Bladder Cancer Cell Line T24 Research Articles

Abstract 389: The functional relevance of decreased ANGPTL4 in human urinary bladder cancer.

Abstract Transitional cell carcinoma is the most common urothelial cancer in the US. Current therapeutic options have been unable to overcome issues with the highly recurrent nature of this tumor, which requires frequent follow-up making urinary bladder cancer, one of the most expensive solid tumors to manage. Microarray-based gene expression analysis in response to capsaicin treatment identified significant upregulation of angiopoietin-like 4 (ANGPTL4) compared with solvent-treated bladder cancer cells. The focus of this study was to determine the functional relevance of ANGPTL4 in normal urothelial and bladder cancer cells. The microarray data was validated in a panel of normal, immortalized and bladder cancer cells of increasing stage by real-time qPCR, immunoblotting, and ELISA. To determine the possible function of ANGPTL4, we knocked down and overexpressed ANGPTL4 in normal urothelial and bladder cancer cells respectively. The role of ANGPTL4 in growth, proliferation and angiogenesis was assessed. Our results show that normal urothelial cells have significantly higher levels of ANGPTL4 when compared to non-tumorigenic and bladder cancer cells. Upon capsaicin treatment ANGPTL4 levels increased in all cell lines tested. Overexpression of ANGPTL4 in the metastatic cell line TCCSUP, led to decrease in cell growth. Further, ANGPTL4 knockdown increased cell growth in the normal urothelial cell line. Capsaicin treatment decreased the expression of VEGF in bladder cancer cell lines UMUC-3 and T24 as well as reduced expression in normal urothelium, albeit initial results in ANGPLT4-knockdown in normal urothelium indicates no change in VEGF expression. Our results thus far suggest that the loss of ANGPTL4 expression may be involved in bladder cancer. The mechanism involved is under investigation. Capsaicin remains an attractive compound for further studies in bladder cancer. Supported by RO3 CA136058 (RG) and NIHT32 T32CA148724 (EC). Citation Format: Eduardo Cardenas, Tiffani Horton, Rita Ghosh. The functional relevance of decreased ANGPTL4 in human urinary bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2013-389

Abstract 4624: Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents.

Abstract The successful use of DNA methyltransferase inhibitors (DNMTis) in Myelodysplastic Syndromes (MDS) therapy has brought epigenetic drugs to the forefront of cancer management. However it has been difficult to find a consistent association between patients’ outcome and DNA methylation changes. This could be attributed to a variety of factors including the choice of DNA methylation markers to track the response to treatment. LINE-1 methylation changes are widely studied as a marker predictive of genome-wide DNA methylation changes; however recent reports have shown that its methylation levels are variable across tissues. In addition, we have found that LINE-1 remethylation after DNMTis withdrawal occurs faster than that of other regions, suggesting that LINE-1 methylation changes might not reflect the overall demethylation effects of DNMTis. Thus, it is imperative to find more sensitive DNA methylation markers to accurately track the methylation change that occurs after DNMTi treatment. Ideally, such markers could be applied to various tumor types and to samples collected by invasive and non-invasive methods. To find improved DNA methylation markers, we took advantage of the well-established Infinium DNA methylation platform and found 1429 probes, which were consistently methylated in both normal/tumor bladder tissue samples as well as white blood cells from healthy donors. The remethylation pattern of those 1439 probes was investigated in the T24 bladder cancer and HL60 leukemia cell lines treated with 5-Aza-CdR for 24 hours. Probe consensus clustering yielded a group of 79 probes that significantly responded to the demethylation treatment and remained demethylated beyond 30 days. In silico analysis of the DNA methylation patterns of the top two probes show consistent hypermethylation in both normal and tumor samples. As a proof of principle, we tested the DNA demethylation levels of these two markers in urine sediments from 7 MDS patients treated with Azacitidine using pyrosequencing. Our results showed that the two markers were significantly demethylated; in contrast, LINE-1 methylation showed no clear decreasing trend. Demethylation of these two markers was also observed in peripheral blood samples from MDS patients treated with Azacitidine. We are in the process of testing more samples to find the association between marker demethylation and patient's outcome. In summary, we have identified and validated two DNA methylation markers, which unlike LINE-1, show consistent demethylation in response to the DNMTi treatment irrespective of the type of sample tested. The wider range of demethylation provided by these markers may offer a more accurate representation of the patient's response to treatment. Citation Format: Xiaojing Yang, Han Han, Marianne B. Treppendahl, Yvonne C. Tsai, Casey O'Connell, Dan Weisenberger, Fides Lay, Kirsten Grønbæk, Gangning Liang, Peter A. Jones. Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2013-4624

Abstract 4429: Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells.

