Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach

  • Authors:
    • Qingdi Quentin Li
    • Jian-Jiang Hao
    • Zheng Zhang
    • Iawen Hsu
    • Yi Liu
    • Zhen Tao
    • Keidren Lewi
    • Adam R. Metwalli
    • Piyush K. Agarwal
  • View Affiliations

  • Published online on: April 7, 2016     https://doi.org/10.3892/ijo.2016.3478
  • Pages: 2591-2607
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Abstract

The Cancer Genome Atlas (TCGA) project recently identified the importance of mutations in chromatin remodeling genes in human carcinomas. These findings imply that epigenetic modulators might have a therapeutic role in urothelial cancers. To exploit histone deacetylases (HDACs) as targets for cancer therapy, we investigated the HDAC inhibitors (HDACIs) romidepsin, trichostatin A, and vorinostat as potential chemotherapeutic agents for bladder cancer. We demonstrate that the three HDACIs suppressed cell growth and induced cell death in the bladder cancer cell line 5637. To identify potential mechanisms associated with the anti-proliferative and cytotoxic effects of the HDACIs, we used quantitative proteomics to determine the proteins potentially involved in these processes. Our proteome studies identified a total of 6003 unique proteins. Of these, 2472 proteins were upregulated and 2049 proteins were downregulated in response to HDACI exposure compared to the untreated controls (P<0.05). Bioinformatic analysis further revealed that those differentially expressed proteins were involved in multiple biological functions and enzyme-regulated pathways, including cell cycle progression, apoptosis, autophagy, free radical generation and DNA damage repair. HDACIs also altered the acetylation status of histones and non-histone proteins, as well as the levels of chromatin modification proteins, suggesting that HDACIs exert multiple cytotoxic actions in bladder cancer cells by inhibiting HDAC activity or altering the structure of chromatin. We conclude that HDACIs are effective in the inhibition of cell proliferation and the induction of apoptosis in the 5637 bladder cancer cells through multiple cell death-associated pathways. These observations support the notion that HDACIs provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

Introduction

Urinary bladder cancer is the second most common malignant tumor of the genitourinary tract and ranks fourth among male cancers (1). Approximately 70% of initially diagnosed tumors are superficial and can be treated by transurethral resection, while the remaining 30% become muscle invasive and are associated with a high risk of metastatic disease (2,3). Systemic chemotherapy is a treatment option for patients with locally advanced or metastatic disease. Despite huge efforts to tackle the disease in the past two decades, current treatments confer only a modest survival benefit upon bladder cancer patients, and long-term survival of patients suffering from metastatic disease does not exceed 20% (4); therefore, there is an urgent need for innovative ideas that deviate from conventional approaches.

The search for novel chemical agents against cancer has long been the mainstay of cancer research. During recent years, it has been shown that epigenetic aberrations are involved in tumorigenesis. Particularly, an imbalance in the equilibrium between histone acetylation and histone deacetylation has 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 acetyltransferases (HATs), and correlates with nucleosome remodeling and transcriptional activation, whereas deacetylation of histone tails is catalyzed by histone deacetylases (HDACs) and induces transcriptional repression through chromatin condensation (5).

Altered expression of different HDACs has been reported in various human cancers (612). Systemic analysis of the expression 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 expression than in corresponding normal tissue. For instance, recent evidence shows that both clinical samples from patients with urinary bladder cancer and tumor tissues from a mouse model have demonstrated a significantly increased HDAC expression compared with surrounding healthy tissue (13). Thus, HDAC inhibition might be an effective option to treat bladder cancer.

Thus far, 18 HDACs have been identified in mammals that are classified into four classes based on their homology to yeast proteins (7,14). Class I HDAC enzymes (HDACs 1, 2, 3 and 8) are widely expressed (12,15), class II HDAC enzymes (HDACs 4, 5, 6, 7, 9 and 10) have tissue-specific distribution and are involved in organ development and function (12), and other classes are less specific in terms of tissue distribution and function. HDAC inhibitors (HDACIs) prevent HDACs from removing acetyl groups, leading to increased acetylation and allowing DNA to remain transcriptionally active (5). There are many known natural and synthetic HDACIs, which can be subdivided into five structural classes, including hydroxamates, cyclic peptides, aliphatic acids, benzamines and electrophilic ketones. The hydroxamate compound trichostatin A (TSA) is a potent nanomolar inhibitor of most class I and class II HDAC enzymes (12,16). Romidepsin (FK228) is the only cyclic peptide HDACI in clinical development (17,18), and it potently inhibits class I HDACs (12,18). Class I HDAC enzymes are overexpressed in many malignancies, and this overexpression is often associated with poor prognosis (12). A number of structurally different HDACIs are in clinical trials for a wide variety of hematologic and solid neoplasms, including cancer of the lung, breast, pancreas, and kidney, melanoma, glioblastoma, leukemias, lymphomas and multiple myeloma (5). Among them, romidepsin and vorinostat (SAHA) have been approved by the Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL). However, the effect and the mechanism of action of HDACIs as chemotherapeutic regimens for bladder cancer remain to be determined.

Herein, we show that the treatment with HDACIs (romidepsin, TSA and SAHA) inhibited cell growth and proliferation in a dose-dependent fashion in the urinary bladder cancer cell line 5637. We further analyzed the protein expression patterns in response to romidepsin and TSA in this cell model system using quantitative proteomic studies and found that the effect of these two HDACIs on growth inhibition and cell death is mediated through modulating the expression of proteins involved in cell cycle progression, apoptosis, autophagy, reactive oxygen species (ROS) generation and DNA damage repair in 5637 bladder cancer cells.

Materials and methods

Chemicals and reagents

The minimum essential medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin (100X), and 0.25% trypsin-EDTA solution (1X) were obtained from Invitrogen Corp., Life Technologies (Carlsbad, CA, USA). Trichostatin A (TSA) (>98% purity) was from Selleckchem (Houston, TX, USA). Romidepsin (FK228) (>98% purity) was purchased from Apexbio Technology LLC (Houston, TX, USA). Vorinostat (SAHA) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). Romidepsin, TSA or SAHA were dissolved in DMSO separately and stored at −20°C. The CellTiter 96 AQueous One Solution Cell Proliferation Assay was from Promega Corp. (Madison, WI, USA).

Cell culture and cell viability assay

The human bladder cancer cell line 5637 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cell line was grown in MEM, supplemented with 10% FBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO2.

The antiproliferative effects of romidepsin, TSA and SAHA were assessed using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-based assay (Promega) as previously described (19). In brief, 5637 bladder carcinoma cells (5×103 cells/well) were evenly distributed in 96-well plates, grown overnight, and then treated with various concentrations of romidepsin, TSA or SAHA at the indicated concentrations (0, 0.1 1, 10 and 100 nM, 1, 10 and 100 μM) for 24 or 72 h. At the end of incubation, 20 μl of CellTiter 96 AQueous One Solution reagent was added to each well of the assay plates containing the treated and untreated cells in 200 μl of culture medium, and the plates were incubated at 37°C and 5% CO2 for 2 h. The optical density at 490 nm was determined using a 96-well iMark™ Microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Proliferation rates were calculated from the optical densities of the HDACI-treated cells relative to the optical density of DMSO-treated control cells with no HDACI exposure (control value, 100%). The half-maximal inhibitory concentration (IC50) values for romidepsin, TSA and SAHA on 24 and 72 h in 5637 cell line were calculated using GraphPad Prism version 6.01 software (GraphPad Software, Inc., La Jolla, CA, USA). IC50 was considered as the drug concentration that decreases the cell count by 50%. Non-linear regression curve fitting was performed. The data were fitted to an exponential first-order decay function.

