Histone Deacetylase Inhibitor-Induced Cell Death in Bladder Cancer Is Associated with Chromatin Modification and Modifying Protein Expression: a Proteomic Approach
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INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2591-2607, 2016 Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach QINgDI QUeNTIN LI1, JIAN-JIANg HAO2, ZHeNg ZHANg2, IaweN HSU1, YI LIU2, ZHeN TAO2, KeIDreN LeWI1, ADAm r. metwalli1 and PIyUSH K. AgArwal1 1Urologic Oncology Branch, Center for Cancer research, National Cancer Institute, National Institutes of Health, Bethesda, mD 20892; 2Poochon Scientific, Frederick, mD 21704, USA received February 6, 2016; Accepted march 17, 2016 DOI: 10.3892/ijo.2016.3478 Abstract. The Cancer Genome Atlas (TCGA) project recently induction of apoptosis in the 5637 bladder cancer cells through identified the importance of mutations in chromatin remod- multiple cell death-associated pathways. These observations eling genes in human carcinomas. These findings imply support the notion that HDACIs provide new therapeutic that epigenetic modulators might have a therapeutic role in options for bladder cancer treatment and thus warrant further urothelial cancers. To exploit histone deacetylases (HDACs) as preclinical exploration. targets for cancer therapy, we investigated the HDAC inhibi- tors (HDACIs) romidepsin, trichostatin A, and vorinostat as Introduction potential chemotherapeutic agents for bladder cancer. We demonstrate that the three HDACIs suppressed cell growth Urinary bladder cancer is the second most common malignant and induced cell death in the bladder cancer cell line 5637. tumor of the genitourinary tract and ranks fourth among To identify potential mechanisms associated with the anti- male cancers (1). Approximately 70% of initially diagnosed proliferative and cytotoxic effects of the HDACIs, we used tumors are superficial and can be treated by transurethral quantitative proteomics to determine the proteins potentially resection, while the remaining 30% become muscle invasive involved in these processes. Our proteome studies identi- and are associated with a high risk of metastatic disease (2,3). fied a total of 6003 unique proteins. Of these, 2472 proteins Systemic chemotherapy is a treatment option for patients with were upregulated and 2049 proteins were downregulated locally advanced or metastatic disease. Despite huge efforts in response to HDACI exposure compared to the untreated to tackle the disease in the past two decades, current treat- controls (P<0.05). Bioinformatic analysis further revealed ments confer only a modest survival benefit upon bladder that those differentially expressed proteins were involved in cancer patients, and long-term survival of patients suffering multiple biological functions and enzyme-regulated pathways, from metastatic disease does not exceed 20% (4); therefore, including cell cycle progression, apoptosis, autophagy, free there is an urgent need for innovative ideas that deviate from radical generation and DNA damage repair. HDACIs also conventional approaches. altered the acetylation status of histones and non-histone The search for novel chemical agents against cancer has proteins, as well as the levels of chromatin modification long been the mainstay of cancer research. During recent years, proteins, suggesting that HDACIs exert multiple cytotoxic it has been shown that epigenetic aberrations are involved in actions in bladder cancer cells by inhibiting HDAC activity or tumorigenesis. Particularly, an imbalance in the equilibrium altering the structure of chromatin. We conclude that HDACIs between histone acetylation and histone deacetylation has are effective in the inhibition of cell proliferation and the been proposed as a driving force, causing normal cells to become malignant. Therefore, modulating acetylation may be an innovative strategy to treat malignant disease. Acetylation is catalyzed by a specific enzyme family, histone acetyltrans- ferases (HATs), and correlates with nucleosome remodeling Correspondence to: Dr Qingdi Quentin Li or Dr Piyush K. Agarwal, and transcriptional activation, whereas deacetylation of National Institutes of Health, Bethesda, mD 20892, USA histone tails is catalyzed by histone deacetylases (HDACs) e-mail: [email protected] and induces transcriptional repression through chromatin e-mail: [email protected] condensation (5). Key words: apoptosis, bladder cancer, HDAC inhibitor, proteomics, Altered expression of different HDACs has been reported in pathway analysis, cell cycle, DNA damage repair various human cancers (6-12). Systemic analysis of the expres- sion levels of HDACs in cultured cancer cell lines, as well as primary cultures of human cancer cells and various human tumor biopsy samples, frequently identifies higher