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Preclinical Development

Chemopreventive and Chemotherapeutic Effects ofIntravesical Silibinin against Bladder Cancer byActing on Mitochondria

Jin Zeng1, Yi Sun1, Kaijie Wu1, Lei Li1, Gang Zhang1, Zenglei Yang1, Zhiqiang Wang1, Dong Zhang1,Yan Xue1, Yule Chen1, Guodong Zhu1, Xinyang Wang2, and Dalin He1,2

AbstractIntravesical chemotherapy is often used to prevent the recurrence of superficial bladder cancer after

transurethral resection. A search for more effective and less toxic intravesical agents is urgently needed. We

previously found the in vitro apoptotic effects of silibinin, a natural flavonoid, on high-risk bladder carcinoma

cells. Here, we further explored the underlying mechanisms and examined the intravesical efficacy in the

prevention and treatment of bladder cancer. Human bladder carcinoma cell line 5637, which has the same

molecular features of high-risk superficial bladder cancer, was used as the model system in vitro and in vivo.

Autochthonous rat model of bladder cancer induced by intravesical N-methyl-N-nitrosourea (MNU) was

used to investigate its intravesical efficacy. Exposure of 5637 cells to silibinin resulted in growth inhibition and

induction of caspase-dependent and -independent apoptosis, which was associated with disruption of

mitochondrial membrane potential and selective release of cytochrome c, Omi/HtrA2, and apoptosis-

inducing factor (AIF) from mitochondria. Silibinin also downregulated survivin and caused nuclear translo-

cation of AIF. Oral silibinin suppressed the growth of 5637 xenografts, which was accompanied with the

activation of caspase-3, downregulation of survivin, and increased translocation of AIF. Furthermore,

intravesical silibinin effectively inhibited the carcinogenesis and progression of bladder cancer in rats initiated

byMNU by reducing the incidence of superficial and invasive bladder lesions without any side effects, which

was accompanied with proapoptotic effects. These findings identify the in vitro and in vivo antitumor efficacy

of silibinin, and suggest silibinin as an effective and novel intravesical agent for bladder cancer. Mol Cancer

Ther; 10(1); 104–16. �2011 AACR.

Introduction

Bladder cancer poses a health problem worldwide.There were 52,810 new cases of bladder cancer occurringin men and 18,170 in women in the United States in 2009(1). Superficial bladder cancer accounts for approxi-mately 70% of all bladder cancer cases. The standardtreatment for patients with superficial bladder cancer istransurethral resection (TUR) of tumors (2). However,

approximately 60% to 70% of these tumors will recur,with 25% showing progression to a higher stage or grade(3). Intravesical chemotherapy is widely used as an adju-vant therapy to prevent recurrence and progression ofsuperficial disease after TUR (4). Although many chemi-cal agents have shown some evidence of activity (4), theirtoxicity and incomplete efficacy have limited their use ascommon intravesical agents. These factors highlight theurgent need to search for novel adjuvant intravesicalagents.

Silibinin, a natural flavonoid, is the major bioactivecomponent of silymarin isolated from milk thistle(Fig. 1A). Accumulating evidence indicates that silibininhas anticancer activity in various tumor cells, includingcancers of prostate (5–9), breast (10, 11), skin (12), colon(13), lung (14), kidney (15, 16), and bladder (17–20). Thereis increasing interest in elucidating the mechanisms ofaction for silibinin as an effective agent for chemopreven-tion and chemotherapy against various types of cancers.Induction of apoptosis is believed to be one of the majormechanisms of action for silibinin against cancer cells(21), although the details are yet to be elucidated. Thereare multiple signaling pathways controlling the fate of

Authors' Affiliations: 1Department of Urology, The First Hospital of Xi’anJiaotong University, and 2Oncology Research Lab, Key Laboratory ofEnvironment and Genes Related to Diseases, Ministry of Education, Xi’an,China

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

J. Zeng and Y. Sun contributed equally to this work.

Corresponding Author: Dalin He, Department of Urology, The FirstHospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an710061, China; Phone: 86-29-8532-3661; Fax: 86-29-8532-3203. E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-10-0577

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(1) January 2011104

cells undergoing apoptosis and determining their sensi-tivity to apoptosis inducers. Mitochondria play a centralrole in apoptosis, undergoing a number of profoundchanges early within the apoptotic cascade (22). The lossof mitochondrial membrane potential (DYm) releasescytochrome c (Cyto c) into the cytosol, which subse-quently forms a complex with apaf-1 and pro-caspase-9 for the activation of caspase-9, resulting in the finalactivation of caspase-dependent DNase that is responsi-ble for DNA degradation (23). In addition, the apoptosis-inducing factor (AIF), the first caspase-independent celldeath effector to be identified, translocates from themitochondria via the cytosol to the nucleus, where itinteracts with DNA and causes nuclear condensation

and DNA fragmentation (24), features seen in apoptoticcells. However, the exact role of AIF in carcinogenesisand cancer therapy is not fully understood. For example,Gallego and colleagues showed that AIF determines thesensitivity of non–small-cell lung carcinomas to che-motherapy (25), and the chemoresistance of non–small-cell lung carcinoma is overcome through restoration ofthe AIF-dependent apoptotic pathway (26). In contrast,Urbano and colleagues reported that AIF suppresseschemical stress-induced apoptosis and maintains thetransformed state of tumor cells, indicating that AIFmay play an important role in tumorigenesis (27).

Several reports suggest that silibinin induces apoptosisthrough caspase activation in human bladder carcinoma

A B

DC

Figure 1. Effect of silibinin on DYm and subcellular/submitochondrial distribution of cyto c, Omi/HtrA2, and AIF in 5637 cells. A, chemical structure of silibinin.B, cells were treated with different doses of silibinin and/or 80 mM z-VAD-fmk for 48 hours and processed for JC-1 staining followed by flow cytometryto measure the fraction of cells with depolarized mitochondrial membrane. In combination treatment, z-VAD-fmk was added 1 hour prior to silibinin treatment.Data are presented as mean � SE of triplicate samples and are representative of 2 independent experiments. SB, silibinin. Error bars represent SEs.*, P < 0.05. C, cytosolic (left), mitochondrial (middle), and nuclear fractions (right) were prepared andWestern blotting was performed to analyze cyto c, Smac/DIABLO, Omi/HtrA2, and AIF in cells incubated with different doses of silibinin for 48 hours. GAPDH, COX IV, and Histone H1 were used as cytosolic,mitochondrial, and nuclear loading control, respectively. Quantitative analyses were carried out under each panel. D, in similar treatments for 48 hours, totalcell lysates were prepared and analyzed for cyto c, Smac/DIABLO, Omi/HtrA2, AIF, GAPDH, COX IV, and Histone H1 by Western blotting. Quantitativeanalyses were carried out under each panel. Blots are representative of 4 separate experiments. Error bars represent SEs. *, P < 0.05; **, P < 0.01.

