Inhibited Bladder Cancer Cell Proliferation Research Articles

Downregulation of GAS5 Promotes Bladder Cancer Cell Proliferation, Partly by Regulating CDK6

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as transcriptional regulation, cell growth and tumorigenesis. However, little is known about whether lncRNA-GAS5 (growth arrest-specific 5) regulates bladder cancer progression. In the present study, we found that the GAS5 expression is commonly downregulated in bladder cancer cell lines and human specimens. Knockdown of GAS5 promotes bladder cancer cell proliferation, whereas forced expression of GAS5 suppresses cell proliferation. We further demonstrated that knockdown of GAS5 increases CDK6 mRNA and protein levels in bladder cancer cells. Expectedly, GAS5 inhibition induces a significant decrease in G0/G1 phase and an obvious increase in S phase. Gain-of-function and loss-of-function studies showed that GAS5 inhibits bladder cancer cell proliferation, at least in part, by regulating CDK6 expression.ConclusionsDownregulated GAS5 promotes bladder cancer cell proliferation, partly by regulating CDK6, and thus may be helpful in the development of effective treatment strategies against bladder cancer.

Emetine Dihydrochloride: A Novel Therapy for Bladder Cancer

Current cisplatin based therapies for stage IV bladder cancer show 4% to 20% 5-year survival, underscoring the need to develop novel therapies for these patients. In the 1970s the natural alkaloid emetine dihydrochloride demonstrated modest anticancer efficacy as a single agent in clinical trials but this was not pursued. Groups recently reported that emetine induced apoptosis in leukemia cell lines, which was enhanced by cisplatin. We determined the antiproliferative effects of emetine with and without cisplatin in bladder cancer cells. Human bladder cancer cell lines and normal human urothelial cell cultures were treated with emetine and/or cisplatin. We measured cell proliferation and evaluated synergy using the Chou-Talalay method. The combination index was calculated. Cell cycle analysis was done and caspase activation was evaluated to assess growth arrest and apoptosis. Emetine and cisplatin individually inhibited bladder cancer cell proliferation. When combined, emetine and cisplatin acted synergistically to inhibit tumor cell proliferation with combination index values reflecting moderate to strong synergy. Normal urothelial cells were relatively resistant to this treatment. Emetine alone and combined with cisplatin appeared to primarily induce tumor cell growth arrest and not apoptosis. To our knowledge this study demonstrates for the first time that emetine has invitro antiproliferative activity against bladder cancer cell lines at nanomolar concentrations but little effect on normal urothelial cells. Moreover, emetine and cisplatin worked synergistically to inhibit tumor cell proliferation. Results suggest that combined emetine and cisplatin based chemotherapy may benefit patients with bladder cancer.

Knockdown BMI1 expression inhibits proliferation and invasion in human bladder cancer T24 cells

B cell-specific moloney murine leukemia virus integration site 1 (BMI1) is a transcriptional repressor of polycomb repressive complex 1, which is involved in the proliferation, senescence, migration, and tumorigenesis of cancer. Experimental researchers have convincingly linked BMI1 to tumorigenesis. However, there is no study about the issue on the role of BMI1 in the proliferation, apoptosis, and migration of bladder cancer. To address this question, we examined the expression of BMI1 in bladder cancer tissues and used siRNA to knockdown BMI1 expression in bladder cancer T24 cells. Then we tested the cell proliferation by CCK8 assay and soft agar colony formation assay, apoptosis by flow cytometry assay, and cell invasiveness by transwell migration assay. Our results revealed that BMI1 promoted proliferation, migration, invasion, and progression in bladder cancer. Over-expression of BMI1 was correlated with tumor clinic-pathological features. BMI1 siRNA effectively inhibited bladder cancer cell proliferation and migration in vitro, and it promoted bladder cancer invasion, maybe by causing epithelial-to-mesenchymal transition. Our findings suggested that BMI1 may represent a novel diagnostic marker and a therapeutic target for bladder cancer, and deserves further investigation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-013-1745-0) contains supplementary material, which is available to authorized users.

