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Rheumatology Advance Access originally published online on February 15, 2008
Rheumatology 2008 47(4):418-424; doi:10.1093/rheumatology/ken003
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Anti-arthritic effects of combined treatment with histone deacetylase inhibitor and low-intensity ultrasound in the presence of microbubbles in human rheumatoid synovial cells

C. Nakamura1, I. Matsushita1, E. Kosaka1, T. Kondo2 and T. Kimura1

1Department of Orthopaedic Surgery and 2Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Science, University of Toyama, Toyama, Japan.

Correspondence to: T. Kimura, Department of Orthopaedic Surgery, University of Toyama, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. E-mail: tkimura{at}med.u-toyama.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. The therapeutic effects of histone deacetylase (HDAC) inhibitor combined with ultrasound (US) (1 MHz, 10% duty factor, 0.1 or 0.2 W/cm2) in RA synovial fibroblasts (RASFs) were examined.

Methods. RASFs were isolated from rheumatoid synovial tissues obtained from patients with RA during total knee arthroplasty. RASFs were treated with an HDAC inhibitor, trichostatin A (TSA), with or without US. Cell viability was estimated using the Trypan blue dye exclusion test and cell cycle was examined by flow cytometry using propidium iodide (PI) staining. Gene expression of cell cycle-related genes cyclin D, cyclin A, cyclin B and p21WAF1/Cip1 was analysed by semi-quantitative RT-PCR. Detection of apoptosis was examined by flow cytometry using annexin V-FITC and PI staining. Microarray analysis was carried out to profile gene expression of inflammation-related genes.

Results. Dose-dependent decreases in cell viability, cell cycle arrest and apoptosis in RASFs due to TSA were observed. US treatment in the presence of microbubbles increased cellular uptake, but did not induce cell cycle arrest or apoptosis. The combination of TSA and US modulated cell cycle-related gene expression and significantly decreased S phase cells and increased G2–M phase cells. US also further enhanced TSA-induced RASF apoptosis and regulated expression of inflammation-related genes.

Conclusions. HDAC inhibitor in combination with US effectively reduces cell viability and induces apoptosis in RASFs. The combination therapy could be useful to control synovial proliferation and inflammation, since US can be easily applied to targeted joints as local physiotherapy.

KEY WORDS: Rheumatoid, Synovial fibroblasts, Histone deacetylase inhibitor, Low-intensity ultrasound, Sonoporation


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
RA is a systemic inflammatory disease that results in the progressive destruction of joints [1]. Joint inflammation includes activation and proliferation of synovial cells, expression of inflammatory cytokines, and recruitment of inflammatory cells. Anti-inflammatory cytokine therapies such as TNF-{alpha} inhibition have recently been showing remarkable efficacy in RA patients [2, 3]. However, neither anti-inflammatory cytokine therapies nor conventional therapies are always effective and even in responders, some joints display prolonged synovial inflammation that is likely to result in progressive joint damage. A targeted or topical intervention to treat such inflammatory joints should thus be considered, in combination with systemic treatment for inflammation.

Acetylation and deacetylation of histones caused by histone acetyltransferases and histone deacetylases play important roles in transcriptional regulation of gene expression [4]. Various HDAC inhibitors that affect the acetylation–deacetylation balance have been found to inhibit cell proliferation, arrest cell cycle progression, induce apoptosis in cultured cells, and inhibit tumour growth in model animals. HDAC inhibitors are thus expected as a new class of anti-cancer agents. HDAC inhibitors have also recently been identified as potential anti-arthritic agents. In RA synovial fibroblasts (RASFs), HDAC inhibitors such as trichostatin A (TSA) and FK228 inhibited cell proliferation and induced apoptosis. In animal models of RA, HDAC inhibitors have inhibited joint swelling, synovial inflammation and subsequent bone and cartilage destruction [5–8]. In addition, HDAC inhibitors not only induced the expression of the cell cycle regulators p21WAF1/Cip1 and p16INK4a, but also inhibited the expression of TNF-{alpha} and IL-1β in affected synovial tissue [8]. These results indicate the potential utility of HDAC inhibitors as anti-rheumatic agents.

