for bladder cancer[J]. Tumori,2007,93(1):4-12.
[22] Kirkali Z, Chan T, Manoharan M, et al. Bladder cancer: epidemiology, staging and grading, anddiagnosis[J]. Urology,2005,66(6Suppl1):4-34.
[23]凌云.膀胱癌的流行病学和预防[J].国外医学(卫生学分册),2002,29(1):31-35.
[24]韩瑞发，潘建刚.中国人群膀胱癌发病危险因素的Meta分析[J].中华泌尿外科杂志,2006,27(4):243-246
[25] Scelo G, Brennan P. The epidemiology of bladder and kidney cancer[J]. Nat Clin Pract Urol,2007,4(4):205-217.
[26] Montironi R, Mazzucchelli R, Scarpelli M, et al. Morphological diagnosis of urothelial neoplasms[J]. J Clin Pathol,2008,61(1):3-10.
[27] Seitz M, Zaak D, Knuchel-Clarke R, et al.[Urinary bladder tumours. The new2004WHOclassification][J]. Urologe A,2005,44(9):1073-1086.
[28] Puppo P, Conti G, Francesca F, et al. New Italian guidelines on bladder cancer, based on the WorldHealth Organization2004classification[J]. BJU Int,2010,106(2):168-179.
[29] Sexton W J, Wiegand L R, Correa J J, et al. Bladder cancer: a review of non-muscle invasivedisease[J]. Cancer Control,2010,17(4):256-268.
[30] Bischoff C J, Clark P E. Bladder cancer.[J]. Curr Opin Oncol,2009,21(3):272-277.
[31] Kaufman D S, Shipley W U, Feldman A S. Bladder cancer.[J]. Lancet,2009,374(9685):239-249.
[32] Sonpavde G, Sternberg C N, Rosenberg J E, et al. Second-line systemic therapy and emergingdrugs for metastatic transitional-cell carcinoma of the urothelium[J]. Lancet Oncol,2010,11(9):861-870.
[33] Stein J P, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladdercancer: long-term results in1,054patients[J]. J Clin Oncol,2001,19(3):666-675.
[34] Babjuk M, Oosterlinck W, Sylvester R, et al. EAU Guidelines on Non-Muscle-Invasive UrothelialCarcinoma of the Bladder, the2011Update[J]. Eur Urol,2011,59(6):997-1008.
[35] Veenema R J. The role of thiotepa instillations in bladder cancer[J]. JAMA,1968,206(12):2725-2726.
[36] Serretta V, Morgia G, Altieri V, et al. A1-year maintenance after early adjuvant intravesicalchemotherapy has a limited efficacy in preventing recurrence of intermediate risk non-muscle-invasive bladder cancer[J]. BJU Int,2010,106(2):212-217.
[37] Cheng C W, Chan S F, Chan L W, et al. Twelve-year follow up of a randomized prospective trialcomparing bacillus Calmette-Guerin and epirubicin as adjuvant therapy in superficial bladdercancer[J]. Int J Urol,2005,12(5):449-455.
[38] Okamura K, Ono Y, Kinukawa T, et al. Randomized study of single early instillation of (2"R)-4'-O-tetrahydropyranyl-doxorubicin for a single superficial bladder carcinoma[J]. Cancer,2002,94(9):2363-2368.
[39] Nilsson S, Ragnhammar P, Glimelius B, et al. A systematic overview of chemotherapy effects inurothelial bladder cancer[J]. Acta Oncol,2001,40(2-3):371-390.
[40] Lamm D L, Riggs D R, Traynelis C L, et al. Apparent failure of current intravesical chemotherapyprophylaxis to influence the long-term course of superficial transitional cell carcinoma of thebladder[J]. J Urol,1995,153(5):1444-1450.
[41] Bohle A, Jocham D, Bock P R. Intravesical bacillus Calmette-Guerin versus mitomycin C forsuperficial bladder cancer: a formal meta-analysis of comparative studies on recurrence and toxicity[J]. J Urol,2003,169(1):90-95.
