Human Bladder Transitional Cell Research Articles

Abstract C182: Novel derivative of doxorubicin, AD198, for treatment of bladder transitional cell carcinomas in vitro

Abstract Human bladder cancer is one of the most expensive cancers to treat due to its high rate of reoccurrence. Development and characterization of animal models for human cancers is important for the improvement of diagnosis and therapy. The bladder transitional cell carcinoma (TCC) of domestic animals resembles human bladder TCC in many aspects, therefore cell lines derived from bladder TCC of dogs are valuable model for studying human bladder TCC. A chemotherapeutic agent, Doxorubicin (Dox), is often used to treat advanced bladder cancer. Despite the success of Dox based therapies, drug resistance and cardio-toxicity are limiting factors in treating any cancer with Dox. N-benzyladriamycin-14-valerate (AD198), a novel derivative of Dox has no measurable cardio-toxic effects in the rodent model of lymphoma. In this study, we focused in comparing the efficacy and mechanisms of Dox and AD198 in human and primary canine bladder transitional cell carcinoma (TCC) cells in vitro. Using human T24 and UMUC-3 TCC and three canine primary bladder K9TCC-Dakota, K9TCC-Lillie, and K9TCC-Molly cell lines, we evaluated the Dox and AD198 efficacy on cell proliferation by MTS assay. Caspase 3/7 assay, reactive oxygen species (ROS) production, and western blot analysis were used to study the mechanisms of Dox- and AD198-induced apoptosis. AD198 was more effective than Dox in inhibition of cell proliferation in tested TCC cells in dose-dependent manner. ROS production was increased when compared to control by both Dox and AD198 leading to apoptosis, which was confirmed by caspase 3/7 activity and cleavage of poly ADP ribose polymerase (PARP) in tested TCC cells. AD198 increased ROS production significantly more than Dox in all tested TCC cells. Both Dox and AD198 increased phosphorylation of pro-apoptotic p38 MAPK, while at the same time increased the activity of anti-apoptotic phosphatidylinositol 3-kinase (PI3K/AKT/mTOR) signal transduction pathway in tested TCC cells. When a PI3K inhibitor, LY294002, was used in combination with either Dox or AD198, cell proliferation was inhibited more effectively than when drugs used alone. In addition, cleaved PAPR was increased when TCC cells were co-treated with LY294002 and in combination with Dox or AD198 as compared to either drug alone. Our data suggest that AD198 as novel derivative of Dox may be a valuable treatment option for bladder TCC cancers. Dox- and AD198-induced AKT phosphorylation that is an indicator of pro-survival and drug-resistance mechanisms of chemotherapies in tested bladder TCC cancer. Combined therapy of Dox or AD198 with inhibitors of PI3K/AKT1 pathway might lead to more effective treatment outcome for human and veterinary patients diagnosed with bladder TCC cancer. Evaluation of new therapeutic and imaging drugs that have shown promise in vitro and in vivo in the rodent model are important; however, pet animals like, dogs and cats with naturally-occurring tumors provide an important step for successful translation from rodents to human clinical application. Citation Format: Dmitriy Smolensky, Kusum Rathore, Maria Cekanova, Maria Cekanova. Novel derivative of doxorubicin, AD198, for treatment of bladder transitional cell carcinomas in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C182.

Effect of TRPM8 on proliferation and apoptosis of human bladder transitional cell carcinoma cell T24

Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24. Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation. The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. Expression of extracellular regulated kinase(ERK), cyclin D1, and Bcl-2 were detected with Western blot. Results TRPM8-targeted shRNA down-regulated TRPM8 expression of T24 cells. MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P<0.01). Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P<0.01). In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group. Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K. It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer. Key words: Transient receptor potential channels/BI/PD; RNA interference; Urinary bladder neoplasms/TH/ME/PP; Cell proliferation/DE; Apoptosis/DE

Epithelial-mesenchymal transition and metastatic bladder cancer profile under vinflunine treatment.