Intravesical Silibinin for Prevention and Therapy of Bladder Cancer

www.aacrjournals.org Mol Cancer Ther; 10(1) January 2011 105

RT4 (a well-differentiated papillary noninvasive tumorphenotype) cells (17, 19, 20). In addition, oral administra-tion of silibinin has been reported to prevent N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced mousebladder carcinogenesis (28), suggesting a value of sili-binin in bladder cancer prevention and therapy. How-ever, as the standard of care in patients with bladdercancer who have high-risk clinical and pathologic fea-tures, intravesical therapy receives more attention byurologists and their patients than oral administration (4).Therefore, the purpose of this studywas to determine theeffects of intravesical silibinin against superficial blad-der cancer. In the present study, using in vitro and in vivobladder cancer models, we also explored the molecularmechanisms of silibinin-induced apoptosis, focusingon 2mitochondrial cell death pathways, namely the cytoc/caspase-dependent and the AIF/caspase-indepen-dent pathways.

Materials and Methods

Cell culture and animalsThe human bladder transitional cell carcinoma 5637

cell line was from the American Type Culture Collection(ATCC) in 2008. The cell line was used in fewer than6 months after receipt or resuscitation. ATCC performsauthentication of cell line via short tandem repeat profil-ing, karyotyping, and cyto c oxidase subunit I testing.Only mycoplasma tests were performed for the cell lineauthentication in our laboratory. We did not carry outadditional testing to authenticate cell line, but its mor-phology and behavior were consistent with ATCCdescriptions. Cells were cultured in RPMI-1640 supple-mented with 10% of fetal bovine serum (Gibco) and 1% ofpenicillin–streptomycin at 37�C, in humidified air con-taining 5% of CO2. Six-week-old athymic BALB/c nu/numice and 6- to 8-week-old female Sprague-Dawley ratswere obtained from the Experimental Animal Center ofXi’an Jiaotong University. Animal care and protocolswere approved by the Institutional Animal Care andUse Committee of Xi’an Jiaotong University; all theanimal experiments were performed in adherence withthe NIH Guidelines on the Use of Laboratory Animals.

ReagentsSilibinin, MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphe-

nyltetrazolium bromide], N-methyl-N-nitrosourea(MNU) and pan caspase inhibitor z-VAD-fmk were pur-chased from Sigma Chemical Co. Antibodies against pro-caspase-3, AIF, Histone H1, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), cyto c, Smac (second mito-chondria-derived activator of caspases)/DIABLO (directIAP-binding protein with low pI), Omi/HtrA2 (humanhigh temperature requirement protein A2), and survivinwere from Santa Cruz Biotechnology, Inc. Antibodiesagainst caspase-9, cleaved caspase-9, cleaved caspase-3,cleaved caspase-8, poly (ADP-ribose) polymerase (PARP),cyto c oxidase IV (COX IV), and peroxidase- or FITC-

conjugated secondary antibodies were from Cell Signal-ing Technology, Inc. The enhanced chemiluminescence(ECL) detection system was obtained from Amersham.

Cell viability assayCell viability was assessed using a tetrazolium-based

assay. Cells (5637) were incubated in the absence or pre-sence of silibinin for the indicated times, and then washedonce and incubated with 0.5 mg/mL of MTT at 37�C for1 hour. The reagent was reduced by living cells to form aninsoluble blue formazan product. After incubation, cellswere lysed with DMSO. Colorimetric analysis using a96-well microplate reader was performed at a wavelengthof 490 nm. The experiments were performed in triplicate.

Quantitative detection of apoptosisCells (5637) were exposed to different doses of silibinin

(50, 100, and 200 mM) and/or z-VAD-fmk for 48 hours.The cells were collected and subjected to annexin V andpropidium iodide (PI) staining using an Annexin V-FITCApoptosis Detection Kit, following the protocol providedby the manufacturer. Apoptotic cells were then analyzedby flow cytometry.

Assay for caspase activityCaspase activity was assessed by a colorimetric system.

Briefly, after treatment with desired doses of silibininand/or z-VAD-fmk, cells were lysed in a lysis buffer byfreeze/thaw cycles. Lysates were clarified by centrifuga-tion at 10,000 g (4�C) for 10 minutes. One hundredmicrograms of protein was incubated with 30 mL ofcaspase assay buffer and 2 mL of caspase-3 (DEVD-pNA) or caspase-9 (LEHD-pNA) colorimetric substrateat 37�C for 4 hours. The optical density of the reactionmixture was quantitated spectrophotometrically at awavelength of 405 nm using a 96-well microplate reader.

Preparation of mitochondria, cytosolic, and nuclearfractions

Preparation of mitochondrial and cytosolic extractshas been previously described (29). Briefly, cells wereincubated in permeabilization buffer [250 mmol/L ofsucrose, 20 mmol/L of HEPES/KOH (pH of 7.4),1 mmol/L of EGTA, 1 mmol/L of EDTA, 1 mmol/Lof DTT, 0.1 mmol/L of PMSF, 1 mg/mL of chymostatin,1 mg/mL of leupeptin, 1 mg/mL of antiparin, and1 mg/mL of pepstatin A]. Cells were homogenizedand centrifuged at 500 g (4�C) for 10 minutes to pelletizethe nucleus and cell debris. The supernatants werefurther centrifuged at 13,000 g for 30 minutes. Cytosolicsupernatants and mitochondrial pellets were collected.The pellets were then suspended in mitochondrial lysisbuffer [150 mmol/L of NaCl, 50 mmol/L of Tris-HCl(pH of 7.4), 1% of NP-40, 0.25% of sodium deoxycholate,and 1 mmol/L of EGTA and protease inhibitor]. After1 minute of vigorous vortex and 15 minutes of centri-fugation at 15,000 g (4�C), the mitochondrial superna-tants were collected.