Abstract Erlotinib is a first-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor and have well-established efficacy in non-small cell lung cancer patients with activating EGFR mutations. Afatinib, on the other hand, is a second-generation EGFR tyrosine kinase inhibitor with anti-HER2 activity and was found to be of benefit to patients with advanced lung adenocarcinoma who failed previous gefetinib or erlotinib. In previous study we have demonstrated the in vitro and in vivo radiosensitising activity of afatinib in a murine bladder cancer model. However, the radiosensitizing effect of afatinib and erlotinib, has never been compared in cancer cells including bladder cancer. We performed RTK (Receptor Tyrosine Kinase) antibody arrays to investigate the relative levels of phosphorylation in T24 human bladder cancer cell line. We found that EGFR and HER2 signals were activated after radiation10 Gy. Afatinib 100 nM suppressed both irradiation-induced EGFR and HER2 signals but erlotinib 100 nM only suppressed irradiation-induced EGFR signal. Clonogenic assay of T24 and NTUB1 human bladder cancer cell lines was then examined. Afatinib (100-500 nM) showed better radiosensitizing effect (radiation dose: 2-10 Gy) than erlotinib (300-1500 nM) in both T24 and NTUB1 cells by significantly decreasing the numbers of colonies 7 days after treatment. The effect is more prominent in high doses of afatinib. Flow cytometry was used to determine the distribution of cells among various cell cycle phases. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 200 nM, erlotinib 200 nM or the combination of radiation and afatinib or erlotinib. When compared with radiation or drug alone, treatment combining radiation and afatinib in T24 cells resulted in a significant increase of cells in sub-G1 phase but the phenomenon was not observed in the combination of erlotinib and radiation. A similar trend was found in NTUB1 cells. The results implicated that afatinib might enhance radiation effect by increasing apoptosis in bladder cancer cells. To study the DNA damage status after treatment, we checked intra-nuclear γH2AX foci by mmunofluorescence microscopy. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 100 nM, erlotinib 100 nM or the combination of radiation and afatinib or erlotinib. Radiation alone significantly increased γH2AX foci and combining radiation with afatinib further increased the foci in both T24 and NTUB1 cells. The enhancement was absent in the combination of radiation and erlotinib. Our data clearly show that afatinib, a new generation EGFR tyrosine kinase inhibitor with anti-HER2 activity, is superior to erlotinib as a radiosensitizer in the treatment of human bladder cancer cells. Citation Format: Yu-Chieh Tsai, Tsung-Fan Tuan, Pei-Yin Ho, Wei-Lin Liu, Liang-Yu Chang, Yeong-Shiau Pu, Ann-Lii Cheng, Jason Chia-Hsien Chen. Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2013-4429

The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells. Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer.

Overexpression of immunoglobulin G prompts cell proliferation and inhibits cell apoptosis in human urothelial carcinoma

Only B lymphocytes can express immunoglobulins according to the traditional immunological theories, and the expression of immunoglobulin G (IgG) messenger RNA (mRNA) and protein was found in certain human cancer cells recently. However, the expression pattern of IgG and its possible role in human urothelial carcinoma are still elusive. In this study, we investigated the expression of IgG in two human urothelial carcinoma cell lines, T24 and BIU-87, and in 56 cases of clinical urothelial carcinoma tissues. The mRNA of IgG was positively detected by in situ hybridization and reverse transcription PCR; furthermore, IgG protein was also positively detected by immunohistochemistry and Western blot. Moreover, blockade of tumor-derived IgG by either antihuman IgG antibody or antisense oligonucleotides increased cell apoptosis and inhibited cell growth in bladder cancer cell lines in vitro, and antihuman IgG antibody could suppress the growth of xenotransplant tumor in vivo. In addition, either antihuman IgG antibody or antisense oligonucleotides enhanced the sensitivity to mitomycin C in bladder cancer cell line T24. Furthermore, blockade of IgG in bladder cancer cell T24 resulted in upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Our results indicated that bladder cancer cells were capable of expressing IgG, and blockade of IgG expression induced cell apoptosis through activation of caspase-dependent pathway. A novel potential targeted therapy for bladder cancer will be possibly developed based on these data.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0717-z) contains supplementary material, which is available to authorized users.