e22021 Background: Given the role of vinflunine (VFL) in the microtubule dynamics and the link between microtubules and cell adhesions through cadherins, we have investigated the possible influence of VFL on adherens junctions through its interaction with microtubules. We have studied the implication of VFL on the reversion of epithelial-mesenchymal transition (EMT) in bladder transitional cell carcinoma and explored a possible novel molecular mechanism. Methods: Four human bladder transitional carcinoma cell lines were used to carry out the following experimental procedure: Cytotoxicity assay by using MTT assay, qRTPCR to analyze mRNA markers of the EMT, Western blotting using specific antibodies for EMT markers, and immunofluorescence images, analyzed by epifluorescence microscopy. Results: Cell growth reduction was detected in human bladder carcinoma cells under VFL treatment compared to control. VFL induces mesenchymal to epithelial phenotype and modulates the EMT markers: E-cadherin and Cytokeratin-19 were enhanced under treatment, while significantly reduction of mRNA mesenchymal markers expression (Vimentin, N-cadherin) and EMT-transcriptional factors (Snail and Zeb1) was detected. Strong reduction of Hakai protein was seen under VFL treatment. Hakai was discovered as an E3 ubiquitin-ligase that mediates the posttranslational downregulation of E-cadherin. Epifluorescence images showed that VFL treatment promotes E-cadherin localization specifically at cell-cell contact; while, Hakai expression decreases its expression in the nuclei and cytoplasm. Conclusions: These results suggest that VFL up-regulates E-cadherin contributing to mesenchymal to epithelial transition, and that Hakai modulation might be the molecular mechanism by which the increasing E-cadherin at cell-cell contacts in bladder carcinoma cell lines is detected. Given the relevant in vitro role of VFL on E-cadherin expression and on the reversion of EMT process, we hypothesized that VFL could exert a clinical benefit in delaying the metastasis in urothelial tumors.

Correlation between transcription factor forkhead box protein Q1 and epithelial-mesenchymal transition in human bladder transitional cell carcinoma

Objective To investigate the relationship between transcription factor forkhead box protein Q1 (FOXQ1) and epithelial mesenchymal transition (EMT) in bladder transitional cell carcinoma (BTCC),and the association between FOXQ1 and clinicopathologic features.Methods Sixty-five BTCC specimens surgically resected were selected.For each specimen,the cancerous tissue,peri-cancerous tissue and the remote normal mucosa were analyzed and compared.The expression of FOXQ1,transforming growth factor (TGF)-βl,E-cadherin and Vimentin genes in resected cancer tissues was detected by using reverse transcription-polymerase chain reaction (RT-PCR),Western blotting and immunohistochemistry,and their association with clinicopathologic data was analyzed.Results (1) The expression rate of FOXQ1 was significantly higher in BTCC tissues than in normal tissues (89.2％ vs 13.8％,P ＜0.05),and that in deeply infiltrating group was significantly higher than that in superficially infiltrating group (97.3％ vs 78.6％,P ＜0.05).The level of TGF-β1 was significantly higher in BTCC tissues than in normal tissues (76.9％ vs 26.2％,P ＜0.05).The expression level of E-cadherin in BTCC tissues was significantly lowered in comparison with that in normal bladder epithelium (27.7％ vs 89.2％,P ＜0.05).The expression rate of Vimentin was higher in BTCC tissues than in normal tissues (83.1％ vs 20.0％,P ＜ 0.05) ; (2)The expression of FOXQ1 serving as one new positive control factor of EMT was inversely correlated to Ecadherin,but positively to TGF-β1 and Vimentin.Conclusion FOXQ1 is related to the differentiation and metastasis of BTCC,and may induce EMT to promote BTCC metastasis. Key words: Epithelial mesenchymal transition; Bladder transitional cell carcinoma; Transcription factor; Forkhead box protein Q1

Expression of Survivin and leucine-rich and immunoglobulin-like domain 2 in human bladder transitional cell carcinoma and its clinical application

