Inhibition of bladder tumour growth by histone deacetylase inhibitor
Akira Ozawa, Nozomu Tanji, Tadahiko Kikugawa, Toyokazu Sasaki, Yutaka Yanagihara, Noriyoshi Miura and Masayoshi Yokoyama
Department of Urology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Accepted for publication 28 May 2009
KEYWORDS : urinary bladder tumour, histone deacetylase 1, valproic acid, epigenetics
OBJECTIVE
To examine the expression profile of histone deacetylase (HDAC)-1 and explore its potential role in the development of bladder cancer, using valproic acid (VPA), a HDAC inhibitor, which reduces tumour growth and metastasis formation in animal models.
MATERIALS AND METHODS
The study comprised clinical samples from patients with urinary bladder cancer, mouse urinary bladder tissue specimens, and two human urinary bladder cancer cell lines (HT- 1376 and 5637). HDAC1 mRNA and protein expression were examined using real-time reverse transcription-polymerase chain.
INTRODUCTION
The genetic code for proteins resides in the base sequence of DNA and the expression of genes is largely regulated by the structure of chromatin (epigenetic gene regulation) [1]. It has recently become clearer that abnormal epigenetics are profoundly involved in many human diseases. Repetitive units of the nucleosome led to the chromatin in which all the human genome is packed. A single nucleosomal core particle is composed of a fragment of DNA (146 bp) wrapped around a histone octamer formed by four histone partners, an H3-H4 tetramer and two H2A- H2B dimmers [2]. Histones are small basic proteins rich in the amino-acids lysine and arginine. Reversible histone acetylation, which occurs at conserved lysine residues clustered near the amino-terminus of core histones, is one of the important mechanisms by which DNA accessibility is controlled.
Acetylation of the core histones has been shown to weaken the histone–DNA reaction and immunohistochemical methods. Female C3H/He mice were given VPA (0, 250, 500 and 750 mg/kg body weight, intraperitoneal, every day) from the start or 4 weeks after 0.05% N-butyl-N-(4- hydroxybutyl)-nitrosamine (BBN) treatment, and were humanely killed and sampled at 8 and 12 weeks.
RESULTS
A significantly higher level of HDAC1 mRNA was expressed in human urinary bladder cancer specimens. The immunohistochemical study showed that HDAC1 was expressed in the cytoplasm and nucleus in the specimens. BBN treatment interactions, thus consequently increasing DNA accessibility [3].
The level of acetylation is controlled by equilibrium between acetylation and deacetylation. Whereas acetylation correlates with nucleosome remodelling and transcriptional activation, the deacetylation of histone tails induces transcriptional repression through chromatin condensation. Acetylation and deacetylation are catalysed by specific enzyme families, histone acetyltransferases and deacetylases (HDACs), respectively. As aberrant histone acetylation has been linked to malignant diseases in some cases, HDAC inhibitors have great potential as new anticancer drugs, due to their ability to modulate transcription and to induce differentiation and apoptosis [4]. Various natural and synthetic HDAC inhibitors have been reported. Several HDAC inhibitors have been successfully introduced into phase I trials as antitumour and differentiating reagents for patients with advanced or increased HDAC1 mRNA expression in the urinary bladder. VPA administration seemed to delay the incidences of BBN-induced mouse urinary bladder tumour, possibly through p21WAF1 protein expression.
CONCLUSION
These results indicate that HDAC might be an effective molecular target for cancer therapy.Bladder cancer is the ninth most commonly diagnosed cancer in men and the 17th commonly diagnosed cancer in woman, and accounted for 5800 deaths in Japan in 2000 [6]. Most newly diagnosed bladder cancer is non-muscle-invasive. Invasive, locally advanced and metastatic bladder cancer remain therapeutic challenges, requiring cystectomy, systemic chemotherapy, radiotherapy or a combination of these methods. A review of the current treatments highlighted deficiencies where alternative therapies might hold promise [7].
The anti-epileptic agent valproic acid (VPA) has been reported to reduce tumour growth and metastasis formation in animal experiments, and to be a class I selective HDAC inhibitor [8]. The class I HDACs include HDAC1, 2, 3 and 8. Class I HDACs play a role in cell death and proliferation pathways,whereas class II HDACs appear to be important for tissue-specific functions [9]. Therefore, the expression profiles of HDAC1 were examined to explore its potential role in the development of bladder cancer, using VPA.