Chemoprevention of BBN-Induced Bladder Carcinogenesis by the Selective Estrogen Receptor Modulator Tamoxifen

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States. Due to a high likelihood of recurrence, effective chemoprevention is a significant unmet need. Estrogen receptors (ERs), primarily ERβ, are expressed in normal urothelium and urothelial carcinoma, and blocking ER function with selective ER modulators such as tamoxifen inhibits bladder cancer cell proliferation in vitro. Herein, the chemoprotective potential of tamoxifen was evaluated in female mice exposed to the bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Carcinogen treatment resulted in a 76% tumor incidence and increased mean bladder weights in comparison to controls. In contrast, mice receiving tamoxifen concurrent (8–20 weeks) or concurrent and subsequent (8–32 weeks) to BBN administration had no change in bladder weight and only 10% to 14% incidence of tumors. Non-muscle-invasive disease was present in animals treated with tamoxifen before (5–8 weeks) or after (20–32 weeks) BBN exposure, while incidence of muscle-invasive bladder carcinoma was reduced. ERβ was present in all mice and thus is a potential mediator of the tamoxifen chemoprotective effect. Surprisingly, ERα expression, which was detected in 74% of the mice exposed to BBN alone but not in any controlmice, was correlated with tumor incidence, indicating a possible role for this receptor in carcinogen-induced urothelial tumorigenesis. Thus, these data argue that both ERα and ERβ play a role in modulating carcinogen-induced bladder tumorigenesis. Administration of tamoxifen should be tested as a chemopreventive strategy for patients at high risk for bladder cancer recurrence.

HepaCAM and p-mTOR Closely Correlate in Bladder Transitional Cell Carcinoma and hepaCAM Expression Inhibits Proliferation via an AMPK/mTOR Dependent Pathway in Human Bladder Cancer Cells

We explored the correlation between hepaCAM and activated p-mTOR in bladder transitional cell carcinoma. We also determined whether the antiproliferation effect of hepaCAM is associated with the AMPK/mTOR pathway. We performed quantitative reverse transcriptase-polymerase chain reaction to determine hepaCAM mRNA expression as well as Western blot to measure hepaCAM and p-mTOR protein levels in 25 men and 5 women. Disease was Ta-T1 in 7 patients, T2-T4 in 23, grade 1 in 13, grade 2 in 9, grade 3 in 8, primary in 13 and recurrent in 17. The WST-8 assay was used to study the effect of hepaCAM on cellular proliferation. p-AMPK, p-mTOR, total AMPK, total mTOR, c-Myc and cyclin D1 were also determined by Western blot. hepaCAM mRNA and protein levels were significantly decreased, while p-mTOR protein was remarkably increased in bladder transitional cell carcinoma compared to adjacent tissues (each p<0.01). Spearman correlation analysis revealed that the hepaCAM decrease was associated with an increase in p-mTOR (r=-0.533, p=0.002). Also, hepaCAM inhibited the proliferation of human bladder transitional cell carcinoma cells. hepaCAM over expression activated AMPK and down-regulated p-mTOR, and its targets c-Myc and cyclin D1. Treatment with the AMPK inhibitor compound C prevented the antiproliferation effect of hepaCAM. Compound C completely blocked hepaCAM induced activation of AMPK and down-regulation of p-mTOR and its targets c-Myc and cyclin D1. Results suggest an important correlation between hepaCAM and p-mTOR. hepaCAM can inhibit bladder cancer cell proliferation through an AMPK/mTOR dependent pathway.