[42] Sylvester R J, van der Meijden A P, Lamm D L. Intravesical bacillus Calmette-Guerin reduces therisk of progression in patients with superficial bladder cancer: a meta-analysis of the publishedresults of randomized clinical trials[J]. J Urol,2002,168(5):1964-1970.
[43] Malmstrom P U, Sylvester R J, Crawford D E, et al. An individual patient data meta-analysis of thelong-term outcome of randomised studies comparing intravesical mitomycin C versus bacillusCalmette-Guerin for non-muscle-invasive bladder cancer[J]. Eur Urol,2009,56(2):247-256.
[44] Jain A, Phillips R M, Scally A J, et al. Response of multiple recurrent TaT1bladder cancer tointravesical apaziquone (EO9): comparative analysis of tumor recurrence rates[J]. Urology,2009,73(5):1083-1086.
[45] Hendricksen K, van der Heijden A G, Cornel E B, et al. Two-year follow-up of the phase II markerlesion study of intravesical apaziquone for patients with non-muscle invasive bladder cancer[J].World J Urol,2009,27(3):337-342.
[46] Mckiernan J M, Masson P, Murphy A M, et al. Phase I trial of intravesical docetaxel in themanagement of superficial bladder cancer refractory to standard intravesical therapy[J]. J ClinOncol,2006,24(19):3075-3080.
[47] Dalbagni G, Russo P, Bochner B, et al. Phase II trial of intravesical gemcitabine in bacilleCalmette-Guerin-refractory transitional cell carcinoma of the bladder[J]. J Clin Oncol,2006,24(18):2729-2734.
[48] De Berardinis E, Antonini G, Peters G J, et al. Intravesical administration of gemcitabine insuperficial bladder cancer: a phase I study with pharmacodynamic evaluation[J]. BJU Int,2004,93(4):491-494.
[49] Alexandroff A B, Nicholson S, Patel P M, et al. Recent advances in bacillus Calmette-Guerinimmunotherapy in bladder cancer[J]. Immunotherapy,2010,2(4):551-560.
[50] Gontero P, Bohle A, Malmstrom P U, et al. The role of bacillus Calmette-Guerin in the treatment ofnon-muscle-invasive bladder cancer[J]. Eur Urol,2010,57(3):410-429.
[51] Colombel M, Saint F, Chopin D, et al. The effect of ofloxacin on bacillus calmette-guerin inducedtoxicity in patients with superficial bladder cancer: results of a randomized, prospective, double-blind, placebo controlled, multicenter study[J]. J Urol,2006,176(3):935-939.
[52] Agrawal M S, Agrawal M, Bansal S, et al. The safety and efficacy of different doses of bacillusCalmette Guerin in superficial bladder transitional cell carcinoma[J]. Urology,2007,70(6):1075-1078.
[53] Gee J R, Jarrard D F, Bruskewitz R C, et al. Reduced bladder cancer recurrence rate withcardioprotective aspirin after intravesical bacille Calmette-Guerin[J]. BJU Int,2009,103(6):736-739.
[54] Pan C W, Shen Z J, Ding G Q. The effect of intravesical instillation of antifibrinolytic agents onbacillus Calmette-Guerin treatment of superficial bladder cancer: a pilot study[J]. J Urol,2008,179(4):1307-1312.
[55] Jimenez-Cruz J F, Vera-Donoso C D, Leiva O, et al. Intravesical immunoprophylaxis in recurrentsuperficial bladder cancer (Stage T1): multicenter trial comparing bacille Calmette-Guerin andinterferon-alpha[J]. Urology,1997,50(4):529-535.
[56] Rajala P, Kaasinen E, Raitanen M, et al. Perioperative single dose instillation of epirubicin orinterferon-alpha after transurethral resection for the prophylaxis of primary superficial bladdercancer recurrence: a prospective randomized multicenter study--FinnBladder III long-termresults[J]. J Urol,2002,168(3):981-985.
[57] Joudi F N, Smith B J, O'Donnell M A. Final results from a national multicenter phase II trial ofcombination bacillus Calmette-Guerin plus interferon alpha-2B for reducing recurrence ofsuperficial bladder cancer[J]. Urol Oncol,2006,24(4):344-348.
