KK47 Cells Research Articles

Dibenzolium induces apoptosis and inhibits epithelial-mesenchymal transition (EMT) in bladder cancer cell lines

Bladder cancer treatments are highly aggressive and have strong side effects. Safer and more effective treatments are needed. In this study, Dibenzolium (DIB), a potent NADPH oxidase inhibitor, was evaluated for its anti-tumor effects. KK-47 (non-invasive), T24 and 5637 (invasive) cells were used in experiments. Cell proliferation, apoptosis and wound healing assays and western blotting were conducted. In addition, DIB was intratumorally administered to mice bearing KK-47, T24 and 5637 tumors, and tumor size and weight were observed over time. After removing tumors, immunohistochemistry (IHC) staining was conducted. Cell proliferation was significantly suppressed in all cell lines, and apoptotic cells increased in the KK-47 and T24 cell lines after DIB. Wound healing was suppressed in all cell lines by DIB. In KK-47 and T24, DIB increased the protein expression of the epithelial marker E-cadherin. In vivo, DIB safely suppressed tumor growth in all cell lines-bearing mice. Cleaved-Caspase-3 and E-cadherin expression increased in KK-47 and T24 tumors after DIB. In conclusion, DIB inhibited tumor growth by inducing apoptosis through the Caspase-3 pathway and reduced migration and invasion by suppressing epithelial mesenchymal transition (EMT) in bladder cancer similarly shown as our previous study of prostate cancer.

Nanaomycin K inhibited epithelial mesenchymal transition and tumor growth in bladder cancer cells in vitro and in vivo

Nanaomycin K, derived from Streptomyces rosa subsp. notoensis OS-3966T, has been discovered to have inhibitory bioactivity on epithelial–mesenchymal transition (EMT), an important mechanism of cancer cell invasion and migration. In this study, we examined the anti-EMT and anti-tumor effect of nanaomycin K in bladder cancer, where EMT has important roles in progression. We treated two bladder cancer lines, non-muscle-invasive KK47 and muscle-invasive T24, with nanaomycin K to determine the effects on cell proliferation, apoptosis and expression of EMT markers in vitro. Wound-healing assays were performed to assess cell invasion and migration. We conducted an in vivo xenograft study in which mice were inoculated with bladder cancer cells and treated with intratumoral administration of nanaomycin K to investigate its anti-tumor and EMT inhibition effects. As the results, nanaomycin K (50 µg/mL) significantly inhibited cell proliferation in KK47 (p < 0.01) and T24 (p < 0.01) in the presence of TGF-β, which is an EMT-inducer. Nanaomycin K (50 µg/mL) also significantly inhibited cell migration in KK47 (p < 0.01) and T24 (p < 0.01), and induced apoptosis in both cell lines in the presence of TGF-β (p < 0.01). Nanaomycin K increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin in both cell lines. Nanaomycin K also decreased expression of Snail, Slug, phospho-p38 and phospho-SAPK/JNK especially in T24. Intratumoral administration of nanaomycin K significantly inhibited tumor growth in both KK47 and T24 cells at high dose (1.0 mg/body) (p = 0.009 and p = 0.003, respectively) with no obvious adverse events. In addition, nanaomycin K reversed EMT and significantly inhibited the expression of Ki-67 especially in T24. In conclusion, we demonstrated that nanaomycin K had significant anti-EMT and anti-tumor effects in bladder cancer cells, suggesting that nanaomycin K may be a therapeutic candidate for bladder cancer treatment.

Long noncoding RNA neuroblastoma-associated transcript 1 gene inhibits malignant cellular phenotypes of bladder cancer through miR-21/SOCS6 axis

Bladder cancer (BC) is one of the most common tumors in the urinary system. Noncoding RNAs are considered to take part in cellular phenotypes and are emerging as diagnostic and prognostic biomarkers of BC. The aim of this study is to investigate the clinical significance of neuroblastoma- associated transcript 1 (NBAT1) gene and its effects on malignant cellular phenotypes in BC. NBAT1 gene was low-expressed in BC tissues and cell lines and its low-expression was related with high pathological grade and metastasis of BC. Upregulation of NBAT1 gene depressed cell viability and invasiveness of KK47 and T24 cells and arrested KK47 and T24 cells at G1 stage. In addition, NBAT1 could target silence the expression of miR-21-5p in RNA-induced silencing complex-dependent manner. KK47 and T24 cells with miR-21-5p knockdown showed reduced cell viability, G1-stage arrest, and depressed invasiveness. MiR-21-5p mediates the regulatory effects of NBAT1 on malignant cellular phenotypes of BC cells. Moreover, SOCS6 gene was a target gene of miR-21-5p, and miR-21-5p modulated malignant cellular phenotypes of KK47 and T24 cells through targeted silencing of SOCS6. In conclusion, low-expression of NBAT1 is associated with the progress and metastasis of BC, and NBAT1 inhibits malignant cellular phenotypes through miR-21-5p/SOCS6 axis in BC. Our findings help to elucidate the tumorigenesis of BC, and future study will provide a novel therapeutic target for BC.