Preparation of protein extraction, separation of proteins and in-gel trypsin digestion

Total protein extraction from cell pellets was prepared by the following method. In brief, cell pellets were lysed in 0.4 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor cocktail pill). After cells were lysed, 50 μl of 10% SDS and 50 μl of 1 M DTT were added into the mixture followed by incubation at 95°C for 10 min. The extraction was then sonicated and centrifuged at 15,000 × g for 10 min. Supernatants were collected and stored at −80°C for further analysis. The protein concentration of the supernatants was determined by a BCA™ reducing reagent compatible assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

Equal amounts of protein (130 μg) from each sample were fractioned by separation on a NuPAGE 4–12% Bis-Tris Gel (Life Technologies, Grand Island, NY, USA). Sixteen gel fractions from each lane representing one sample were treated with DTT for reduction, then iodoacetamide for alkylation, and further digested by trypsin in 25 mM NH4HCO3 solution. The digested protein was extracted, and the extracted peptides were dried and reconstituted in 20 μl of 0.1% formic acid before nanospray LC/MS/MS analysis was performed.

Nanospray LC/MS/MS analysis

Sixteen tryptic peptide fractions from one cell sample were analyzed sequentially using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a Thermo Dionex UltiMate 3000 RSLCnano system. Tryptic peptide samples were loaded onto a peptide trap cartridge at a flow rate of 5 μl/min. The trapped peptides were eluted onto a reversed-phase 25-cm C18 Picofrit column (New Objective, Woburn, MA, USA) using a linear gradient of acetonitrile (3–36%) in 0.1% formic acid. The elution duration was 110 min at a flow rate of 0.3 μl/min. Eluted peptides from the Picofrit column were ionized and sprayed into the mass spectrometer, using a Nanospray Flex Ion Source ES071 (Thermo Fisher Scientific) under the following settings: spray voltage, 1.6 kV and capillary temperature, 250°C. The Q Exactive instrument was operated in the data dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300–2000) was acquired in the Orbitrap with 70,000 resolution (m/z 200) after accumulation of ions to a 3×106 target value based on predictive AGC from the previous full scan. Dynamic exclusion was set to 20 sec. The 12 most intense multiply-charged ions (z ≥2) were sequentially isolated and fragmented in the Axial higher energy collision-induced dissociation (HCD) cell using normalized HCD collision energy at 25% with an AGC target 1e5 and a maxima injection time of 100 ms at 17,500 resolution.

LC/MS/MS data analysis

The raw MS files were analyzed using the Thermo Proteome Discoverer 1.4.1 platform (Thermo Fisher Scientific, Bremen, GmbH) for peptide identification and protein assembly. For each cell sample, 16 raw MS files obtained from 16 sequential LC/MS analyses were grouped for a single database search against the Human UniProtKB/Swiss-Prot human protein sequence databases (20597 entries, 12/20/2013) based on the SEQUEST and percolator algorithms through the Proteome Discoverer 1.4.1 platform. Carbamidomethylation of cysteines was set as a fixed modification. The minimum peptide length was specified to be five amino acids. The precursor mass tolerance was set to 15 ppm, whereas fragment mass tolerance was set to 0.05 kDa. The maximum false peptide discovery rate was specified as 0.01. The resulting Proteome Discoverer Report contains all assembled proteins (a proteome profile) with peptides sequences and matched spectrum counts. Three proteome profiles were generated for the untreated control cells and two HDACI-treated cell samples.

Protein quantification

Protein quantification used the normalized spectral abundance factors (NSAFs) method to calculate the protein relative abundance (20,21). To quantitatively describe the relative abundance, the ppm (part per million) was chosen as the unit and the 1,000,000 ppm value was assigned to each proteome profile. A ppm value at the range of 0–1,000,000 ppm for each identified protein in each proteome profile was calculated based on its normalized NSAF.

The ppm was calculated as follows: RCN = 106 × NSAFN, where RCN is the relative concentration of protein N in the proteome of test sample, NSAFN is the protein's normalized spectral abundance factor and N is the protein index.

NSAFs were calculated as follows: NSAFN = (SN/LN)/(∑ni=1Si/Li), where N is the protein index, SN is the number of peptide spectra matched to the protein, LN is the length of protein N (number of amino acid residues), and n is the total number of proteins in the input database (proteome profile for one cell sample). The ratio of HDACI treated vs. untreated control was defined as 1,000 if the protein was not identified in untreated control, or as 0.001 if the protein was not identified in HDACI-treated sample.

Signaling pathway analysis

The cell functions are executed and regulated by the entire sets of proteins (the proteome). The regulation of different cellular functions has been categorized into a number of pathways, such as cell cycle and apoptosis signaling pathways. In each pathway, the components according to their functions are generally named as ligands, receptors, activating regulators, inhibitory regulators and effectors. In order to measure the activation strength of a pathway, the protein molecules that belong to ligands, receptors, activating regulators, inhibitory regulators, or effectors were grouped and their relative abundances (ppm) were summed. The protein list for all analyzed pathways and processes was obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/pathway.html), and their functional annotations were manually confirmed using the UniProtKB protein database and the NCBI protein database or available publications.

Statistical data analysis

All quantitative values are presented as means ± SD. Data were statistically analyzed using two-way analysis of variance (ANOVA) for comparison among groups. Student's t-test was used to analyze the statistical significance of differences between untreated controls and HDACI-treated groups. All P-values were determined using a two-sided test, and P-values <0.05 were considered to indicate significance.

Results

HDACIs inhibit cell proliferation and induce cytotoxicity in human bladder cancer cells

To investigate the effect of HDACIs on bladder cancer cell growth and proliferation, we selected human bladder cancer 5637 cells, a cell line commonly used as a model for studying bladder carcinoma. The dose-response of romidepsin, TSA and SAHA inhibition of the growth of 5637 cell line was characterized in vitro using the MTS assay. Romidepsin, TSA or SAHA at concentrations of 0.1 nM to 100 μM caused dose-dependent inhibition of the proliferation of 5637 cells at 72 h (Fig. 1A). The half-maximal inhibitory concentration (IC50) values of romidepsin, TSA and SAHA at 72 h in this line were 1.0±0.1 nM, 100±3.5nM and 1.9±0.1 μM, respectively. These results indicate that HDACIs can potently inhibit cell proliferation and induce cell toxicity in bladder cancer cells.