[58] Gelabert-Mas A, Arango T O, Bielsa G O, et al.[A prospective and randomized study of thecomplete response, index of recurrences and progression in superficial bladder carcinoma treatedwith mitomycin C alone versus mitomycin C and BCG alternatively][J]. Arch Esp Urol,1993,46(5):379-382.
[59] Witjes J A, Caris C T, Mungan N A, et al. Results of a randomized phase III trial of sequentialintravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C alone inpatients with superficial bladder cancer[J]. J Urol,1998,160(5):1668-1672.
[60] Solsona E, Iborra I, Ricos J V, et al. Clinical panurothelial disease in patients with superficialbladder tumors: therapeutic implications[J]. J Urol,2002,167(5):2007-2011.
[61] Flora K, Hahn M, Rosen H, et al. Milk thistle (Silybum marianum) for the therapy of liverdisease.[J]. Am J Gastroenterol,1998,93(2):139-143.
[62] Jacobs B P, Dennehy C, Ramirez G, et al. Milk thistle for the treatment of liver disease: asystematic review and meta-analysis.[J]. Am J Med,2002,113(6):506-515.
[63] Deep G, Agarwal R. Antimetastatic efficacy of silibinin: molecular mechanisms and therapeuticpotential against cancer[J]. Cancer Metastasis Rev,2010,29(3):447-463.
[64] Ramasamy K, Agarwal R. Multitargeted therapy of cancer by silymarin.[J]. Cancer Lett,2008,269(2):352-362.
[65] Kaur M, Agarwal R. Silymarin and epithelial cancer chemoprevention: how close we are tobedside?[J]. Toxicol Appl Pharmacol,2007,224(3):350-359.
[66] Gazak R, Walterova D, Kren V. Silybin and silymarin--new and emerging applications inmedicine[J]. Curr Med Chem,2007,14(3):315-338.
[67] Wang H J, Tashiro S, Onodera S, et al. Inhibition of insulin-like growth factor1receptor signalingenhanced silibinin-induced activation of death receptor and mitochondrial apoptotic pathways inhuman breast cancer MCF-7cells.[J]. J Pharmacol Sci,2008,107(3):260-269.
[68] Son Y G, Kim E H, Kim J Y, et al. Silibinin sensitizes human glioma cells to TRAIL-mediatedapoptosis via DR5up-regulation and down-regulation of c-FLIP and survivin.[J]. CancerRes,2007,67(17):8274-8284.
[69] Yoo H G, Jung S N, Hwang Y S, et al. Involvement of NF-kappaB and caspases in silibinin-induced apoptosis of endothelial cells.[J]. Int J Mol Med,2004,13(1):81-86.
[70] Cui W, Gu F, Hu K Q. Effects and mechanisms of silibinin on human hepatocellular carcinomaxenografts in nude mice.[J]. World J Gastroenterol,2009,15(16):1943-1950.
[71] Bang C I, Paik S Y, Sun D I, et al. Cell growth inhibition and down-regulation of survivin bysilibinin in a laryngeal squamous cell carcinoma cell line.[J]. Ann Otol Rhinol Laryngol,2008,117(10):781-785.
[72] Li L, Gao Y, Zhang L, et al. Silibinin inhibits cell growth and induces apoptosis by caspaseactivation, down-regulating survivin and blocking EGFR-ERK activation in renal cell carcinoma.[J]. Cancer Lett,2008,272(1):61-69.
[73] Hubbard S R, Miller W T. Receptor tyrosine kinases: mechanisms of activation and signaling.[J].Curr Opin Cell Biol,2007,19(2):117-123.
[74] Gschwind A, Fischer O M, Ullrich A. The discovery of receptor tyrosine kinases: targets for cancertherapy.[J]. Nat Rev Cancer,2004,4(5):361-370.
[75] Sharma Y, Agarwal C, Singh A K, et al. Inhibitory effect of silibinin on ligand binding to erbB1and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostatecarcinoma cells.[J]. Mol Carcinog,2001,30(4):224-236.
[76] Tyagi A, Sharma Y, Agarwal C, et al. Silibinin impairs constitutively active TGFalpha-EGFRautocrine loop in advanced human prostate carcinoma cells.[J]. Pharm Res,2008,25(9):2143-2150.