Objective To investigale the expression of Survivin and leucine-rich and immunoglobulin-like domain 2 (LRIG2) in bladder transitional cell carcinoma ( BTCC ),and their association with clinicopathologic features.Methods Seventy-two BTCC specimens surgically resected were selected.For each specimen,the cancerous tissue,peri-cancer tissue and its remote normal mucosa were analyzed and compared.Immunohistochemistv was used to detect the expression of Survivin and LRIG2 in 72 cases of BTCC tissues.Results The immunohistochemisty revealed that the expression of Sruvivin in BTCC was much higher than that in peri-cancer tissues ( 90.28％ vs.33.33 ％,P ＜ 0.05 ),but no expression was detectable in normal bladder tissues.Among the pathological grades of G1,G2 and G3,the positive rate of Survivin respectively was 72.22％,90.48％ and 100.00％.In the clinical stages of Ta-Tis and T1-T2,the positive rate of Survivin respectively was 81.25％ and 97.50％,and in the two groups with lymphatic metastasis and negative lymphatic metastasis,it respectively was 96.00％ and 85.11％. Moreover,with the advancing grades and clinical stages and appearances of lymph node metastasis,the expression of Survivin was increased gradually.There were significant diffrences in each group ( P ＜ 0.05 ).The expression rate of LRIG2 was significantly lower in BTCC tissues than in peri-cancer tissues and normal tissues (30.56％,66.67％ and 87.50％ respectively).Among the pathological grades of G1,G2 and G3,the positive rate of LRIG2 respectively was 33.33％,19.05％,and 9.38％.With the advancing grades and clinical stages and appearances of lymph node metastasis,the expression of LRIG2 was decreased gradually,and there were significant differences in each group (P ＜ 0.05).Moreover,the expression of Survivin was inversely correlated to LRIG2. Conclusion The expression of Survivin and LRIG2 is related to the clinicopathologic features of BTCC with differentiation and metastasis.The over-expression of Survivin and genetic mutation or missing of LRIG2 may play an important role in carcinogenesis and progress of BTCC.Detection of Survivin and LRIG2 protein will be helpful to the early diagnosis and treatment of BTCC and evaluate its biologic behaviors. Key words: Survivin; Leucine-rich and immunoglobulin-like domain 2; Bladder transitional cell carcinoma

Abstract 2569: Lyc-O-Mato causes cell cycle arrest and apoptosis in human bladder transitional carcinoma cells

Abstract Approximately two thirds of new bladder cancer cases in the United States are superficial papillary transitional cell carcinoma (TCC), the majority of which are low-grade neoplasms. Data suggest that bladder carcinogenesis is a multi-step, multi-focal (field effect) process, possibly involving the spread of pre-malignant clones-all of which are prerequisites for effective preventive and/or interventional approaches. Epidemiological data suggests an inverse relationship between serum levels of carotenoids and risk of bladder cancer which has encouraged further investigation. In this study, we evaluated the effect of Lyc-O-Mato® (a crude tomato extract containing 10% lycopene and other tomato phytonutrients viz. tocopherols, phytoene, phytofluene, beta-carotene, phospholipids and phytosterols) on human bladder cancer cell growth, cell cycle modulation, induction of apoptosis, and associated molecular alterations. Treatment of bladder cancer cell lines (T-24, high-grade TCC and TCC-SUP, high-grade invasive tumor) with Lyc-O-Mato® at doses of 1-60µM resulted in inhibition of cell growth along with a G1 arrest in T24 cells and G2/M arrest in TCCSUP cells at 24 h post-treatment. In other studies, Lyc-O-Mato® treatment strongly induced the expression of p21/waf1 and p27/kip1, and resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins involved in G1 and G2/M progression in T24 and TCC SUP cells. Lyc-O-Mato® exposure to these cells showed an increased interaction between cyclin dependent kinase inhibitors (CDKIs)-cyclin-dependent kinases (CDKs) and decrease in the interaction of cyclins and CDKs with their respective stages of the cell cycle. Furthermore, Lyc-O-Mato® treatment significantly reduced the phosphorylation of Retinoblastoma at Serine780, although no significant changes were observed in the total levels of this protein. In additional studies, Lyc-O-Mato® showed a dose- and time-dependent apoptotic death in both T24 and TCC SUP cells which was associated with significant downregulation of survivin along with the enhancement of pro-apoptotic protein, Bax. These observations provide a strong rationale for further evaluating preventive and/or intervention strategies for Lyc-O-Mato® in pre-clinical models of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2569. doi:1538-7445.AM2012-2569

Effect of shRNA-mediated survivin gene silencing on the biological behavior of human bladder transitional cell carcinoma T24 cells

To construct the expression vector of the survivin-specific short hairpin RNA (shRNA) and to investigate its effect on the biological behavior of human bladder transitional cell carcinoma T24 cells. Survivin-specific short oligonucleotides were designed, synthesized, and cloned into the PTZU6+1 vector, resulting in a survivin-specific shRNA expression construct, which was then transfected into T24 cells. The mRNA and protein levels of survivin were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Tumor cell growth, movement and neoplasm invasiveness were detected by a cell growth curve, a random motion experiment, and a Matrigel transmembrane assay. Target DNA fragments containingpsurvivin-shRNA vectors was verified by EcoR I / Hind III double digestion and sequencing and successfully transfected into T24 cells. Transfection of psurvivin-shRNA cellssignificantly inhibited mRNA levels by 61.73% and protein expression by 73.37%. The ability of cell invasion and movement was significantly decreased. The inhibition rates of tumor cell growth were 59.13% on day 3 and 83.86% on day 5. A survivin-specific shRNA expression vector was constructed based on the PTZU6 +1 vector and was transfectedinto T24 cells, which resulted in the inhibition of T24 cell proliferation and movement. Key words: Survivin, short hairpin RNA, Neoplasm invasiveness, bladder neoplasms.