Ultrasound (US) has been widely used for diagnosis and therapy in clinical fields. Biophysical modes of ultrasonic action are classified as thermal and non-thermal effects. While high-intensity-focused US has been used for tissue ablation due to thermal effects, low-intensity pulsed US has been used for healing of fractures due to non-thermal effects. Non-thermal effects are further classified as cavitational and non-cavitational effects, and ultrasonic cavitation induces a variety of reversible and non-reversible biological effects. One such effect is an increase in the permeability and formation of transient pores in cell membranes facilitating the uptake of various low molecular weight substances, which is known as ‘sonoporation’ [9]. Although US alone increases cell permeability, presence of echo contrast microbubble agents that act as cavitation nuclei further increases the transfection efficiency of US [10, 11]. Ultrasonic cavitation and the collapse of microbubbles produce high-velocity fluid microjets that penetrate cell membranes, leading to reversible permeabilization and increased intracellular uptake of molecules [12]. Thus, US combined with echo contrast microbubble agent can be utilized to enhance uptake of gene constructs or drugs into targeted tissues, such as the heart, pancreas, skeletal muscle and synovial tissue [13–16]. In addition, certain level of US is also known to induce a gene-regulated cell death and apoptosis, and affects the expression of various genes [17, 18]. US thus appears to be a useful tool for enhancing the regional effects of drugs, since application of US is relatively simple for the targeted area.

Here, to investigate the possibility of enhancement of the effects of HDAC inhibitor as an anti-arthritic agent for targeting joints, we performed the combined treatment using US with an echo contrast microbubble agent, Levovist, and an HDAC inhibitor, TSA, in RASFs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Isolation and culture of human RASFs
RASFs were isolated from rheumatoid synovial tissues obtained from RA patients during total knee arthroplasty with the patients’ permission. All patients fulfilled the criteria of the American College of Rheumatology. The patients had been treated with methotrexate (6–10 mg/week) but showed active and sustained synovitis in the knee joints at surgery. Fresh synovial tissues were minced into small pieces and immediately digested with 1 mg/ml of collagenase (Wako, Osaka, Japan) in serum-free DMEM (Sigma-Aldrich, St Louis, MO, USA) for 2 h at 37°C. After filtering through a nylon mesh, cells were extensively washed, suspended and cultured in DMEM supplemented with 10% fetal bovine serum (FBS; ICN Biochemicals, Aurora, OH, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2. After culturing overnight, non-adherent cells were removed, and incubation of adherent cells was continued in fresh medium. At confluence, cells were trypsinized and passaged at a 1 : 3 split ratio. Medium was changed twice each week, and fourth to fifth passage RASFs were used in the following experiments.

All patient samples were obtained with informed consent according to the Declaration of Helsinki and ethics approval for the study was obtained from the institutional review board.

US apparatus and intensity measurement
An ultrasonic apparatus (Sonicmaster ES-2, OG Giken, Okayama, Japan) with a resonance frequency of 1 MHz with 100 Hz pulse repetition frequency was used in all experiments. This device is equipped with an intensity regulator, duty factor (DF) controller and built-in digital timer. Settings for US were as follows: intensity, 0, 0.1 and 0.2 W/cm2; duty cycle, 10%; sonication duration, 1 min. The transducer (diameter, 5.0 cm) was fixed to the stand to keep the transducer facing upward and horizontally. Using an US power meter (UPM-DT-10E, Ohmic Instrument Co., Easton, MD, USA), the spatial-average–temporal-average intensities (ISATA) of 0.1 and 0.2 W/cm2 (device-indicated value) at 10% DF were 0.048 and 0.072 W/cm2, respectively and the peak acoustic pressures were 0.061 and 0.105 MPa, respectively, as reported previously [19]. In this article, we used device-indicated intensities to refer to these values.

