Inhibits Bladder Cancer Research Articles

Abstract 3692: Allyl isothiocyanate arrests bladder cancer cells in mitosis, and mitotic arrest in turn leads to apoptosis via Bcl-2 phosphorylation

Abstract Allyl isothiocyanate (AITC) occurs in many common cruciferous vegetables, and was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in an orthotopic rat bladder cancer model. Here, we show that AITC arrests human bladder cancer cells in mitosis and induces apoptosis. Mitotic arrest by AITC was associated with significant down regulation of both α-tubulin and β-tubulin which are essential for mitosis. AITC also induced mitochondria-mediated apoptosis, including cytochrome c release from mitochondria to cytoplasm, cleavage of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP) and lamin B1, and formation of TUNEL-positive cells. Both a general caspase inhibitor (Z-VAD-FMK) and a caspase-9 inhibitor (Z-LEHD-FMK) attenuated AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC resulted from mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser70. Thus, AITC-induced mitotic arrest was accompanied by a strong Bcl-2 phosphorylation, while Bcl-2 expression was not affected. Pre-arresting cells in G1 phase using hydroxyurea abrogated both mitotic arrest and Bcl-2 phosphorylation in cells treated with AITC. Overexpression of a Bcl-2 mutant (loop domain deleted, removing Ser70) prevented AITC from inducing apoptosis. AITC-induced Bcl-2 phosphorylation appears to be mediated by c-Jun N-terminal kinase (JNK), as AITC activated JNK but a JNK inhibitor (SP600125) abolished Bcl-2 phosphorylation in AITC-treated cells. In summary, AITC is a highly promising agent for bladder cancer prevention and treatment, and the present study reveals a novel anticancer mechanism of AITC in human bladder cancer cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2011-3692

Paeonia lactiflora Pall inhibits bladder cancer growth involving phosphorylation of Chk2 in vitro and in vivo

Extracts of Paeonia lactiflora Pall (RPA), a traditional Chinese medicines has been shown to treat cancers. The purpose of this study is to evaluate the anticancer effect of RPA in urinary bladder carcinoma in vitro and in vivo. The cell viability was analyzed with DAPI. Flow cytometry and Western blot were used to study the apoptosis and cell cycle related mechanism. A rat model of bladder cancer was induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Tumors were analyzed with immunohistochemical analysis. Our data suggested that RPA inhibits growth of bladder cancer via induction of apoptosis and cell cycle arrest. Treatment of TSGH-8301 cells with RPA resulted in G2-M phase arrest that was associated with a marked decline in protein levels of cdc2, cyclin B1, cell division cycle 25B (Cdc25B) and Cdc25C. We also reported that RPA-mediated growth inhibition of TSGH-8301 cells was correlated with activation of checkpoint kinase 2 (Chk2). Herein, we further evaluated urinary bladder cancer using a model of bladder cancer induced by OH-BBN. Analysis of tumors from RPA-treated rats showed significant decrease in the expression of Bcl2, cyclin D1, and PCNA, and increase in the expression of p-Chk2 (Thr-68), Bax, and Cip1/p21. Our data provide the experimental evidence that RPA could modulate apoptosis in models of bladder cancer.

Overexpression of Pim-1 in bladder cancer

BackgroundPim-1 is a serine-threonine kinase which promotes early transformation, cell proliferation and cell survival during tumorigenesis. Several studies have demonstrated that Pim-1 kinase play a role in different cancer types, however, the function of Pim-1 in bladder cancer is poorly understood.MethodsExpression and localization of Pim-1 in human normal and malignant bladder specimens were examined by Immunohistochemistry and Pim-1 staining score was compared with several clinicopathologic parameters. To further demonstrate the biological function of Pim-1 in bladder cancer, its expression was validated in five bladder cancer cell lines by western blot and immunohistochemistry analyses. Subsequent knockdown of Pim-1 was achieved by lentivirus encoding small interfering RNA, and the effect of Pim-1 on bladder cell survival and drug sensitivity were further assessed by colony formation and cell proliferation assays.ResultsWhen compared with normal epithelium, Pim-1 was overexpressed in bladder cancer epithelium, and the expression level was higher in invasive bladder cancer than Non-invasive bladder cancer specimens. Pim-1 was also detected in all the bladder cancer cell lines examined in our study. Moreover, the knockdown of Pim-1 significantly inhibited bladder cancer cell growth and also sensitized cells to chemotherapeutic drugs in vitro.ConclusionsOur results in this study suggest that Pim-1 may play a role in bladder cancer initiation and progression. Since Pim-1 is also involved in bladder cancer cell survival and drug resistance, Pim-1 is a potential candidate for targeted therapy in bladder cancer.