MATERIALS AND METHODS
This study was approved by the Ehime University Hospital Ethical Committee. All patients had signed an informed consent. Primary tumour tissues and corresponding normal tissues were collected at surgery in the Ehime University Hospital between March and July 2005, from 10 patients with urinary bladder cancer who had been diagnosed histologically. Fresh tissue specimens were collected, snap-frozen in liquid nitrogen and stored at 80 C until use. For histological examination, specimens were fixed in 10% buffered formalin, embedded in paraffin and cut into 5-m sections. Tumours were graded and staged according to the TNM classification (1997).
For quantitative real-time reverse transcription (RT)-PCR, total RNA was extracted from snap-frozen tissue samples using TRI reagent (Sigma-Aldrich, St Louis, MO, USA) according to the instructions of the reported as the fold difference relative to the -actin gene expression.
For immunohistochemistry, after deparaffinization and rehydration, the tissue sections were treated using 2% H2O2 in 50% methanol for 15 min to eliminate endogenous peroxidase. The sections were subjected to antigen retrieval by 10 mM citrate buffer (pH 6.0). The sections were incubated with rabbit polyclonal antihuman HDAC1 antibodies (1/50 dilution; Cell Signalling Technology, Danvers, MA, USA) overnight at 4 C. After washing with PBS the sections were incubated for 1 h with biotinylated goat antirabbit IgG (Vector, Peterborough, UK) and incubated for 30 min with avidin-biotin complex (Vector) at room temperature.
FIG. 1. The experimental design for evaluating HDAC1 expression and the efficacy of VPA in BBN- induced mouse urinary bladder tumour; 0.05% BBN was given in the drinking water and VPA was given i.p. to the mice every day.
RNA was reverse transcribed at 37 C for 90 min in a 30-L reaction volume and then heated to 100 C for 5 min. The resultant cDNA was used for PCR. Appropriate dilutions of each single-strand cDNA were prepared, followed by the normalization of the cDNA content using -actin as a quantitative control, with PCR using single-strand cDNA as the PCR templates. Quantitative PCR amplification was done with a 20-L final reaction mixture consisting of 0.5 L of reverse transcription reaction mixture, 0.3 M of the each sense and antisense primers (Table 1) and 2 L of LightCycler Fast Start DNA Master SYBR Green I (Roche Diagnostics GmbH, Germany). The conditions were: initial denaturation at 95 C for 10 min; 40 cycles of denaturation at 95 C for 10 s; annealing at 58 C for 10 s for human and 55 C for 10 s for mouse and elongation at 72 C for 10 s. Real-time quantitative PCRs were conducted using a LightCycler Quick System 350.
The quantitative data were analysed by LightCycler Software Version 3.5. The HDAC1 expression in the test samples was normalized to the corresponding -actin level and is 3, 3-diaminobenzidine (Sigma-Aldrich). All sections were lightly counterstained using haematoxylin.
The negative controls were serial sections that were stained using equivalent concentrations of non-immune rabbit IgG in the place of the primary antibodies.In all, the study included 105 7-week-old female C3H/He mice (Clea Japan, Osaka, Japan), quarantined for a week before beginning the experiment, then housed in the cages and maintained at 25 C under controlled lighting conditions (12 h light/12 h darkness). The mice allowed free access to water and pellet food.
The experimental protocol is shown in Fig. 1; 105 mice at 8 weeks were divided into four groups (1–4), and 20 in Group 1 were given 0.05% N-butyl-N-(4-hydroxybutyl)- nitrosamine (BBN; Sigma-Aldrich) in their drinking water for 4, 8, 12 and 16 weeks (five mice each). Twenty-five mice in Group 2 (control) received tap water ad libitum for 0, 4, 8, 12 and 16 weeks (five mice each). VPA (250, Period of treatment, weeks 500 and 750 mg/kg body weight (BW) was administered intraperitoneally every day to mice from 4 weeks after the start of BBN administration, to examine the antitumoral effect of VPA (Group 3) and killed 4 and 8 weeks later (five mice each). In addition, to examine the preventive effect, VPA (250, 500 and 750 mg/kg BW) was administered every day to mice from the start of BBN administration and 8 and 12 weeks later (five mice each).
The urinary bladders were removed when the mice were killed. They were fixed in 10% buffered formalin and embedded in paraffin for histological examination. Transverse sections (5 m thick) were prepared and stained with haematoxylin and eosin to assess the proliferation of mucosal epithelial cells.Human urinary bladder cancer cell lines, HT-1376 and 5637, were obtained from Dainippon Sumitomo Pharma (Osaka, Japan).