Sonication with TSA and a microbubble, Levovist
RASFs were treated with TSA and/or US exposure in the presence of microbubble agent under the same condition as reported previously [19]. First, RASFs (5 x 104) were seeded into 35-mm culture dishes containing 2 ml of DMEM supplemented with recombinant human TNF-{alpha} (1 ng/ml) and IL-1β (10 ng/ml) (both from R&D Systems, Minneapolis, MN, USA) and cultured for 3 days until sub-confluence. RASFs were then treated with TSA and/or US. In preparation for sonication, RASFs were incubated with 1.8 ml of medium containing 0, 0.1 or 1.0 µM TSA (Wako) at 37°C in a humidified atmosphere containing 5% CO2 for 15 min. Just before sonication, 200 µl of freshly prepared stock solution of an echo contrast microbubble, Levovist (Schering AG, Berlin, Germany), with medium was added to cultures at a final concentration of 1 mg/ml. Culture dishes were then placed onto the transducer and exposed to US at various intensities.

Measurement of cellular uptake and viability
To assess transient cell membrane permeabilization and cell viability after US exposure, we used calcein (Sigma-Aldrich) and propidium iodide (PI; Sigma-Aldrich) as reagents for cell staining. Calcein is a green-fluorescent and cell membrane-impermeable molecule used to quantify transport of molecules into viable cells. RASF cultures were exposed to US in the presence of calcein at a final concentration of 1 mg/ml and Levovist as above. After exposure, samples were kept at room temperature for 15 min and washed twice with phosphate-buffered saline (PBS), and PI was added at a final concentration of 0.1 mg/ml. Calcein- and/or PI-positive cells were then examined under fluorescent microscopy. Transiently sonoporated viable cells appear green with calcein under fluorescence excitation, whereas permanently damaged non-viable cells appear red due to PI staining.

Measurement of cell viability by Trypan blue dye exclusion test
Cell viability was examined by Trypan blue dye exclusion test. RASFs were treated with TSA and/or US as above, and incubated for 24 h. Cells were then harvested with trypsin/EDTA, suspended in PBS and mixed with an equal amount of 0.4% Trypan blue stain (Invitrogen). The number of cells excluding Trypan blue, representing viable cells, was counted using a Burker-Turk haemocytometer (Erma, Tokyo, Japan). The count for non-treated cells was considered 100%, while the decrease in number of viable cells after treatment was considered as loss of viability.

Flowcytometric analysis of cell cycle distribution
Distribution of cell cycle was analysed using PI staining. RASFs were treated with TSA and/or US as above and incubated for 24 h. Cells were harvested with trypsin/EDTA and washed twice with cold PBS. Detached cells were resuspended in 75% cold ethanol and fixed overnight at 4°C. For staining, fixed cells were washed once with PBS and incubated in 0.025% RNase A for 30 min at 37°C. Nuclear DNA was then stained with PI (500 µg/ml) for 30 min at 4°C. The distribution of cellular DNA content was analysed by FACSCalibur (BD Biosciences, San Jose, CA, USA), using CellQuest and Modifit data analysis software (BD Biosciences, San Jose, CA, USA).