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Mol Cancer Ther; 10(1) January 2011 Molecular Cancer Therapeutics106

Preparation of nuclear extracts was carried out asdescribed previously (30) with some modifications.Briefly, cells were incubated with buffer A [10 mmol/Lof HEPES/KOH (pH of 7.9), 10 mmol/L of KCl,0.1 mmol/L of EDTA, 1 mmol/L of DTT and 0.4% ofNP-40]. The lysates were placed on ice for 30 minutes andthen centrifuged at 15,000 g (4�C) for 5minutes. The pelletwas resuspended in buffer B [20 mmol/L of HEPES (pHof 7.9), 400 mmol/L of NaCl, 1 mmol/L of EDTA,1 mmol/L of DTT and 10% of glycerol] and incubatedfor 60 minutes on ice, and then further centrifuged at15,000 g (4�C) for 15 minutes. The supernatants contain-ing the nuclear fraction were collected. All the sampleswere stored at �80�C until use.

Western blottingCell lysates were prepared in lysis buffer [10 mmol/L

of Tris-HCl (pH of 7.4), 150mmol/L of NaCl, 0.1% of SDS,1 mmol/L of EDTA, 1 mmol/L of EGTA, 0.3 mmol/L ofPMSF, 0.2 mmol/L of sodium orthovanadate, 1% of NP-40, 10 mg/mL of leupeptin and 10 mg/mL of aprotinin].For immunoblot analyses, 30- to 60-mg samples of protein(total lysates, mitochondrial, cytosolic, and nuclear frac-tions) were subjected to SDS-PAGE on 12% or 16% Tris-glycine gels, and separated proteins were transferredonto nitrocellulose membranes by Western blotting.The membranes were blocked with 5% milk for 1 hourat room temperature and probedwith primary antibodiesagainst desired molecules overnight at 4�C followed byperoxidase-conjugated secondary antibody for 1 hour atroom temperature (25�C). The bands of proteins of inter-est were visualized using the ECL detection systemfollowed by exposure to X-ray film. The relative intensityof each band was determined by using Glyko BandScansoftware.

Mitochondrial membrane potential assayMitochondrial membrane potential collapse is a critical

step that occurs in cells undergoing apoptosis (31). In thepresent study, DYm was determined using JC-1 probes.Cells were harvested after 48 hours of exposure to DMSOor desired doses of silibinin and centrifuged at 400 g(4�C) for 5 minutes. The cell pellet was resuspended in0.5 mL of JC-1 solution and incubated at 37�C for20 minutes. After rinsing, the cells were analyzed by flowcytometry. A dot plot of red fluorescence (FL2, living cellswith intact DYm) versus green fluorescence (FL1, cellswith lost DYm) was recorded.

Immunofluorescence stainingCells were plated on slides overnight and then treated

with 200 mM of silibinin and/or z-VAD-fmk for 48 hours.The cells were fixed in 4% paraformaldehyde for25 minutes at room temperature. They were rinsed withPBS and blocked for 1 hour in 3% bovine serum albuminat room temperature, incubated with anti-AIF antibody(1:200) at 37�C for 2 hours. Then, the cells were incubatedwith secondary antibody (antigoat-FITC, 1:100) in the

dark at 37�C for 50 minutes. Finally, stained cells werevisualized under Olympus IX-50 fluorescence invertedmicroscope.

Bladder cancer xenograft model and in vivo therapywith silibinin

The cultured 5637 cells were detached by trypsiniza-tion,washed, and resuspended in serum-free RPMI-1640medium. Cells (1� 106 cells in 100 mL)were then injectedsubcutaneously into the right flank of athymicBALB/c nu/nu mice to initiate tumor growth. After 7days, 98% of mice grew visible tumors. Tumor-bearingmice were randomized to control and various treat-ment groups, and gavaged with the following prepara-tions of silibinin in physiologic saline (0.9%NaCl): (a) thecontrol group (n ¼ 8), saline; (b) silibinin-100 (n ¼ 10),100 mg/kg/day of silibinin; (c) silibinin-200 (n ¼ 10),200 mg/kg/day of silibinin; (d) silibinin-300 (n ¼ 10),300 mg/kg/day of silibinin. All treatments were admi-nistered for 32 days. Body weight and diet consumptionwere recorded twice weekly throughout the study. Thetumor volume was measured twice a week and wascalculated by the formula 0.5236 ab2, wherein a repre-sents the long axis and b represents the short axis of thetumor. At the end of experiment, tumors were excised,weighed, and then fixed in 4% of paraformaldehyde forfurther analyses. Thenecropsieswere carried out after allthe mice were sacrificed. Gross and histologic surveywas performed in liver, lung, spleen, and kidney.

Chemically induced bladder cancer model in ratN-methyl-N-nitrosourea-induced rat bladder tumor

model utilized has been previously described with somemodifications (32). Briefly, female Sprague-Dawley ratswere anesthetized with intraperitoneal chloral hydrate.After the bladder was drained, 0.2 mL (2 mg) of MNU insaline was instilled intravesically via a 22-gauge Teflonangiocatheter every other week, for a total of 4 doses(weeks 2, 4, 6, and 8). The rats were divided randomlyinto 7 groups. Groups 1 to 3 (25 rats/group) receivedintravesical MNU every other week for 4 doses, andsilibinin dissolved in DMSO (200 mg/kg for Group 1,MNUþ silibinin-200, and400mg/kg forGroup2,MNUþsilibinin-400) or DMSO alone (Group 3, vehicle control,MNU þ DMSO) weekly beginning week 1 for 17 weeks.Group 4 (MNU, 25 rats) received intravesical MNU only.Group 5 (silibinin-400, 15 rats) received no MNU treat-ment, but 400mg/kgof intravesical silibininweekly for 17weeks. Group 6 (DMSO þ saline, 15 rats) received noMNU treatment, but intravesical saline andDMSO once aweek for 17weeks.Group 7 (10 rats) receivedno treatmentand served as normal control. When silibinin and MNUwere instilled in the same week, they did not presenttogether in the same intravesical solutions on the sameday instead of separation for at least 3 days.

In another cohort study, silibinin was instilled intrave-sically beginning at week 10 for 8 weeks after 4-doseinstillation of MNU with the same grouping, animal



numbers, and doses as the first study above (Supplemen-tary Fig. S1). For the treatment, the rats remained anesthe-tized for 2 hours after catheterization without urination.All animals received medicated water (a combination oftrimethoprim sulfamethoxazole, neomycin, and poly-myxin B; ref 32). Body weight and diet consumption wererecorded weekly throughout the study.

HistopathologyAll rats were killed at week 18 and necropsy was

performed. The bladders were excised and immediatelyfixed in 4% paraformaldehyde for at least 24 hours,embedded in paraffin, and submitted for histopathology.Sectionswere cut transversely through themid-portion ofthe bladder and stained with hematoxylin and eosin(H&E).

