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Research Articles: Therapeutics, Targets, and Development
Histone deacetylase inhibitor enhances 5-fluorouracil cytotoxicity by down-regulating thymidylate synthase in human cancer cells
1 National Research Laboratory for Cancer Epigenetics, Cancer Research Institute; Departments of 2 Tumor Biology and 3 Internal Medicine, Seoul National University College of Medicine, Seoul, Korea; and 4 Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea
Requests for reprints: Tae-You Kim, Division of Hematology/Medical Oncology, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea. Phone: 82-2-2072-3943; Fax: 82-2-762-9662. E-mail: kimty{at}snu.ac.kr
Abstract
Thymidylate synthase (TS) overexpression is a key determinant of 5-fluorouracil (5-FU) resistance in human cancer cells. TS is also acutely up-regulated with 5-FU treatment, and, thus, novel strategies targeting TS down-regulation seem to be promising in terms of modulating 5-FU resistance. Here, we report that histone deacetylase inhibitors can reverse 5-FU resistance by down-regulating TS. By using cDNA microarrays and validation experiments, we found that trichostatin A reduced the expression of both TS mRNA and TS protein. Cotreatment with trichostatin A and cycloheximide restored TS mRNA expression, suggesting that TS mRNA is repressed through new protein synthesis. On the other hand, TS protein expression was significantly reduced by lower doses of trichostatin A (50 nmol/L). Mechanistically, TS protein was found to interact with heat shock protein (Hsp) complex, and trichostatin A treatment induced chaperonic Hsp90 acetylation and subsequently enhanced Hsp70 binding to TS, which led to the proteasomal degradation of TS protein. Of note, combined treatment with low-dose trichostatin A and 5-FU enhanced 5-FU–mediated cytotoxicity in 5-FU–resistant cancer cells in accordance with TS protein down-regulation. We conclude that a combinatorial approach using histone deacetylase inhibitors may be useful at overcoming 5-FU resistance. [Mol Cancer Ther 2006;5(12):3085–95]
Introduction
Thymidylate synthase (TS; EC 2.1.1.45) is a folate-dependent enzyme that catalyzes the enzymatic reaction to produce intracellular de novo source of thymidylate, an essential precursor of DNA biosynthesis (1). TS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate by transferring a methylene group from the cofactor CH2H4 folate to generate deoxythymidine-5'-monophosphate, which is subsequently phosphorylated to 2'-deoxythymidine-5'-triphosphate (2). 5-Fluorouracil (5-FU) is widely used to treat colorectal, breast, and aerodigestive tract cancers (3), and is converted intracellularly to various active metabolites, which inhibit RNA synthesis and TS activity. In particular, the 5-FU metabolite 5-fluoro-deoxyuridine-monophosphate binds to TS and forms a stable tertiary complex composed of TS, 5-fluoro-2'-deoxyuridine-monophosphate, and tetrahydrofolate, and thereby inhibits TS (2). This inhibition of TS induces an accumulation of 2'-deoxyuridine-5'-monophosphate, which may be subsequently incorporated into DNA, and causes DNA strand breakage and cell death.
Several studies indicate that TS expression or activity functions as a key determinant of 5-FU sensitivity, and preclinical in vitro and in vivo studies have shown an inverse relationship between TS expression in cancer cells and 5-FU sensitivity (4–10). Importantly, these findings have been extended to clinical settings where low tumoral TS expression is predictive of a response to 5-FU–based chemotherapy for colorectal and breast cancer (4, 8). Conversely, increased TS expression and activity are viewed as being mechanistic drivers of 5-FU resistance in cancer cells (11, 12). Given that TS determines response to 5-FU, various efforts have been made to enhance 5-FU response or overcome 5-FU resistance. In this context, the use of reduced folate leucovorin in combination with 5-FU increased ternary complex stability (13). Moreover, in vitro studies have confirmed that 5-FU cytotoxicity is significantly enhanced in the presence of added leucovorin (14). The majority of clinical trials have also shown that the 5-FU and leucovorin combination is associated with a better response than 5-FU alone (15). In addition, newer, more potent TS inhibitors than 5-FU, such as raltitrexed, nolatrexed, and ZD9331, have been developed (16–18). However, despite their potent and specific TS inhibitory effects, these inhibitors acutely up-regulate TS expression when they are administered to cancer cells (19), and this reaction is regarded to be the main reason for the development of clinical resistance to 5-FU.