[77] Qi L, Singh R P, Lu Y, et al. Epidermal growth factor receptor mediates silibinin-inducedcytotoxicity in a rat glioma cell line.[J]. Cancer Biol Ther,2003,2(5):526-531.
[78] Pollak M. Insulin and insulin-like growth factor signalling in neoplasia.[J]. Nat Rev Cancer,2008,8(12):915-928.
[79] Singh R P, Sharma G, Dhanalakshmi S, et al. Suppression of advanced human prostate tumorgrowth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increasedapoptosis, and inhibition of angiogenesis.[J]. Cancer Epidemiol Biomarkers Prev,2003,12(9):933-939.
[80] Raina K, Blouin M J, Singh R P, et al. Dietary feeding of silibinin inhibits prostate tumor growthand progression in transgenic adenocarcinoma of the mouse prostate model.[J]. CancerRes,2007,67(22):11083-11091.
[81] Cheung C W, Taylor P J, Kirkpatrick C M, et al. Therapeutic value of orally administered silibininin renal cell carcinoma: manipulation of insulin-like growth factor binding protein-3levels.[J]. BJUInt,2007,100(2):438-444.
[82] Klampfer L. Signal transducers and activators of transcription (STATs): Novel targets ofchemopreventive and chemotherapeutic drugs.[J]. Curr Cancer Drug Targets,2006,6(2):107-121.
[83] Turkson J. STAT proteins as novel targets for cancer drug discovery.[J]. Expert Opin TherTargets,2004,8(5):409-422.
[84] Agarwal C, Tyagi A, Kaur M, et al. Silibinin inhibits constitutive activation of Stat3, and causescaspase activation and apoptotic death of human prostate carcinoma DU145cells.[J].Carcinogenesis,2007,28(7):1463-1470.
[85] Singh R P, Raina K, Deep G, et al. Silibinin suppresses growth of human prostate carcinoma PC-3orthotopic xenograft via activation of extracellular signal-regulated kinase1/2and inhibition ofsignal transducers and activators of transcription signaling.[J]. Clin Cancer Res,2009,15(2):613-621.
[86] Tyagi A, Singh R P, Ramasamy K, et al. Growth inhibition and regression of lung tumors bysilibinin: modulation of angiogenesis by macrophage-associated cytokines and nuclear factor-kappaB and signal transducers and activators of transcription3.[J]. Cancer Prev Res (PhilaPa),2009,2(1):74-83.
[87] Annunziata C M, Davis R E, Demchenko Y, et al. Frequent engagement of the classical andalternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma.[J]. CancerCell,2007,12(2):115-130.
[88] Naugler W E, Karin M. NF-kappaB and cancer-identifying targets and mechanisms.[J]. Curr OpinGenet Dev,2008,18(1):19-26.
[89] Van Waes C. Nuclear factor-kappaB in development, prevention, and therapy of cancer.[J]. ClinCancer Res,2007,13(4):1076-1082.
[90] Dhanalakshmi S, Singh R P, Agarwal C, et al. Silibinin inhibits constitutive and TNFalpha-inducedactivation of NF-kappaB and sensitizes human prostate carcinoma DU145cells to TNFalpha-induced apoptosis.[J]. Oncogene,2002,21(11):1759-1767.
[91] Tyagi A, Raina K, Singh R P, et al. Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder carcinogenesis in male ICR mice.[J]. MolCancer Ther,2007,6(12):3248-3255.
[92] Singh R P, Dhanalakshmi S, Agarwal C, et al. Silibinin strongly inhibits growth and survival ofhuman endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-kappaB:implications for angioprevention and antiangiogenic therapy.[J]. Oncogene,2005,24(7):1188-1202.
[93] Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.[J]. Nat RevCancer,2009,9(3):153-166.
[94] Lapenna S, Giordano A. Cell cycle kinases as therapeutic targets for cancer.[J]. Nat Rev DrugDiscov,2009,8(7):547-566.
[95] Meeran S M, Katiyar S K. Cell cycle control as a basis for cancer chemoprevention through dietaryagents.[J]. Front Biosci,2008,13:2191-2202.