In situ apoptosis detection by TUNEL stainingAfter desired treatment, the cells and paraffin-

embedded sections of samples were studied by terminaldeoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay. Staining was carried outaccording to the protocol provided by the supplier.Apoptosis was evaluated by counting the positive cellsas well as the total number of cells at 10 arbitrarilyselected fields at 400� magnification in a blinded man-ner and quantified as number of apoptotic cells � 100/total number of cells.

Immunohistochemistry stainingImmunohistochemistry (IHC) was conducted using a

Dako Autostainer Plus system. Tissues were de-paraffi-nized, rehydrated, and subjected to 5 minutes of pressurecooking antigen retrieval, 15 minutes of endogenousenzyme block, 60 minutes of primary antibody incuba-tion, and 30 minutes of DakoCytomation EnVision-HRPreagent incubation for mouse and rabbit antibodies, or15 minutes of biotinylated link coupled with 15 minutesof streptavidin-HRP incubation for goat antibodies.Signals were detected by adding substrate hydrogenperoxide using diaminobenzidine (DAB) as a chromogenfollowed by hematoxylin counterstaining. Negative con-trol slices were prepared by omitting the primary anti-body. Stained (brown) cells were quantified as number ofpositive cells � 100/total number of cells in 10 randommicroscopic (400�) fields in each slice.

Statistical analysisAll statistical analyseswere performed using SPSS 15.0.

Quantitative data are presented as mean � SE and thedifferences among the control and various treatmentgroups were compared by 1-way ANOVA, followed byDunnett’s t test for separate comparisons. When thecomparison involved only 2 groups, Student’s t testwas used. Differences in the incidence of rat bladderlesions among groupswere analyzed by c2 test or Fisher’sexact probability test. P < 0.05 was considered statisticallysignificant.

Results

Silibinin inhibits cancer cell growth and inducesapoptosis

First, we investigated the growth-inhibitory and apop-totic effects of silibinin onbladder cancer cells.As shown inSupplementary Fig. S2A, silibinin inhibited the growth of5637 cells in a dose- and time-dependent manner. Silibininat 100, 200, and 400 mM exerted significant inhibitoryeffects on cell growth (P < 0.05). The effects of silibininon the cell apoptosis, as measured by flow cytometry, areshown in Supplementary Fig. S2B, with the 48-hour silibi-nin treatments at 50, 100, and 200 mM resulting in 5.7%,30.23%, and 47.55% of apoptotic cells, respectively, and thebaseline of the control cells being 3.88% (P < 0.05). Cor-respondingly, the number of TUNEL-positive apoptoticcells increased significantly, following treatment with 100and 200 mMof silibinin for 48 hours comparedwith controland 50 mM of silibinin (Supplementary Fig. S2C and S2D).

Silibinin disrupts mitochondrial membranepotential and results in release of cyto c, AIF, andOmi/HtrA2

Mitochondrial membrane potential collapse plays animportant role in triggering various apoptotic pathways(33). Using a cationic lipophilic dye JC-1 as a marker ofDYm, flow cytometric studies revealed that silibinininduced a dose-dependent loss of DYm (Fig. 1B). Inaddition, 80 mM of z-VAD-fmk, a pan caspase inhibitor,showed no protective effects on the dissipation of DYm inthe cells treated with 200 mM silibinin for 48 hours.

The role of mitochondrial disruption in apoptosis is sug-gested to be mediated by the releasing of apoptogenicmolecules (33). In the present study, we found that the pro-tein levels of mitochondrial cyto c, Omi/HtrA2 and AIFdecreased in silibinin-treated cells (Fig. 1C, center). Conco-mitantly, compared to untreated group, silibinin treatmentstrongly increased the amount of cyto c, Omi/HtrA2, andAIF in the cytosolic fraction, indicating a release from themitochondria into the cytosol (Fig. 1C, left). However, norelease of Smac/DIABLO was observed. In addition, silibi-nin had no effects on total levels of cyto c, AIF, Omi/HtrA2,Smac/DIABLO, and 3 individual loading controls (COX IV,GAPDH, and Histone H1 for mitochondrial, cytosolic,and nuclear loading control, respectively, Fig. 1D).

Silibinin induces both caspase-dependent and-independent apoptosis, downregulates survivin,and causes nuclear translocation of AIF

Based on increased apoptosis and release of cyto c intocytosol in silibinin-treated cells, our next aim was toexamine the involvement of caspases that are consideredto be executioners of apoptosis. As shown in Fig. 2A,silibinin activated caspase-3 and caspase-9, as observedby its increased cysteine protease activity for individualsubstrate, in a dose-dependent manner. In addition, sili-binin increased the cleaved subunits of caspase-3 (17 and19 kDa), caspase-9 (35 kDa), caspase-8 (10 kDa), and

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PARP (89 kDa), indicating that silibinin induced theactivation of caspase cascade (Fig. 2B). In this study,we also found that silibinin resulted in a decreasein survivin protein level (Fig. 2B). Furthermore,z-VAD-fmk did not completely reverse silibinin-inducedapoptosis (Fig. 2C). The specificity and efficacy ofz-VAD-fmk activity in 5637 cells was confirmed by cas-pase-3 activity assay that clearly demonstrated that pre-treatment with z-VAD-fmk decreased caspase-3 activity(Fig. 2D). These findings suggested that silibinin-inducedapoptosis in 5637 cells was partially caspase dependent.Because we had observed the release of AIF from mito-

chondria to cytosol, we next analyzedAIF levels in nuclearprotein extracts. We found that 48 hours of exposure tosilibinin increased the amount of AIF in the nuclear frac-tions (Fig. 1C, right).Our results from immunofluorescencestaining (Supplementary Fig. S3) also supported the find-ings. Untreated cells had AIF mainly localized to the cyto-sol, and the treatment with 200 mMof silibinin for 48 hours

resulted in remarkable translocation of AIF to the nucleus.Pretreatment with pan caspase inhibitor was unable toprevent AIF localization to the nucleus. These results sup-ported the contention that silibinin was able to inducecaspase-independent apoptosis in the bladder cancer cells.