599 EMETINE DIHYDROCHLORIDE: A NOVEL THERAPY FOR BLADDER UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013599 EMETINE DIHYDROCHLORIDE: A NOVEL THERAPY FOR BLADDER UROTHELIAL CARCINOMA Kimberly Foreman, John Jesse, and Gopal Gupta Kimberly ForemanKimberly Foreman Maywood, IL More articles by this author , John JesseJohn Jesse Maywood, IL More articles by this author , and Gopal GuptaGopal Gupta Maywood, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1995AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There are currently several cisplatin-based combination therapies for metastatic bladder cancer. These regimens demonstrate a 5-year survival of about 4-20%, underscoring the urgent need to develop novel therapies for these patients. Emetine, a natural product alkaloid, demonstrated anti-cancer efficacy in clinical trials in the early 1970's, but was not pursued in favor of other agents. Recently, studies showed emetine induces caspase-dependent apoptosis in leukemia cells, which was markedly enhanced when combined with cisplatin. We sought to determine the anti-cancer effects of emetine as a single agent and combined with cisplatin on human bladder cancer cell lines. METHODS Muscle invasive human bladder cancer cell lines (UMUC3, HT1376) or normal human urothelial cells were treated for 48 hours with emetine, cisplatin, or both. Cell proliferation was measured with the MTT assay. Synergy was evaluated using constant drug ratios, and the combination index (CI) calculated using Calcusyn software. Cell cycle analysis and caspase activation were evaluated by flow cytometry. RESULTS Emetine and cisplatin each inhibited bladder cancer cell proliferation, while normal urothelial cells were more resistant. Bladder cancer cells had an average IC50 of 19 nM and 4 μM for emetine and cisplatin, respectively, while normal cells had an average IC50 of 127 nM and 36 μM, respectively. When combined, emetine and cisplatin acted synergistically to inhibit bladder cancer cell proliferation with CI values reflecting moderate to strong synergy. Normal urothelial cells were relatively uneffected under the same conditions. Interestingly, emetine alone induced growth arrest, but not apoptosis in bladder cancer cells. However, when combined, emetine enhanced cisplatin-induced apoptosis. Both caspase-3 and -8 were activated in the treated cells. CONCLUSIONS Ours is the first study to evaluated emetine with or without cisplatin as a potential therapy for muscle invasive bladder cancer. We showed emetine has in vitro anti-tumor activity against two bladder cancer cell lines at nanomolar concentrations, but had little effect on normal urothelial cells. Moreover, emetine and cisplatin worked synergistically to inhibit tumor cell proliferation. When combined, emetine and cisplatin increased apoptosis compared to either drug alone, and changes in the DNA/PI profile suggest growth arrest is also involved. Our results suggest a combined therapy of emetine and cisplatin may benefit bladder cancer patients. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e245 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kimberly Foreman Maywood, IL More articles by this author John Jesse Maywood, IL More articles by this author Gopal Gupta Maywood, IL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer

Growing evidence suggests that microRNAs (miRNAs) are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the mechanism of carcinogenesis. However, the genetic or epigenetic regulations of these miRNAs have not yet been fully elucidated in bladder cancer (BC). Chromosomal alterations of cancer cells give us important information for the identification of tumor suppressor genes. Our miRNA array-comparative genomic hybridization (CGH) analysis showed several miRNAs to be candidate tumor suppressors of BC. Our array-CGH analysis revealed that chromosome 4 was lost in all BC cell lines. We selected 19 miRNAs located on chromosome 4 and evaluated their expression levels in cancer cell lines as well as clinical samples. Gain-of-function analysis revealed that miR-218 inhibited BC cell proliferation, migration and invasion. Furthermore, flow cytometry analysis showed that it induced BC cell apoptosis. Genome-wide gene expression analysis showed that it targeted multiple oncogenes in BC. Our study is the first to demonstrate that miR-218 located on chrosomosme 4p15.31 is a tumor suppressive miRNA in BC. The identification of tumor suppressive miRNAs and their target genes on the basis of array-CGH analysis could provide new insights into the mechanisms of BC carcinogenesis.