In Vitro Regulation of Cell Growth and Angiogenesis by Inositol Hexaphosphate in Bladder Cancer

Background: Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is found in food sources high in fiber content. We hypothesized that IP6 would inhibit the cell growth rate of bladder cancer in vitro. Methods: T24 and TCCSUP bladder cancer cell lines were treated with titrating doses of IP6 (0.3, 0.6 and 0.9 mM/well). Cell viability and vascular endothelial growth factor levels were measured. Results: Significant reductions (p < 0.001) in cellular growth were noted in both cell lines at all doses and time points tested, with the exception of 0.3 mM IP6 at 24 hours in the T24 cell line. The percent inhibition of vascular endothelial growth factor was significantly higher than that observed in the TCCSUP cell line at 48 and 72 hours with 0.3 mM IP6 (p < 0.001). The T24 cells exhibited the same level of inhibition at 24 and 48 hours with 0.6 m<smlcap>M</smlcap> dose of IP6 and at 72 hours with the 0.3 mM dose (p < 0.001). Conclusions: In vitro treatment of bladder cancer with the common dietary polyphosphorylated carbohydrate IP6 significantly decreased cellular growth by anti-angiogenic mechanisms. We feel that this data warrants further investigation and consideration for initiation of clinical trials to evaluate the safety and clinical utility of this agent.

Expression of a tumor-associated gene, LASS2, in the human bladder carcinoma cell lines BIU-87, T24, EJ and EJ-M3

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), a metastasis suppressor gene of human cancer, is the most abundantly expressed member of the ceramide synthase gene family. Expression of LASS2 has been reported in carcinomas of the prostate, liver and breast. However, there has been no report on the expression of LASS2 in human bladder cancer cell lines. In order to investigate the expression and potential role of this new tumor metastasis supressor gene in human bladder cancer, we compared the proliferation, metastasis and invasion among the BIU-87, T24, EJ and EJ-M3 human bladder cancer cell lines. The mRNA expression levels of the LASS2 gene were examined using real-time quantitative PCR (qPCR). The expression levels of LASS1 and LASS3 mRNA were used as references. The protein expression level of the LASS2 gene was detected using western blotting. The most aggressive of these four human cancer cell lines was observed to be EJ-M3. The expression of LASS2 mRNA was significantly correlated with diverse proliferation, metastasis and invasion. The expression levels of LASS1 and LASS3 mRNA were not correlated with these parameters. At the protein level, we observed that the more aggressive the cancer cell line, the lower the LASS2 protein expression level. Therefore, LASS2 expression may be correlated with the development and progression of human bladder cancer and may be a prognostic indicator for this cancer.

Predictive value of Sox2 expression in transurethral resection specimens in patients with T1 bladder cancer

Sox2 is thought to be an important regulator of self-renewal in embryonic stem cell. According to the cancer stem cell (CSC) theory, the overexpression of Sox2 is potentially involved in carcinogenesis and could affect tumor recurrence and metastasis. Previous study proved Sox2 might be prognostic marker for multiple human malignancies. The purpose of this study was to investigate the clinicopathological significance of Sox2 expression in human non-muscle-invasive bladder cancer. We examined Sox2 expression in 32 paired non-muscle-invasive bladder cancer tissues and adjacent non-cancerous tissues by quantitative real-time RT-PCR (qrtRT-PCR). In addition, we analyzed Sox2 and Ki-67 expression in 126 non-muscle-invasive bladder cancer samples and bladder cancer cell line T24 by immunohistochemistry and immunofluorescence assays. The recurrence-free survival was determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for univariate and multivariate analyses of prognostic factors. The expression of Sox2 was significantly increased in non-muscle-invasive bladder cancer tissues. Sox2 expression was significantly correlated with that of Ki-67 (P < 0.001). The expression of Sox2 was significantly associated with tumor size (P = 0.006), tumor number (P = 0.037), and tumor grade (P < 0.001). Patients with high Sox2 expression had significantly poorer recurrence-free survival (P = 0.0002) when compared with patients with the low expression of Sox2. On multivariate analysis, Sox2 expression and tumor grade were found to be independent prognostic factors for recurrence-free survival (P < 0.05). Our data suggested for the first time that the high expression of Sox2 may contribute to the development of non-muscle-invasive bladder cancer and serve as a novel prognostic marker in patients with T1 bladder cancer.