Silibinin inhibits human bladder tumor xenograftgrowth in athymic nude mice

To extend the observationsmade in cultured cells and toassess the in vivo efficacy of silibinin, its effects on thegrowth of 5637 human bladder tumor xenografts weredetermined in athymic nude mice. All mice survived atthe end of the experiment. As shown in Fig. 3A, the growthof5637 tumorxenograftswas inhibitedsignificantly follow-ing oral silibinin treatment at the dose levels of 200 and 300mg/kg. The average tumormasses in the control rats were2- to 3-fold (P< 0.05) greater than that of 200 and300mg/kgofsilibinin-treatedmice(Fig. 3B).Theaveragebodyweightsand diet consumption of the control and silibinin-treated

AB

C D

Figure 2. Effect of silibinin on caspase-dependent and -independent apoptosis in 5637 cells. A, cells were treated with different doses of silibinin for48 hours and cell extracts were prepared for caspase-3 and caspase-9 activity assay. B, in similar treatment, total lysates were analyzed for caspase cascadeactivation as indicated in the figure. Blots are representative of 4 separate experiments. Cells were treated with 200 mM of silibinin (SB-200) and/or80 mM of z-VAD-fmk for 48 hours; in combination treatment, z-VAD-fmk was added 1 hour prior to silibinin treatment. At the end of treatment, the cells wereanalyzed for apoptosis by flow cytometry (C) and cell extracts were prepared for caspase-3 activity assay (D). Error bars represent SEs. *, P < 0.05.



mice showed no difference throughout the experiment(Fig. 3C and D). Gross and microscopic examination ofthe liver, lung, spleen, and kidney did not reveal anyevidence of change (data not shown). These results sug-gested the in vivo antitumor efficacy of oral silibinin againsthuman bladder tumor without host toxicity.

Silibinin induces apoptosis, increases translocationof AIF, and decreases survivin expression in vivo

As shown in Fig. 4A–D, TUNEL staining showed thenumber of apoptotic cells increased from 8.5% in thecontrol group to 12.5%, 21% (P < 0.05), and 37.5% (P <0.05) in 100, 200, and 300 mg/kg of silibinin-treatedgroups, respectively, demonstrating significant effectsof silibinin on apoptosis in vivo. Furthermore, the micro-scopic examination of stained tumor sections showedstrong immunoreactivity for survivin (brown color) inthe control group that was decreased in the silibinin-treated groups (Fig. 4I–L). The increased number ofcleaved caspase-3–staining cells was also observed in

the silibinin-fed group of tumors (Fig. 4E–H). In addition,silibinin treatment at the dose levels of 200 and 300mg/kgstrongly increased the nuclear AIF expression as com-pared with control and the 100 mg/kg dose of silibiningroup (Fig. 4M–P). Taken together, the results clearlyshowed that silibinin induced both caspase-dependentand -independent apoptosis in vivo.

Intravesical silibinin inhibits carcinogenesis andprogression of MNU-induced bladder cancer in rats

Because intravesical administration of chemothera-peutic agent is widely used as an adjuvant therapy toprevent recurrence and progression of superficial blad-der cancer after TUR, and silibinin was observed to beeffective in inhibiting the growth of bladder cancer cellsin vitro and in vivo, we next investigated the intravesicalefficacy of silibinin in an autochthonous animal modelof bladder cancer induced by intravesical instillation ofMNU. Approximately 6% (8 of 140) of all animals didnot survive all doses in this study. The causes of death

AB

C D

Figure 3. In vivo antitumor effect of oral silibinin on bladder cancer xenograft model. Athymic nude mice were injected with a 100 mL cell suspensioncontaining 1�106 5637-tumorigenic cell line into the flank. After 7 days, mice were fed with saline (control) or 100, 200, and 300 mg/kg/day doses of silibinin(SB) for 32 days by oral gavage. Effects of silibinin on tumor volume (A), tumor weight (B) body weight (C), and average diet consumption (D) were recorded.Data shown are mean of 8 to 10 mice in each group. Error bars represent SEs. *, P < 0.05.

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were related to the ulceration of the bladder or urosep-sis secondary to urethral structure formation and urin-ary obstruction. Four doses of intravesical MNU to ratsresulted in the induction of hyperplasia, papillarydysplasia, atypia, superficial, and muscle invasive blad-der carcinoma at the end of the 17-week study (Fig. 5C,Table 1). There were significant differences in histo-pathologic changes among the silibinin-treated groupsand the MNU or MNU þ DMSO group (P < 0.05). Theincidences of superficial and invasive transitionalcell carcinoma were reduced with 200 mg/kg and

400 mg/kg silibinin treatments when compared toMNU or MNU þ DMSO group (Table 1). Importantly,there were no invasive lesions in the 400 mg/kg silibi-nin treatment group. Intravesical silibinin alone did nothave any effects on bladder histology (Fig. 5C and D,Table 1). We did not observe any gross sign of toxicitywith intravesical silibinin as measured by body weightand diet consumption throughout the study, wherethere was no significant change in body weight gain(Fig. 5B) and diet intake (Supplementary Fig. S4A) pro-files among each group. In addition, in vivo apoptotic

Figure 4. Effects of silibinin on cell apoptosis, cleaved caspase-3, survivin, and AIF levels in 5637 tumor xenograft. Tumor sections were processedimmunohistochemistry for TUNEL (A–C), cleaved caspase-3 (E–G), surviving (I–K), and AIF (M–O) staining. The scale bar represents 10 mm. D, H, L, and P,quantitative data for percentage of TUNEL, cleaved caspase-3, survivin, and AIF-positive cells in tumors, respectively. Data are mean of 8 samples fromindividual mice in each group. Error bars represent SEs, and SB, silibinin. *P < 0.05.



A B

C

D

Figure 5. Effects of intravesical silibinin on bladder tumorigenecity in a rat bladder model. A, experimental design and protocol to study the chemopreventiveeffects of intravesical silibinin on MNU-induced bladder carcinogenesis in female Sprague-Dawley rats. B, the body weight of each rat was recordedweekly during the experiment. C and D, sections were processed with H&E staining for histopathology (C) or with TUNEL staining for apoptosis (D).MNU-treated rat bladder showed superficial papillary or muscle invasive transitional cell carcinoma. MNU þ SB-200 and MNU þ SB-400 groupshowed hyperplasia or atypia. The scale bar represents 20 mm (scale bar in the picture of MNU group showing superficial papillary transitional cellcarcinoma represents 100 mm). Error bars represent SEs; SB, silibinin.

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effects of intravesical silibinin on MNU-inducedbladder cancer were observed by TUNEL staining(Fig. 5D and Supplementary Fig. S4B).In another cohort study arming to investigate the

chemotherapeutic efficacy of intravesical silibinin,approximately 7% (10 of 140) of all animals did notsurvive all doses in this study. As shown in Table 2, only400 mg/kg intravesical silibinin treatment beginning atweek 10 effectively reduced the incidence of superficialand invasive bladder lesions initiated by MNU whencompared to MNU or MNU þ DMSO group (P < 0.05).However, there was still one rat (4%) with invasivebladder lesions in this group. Similarly, intravesical sili-binin also significantly increased the number of TUNEL-positive apoptotic cells compared with the control group(data not shown).Taken together, these results showed the chemopre-

ventive and chemotherapeutic effects of intravesical sili-binin against MNU-induced bladder carcinogenesis in

rats, which may be associated with its proapoptoticeffects.