In addition to the well-established concept that TS is a target of fluoropyrimidine, a recent study suggested that TS may function as an oncogene (20). It was shown that the forced overexpression of human TS transforms immortalized murine cells to a malignant phenotype. In general, TS protein and mRNA expressions are higher in cervical, breast, kidney, bladder, lung, and gastric tumor tissues than in their normal counterparts (4–10), and patients showing high tumor TS expression have significantly poorer prognoses (21–24). These findings suggest that the overexpression of TS not only reflects drug resistance to fluoropyrimidine but also indicates the biological aggressiveness of cancer cells. Therefore, the development of a new therapeutic strategy that reduces TS expression would be of much interest.
Histone deacetylase inhibitors (HDACI) have been shown to acetylate the nucleosomal histones of condensed chromatin, and thus to cause the reactivation of genes silenced by hyperacetylating histones (25). HDACIs induce cell cycle arrest at G1 and/or at G2-M, differentiation, and apoptosis; they also have a potent antitumor activity against a variety of tumor cells both in vitro and in vivo (25–27). Several HDACIs are currently undergoing clinical trials and some of these show promising antitumor activity (28–33). In addition to histones, HDACIs acetylate nonhistone protein targets involved in transcription, cytoskeletal formation, DNA repair, signal transduction, and chaperone heat shock protein (Hsp; refs. 25, 34–37). The acetylation of nonhistone proteins seems to be an important requirement for the antitumor effects of HDACIs. In particular, recent studies have shown that HDACIs cause the depletion of several oncoproteins that are stabilized by chaperonic Hsp. In addition, Hsp90 is a key regulator of several client oncoproteins, such as, ErbB2, Akt, Bcr-Abl, and Raf. HDACI depsipeptide treatment promotes Hsp90 acetylation in parallel with the time-dependent stabilization of Hsp90-client protein complexes (37). Similarly, LAQ824 down-regulates ErbB2 by acetylating Hsp90 and, further, sensitizes breast cancer cells to chemotherapeutic agents (38).
In the present study, we show that HDACI can reverse 5-FU resistance by down-regulating TS. Trichostatin A treatment acetylated Hsp90 and subsequently enhanced the proteasomal degradation of TS protein, which sensitized cancer cells to 5-FU. Thus, combined treatment with trichostatin A and 5-FU is potentially promising in terms of overcoming 5-FU resistance.
Materials and Methods
Cell Culture
Human gastric cancer cells (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, SNU-668, and SNU-719) were purchased from the Korean Cell Line Bank (Seoul, Korea; ref. 39). All cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (WelGENE, Inc., Daegu, Korea) and gentamicin (10 µg/mL). Human breast cancer cells (SkBr3, MCF7) and colon cancer cells (LOVO, HCT116) were purchased from the American Type Culture Collection (Manassas, VA), and grown in RPMI 1640 (SkBr3, MCF7, LOVO) or DMEM (HCT116) supplemented with 10% fetal bovine serum and gentamicin (10 µg/mL). All cancer cells were incubated under standard culture condition (20% O2 and 5% CO2 at 37°C).
Reagents
Anti-Hsp70 and anti-Hsp90 monoclonal antibodies were purchased from Stress-Gen Biotechnologies Corp. (Victoria, British Columbia, Canada). Anti–TS monoclonal antibody was purchased from NeoMarkers (Fremont, CA). Antibodies to acetylated histone H3, acetylated histone H4, and dimethyl-histone H3 K9 were purchased from Upstate Biotechnology (Waltham, MA). Normal rabbit IgG antibody (a negative control) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antiacetylated lysine polyclonal antibody was from Cell Signaling (Beverly, MA). Trichostatin A, cycloheximide, and MG-132 were purchased from Sigma (St. Louis, MO), and 5-FU was from Choong Woe Co., Ltd (Seoul, Korea). Suberoylanilide hydroxamic acid (SAHA), oxamflatin, valproic acid, and sodium butyrate were kindly provided by Dr. Dae-Kee Kim (In2Gen, Seoul, Korea).