The Effect of Gartanin, a Naturally Occurring Xanthone in Mangosteen Juice, on the mTOR Pathway, Autophagy, Apoptosis, and the Growth of Human Urinary Bladder Cancer Cell Lines

Garcinia mangostana, often referred to as mangosteen, is a fruit grown in Southeast Asia and has been used for centuries as a local beverage and natural medicine. Its bioactive compounds, xanthones (i.e., gartanin, α-mangostin, etc), have reported effects on ailments ranging from skin infections and inflammation to urinary tract infections. We demonstrate that mangosteen xanthones (i.e., gartanin and α-mangostin) at pharmacologically achievable concentrations inhibit the growth of cancer cell lines from different stages of human urinary bladder cancer. The growth inhibitory effects of gartanin in mouse embryonic fibroblasts are at least in part dependent on the existence of p53 or TSC1. Indeed, further studies have shown that gartanin treatment of bladder cancer cell lines T24 and RT4 resulted in a marked suppression of p70S6 and 4E-BP1 expression and induction of autophagy, suggesting the inhibition of the mTOR pathway. In addition, gartanin downregulated the expression of Bcl-2 and activated the p53 pathway leading to apoptosis induction. Together, these results suggested that gartanin is a multiple targeting agent that is suitable for further study into its chemopreventive properties for human urinary bladder cancer.

Vitamin C and K3 Combination Causes Enhanced Anticancer Activity against RT-4 Bladder Cancer Cells

Introduction: Vitamin C (VC), Vitamin K3 (VK3) and the combination (VC:VK3) were evaluated against human bladder cancer cell lines RT-4 and T24 to evaluate their synergistic anticancer activity. Methods/Results: An MTT assay compared a 1hr pulsed versus a 5hr continuous exposure. VC:VK3 was synergistic, increasing the antitumor activity 12- to 24 fold for RT-4 cells. VC:VK3 pulsed versus continuous exposure produced comparable CD50 values, indicating a triggered response involving a catalase reversible redox mechanism generating hydrogen peroxide. Hydrogen peroxide production caused lipid peroxidation and depletion of cellular thiols. ATP levels were measured over 5hrs to determine metabolic effects where VC:VK3 caused a unique spike in ATP levels. Though the cause of the ATP spike is unknown a possible mechanism is a shunt formed around a defective region of complex III of the ETC from coenzyme Q to cytochrome c, producing a shift from glycolytic to oxidative metabolism and a diminution of lactic acidosis. Analysis of mitochondrial and extra mitochondrial calcium levels revealed a unique calcium pattern for RT4 cells treated with CD90 doses of VC, VK3 or VC:VK3. Conclusion: VC:VK3 was able to cause autoschizic cell death through oxidative stress, thiol depletion, lipid peroxidation, modification of ATP levels and calcium regulation. Because of these results, VC:VK3 was granted orphan drug status for the treatment of metastatic or locally advanced, inoperable transitional cell carcinoma of the urothelium (stage III and IV bladder cancer). Efforts are underway to conduct a phase II clinical trial for this indication.

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53

OBJECTIVES:Bladder cancer represents 3% of all carcinomas in the Brazilian population and ranks second in incidence among urological tumors, after prostate cancer. The loss of p53 function is the main genetic alteration related to the development of high-grade muscle-invasive disease. Prima-1 is a small molecule that restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Our aim was to investigate the ability of Prima-1 to induce apoptosis after DNA damage in bladder cancer cell lines.METHOD:The therapeutic effect of Prima-1 was studied in two bladder cancer cell lines: T24, which is characterized by a p53 mutation, and RT4, which is the wild-type for the p53 gene. Morphological features of apoptosis induced by p53, including mitochondrial membrane potential changes and the expression of thirteen genes involved in apoptosis, were assessed by microscopic observation and quantitative real-time PCR (qRT-PCR).RESULTS:Prima-1 was able to reactivate p53 function in the T24 (p53 mt) bladder cancer cell line and promote apoptosis via the induction of Bax and Puma expression, activation of the caspase cascade and disruption of the mitochondrial membrane in a BAK-independent manner.CONCLUSION:Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo may be conducted to test this molecule as a new therapeutic agent for urothelial carcinomas of the bladder, which characteristically harbor p53 mutations.