FIG. 2. (A) Comparison of histone deacetylase 1 (HDAC1) mRNA using quantitative real-time RT-PCR between in primary tumour tissues and corresponding normal tissues from 10 patients with urinary bladder cancer. The mRNA expression normalized to the corresponding actin level of tumour tissues was significantly higher than that of neighboring normal tissues. The height of each bar indicates the mean / SE of 4 rats. *, P 0.05, significant difference from the value of neighboring normal tissues, according to Student’s t-test. (B) Immunohistochemical results show that the immunoreactivity against HDAC1 was weak in the cytoplasm of normal epithelium. (C) In cancerous epithelium, HDAC1 was weakly expressed in the cytoplasm and intensely expressed in the nuclei. The immunoreactivity was absent in the neighbouring mesenchyme of both normal and cancerous tissues. Original 200.
FIG. 3. Macroscopic and microscopic effect of BBN treatment on the mouse urinary bladder. (A) An isolated mouse urinary bladder at 24 weeks is small and thin; the bar indicates 5 mm. (B) Treatment for 16 weeks induced enlargement and many tumours inside the bladder. (C) Histological results showed that the normal mucosa consists of two to three layers of transitional cells. Original 400. (D) BBN treatment for 16 weeks induced cancerous epithelium. Original 400.
For Western blot analysis, the HT-1376 cells were homogenized in CelLyttc-M Mammalian cell lysis/extraction reagent and CelLyttc-M Mammalian Tissue lysis/extraction reagent (Sigma-Aldrich), respectively, according to the manufacturer’s instructions. An aliquot of the homogenate (equivalent to 20 g protein) was dissolved in Laemmli Sample Buffer (Bio- Rad, Hercules, CA, USA) and separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Bio- Rad) and immunoblotted using rabbit polyclonal anti-p21WAF1 antibody (1 g/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal anti-actin antibody (0.5 g/mL; Santa Cruz). Horseradish peroxidase-conjugated secondary antibody (1:20 000 dilution; Amersham Bioscience, Amersham, UK) and the ECL Plus detection system (Amersham) was used to visualize the immunoreactions. The band density was quantified using the ImageJ software program.
All values are expressed as the mean (SE) and were analysed statistically using Student’s t-test, paired t-test or Fisher’s protected least- significant difference test, with P 0.05 considered to indicate statistical significance.
RESULTS
The cells were maintained in RPMI 1640 (Nikken Bio Medical Laboratory, Kyoto, Japan), supplemented with 10% fetal bovine serum. HT-1376 and 5637 cells were seeded in quadruplicate on 24-well plates and incubated for 48 h under standard conditions. The cells were treated with VPA (0.5–10.0 mM) for 4 and 48 h.
Cell proliferation was measured using the methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) rapid colorimetric assay, conducted by replacing the standard medium with 100 L of serum-free medium containing MTT (0.5 mg/mL) and incubated at 37 C for 3 h. After incubation, 20 L of dimethyl sulphoxide (Sigma-Aldrich) was added to each well and mixed thoroughly. The plates were then measured at 490 nm using a spectrophotometer (Immuno Mini NJ-2300, Nulge Nunc International, Tokyo, Japan).
For cell-cycle analysis, floating and attached cells dispersed with trypsin-EDTA were pelleted, washed and incubated with CycleTestTM plus DNA Reagents (Becton Dickinson, San Jose, CA, USA) according to the instructions of the manufacturer. All samples were analysed within 6 h on a flow cytometer (FACSCalibur; Beckton Dickinson).
In the present study, primary tumour tissues and corresponding normal tissues from 10 patients with urinary bladder cancer were available for analysis of HDAC1 mRNA expression using real-time RT-PCR. The mRNA expression normalized to the corresponding -actin level of the tumour tissues was significantly higher (P 0.05, Student’s t-test) than that of neighbouring normal tissues, with values of 2.08 (0.47) vs 0.94 (0.38) (Fig. 2A).
The immunoreactivity of HDAC1 was weakly identified in the cytoplasm of normal epithelia (Fig. 2B). No immunoreactivity was detected in the nucleus. In cancerous epithelia, HDAC1 was weakly expressed in the cytoplasm and intensely expressed in the nuclei (Fig. 2C).
HDAC1 immunoreactivity was absent in the neighbouring mesenchyme of both the normal and cancerous tissues. Control slides incubated with non-immune rabbit IgG showed no immunoreactivity in any cases (data not shown).