Western Blot Analysis
Cells were collected after trichostatin A and/or 5-FU treatment, washed with ice-cold PBS, and suspended in an extraction buffer [20 mmol/L Tris-Cl (pH 7.4), 100 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 5 mmol/L MgCl2, 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L pepstatin A, 0.1 mmol/L antipain, 0.1 mmol/L chymostatin, 0.2 mmol/L leupeptin, 10 mg/mL aprotinin, 0.5 mg/mL soybean trypsin inhibitor, and 1 mmol/L benzamidine] on ice for 15 min. Lysates were cleared by centrifugation at 13,000 rpm for 20 min. Equal amounts of cell extracts were resolved on 10% SDS-polyacrylamide denaturing gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), and probed with an appropriate primary antibody and horseradish peroxidase–conjugated secondary antibody. Detection was done using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ; ref. 40).
Immunoprecipitation
Cells were collected after trichostatin A treatment and washed with PBS. They were then suspended in an immunoprecipitation extraction buffer [50 mmol/L Tris-Cl (pH 7.5), 250 mmol/L NaCl, 0.1% NP40, 5 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L NaVO4, 100 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L leupeptin, 10 µg/mL aprotinin, 0.1 mmol/L pepstatin A, and 0.1 mmol/L antipain] and incubated on ice for 15 min. One microgram of each antibody (Hsp90 and TS) was added to 500 µg of each cell extract in 700 µL of extraction buffer and incubated overnight at 4°C with continuous agitation. To collect immune complexes, 30 µL of protein A/G agarose were added to mixtures, which were then incubated for 4 h. Immune complexes were centrifuged at 1,200 rpm for 2 min, and precipitates were washed thrice with extraction buffer. Proteins were eluted with SDS sample loading buffer before immunoblot analysis (40).
Reverse Transcription-PCR and Quantitative Real-time Reverse Transcription-PCR Analysis
Total RNA was isolated with TRI reagent (Molecular Research Center, Inc. Cincinnati, OH), and cDNAs were synthesized from 1 µg of total RNA using ImProm-II Reverse Transcriptase (Promega Corporation, Madison, WI) using random hexamers. The primers used in the PCR reaction were as follows: TS, forward primer 5'-TCTGGAAGGGTGTTTTGGAG-3' and reverse primer 5'-CCTCCACTGGAAGCCATAAA-3'; and ß-actin, forward primer 5'-CCACACTGTGCCCATCTACG-3' and reverse primer 5'-AGGATCTTCATGAGGTAGTCAGTCAG-3'. Amplification reactions were done in 20 µL volumes for 94°C for 5 min, 25 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and finally 10 min at 72°C. To estimate the efficiency of cDNA synthesis from each cell line, ß-actin was used as an internal control. PCR products were resolved on 1.2% SeaKem agarose gels, and quantitative PCR was done using an iCycler IQ detection system (Bio-Rad Laboratories, Hercules, CA) using SYBR green I as a double-stranded, DNA-specific binding dye. Thermocycling was done in a final volume of 20 µL containing 4 µL cDNA sample, 10 pmol of each primer, 0.125 mmol/L deoxynucleotide triphosphate mixture, 0.25 mg/mL bovine serum albumin, 0.05% Tween 20, 1x rTaq reaction buffer containing 1.5 mmol/L MgCl2 (Takara Bio., Otsu, Japan), 1 unit rTaq DNA polymerase (Takara), and 1x SYBR green I (Roche Diagnostics, Mannheim, Germany). After an initial denaturation at 95°C for 10 min, 40 cycles at 94°C for 30 s, annealing at 53°C for 30 s, and 72°C for 30 s were carried out. All cDNA samples were synthesized in parallel, and PCR reactions were run in triplicate. mRNA levels were derived from standard curves and are expressed as relative changes after normalization versus ß-actin mRNA levels.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation was done using antibodies specific for acetylated histone H3 and H4, as described previously (41). Briefly, SNU-484 cells, in a 10-cm dish, were fixed by adding formaldehyde to a final concentration of 1% and then incubated for 10 min at 37°C. Glycine (0.125 mol/L) was then added to quench the reaction (5 min at room temperature). The cells were suspended in SDS lysis buffer [1% SDS/10 mmol/L EDTA/50 mmol/L Tris-HCl (pH 8.1)], and the lysates were sonicated to shear the DNA into lengths of 200 to 500 bp. For preclearing, 50% protein A/G Sepharose slurry, containing 20 µg of sonicated salmon sperm DNA and 1 mg/mL bovine serum albumin, was added and the mixture was incubated by rocking for 30 min at 4°C. Precleared supernatants were immunoprecipitated using antibodies specific for acetylated histone H3, acetylated histone H4, or normal rabbit IgG (5 µg of each) and incubated overnight at 4°C. No antibody controls were included in the chromatin immunoprecipitation assays, and no precipitation was observed. Antibody/protein complexes were collected as salmon sperm DNA/protein A agarose slurries for 2 h at 4°C, washed five times for 5 min, and eluted twice with 250 µL of elution buffer (0.1 mol/L NaHCO3/1% SDS). RNase A (0.03 mg/mL) and NaCl (0.3 mol/L) were added, and crosslinks were reversed by incubation for 4 h at 65°C. Samples were digested with Proteinase K (0.24 mg/mL) for 1 h at 45°C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. The recovered DNA was analyzed by PCR using TS promoter–specific primer pairs, namely, forward primer 5'-CCTGGGCTCCGTTCTGTG-3' and reverse primer 5'-CCGCGCCATGCCTGTGGCCGGCTCGGAGCT-3'. The size of the amplified product was 270 bp. The conditions used were 95°C for 5 min, 35 cycles at 96°C for 1 min, annealing at 60°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min.