[96] Agarwal C, Singh R P, Dhanalakshmi S, et al. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinomaHT-29cells.[J]. Oncogene,2003,22(51):8271-8282.
[97] Kaur M, Velmurugan B, Tyagi A, et al. Silibinin suppresses growth and induces apoptotic death ofhuman colorectal carcinoma LoVo cells in culture and tumor xenograft.[J]. Mol Cancer Ther,2009,8(8):2366–2374
[98] Yang S H, Lin J K, Huang C J, et al. Silibinin inhibits angiogenesis via Flt-1, but not KDR,receptor up-regulation[J]. J Surg Res,2005,128(1):140-146.
[99] Gallo D, Giacomelli S, Ferlini C, et al. Antitumour activity of the silybin-phosphatidylcholinecomplex, IdB1016, against human ovarian cancer[J]. Eur J Cancer,2003,39(16):2403-2410.
[100] Singh R P, Raina K, Sharma G, et al. Silibinin inhibits established prostate tumor growth,progression, invasion, and metastasis and suppresses tumor angiogenesis and epithelial-mesenchymal transition in transgenic adenocarcinoma of the mouse prostate model mice.[J]. ClinCancer Res,2008,14(23):7773-7780.
[101] Singh R P, Deep G, Chittezhath M, et al. Effect of silibinin on the growth and progression ofprimary lung tumors in mice.[J]. J Natl Cancer Inst,2006,98(12):846-855.
[102] Velmurugan B, Gangar S C, Kaur M, et al. Silibinin exerts sustained growth suppressive effectagainst human colon carcinoma SW480xenograft by targeting multiple signaling molecules[J].Pharm Res,2010,27(10):2085-2097.
[103] Thiery J P, Acloque H, Huang R Y, et al. Epithelial-mesenchymal transitions in development anddisease.[J]. Cell,2009,139(5):871-890.
[104] Gupta S C, Kim J H, Prasad S, et al. Regulation of survival, proliferation, invasion, angiogenesis,and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals[J].Cancer Metastasis Rev,2010,29(3):405-434.
[105] Raina K, Rajamanickam S, Singh R P, et al. Stage-specific inhibitory effects and associatedmechanisms of silibinin on tumor progression and metastasis in transgenic adenocarcinoma of themouse prostate model[J]. Cancer Res,2008,68(16):6822-6830.
[106] Mokhtari M J, Motamed N, Shokrgozar M A. Evaluation of silibinin on the viability, migration andadhesion of the human prostate adenocarcinoma (PC-3) cell line[J]. Cell Biol Int,2008,32(8):888-892.
[107] Wu K, Zeng J, Li L, et al. Silibinin reverses epithelial-to-mesenchymal transition in metastaticprostate cancer cells by targeting transcription factors.[J]. Oncol Rep,2010,23(6):1545-1552.
[108] Wu K J, Zeng J, Zhu G D, et al. Silibinin inhibits prostate cancer invasion, motility and migrationby suppressing vimentin and MMP-2expression.[J]. Acta Pharmacol Sin,2009,30(8):1162-1168.
[109] Chen P N, Hsieh Y S, Chiou H L, et al. Silibinin inhibits cell invasion through inactivation of bothPI3K-Akt and MAPK signaling pathways[J]. Chem Biol Interact,2005,156(2-3):141-150.
[110] Lee S O, Jeong Y J, Im H G, et al. Silibinin suppresses PMA-induced MMP-9expression byblocking the AP-1activation via MAPK signaling pathways in MCF-7human breast carcinomacells.[J]. Biochem Biophys Res Commun,2007,354(1):165-171.
[111] Hsieh Y S, Chu S C, Yang S F, et al. Silibinin suppresses human osteosarcoma MG-63cellinvasion by inhibiting the ERK-dependent c-Jun/AP-1induction of MMP-2.[J]. Carcinogenesis,2007,28(5):977-987.
[112] Baguley B C. Multiple drug resistance mechanisms in cancer[J]. Mol Biotechnol,2010,46(3):308-316.
[113] Higgins C F. Multiple molecular mechanisms for multidrug resistance transporters[J]. Nature,2007,446(7137):749-757.