levels of 2592 LI et al: ProteOmic analysis of HDACI-TreateD bladder cancer Cells expression than in corresponding normal tissue. For instance, TSA or SAHA were dissolved in DmSO separately and recent evidence shows that both clinical samples from patients stored at -20˚C. The CellTiter 96 AQueous One Solution Cell with urinary bladder cancer and tumor tissues from a mouse Proliferation Assay was from Promega Corp. (madison, WI, model have demonstrated a significantly increased HDAC USA). expression compared with surrounding healthy tissue (13). Thus, HDAC inhibition might be an effective option to treat Cell culture and cell viability assay. The human bladder bladder cancer. cancer cell line 5637 was purchased from the American Type Thus far, 18 HDACs have been identified in mammals Culture Collection (ATCC; manassas, VA, USA). The cell line that are classified into four classes based on their homology was grown in MEM, supplemented with 10% FBS, 50 IU/ml to yeast proteins (7,14). Class I HDAC enzymes (HDACs penicillin, and 50 µg/ml streptomycin, at 37˚C in a humidified 1, 2, 3 and 8) are widely expressed (12,15), class II HDAC atmosphere with 5% CO2. enzymes (HDACs 4, 5, 6, 7, 9 and 10) have tissue-specific The antiproliferative effects of romidepsin, TSA and SAHA distribution and are involved in organ development and func- were assessed using an mTS (3-(4,5-dimethylthiazol-2-yl)-5- tion (12), and other classes are less specific in terms of tissue (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)- distribution and function. HDAC inhibitors (HDACIs) prevent based assay (Promega) as previously described (19). In brief, HDACs from removing acetyl groups, leading to increased 5637 bladder carcinoma cells (5x103 cells/well) were evenly acetylation and allowing DNA to remain transcriptionally distributed in 96-well plates, grown overnight, and then treated active (5). There are many known natural and synthetic with various concentrations of romidepsin, TSA or SAHA at HDACIs, which can be subdivided into five structural the indicated concentrations (0, 0.1 1, 10 and 100 nm, 1, 10 classes, including hydroxamates, cyclic peptides, aliphatic and 100 µm) for 24 or 72 h. At the end of incubation, 20 µl of acids, benzamines and electrophilic ketones. The hydroxa- CellTiter 96 AQueous One Solution reagent was added to each mate compound trichostatin A (TSA) is a potent nanomolar well of the assay plates containing the treated and untreated inhibitor of most class I and class II HDAC enzymes (12,16). cells in 200 µl of culture medium, and the plates were incubated romidepsin (FK228) is the only cyclic peptide HDACI in at 37˚C and 5% CO2 for 2 h. The optical density at 490 nm clinical development (17,18), and it potently inhibits class I was determined using a 96-well imark™ microplate reader HDACs (12,18). Class I HDAC enzymes are overexpressed (Bio-rad Laboratories, Hercules, CA, USA). Proliferation in many malignancies, and this overexpression is often rates were calculated from the optical densities of the HDACI- associated with poor prognosis (12). A number of structur- treated cells relative to the optical density of DmSO-treated ally different HDACIs are in clinical trials for a wide variety control cells with no HDACI exposure (control value, 100%). of hematologic and solid neoplasms, including cancer of the The half-maximal inhibitory concentration (IC50) values for lung, breast, pancreas, and kidney, melanoma, glioblastoma, romidepsin, TSA and SAHA on 24 and 72 h in 5637 cell line leukemias, lymphomas and multiple myeloma (5). Among were calculated using graphPad Prism version 6.01 software them, romidepsin and vorinostat (SAHA) have been approved (graphPad Software, Inc., La Jolla, CA, USA). IC50 was consid- by the Food and Drug Administration for the treatment of ered as the drug concentration that decreases the cell count by cutaneous T-cell lymphoma (CTCL). However, the effect and 50%. Non-linear regression curve fitting was performed. The the mechanism of action of HDACIs as chemotherapeutic data were fitted to an exponential first-order decay function. regimens for bladder cancer remain to be determined. Herein, we show that the treatment with HDACIs Preparation of protein extraction, separation of proteins and (romidepsin, TSA and SAHA) inhibited cell growth and in-gel trypsin digestion. Total protein extraction from cell proliferation in a dose-dependent fashion in the urinary pellets was prepared by the following method. In brief, cell bladder cancer cell line 5637. We further analyzed the protein pellets were lysed in 0.4 ml lysis buffer (20 mm Tris-HCl, expression patterns in response to romidepsin and TSA in pH 7.5, 150 mm NaCl, 1 mm Na2eDTA, 1 mm EGTA, 1% this cell model system using quantitative proteomic studies Triton X-100, protease inhibitor cocktail