Cell Growth Inhibition Assay
Cells (5 x 103–7 x 103 in 50 µL/well) were seeded on 96-well plates and incubated for 24 h at 37°C and treated with drugs (i.e., trichostatin A or 5-FU) for 1 to 4 days at 37°C. After drug treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, cells were incubated for 4 h at 37°C, and the medium was then removed. DMSO was then added and shaken for 30 min at room temperature. Absorbance was analyzed using a microplate reader (Molecular Devices, Sunnyvale, CA; ref. 42). In some experiments, proliferating cancer cells were incubated in the presence of 50 nmol/L trichostatin A for 24 h, and then medium was removed. Cells were then washed thoroughly with PBS, and fresh medium containing 1 µmol/L 5-FU was added. The cells were then incubated for an additional 3 days. Graphs were generated by nonlinear regression of the data points to a four-variable logistic curve using SigmaPlot software (Statistical Package for the Social Sciences, Inc., Chicago, IL).
Colony Formation Assay
For colony formation assays, cells were grown exponentially to 
40% confluency and exposed to trichostatin A for 24 h or mock-treated with DMSO. After 24 h, cells were trypsinized, washed in PBS, counted, and plated in growth medium in six-well plates at densities of 2 x 103 or 4 x 103 cells per well for SNU-484 or SNU-601, respectively. Cells were then incubated for 18 h until cells attach; 5-FU was added at concentrations of 0, 2, 4, 6, 8, or 10 µmol/L for 24 h; and drug-containing medium was decanted. Cells were then washed twice with PBS and incubated in growth medium for 10 to 12 days. Colonies produced were stained with 0.1% Coomassie brilliant blue R-250 (Bio-Rad Laboratories) in 30% methanol and 10% acetic acid. Colonies (>500 cells or 1 mm in diameter) were counted by two individuals. Graphs were constructed using SigmaPlot software (Statistical Package for the Social Sciences).
Combination Index by IC50 Isobologram
The effect of combined trichostatin A and 5-FU on cell growth inhibition was analyzed by isobologram method of Steel and Peckham (43). Based on dose-response curves of trichostatin A in combination with 5-FU, three isoeffect curves (modes I, IIa, and IIb) were drawn and the envelope of additivity was surrounded by modes I and II (44). When the data points of the combination fell within the envelope, the combination was considered additive. If the data points fell below the envelope of additivity, the combination was considered synergistic. Data points on the right side of the envelope were regarded as antagonistic. Sequential treatment was done in the same way used in cell growth inhibition assay. For concurrent treatment, cells were incubated for 3 days in the presence of both trichostatin A and 5-FU.
Statistical Analyses
Data were expressed as mean ± SE. Comparisons used Student's t test or ANOVA, as appropriate. P values of <0.05 were assigned significant.