Previous study has demonstrated that HDACIs increase histone acetylation levels in human bladder cancer cells and that these levels peak at 24 h and decrease gradually over 48–72 h (22). Therefore, we chose 24-h treatment with HDACIs for this in vitro study. To establish the appropriate HDACI treatment concentration for our proteomic studies, we performed cytotoxicity assays in 5637 cells in response to romidepsin, TSA or SAHA treatment at different concentrations. As shown in Fig. 1B, with dose-increased HDACI treatment for 24 h, the viability of 5637 cells correspondingly decreased, and the romidepsin, TSA and SAHA working concentrations resulting in 50% cell viability were 50±3.5 nM, 200±20 nM and 7.5±0.5 μM, respectively. Since the activity of romidepsin and TSA was much more potent than SAHA in cytotoxicity in 5637 cells (Fig. 1), we therefore, finally used the working concentrations of 50 and 200 nM for 24-h treatment for romidepsin and TSA, respectively, for the following proteomic experiments.

Quantitative proteomic analysis of bladder cancer cells following HDACI treatment

To analyze the mechanisms responsible for the effect of HDACIs on cell proliferation and cytotoxicity in bladder cancer cells, the whole cell proteome profiles of the HDACI-treated and untreated 5637 cells were compared using quantitative proteomic studies. Differentially expressed proteins were identified and quantified by nanospray LC/MS/MS mass spectrometry. The selection criteria for deregulation were the same for all the samples: identification based on at least two unique peptides and fold difference >2.0 or <−2.0.

Using the nanospray LC/MS/MS analysis, a total of 6003 non-redundant proteins were identified in both HDACI treated and untreated 5637 cells. Of these, 4865, 4618 and 4674 were quantified in romidepsin-treated, TSA-treated and untreated cells, respectively. A total of 3518 proteins were common to the two HDACI-treated cells and untreated cells.

Compared with the untreated control, there were 5698 differentially expressed proteins in romidepsin-treated 5637 cells, including 2969 upregulated proteins (1845 ≥2-fold upregulated proteins) and 2729 downregulated proteins (1626 ≥2-fold down regulated proteins). The fold changes ranged from 45.51 to -35.99 and 1979 of these proteins (both upregulated and downregulated proteins) showed >10-fold deregulation. For the TSA-treated 5637 cells, a total of 5497 proteins were differentially regulated; 2808 were upregulated (1709 ≥2-fold upregulated) and 2689 downregulated (1563 ≥2-fold down-regulated). The fold changes ranged from 36.18 to −26.83 and 1826 of these proteins (both upregulated and downregulated proteins) showed more than 10-fold deregulation. A total of 1082 ≥2-fold upregulated proteins and 1140 ≥2-fold down-regulated proteins were common to both romidepsin-treated and TSA-treated 5637 cells.

Functional classification of differentially expressed proteins in HDACI-treated bladder cancer cells

To gain an initial understanding of the role and function of the identified proteins between the HDACI treated and untreated 5637 bladder cancer cells, we merged the protein datasets and used pathway software to provide a descriptive analysis. The functional correlation analysis of the differentially regulated proteins was done by database search using UniProt, Swiss-Prot and PANTHER. The categorization of differentially expressed proteins (≥2-fold upregulated and downregulated proteins) according to their molecular function, biological process and cellular component is shown in Fig. 2. These data are based on a compilation of proteins from the romidepsin-treated cell samples and are presented to demonstrate the range of molecular functions (Fig. 2A) and biological processes (Fig. 2B) represented by the identified proteins, and the cellular component (Fig. 2C) to which the proteins belong. According to cellular component, the analysis revealed a high percentage of proteins corresponding to the cell part, organelle, macromolecular complex, extracellular region and extracellular matrix (Fig. 2C). Based on molecular function, the most general categories of proteins were catalytic activity, binding activity, structural molecule activity, nucleic acid transcription factor activity, enzyme regulator activity, transporter activity and receptor activity (Fig. 2A). Differentially expressed proteins related to 13 biological processes, including metabolic process, cellular process, localization, biological regulation, developmental process, cellular component organization or biogenesis, response to stimulus and apoptotic process (Fig. 2B).

A majority of the molecular functions and biological processes were affected in both romidepsin-treated and TSA-treated bladder cancer cells. Although romidepsin caused more differentially expressed proteins (3471 ≥2-fold upregulated and downregulated) than those caused by TSA (3272 ≥2-fold upregulated and downregulated proteins), the percentages of proteins in each category of the molecular function and biological process were similar between the romidepsin-treated (Fig. 2A and B) and TSA-treated (data not shown) 5637 cells. There were 1845 and 1709 ≥2-fold upregulated proteins and 1626 and 1563 ≥2-fold downregulated proteins in romidepsin-treated and TSA-treated cell samples, respectively. We also compared and showed that in either the upregulated proteins or the downregulated proteins, there was no significant difference for the percentages of proteins in each category of the molecular function and biological process between the romidepsin-treated cells and TSA-treated cells (data not shown), suggesting that both romidepsin and TSA exert the same or similar actions on functional categories in our cell model of bladder cancer.

Biological pathway analysis according to the Kyoto Encyclopedia of Genes and Genomes (KEGG)

Next, we used KEGG pathway analysis to identify the biological pathways of the proteins that were significantly differentially expressed (≥2-fold upregulated and downregulated) in the HDACI-treated 5637 bladder cancer cells. Pathway analysis using KEGG database by DAVID bioinformatics resources tool (DAVID v6.7, the Database for Annotation, Visualization and Integrated Discovery) showed that the downregulated proteins were associated with multiple pathways, such as cell cycle, bladder cancer, lysine degradation, valine, leucine, and isoleucine degradation, and all major annotated lipid metabolism pathways including glycerophospholipid metabolism, steroid biosynthesis, glycerolipid metabolism, sphingolipid metabolism and ether lipid metabolism (Table I). We also performed the same analysis for HDACI upregulated proteins, which were enriched in the DNA replication and nucleotide related processes, including ribosome, amino sugar and nucleotide sugar metabolism, mismatch repair, basal transcription factors, nucleotide excision repair, purine metabolism, pyrimidine metabolism and RNA polymerase (Table I).

Table I

Main metabolic and enzymatic pathways associated with the upregulated and downregulated proteins in romidepsin-treated 5637 cells as analyzed by the Kyoto Encyclopedia of Genes and Genomes.a

Table I

Main metabolic and enzymatic pathways associated with the upregulated and downregulated proteins in romidepsin-treated 5637 cells as analyzed by the Kyoto Encyclopedia of Genes and Genomes.a

