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  • 1
    Language: English
    In: Targeted Oncology, 2016, Vol.11(6), pp.783-798
    Description: To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1007/s11523-016-0444-7 Byline: Maria Pinkerneil (1), Michele J. Hoffmann (1), Hella Kohlhof (2), Wolfgang A. Schulz (1), Gunter Niegisch (1) Abstract: Background Targeting of class I histone deacetylases (HDACs) exerts antineoplastic actions in various cancer types by modulation of transcription, upregulation of tumor suppressors, induction of cell cycle arrest, replication stress and promotion of apoptosis. Class I HDACs are often deregulated in urothelial cancer. 4SC-202, a novel oral benzamide type HDAC inhibitor (HDACi) specific for class I HDACs HDAC1, HDAC2 and HDAC3 and the histone demethylase LSD1, shows substantial anti-tumor activity in a broad range of cancer cell lines and xenograft tumor models. Aim The aim of this study was to investigate the therapeutic potential of 4SC-202 in urothelial carcinoma (UC) cell lines. Methods We determined dose response curves of 4SC-202 by MTT assay in seven UC cell lines with distinct HDAC1, HDAC2 and HDAC3 expression profiles. Cellular effects were further analyzed in VM-CUB1 and UM-UC-3 cells by colony forming assay, caspase-3/7 assay, flow cytometry, senescence assay, LDH release assay, and immunofluorescence staining. Response markers were followed by quantitative real-time PCR and western blotting. Treatment with the class I HDAC specific inhibitor SAHA (vorinostat) served as a general control. Results 4SC-202 significantly reduced proliferation of all epithelial and mesenchymal UC cell lines (IC.sub.50 0.15--0.51 [micro]M), inhibited clonogenic growth and induced caspase activity. Flow cytometry revealed increased G2/M and subG1 fractions in VM-CUB1 and UM-UC-3 cells. Both effects were stronger than with SAHA treatment. Conclusion Specific pharmacological inhibition of class I HDACs by 4SC-202 impairs UC cell viability, inducing cell cycle disturbances and cell death. Combined inhibition of HDAC1, HDAC2 and HDAC3 seems to be a promising treatment strategy for UC. Author Affiliation: (1) Department of Urology, Medical Faculty, Heinrich-Heine-University, Moorenstr. 5, 40225, Duesseldorf, Germany (2) 4SC AG, Martinsried, Germany Article History: Registration Date: 10/05/2016 Online Date: 02/06/2016 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s11523-016-0444-7) contains supplementary material, which is available to authorized users.
    Keywords: Cell Death – Analysis ; Cell Death – Health Aspects ; Carcinoma – Analysis ; Carcinoma – Health Aspects;
    ISSN: 1776-2596
    E-ISSN: 1776-260X
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  • 2
    Language: English
    In: Current Medicinal Chemistry, 2017, Vol.24(37), p.4151-4165
    Description: Histone deacetylases (HDACs) influence diverse cellular processes and may contribute to tumor development and progression by multiple mechanisms. Class I HDACs are often overexpressed in cancers contributing to a genome-wide epigenetic state permitting increased proliferation, and diminished apoptosis and cell differentiation. Class IIA and IIB isoenzymes may likewise contribute to tumorigenesis as components of specific intranuclear repressor complexes or regulators of posttranslational protein modifications. As HDAC inhibitors may counteract these tumorigenic effects several of these compounds are currently tested in clinical trials. 〈/P〉〈P〉 HDAC inhibitors are also considered for urothelial carcinoma, where novel therapeutic drugs are urgently required. However, only modest antineoplastic activity has been observed with isoenzyme-unspecific pan-HDAC inhibitors. Therefore, inhibition of specific HDAC isoenzymes might be more efficacious and tumor-specific. Here, we systematically review knowledge on the expression, function and suitability as therapeutic targets of the 11 classical HDACs in UC. Overall, the class I HDACs HDAC1 and HDAC2 are the most promising targets for antineoplastic treatment. In contrast, targeting HDAC8 and HDAC6 is likely to be of minor relevance in urothelial carcinoma. Class IIA HDACs like HDAC4 require further study, since their downregulation rather than upregulation could be involved in urothelial carcinoma pathogenesis.
    Keywords: Urothelial Carcinoma Bladder Cancer Hdacss Class I Hdacs Targeted Therapy.