Overexpression of REIC/Dkk-3 suppresses the expression of CD147 and inhibits the proliferation of human bladder cancer cells.

Our group previously developed an adenoviral vector encoding the REIC/Dkk-3 gene (Ad-REIC), a tumor suppressor, for cancer gene therapy. The Ad-REIC agent induces apoptosis and inhibits invasion in a number of cancer cell lines; however, the molecular mechanisms underlying its effects remain unclear. Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a key molecule that promotes cancer proliferation and invasion. In order to elucidate the therapeutic mechanism of Ad-REIC, its effect on the expression of CD147 in human bladder cancer KK47 cells was investigated. Treatment with Ad-REIC markedly downregulated the expression of CD147 and significantly inhibited cellular proliferation. Since the expression of CD147 is reported to be under the positive control of mitogen-activated protein kinase (MAPK) signaling and the c-Myc protein, the correlations between the expression of CD147 and the activation of MAPKs or the expression of c-Myc were examined. Unexpectedly, no positive correlation was observed between the level of CD147 and the potential regulators that were assessed, indicating that another signaling pathway is responsible for the downregulation of CD147. The results from the present study demonstrate that Ad-REIC treatment can significantly downregulate the expression of CD147 in bladder cancer cells. Downregulation of the cancer-progression factor CD147 may be a novel mechanism that underlies the therapeutic effects of Ad-REIC treatment.

Quantitative Analysis of Differential Proteome Expression in Epithelial-to-Mesenchymal Transition of Bladder Epithelial Cells Using SILAC Method.

Epithelial-to-mesenchymal transition (EMT) is an essential biological process involved in embryonic development, cancer progression, and metastatic diseases. EMT has often been used as a model for elucidating the mechanisms that underlie bladder cancer progression. However, no study to date has addressed the quantitative global variation of proteins in EMT using normal and non-malignant bladder cells. We treated normal bladder epithelial HCV29 cells and low grade nonmuscle invasive bladder cancer KK47 cells with transforming growth factor-beta (TGF-β) to establish an EMT model, and studied non-treated and treated HCV29 and KK47 cells by the stable isotope labeling amino acids in cell culture (SILAC) method. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography/LTQ Orbitrap mass spectrometry. Among a total of 2994 unique identified and annotated proteins in HCV29 and KK47 cells undergoing EMT, 48 and 56 proteins, respectively, were significantly upregulated, and 106 and 24 proteins were significantly downregulated. Gene ontology (GO) term analysis and pathways analysis indicated that the differentially regulated proteins were involved mainly in enhancement of DNA maintenance and inhibition of cell-cell adhesion. Proteomes were compared for bladder cell EMT vs. bladder cancer cells, revealing 16 proteins that displayed similar changes in the two situations. Studies are in progress to further characterize these 16 proteins and their biological functions in EMT.

Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells.

Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles.

Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method

The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2014MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL Yuki Tobisawa, Shingo Hatakeyama, Tohru Yoneyama, Teppei Okamoto, Hayato Yamamoto, Kazuyuki Mori, Akiko Okamoto, Atsushi Imai, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, and Chikara Ohyama Yuki TobisawaYuki Tobisawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Teppei OkamotoTeppei Okamoto More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Kazuyuki MoriKazuyuki Mori More articles by this author , Akiko OkamotoAkiko Okamoto More articles by this author , Atsushi ImaiAtsushi Imai More articles by this author , Takahiro YoneyamaTakahiro Yoneyama More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , Takuya KoieTakuya Koie More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.836AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Platelet aggregation together with cancer cell is one of the key factors for cancer progression and metastasis. Podoplanin (PDPN), which is known as a lymphatic endothelial marker, expressed on cancer cell surface has a potential of platelet aggregation through interaction with CLEC-2 expressed on Platelet. Core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) is a key enzyme for O-glycosylation of membrane proteins. Although PDPN has a core2 O-glycosylation site on their mucin domain, its roll in cancer progression is still unclear. Here, we examine the roles of PDPN and their O-glycosylation in bladder cancer (BCa) progression and invasion. METHODS YTS-1 is a BCa cell line established from muscle invasive BCa and highly positive for PDPN and C2GnT. KK-47 is a BCa cell line established from non-muscle invasive BCa and expresses a faint level of PDPN and C2GnT. YTS-1 cells were transfected with PDPN-siRNA or C2GnT-siRNA to silence of PDPN or C2GnT expression. KK47 cells were transfected with pcDNA3.1-PDPN and/or pcDNA3.1-C2GnT to make KK47-PDPN, KK47-C2GnT and KK47-C2PDPN which are highly positive for PDPN, C2GnT or both of them. These cells were subjected to growth factor assays and invasion assay. Co-culture system of BCa cells and platelet was used to evaluate growth kinetics of the BCa cells and production of growth factors. In vitro invasion assay was used to evaluate invasion potential of the BCa cells. RESULTS In the co-culture system of BCa cells and platelet, BCa cells culturing with platelet had higher growth activity than without platelet (p<0.05). YTS-1 produced significantly higher amounts of bFGF, EGF, NGF beta, PDGF-BB, TGF-beta and VEGF when they are co-cultured with platelet than they are cultured without platelet (p<0.05). O-glycosylation of PDPN on BCa cells significantly increased the production of growth factors (p<0.05). When PDPN is knocked-down, invasive potential of YTS-1 was markedly reduced. (p=0.005). CONCLUSIONS These results suggest that expression of PDPN and its O-glycosylation enhance growth factors production, proliferation activity, and invasion potential of bladder cancer cells through interaction with platelet. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e228 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yuki Tobisawa More articles by this author Shingo Hatakeyama More articles by this author Tohru Yoneyama More articles by this author Teppei Okamoto More articles by this author Hayato Yamamoto More articles by this author Kazuyuki Mori More articles by this author Akiko Okamoto More articles by this author Atsushi Imai More articles by this author Takahiro Yoneyama More articles by this author Yasuhiro Hashimoto More articles by this author Takuya Koie More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In Vivo Selection of High-Metastatic Subline of Bladder Cancer Cell and its Characterization

The majority of deaths associated with solid tumors are caused by tumor metastasis. To prevent metastasis, it is vital to understand its detailed process. In hematogenous metastasis of bladder cancer, some cancer cells disseminating into blood circulation extravasate into the lung tissues to form metastases. To study the molecular basis of the lung metastasis of bladder cancer, we employed an in vivo selection system that mimics hematogenous metastasis of bladder cancer on a low-metastatic bladder cancer cell line (KK-47). We have successfully isolated a high-metastatic bladder cancer subline, KK-47HM4, from KK-47 cells. We characterized KK-47HM4 in in vitro experimental systems. No significant difference in growth rate and susceptibility to NK cell attack between KK-47 and KK-47HM4 cells was observed. However, KK-47HM4 exhibited the higher capacities of Matrigel Matrix invasion and transendothelial invasion than KK-47. These results suggest that the extravasation of KK-47HM4 cells was enhanced among the multiple steps of the lung metastasis of bladder cancer. Our cDNA microarray analysis identified 67 genes whose expression was up- or downregulated in KK-47HM4 cells compared with KK-47 cells. This analysis data implied that one possible cause for enhanced extravasation of KK-47HM4 is its higher adhesion to extracellular matrix proteins. KK-47HM4 is the first bladder cancer subline with enhanced extravasation potential using the in vivo selection system. The information provided by our cDNA microarray analysis using KK-47HM4 will be useful for further investigation into the molecular basis of extravasation of cancer cells.