RNA isolation and semi-quantitative RT-PCR
Semi-quantitative RT-PCR was performed to examine mRNA expression levels for cell cycle-related genes cyclin D, cyclin A, cyclin B and p21WAF1/Cip1. RASFs were treated with TSA and/or US as above and incubated for 6 or 24 h. Total RNA was extracted from cells using Isogen (Nippon Gene, Tokyo, Japan) and an RNeasy mini kit (Qiagen, Valencia, CA, USA), then converted to cDNA using M-MLV reverse transcriptase (Invitrogen) and oligo (dT) primer (Invitrogen) in accordance with the instructions of the manufacturer. PCR amplification was performed using TaKaRa Ex Taq (Takara Bio, Otsu, Japan) and specific primers. Primer sequences for β-actin, cyclin D, cyclin A, cyclin B and p21WAF1/Cip1 were as follows: β-actin, 5'-CGT ACC ACT GGC ATC GTG AT-3', 5'-GTG TTG GCG TAC AGG TCT TTG-3'; cyclin D, 5'-AAC AGA AGT GCG AGG AGG AG-3', 5'-CTG GCA TTT TGG AGA GGA AG-3'; cyclin A, 5'-ATT AGT TTA CCT GGA CCC AG-3', 5'-CAC AAA CTC TGC TAC TTC TG-3'; cyclin B, 5'-CCT GAG CCA GAA CCT GAG CC-3', 5'-AGT CAC CAA TTT CTG GAG GG-3'; and p21WAF1/Cip1, 5'-GTG AGC GAT GGA ACT TCG A-3', 5'-AAT CTG TCA TGC TGG TCT GC-3'. To compare levels of each mRNA, concentrations of cDNA were normalized to yield PCR products that were approximately equivalent to those of β-actin. All mRNA levels were quantified in the exponential phase of amplification, which was determined by analysing PCR products synthesized with different numbers of cycles, using a fixed amount of cDNA. Thermal cycle condition was 5 min at 94°C, followed by 30 s at 94°C, 30 s at 60°C (β-actin, cyclin B and p21WAF1/Cip1), 62°C (cyclin D) or 59°C (cyclin A) and 30 s at 72°C for the respective cycles, followed by 7 min at 72°C for final extension. PCR products were electrophoresed in 2% agarose gel and stained using ethidium bromide.

Assessment of apoptosis
Apoptosis was assessed by annexin V-FITC Detection Kit I (BD Biosciences, Pharmingen, San Diego, CA, USA) following the instructions of the manufacturer. RASFs were treated with TSA and/or US as above and incubated for 24, 48, 72 or 96 h. At each time after treatment, supernatants containing detached cells and residual attached cells were harvested with trypsin/EDTA, washed with cold PBS, and incubated with annexin V-FITC and PI for 15 min at room temperature in the dark. Samples were then analysed by FACSCalibur (BD Biosciences, San Jose, CA, USA). Percentages of stained cells in each quadrant were quantified using CellQuest (BD Biosciences, San Jose, CA, USA) software.

Microarray analysis of inflammation-related genes
RASFs were treated with or without TSA at a concentration of 1 µM and/or US at an intensity of 0.2 W/cm2 and incubated for 24 h. Total RNA was extracted from harvested cells using Isogen and an RNeasy mini kit. Labelling of RNA was performed using an Amino Allyl MessageAmp aRNA Kit (Ambion, Austin, TX, USA) according to the instructions of the manufacturer. For mRNA expression profiling, we used an AceGene (Human Oligo Chip 30K, DNA Chip Research and Hitachi Software, Yokohama, Japan). Arrays were scanned by ScanArray Express (PerkinElmer, Waltham, MA, USA) and changed to numerical values by ImaGene (BioDiscovery, El Segundo, CA, USA). Numerical data were normalized using the LOWESS method by GeneSight (BioDiscovery). Data were analysed using Microsoft Office Excel 2003. Each gene expression level of RASFs treated with TSA and/or US was determined by comparison with that from RASFs without TSA and US. Fold-changes were indicated as log-transformed data.

Statistical analysis
Data are expressed as mean ± S.D. Statistical analysis was performed by one-way analysis of variance and subsequent Fisher's protected least significant difference test. Values of P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effects of US on uptake of reagent into RASFs
To confirm sonoporation due to US exposure for RASFs, uptake of membrane-impermeable calcein was examined. RASFs without US exposure showed no uptake of calcein or PI (Fig. 1A). In contrast, when RASFs were treated with US, slight uptake of calcein was observed at 0.1 W/cm2 (Fig. 1B). The uptake was further increased by US exposure at 0.2 W/cm2 (Fig. 1C), indicating enhanced membrane permeability at this intensity. US exposure induced partial detachment of RASFs, particularly at 0.2 W/cm2, but not at 0.1 W/cm2. When number of PI-positive cells was counted after US exposure at 0.1 W/cm2, only small numbers of PI-positive cells were found (Fig. 1B). On the other hand, US exposure at 0.2 W/cm2 indicated slight increase in PI-positive cells (Fig. 1C).