Methylation and aberrant expression of Wnt antagonist SFRP1 in bladder cancer

Objective To measure the expression of SFRP1 in bladder cancer and explore the corresponding mechanism,in order to study the roles of SFRP1 in the pathogenesis of bladder cancer.Methods SFRP1 mRNA was detected by RT-PCR.Methylation status of SFRP1 was detected by methylation specific PCR.SFRP1 protein was determined by Western blotting.Results SFRP1 was methylated in bladder cancer cell lines T24 and 5637,not in SCaBER.SFRP1 mRNA and protein were detected in SCaBER,but not in T24 and 5637.Six hours after treating T24 and 5637 with 5'-aza-deoxycytidine,both T24 and 5637 expressed SFRP1 mRNA and protein.In 45 bladder cancers,methylation of SFRP1 was detected in 28 (62.2％).In matched cancer adjacent tissues,6 (13.3％) were found with SFRP1 methylation.The methylation rate in bladder cancers was significant higher than that in matched cancer adjacent tissues (P ＜ 0.01).Conclusions SFRP1 is downregulated in some bladder cancers due to methylation.SFRP1 methylation and aberrant expression may be involved in the pathogenesis of bladder cancers. Key words: Secreted frizzled-related protein 1; Methylation; Bladder cancer

Effects of combined use of Ad-tumor necrosis factor-related apoptosis-inducing ligand with Epirubicin on proliferation and apoptosis of bladder cancer cell line T24

Objective To observe the effects of the combined use of Ad-tumor necrosis factor-related apoptosis-inducing ligand (Ad-TRAIL) with Epirubicin on proliferation and apoptosis of bladder cancer cell line T24.Methods T24 cells were divided into 4 groups:Ad-TRAIL plus EPB group,Ad-TRAIL group,EPB group and PBS group.Cytotoxic effect was determined by using violet crystal staining method.TRAIL protein was detected by using Western blotting.The inhibitory rate of cells was assessed by using cell counting kit-8 (CCK-8) assay.Cell apoptosis was analyzed by AnnexinV and TdT-mediated dUTP nick end labeling (TUNEL) assay,respectively.Results Violet Crystal staining method indicated the cytotoxicity to T24 cells in Ad-TRAIL plus EPB group was significantly increased as compared with Ad-TRAIL group and EPB group.Western blotting confirmed that T24 cells treated with Ad-TRAIL plus EPB could express TRAIL with high efficiecy.CCK-8 assay revealed the inhibitory rate after 48 h in Ad-TRAIL plus EPB group [(56.8 ± 3.5) ％] was significandy higher than in Ad-TRAIL group and EPB group (P ＜0.01).TUNEL demonstrated that the apoptosis rate after 48 h in Ad-TRAIL plus EPB group [(53.4 ±3.6)％] was significantly higher than in Ad-TRAIL group [(34.1 ±3.3)％] and EPB group [(21.3 ±2.2) ％].Conclusion The combined use of Ad-TRAIL with EPB could increase cytotoxic activity and apoptosis rate synergistically,which may provide novel strategies for the treatment of bladder cancer. Key words: Tumor necrosis factor-related apoptosis-inducing ligand ; Epirubicin; Bladder cancer

Replacement treatment with microRNA-143 and -145 induces synergistic inhibition of the growth of human bladder cancer cells by regulating PI3K/Akt and MAPK signaling pathways