555 NANOPARTICLE BCG HAS IDENTICAL DIRECT ANTITUMOR EFFECT AS LIVE BCG, BUT HAS A DIFFERENT ANTITUMOR MECHANISM

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2012555 NANOPARTICLE BCG HAS IDENTICAL DIRECT ANTITUMOR EFFECT AS LIVE BCG, BUT HAS A DIFFERENT ANTITUMOR MECHANISM Kazuyuki Mori, Tohru Yoneyama, Yuki Tobisawa, Yusuke Ishibashi, Hiromi Murasawa, Hayato Yamamoto, Hirofumi Ishimura, Shingo Hatakeyama, Shigemasa Kudo, Takahiro Yoneyama, Takuya Koie, Noritaka Kamimura, and Chikara Ohyama Kazuyuki MoriKazuyuki Mori Hirosaki, Japan More articles by this author , Tohru YoneyamaTohru Yoneyama Hirosaki, Japan More articles by this author , Yuki TobisawaYuki Tobisawa Hirosaki, Japan More articles by this author , Yusuke IshibashiYusuke Ishibashi Hirosaki, Japan More articles by this author , Hiromi MurasawaHiromi Murasawa Hirosaki, Japan More articles by this author , Hayato YamamotoHayato Yamamoto Hirosaki, Japan More articles by this author , Hirofumi IshimuraHirofumi Ishimura Hirosaki, Japan More articles by this author , Shingo HatakeyamaShingo Hatakeyama Hirosaki, Japan More articles by this author , Shigemasa KudoShigemasa Kudo Hirosaki, Japan More articles by this author , Takahiro YoneyamaTakahiro Yoneyama Hirosaki, Japan More articles by this author , Takuya KoieTakuya Koie Hirosaki, Japan More articles by this author , Noritaka KamimuraNoritaka Kamimura Hirosaki, Japan More articles by this author , and Chikara OhyamaChikara Ohyama Hirosaki, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.630AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In our previous report on AUA2007, we generated nanoparticle BCG, which exerts a direct antitumor effect identical to that by live BCG against J82 and KK47 bladder cancer cells, however, we did not analyze the mechanism. In this study, we performed cell cycle analysis and detection of apoptosis. METHODS The Tokyo 172 BCG sub-strain suspension was disrupted by a French press, and the particle distribution was determined in a particle analyzer. After removing the non-disrupted bacteria by centrifugation at 6800 ×g, the supernatant was centrifuged at 18,000 ×g. The supernatant (CM) and the precipitate (CW) were lyophilized to obtain nanoparticle BCG. The bladder cancer cell lines J82 and KK47 were maintained in RPMI1640 medium. Live BCG, CW, CM, and CW+CM (mix) were added to the culture medium at a final concentration of 0.1 mg/mL and cultured for 5 days, and the viable cells were counted using trypan blue staining. Cells and culture sup were collected and stained with propidium iodide, and cell cycle analysis and detection of apoptosis using flow cytometry was performed. RESULTS After 5 days of culturing, the viability of cells treated with BCG and nanoparticle BCG significantly decreased to 72.4% and 73.4% (J82 cells) and 68.2% and 67.0% (KK47 cells) of control cells, respectively. The percentage of apoptosis in control cells, cells treated with BCG, and cells treated with nanoparticle BCG was 3.61%, 7.61%, and 4.02% in J82 cells, and 1.53%, 7.14%, and 0.07% in KK47 cells, respectively. BCG induced apoptosis, but nanoparticle BCG did not. The percentage of total S phase in control cells, cells treated with BCG, and cells treated with nanoparticle BCG was 26.19%, 32.39%, and 27.48% in J82 cells, and 27.88%, 26.78%, and 27.48% in KK47 cells, respectively. In J82 cells, BCG significantly increased the percentage of total S phase cells, but nanoparticle BCG did not. CONCLUSIONS Nanoparticle BCG had identical direct antitumor effect as live BCG; however, it did not induce apoptosis. These results suggest that nanoparticle BCG can be used to develop new therapeutic drugs through the different antitumor mechanism against live BCG-failure cases, invasive bladder cancer, or other tissue cancer. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e227 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kazuyuki Mori Hirosaki, Japan More articles by this author Tohru Yoneyama Hirosaki, Japan More articles by this author Yuki Tobisawa Hirosaki, Japan More articles by this author Yusuke Ishibashi Hirosaki, Japan More articles by this author Hiromi Murasawa Hirosaki, Japan More articles by this author Hayato Yamamoto Hirosaki, Japan More articles by this author Hirofumi Ishimura Hirosaki, Japan More articles by this author Shingo Hatakeyama Hirosaki, Japan More articles by this author Shigemasa Kudo Hirosaki, Japan More articles by this author Takahiro Yoneyama Hirosaki, Japan More articles by this author Takuya Koie Hirosaki, Japan More articles by this author Noritaka Kamimura Hirosaki, Japan More articles by this author Chikara Ohyama Hirosaki, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A3 Adenosine Receptor Mediates Apoptosis in 5637 Human Bladder Cancer Cells by Gq Protein/PKC-Dependent AIF Upregulation