Figure 1
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  		FIG. 1. Effect of sonoporation by US exposure on RASFs. (A–C) Phase-contrast and fluorescent photomicrographs of synovial cells treated with or without US in the presence of calcein and PI. (A) RASFs without US show no uptake of calcein and PI. RASFs treated with US at an intensity of 0.1 W/cm2 (B) and 0.2 W/cm2 (C) show uptake of calcein into the cell depending on the intensity of US. PI-positive cells are slightly increased by US exposure.

 
Effects of TSA and US on cell viability
We examined cell viability 24 h after treatment with TSA and/or US. When RASFs were exposed to US at 0.2 W/cm2, significant decrease in cell viability was found, but not at 0.1 W/cm2. RASFs treated with TSA showed dose-dependent decreases in cell viability (Fig. 2). When the combined treatment with US at 0.1 W/cm2 and TSA was applied for RASFs, little change in cell viability was observed. On the other hand, US at 0.2 W/cm2 with TSA resulted in significant decrease in cell viability (Fig. 2) due to US effect on cells and membrane permeability. Based on these results, we used the intensity of 0.2 W/cm2 for further experiments, because the combination with 0.2 W/cm2 showed the enhancement of decrease in cell viability.


Figure 2
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  		FIG. 2. Effect of TSA and/or US on cell viability of RASFs. RASFs were treated at the indicated TSA concentration and US intensity. Cell viability was measured by Trypan blue dye exclusion test 24 h after treatment. Values are expressed as percentage of control value of RASFs without TSA or US. Values represent the mean of four independent experiments and S.D.

 
Cell cycle arrest by treatment with TSA and US
Distribution of cell cycle phase was analysed 24 h after US exposure at 0.2 W/cm2. US alone did not affect the cell cycle (Fig. 3A). When the cell cycle was analysed 24 h after treatment with TSA at a concentration of 0.1 µM, significant increase in G0–G1 phase and decrease in S phase were observed (Fig. 3A and B). When US at 0.2 W/cm2 was combined with TSA, significant further decrease in S phase and increase in G2–M phase were found (Fig. 3B). When RASFs were treated with 1 µM TSA, further decrease in S phase and increase in G2–M phase, compared with cells treated with 0.1 M TSA, were observed. However, US exposure did not induce significant change of the cell cycle (Fig. 3C). US exposure to RASF thus enhanced cell cycle arrest when combined with a low TSA concentration but not a high concentration of TSA.


Figure 3
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  		FIG. 3. Effect of TSA and/or US on cell cycle distribution of RASFs. RASFs were treated at the indicated TSA concentration and US intensity. Cell cycle distribution was analysed by flow cytometry using PI staining 24 h after treatment. Values represent the mean of four independent experiments and S.D. (A) Control RASFs treated with or without US. (B) TSA (0.1 µM)-treated RASFs with or without US. (C) TSA (1 µM)-treated RASFs with or without US.

 
Influence on expression of cell cycle-related genes of treatment with TSA and US
To examine expression of cell cycle-related genes after treatment with TSA and/or US, semi-quantitative RT-PCR was performed at 6 and 24 h. TSA treatment induced dose-dependent increases in p21WAF1/Cip1 and decreases in cyclin D and A at 6 h (Fig. 4A). Similarly, US exposure alone up-regulated expression of p21WAF1/Cip1 and down-regulated cyclin A, whereas cyclin D expression was enhanced. Combined treatment with TSA and US exposure resulted in further increases in expression of p21WAF1/Cip1. After 24 h of TSA treatment, decreased expression of cyclin B was apparent, in addition to cyclin D and cyclin A, although p21WAF1/Cip1 expression was maintained independent of TSA concentration (Fig. 4B). The effect of US exposure on cell cycle-related genes was not marked at this stage.