Discussion

The goals of the present study were to show the in vivochemopreventive and chemotherapeutic intravesicaleffects of silibinin on bladder cancer and to elucidatethe novel underlying mechanisms of action. Collectively,we have made the following major discoveries. First,silibinin activated the mitochondrial pathways andinduced both caspase-dependent and -independentapoptosis in human high-risk bladder carcinoma cells.Silibinin-induced apoptosis involved (a) dissipation ofDYm; (b) selective releases of cyto c, Omi/HtrA2, and AIFfrom mitochondria into cytosol; (c) downregulation ofsurvivin; (d) activation of caspases and PARP cleavage,and (e) translocation of AIF to the nucleus. Second, oraladministration of silibinin inhibited the growth of human

Table 1. Chemopreventive effect of intravesical silibinin on histopathologic changes in Sprague-Dawleyrat bladders of different groups

Group No. of ratssacrificed

Normal (%) Hyperplasia, oratypia/dysplasia (%)

Superficial bladdercancer (%)

Muscle invasivebladder cancer (%)

Control 10 10 0 0 0MNU 24 0 9 (37.5) 9 (37.5) 6 (25)MNU þ DMSO 23 0 8 (35) 10 (43) 5 (22)MNU þ SB-200a 24 0 17 (71) 6 (25) 1 (4)MNU þ SB-400a,b 23 2 (9) 17 (74) 4 (17) 0SB-400 14 13 (93) 1 (7) 0 0DMSO þ saline 14 12 (86) 2 (14) 0 0aThe observed differences for histopathologic change in MNU þ SB-200 and MNU þ SB-400 groups versus the MNU or MNU þDMSO group were statistically significant with P < 0.05.bSignificantly different from the MNU þ SB-200 group (P < 0.05).SB, silibinin. MNU, N-methyl-N-nitrosourea.

Table 2. Chemotherapeutic effect of intravesical silibinin on histopathologic changes in Sprague-Dawleyrat bladders of different groups

Group No. of ratssacrificed

Normal (%) Hyperplasia, oratypia/dysplasia (%)

Superficial bladdercancer (%)

Muscle invasivebladder cancer (%)

Control 10 10 0 0 0MNU 21 0 7 (33) 9 (43) 5 (24)MNU þ DMSO 24 0 7 (29) 12 (50) 5 (21)MNU þ SB-200 22 0 10 (45) 9 (41) 3 (14)MNU þ SB-400a 24 1 (4) 19 (79) 3 (13) 1 (4)SB-400 15 13 (87) 2 (13) 0 0DMSO þ saline 14 13 (93) 1 (7) 0 0aSignificantly different from the MNU or MNU þ DMSO group (P < 0.05).SB, silibinin. MNU, N-methyl-N-nitrosourea.



bladder tumor xenografts in athymic nude mice withoutany side effects. Third, intravesical administration ofsilibinin effectively inhibited the carcinogenesis and pro-gression of bladder cancer in rats initiated by MNU.Finally, the in vivo anticancer effects of silibinin wereshown to link to the in vivo induction of apoptosis andprotein expression similar to that seen in vitro. Our studiesthusprovide a rationale for thedevelopment of silibinin asa chemopreventive and chemotherapeutic intravesicalagent against bladder cancer in the clinical setting.

It is becoming clear that apoptosis plays a vital role inefficacy of anticancer agents. There are 2 major pathwaysthat initiate apoptosis: one is mediated by death receptorson the cell surface, and the other is mediated by mito-chondria (23). The mitochondria have been considered toact as a point of integration for apoptotic signals originat-ing from both of these 2 apoptotic pathways. Amongvarious proapoptotic events and factors, the disruption ofDYm and release of cyto c are considered critical inmitochondria-mediated apoptosis pathways (34). In thisstudy, silibinin showed a dissipation of DYm in bladdercancer cells as evidenced by JC-1 dye staining, which wasaccompanied by the release of cyto c from mitochondriato cytosol, as evidenced by immunoblotting, and furtheractivation of downstream effectors caspase-9, caspase-3and resultant PARP cleavage. Activation of caspase-8, akey event in death receptor-mediated apoptosis, also hasbeen shown to lead to the release of cyto c from mito-chondria (35). In this study, activation of caspase-8 wasalso observed. Furthermore, studies are needed to inves-tigate the relationship between caspase-8 activation andthe intrinsic mitochondrial pathway in silibinin-treatedbladder cancer cells.

Survivin, a member of the inhibitors of apoptosisproteins (IAP) family, has been considered as a molecularsignature of unfavorable outcome, a diagnostic biomar-ker of tumor onset and recurrence, and a validated targetfor cancer drug discovery (36, 37). Survivin is highlyexpressed in human bladder carcinomas, and its expres-sion has been correlated with resistance against therapyand abbreviated patient survival (38). Our data showed astrong downregulation of survivin by silibinin. Further-more, studies are needed to define the underlying mole-cular mechanisms.

We also found that the inhibitor of caspases, z-VAD-fmk, at a concentration that nearly completely blockedcaspase activity, failed to block the silibinin-induceddisruption of DYm in bladder cancer cells, suggestingcaspase-independent pathways of silibinin-inducedapoptosis. It is reported that pretreatment with z-VAD-fmk almost completely reversed silibinin-caused celldeath in human bladder carcinoma RT4 cells (19). Suchdisparate findings from our results could be due to thedifference in cell type used, implying that there may befundamental differences of the individual gene expres-sion pattern among various cell lines. Therefore, precau-tions should be taken when extrapolating results fromone to another study.