Background/Aims: A₃ adenosine receptor mediates apoptosis in a variety of cancer cells via diverse signaling pathways. The present study was conducted to assess A₃ adenosine receptor-mediated apoptosis in human bladder cancer cell lines and to understand the underlying mechanism. Methods: Human bladder cancer cell lines such as 253J, 5637, KK-47, TCCSUP, T24, and UMUC-3 cells were cultured. The siRNA to silence the A₃ adenosine receptor-targeted gene was constructed and transfected into cells. MTT assay, TUNEL staining, Western blotting, and real-time RT-PCR were carried out. Results: For all the investigated cell types adenosine induced apoptosis in a concentration (0.01-10 mM)- and treatment time (24-48 h)-dependent manner. Adenosine-induced 5637 cell death was significantly inhibited by the A₃ adenosine receptor inhibitor MRS1191 or knocking-down A₃ adenosine receptor, and the A₃ adenosine receptor agonist 2-Cl-IB-MECA mimicked the adenosine effect. The adenosine effect was prevented by GF109203X, an inhibitor of protein kinase C (PKC), but it was not affected by forskolin, an activator of adenylate cyclase. Adenosine-induced 5637 cell death, alternatively, was not inhibited by the pan-caspase inhibitor Z-VAD. Adenosine upregulated expression of apoptosis-inducing factor (AIF), that is suppressed by knocking-down A₃ adenosine receptor, and accumulated AIF in the nucleus. Conclusion: The results of the present study show that adenosine induces 5637 cell apoptosis by upregulating AIF expression via an A₃ adenosine receptor-mediated G_q protein/PKC pathway.

Foxo3a Suppression of Urothelial Cancer Invasiveness through Twist1, Y-Box–Binding Protein 1, and E-Cadherin Regulation

Invasion and metastasis are key steps in the progression of urothelial cancer (UC) into a critical disease. Foxo3a is a member of the Foxo transcription factor family that modulates the expression of various genes. We aimed to elucidate the role of Foxo3a in UC invasion. Foxo3a mRNA and protein expressions in UC samples were investigated by gene expression assays and immunohistochemistry, respectively. Foxo3a expression was compared with clinicopathologic characteristics and patient prognoses based on UC samples. Quantitative real-time polymerase chain reaction, Western blotting, and migration assays were also conducted in UC cells. Foxo3a expression decreased in invasive UC; patients with low Foxo3a expression had poor disease-free survival, cancer-specific survival, and overall survival; Foxo3a knockdown in UC cells increased cellular motility. Foxo3a negatively regulated Twist1 and Y-box-binding protein 1 (YB-1), and positively regulated E-cadherin in KK47 and TCCsup cells that expressed Twist1, but not in T24 cells that did not express Twist1. Foxo3a-associated acetyltransferase p300 and Foxo3a acetylation status also affected UC motility. The results of this study indicate that Foxo3a regulates motility of UC through negative regulation of Twist1 and YB-1, and through positive regulation of E-cadherin. This suggests that Foxo3a could act as an independent prognostic factor in UC and could represent a promising molecular target for cancer therapeutics.

Twist1 and Y‐box‐binding protein‐1 promote malignant potential in bladder cancer cells

To investigate the roles of Twist1 and Y-box binding protein-1 (YB-1) and their potential as therapeutic targets in bladder cancer (BC), as both have been suggested to play important roles in tumour growth, invasion and drug resistance. Bladder cancer cell lines (TCCsup, UMUC3, T24 and KK47 cells) were used. Twist1 and YB-1 expression levels were assessed by luciferase reporter assay, quantitative real-time polymerase chain reaction (PCR) and western blot analysis. Tumour growth and cell cycle were analysed by cell proliferation assay and flow cytometry, respectively. Invasive and motile abilities were investigated by scratch-wound test and migration assay, respectively. Cytotoxicity assay was performed to determine drug sensitivity. The findings showed that Twist1 regulated YB-1 expression in BC cells. Both Twist1 and YB-1 were involved in cell growth, invasion, motility and resistance to cisplatin and doxorubicin, but not to 5-fluorouracil (5-FU). The present study showed that Twist1 regulates YB-1 expression and that both Twist1 and YB-1 promote malignant potentials, including tumour growth, invasion and anti-cancer-drug resistance, indicating that both Twist1 and YB-1 are novel molecular targets in BC.