Telomerase suppression initiates PML-dependent p53 activation to inhibit bladder cancer cell growth

Most human cancer cells maintain telomere to immortalization through telomerase activity. Inhibition of telomerase activity is a powerful strategy for cancer therapy; however, the potential molecular signals following telomerase suppression are still not clear. Promyelocytic leukemia protein (PML) is an essential component of PML nuclear bodies and a tumor suppressor in bladder cancer. In this study, using mutant human telomerase reverse transcriptase (hTERT) or shRNA to inhibit telomerase activity, we found telomerase suppression increased the expression of PML and resulted in its translocation to the nucleus in bladder cancer T24 cells. Additionally, we found that p53 was recruited into nucleus and colocalized with PML after telomerase suppression. Subsequently, there was a decrease in cell growth in vitro and in vivo and an increase in cell cycle arrest and apoptosis. Furthermore, we showed here that PML is indispensable for p53 nuclear translocation and p21 induction after telomerase inhibition. Therefore, our data indicate that suppression of telomerase could activate the PML-dependent p53 signaling pathway and inhibit bladder cancer cell growth, and also provide new insight into the potential crosstalk between PML and hTERT in bladder cancer cells.

Allyl isothiocyanate-rich mustard seed powder inhibits bladder cancer growth and muscle invasion

Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G(2)/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 μmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.

Combining siRN A-mediated hTR and hTERT expression in bladder uroepithelium cancer BIU-87 cell lines

Objective To perform a DNA microarray analysis of combining small interfering RNA (siRNA) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) gene in bladder transitional cell carcinoma cell line BIU-87 telomere Inhibition of enzyme activity. Methods According to the siRNA design principles and TERT.TR gene mRNA coding sequence,respectively,were designed and synthesized three pairs of different and can interfere with TR and TERT gene expression in siRNA sequence and filter out the most efficient suppression sequence ( pRNAT-TERT-Ⅲ: GTACAG-GTTTCACGCATGT gene No. 3229, pRNAT-TR- Ⅲ: CGAAGGTGCCTTGGAATAT gene No. 306). Constructing the hTR,hTERT gene as a target of the joint RNA interference ( RNAi) device pRNAT-TR-Ⅲ+ pRNAT-TERT-Ⅲ(liposome) and transfected into bladder transitional cell carcinoma of BIU-87 cell line, using fluorescent quantitative polymerase chain reaction (PCR) analysis of hTR and hTERT mRNA expression (in the target gene and GAPDH copy number ratios for the relative quantitative analysis) , TRAP-ELJSA detection of telomerase activity (measured A value) ,MTT assay BIU-87 cell growth inhibition (by MTT method Detection of the survival rate of transfected cells) ,and before and after co-RNAi gene chip a-nalysis,using RNA extraction,RNA quality control,cRNA marking and synthesis,chip hybridization, chemi-luminescence detection,image acquisition and data analysis,and further use of part of the reverse transcription (RT)-PCR validation of differentially expressed genes. Results The combined RNA interference hTR and hTERT gene, pRNAT-TERT-Ⅲ+ pRNAT-TR-Ⅲcombined RNAi can specifically inhibit the bladder transitional cell carcinoma cell line BIU-87 in TERT and TR expression (pRNAT-TERT- Ⅲ+ pRNAT-TR-I TERT:0.300 ±0.033 ,P <0.05;TR:0.430 ±0.028,P <0.05). After the combined RNAi and bladder transitional cell carcinoma of BIU-87 cell line growth and telomerase activity were significantly inhibited (pRNAT-TERT-Ⅲ+ pRNAT-TR- Ⅲ: 1.250 ± 0.012, P < 0.05 ). Before and after the joint siRNA gene chip analysis found 21 genes down ( ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PRKDC, SERP1NE1, THBS1, TNFRSF1A, UCCl). Conclusion The combined RNA interference with hTERT and hTR gene can significantly inhibit bladder cancer BIU-87 cell line growth;joint siRNA inhibited bladder transitional cell carcinoma of BIU-87 cell line causes growth of 21 genes were cut. Key words: Telomerase; siRNA; Bladder transitional cell carcinoma

Antisense oligonucleotide targeting Livin induces apoptosis of human bladder cancer cell via a mechanism involving caspase 3