Biological pathway%P-valueBenjamini
1845 ≥2-fold upregulated proteins
 Ribosome1.8 4.0×10−9 7.1×10−7
 Oxidative phosphorylation2.1 5.8×10−7 3.4×10−5
 Ubiquitin mediated proteolysis2.0 6.1×10−6 2.1×10−4
 Lysosome1.5 7.1×10−4 1.5×10−2
 Amino sugar and nucleotide sugar metabolism0.8 1.3×10−3 2.2×10−2
 Mismatch repair0.5 6.8×10−3 8.8×10−2
 Basal transcription factors0.6 7.6×10−3 9.1×10−2
 DNA replication0.6 9.3×10−3 1.0×10−2
 Nucleotide excision repair0.6 3.3×10−2 2.8×10−2
 Purine metabolism1.5 4.1×10−2 3.2×10−2
 Pyrimidine metabolism1.0 6.2×10−2 4.0×10−2
 RNA polymerase0.4 6.2×10−2 3.9×10−2
1626 ≥2-fold downregulated proteins
 N- and O-Glycan biosynthesis1.3 5.4×10−5 1.2×10−4
 Glycerophospholipid metabolism1.0 4.7×10−3 1.5×10−2
 Cell cycle1.4 4.8×10−3 1.3×10−2
 Lysine degradation0.7 1.1×10−2 2.0×10−2
 Valine, leucine and isoleucine degradation0.6 3.2×10−2 3.8×10−2
 Glycerolipid metabolism0.6 3.6×10−2 3.9×10−2
 Sphingolipid metabolism0.5 4.7×10−2 4.1×10−2
 Biosynthesis of unsaturated fatty acids0.4 3.2×10−2 3.1×10−2
 Steroid biosynthesis0.3 5.3×10−2 4.0×10−2
 Bladder cancer0.5 6.6×10−2 4.1×10−2
 Ether lipid metabolism0.5 7.6×10−2 4.3×10−2
 Methane metabolism0.2 8.8×10−2 4.7×10−2

a Similar results were observed in trichostatin A-treated 5637 bladder cancer cells.

HDACI-induced cell death in bladder cancer cells is mediated via modulating cell cycle progression, apoptosis and DNA damage repair

Given that HDACIs have been shown to exert a variety of anticancer activities in different types of tumors and that both romidepsin and TSA induced cell growth inhibition and cell death in our bladder cancer cells (Fig. 1), we elucidated the mechanism underlying the effect of HDACIs on cell proliferation and cytotoxicity in this model system. We performed pathway-clustering analyses of the HDACI-responsive proteome for pathways involved in cell death. The results showed that cell cycle, apoptosis, oxidative stress, autophagy, and DNA damage repair were the most prominent pathways enriched with altered protein levels in HDACI-treated cells (Fig. 3), suggesting that these pathways are involved in HDACI-induced cell death in 5637 bladder cancer cells.

To understand more about the mechanisms of HDACI-induced cell death in our bladder cancer cell model, we identified the differentially expressed proteins related to cell death in these pathways in response to HDACI treatment. Table II shows part of the differentially expressed proteins involved in cell death in both romidepsin and TSA treated cells. These include 37 proteins involved in cell cycle progression, 19 proteins associated with apoptosis process, 30 proteins in various DNA damage repair pathways and 11 proteins involved in ROS generation and autophagy regulation. The functions and levels of the proteins in each pathway are listed in the table.

Table II

Alterations in the levels of the proteins associated with cell death in bladder cancer cells in response to romidepsin or trichostatin A (TSA) treatment.

Table II

Alterations in the levels of the proteins associated with cell death in bladder cancer cells in response to romidepsin or trichostatin A (TSA) treatment.

Protein level (ppm)