Results
Transcriptional Down-Regulation of TS mRNA by Trichostatin A
To identify novel target genes regulated by HDACIs, we previously did cDNA microarrays to determine the effects of trichostatin A in SNU-601 gastric cancer and MDA-MB-231 breast cancer cells. It was found that trichostatin A significantly repressed TS mRNA by 9-fold in both cell lines (data not shown). To validate these findings, we first examined the expression of TS mRNA in gastric cancer cells. Semiquantitative reverse transcription-PCR (RT-PCR) showed that TS mRNA was expressed in all cancer cells, but its expression was at a higher level in SNU-1, SNU-484, and SNU-719 cells (Fig. 1A
). To further validate the microarray results, TS mRNA expression was analyzed in SNU-484 cells after trichostatin A treatment. These cells were treated for the indicated concentrations (left) for 24 h or indicated times with 300 nmol/L trichostatin A (Fig. 1B, right). Treatment with trichostatin A was found to reduce TS gene expression in a dose- and time-dependent manner, and TS mRNA expression was reduced below 20% of baseline by trichostatin A at 250 nmol/L. When cells were treated with 300 nmol/L trichostatin A, TS mRNA levels were observed to decrease after 12 h of treatment. With regard to transcriptional regulation, trichostatin A is known to activate gene transcription by increasing the acetylation of histones tethered to promoter regions. To determine whether TS promoter is directly associated with histone acetylation, we did chromatin immunoprecipitation assays using various antihistone antibodies after treatment with 300 nmol/L of trichostatin A for 24 h. It was found that TS promoter is associated with the acetylation of histones H3 and H4 even in the absence of trichostatin A, and that trichostatin A treatment did not alter the association between TS promoter and acetylated histones H3 and H4 in SNU-484 cells (Fig. 1C). Considering that trichostatin A tends to reduces TS gene transcription, these findings suggest that trichostatin A does not directly affect the histone acetylation of TS promoter, but rather that it indirectly regulates TS gene transcription. One possible explanation for this is that trichostatin A may induce a transcriptional factor that represses TS gene expression. To address this possibility, we tested the effect of cycloheximide on TS gene expression. As shown in Fig. 1D, cycloheximide, which inhibits new protein synthesis, effectively restored TS mRNA levels after trichostatin A treatment, which supports the indirect regulatory effect of trichostatin A on TS mRNA expression (i.e., that an unknown transcriptional repressor is induced by trichostatin A treatment and that it inhibits TS transcription).
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Trichostatin A Promotes the Proteasomal Degradation of TS Protein by Acetylating Chaperonic Hsp
It was recently reported that HDACIs acetylate Hsp90, and that this results in the degradation of client oncoproteins via the proteasomal pathway (37). Therefore, we attempted to determine whether TS is similarly affected by the acetylation of chaperonic Hsp. First, we found that TS protein interacts with Hsp90 and Hsp70 in vivo (Fig. 3A–C
), and it was also found that trichostatin A affects the acetylation of Hsp90, but not that of Hsp70 (Fig. 3B and C). Immunoprecipitation also showed that trichostatin A treatment increases binding between Hsp70 and TS in accordance with the down-regulation of TS protein. Time-dependent observations more clearly showed increases in the amount of bound Hsp70, which is subject to proteasomal degradation of the client TS protein in cancer cells (Fig. 3D). Taken together, these findings show that TS protein is degraded by proteasome that is activated by Hsp90 acetylation after trichostatin A treatment. Overall, we conclude that trichostatin A regulates TS expression via the indirect transcriptional repression of the TS gene or via the proteasomal degradation of TS protein mediated by Hsp90 acetylation.
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80% with 50 nmol/L trichostatin A treatment, which reduced TS protein expression by 50%. Therefore, 50 nmol/L trichostatin A seems to be appropriate in terms of TS down-regulation and toxicity. We next determined the effects of trichostatin A on the cytotoxic effects of 5-FU on cancer cells. In HCT-116 colon cancer cells, which are resistant to 5-FU, sequential treatment with 50 nmol/L trichostatin A followed by 5-FU enhanced 5-FU cytotoxicity. Compared with untreated controls, 50% of HCT-116 cells survived trichostatin A (50 nmol/L) and 5-FU (1 µmol/L) treatment (P = 0.02), whereas 60% of 5-FU–sensitive LOVO cells survived 5-FU treatment (P < 0.01). As expected, TS protein expression was reduced in HCT-116 cells after trichostatin A treatment (Fig. 5B). Therefore, the sequential use of low-dose trichostatin A seemed to make 5-FU–resistant cells sensitive to 5-FU. Similarly, colony formation experiments showed that the sequential use of 50 nmol/L trichostatin A and various doses of 5-FU reduced colony formation by >30% versus 5-FU alone in 5-FU–resistant SNU-484 gastric cancer cells (Fig. 5C). Moreover, the growth-inhibitory effect of sequential trichostatin A and 5-FU in SNU-484 cells was found to be similar to that of 5-FU alone in 5-FU–sensitive SNU-601 cells. IC50 values were reduced to 4.63 µmol/L in SNU-484 cells, whereas the IC50 value of 5-FU was 13.22 µmol/L; the combination of trichostatin A potentiated the cytotoxicity of 5-FU to 3-fold. Isobologram analyses were used to determine the effects of sequential or concurrent treatment of trichostatin A and 5-FU in 5-FU–resistant SNU-484 cells. When SNU-484 cells were exposed to trichostatin A and varying doses of 5-FU (0.25–8 µmol/L) as a sequential or concurrent approach, the data points fell to the left side of envelope of additivity, indicating that each combination was synergistic (Fig. 5D). Taken together, these findings show that this combinatorial approach of HDACI and 5-FU seems to be effective in enhancing 5-FU cytotoxicity by down-regulating TS in 5-FU–resistant cancer cells.