P300/CBP‐associated factor regulates Y‐box binding protein‐1 expression and promotes cancer cell growth, cancer invasion and drug resistance

Twist1 has been proposed to have oncogenic properties. Although Twist1 was reported to interact with p300/CBP-associated factor (PCAF) and to inhibit the functions of PCAF, it remains unclear how PCAF affects the functions of Twist1, cell growth, invasive ability, and cellular sensitivity to anticancer agents. We found that PCAF, Twist1, and Y-box binding protein-1 (YB-1) expressions were elevated in cisplatin- and doxorubicin-resistant cancer cells. Luciferase reporter assays revealed that PCAF manipulation modulated YB-1 transcription in a Twist1-dependent manner. In addition, PCAF regulated the Twist1 intracellular localization and the Twist1 transcriptional activity through its acetylation function to the Twist1. Suppression of PCAF expression reduced YB-1 expression in human urothelial cancer KK47 cells. As a result, the cell growth and invasive ability of KK47 cells was retarded by PCAF knockdown, and PCAF knockdown rendered KK47 cells sensitive to cisplatin and doxorubicin, but not to 5-fluorouracil. The present data suggest that Twist1 and YB-1 as well as PCAF may be promising molecular therapeutic targets.

Midkine Promoter-Based Conditionally Replicative Adenovirus for Targeting Midkine-Expressing Human Bladder Cancer Model

To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-E1a-a midkine (MK) promoter-regulated, conditionally replicating, adenovirus. We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative polymerase chain reaction, MK promoter activity, after plasmid transfection, using a luciferase assay, and the transduction efficiency by co-transfection with the cytomegalovirus-beta-gal plasmid. In these cells, we assessed the cell type-specific replication of Ad-MK-E1a virus by measuring the E1a DNA copy number by real-time polymerase chain reaction and the cell growth inhibition due to this virus using the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with phosphate-buffered saline or Ad5-CMV-LacZ or Ad-MK-E1a. The MK mRNA expression level and MK promoter-driven luciferase activity were relatively greater and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells. After Ad-MK-E1a infection, the E1a DNA copy number increased more significantly in the KK47, 5637, and A549 cells than in the T24 and H891 cells. At a multiplicity of infection of 0.01, Ad-MK-E1a significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth. We have demonstrated the antitumor effect of Ad-MK-E1a in a human bladder cancer model overexpressing MK mRNA.

Centrosome Hyperamplification and Chromosomal Damage after Exposure to Radiation

Objective: In order to elucidate the effects of radiation on centrosome hyperamplification (CH), we examined the centrosome duplication cycle in KK47 bladder cancer cells following irradiation. Methods: KK47 cells were irradiated with various doses of radiation and were examined for CH immunostaining for γ-tubulin. Results: Nearly all control cells contained one or two centrosomes, and mitotic cells displayed typical bipolar spindles. The centrosome replication cycle is well regulated in KK47. Twenty-four hours after 5-Gy irradiation, ∼80% of irradiated cells were arrested in G2 phase, and at 48 h after irradiation, 56.9% of cells contained more than two centrosomes. Laser scanning cytometry performed 48 h after irradiation showed the following two pathways: (1) unequal distribution of chromosomes to daughter cells, or (2) failure to undergo cytokinesis, resulting in polyploidy. With mitotic collection, M-phase cells with CH could be divided into G1 cells with micronuclei and polyploidal cells. Fluorescence in situ hybridization analysis showed clear signs of chromosomal instability (CIN) at 48 h after irradiation. The present study had two major findings: (1) continual duplication of centrosomes occurred in the cell cycle-arrested cells upon irradiation, leading to centrosome amplification; (2) cytokinesis failure was due to aberrant mitotic spindle formation caused by the presence of amplified centrosomes. Abnormal mitosis with amplified centrosomes was detected in the accumulating G2/M population after irradiation, showing that this amplification of centrosomes was not caused by failure to undergo cytokinesis, but rather that abnormal mitosis resulting from amplification of centrosomes leads to cytokinesis block. Conclusion: These results suggest that CH is a critical event leading to CIN following exposure to radiation.

Granulocyte colony-stimulating factor may promote proliferation of human bladder cancer cells mediated by basic fibroblast growth factor.