Accession no.Protein nameSymbolProtein functionUntreatedRomidepsinTSA
Regulation of cell cycle
116176G2/mitotic-specific cyclin-B1CCNB1Cyclin7.1900
5921731G2/mitotic-specific cyclin-B2CCNB2Cyclin7.8300
218511966Cyclin-KCCNKCyclin37.5723.287.47
74753368Cyclin-L1CCNL1Cyclin11.846.410
9296942Cyclin-T1CCNT1Cyclin12.8700
6226784Cyclin-dependent kinase 10CDK10CDK25.9500
205371737Anaphase-promoting complex subunit 4APC4Mitosis factor15.428.365.36
37537861Anaphase-promoting complex subunit 5APC5Mitosis factor4.1200
37537762Cell division cycle protein 20 homologCDC20Mitosis factor12.4800
37537763Cell division cycle protein 16 homologCDC16Mitosis factor20.0916.330
254763423Cell division cycle protein 23 homologCDC23Mitosis factor15.6507.26
12644198Cell division cycle protein 27 homologCDC27Mitosis factor7.5600
12230256Mitotic spindle assembly checkpoint protein MAD2AMD2L1Mitosis factor45.58021.14
729143Cyclin-dependent kinase inhibitor 1CDN1ACDK inhibitor020.580
3041660Cyclin-dependent kinase inhibitor 2ACD2A1CDK inhibitor59.89173.1155.56
172047302Cyclin-D1-binding protein 1CCNDBP1CDK inhibitor09.3812.04
1709658 Serine/threonine-protein kinase PLK1PLK1Positive regulator30.995.600
68571766DNA replication licensing factor MCM4MCM4Positive regulator21.657.8215.06
19858646DNA replication licensing factor MCM5MCM5Positive regulator67.8959.7959.04
76803807Origin recognition complex subunit 1ORC1Positive regulator7.233.925.03
6174924Origin recognition complex subunit 5ORC5Positive regulator21.487.7619.92
25091097Double-strand-break repair protein rad21 homologRAD21Positive regulator167.8132.1027.47
13633914Mothers against decapentaplegic homolog 2SMAD2Positive regulator13.347.239.28
51338669Mothers against decapentaplegic homolog 3SMAD3Positive regulator51.297.9410.20
29336622Structural maintenance of hromosomes protein 1ASMC1APositive regulator434.44177.95207.35
29337005Structural maintenance of chromosomes protein 3SMC3Positive regulator442.71183.07188.71
209572720Cohesin subunit SA-1STAG1Positive regulator17.3310.733.44
73621291Cohesin subunit SA-2STAG2Positive regulator43.0110.970
135674Transforming growth factor β-1TGFB1Positive regulator23.9600
132164 Retinoblastoma-associated proteinRBPositive regulator3.3600
134559014-3-3 protein β/αYWHABNegative regulator468.41644.94827.91
5170221014-3-3 protein ɛYWHAENegative regulator708.351006.081053.59
134559314-3-3 protein ηYWHAHNegative regulator215.22343.05563.68
4842872114-3-3 protein γYWHAGNegative regulator428.69464.67719.29
11269014-3-3 protein θYWHAQNegative regulator483.03771.58937.41
5200088714-3-3 protein ζ/δYWHAZNegative regulator699.13771.581025.84
39895314-3-3 protein σSFNNegative regulator452.08476.40698.92
Regulation of apoptosis
6094511Tumor necrosis factor receptor type 1-associated DEATH domain proteinTRADDPro-apoptosis010.820
20141188Apoptotic protease-activating factor 1APAFPro-apoptosis05.410
18203316Diablo homolog, mitochondrialDBLOHPro-apoptosis195.46211.86253.83
17376879Serine protease HTRA2, mitochondrialHTRA2Pro-apoptosis54.4073.7075.69
728945Apoptosis regulator BAXBAXPro-apoptosis97.32351.63203.12
2493274Bcl-2 homologous antagonist/killerBAKPro-apoptosis29.5263.9941.07
2493285BH3-interacting domain death agonistBIDPro-apoptosis31.94207.7388.89
23396740Bcl-2-like protein 13B2L13Pro-apoptosis12.8420.8826.80
2810997DNA fragmentation factor subunit αDFFAPro-apoptosis9.4140.7939.27
575773389Serine-protein kinase ATMATMPro-apoptosis1.021.102.84
77416852Caspase-3CASP3Caspase11.2424.3746.93
115612Calpain small subunit 1CPNS1 Calpain-calcium81.34151.15129.35
62906858Interleukin-1 βIL1BPro-survival46.31016.11
125987833Interleukin-1 receptor-associated kinase-like 2IRAK2Pro-survival14.9500
18202671Myeloid differentiation primary response protein MyD88MYD88Pro-survival10.5200
21542418Nuclear factor NF-kappa-B p105 subunitNFKB1Pro-survival9.6500
125193cAMP-dependent protein kinase type I-α regulatory subunitPRKAR1APro-survival147.138.8634.12
229463042cAMP-dependent protein kinase type I-β regulatory subunitPRKAR1BPro-survival32.7000
125198cAMP-dependent protein kinase type II-α regulatory subunitPRKAR2APro-survival92.5075.2085.81
Regulation of DNA damage repair
73921676DNA-(apurinic or apyrimidinic site) lyase 2APEX2Base excision repair12.0200
251757259DNA ligase 3LIG3Base excision repair37.0423.4217.18
317373290DNA repair protein XRCC1XRCC1Base excision repair49.2016.0041.07
130781Poly [ADP-ribose] polymerase 1PARP1Base excision repair380.84236.36311.96
17380230Poly [ADP-ribose] polymerase 2PARP2Base excision repair26.715.790
296453081DNA repair protein complementing XP-C cellsXPCNucleotide excision repair3.3100
12643730DNA damage-binding protein 1DDB1Nucleotide excision repair188.50109.56148.24
12230033DNA damage-binding protein 2DDB2Nucleotide excision repair29.17010.15
119541TFIIH basal transcription factor complex helicase XPB subunitERCC3Nucleotide excision repair31.8617.2711.08
17380326General transcription factor IIH subunit 2GTF2H2Nucleotide excision repair31.548.550
50403772General transcription factor IIH subunit 3GTF2H3Nucleotide excision repair30.3321.9214.07
17380328General transcription factor IIH subunit 4GTF2H4Nucleotide excision repair74.157.3156.28
1706232Cyclin-HCCNHNucleotide excision repair28.9310.4526.83
25091548Pre-mRNA-splicing factor SYF1XAB2Nucleotide excision repair87.4251.3335.48
108936013Cullin-4ACUL4ANucleotide excision repair36.9322.2422.84
60392986DNA repair protein RAD50RAD50Homologous recombination71.2151.4636.33
17380137Double-strand break repair protein MRE11AMRE11AHomologous recombination39.594.7724.48
74762960NibrinNBNHomologous recombination41.3013.45.75
116242745DNA endonuclease RBBP8RBBP8Homologous recombination3.4700
166898077Crossover junction endonuclease MUS81MUS81Homologous recombination5.6500
2501242DNA topoisomerase 3-αTOP3AHomologous recombination3.1100
38258929DNA-dependent protein kinase catalytic subunitPRKDCNon-homologous end-joining521.31295.21363.21
125731X-ray repair cross-complementing protein 5XRCC5Non-homologous end-joining582.87387.37443.99
125729X-ray repair cross-complementing protein 6XRCC6Non-homologous end-joining772.18454.52619.04
74760390WD repeat-containing protein 48WDR48Fanconi anemia4.6000
48428038AprataxinAPTXEditing and processing nuclease43.7400
146325723E3 ubiquitin-protein ligase SHPRHSHPRHUbiquitination and modification1.8500
68565701Telomere-associated protein RIF1RIF1Other related51.6515.028.76
1705919Dual specificity protein kinase CLK2CLK2Other related12.4800
55976619Pre-mRNA-processing factor 19PRPF19Other related463.44241.12335.32
ROS generation
14916998Glutathione reductaseGSHRReductase17.906.478.30
182705230Thioredoxin reductase 2TRXR2Reductase17.8300
2506326Xanthine dehydrogenase/oxidaseXDHOxidase02.672.53
Regulation of autophagy
254763436Protein kinase, AMP-activated, α 1 catalytic subunitPRKAA1Autophagy22.2836.2346.51
20178289Interferon, α 21IFNA21Autophagy017.860
74762700 Phosphoinositide-3-kinase, regulatory subunit 4PIK3R4Autophagy04.973.19
74730233 Phosphatidylinositol 3-kinase, catalytic subunit type 3PIK3C3Autophagy03.814.89
62286592Autophagy related 7ATGAutophagy22.1562.4212.33
62510482Autophagy related 16-like 1 (S. cerevisiae)ATG16L1Autophagy5.1311.120
44888808GABA(A) receptor-associated protein-like 2GABARAPLAutophagy028.85111.11
61212142Autophagy related 3ATG3Autophagy010.7555.20

[i] ROS, reactive oxygen species.

For example, our data showed that multiple autophagy-associated proteins, such as ATG3, PRKAA1, GABARAPL and ATG7, were highly upregulated (Table II), suggesting that these proteins might have important roles in HDACI-induced autophagy in bladder carcinoma.

HDACIs enhance global histone and non-histone protein acetylation levels and induce deregulation of chromatin modification proteins

Since both romidepsin and TSA are HDACIs, we assessed the effect of the two HDACIs on lysine acetylation in 5637 cells. We first verified whether inhibition of histone deacetylation by the HDACIs altered global acetylation in our model system. We searched the whole cell proteome and identified the non-redundant peptides containing the acetylated lysine residues. As shown in Table III, both romidepsin and TSA significantly increased global histone and non-histone lysine acetylation levels compared to the untreated control. Romidepsin induced ~2.5-fold and 2-fold increases in histone and non-histone protein acetylation levels, respectively (P<0.01), while TSA increased global lysine acetylation levels 63 and 50% in histone and non-histone proteins, respectively (P<0.05), indicating that romidepsin exerts a more potent effect than TSA on the lysine acetylated profile of both non-histone substrates and core histones in 5637 cells.

Table III

Histone deacetylase inhibitors induce enhanced global lysine acetylation in histones and non-histone proteins in 5637 bladder cancer cells as determined by proteomic analysis.

Table III

Histone deacetylase inhibitors induce enhanced global lysine acetylation in histones and non-histone proteins in 5637 bladder cancer cells as determined by proteomic analysis.

TreatmentHistone proteinNon-histone protein
Untreated172426
Romidepsin422841
Trichostatin A280638

Next, the overall increased lysine acetylation levels in histone proteins prompted us to further investigate the impact of HDACIs on site-specific histone lysine acetylation. To this end, we applied the quantitative proteomics to profile histone lysine acetylation in 5637 cells after romidepsin or TSA treatment, followed by protein sequence database search for peptide identification and post-translational modification site mapping. The diagram of Fig. 4 shows that a total of 23 lysine acetylation (Kac) sites in core histones were identified, in most of them Kac sites were previously reported in core histones in mammalian cells. Importantly, we identified two new histone marks, including H2AK118ac and H2BK34ac in both romidepsin and TSA treated 5637 cells (Fig. 4A and B). The representative spectra of histone lysine acetylated peptides are shown in Fig. 4C and D, including the spectra for peptides of H2AK118ac and H2BK34ac. In addition, the sequences of the identified lysine acetylated peptides in core histones in romidepsin and TSA treated cells are listed in Table IV.