We recently reported that both microRNA (miR)-143 and -145 are downregulated in human bladder cancer T24 cells and that miR-143 targets ERK5. In this study, we assessed the anti-tumor effects of combination treatment with miR-143 and -145 on bladder cancer cell lines T24, SNK57, and NKB1, in which the expression levels of miR-143 and -145 are downregulated. The ectopic expression of both miR-143 and -145 led to a significantly synergistic growth inhibition of T24 and NKB1 cells, but not that of SNK57 cells with the levels of miR-143 and -145 higher than those in T24 and NKB1 cells. The MAPK signaling pathway in NKB1 cells and both PI3K/Akt and MAPK signaling pathways in T24 cells were synergistically repressed by the co-treatment with miR-143 and -145. We newly elucidated that miR-143 targeted akt and that miR-145 targeted integrin-linked kinase (ilk) in T24 cells based on the results of a luciferase activity assay. Silencing of ilk significantly inhibited the growth of all the bladder cancer cells tested. Also, the level of phosphorylated ERK1/2 in T24 cells and that of phosphorylated Akt in SNK57 and NKB1 cells were decreased by ilk silencing. This study has provided novel important evidence with regard to the functions of anti-oncogenic miR-143 and -145 and also suggests the possible use of miR-143 and -145 for combination replacement therapy in cancers in which both miRNAs are downregulated.

Antitumor Effects of Exogenous Ganglioside GM3 on Bladder Cancer in an Orthotopic Cancer Model

To investigate the therapeutic effects of exogenous gangliosides GM3 on human bladder cancer cell lines and the severe combined immunodeficiency mouse model of orthotopic bladder cancer. Human bladder cancer cell lines YTS-1, T24, 5637, and KK47 were used in the study. Invitro cytotoxicity of GM3 was assessed using the cell counting kit-8. Cell adhesion was determined using a spreading assay. Phosphorylation of epidermal growth factor receptor was determined by Western blotting. Invivo, the orthotopic bladder cancer model was established using severe combined immunodeficiency mice and GM3 was administered intravesically by way of a transurethral catheter. GM3 inhibited the proliferation of all the bladder cancer cell lines tested. The addition of GM3 decreased cell adhesion and epidermal growth factor-dependent phosphorylation of epidermal growth factor receptor. Direct instillation of GM3 into the bladder of the orthotopic model significantly inhibited tumor growth. Our results suggest exogenous GM3 as a potential therapeutic agent for treating bladder cancer.

Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: A potential improvement of intravesical chemotherapy in bladder cancer

Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporation improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01–2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3F cells, the RRs of survival were: 28%, 29%, and 33%, by concentrations 0μM, 500μM and 1000μM respectively. In conclusion, electroporation and mitomycin together are about 30% more effective than mitomycin alone. The results help to elucidate the additive effect of mitomycin and electric pulses and support the use of this combination in the treatment of bladder cancer.

Synthesis, structure, spectroscopic studies and cytotoxic effect of novel palladium(II) complexes with 2-formylpyridine-4-Nethyl-thiosemicarbazone: Potential antitumour agents

New palladium(II) complexes have been synthesized by the reaction of Pd(II) with 2-formylpyridine-4-Nethyl-thiosemicarbazone, HFo4NEt, 1. The complexes [Pd(Fo4NEt)Cl], 2, [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and [Pd(Fo4NEt)2], 4 have been characterised by elemental analyses and spectroscopic studies. The crystal structure of the complex [Pd(H2Fo4NEt)(Fo4NEt)Cl2], 3 and the protonated ligand [(H2Fo4NEt)Cl], 5 have been solved by single-crystal X-ray diffraction. The cytotoxic activities of 1–4 have been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). The compound 3 display IC50 values in a μM range better than that of the antitumor drug cis-platin against MCF-7 and T-24 cell lines and is considered as agent with potential antitumor activity candidates for further stages of screening in vitro and/or in vivo.

Formation and cytotoxicity of a new disinfection by-product (DBP) phenazine by chloramination of water containing diphenylamine