Background and Aimin recent years, Livin, a new member of IAPs family, is found to be a key molecule in cancers. Researchers consider Livin may become a new target for tumor therapy; however, the role of it in bladder cancer is still unclear. The purpose of this article is to investigate Antisense Oligonucleotide (ASODN) of Livin on treating bladder cancer cell and underlying mechanisms.MethodsPhosphorathioate modifying was used to synthesize antisense oligonucleotides targeting Livin, followed by transfection into human bladder cancer cell 5637. After transfection, Livin mRNA and protein level, cell proliferation and apoptosis changes, caspase3 level and its effect on human bladder cancer transplantable tumor in nude mice were measured.Resultresults showed Livin ASODN effectively inhibited Livin expression and tumor cell proliferation, and these effects probably through enhanced caspase3 activity and apoptosis of tumor cells. In nude mice transplantable tumor model, Livin expressions were inhibited meanwhile caspase3 expression was increased. Tumor growth slowed down and apoptosis was enhanced.ConclusionOur data suggest that Livin plays an important role in inhibiting apoptosis of bladder cancer cells. Livin ASODN may promote cell apoptosis, inhibit bladder cancer growth, and become one of the methods of gene therapy for bladder cancer.

Drugs that Target Specificity Proteins Downregulate Epidermal Growth Factor Receptor in Bladder Cancer Cells

The epidermal growth factor receptor (EGFR) is an important chemotherapeutic target for tyrosine kinase inhibitors and antibodies that block the extracellular domain of EGFR. Betulinic acid (BA) and curcumin inhibited bladder cancer cell growth and downregulated specificity protein (Sp) transcription factors, and this was accompanied by decreased expression of EGFR mRNA and protein levels. EGFR, a putative Sp-regulated gene, was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3, and Sp4, and RNA interference with individual Sp knockdown indicated that EGFR expression was primarily regulated by Sp1 and Sp3. BA, curcumin, and iSp also decreased phosphorylation of Akt in these cells, and downregulation of EGFR by BA, curcumin, and iSp was accompanied by induction of LC3 and autophagy, which is consistent with recent studies showing that EGFR suppresses autophagic cell death. The results show that EGFR is an Sp-regulated gene in bladder cancer, and drugs such as BA and curcumin that repress Sp proteins also ablate EGFR expression. Thus, compounds such as curcumin and BA that downregulate Sp transcription factors represent a novel class of anticancer drugs that target EGFR in bladder cancer cells and tumors by inhibiting receptor expression.

Arsenic trioxide downregulates specificity protein (Sp) transcription factors and inhibits bladder cancer cell and tumor growth

Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, ≤ 5 μM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 μM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide.

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 ...

Androgen Receptor Is a Potential Therapeutic Target for Bladder Cancer

To investigate whether androgen receptor (AR) could serve as a potential molecular target for the treatment of bladder cancer. Cell proliferation, apoptosis, and migration capacity were determined in human transitional carcinoma cell lines T24 and 253-J treated with small interfering RNA directed against AR, and expression levels of growth- and metastasis-related genes were assessed using quantitative reverse transcriptase-polymerase chain reaction. Tumor cell growth and apoptosis were also evaluated in vivo in T24 tumor-bearing nude mice receiving electroporation-assisted administration of anti-AR small interfering RNA. AR expression knockdown produced increased apoptosis, decreased proliferation, and migration of bladder cancer cells. Cyclin D1, Bcl-x(L), and matrix metallopeptidase-9 gene expression were also reduced with AR knockdown, which might have contributed to the altered biological behavior of cancer cells. In vivo experiments showed that silencing AR expression, by interference aided by electroporation, significantly suppressed AR-positive bladder tumor growth with decreased cell proliferation and increased apoptotic rates. Downregulation of AR expression inhibits bladder cancer cell growth in vitro and in vivo, implying that its use might be a potential therapeutic target for the treatment of bladder cancer.