    ISSN: 0929-8673
    E-ISSN: 1875-533X
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  • 3
    Language: English
    In: Urologic Oncology: Seminars and Original Investigations, October 2017, Vol.35(10), pp.612-613
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.urolonc.2017.06.017 Byline: Alexander Holscher, Wolfgang A. Schulz, Maria Pinkerneil, Michele J. Hoffmann
    Keywords: Medicine
    ISSN: 1078-1439
    E-ISSN: 1873-2496
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  • 4
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2018, Vol.1655, pp.289-317
    Description: Mutations, dysregulation, and dysbalance of epigenetic regulators are especially frequent in urothelial carcinoma (UC) compared to other malignancies. Accordingly, targeting epigenetic regulators may provide a window of opportunity particularly in anticancer therapy of UC. In general, these epigenetic regulators comprise DNA methyltransferases and DNA demethylases (for DNA methylation), histone methyltransferases, and histone demethylases (for histone methylation) as well as acetyl transferases and histone deacetylases (for histone and non-histone acetylation).As epigenetic regulators target a plethora of cellular functions and available inhibitors often inhibit enzymatic activity of more than one isoenzyme or may have further off-target effects, analysis of their functions in UC pathogenesis as well as of the antineoplastic capacity of according inhibitors should follow a multidimensional approach.Here, we present our standard approach for the analysis of the cellular and molecular functions of individual HDAC enzymes, their suitability as treatment targets and for the evaluation of isoenzyme-specific HDAC inhibitors regarding their antineoplastic efficacy. This approach may also serve as prototype for the preclinical evaluation of other epigenetic treatment approaches.
    Keywords: Epigenetics ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; Targeted Therapy ; Urothelial Carcinoma ; Antineoplastic Agents -- Therapeutic Use ; Epigenesis, Genetic -- Drug Effects ; Urologic Neoplasms -- Drug Therapy
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 5
    Language: English
    In: Clinical Epigenetics, 01 January 2018, Vol.10(1), pp.1-14
    Description: Abstract Background New efficient therapies for urothelial carcinoma (UC) are urgently required. Small-molecule drugs targeting chromatin regulators are reasonable candidates because these regulators are frequently mutated or deregulated in UC. Indeed, in previous work, Romidepsin, which targets class I histone deacetylases (HDAC), efficiently killed UC cells, but did not elicit canonical apoptosis and affected benign urothelial cells indiscriminately. Combinations of HDAC inhibitors with JQ1, an inhibitor of bromodomain-containing acetylation reader proteins like BRD4, which promote especially the transcription of pro-tumorigenic genes, have shown efficacy in several tumor types. We therefore investigated the effects of combined Romidepsin and JQ1 treatment on UC and benign urothelial control cells. Results JQ1 alone induced cell cycle arrest, but only limited apoptosis in eight UC cell lines with strongly varying IC50 values between 0.18 and 10 μM. Comparable effects were achieved by siRNA-mediated knockdown of BRD4. Romidepsin and JQ1 acted in a synergistic manner across all UC cell lines, efficiently inhibiting cell cycle progression, suppressing clonogenic growth, and inducing caspase-dependent apoptosis. Benign control cells were growth-arrested without apoptosis induction, but retained long-term proliferation capacity. In UC cells, anti-apoptotic and oncogenic factors Survivin, BCL-2, BCL-XL, c-MYC, EZH2 and SKP2 were consistently downregulated by the drug combination and AKT phosphorylation was diminished. Around the transcriptional start sites of these genes, the drug combination enhanced H3K27 acetylation, but decreased H3K4 trimethylation. The cell cycle inhibitor CDKN1C/p57KIP2 was dramatically induced at mRNA and protein levels. However, Cas9-mediated CDKN1C/p57KIP2 knockout did not rescue UC cells from apoptosis. Conclusion Our results demonstrate significant synergistic effects on induction of apoptosis in UC cells by the combination treatment with JQ1 and Romidepsin, but only minor effects in benign cells. Thus, this study established a promising new small-molecule combination therapy approach for UC.
    Keywords: Bet Inhibitor ; Jq1 ; Hdaci ; Romidepsin ; Bladder Cancer ; Apoptosis ; Zoology
    ISSN: 1868-7075
    E-ISSN: 1868-7083
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  • 6
    Language: English
    In: International Journal of Molecular Sciences, 01 July 2017, Vol.18(7), p.1449
    Description: Disturbances in histone acetyltransferases (HATs) are common in cancers. In urothelial carcinoma (UC), p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF (General Control Nonderepressible 5, p300/CBP-Associated Factor) are often upregulated. Here, we explored the effects of specific siRNA-mediated knockdown of GCN5, PCAF or both in four UC cell lines (UCCs). Expression of various HATs and marker proteins was measured by qRT-PCR and western blot. Cellular effects of knockdowns were analyzed by flow cytometry and ATP-, caspase-, and colony forming-assays. GCN5 was regularly upregulated in UCCs, whereas PCAF was variable. Knockdown of GCN5 or both GNATs, but not of PCAF alone, diminished viability and inhibited clonogenic growth in 2/4 UCCs, inducing cell cycle changes and caspase-3/7 activity. PCAF knockdown elicited GCN5 mRNA upregulation. Double knockdown increased c-MYC and MDM2 (Mouse Double Minute 2) in most cell lines. In conclusion, GCN5 upregulation is especially common in UCCs. GCN5 knockdown impeded growth of specific UCCs, whereas PCAF knockdown elicited minor effects. The limited sensitivity towards GNAT knockdown and its variation between the cell lines might be due to compensatory effects including HAT, c-MYC and MDM2 upregulation. Our results predict that developing drugs targeting individual HATs for UC treatment may be challenging.