[114] Tyagi A K, Agarwal C, Chan D C, et al. Synergistic anti-cancer effects of silibinin withconventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breastcarcinoma MCF-7and MDA-MB468cells[J]. Oncol Rep,2004,11(2):493-499.
[115] Dhanalakshmi S, Agarwal P, Glode L M, et al. Silibinin sensitizes human prostate carcinomaDU145cells to cisplatin-and carboplatin-induced growth inhibition and apoptotic death[J]. Int JCancer,2003,106(5):699-705.
[116] Rho J K, Choi Y J, Jeon B S, et al. Combined treatment with silibinin and epidermal growth factorreceptor tyrosine kinase inhibitors overcomes drug resistance caused by T790M mutation[J]. MolCancer Ther,2010,9(12):3233-3243.
[117] Agarwal R, Agarwal C, Ichikawa H, et al. Anticancer potential of silymarin: from bench to bedside.[J]. Anticancer Res,2006,26(6B):4457-4498.
[118] Tyagi A, Singh R P, Agarwal C, et al. Silibinin activates p53-caspase2pathway and causescaspase-mediated cleavage of Cip1/p21in apoptosis induction in bladder transitional-cell papillomaRT4cells: evidence for a regulatory loop between p53and caspase2.[J]. Carcinogenesis,2006,27(11):2269-2280.
[119] Santiago A R, Cristovao A J, Santos P F, et al. High glucose induces caspase-independent celldeath in retinal neural cells.[J]. Neurobiol Dis,2007,25(3):464-472.
[120] Thornberry N A, Lazebnik Y. Caspases: enemies within.[J]. Science,1998,281(5381):1312-1316.
[121] Shankar S L, Mani S, O G K, et al. Survivin inhibition induces human neural tumor cell deaththrough caspase-independent and-dependent pathways.[J]. J Neurochem,2001,79(2):426-436.
[122] Joza N, Susin S A, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor inprogrammed cell death.[J]. Nature,2001,410(6828):549-554.
[123] Jr Hail N. Mitochondria: A novel target for the chemoprevention of cancer.[J]. Apoptosis,2005,10(4):687-705.
[124] Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase8mediates the mitochondrial damage in theFas pathway of apoptosis.[J]. Cell,1998,94(4):491-501.
[125] Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl2interacting protein, mediates cytochrome c releasefrom mitochondria in response to activation of cell surface death receptors.[J]. Cell,1998,94(4):481-490.
[126] Wang C, Youle R J. The role of mitochondria in apoptosis.[J]. Annu Rev Genet,2009,43:95-118.
[127] Kroemer G, Reed J C. Mitochondrial control of cell death.[J]. Nat Med,2000,6(5):513-519.
[128] Lorenzo H K, Susin S A. Therapeutic potential of AIF-mediated caspase-independent programmedcell death.[J]. Drug Resist Updat,2007,10(6):235-255.
[129] Kuranaga E. Caspase signaling in animal development[J]. Dev Growth Differ,2011,53(2):137-148.
[130] Ghobrial I M, Witzig T E, Adjei A A. Targeting apoptosis pathways in cancer therapy.[J]. CACancer J Clin,2005,55(3):178-194.
[131] Crawford E D, Wells J A. Caspase Substrates and Cellular Remodeling[J]. Annu Rev Biochem,2011,80:1055-1087.
[132] Ledgerwood E C, Morison I M. Targeting the apoptosome for cancer therapy.[J]. Clin Cancer Res,2009,15(2):420-424.
[133] Olsson M, Zhivotovsky B. Caspases and cancer[J]. Cell Death Differ,2011,18(9):1441-1449.
[134] van Loo G, van Gurp M, Depuydt B, et al. The serine protease Omi/HtrA2is released frommitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhancedcaspase activity.[J]. Cell Death Differ,2002,9(1):20-26.
[135] Daugas E, Nochy D, Ravagnan L, et al. Apoptosis-inducing factor (AIF): a ubiquitousmitochondrial oxidoreductase involved in apoptosis.[J]. FEBS Lett,2000,476(3):118-123.
[136] Susin S A, Lorenzo H K, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor.[J]. Nature,1999,397(6718):441-446.