Disinfection by-products (DBPs) in drinking water have caused worldwide concern due to their potential carcinogenic effects. The formation of phenazine from diphenylamine (DPhA) chloramination was studied and its cytotoxicities for two human cancer cells were also investigated. Phenazine was detected synchronously with the consumption of DPhA by chloramination, which further confirmed that the new DBP phenazine can be produced along with N-nitrosodiphenylamine (NDPhA) from DPhA chloramination. The formation of phenazine had a maximum molar yield with solution pH increasing from 5.0 to 9.0, with phenazine as the main product for DPhA chloramination at lower pH, but higher pH favored the formation of NDPhA. Thus, solution pH is the key factor in controlling the formation of phenazine and NDPhA. Both the initial DPhA and chloramine concentrations did not show a significant effect on the molar yields of phenazine, although increasing the chloramine concentration could speed up the reaction rate of DPhA with chloramines. The cytotoxicity assays showed that phenazine had significant cell-specific toxicity towards T24 (bladder cancer cell lines) and HepG2 (hepatic tumor cell lines) cells with IC50 values of 0.50 and 2.04 mmol/L, respectively, and T24 cells being more sensitive to phenazine than HepG2 cells. The IC50 values of phenazine, DPhA, and NDPhA for T24 cells were of the same order of magnitude and the cytotoxicity of phenazine for T24 cells was slightly lower than that of NDPhA (IC50, 0.16 mmol/L), suggesting that phenazine in drinking water may have an adverse effect on human health.

Arctigenin Anti‐Tumor Activity in Bladder Cancer T24 Cell Line Through Induction of Cell‐Cycle Arrest and Apoptosis

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer.

Abstract A22: Effectiveness of ErbB inhibitors in the treatment of bladder cancer

Abstract Background: It is estimated that close to 70,000 men and women will be diagnosed with bladder cancer every year and, of those, close to 15,000 will die from the disease. This makes bladder cancer the ninth most common cancer among women and the fourth most common cancer amongst men. Current treatments focus on surgery and radiation with total bladder removal being common with more advanced disease. Chemotherapy including cisplatin offers an alternative treatment for patients who do not want to undergo such a drastic, life-changing operation. While these treatments are initially effective, chemoresistance is common, thereby increasing the need for newer therapies. Recent studies indicated the efficacy of epidermal growth factor receptors, alone or in combination with ErbB2 inhibitors in bladder cancer cell lines – hence we investigated the efficacy of these inhibitors in cisplatin-resistant and -sensitive cells. Methods: We used the cisplatin-resistant T24 bladder cancer cell line along with the cisplatin-sensitive TCCSUP bladder cancer cell line to evaluate the use of the single ErbB inhibitors Herceptin (trastuzumab) and Tarceva (erlotinib), which was purchased from LC Laboratories, along with the dual ErbB inhibitors Tykerb (lapatinib), which was purchased from LC Laboratories, and dacomitinib (PF00299804). Cells were plated into 24-well plates and treated as indicated. Cell growth was estimated by MTT assay. Protein expression was determined by Western blotting after incubation with the indicated treatment for 72 hours. Results: MTTs with varying concentrations of Herceptin indicated that this drug was an ineffective growth inhibitor in both cell lines while Tarceva treatment was more effective, especially in TCCSUP cells. Literature suggests that dual-ErbB inhibition in various cancers is more effective than single target therapy and so we opted to explore drugs that target multiple ErbB family members. Lapatinib, which targets ErbB2 at low doses along with EGFR at higher doses, was relatively ineffective in inhibiting growth of bladder cancer cells, especially at low doses. On the other hand, dacomitinib, which targets EGFR at low doses in addition to ErbB2 at higher doses, was effective at inhibiting growth of bladder cancer cells at a much lower dose. Upon examination of the relative levels of ErbB expression we discovered that both cell lines had increased expression of EGFR but much lower expression of ErbB3 when compared to an androgen-independent prostate cancer cell line. However, treatment with lapatinib, dacomitinib, and Tarceva, alone or in combination, showed dephosphorylation of EGFR and AKT when cells were stimulated with EGF. Furthermore, treatment with Lapatinib increased expression of EGFR and ErbB2. Finally, we assayed the growth of both cell lines when treated with these drugs, alone or in combination, and found that the combination of Tarceva and dacomitinib was most effective at inhibiting the growth of bladder cancer cells while lapatinib had little effect on its own or in any combination. Conclusions: Herceptin was ineffective as a bladder cancer treatment while dacomitinib and Tarceva, in combination, have a synergistic effect which inhibits growth of cisplatin-resistant as well as sensitive bladder cancer cell lines. These studies indicate the efficacy of EGFR inhibitors, but not ErbB2 inhibitors in cisplatin-resistant bladder cancer lines. Acknowledgements: We would like to thank Dr. deVere White for the gift of T24 and TCCSUP cells. We would also like to thank Pfizer Inc. for the gift of dacomitinib and Genentech Inc. for the gift of Herceptin.