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In the present study, we showed that low-dose trichostatin A down-regulates TS protein and as a result sensitizes 5-FU–resistant cancer cells to 5-FU. In terms of the molecular basis of TS down-regulation, we observed for the first time that following trichostatin A treatment, client TS protein of chaperonic Hsp90 is destabilized via the acetylation of Hsp90 and the subsequent recruitment of Hsp70. This finding is important in terms of overcoming 5-FU resistance. These observations suggest that HDACIs may be used in combination therapies with fluoropyrimidine to overcome the drug resistance associated with reduced TS protein expression in cancer cells.
5-FU was developed some 50 years ago but still remains one of the main anticancer agents used to treat colorectal, breast, gastric, esophageal, pancreatic, and head and neck cancers (4–10). In addition to 5-FU, various TS inhibitors have been developed that more specifically and potently inhibit TS. These inhibitors include raltitrexed (tomudex, ZD1674), pemetrexed (LY231514), nolatrexed (AG337), and ZD9331 (16–18), and all show potent inhibitory effect on TS and promising antitumor activity against different cancer cells. However, although some of these have shown improved efficacies in 5-FU–resistant cancer cells, TS overexpression also presents a main mechanism of resistance to these TS inhibitors (19). Moreover, cancer cells exposed to these TS inhibitors acutely up-regulate TS, which may eventually lead to drug resistance (2, 19). Therefore, a novel strategy aimed at inhibiting TS in a manner that does not induce TS may be a more reasonable approach to the overcoming drug resistance.
It is well known that TS is frequently overexpressed in cancer cells compared with normal tissues, and that tumors with high TS expression are generally resistant to fluoropyrimidine anticancer agents (11, 47). The present study also shows that high TS protein expressions in SNU-484 (gastric cancer), HCT-116 (colon cancer), and SkBr3 (breast cancer) are related with 5-FU resistance in vitro (Fig. 4). Moreover, preclinical and clinical studies have shown close relationships between TS mRNA/TS protein levels and response to therapy in colorectal cancer patients treated with fluoropyrimidine (5). Using a cDNA microarray approach, we previously observed that TS mRNA expression is down-regulated after trichostatin A treatment in breast cancer cells. In the present study, we confirmed that TS mRNA and TS protein expressions are decreased by trichostatin A treatment in a time- and dose-dependent manner in cancer cells (Figs. 1B and 2B). However, the mechanisms underlying the down-regulation of TS mRNA and TS protein seem to differ. More specifically, whereas trichostatin A did not alter the histone acetylation of TS promoter, it did repress TS gene transcription, possibly by inducing an unknown transcriptional factor that represses TS gene expression. Moreover, TS protein was down-regulated by <100 nmol/L of trichostatin A, but TS mRNA was not significantly affected at this dose. In theory, the down-regulation of TS protein, which can be achieved by using antisense oligodeoxynucleotides or small interference RNA, may sensitize cancer cells to TS-directed chemotherapeutic agents (48). In the present study, we showed that HDACIs deplete TS and sensitize cancer cells to 5-FU. Therefore, HDACIs may represent a novel class of TS inhibitors that deplete TS mRNA and TS protein and thereby inhibit TS function.