Gene expression profiles in human bladder epithelial cells stimulated with uropathogenic Escherichia coli

Objective To investigate the interaction between uropathogenic Escherichia coli (UPEC) and host uroepithelial cells, define the role uroepithelial cells play in initiating and modulating the host response to infection with UPEC strain. Methods The human bladder transitional epithelial EJ cells were evaluated for their capacities to allow the adherence and invasion by UPEC132, a clinical strain isolated from Tianjin, China, and a cDNA microarray for 22 000 human genes was used to identify the gene expression differences between EJ cells infected with UPEC132 and uninfected EJ cells. Results Microscope observation showed that UPEC132 could adhere to EJ cells with the adherence rate of (73.20 ± 5.26)%. And visualization by confocal microscope revealed that this microorganism could be seen within the cells. EJ cells infected with UPEC132 changed mRNA expression of a total of 29 genes, including 28 genes up-regulated and 1 gene down-regulated. Of these, regulators of growth and proliferation, cytokines, and modulators of apoptotic responses were the most prominent. Conclusion The gene expression profiling of EJ cells is affected by the infection of UPEC strain. The differentially expressed genes may contribute to further investigate the interaction of UPEC and uroepithelial cells. Key words: Uropathogenic Escherichia coli; Gene expression; Bladder epithelial cell; cDNA microarray

Expressions of PKM2 and PTB and their clinical significance in human bladder transitional cell carcinoma

Objective To explore the expressions of tumor M2 pyruvate kinase (PKM2) and polypyrimidine tract binding protein (PTB) and their clinical significance in bladder transitional cell carcinoma (BTCC). Methods The changes of PKM2 and PTB mRNA levels in different grades of BTCC and cell lines were identified by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the changes of PKM1, PKM2 and PTB protein levels were validated by Western blotting. The expressions of PKM1, PKM2 and PTB were also studied by using the SP method of immunohistochemistry in BTCC samples and normal bladder tissues. Results The mRNA levels and the protein levels of PKM2 and PTB increased in the BTCC tissues, obviously in high-grade tumors and cell lines in comparison with those in normal bladder epithelium. The positive rate of PKM2 was 84.4% (38/45) in BTCC tissues, which was significantly higher than that in normal tissues 60.0% (27/45) ( P ＜ 0. 05 ). The expression of PTB was higher in the BTCC tissues (95.6%, 43/45) than that in normal tissues ( 55.6%, 25/45, P ＜ 0. 05 ).There was a positive correlation between the expressions of PKM2 and PTB. Conclusion PTB may play an important role in the alternative processing of PKM2 to regulate the energy metabolism of bladder cancer cells. Key words: Energy metabolism; Bladder transitional cell carcinoma; PKM2; PTB

The reverse effect ofthymosin β 4 gene silencing onepithelial-mesenchymal transition in human bladder transitional cell carcinoma

：Objective To investigatethe reverse effect of epithelial-mesenchymal transition (EMT) by silencing human genethymosin β4(Tβ4) ,and further reseach forits role on invasion and me-tastasis of cancer. Methods Lentiviral shRNA vector encodingTIM was transfected into T24 cell hne. The expression of Tβ4, ILK and epithelial markersE-cadherin, β-cateniu were detected by real-time reversetranscription-polymerase chain reaction (RT-PCR) and Western blotting; the expression ofintegrin-linked kinase (ILK) ,E-cadherin and β-catenin in T24 cells weredetected by immunofluorescence after transfec-tion via lentiviral vector;the metastaticpotential and apoptosis were examined by in vitro cell wound model, Boyden chamberinvasion assay and acridine orange-ethidium bromide fluorescent staining. Results Theexpression of Tβ4, ILK and β-catenin were decreased inthe T24 cells after transfected with Tβ4 shRNA, and the expressionof E-cadherin was significantly up-regulated (P <0. 05 ) ;the level of ILK in cytoplasmand level of β-catenin in nuclear were obviously decreased by immunofluorescenceanalysis, and the higher level of E-cadherin was observed, the morphology of T24 Cells wastransformed into a normal epithelial phe-notype ; the motility, invation of T24 cells[(10.4 ± 1.2) % vs (73.5 ± 1.4 ) %] were inhibited and apop-tosis (12. 3% vs 36. 6% )was enhanced. Conclusion The silencing of Tβ4 may reverse fibroblastoidmorphology into a normal epithelial phenotype, and suppress tumor metastatic potential.