    Keywords: Histone Acetyltransferases ; Urothelial Carcinoma ; Bladder Cancer ; Gcn5 ; Pcaf ; P300 ; Cbp ; Mdm2 ; C-Myc ; Epigenetic Drugs ; Biology
    E-ISSN: 1422-0067
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  • 7
    Language: English
    In: Molecular cancer therapeutics, February 2016, Vol.15(2), pp.299-312
    Description: Class I histone deacetylases HDAC1 and HDAC2 contribute to cell proliferation and are commonly upregulated in urothelial carcinoma. To evaluate whether specific inhibition of these enzymes might serve as an appropriate therapy for urothelial carcinoma, siRNA-mediated knockdown and specific pharmacologic inhibition of HDAC1 and HDAC2 were applied in urothelial carcinoma cell lines (UCC) with distinct HDAC1 and HDAC2 expression profiles. HDACs and response marker proteins were followed by Western blotting and qRT-PCR. Effects of class I HDAC suppression on UCCs were analyzed by viability, colony forming, and caspase-3/7 assays; flow cytometry, senescence and lactate dehydrogenase cytotoxicity assays; and immunofluorescence staining. Whereas single knockdowns of HDAC1 or HDAC2 were impeded by compensatory upregulation of the other isoenzyme, efficient double knockdown of HDAC1 and HDAC2 reduced proliferation by up to 80% and induced apoptosis-like cell death in all UCCs. Clonogenic growth was cell line- and HDAC-dependently reduced, with double knockdown of HDAC1 and HDAC2 being usually most efficient. Class I HDAC-specific inhibitors, especially the more specific HDAC1/2 inhibitors romidepsin and givinostat, significantly reduced proliferation of all UCCs (IC50, 3.36 nmol/L-4.59 μmol/L). Romidepsin and givinostat also significantly inhibited clonogenic growth of UCCs, with minor effects on nontumorigenic controls. Intriguingly, these compounds induced primarily S-phase disturbances and nonapoptotic cell death in UCCs. Thus, although both ways of inhibiting HDAC1/2 share mechanisms and efficaciously inhibit cell proliferation, their modes of action differ substantially. Regardless, combined inhibition of HDAC1/2 appears to represent a promising strategy for urothelial carcinoma therapy. Mol Cancer Ther; 15(2); 299-312. ©2016 AACR.
    Keywords: Carbamates -- Pharmacology ; Depsipeptides -- Pharmacology ; Histone Deacetylase 1 -- Metabolism ; Histone Deacetylase 2 -- Metabolism ; Histone Deacetylase Inhibitors -- Pharmacology ; Urinary Bladder Neoplasms -- Enzymology
    ISSN: 15357163
    E-ISSN: 1538-8514
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  • 8
    In: Isono, Makoto and Hoffmann, Michèle J and Pinkerneil, Maria and Sato, Akinori and Michaelis, Martin and Cinatl, Jindrich and Niegisch, Günter and Schulz, Wolfgang A (2017) Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine. Journal of experimental & clinical cancer research : CR, 36 (1). p. 1.
    Description: BACKGROUND More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. METHODS Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. RESULTS Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. CONCLUSIONS AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
    Keywords: RM Therapeutics. Pharmacology
    ISSN: 1756-9966
    Source: University of Kent
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  • 9
    Language: English
    In: Journal of experimental & clinical cancer research : CR, 03 January 2017, Vol.36(1), pp.1
    Description: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
    Keywords: Azd7762 ; Bladder Cancer ; Checkpoint Kinase ; Gemcitabine ; Urothelial Carcinoma ; Antimetabolites, Antineoplastic -- Pharmacology ; Carcinoma, Transitional Cell -- Metabolism ; Deoxycytidine -- Analogs & Derivatives ; Protein Kinase Inhibitors -- Pharmacology ; Thiophenes -- Pharmacology ; Urea -- Analogs & Derivatives ; Urinary Bladder Neoplasms -- Metabolism
    ISSN: 03929078
    E-ISSN: 1756-9966
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  • 10
    In: Bladder Cancer (Amsterdam, Netherlands), Vol.2(4), p.449-463
    Description: Background: Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells. Objective: To characterize utility and limitations of HBLAK cells as an urothelial cell culture model. Methods: Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting. Results: HBLAK cells proliferated for 〉50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in p16 INK 4 A loss. A C228T TERT promoter mutation was present, but no other mutation typical of urothelial carcinoma. TP53 was wild-type and the cell cycle was arrested in response to genomic stress. Conclusions: HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial in vitro carcinogenesis model.
    Keywords: Research Report ; Immortalization ; Normal Urothelial Cells ; Bladder Cancer ; Cell Culture Model ; Telomerase ; Urothelial Differentiation ; Cdkn2a ; P53
    ISSN: 2352-3727
    E-ISSN: 2352-3735
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