Figure 4
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  		FIG. 4. RT-PCR analysis of cell cycle-related genes. RASFs were treated at the indicated TSA concentration and US intensity for 6 h (A) or 24 h (B). Expressions of indicated cell cycle-related genes are shown.

 
Induction of apoptosis by treatment with TSA and US exposure
We studied apoptosis of RASFs at 24–96 h after treatment with TSA and/or US by flowcytometric analysis using annexin V-FITC and PI staining. Induction of apoptosis was not observed in RASFs treated with TSA at a low concentration of 0.1 µM or US exposure at 0.2 W/cm2. Conversely, RASFs treated with TSA at 1 µM showed time-dependent induction of apoptosis, and combined treatment with US significantly enhanced induction of apoptosis (Fig. 5).


Figure 5
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  		FIG. 5. Flowcytometric analysis of apoptosis in RASFs treated with TSA and/or US exposure. RASFs were treated at the indicated TSA concentration and US intensity, and incubated for up to 96 h. Apoptosis was examined by flowcytometry using annexin V-FITC and PI staining. Values represent the mean of four independent experiments and S.D.

 
Microarray analysis of inflammation-related genes
For better understanding the effects of US and TSA on expressed genes, we performed microarray analysis 24 h after treatment with 1 µM TSA and/or 0.2 W/cm2 US exposure. US exposure alone did not significantly change expression of most inflammatory genes. Treatment with 1 µM TSA showed modest alterations in expression of most inflammatory genes (Table 1), although significantly decreased mRNA expression of IL-6 and vascular cell adhesion molecule-1 (VCAM-1) was observed. In RASFs treated with TSA and US, expression of pro-inflammatory cytokine genes IL-15 and IL-18 was markedly reduced in addition to IL-6 and VCAM-1. Moderate down-regulation of GM-CSF, MMP-2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) and cathepsin L (CTSL) were also apparent. In contrast, tissue inhibitor of metalloproteinase-1 (TIMP1) showed increased expression. These results suggest that treatment of RASFs with TSA and US favourably support regulation of inflammatory genes.


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  		TABLE 1. Inflammatory-related gene expression profile in RASFs treated with TSA and/or US

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study demonstrated that an HDAC inhibitor, TSA, induced dose-dependent decreases in cell viability, cell cycle arrest and apoptosis in RASFs. Our results also indicate that combined treatment with US further enhanced the effect of TSA by promoting cellular uptake in the presence of microbubbles. Combination treatment with US also modulated the gene expression profile and enhanced anti-inflammatory effects in RASFs.

Post-translational modifications of histones by acetylation and deacetylation play critical roles in regulating gene expression, and modification of the histone acetylation–deacetylation balance should accompany alterations in cell function. HDAC inhibitors have recently been shown to represent good therapeutic candidates not only for malignancy, but also for inflammatory diseases. HDAC inhibitors reduce inflammatory cytokines [20–22] and inhibit activation of transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B), which are crucial for the production of inflammatory mediators [23]. HDAC inhibitor has demonstrated therapeutic effects in animal models of RA and lupus erythematosus [5, 8, 24]. HDAC inhibitors may also show various adverse effects, including enhanced airway, neural and high glucose-induced inflammation [25–27]. Adverse effects of HDAC inhibitors have also been documented in initial anti-cancer clinical trials [28, 29]. With systemic administration of HDAC inhibitors, adverse events inducing haematological toxicity and gastrointestinal symptoms may increase in a dose-dependent manner. Strategies for the use of HDAC inhibitors thus need to be developed to achieve higher intracellular delivery and to target specific sites or organs, to reduce systemic toxicity. The present results suggest that use of US could enhance intracellular delivery and help HDAC inhibitors to exhibit effects on RASFs at relatively low concentrations. With clinical setting in mind, HDAC inhibitors could be administered in a systemic manner or locally by intra-articular injection. We think intra-articular injection of HDAC inhibitor and local US exposure is an easily applicable treatment to a specific joint with sustained synovitis. In addition to small joints, larger joints may be treated with US exposure at multiple sites, if necessary, after intra-articular injection. We also expect that local US exposure may help to increase the local uptake of HDAC inhibitor after systemic administration.