AIF translocates from mitochondria via the cytosol tothe nucleus, where it induces chromatin condensationand DNA fragmentation, and is a hallmark of caspase-independent apoptosis (39). Our data showed that sili-binin treatment induced AIF release from mitochondriaand translocation to the nucleus, supporting that silibi-nin is able to induce caspase-independent apoptosis.Thus, the mitochondrial apoptotic pathway involvesboth cyto c (caspase dependent) and AIF (caspase inde-pendent) processes in silibinin-induced apoptotic celldeath. In addition, the release of cyto c is usuallyassociated with Smac/DIABLO and Omi/HtrA2 (40),which are also mitochondrial intermembrane spaceproteins in the apoptotic process. Smac/DIABLO andOmi/HtrA2 have the ability to bind and antagonize theactions of IAPs, which then facilitate the activation ofcaspases. Cytosolic Omi/HtrA2 also contributes to bothcaspase-dependent and -independent apoptosis (41).However, in our study we failed to detect Smac/DIA-BLO release, whereas cyto c and Omi/HtrA2 weretranslocated to cytosol in silibinin-treated cells. Theseobservations are surprising because several papershave reported that the mitochondrial intermembraneproteins, cyto c, Smac/DIABLO and Omi/HtrA2 arereleased together with the same or similar kinetic pat-tern (40,42). Furthermore, studies are needed to identifythe relationship between cyto c and Omi/HtrA2 releas-ing in silibinin-treated cells.

The possibility that bladder cancer cells may be leftbehind, whether from implantation or incomplete resec-tion, has led many urologists to believe that TUR,though effective, will not be a sufficient treatment alonefor all cases of superficial bladder cancer (43). As aconsequence, intravesical chemotherapy becomes animportant treatment for superficial transitional cell car-cinoma. There are 3 fundamental purposes of treatmentof superficial bladder cancer with intravesical therapy:(a) eradicating existing lesions, (b) prevention of recur-rence, and (c) prevention of tumor progression. Theautochthonous animal model of bladder cancer inducedby intravesical instillation of MNU utilized in this studyhas been previously characterized (32). Because DMSOis a urothelium permeability enhancer that was provedto promote the penetration of water-soluble drugs(e.g., cisplatin, pirarubicin, and doxorubicin) and alipophilic drug paclitaxel across the urothelium in dogsor rats (4), we thus used silibinin dissolved in DMSO asthe intravesical agent. In this study, intravesical silibi-nin beginning either at week 1 or at week 10 effectivelyinhibited carcinogenesis and progression of bladdercancer in rats by reducing the incidence of superficialand invasive bladder lesions, suggesting its chemopre-ventive and chemotherapeutic effects against bladdercancer. No toxicity, locally or systemically, wasobserved in rats receiving intravesical silibinin alone.Intravesical silibinin alone induced 7% and 13% ofordinary hyperplasia instead of atypia hyperplasia ordysplasia in chemopreventive and chemotherapeutic

Zeng et al.


study, respectively. All the chronic cystitis, ulceration ofbladder, and urosepsis due to repeatedly intravesicalinstillation may contribute to this ordinary hyperplasia.As seen in other chemically induced tumor models, themodel used in the present study may not completelyreflect the clinically observed tumors, yet these arespontaneous, not implanted tumors, and histologicallyequivalent to the human transitional cell carcinomaseen clinically. Our results showed that intravesicalsilibinin was both safe and effective in the preventionand therapy of MNU-induced rat bladder cancer.In summary, our in vitro and in vivo results demon-

strated that silibinin induced-apoptosis in human blad-der cancer was mediated by the activation of 2mitochondrial death pathways, namely the cyto c/cas-pase-dependent and the AIF/caspase-independent path-ways involving selective translocation of Omi/HtrA2,and by the downregulation of survivin that is a moleculartarget of human bladder cancer. Silibinin may prove to bea new form of intravesical chemotherapy in the inhibitionof carcinogenesis and progression of bladder cancer,

providing a basis for future clinical trials of intravesicalsilibinin used in patients with bladder cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. Erwin GVanMeir, PaulaM. Vertino (Emory UniversitySchool of Medicine, Atlanta, GA) and Jer-Tsong Hsieh (University ofTexas Southwestern Medical Center, Dallas, TX) for helpful discussion.

Grant Support

The "13115" Scientific and Technological Innovation Project of ShaanxiProvince, China (2008ZDKJ-63)

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 21, 2010; revised October 6, 2010; accepted November 1,2010; published online January 10, 2011.

References1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics.

Cancer J Clin 2009;59:225–49.2. Bischoff CJ, Clark PE. Bladder cancer. Curr Opin Oncol 2009;21:272–7.3. Schenk-Braat EA, BangmaCH. Immunotherapy for superficial bladder

cancer. Cancer Immunol Immunother 2005;54:414–23.4. Shen Z, Shen T, Wientjes MG, O’Donnell MA, Au JL. Intravesical

treatments of bladder cancer: review. Pharm Res 2008;25:1500–10.5. Wu KJ, Zeng J, Zhu GD, Zhang LL, Zhang D, Li L, et al. Silibinin

inhibits prostate cancer invasion, motility and migration by suppres-sing vimentin and MMP-2 expression. Acta Pharmacol Sin 2009;30:1162–8.

6. Roy S, Kaur M, Agarwal C, Tecklenburg M, Sclafani RA, Agarwal R.p21 and p27 induction by silibinin is essential for its cell cycle arresteffect in prostate carcinoma cells. Mol Cancer Ther 2007;6:2696–707.

7. Raina K, Blouin MJ, Singh RP, Majeed N, Deep G, Varghese L, et al.Dietary feeding of silibinin inhibits prostate tumor growth and pro-gression in transgenic adenocarcinoma of the mouse prostate model.Cancer Res 2007;67:11083–91.

8. Singh RP, Raina K, Sharma G, Agarwal R. Silibinin inhibits establishedprostate tumor growth, progression, invasion, and metastasis andsuppresses tumor angiogenesis and epithelial-mesenchymal transi-tion in transgenic adenocarcinoma of themouse prostate model mice.Clin Cancer Res 2008;14:7773–80.

9. Singh RP, Raina K, Deep G, Chan D, Agarwal R. Silibinin suppressesgrowth of human prostate carcinoma PC-3 orthotopic xenograft viaactivation of extracellular signal-regulated kinase 1/2 and inhibition ofsignal transducers and activators of transcription signaling. ClinCancer Res 2009;15:613–21.

10. Tyagi AK, Agarwal C, Chan DC, Agarwal R. Synergistic anti-cancereffects of silibinin with conventional cytotoxic agents doxorubicin,cisplatin and carboplatin against human breast carcinomaMCF-7 andMDA-MB468 cells. Oncol Rep 2004;11:493–9.

11. Lee SO, Jeong YJ, Im HG, Kim CH, Chang YC, Lee IS. Silibininsuppresses PMA-induced MMP-9 expression by blocking the AP-1activation via MAPK signaling pathways in MCF-7 human breastcarcinoma cells. Biochem Biophys Res Commun 2007;354:165–71.