Macroscopically, the mouse urinary bladder is very small and thin (Fig. 3A). BBN treatment induced an enlargement of the urinary bladder and the bladder developed many tumours inside (Fig. 3B). A histological examination showed that normal bladder mucosa consists of two or three layers of transitional epithelial cells lining the urinary bladder (Fig. 3C). After administering BBN for 16 weeks, there were preneoplastic lesions, including simple dysplasia or papillary or nodular dysplasia, in the urinary bladder; cancerous epithelia were also detected (Fig. 3D).
The time course of the effects of BBN treatment on HDAC1 mRNA expression in mouse urinary bladder is shown in Fig. 4. HDAC1 mRNA expression level normalized to the corresponding -actin level in the urinary bladder at 8 weeks, which was 1.23 (0.10) at the beginning of the treatment, and increased by more than three times after 16 weeks of BBN treatment. In mice not administered BBN the ratio did not change from the beginning.
FIG. 4. Time-course effect of BBN treatment on HDAC1 mRNA expression in mouse urinary bladder using quantitative RT-PCR. The expression did not increase in mice given only tap water. BBN treatment significantly increased the expression time- dependently. *P 0.05, significant difference from the value of control, Student’s t-test.
FIG. 5. (A) Effect of VPA on the proliferation of HT- 1376 cells. The MTT assay showed that exposure to VPA for 48 h decreased the survival of cells in a dose- dependent manner. The effect of VPA is shown for 4 (B, D) and 48 h (C, E) on cell-cycle distribution of HT- 1376 cells. Cell-cycle analysis showed a decrease in the G2/M fraction and an increase in the sub-G1 fraction in the cells treated with 10 mM VPA for 48 h (E). The % inhibition (1 – average optical density value of experimental wells/average optical density of control wells) 100 (*P 0.05, significant difference from the value after the exposure for 4 h, paired t-test).
Table 2 summarizes the data on the incidence of papillary or nodular dysplasia and TCC in the urinary bladder after BBN with or without VPA administration. Papillary or nodular dysplasia developed in three of five mice at 8 weeks and TCC developed in two of five mice at 12 weeks in Group 1. First, to examine the therapeutic effects of VPA against BBN- induced bladder lesions, VPA administration started 4 weeks after BBN treatment in Group 3. VPA (250 and 500 mg/kg BW) did not suppress the incidence. All mice given 750 mg/kg BW every day died by 8 weeks.
Second, to examine the preventive effects of VPA, VPA was started at the same time that BBN was started in Group 4. In mice given 250 and 500 mg/kg BW, respectively, papillary or nodular dysplasia developed in one and none of each five mice at 8 weeks, and in four of each five mice at 12 weeks. In addition, TCC developed in one and none of each five mice given 250 and 500 mg/kg BW, respectively, at 12 weeks. VPA administration seemed to delay the incidence in mice treated with BBN.
Human urinary bladder cancer HT-1376 and 5637 cells were used to clarify the mechanism of VPA as an HDAC inhibitor. The MTT assay showed that adding VPA to the medium decreased the survival of the cells in a dose- dependent manner. The surviving ratio of HT- 1376 cells after exposure to 0.5, 1.0, 5.0 and 10.0 mM VPA for 4 h vs 48 h were 97.9%, 96.7%, 96.1% and 95.0%, vs 90.7%, 82.1%, 47.6% and 29.4%, respectively (Fig. 5A).
Exposure at 0.5 mM VPA significantly decreased the survival of the cells. The survival ratio of 5637 cells after exposure to 0.5, 1.0, 5.0 and 10.0 mM VPA for 4 h vs 48 h were 97.6%, 96.8%, 95.8% and 95.1%, vs 93.4%, 62.5%, 55.9% and 50.9%, respectively. Exposure at 1.0 mM VPA significantly decreased the survival of the cells.
As shown in Fig. 5B–E, the effects of adding VPA to the medium on cell-cycle distribution of HT1376 cells were analysed by flow cytometric analysis. In the cells treated with 10 mM VPA for 48 h, there was a decrease in the G2/M population and an increase in the sub-G1 population (Fig. 5E), indicating that the cells were dying.
Figure 6 shows p21WAF1 protein expression in HT-1376 and 5637 cells. While p21WAF1 protein was faintly expressed in the untreated cells,
5.0 and 10.0 mM VPA treatment increased the expression dose-dependently in both cell types.