US in the presence of echo contrast microbubble agents has recently been investigated to improve intracellular delivery of drugs or genes due to ultrasonic cavitation. The cavitation observed in low-intensity US includes creation, vibration and collapse of small gas-filled bubbles due to the interaction of US with liquid. In the present study, we performed US exposure with an echo contrast microbubble agent, Levovist, to RASFs. To examine ultrasonic cavitation effects causing reversible and non-reversible biological effects, we investigated cellular uptake of calcein and cell viability. At US intensity 0.1 W/cm2, RASFs showed slight uptake of calcein and little change of cell viability. On the other hand, when US intensity 0.2 W/cm2 was used, RASFs showed obvious uptake of calcein and significant decrease of cell viability. This US intensity was in accord with the intensity to produce cavitation [19] and there is an apparent threshold for cavitation occurrence depending on the US intensity [30].

The primary components regulating cell cycle progression are cyclins, cyclin-dependent kinases (CDKs) and other CDK regulators. HDAC inhibitors can induce p21WAF1/Cip1, an inhibitor of CDK, and arrest tumour cells at G1 or G2–M phase [31, 32]. In addition, recent studies have found that HDAC inhibitors regulate the expression of other cell cycle-related genes such as p27Kip1, p16INK4a and cyclin A, B, D and E [33–36]. The present study confirmed that TSA treatment induced dose-dependent increases in p21WAF1/Cip1, and demonstrated decreases in cyclin D, A and B at 6 or 24 h after treatment. In addition, US exposure up-regulated expression of p21WAF1/Cip1 and cyclin D and down-regulated expression of cyclin A. Such effects of US on expression of cell cycle-related genes were not marked after 24 h, whereas the dose-dependent effect of TSA was still maintained, enhancing cell cycle arrest at G1 or G2–M phase. The sonochemical effects of low-intensity US itself on expression of cell cycle-related genes are thus relatively transient.

Another effect of HDAC inhibitors is to induce apoptosis by activating death signalling pathways, such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and Fas signals or intrinsic apoptotic pathways [6, 7, 37]. Our study also demonstrates that TSA induces apoptosis in RASFs. Although sensitivity to the induction of apoptosis by TSA is dependent on cell type, RASFs were moderately sensitive to 1 µM TSA, showing 14% apoptotic RASFs after 96 h. US exposure augmented the effects of TSA and increased RASF sensitivity, showing an increase of about 2.5-fold in apoptosis. Combined treatment with TSA and US is thus also effective to reduce viable RASFs by enhancing apoptosis. In addition to these bioeffects of HDAC inhibitor and US, cDNA-chip analysis indicated a regulatory role on inflammation-regulated genes. Expression profiling data revealed marked decreases in expression of IL-6, IL-15, IL-18, VCAM-1, MMP-2 and ADAMTS-5. Taken together, combination treatment with HDAC inhibitor and US resulted in further favourable responses by RASFs.

In summary, HDAC inhibitor in combination with US effectively affects cell cycle of RASFs, reduces viability and induces apoptosis. Such combination therapy could be useful for controlling synovial proliferation and inflammation in a targeted manner under relatively low-toxicity concentrations of the HDAC inhibitor.

Formula

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 11 October 2007; revised version accepted 2 January 2008.
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