Chemopreventive And Chemotherapeutic Effects Of Intravesical Silibinin Against Bladder Cancer In Rats And Its Apoptotic Effects On Bladder Cancer Cells

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.