Table IV

The identified lysine acetylation (Kac) sites in core histones and the lysine-acetylated peptide sequences in histone deacetylase inhibitor-treated bladder cancer 5637 cells.

Table IV

The identified lysine acetylation (Kac) sites in core histones and the lysine-acetylated peptide sequences in histone deacetylase inhibitor-treated bladder cancer 5637 cells.

Modified histone siteModified peptide sequence
Romidepsin treatment
H2AK118ac _VTIAQGGVLPNIQAVLLPK(ac)K(ac)TESHHK_
H2AK119ac _VTIAQGGVLPNIQAVLLPK(ac)K(ac)_
H2BK16ac _K(ac)AVTK(ac)AQK_
H2BK20ac _K(ac)AVTK(ac)AQK_
H2BK34ac _K(ac)ESYSIYVYK_
H2BK46ac _VLK(ac)QVHPDTGISSK_
H3K9ac _K(ac)STGGK(ac)APR_
H3K14ac _K(ac)STGGK(ac)APR_
H3K18ac _K(ac)QLATK(ac)AAR_
H3K23ac _K(ac)QLATK(ac)AAR_
H3K27ac _K(ac)SAPATGGVKKPHR_
H3K56ac _YQK(ac)STELLIR_
H3K79ac _EIAQDFK(ac)TDLR_
H3K122ac _VTIMPK(ac)DIQLAR_
H4K5ac _GK(ac)GGK(ac)GLGK_
H4K8ac _GK(ac)GGK(ac)GLGK_
H4K12ac _GLGK(ac)GGAK(ac)R_
H4K16ac _GLGK(ac)GGAK(ac)R_
H4K20ac _K(ac)VLRDNIQGITKPAIR_
H4K31ac _DNIQGITK(ac)PAIR_
H4K79ac _K(ac)TVTAMDVVYALKR_
Trichostatin A treatment
H2AK118ac _VTIAQGGVLPNIQAVLLPK(ac)K(ac)TESHHK_
H2AK119ac _VTIAQGGVLPNIQAVLLPK(ac)K(ac)_
H2BK11ac _SAPAPK(ac)K(ac)GSK_
H2BK12ac _SAPAPK(ac)K(ac)GSK_
H2BK16ac _K(ac)AVTK(ac)AQK_
H2BK20ac _K(ac)AVTK(ac)AQK_
H2BK34ac _K(ac)ESYSIYVYK_
H2BK46ac _VLK(ac)QVHPDTGISSK_
H3K9ac _K(ac)STGGK(ac)APR_
H3K14ac _K(ac)STGGK(ac)APR_
H3K18ac _K(ac)QLATK(ac)AAR_
H3K23ac _K(ac)QLATK(ac)AAR_
H3K27ac _K(ac)SAPATGGVKKPHR_
H3K79ac _EIAQDFK(ac)TDLR_
H3K122ac _VTIMPK(ac)DIQLAR_
H4K12ac _GLGK(ac)GGAK(ac)R_
H4K16ac _GLGK(ac)GGAK(ac)R_
H4K20ac _K(ac)VLRDNIQGITKPAIR_
H4K79ac _K(ac)TVTAMDVVYALKR_

Finally, we quantified dynamic change in global protein abundance of the chromatin modifying proteins in HDACI-induced bladder cancer cells. Unexpectedly, we found that the protein levels of HDAC1, HDAC2 and HDAC3 in the deacetylation complexes of Mi-2/NuRD, CoREST, NcoR, SMRT and Sin3 were all downregulated in both romidepsin and TSA treated cells. As seen in Table V, treatment with romidepsin and TSA induced 2- and 1.7-fold downregulation for HDAC1, 3.2- and 2-fold downregulation for HDAC2, and 5.5- and 2.2-fold downregulation for HDAC3, respectively. The levels of Sin3 histone deacetylase corepressor complex component SDS3, a regulatory protein that augments histone deacetylase activity of HDAC1, were also reduced in response to exposure to romidepsin or TSA (Table V). Additionally, romidepsin and TSA decreased the levels of histone deacetylase complex subunit SAP130 by 2.8- and 2.2-fold, respectively, in the 5637 cells. In contrast, the levels of the lysine acetyltransferase KAT6A and histone acetyltransferase type B catalytic subunit, the latter acetylates histone H4 at H4K5ac and H4K12ac, were both elevated following the HDACI induction (Table V). These data suggest that romidepsin and TSA induced global acetylation in core histones and non-histone proteins are mediated partly through the elevated levels of HATs and reduced levels of HDACs in 5637 bladder cancer cells.

Table V

The differentially expressed chromatin modifying proteins in response to histone deacetylase inhibitor treatment in bladder cancer 5637 cells.

Table V

The differentially expressed chromatin modifying proteins in response to histone deacetylase inhibitor treatment in bladder cancer 5637 cells.

Accession no.Protein nameSymbolComplexProtein functionProtein level (ppm)

UntreatedRomidepsinTSA
2498443Histone deacetylase 1HDAC1Mi-2/NuRD; CoREST; Sin 3Lysine deacetylase374.75189.09224.75
68068066Histone deacetylase 2HDAC2Mi-2/NuRD; CoREST; Sin 3Lysine deacetylase421.20131.42213.11
3334210Histone deacetylase 3HDAC3Mi-2/NuRD; NcoR/SMRTLysine deacetylase87.3215.7740.49
74717977Histone deacetylase complex subunit SAP130SP130Sin 3Repressor17.836.448.26
68053233Sin3 histone deacetylase corepressor complex component SDS3SDS3Sin 3Corepressor37.98026.42
3334209Histone acetyltransferase type B catalytic subunitHAT1KATsLysine acetyltransferase52.0248.34103.42
215274095Histone acetyltransferase KAT6AKAT6AKATsLysine acetyltransferase1.553.372.16

[i] TSA, trichostatin A.

Discussion

Although HDACIs such as romidepsin and vorinostat (SAHA) have been approved for the treatment of CTCL, there is no currently approved HDACI for any solid tumor indication; therefore, we explored the potential for the development of HDACI as a novel therapeutic for bladder urothelial carcinoma. In the present study, we have demonstrated that romidepsin, SAHA and TSA suppressed cell growth and caused cell death in 5637 bladder cancer cells in vitro. Furthermore, our quantitative proteomic studies showed that 2472 proteins were 2-fold upregulated and 2049 proteins were 2-fold downregulated in this model in response to romidepsin and TSA exposure, among them 1082 ≥2-fold upregulated proteins and 1140 ≥2-fold downregulated proteins were common to both romidepsin and TSA treatment, as compared to the untreated controls (P<0.05). The subsequent bioinformatic analysis revealed that those differentially expressed proteins were mainly involved in biological and metabolic functions and cell death associated pathways. HDACI exposure also enhanced global acetylation levels in both histone and non-histone proteins. Twenty-three lysine acetylation marks were detected on core histones in HDACI-treated bladder cancer cells including two newly identified histone Kac sites (H2AK118ac and H2BK34ac). These data suggest that HDACI-induced alterations in protein expression is mediated, at least in part, through histone modification, leading to changes in biological and metabolic functions and cell death in bladder cancer cells. By establishing the link between histone modification and whole proteome in response to HDACI treatment, this study may deepen our understanding of HDACI-mediated therapeutics in bladder cancer.