Abstract A73: Bladder cancer inhibition by allyl isothiocyante

Abstract Nearly all cancers of the urinary bladder originate from its inner surface (epithelium). While most bladder cancers are diagnosed at an early stage, recurrence rate is extremely high. There is an urgent need for agents capable of inhibiting bladder cancer development and recurrence. Allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, was previously shown to kill bladder cancer cells in vitro. Our objective was to evaluate the chemopreventive potential of AITC against bladder cancer. The anticancer activities of AITC were assessed using multiple bladder cancer cell lines in vitro and two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). Its toxicity toward normal human bladder epithelial (NHBE) cells was also examined. Pharmacokinetic study of AITC and histological examination of normal and malignant bladder tissues of AITC-treated rats were undertaken as well. Proliferation of bladder cancer cells was potently inhibited by AITC (IC50 of approximately 3 µM), which was associated with G2/M arrest and apoptosis. AITC was markedly less toxic to NHBE cells (IC50 of 69 µM). Low oral dose of AITC (1 mg/kg), starting one day after cancer cell inoculation significantly inhibited the development of orthotopic bladder cancer, but was ineffective against subcutaneous xenografts of the same cancer cells. This differential effect was explained by our finding that urinary concentrations of AITC equivalent were 2–3 orders of magnitude higher than that in the plasma, and that its levels in the orthotopic bladder cancer tissues were also 3 orders of magnitude higher than that in the subcutaneous cancer tissues. Thus, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development in vivo. Besides its potential use for prevention of primary bladder cancer, AITC appears to represent a novel and very attractive approach in the management of recurrence of superficial bladder cancer, since all currently available intravesicle therapies against bladder cancer recurrence mandate urethral catherization. In addition, this new knowledge opens the possibility for adjuvant or multimodality combination approaches for the prevention and treatment of bladder cancer. Supported by NCI grant CA124627. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A73.

Vitamin E succinate induced apoptosis and enhanced chemosensitivity to paclitaxel in human bladder cancer cells in vitro and in vivo

There have been several studies on the antitumor activities of vitamin E succinate (alpha-TOS) as complementary and alternative medicine. In the present study, we investigated the cytotoxic effect of alpha-TOS and the enhancement of chemosensitivity to paclitaxel by alpha-TOS in bladder cancer. KU-19-19 and 5637 bladder cancer cell lines were cultured in alpha-TOS and/or paclitaxel in vitro. Cell viability, flow cytometric analysis, and nuclear factor-kappa B (NF-kappaB) activity were analyzed. For in vivo therapeutic experiments, pre-established KU-19-19 tumors were treated with alpha-TOS and/or paclitaxel. In KU-19-19 and 5637 cells, the combination treatment resulted in a significantly higher level of growth inhibition, and apoptosis was significantly induced by the combination treatment. NF-kappaB was activated by paclitaxel; however, the activation of NF-kappaB was inhibited by alpha-TOS. Also, the combination treatment significantly inhibited tumor growth in mice. In the immunostaining of the tumors, apoptosis was induced and proliferation was inhibited by the combination treatment. Combination treatment of alpha-TOS and paclitaxel showed promising anticancer effects in terms of inhibiting bladder cancer cell growth and viability in vitro and in vivo. One of the potential mechanisms by which the combination therapy has synergistic cytotoxic effects against bladder cancer may be that alpha-TOS inhibits NF-kappaB induced by chemotherapeutic agents.

Inhibition of bladder cancer development by allyl isothiocyanate

Bladder cancer is one of the common human cancers and also has a very high recurrence rate. There is a great need for agents capable of inhibiting bladder cancer development and recurrence. Here, we report that allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, potently inhibited the proliferation of bladder carcinoma cell lines in vitro [half maximal inhibitory concentration (IC(50)) of 2.7-3.3 microM], which was associated with profound G(2)/M arrest and apoptosis. In contrast, AITC was markedly less toxic to normal human bladder epithelial cells (IC(50) of 69.4 microM). AITC was then evaluated in two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). The orthotopic model closely mimics human bladder cancer development and recurrence. We show that a low oral dose of AITC (1 mg/kg) significantly inhibited the development and muscle invasion of the orthotopic bladder cancers but was ineffective against the subcutaneous xenografts of the same cancer cells in the same animals. This differential effect was explained by our finding that urinary levels of AITC equivalent were two to three orders of magnitude higher than that in the plasma and that its levels in the orthotopic cancer tissues were also three orders of magnitude higher than that in the subcutaneous cancer tissues. Moreover, we show that AITC is a multi-targeted agent against bladder cancer. In conclusion, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development and invasion.

Construction of Urothelium-Specific Recombinant Adenovirus and Its Inhibition in Bladder Cancer Cell

Aim: To construct urothelium-specific recombinant adenovirus and investigate its inhibition in bladder cancer cell. Methods: RT-PCR analysis was used to determine expression patterns of hUPII and coxsackie adenovirus receptor on multiple cell lines. Transient transfection and luciferase detecting assay were used to detect tissue specificity of the hUPII promoter. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed. Restrictive enzyme digestion assay and PCR confirmed the correct construction. The adenovirus E1A protein expressed in BIU-87 was tested by Western blot after cells were infected with recombinant adenovirus. Recombinant adenovirus Ad-UPII-E1A was tested for its inhibition in bladder cancer cell line BIU-87. Results: HUPII and CAR were expressed and the hUPII promoter is highly active in bladder cancer cell line BIU-87. Using homologous recombination in bacteria technology, the hUPII promoter and E1A gene were inserted into the genome of type 5 recombinant adenovirus. The E1A protein was markedly positive in the samples of BIU-87 cells infected with recombinant adenovirus Ad-UPII-E1A. MTT assay demonstrated recombinant adenovirus Ad-UPII-E1A inhibited bladder cancer cell BIU-87 growth. Conclusion: The hUPII promoter shows high tissue specificity. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed and confirmed. Recombinant adenovirus Ad-UPII-E1A is effective in inhibition in bladder cancer cell line BIU-87.