DISCUSSION
In the present study we investigated HDAC1 mRNA expression in urinary bladder tissues and compared it between cancerous and non- cancerous tissues. HDAC1 was selected based on previous reports of greater HDAC1 levels in transformed than non-malignant cell lines [10], including those from the urinary bladder might have an important role in cell aggressiveness and dedifferentiation. In addition, the distinct HDAC1 expression profile between epithelia and mesenchyme which was shown in the study was described in the report on human prostate cancer [16].
FIG. 6. Effect of VPA on p21WAF1 protein expression in HT-1376 and 5637 cells. Western blotting showed that adding VPA to the medium increased p21WAF1 expression in both cell lines, while the protein was faintly expressed in those cells cultured in medium without VPA. The density of a band corresponding p21WAF1 (21 kDa) and actin (45 kDa) was quantified. The ratios of the density (p21WAF1 protein expression/ actin protein expression) were compared. Treatment with VPA increased the value of the ratio in both cell lines compared with those in the cell lines cultured without VPA. *P 0.05, significant difference from the value in each cell line cultured in the medium without VPA, Fisher’s protected least-significant difference test.
Most importantly, the current study showed the potential preventive efficacy of an HDAC inhibitor against urinary bladder cancer, by showing the inhibitory effect of VPA on BBN- induced epithelial changes, including cancer. VPA is reported to alter growth properties in some subcutaneous xenograft models [17,18]. However, no experiments have been conducted so far using a chemical-induced cancer model. One of the advantages of the urinary bladder cells. Indeed, HDAC inhibitors can cause growth arrest, differentiation or cell death of a variety of solid tumour cells in vitro, including kidney, prostate, ovary, breast, lung and colon [6]. The present study showed that exposure to VPA for 48 h decreased the survival of cells in a dose-dependent manner. VPA-induced inhibition of cell survival in vitro could be attributed to increased levels of p21WAF1 protein. The cyclin-dependent kinase inhibitor p21WAF1, which can lead to the arrest of cells in G1, is one of the most commonly induced genes [19]. The relationship between HDAC inhibition and p21WAF1 transcription is based on the results that HDAC inhibitors release HDAC from the p21 promoter and accumulate acetylation of the gene-therapeutic purposes. In the present study, the incidences of BBN-induced mouse urinary bladder pre-cancerous and cancerous regions seemed to be delayed in mice treated with VPA every day from the start of BBN administration (Group 4), although there were too few mice in each group for a valid statistical analysis. When VPA was induced after RNA interference treatment against HDAC1 [21].
Therefore, HDAC has been considered to be a molecular target for cancer therapy. However, it is still necessary to establish optimal dosing, to determine whether different cancers respond to different schedules of start of BBN treatment the incidence seemed to be same as those in mice treated without VPA, indicating that the antitumoral effect of VPA is very weak. HDAC inhibitors that have been reported to date can be divided combinations of therapeutic agents are most effective in arresting and selectively inducing the death of cancer cells, and with acceptable side-effects.
Increased expression of HDAC1 has been detected in gastric cancers, oesophageal squamous cell carcinoma and hormone- refractory prostate cancer [12–14]. The current results show that HDAC1 mRNA levels were higher in bladder cancer tissues of both clinical samples and a mouse model than in each of the control specimens. These data are therefore indicative of a potential link between HDAC1 and the malignant phenotypes.
Nuclear expression of HDAC1 in the cancerous epithelia was shown in most cases. Interestingly, a recent immunohistochemical study in gastric cancer showed HDAC1 overexpression in the nuclei of highly neoplastic cells, whereas neighbouring normal glandular epithelial cells had only weak and focal HDAC1 expression [12].Furthermore, nuclear HDAC1 protein expression is associated with postoperative survival in patients with pancreatic cancer [15]. A high rate of HDAC1 protein expression into several structural classes, including hydroxamates, cyclic peptides, aliphatic acids and benzamides. The aliphatic acids, such as VPA, tend to be relatively weak HDAC inhibitors, in the high micromolar or low millimolar range, and not selective. The concentrations which we used in both the in vivo and in vitro studies were relatively high, 10 times the human dose (therapeutic dose 0.3–1.2 mM). However, it has been used as an anti-epileptic agent for 30 years. As to safety, it is of no importance except for the teratogenicity, because it has an extensive safety history and well-established pharmacokinetics. Moreover, it is unclear how the mice metabolize VPA compared to humans, and many drugs are used at a higher dose in mice to achieve comparable results. Phase I and II clinical trials are presently ongoing to evaluate this drug as an antitumour agent.
CONFLICT OF INTEREST
None declared.
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