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Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines

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Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines Targ Oncol DOI 10.1007/s11523-016-0444-7 ORIGINAL RESEARCH ARTICLE Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines Maria Pinkerneil1 & Michèle J. Hoffmann1 & Hella Kohlhof2 & Wolfgang A. Schulz1 & Günter Niegisch1 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Results 4SC-202 significantly reduced proliferation of all ep- Background Targeting of class I histone deacetylases ithelial and mesenchymal UC cell lines (IC50 0.15–0.51 μM), (HDACs) exerts antineoplastic actions in various cancer types inhibited clonogenic growth and induced caspase activity. by modulation of transcription, upregulation of tumor sup- Flow cytometry revealed increased G2/M and subG1 fractions pressors, induction of cell cycle arrest, replication stress and in VM-CUB1 and UM-UC-3 cells. Both effects were stronger promotion of apoptosis. Class I HDACs are often deregulated than with SAHA treatment. in urothelial cancer. 4SC-202, a novel oral benzamide type Conclusion Specific pharmacological inhibition of class I HDAC inhibitor (HDACi) specific for class I HDACs HDACs by 4SC-202 impairs UC cell viability, inducing cell HDAC1, HDAC2 and HDAC3 and the histone demethylase cycle disturbances and cell death. Combined inhibition of LSD1, shows substantial anti-tumor activity in a broad range HDAC1, HDAC2 and HDAC3 seems to be a promising treat- of cancer cell lines and xenograft tumor models. ment strategy for UC. 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 KeyPoints MTT assay in seven UC cell lines with distinct HDAC1, 4SC-202 exerts significant antineoplastic effects on HDAC2 and HDAC3 expression profiles. Cellular effects urothelial carcinoma (UC) cells. were further analyzed in VM-CUB1 and UM-UC-3 cells by colony forming assay, caspase-3/7 assay, flow cytometry, se- 4SC-202 induces strong mitotic disturbances in UC cells. nescence assay, LDH release assay, and immunofluorescence staining. Response markers were followed by quantitative The 4SC-202 mediated effects are characterised by real-time PCR and western blotting. Treatment with the class combined induction of apoptotic and necrotic cell death. I HDAC specific inhibitor SAHA (vorinostat) served as a general control. Electronic supplementary material The online version of this article 1 Introduction (doi:10.1007/s11523-016-0444-7) contains supplementary material, which is available to authorized users. The efficacy of systemic treatment in patients suffering from * Günter Niegisch metastatic urothelial carcinoma (UC) is limited. Although [email protected] about half of the patients respond initially to platinum-based polychemotherapy, up to 90 % of patients will present with 1 Department of Urology, Medical Faculty, tumor relapse within less than 5 years [1–3]. Following the Heinrich-Heine-University, Moorenstr. 5, successful integration of Btargeted therapeutics^, which in- 40225 Duesseldorf, Germany hibits distinct cancer pathways, e.g. MAP kinase or PIK3 2 4SC AG, Martinsried, Germany kinase/Akt signaling, into modern oncological treatment, Targ Oncol according approaches have also been tested in UC [4–6]. 2 Material and Methods However, up to now, none of these attempts has been success- ful [7, 8]. Inefficacy of targeted therapeutics may be due to 2.1 Cell Culture and Treatment various resistance mechanisms by which UC cells circumvent drug-induced inactivation of essential signaling pathways [9]. Experiments were performed in the urothelial cancer cell lines As cancer pathways generally ultimately exert their effects by (UCCs) VM-CUB1, RT-112, SW-1710, 639-V, UM-UC-3, regulating gene expression, a more promising treatment strategy 5637 and T-24 (provided by Dr. M. A. Knowles, Leeds, UK; might consist of targeting gene expression more directly. This Dr. J. Fogh, New York, USA; Dr. B. Grossmann, Houston, could be achieved, among others, by inhibition of histone USA; DSMZ, Braunschweig, Germany) representing the het- deacetylases (HDACs). The HDAC family consists of 18 isoen- erogeneous phenotypes of urothelial carcinoma and different zymes classified into so-called Bclassical^ HDACs (HDAC1-11; HDAC expression patterns [20, 24]. To investigate the tumor class I, class II and class IV) and sirtuins (Sirt1-7; class III) specificity of 4SC-202, experiments were further performed in [10–12]. Especially, class I HDACs (HDAC1, HDAC2, two different urothelial control cell lines, HBLAK (spontane- HDAC3, and HDAC8) act as transcriptional repressors and their ously immortalized from primary human bladder epithelial expression profiles are prognostic in several malignancies