775 THE MTOR PATHWAY IMPACTS PROLIFERATION AND CHEMOSENSITIVITY OF UROTHELIAL CARCINOMA CELLS IN VITRO AND IS HIGHLY EXPRESSED IN A SUBSET OF HUMAN BLADDER CANCERS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2010775 THE MTOR PATHWAY IMPACTS PROLIFERATION AND CHEMOSENSITIVITY OF UROTHELIAL CARCINOMA CELLS IN VITRO AND IS HIGHLY EXPRESSED IN A SUBSET OF HUMAN BLADDER CANCERS Stephen Boorjian, Jiaru Zhang, Igor Makhlin, Christopher Long, Karthik Devarajan, Yan Zhou, Min Huang, and Jonathan Chernoff Stephen BoorjianStephen Boorjian Rockledge, PA More articles by this author , Jiaru ZhangJiaru Zhang Philadelphia, PA More articles by this author , Igor MakhlinIgor Makhlin Philadelphia, PA More articles by this author , Christopher LongChristopher Long Philadelphia, PA More articles by this author , Karthik DevarajanKarthik Devarajan Philadelphia, PA More articles by this author , Yan ZhouYan Zhou Philadelphia, PA More articles by this author , Min HuangMin Huang Philadelphia, PA More articles by this author , and Jonathan ChernoffJonathan Chernoff Philadelphia, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1424AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Although phosphatidylinositol 3-kinase/AKT signaling has been found to be active in urothelial carcinoma (UC), the importance of the mammalian target of rapamycin (mTOR), a downstream target of this pathway, remains to be established. Here, then, we investigated the relevance of mTOR in human bladder cancer cell lines and patient tissue specimens. METHODS The human bladder cancer cell lines UMUC-3, 253J, TCC-SUP, T24, and 1365 were evaluated for expression of mTOR and phospho-mTOR using Western blot analysis. Cells were treated with different concentrations of the mTOR inhibitor rapamycin, and viability was assessed using the Cell Titer Blue assay. Propidium idodide staining and fluorescence-activated cell sorting were used to determine the impact of treatment on the cell cycle. Apoptosis was studied by measurement of PARP cleavage. The effect of mTOR inhibition on chemosensitivity was investigated by treating cells with rapamycin (5nM), alone or together with cisplatin (5μM). In addition, expression of phospho-mTOR in human bladder tissue was assessed using a tissue microarray (TMA) constructed from 328 UC patient tissue specimens. RESULTS High levels of baseline expression of mTOR and phospho-mTOR were noted in all 5 cell lines. Treatment with rapamycin significantly decreased cell viability at 48 and 72 hours in UMUC-3 (p=0.004) and 253J (p<0.001) cells at all doses tested. Cell cycle analysis showed that rapamycin induced arrest in the G0-G1 phase in these cells. Consistent with its known mechanism of action, rapamycin decreased protein expression of phospho-mTOR and its downstream effector, p-S6K, in both cell lines. Moreover, treatment with rapamycin increased the ability of cisplatin to inhibit cell viability in UMUC-3 (p=0.002) and 253J (p=0.03) cells. No evidence for increased PARP cleavage was noted after treatment with rapamycin, alone or in combination with cisplatin. Interestingly, despite high expression in all bladder cancer cell lines tested, phospho-mTOR expression in patient UC specimens was increased compared to non-tumor bladder tissue in only 65/203 (32.0%) tumors on the TMA analysis. CONCLUSIONS Blockade of the mTOR signaling pathway inhibits bladder cancer cell proliferation and enhances the effectiveness of cisplatin in vitro, likely through cell-cycle arrest rather than direct cytotoxicity. Suppression of the mTOR pathway thus has the potential to be a therapeutic target in bladder cancer for select patients and merits further investigation. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e303 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stephen Boorjian Rockledge, PA More articles by this author Jiaru Zhang Philadelphia, PA More articles by this author Igor Makhlin Philadelphia, PA More articles by this author Christopher Long Philadelphia, PA More articles by this author Karthik Devarajan Philadelphia, PA More articles by this author Yan Zhou Philadelphia, PA More articles by this author Min Huang Philadelphia, PA More articles by this author Jonathan Chernoff Philadelphia, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1154 8-METHYL-N-VANILLYL-6-NONENAMIDE (CAPSAICIN) INDUCES APOPTOSIS IN BLADDER CANCER CELLS INDEPENDENT OF P53 STATUS: IMPLICATION FOR THERAPY?