To investigate whether granulocyte colony-stimulating factor (G-CSF) promotes the proliferation of two bladder cancer cell lines, and to assess the mechanism of tumor proliferation in terms of cytokine expression. The proliferation of two bladder cancer cell lines derived from transitional cell carcinoma (KK-47 and T-24) was assessed by using the double-layer soft agarose colony assay in combination with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Seven cytokines were measured in the culture supernatant. Expression of granulocyte colony-stimulating factor (G-CSF) receptor and fibroblast growth factor (FGF) receptor mRNA was studied by means of reverse transcriptase polymerase chain reaction (RT-PCR). Recombinant human G-CSF (rhG-CSF) caused greater induction of the proliferation of KK-47 cells in the presence than in the absence of peripheral blood mononuclear cells (PBMCs) and its effect was dose-dependent. In contrast, rhG-CSF did not stimulate the proliferation of T-24 cells. Among several cytokines measured, only basic FGF was elevated in cultures of KK-47 cells with or without PBMCs. The basic FGF level was significantly increased by rhG-CSF stimulation in a dose-dependent manner. Specific PCR products for the G-CSF and FGF receptors were observed in KK-47 cells as well as PBMC, while no G-CSF receptor was detected in T-24 cells. rhG-CSF may promote the proliferation of KK-47 cells, probably via an increase in basic FGF production.

Enhancement by cyclosporin A of taxol-induced apoptosis of human urinary bladder cancer cells.

Taxol is a microtubule-stabilizing agent which induces apoptosis in various cancer cells. In this study, we found that T24 cells derived from high grade human urinary bladder cancer were relatively resistant to taxol and that the IC50 value determined by a colorimetric WST-1 assay was 406.0 nM. Interestingly, cyclosporin A (CsA), an immunosuppressive drug, dramatically enhanced sensitivity to taxol, and the IC50 value was decreased to 47.5 nM in the presence of 1 microM CsA. KK47 cells derived from low grade human urinary bladder cancer showed high sensitivity to taxol with an IC50 value of 78.8 nM which decreased to 14.4 nM in the presence of 1 microM CsA. FK506, another immunosuppressive drug, also enhanced sensitivity to taxol. Furthermore, a concomitant loss of calcineurin activity was observed after the treatment of both cell lines with both CsA and FK506. Taxol induced apoptosis of the cells, as assessed by Hoechst 33258 staining and by the measurement of caspase 3 activity. Immunoblot analysis with an antibody against Bcl-2 phosphorylated at serine 70 demonstrated that taxol induced the phosphorylation of Bcl-2 with its enhancement in the presence of CsA. In addition, treatment of the cells with CsA significantly decreased the expression of Bcl-2 at both the protein and mRNA levels. These results suggest that the enhancement of taxol-induced apoptosis by immunosuppressive drugs is at least partly due to the inhibition of calcineurin activity and the loss of the antiapoptotic function of Bcl-2 via the enhancement of phosphorylation and the reduction of expression.

Uroplakin Gene Expression by Normal and Neoplastic Human Urothelium

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage.

The novel antifolate lometrexol (5,10-dideazatetrahydrofolate) inhibits de novo purine biosynthesis, and co-incubation with hypoxanthine abolishes its cytotoxicity. The prevention of hypoxanthine rescue from an antipurine antifolate by the nucleoside transport inhibitor dipyridamole was investigated for the first time in nine human and rodent cell lines from seven different tissues of origin. In A549, HeLa and CHO cells, dipyridamole prevented hypoxanthine rescue and so growth was inhibited by the combination of lometrexol, dipyridamole and hypoxanthine, but in HT29, HCT116, KK47, MDA231, CCRF CEM and L1210 cells dipyridamole had no effect and the combination did not inhibit growth. Dipyridamole inhibited hypoxanthine uptake in A549 but not in CCRF CEM cells. Dipyridamole prevented the hypoxanthine-induced repletion of dGTP pools, depleted by lometrexol, in A549 but not in CCRF CEM cells. Thus, the selective growth-inhibitory effect of the combination of lometrexol, dipyridamole and hypoxanthine is apparently due to the dipyridamole sensitivity (ds) or insensitivity (di) of hypoxanthine transport. Both the human and murine leukaemic cells are of the di phenotype. If this reflects the transport phenotype of normal bone marrow it would suggest that the combination of lometrexol, dipyridamole and hypoxanthine might be selectively toxic to certain tumour types and have reduced toxicity to the bone marrow.