A major goal of the chemotherapy of human malignancies is the inhibition of cell proliferation, and drug-induced cancer cell growth arrest is mediated partly by blocking cell cycle progression (23). The eukaryotic cell cycle is regulated via the sequential activation and inactivation of cyclin-dependent kinases (CDKs) that drive cell cycle progression through the phosphorylation and dephosphorylation of regulatory proteins (2426). The activities of CDKs are positively regulated by cyclins and negatively regulated by CDK inhibitors (CKIs). Thus, the cell cycle is regulated by cyclins, CDKs and CKIs. Changes in the expression of specific CDKs or their regulatory proteins such as cyclins and CKIs can lead to uncontrolled cell proliferation and eventually to carcinogenesis (25,27). Whereas, downregulating the levels of cyclins or upregulating CKIs lead to blockade of cell cycle progression.

In this study, we showed that romidepsin and TSA downregulated the protein expression of cyclins B1/B2 and upregulated the expression of anaphase promoting complex-1 (APC1) and 14-3-3 proteins in 5637 cells. Cyclin B binds to and activates CDK1. The complex of cyclin B and CDK1 is responsible for the control of G2/M checkpoint, while APC1 acts by mediating ubiquitination and degradation of cyclin B and subsequent inactivation of CDK1. On the other hand, CDK1 activity is suppressed via phosphorylation of Thr-14 and Tyr-15 by the Wee-1 protein kinase (28) and is activated by CDC25 protein phosphatases, which function to remove the inhibitory phosphates from CDK1 (29). 14-3-3 proteins are involved in the regulation of G2/M checkpoint by 14-3-3-mediated CDC25 inactivation and Wee-1 activation. Romidepsin and TSA caused reduced levels of cyclin B and elevated levels of APC1 and 14-3-3 proteins in 5637 cells, suggesting that romidepsin and TSA suppress bladder cancer cell proliferation through cell cycle blockade at the G2/M phase, and that this occurs via the HDACI downregulation of cyclin B and upregulation of APC1 and 14-3-3 proteins, leading to cell cycle arrest and cell growth suppression in bladder cancer cells.

The other goal of cancer chemotherapy is to commit tumor cells to death or apoptosis following exposure to anticancer agents. Apoptosis is a highly regulated cellular process between cell proliferation and cell death and drug-induced cell death is mediated, at least in part, by apoptotic cell death (30).

In the present study, we found that the levels of caspase-3 were significantly increased in 5637 cells following treatment with romidepsin or TSA. In addition, both romidepsin and TSA enhanced Bax and Bak expression and triggered phosphorylation of Bcl-2 at Ser-70. It is known that the expression of pro-apoptotic proteins is mediated through p53-dependent and -independent pathways. In this study, we showed that the levels of p53 protein as well as other p53-pathway proteins, such as DNA-dependent protein kinase (PRKDC), were not elevated in response to HDACI exposure (Fig. 3A), and we confirmed by DNA sequencing that p53 gene is mutated in this cell line (data not shown), suggesting that the increased expression of apoptosis-associated proteins is not under direct control by p53 in 5637 bladder carcinoma cells. Additionally, it has been shown that phosphorylation of Bcl-2 is induced by several drugs in a panel of cancer cell lines derived from leukemia, lymphoma, and breast and prostate cancer (3135). Phosphorylation of Bcl-2 is cell cycle-dependent, occurs at G2/M (34) and results in concomitant apoptosis (34,36). Interestingly, treatment with HDACIs was found to induce Bcl-2 Ser-70 phosphorylation at the G2/M phase of the cell cycle, with concomitant apoptosis in bladder cancer cells. This is consistent with the literature reporting that Bcl-2 phosphorylation at Ser-70 and loss of anti-apoptotic function in response to antitumor drugs and subsequent elimination of tumor cells via apoptosis (35). These results suggest that a similar mechanism (Bcl-2 phosphorylation at G2/M) may be involved with induction of apoptosis by HDACIs in this model system. Taken together, these data strongly support the involvement of Bcl-2 family proteins in HDACI-induced apoptosis, possibly acting through a p53-independent, mitochondria-dependent intrinsic apoptotic pathway in human bladder cancer 5637 cells.

A wide range of DNA damage can be inflicted, both from extracellular agents including some antitumor drugs and via endogenous mechanisms (37). Genotoxic cancer therapeutics such as cisplatin and mitomycin C bind to DNA, forming adducts that in turn can be repaired by the DNA repair machinery or lead to permanent DNA damage. Anticancer agent-induced DNA damage leads to transient arrest in the G1, S, G2 and M phases of the cell cycle, allowing cells to have sufficient time to repair damaged DNA before resuming cell cycle progression. However, severe DNA injury that is too extensive for intracellular repair mechanisms will lead to activation of intrinsic apoptosis pathway and cell death. Although DNA damage also affects normal cells, tumor cells are often more vulnerable because of defects in DNA repair pathways or critical cell cycle checkpoints.

Five main mechanisms are involved in DNA repair: i) base excision repair, which corrects non-bulky damage; ii) nucleotide excision repair, which corrects lesions that disrupt the double helical structure of DNA; iii) mismatch repair, which corrects replication errors; iv) double-strand break repair, which corrects double-strand breaks through two different pathways, homologous recombination and non-homologous end-joining; and v) direct repair, which corrects methylated or alkylated bases (38). Although the DNA lesions induced directly by HDACIs or indirectly via endogenous mechanisms such as the generation of free radicals, as well as the relevant DNA repair mechanisms responsible for the removal of those lesions in 5637 cells are still not known, our proteomic analysis revealed that the levels of multiple DNA repair proteins in multiple repair mechanisms were decreased in HDACI-treated bladder cancer cells. For example, the protein expression of XRCC1 and PARP1/2 in base excision repair, XPC and ERCC3 in nucleotide excision repair, RAD50 and MRE11A in homologous recombination, and XRCC5 and XRCC6 in non-homologous end-joining were all reduced after romidepsin and TSA treatment. The downregulation of DNA repair protein expression by HDACIs significantly impairs cellular DNA repair activity and DNA damage response, which in turn results in inhibition of transcription, replication, and chromosome segregation leading to blockade of cell cycle progression or apoptosis in bladder carcinoma cells.

Studies suggest oxidative stress as a mechanism to the primary modes of action of antitumor agents. Oxidative stress is a redox (reduction-oxidation) disequilibrium state, in which the generation of ROS overwhelms the antioxidant defense mechanisms (39). ROS such as superoxide and hydroxyl radicals are highly toxic, as a result of their actions as oxidizing agents and can have damaging effects on cell physiology. Under conditions that can cause oxidative stress, cells are exposed to excessive ROS that can oxidize membrane fatty acids, initiating lipid peroxidation, oxidize proteins (40) and cause DNA damage (41). At high level, excessive ROS may cause severe damage to cells, including necrosis and apoptosis (42).