Is Vascular Endothelial Growth Factor Modulation a Predictor of the Therapeutic Efficacy of Gefitinib for Bladder Cancer?

The epidermal growth factor receptor inhibitor gefitinib (Iressa) is currently being studied in patients with bladder cancer and it has significant anti-angiogenic activity. We investigated the relationship between the modulation of vascular endothelial growth factor (Santa Cruz Biotechnology, Santa Cruz, California) expression and the biological efficacy of gefitinib for bladder cancer. In vitro the 4 bladder cancer cell lines 253JB-V, UMUC-3, KU-7 and UMUC-13 were treated with gefitinib and vascular endothelial growth factor secretion was measured. The effects of gefitinib on vascular endothelial growth factor promoter, proliferation, cell cycle and downstream signals were evaluated. In vivo 253JB-V and UMUC-13 were injected into nude mice and tumors were treated with 2 mg gefitinib per day. Tumor kinetics were determined and the levels of phospho-epidermal growth factor receptor (Biosource), vascular endothelial growth factor, phospho-vascular endothelial growth factor (Cell Signaling Technology), angiogenesis and apoptosis were measured. Epidermal growth factor receptor (Neomarkers, Fremont, California) phosphorylation was blocked efficiently in all cell lines at concentrations of 0.5 microM or greater. Gefitinib (1 microM) induced an accumulation of cells in G0/G1 without apoptosis in 253J B-V cells, whereas it had no effect in other cell lines. Gefitinib inhibited vascular endothelial growth factor secretion in 253JB-V and UMUC-13 (concentration inhibiting a 50% response 0.5 and 0.1 microM, respectively) but not in UMUC-3 or KU-7. Gefitinib decreased vascular endothelial growth factor promoter activity in 253JB-V and UMUC-13 by 40% to 60%. In vivo the growth of 253JB-V tumors was significantly inhibited by gefitinib, whereas no effect was demonstrated in UMUC-13 tumors. Vascular endothelial growth factor expression and vascular endothelial growth factor receptor activation were significantly decreased in 253JB-V tumors and to a greater extent in resistant UMUC-13 tumors. Gefitinib inhibited angiogenesis and induced apoptosis in sensitive 253JB-V tumors only. Epidermal growth factor receptor blockade exerts an anti-angiogenic effect on bladder cancer cells, in part by modulating vascular endothelial growth factor expression. However, down-regulation of vascular endothelial growth factor expression is not sufficient to inhibit bladder cancer growth and it should not be used as a predictor of the therapeutic efficacy of gefitinib.

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.

Inhibition of Urinary Bladder Carcinogenesis by Broccoli Sprouts

Isothiocyanates are a well-known class of cancer chemopreventive agents, and broccoli sprouts are a rich source of several isothiocyanates. We report herein that dietary administration to rats of a freeze-dried aqueous extract of broccoli sprouts significantly and dose-dependently inhibited bladder cancer development induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. The incidence, multiplicity, size, and progression of bladder cancer were all inhibited by the extract, while the extract itself caused no histologic changes in the bladder. Moreover, inhibition of bladder carcinogenesis by the extract was associated with significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes that are important protectants against oxidants and carcinogens. Isothiocyanates are metabolized to dithiocarbamates in vivo, but dithiocarbamates readily dissociate to isothiocyanates. We found that >70% of the isothiocyanates present in the extract were excreted in the urine as isothiocyanate equivalents (isothiocyanates + dithiocarbamates) in 12 h after a single p.o. dose, indicating high bioavailability and rapid urinary excretion. In addition, the concentrations of isothiocyanate equivalents in the urine of extract-treated rats were 2 to 3 orders of magnitude higher than those in plasma, indicating that the bladder epithelium, the major site of bladder cancer development, is most exposed to p.o. dosed isothiocyanate. Indeed, tissue levels of isothiocyanate equivalents in the bladder were significantly higher than in the liver. In conclusion, broccoli sprout extract is a highly promising substance for bladder cancer prevention and the isothiocyanates in the extract are selectively delivered to the bladder epithelium through urinary excretion.

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.