[137] Kanwar J R, Kamalapuram S K, Kanwar R K. Targeting survivin in cancer: the cell-signallingperspective[J]. Drug Discov Today,2011,16(11-12):485-94
[138] Carrasco R A, Stamm N B, Marcusson E, et al. Antisense inhibition of survivin expression as acancer therapeutic[J]. Mol Cancer Ther,2011,10(2):221-232.
[139] Li F, Ambrosini G, Chu E Y, et al. Control of apoptosis and mitotic spindle checkpoint bysurvivin[J]. Nature,1998,396(6711):580-584.
[140] Margulis V, Lotan Y, Shariat S F. Survivin: a promising biomarker for detection and prognosis ofbladder cancer.[J]. World J Urol,2008,26(1):59-65.
[141] Altieri D C. Survivin, cancer networks and pathway-directed drug discovery[J]. Nat RevCancer,2008,8(1):61-70.
[142] Cui L, Shi Y, Dai G, et al. Modification of N-Methyl-N-Nitrosourea initiated bladdercarcinogenesis in Wistar rats by terephthalic acid.[J]. Toxicol Appl Pharmacol,2006,210(1-2):24-31.
[143]钱立新，刘训良，丁鸿，等. N-甲基亚硝基脲诱导大鼠膀胱肿瘤作用的动态观察[J].中华实验外科杂志,2004,21(5):563-564.
[144]刘红耀，李晓东，米振国，等. N-甲基亚硝基脲的制备及其诱发膀胱肿瘤的作用[J].中华实验外科杂志,2000,17(6):548-549
[145] Steinberg G D, Brendler C B, Squire R A, et al. Experimental intravesical therapy for superficialtransitional cell carcinoma in a rat bladder tumor model.[J]. J Urol,1991,145(3):647-653.
[146] Steinberg G D, Brendler C B, Ichikawa T, et al. Characterization of an N-methyl-N-nitrosourea-induced autochthonous rat bladder cancer model.[J]. Cancer Res,1990,50(20):6668-6674.
[147] Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases.[J].Drugs,2001,61(14):2035-2063.
[148] Crallan R A, Georgopoulos N T, Southgate J. Experimental models of human bladdercarcinogenesis[J]. Carcinogenesis,2006,27(3):374-381.
[149]颜汝平，王剑松，徐鸿毅.膀胱癌的动物实验模型[J].国外医学.泌尿系统分册,2005,25(4):446-449.
[150]刘俊国，韩瑞发.动物膀胱癌原位模型建立和治疗实验研究进展[J].国际泌尿系统杂志,2006,26(6):748-752.
[151] Schalken J A, van Moorselaar R J, Bringuier P P, et al. Critical review of the models to study thebiologic progression of bladder cancer[J]. Semin Surg Oncol,1992,8(5):274-278.
[152]赵献，颜汝平，李翀，等. OH-BBN诱导不同鼠类膀胱肿瘤的研究现状[J].国际泌尿系统杂志,2010,30(5):649-652
[153] Herman C J, Vegt P D, Debruyne F M, et al. Squamous and transitional elements in rat bladdercarcinomas induced by N-butyl-N-4-hydroxybutyl-nitrosamine (BBN). A study of cytokeratinexpression[J]. Am J Pathol,1985,120(3):419-426.
[154]李云，许传亮，李志华，等.两种构建大鼠膀胱癌模型方法的比较研究[J].临床泌尿外科杂志,2008,23(10):783-784.
[155] Arentsen H C, Hendricksen K, Oosterwijk E, et al. Experimental rat bladder urothelial cellcarcinoma models[J]. World J Urol,2009,27(3):313-317.
[156] Oliveira P A, Colaco A, De la Cruz P L, et al. Experimental bladder carcinogenesis-rodentmodels[J]. Exp Oncol,2006,28(1):2-11.
[157] Drago J R. The Noble rat bladder cancer model. FANFT-induced tumors[J]. Cancer,1984,53(5):1093-1099.
[158] Hicks R M, Wakefield J S. Rapid induction of bladder cancer in rats with N-methyl-N-nitrosourea.I. Histology[J]. Chem Biol Interact,1972,5(2):139-152.