Redox state in the cell is regulated by redox proteins. In this study, we found that romidepsin and TSA downregulated the expression of glutathione reductase (GSHR) and thioredoxin reductase 2 (TRXR2) and upregulated the expression of xanthine dehydrogenase/oxidase (XDH), which lead to ROS formation in 5637 cells. These findings suggest that oxidative stress is involved in the antitumor effects of HDACIs in bladder cancer and that exposure to HDACIs may alter the antioxidant defense system and redox mechanisms in cells. This notion is supported by the reports from other researchers demonstrating that HDACIs induce cell death through ROS production (43).

Although the mechanism for the link between HDACI-induced oxidative stress and cell death is not well understood, several lines of evidence suggest that HDACIs induce cell death via ROS generation by the following mechanisms: i) excess of ROS may facilitate the detachment of cytochrome c from the mitochondrial membrane and increases the mobilized pool of cytochrome c, which is a prerequisite for its release into the cytoplasm through the pores created by pro-apoptotic Bcl-2 family members such as Bax and Bak; ii) ROS may also directly damage mitochondrial membrane and induce membrane potential loss that favors cytochrome c release; iii) death receptor aggregation may also result from ROS production and induce cell death through a different pathway; iv) downregulation of anti-apoptotic molecules and/or upregulation of pro-apoptotic signals are involved in ROS-induced cell death (44); and v) HDACI-induced ROS causes oxidative DNA damage (43), as evidenced by the levels of phosphorylated histone H2AX (γ-H2AX) and ataxia telangiectasia mutated (ATM), early markers of DNA damage, significantly increase after the administration of HDACIs (45,46). Cellular oxidative DNA damage induced by endogenous ROS production via HDACI treatment can lead to bladder cancer cell death (47).

Combining our results discussed above, we propose a possible mechanism by which HDACIs cause bladder cancer cell growth arrest and cell death as shown in Fig. 5. In this model, HDACIs alter changes in the levels and activities of proteins involved in the signaling pathways of cell cycle progression, apoptotic cell death, DNA damage repair, ROS generation, endoplasmic reticulum (ER) stress (4850) and autophagy regulation (51), which are associated with cell death. In the proposed pathways depicted here (Fig. 5), romidepsin and TSA induce cell cycle arrest and apoptotic cancer cell death; cell cycle blockade not only causes cancer cell growth arrest, but prolonged cell cycle arrest also triggers cell suicide, usually in the form of apoptosis. In addition, the HDACIs increase DNA damage directly or indirectly through ROS production, which in turn promotes apoptosis. On the other hand, romidepsin and TSA mediate cancer cell death via inducing ER stress and autophagy. Because HDACIs target cell survival and cell death through multiple closely related but distinct mechanisms, they may act collaboratively or synergistically to promote apoptotic death of bladder cancer cells through these signaling pathways and their downstream molecular events.

Finally, our data indicate that dysregulation of protein expression in HDACI-treated 5637 cells was associated with enhanced lysine acetylation in histone and non-histone proteins as well as alterations in the levels of chromatin modifying proteins, suggesting a role for epigenetic modification.

The three main epigenetic mechanisms (DNA methyltion, histone modifications and RNA-mediated gene silencing) have been studied primarily in the context of gene expression (52,53). The second epigenetic mechanism encompasses various histone modifications, including acetylation, glycosylation, methylation, phosphorylation and ubiquitination of specific residues in the N-terminal tails of histones (54). Histone modifications are post-translational alterations of histone proteins that interact with DNA to form a complex known as chromatin (54). Besides regulating several cellular processes including gene transcription, proliferation, and autophagy, histone modifications also affect many other chromatin-based processes such as DNA repair, replication and recombination (54).

The best-studied histone modification is lysine acetylation. The acetylation of histone modulates transcription by altering the accessibility of DNA to proteins, such as transcriptional regulators (transcriptional activators and repressors), and binding of regulatory proteins (transcription factors or repressors) to the promoter sequence of a gene resulting in activation or blocking of transcription. Furthermore, the activity of non-histone proteins, such as transcription factors and repressors, can also be modulated by post-translational protein modifications (e.g., acetylation, phosphorylation or glycosylation), and these modifications could change protein conformation and lead to changes in activity.

As the variety of gene expression profiles is determined by distinct sets of transcriptional regulators (transcription factors or repressors) that control and determine which genes are switched on or off, HDACIs may upregulate or downregulate gene expression via altering the activity of transcription factors or repressors by post-translational modifications (PTMs) in our bladder tumor cells. Additionally, since the majority of cellular functions are carried out by proteins, HDACIs may modulate biological changes not only through alterations at the protein level but also by PTMs in bladder carcinoma cells. However, the role of the two newly identified histone markers (H2AK118ac and H2BK34ac) in the antitumor activity of HDACIs in bladder cancer, as well as the precise mechanism for how HDACIs upregulate or downregulate specific gene and protein expression through histone modifications and PTMs will require further investigation.

In summary, we have profiled the antitumor activity of HDACIs in association with enhanced lysine acetylation in histone and non-histone proteins as well as altered levels of chromatin modifying proteins in bladder cancer cells. Proteomic data analysis further revealed dysregulation of protein expression involved in multiple biological functions and cell death associated pathways in romidepsin and TSA treated 5637 cells. These results suggest that the antitumor effect of HDACIs in bladder carcinoma is mediated through modulation of these pathways by histone modifications and PTMs, leading to cancer cell growth arrest and cell death. Our findings may be helpful for developing HDCAIs in combination with other therapeutics targeted at modulating relevant cell death pathways or at inhibiting cell proliferation in tumors. Further studies are needed to investigate the anticancer activity of HDACIs in bladder cancer via the modulation of signaling pathways (e.g., PI3K-PTEN-mTOR pathway) (55) or the inhibition of regulatory enzymes in histone modifications and PTMs (56) influencing cell survival and death.

Acknowledgements

The present study was supported by the Intramural Research Program of the U.S. National Cancer Institute, the National Institutes of Health.

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June-2016
Volume 48 Issue 6

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Online ISSN:1791-2423

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Copy and paste a formatted citation
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Spandidos Publications style
Li QQ, Hao J, Zhang Z, Hsu I, Liu Y, Tao Z, Lewi K, Metwalli AR and Agarwal PK: Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach. Int J Oncol 48: 2591-2607, 2016
APA
Li, Q.Q., Hao, J., Zhang, Z., Hsu, I., Liu, Y., Tao, Z. ... Agarwal, P.K. (2016). Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach. International Journal of Oncology, 48, 2591-2607. https://doi.org/10.3892/ijo.2016.3478
MLA
Li, Q. Q., Hao, J., Zhang, Z., Hsu, I., Liu, Y., Tao, Z., Lewi, K., Metwalli, A. R., Agarwal, P. K."Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach". International Journal of Oncology 48.6 (2016): 2591-2607.
Chicago
Li, Q. Q., Hao, J., Zhang, Z., Hsu, I., Liu, Y., Tao, Z., Lewi, K., Metwalli, A. R., Agarwal, P. K."Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach". International Journal of Oncology 48, no. 6 (2016): 2591-2607. https://doi.org/10.3892/ijo.2016.3478