Correlation betweenthymosin β 4 and epithelial-mesenchymaltransition in human bladder transi-tional cell carcinoma

：Objective To investigatethe relationship between human thymosin β4 (Tβ4)and epi-thelial-mesenchymal transition (EMT) in bladder transitional cell carcinoma(BTCC),explore the associa-tion with chnicopathologic features. Methods 60 BTCC specimens fromsurgically resected were selected. For each specimen,the cancerous tissue, peri-cancertissue and its remote normal mucosa were analyzed and compared. The expression of Tβ4,integrin-linked kinese (ILK), E-cadherin and β-catenin proteins inre-sected cancer tissues were detected by using real-time reverse transcription-polymerasechain reaction (RT-PCR), Western blotting and immunohistochemistry, and compared withchnicopathologic data. Results (1 ) The expression rate of Tβ4 was significantly higher inBTCC tissues than in normal tissues (91.7% vs 15. 0% ,P <0. 05 ) ,and the expressionrate of Tβ4 in deeply infiltrating group was significantly higher than thatin superficially infiltrating group (97.4% vs 81% ,P <0. 05 ) ;the level of ILK wassignificantly higher in BTCC than in normal tissues(80. 0% vs 18. 3% ,P <0. 05 ) ;theexpression rate of β-catenin was higher in BTCC tissues than in normaltissues (76. 7% vs 26. 7%, P < 0. 05 ) ; the expression of E-cadherin in BTCC tissueswere significantly lowered in comparison with those in normal bladder epithelium (30. 0%vs 90. 0% ,P < 0. 05 );(2) Tβ4 expression was inverselycorrelated to E-cadherin, but positively to ILK and β-catenin. Conclusion Thymosinβ4 is related to the differentiation and metastasis of bladder transitional cellcarcinoma,and may induce EMT to promote BTCC metastasis.

Basic Science Dietary flavones enhance the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on bladder cancer cells

Introduction. Flavonoids, naturally occurring in fruits and vegetables, demonstrate cancer chemopreventive properties. Flavones, a subclass of flavonoids, promote growth inhibition and apoptosis in bladder cancer cells. TRAIL plays an important role in immune surveillance and the defense mechanism against tumor cells. However, not all tumor cells are sensitive to TRAIL. TRAIL-resistant cancer cells can be sensitized to TRAILinduced apoptosis by anticancer agents. We investigated the effect of TRAIL in combination with dietary flavones on bladder cancer cells. Materials and methods. Two human bladder transitional cancer cell (TCC) lines: SW780 and RT112 were incubated with TRAIL and/or flavones (flavone, chrysin, apigenin, luteolin). Cytotoxicity was determined by MTT and LDH assays. results. Our study confirmed that RT112 bladder cancer cells were resistant to TRAIL, whereas SW780 cells were sensitive to TRAIL. We therefore examined the cytotoxic effect of TRAIL in combination with flavones on bladder cancer cells. We showed for the first time that flavones markedly augmented TRAIL mediated cytotoxicity against TCC cells. The strongest cytotoxic effect in combination with TRAIL was exhibited by luteolin. Co-treatment of bladder cancer cells with TRAIL and dietary flavones, especially luteolin, significantly sensitized bladder cancer cells to TRAIL induced cytotoxicity. Conclusion. The tested flavones enhance the cytotoxic effect of TRAIL on bladder cancer cells. The obtained results suggest that dietary flavones (in particular luteolin) supported TRAIL-induced cytotoxicity in TCC cells and thus they might be promising chemopreventive agents in bladder cancer.