[159] Hamamoto Y, Hamamoto N, Nakajima T, et al. Morphological changes of epithelial rests ofMalassez in rat molars induced by local administration of N-methylnitrosourea.[J]. Arch OralBiol,1998,43(11):899-906.
[160] Knowles M A, Jani H, Hicks R M, et al. N-methyl-N-nitrosourea induces dysplasia and cell surfacemarkers of neoplasia in long-term rat bladder organ cultures.[J]. Carcinogenesis,1985,6(7):1047-1054.
[161] Irving C C, Cox R, Murphy W M. Influence of dose of N-methyl-N-nitrosourea on the induction ofurinary bladder cancer in rats[J]. Cancer Lett,1979,8(1):3-7.
[162]刘红耀，庞东梓，李梦强，等. MNU诱导的近交系大鼠膀胱肿瘤模型[J].肿瘤研究与临床,2005,17(2):89-90.
[163]罗春丽，胡宏波，蔡晓钟，等. MNU诱发SD大鼠膀胱肿瘤原位模型[J].重庆医学,2007,36(14):1352-1354.
[164] Severs N J, Barnes S H, Wright R, et al. Induction of bladder cancer in rats by fractionatedintravesicular doses of N-methyl-N-nitrosourea[J]. Br J Cancer,1982,45(3):337-351.
[165] Chen D, Song D, Wientjes M G, et al. Effect of dimethyl sulfoxide on bladder tissue penetration ofintravesical paclitaxel[J]. Clin Cancer Res,2003,9(1):363-369.
[166] Schoenfeld R H, Belville W D, Jacob W H, et al. The effect of dimethyl sulfoxide on the uptake ofcisplatin from the urinary bladder of the dog: a pilot study[J]. J Am Osteopath Assoc,1983,82(8):570-573.
[167] Hashimoto H, Tokunaka S, Sasaki M, et al. Dimethylsulfoxide enhances the absorption ofchemotherapeutic drug instilled into the bladder[J]. Urol Res,1992,20(3):233-236.
[168] See W A, Xia Q. Regional chemotherapy for bladder neoplasms using continuous intravesicalinfusion of doxorubicin: impact of concomitant administration of dimethyl sulfoxide on drugabsorption and antitumor activity[J]. J Natl Cancer Inst,1992,84(7):510-515.
[169] Hanno P M, Burks D A, Clemens J Q, et al. AUA Guideline for the Diagnosis and Treatment ofInterstitial Cystitis/Bladder Pain Syndrome[J]. J Urol,2011,185(6):2162-2170
[170] Fall M, Baranowski A P, Elneil S, et al. EAU guidelines on chronic pelvic pain[J]. Eur Urol,2010,57(1):35-48.
[171] Michaud D S. Chronic inflammation and bladder cancer[J]. Urol Oncol,2007,25(3):260-268.
[172] Kantari C, Walczak H. Caspase-8and bid: caught in the act between death receptors andmitochondria[J]. Biochim Biophys Acta,2011,1813(4):558-563.
[173] Beere H M, Wolf B B, Cain K, et al. Heat-shock protein70inhibits apoptosis by preventingrecruitment of procaspase-9to the Apaf-1apoptosome[J]. Nat Cell Biol,2000,2(8):469-475.
[174] Saleh A, Srinivasula S M, Balkir L, et al. Negative regulation of the Apaf-1apoptosome byHsp70[J]. Nat Cell Biol,2000,2(8):476-483.
[175] Gurbuxani S, Schmitt E, Cande C, et al. Heat shock protein70binding inhibits the nuclear importof apoptosis-inducing factor[J]. Oncogene,2003,22(43):6669-6678.
[176] Ravagnan L, Gurbuxani S, Susin S A, et al. Heat-shock protein70antagonizes apoptosis-inducingfactor[J]. Nat Cell Biol,2001,3(9):839-843.
[177] Raina K, Agarwal R. Combinatorial strategies for cancer eradication by silibinin and cytotoxicagents: efficacy and mechanisms.[J]. Acta Pharmacol Sin,2007,28(9):1466-1475.