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101154 8-METHYL-N-VANILLYL-6-NONENAMIDE (CAPSAICIN) INDUCES APOPTOSIS IN BLADDER CANCER CELLS INDEPENDENT OF P53 STATUS: IMPLICATION FOR THERAPY? Nathaniel Polnaszek Nathaniel PolnaszekNathaniel Polnaszek More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.653AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mutations in p53 are considered to be central in urothelial carcinogenesis. Missense mutations are significantly correlated with stage and grade since they form hetero oligomers and block wild type p53 binding to p53 responsive elements producing a dominant negative effect. The International Study Initiative on Bladder Cancer analyzed data sets from 25 centers and found positive p53 immunostaining in 48% of muscle invasive tumors. Retrospective analysis of patients undergoing radical cystectomy showed increased risk of recurrence and decreased overall survival with mutant p53 overexpression. Recently a more comprehensive evaluation showed that the combination of p53 gene and protein status may be linked to clinical outcome. Since p53 is involved in cell cycle checkpoint and apoptosis signaling; our objective was to target p53 function either by (a) inhibiting the activity of mutant p53 or (b) work through p53-independent mechanisms to inhibit bladder cancer cell proliferation and induce apoptosis. METHODS Human bladder cancer cells that harbor wild type p53 (RT4), or mutations in the transactivation domain (UMUC3, TCCSUP) or DNA binding domain (T24) were grown as per vendor recommendations. Cells were treated with increasing doses of capsaicin (16, 32 and 64 fÝM) and proliferation inhibition, cell cycle progression, apoptosis induction and the mechanism of capsaicin-induced apoptosis were determined. RESULTS Capsaicin caused a dose responsive inhibition of proliferation and the IC50 was not dependent on p53 status. At low doses of capsaicin cells were arrested in G1 phase and in G2 phase at high doses. We also found a dose-dependent increase in induction of apoptosis following capsaicin treatment that was independent of the status of p53. Examination of the mechanism of apoptosis induction shows that capsaicin disrupts the interactions between proteins in the death receptor pathway and there is crosstalk between the death receptor and mitochondrial pathway in the more advanced bladder cancer cells. CONCLUSIONS This study demonstrates that capsaicin could be a good candidate therapeutic agent for bladder cancer given the putative oncogenic role for p53 in bladder cancer these results are highly significant. San Antonio, TX© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e447 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nathaniel Polnaszek More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells

BackgroundThis study evaluated the cytotoxic and antiproliferative efficacy of two well-characterized members of the Cecropin-family of antimicrobial peptides against bladder tumor cells and benign fibroblasts.MethodsThe antiproliferative and cytotoxic potential of the Cecropins A and B was quantified by colorimetric WST-1-, BrdU- and LDH-assays in four bladder cancer cell lines as well as in murine and human fibroblast cell lines. IC50 values were assessed by logarithmic extrapolation, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was performed to visualize the morphological changes induced by Cecropin A and B in bladder tumor cells and fibroblasts.ResultsCecropin A and B inhibit bladder cancer cell proliferation and viability in a dose-dependent fashion. The average IC50 values of Cecropin A and B against all bladder cancer cell lines ranged between 73.29 μg/ml and 220.05 μg/ml. In contrast, benign fibroblasts were significantly less or not at all susceptible to Cecropin A and B. Both Cecropins induced an increase in LDH release from bladder tumor cells whereas benign fibroblasts were not affected. SEM demonstrated lethal membrane disruption in bladder cancer cells as opposed to fibroblasts.ConclusionCecropin A and B exert selective cytotoxic and antiproliferative efficacy in bladder cancer cells while sparing targets of benign murine or human fibroblast origin. Both peptides may offer novel therapeutic strategies for the treatment of bladder cancer with limited cytotoxic effects on benign cells.

Antitumor effect of dsRNA-induced p21WAF1/CIP1 gene activation in human bladder cancer cells

We recently reported that synthetic dsRNAs targeting promoter regions can induce gene expression in a phenomenon referred to as dsRNA-induced gene activation/RNA activation (RNAa) [Li et al. Proc Natl Acad Sci U S A 2006;103:17337-42]. The present study investigates the in vitro antitumor activity RNAa can elicit through triggering the expression of cell cycle repressor protein p21(WAF1/CIP1) (p21) in human bladder cancer cells. Transfection of a 21-nucleotide dsRNA targeting the p21 promoter (dsP21) was used to induce p21 expression in T24 and J82 bladder cancer cell lines. Reverse transcription-PCR and Western blot analysis accessed the increase p21 mRNA and protein levels, respectively, in transfected cells. In association to p21 induction, dsP21 transfection significantly inhibited bladder cancer cell proliferation and clonogenicity. Further analysis of cell viability and cell cycle distribution revealed that dsP21 transfection also enhanced apoptotic cell death and caused an accumulation in the G(1) phase in both cell lines. In conclusion, p21 activation by RNAa has antitumor activity in vitro in bladder cancer cells. These results suggest that RNAa could be used for cancer treatment by targeted activation of tumor suppressor genes.