cells, provided by CELLnTEC, Bern, Switzerland) and [13–17]. HDAC inhibitors (HDACi) exhibit therapeutic efficacy TERT-NHUC (TERT-immortalized normal human urothelial in some hematological and solid cancers, and several isoenzyme- cells, provided by Dr. M. A. Knowles, Leeds, UK), and in the unspecific HDACi (pan-HDACi) are approved for the treatment non-malignant, non-urothelial cell line HEK-293 (provided of specific hematological malignancies [18, 19]. In UC cell lines, by Dr. V. Kolb-Bachofen, Duesseldorf, Germany) as previ- pan-HDACi are also active by inducing apoptosis and cell cycle ously described [24]. UCCs and HEK-293 cells were cultured arrest [20, 21]. However, the observed preclinical effects of pan- in DMEM GlutaMAX-I (Gibco, Life Technologies, HDACi are limited overall, perhaps because effects on different Darmstadt, Germany) supplemented with 10 % fetal calf se- isoenzymes counterbalance each other. rum (Biochrom, Berlin, Germany) at 37 °C and 5 % CO2. Isoenzyme-specific inhibition of distinct HDACs HBLAK cells were cultured in CnT-Prime Epithelial Culture might be more efficient. For example, selective inhibition Medium (CELLnTEC, Bern, Switzerland). TERT-NHUC of HDAC8 inhibited cell proliferation and clonogenic cells were cultured in supplemented keratinocyte serum-free growth in a preclinical neuroblastoma cell culture model medium (KSFM, Gibco) [26]. KSFM-Medium was supple- and, albeit less efficiently, in urothelial cancer cell lines mented with 0.25 ng/ml EGF (epidermal growth factor; [22, 23]. In a recent analysis on selective inhibition of Gibco), 12.5 μg/ml BPE (bovine pituitary extract; Gibco) class I HDACs, simultaneous and selective inhibition of and 1 % ITS (insulin transferrin selenium; Life the class I HDACs HDAC1 and HDAC2 resulted in sig- Technologies, Darmstadt, Germany), 0.35 μg/ml N-epineph- nificant decreases of cell viability, proliferation and rine (Sigma Aldrich, St. Louis, MO) and 0.33 mg/ml hydro- clonogenicity associated with accumulation of cells in cortisone (Sigma Aldrich). Differentiation of HBLAK cells the G2/M cell cycle phase [24]. was performed after cells reached 90 % confluence according 4SC-202 is a novel isotype-specific HDAC inhibitor that to a published protocol [27]. Briefly, cells were treated with also inhibits KDM1A/LSD1 (Lysine (K)-specific demethylase 2 mM CaCl2 and medium was supplemented with 5 % FCS. 1A). It has been tested in a phase I trial (TOPAS) for the Cells were maintained under these conditions for 7 days, treatment of advanced hematological malignancies [25]. while medium was changed every 3 days. Subsequently, cells 4SC-202 is a benzamide type inhibitor with strong activity were treated with 0.5 μM, 2.5 μM 4SC-202 or DMSO as a against HDAC1 (IC50: 0.16 μM), HDAC2 (0.37 μM) and control for 72 h. HDAC3 (0.13 μM), without affecting other HDAC enzymes For all cell lines, short tandem repeat (STR) profiling was at clinically relevant concentrations (IC50: HDAC4, HDAC5, recently performed by standard DNA fingerprint analysis HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, (analysis reports available upon request; supplemental HDAC11 > 15 μM) (updated, unpublished data, personal Table S1 in Online Resource 1). communication by H.K., detailed data available upon re- Inhibitor experiments were performed 24 h after cell quest). The reported IC50 for KDM1A/LSD1 ranges in clini- seeding with a single dose of 4SC-202 (provided by 4SC cally relevant concentrations from 0.6 to 1.2 μM (Data pre- AG, Martinsried, Germany), vorinostat (2.5 μM, SAHA sented at Sixth Annual EpiCongress, Boston, USA, July 2015, [suberoylanilide hydroxamic acid]; #1009929, Cayman data available online at https://4SC.de). Chemicals, Ann Arbor, MI), romidepsin (3 nM, S3020, In this context, we evaluated the efficacy of 4SC-202 in UC Selleck Chemicals, Munich, Germany), givinostat (0.5 μM, cells. To our knowledge, this is the first detailed preclinical S2170, Selleck Chemicals) or the LSD1 inhibitor (LSD1i) characterization of the novel isotype-specific HDAC inhibitor SP2509 (VM-CUB1: 0.75 μM, UM-UC-3: 0.5 μM, S7680, 4SC-202 in UC. Selleck Chemicals). Throughout, the previous characterized Targ Oncol pan-HDACi SAHA served as control HDACi [20, 24]. The Merck Millipore, Berlin, Germany), fixed in methanol and previously characterized class I HDACi romidepsin (histone stained with Giemsa (Merck, Darmstadt, Germany). acetylation, morphology, class I HDAC expression after HDACi treatment) and givinostat (morphology) were used as additional controls or for comparison with 4SC-202 [24]. 2.4 Flow Cytometry As a positive control in LDH (lactate dehydrogenase) mea- μ surements, cells were treated with bortezomib (50 nM, S1013, UCCs or control cells were cultured
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