12. Gu M, Singh RP, Dhanalakshmi S, Agarwal C, Agarwal R. Silibinininhibits inflammatory and angiogenic attributes in photocarcinogen-esis in SKH-1 hairless mice. Cancer Res 2007;67:3483–91.

13. Singh RP, Gu M, Agarwal R. Silibinin inhibits colorectal cancer growthby inhibiting tumor cell proliferation and angiogenesis. Cancer Res2008;68:2043–50.

14. Singh RP, Deep G, ChittezhathM, Kaur M, Dwyer-Nield LD, MalkinsonAM, et al. Effect of silibinin on the growth and progression of primarylung tumors in mice. J Natl Cancer Inst 2006;98:846–55.

15. Cheung CW, Taylor PJ, Kirkpatrick CM, Vesey DA, Gobe GC, Win-terford C, et al. Therapeutic value of orally administered silibinin inrenal cell carcinoma: manipulation of insulin-like growth factor bindingprotein-3 levels. Bju Int 2007; 100:438–44.

16. Li L, Gao Y, Zhang L, Zeng J, He D, Sun Y. Silibinin inhibits cell growthand induces apoptosis by caspase activation, down-regulating sur-vivin and blocking EGFR-ERK activation in renal cell carcinoma.Cancer Lett 2008;272:61–9.

17. Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM, Agarwal R.Silibinin down-regulates survivin protein and mRNA expression andcauses caspases activation and apoptosis in human bladder transi-tional-cell papilloma RT4 cells. Biochem Biophys Res Commun2003;312:1178–84.

18. Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. Silibinincauses cell cycle arrest and apoptosis in human bladder transitionalcell carcinoma cells by regulating CDKI-CDK-cyclin cascade,and caspase 3 and PARP cleavages. Carcinogenesis 2004;25:1711–20.

19. Tyagi A, Singh RP, Agarwal C, Agarwal R. Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4cells: evidence for a regulatory loop between p53 and caspase 2.Carcinogenesis 2006;27:2269–80.

20. Singh RP, Tyagi A, Sharma G, Mohan S, Agarwal R. Oral silibinininhibits in vivo human bladder tumor xenograft growth involvingdown-regulation of survivin. Clin Cancer Res 2008;14:300–8.

21. Li L, Zeng J, Gao Y, He D. Targeting silibinin in the antiproliferativepathway. Expert Opin Invest Drugs 2010;19:243–255.

22. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptoticprogram in cell-free extracts: requirement for dATP and cytochrome c.Cell 1996;86:147–57.

23. Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathways incancer therapy. Cancer J Clin 2005;55:178–94.



24. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,et al. Molecular characterization of mitochondrial apoptosis-inducingfactor. Nature 1999;397:41–6.

25. Gallego MA, Joseph B, Hemstrom TH, Tamiji S, Mortier L, Kroemer G,et al. Apoptosis-inducing factor determines the chemoresistance ofnon-small-cell lung carcinomas. Oncogene 2004;23:6282–91.

26. Gallego MA, Ballot C, Kluza J, Hajji N, Martoriati A, Castera L, et al.Overcoming chemoresistance of non-small cell lung carcinomathrough restoration of an AIF-dependent apoptotic pathway. Onco-gene 2008;27:1981–92.

27. Urbano A, Lakshmanan U, Choo PH, Kwan JC, Ng PY, Guo K, et al.AIF suppresses chemical stress-induced apoptosis and maintains thetransformed state of tumor cells. EMBO J 2005;24:2815–26.

28. Tyagi A, Raina K, Singh RP, Gu M, Agarwal C, Harrison G, et al.Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl)nitrosamine induced urinary bladder carcinogenesis inmale ICR mice. Mol Cancer Ther 2007;6:3248–55.

29. Santiago AR, Cristovao AJ, Santos PF, Carvalho CM, Ambrosio AF.High glucose induces caspase-independent cell death in retinal neuralcells. Neurobiol Dis 2007;25:464–72.

30. Levrand S, Pesse B, Feihl F, Waeber B, Pacher P, Rolli J, et al.Peroxynitrite is a potent inhibitor of NF-{kappa}B activation trig-gered by inflammatory stimuli in cardiac and endothelial cell lines.J Biol Chem 2005;280:34878–87.

31. Susin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, LoefflerM, et al. Two distinct pathways leading to nuclear apoptosis. J ExpMed 2000;192:571–80.

32. Steinberg GD, Brendler CB, Ichikawa T, Squire RA, Isaacs JT. Char-acterization of an N-methyl-N-nitrosourea-induced autochthonous ratbladder cancer model. Cancer Res 1990;50:6668–74.

33. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med2000;6:513–9.

34. Ledgerwood EC, Morison IM. Targeting the apoptosome for cancertherapy. Clin Cancer Res 2009;15:420–4.

35. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2interacting protein, mediates cytochrome c release frommitochondriain response to activation of cell surface death receptors. Cell 1998;94:481–90.

36. Altieri DC. Validating survivin as a cancer therapeutic target. Nat RevCancer 2003;3:46–54.

37. Altieri DC. Survivin, cancer networks and pathway-directed drugdiscovery. Nat Rev Cancer 2008;8:61–70.

38. Margulis V, Lotan Y, Shariat SF. Survivin: a promising biomarker fordetection and prognosis of bladder cancer. World J Urol 2008;26:59–65.

39. Daugas E, Nochy D, Ravagnan L, Loeffler M, Susin SA, Zamzami N,et al. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrialoxidoreductase involved in apoptosis. FEBS Lett 2000;476:118–23.

40. van LooG, vanGurpM, Depuydt B, Srinivasula SM,Rodriguez I, AlnemriES, et al. The serine protease Omi/HtrA2 is released from mitochondriaduringapoptosis.Omi interactswith caspase-inhibitor XIAPand inducesenhanced caspase activity. Cell Death Differ 2002;9:20–6.

41. Cassens U, Lewinski G, Samraj AK, von Bernuth H, Baust H, KhazaieK, et al. Viral modulation of cell death by inhibition of caspases. ArchImmunol Ther Exp 2003;51:19–27.

42. Munoz-Pinedo C, Guio-Carrion A, Goldstein JC, Fitzgerald P, New-meyer DD, Green DR. Different mitochondrial intermembrane spaceproteins are released during apoptosis in amanner that is coordinatelyinitiated but can vary in duration. Proc Natl Acad Sci U S A 2006;103:11573–8.

43. Malmstrom PU. Intravesical therapy of superficial bladder cancer. CritRev Oncol Hematol 2003;47:109–26.

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