Effects of gene Livin transfection affect on the apoptosis in bladder carcinoma cells

To investigate the effect of gene Livin transfection on the apoptosis of human bladder transitional cell carcinoma (BTCC) cells. Target fragment containing Livin full-length cDNA was obtained from the breast cancer cells of the line MCF7. Vector pcDNA3. 1 (+)-Livin was constructed and transfected into the bladder transitional cell carcinoma (BTCC) cells of the line T24 [T24/ Livin+ cells]. Another T24 cells were transfected with the eukaryotic expression vectors pcDNA3.1 (+) [T24/pcDNA3.1 (+) cells]. T24 cells without transfection were used as blank control group. These T24 cells underwent G418 selection so as to screen the T24/Livin+ cells stably expressing the target fragment. RT-PCR was used to detect the Livin expression level in the transfected cells. After the cells were treated with mitomycin C (MMC) for 24 h, MTT method was used to detect the inhibition rate. Acridine orange (AO) staining was used to observe the apoptotic cells. The apoptotic rate was observed by flow cytometry. RT-PCR results indicated that expression of Livin was positive in the T24/Livin+ cells, while were negative in the T24/pcDNA3.1 (+) and T24 cells. After being treated with MMC for 24 h, the apoptotic rate of the T24/Livin+ cell was (8.7 +/- 1.5)%, significantly lower than those of the T24/pcDNA3.1 (+) cells and T24 cells [(21.4 +/- 2.3)% and (19.6 +/- 2.3)% respectively, both P < 0.01]. The gene Livin increases the anti-apoptosis ability of tumor cells and the cell apoptosis induced by chemotherapy.

PTEN induces anoikis through its phosphatase activity in human bladder transitional carcinoma cells BIU-87

Objective To investigate the effects and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of human bladder transitional carcinoma cells BIU-87.

Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2

Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-alpha, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 siRNA. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspase-mediated cleavage of Cip1/p21.

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective: Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma (BTCC). However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determine a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cells from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods: Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results: That RNA integrity was good after LCM was confirmed by control experiment I; By control experiment II, the correlation between the number of LCMshooting and RNA quantity under arranged conditions was preliminarily confirmed. About 0.5-2.5 kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion: Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.

Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells

Bladder cancer is the fourth and eighth most common cancer in men and women in the United States, respectively. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is deregulated in a wide range of malignancies, including carcinoma of the bladder urothelium. Recent advances have identified survivin as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Silibinin is a naturally occurring flavanone, isolated from milk thistle extract, and has been shown to possess cancer prevention/intervention potential against various cancers. In several animal and human studies, it is found to be safe and non-toxic. Human bladder transitional-cell papilloma RT4 cells were treated with silibinin and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Silibinin treatment of cells for 24 h at 100 μM dose resulted in ∼50% decrease in survivin protein level; however, treatment at 200 μM dose for 24 and 48 h showed a complete loss in survivin protein without any change in actin used as loading control. Employing RT-PCR analysis we also observed that silibinin causes a strong to complete decrease in survivin mRNA levels. In other studies, down-regulation of survivin by silibinin was associated with a very strong and prominent caspases-9 and -3 activation as well as PARP cleavage. Quantitative apoptotic assay showed that silibinin decreased survivin levels and caspases-PARP cleavages, in accord with a strong apoptotic death and growth inhibition of RT4 cells. Together, these findings suggest that more studies are needed to investigate in vivo effect of silibinin on survivin expression and associated biological effects in bladder cancer that could provide useful information for silibinin efficacy in the prevention/intervention of human bladder cancer.

P21~(WAF1)、cyclinD1和pRb在膀胱移行细胞癌中的表达及其意义

目的探讨p21WAF1、细胞周期蛋白D1(cyclin D1)和pRb在膀胱移行细胞癌(BTCC)中的表达及相互关系和其意义.方法应用免疫组织化学SP法检测57例BTCC患者癌组织中p21WAF1、Cyclin D1和pRb的蛋白表达.结果 p21WAF1、cyclin D1和pRb的阳性表达率分别为36.8%、49.1%和45.6%,p21WAF1随病理分级升高阳性率显著下降,cyclin D1和pRb的表达与BTCC的病理分级、临床分期和有无转移均相关,p21WAF1与pRb的表达呈负相关,cyclin D1和pRb的表达呈正相关,而p21WAF1与cyclin D1的表达无关.结论 p21WAF1/cyclin D1/pRb通路异常与BTCC的发生发展密切相关,p21WAF1的改变可能为癌变的早期事件,联合检测p21WAF1、cyclin D1和pRb可较准确地评价BTCC的生物学特性,估计预后,指导治疗.

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas.

Galectin-1 and galectin-3 are galactoside-binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional-cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin-1 mRNA levels were highly increased in most high-grade tumors compared with normal bladder or low-grade tumors. Western blot and immuno-histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin-1 in tumors. Galectin-3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade.

Characterization of cell lines derived from a multiply aneuploid human bladder transitional‐cell carcinoma, UCRU‐BL‐13

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.