Then, how do HDACIs deplete TS protein levels? Studies have reported that HDACIs cause acetylation of several nonhistone proteins (25, 34–37). Of these, the main focus is on the acetylation of Hsp, because several prominent Hsp client proteins were intimately linked to human cancer. For example, cotreatment with SAHA and imatinib mesylate induced Bcr-Abl down-regulation and apoptosis more so than SAHA alone. LAQ824 lowered the expression, and promoted the proteasomal degradation, of Bcr-Abl. It also induced the apoptosis of imatinib refractory chronic myelogenous leukemia (49, 50). Similarly, the present study shows that TS binds to Hsp chaperonic complex (Fig. 3A–C) and that treatment with trichostatin A acetylated Hsp90 but not Hsp70. Moreover, along with Hsp90 acetylation, trichostatin A recruited Hsp70 to Hsp chaperonic complex containing client TS and resulted in the degradation of TS via a proteasomal pathway (Fig. 3B–D). Therefore, taken together, we believe that trichostatin A regulates TS expression in two ways—by indirect transcriptional repression of the TS gene and by the proteasomal degradation of TS protein mediated by Hsp90 acetylation. These results strongly suggest that HDACIs, like trichostatin A, can be used as a chemosensitizer of TS inhibitors. However, considering its significant toxicity, trichostatin A might not be suitable as a 5-FU modulator. About this, even low and relatively nontoxic dose of trichostatin A (<50 nmol/L) was found to down-regulate TS protein and sensitize drug-resistant cells to 5-FU (Fig. 5A–C). Besides low-dose trichostatin A, we found that relatively nontoxic SAHA and oxamflatin under clinical developments also down-regulate TS protein expression (Fig. 2D). Combination index from IC50 isobologram analysis showed that combinational treatments with trichostatin A and 5-FU, either sequential or concurrent, are synergistic in 5-FU–resistant SNU-484 cancer cells (Fig. 5D). Therefore, we expect that this combinatorial approach of HDACI and 5-FU can be potentially translated into clinical purpose.
In conclusion, the present study shows that HDACIs, in addition to regulating transcription, also exert nontranscriptional effects on nonhistone targets such as TS. Although TS inhibitors directly inhibit the enzymatic function of TS, HDACIs down-regulate TS expression and thus sensitize resistant cancer cells to 5-FU. In the aspect of enhancing 5-FU cytotoxicity, combined HDACI and 5-FU treatment seem to be a potentially promising strategy and clinical investigation using this approach is warranted.
Footnotes
Grant support: Korean Ministry of Science and Technology through the National Research Laboratory Program for Cancer Epigenetics, and by the 2002 BK 21 Project for Medicine, Dentistry and Pharmacy.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Presented in part at the annual meeting of AACR, Anaheim, California (2005).
Received 7/18/06; revised 9/16/06; accepted 10/20/06.
References
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-Tubulin was used as a loading control. B, for Western blotting, SNU-484 cells were treated for the indicated concentrations for 24 h (left) or for indicated times with 300 nmol/L trichostatin A (right). Graphs were prepared by quantifying immunoblot bands using TINA 2.0 software (Ray tests). C, trichostatin A promotes the proteasomal degradation of TS. Samples for Western blotting were prepared from cells treated with 300 nmol/L trichostatin A and/or various doses of MG132 for 24 h. D, SNU-484 cells were exposed to trichostatin A (300 nmol/L), SAHA (1 µmol/L), oxamflatin (1 µmol/L), sodium butyrate (1 µmol/L), and valproic acid (1 µmol/L) for 12 or 24 h. 
5-FU (P = 0.02) in HCT-116 cells. Columns, mean of three independent experiments; bars, SE. *, P < 0.05. **, P < 0.01. C, SNU-484 (5-FU–resistant) and SNU-601 (5-FU–sensitive) cells were exposed to increasing concentrations of 5-FU for 24 h, when medium was exchanged for growth medium. The cells were then allowed to form colonies over a period of 7 to 10 d. For combinatorial treatments, after pretreatment with 50 nmol/L trichostatin A for 24 h, trypsinized cells were attached by incubation (18 h) and then exposed to different doses of 5-FU for 24 h. Plates were then washed twice with growth medium and cells were allowed to form colonies over a period of 7 to 10 d. Points, mean of three independent experiments; bars, SD. **, P < 0.01. D, IC50 isobolograms in combination of trichostatin A and 5-FU in SNU-484 cells. Left, results of trichostatin A followed by 5-FU; right, effects of concurrent trichostatin A and 5-FU. These data points fell into the left of the envelope, indicating that each combination was synergistic. Doses of 5-FU: 
, 0.25 µmol/L; 
, 0.5 µmol/L; 
, 1 µmol/L; 
, 2 µmol/L; 
, 4 µmol/L; 
, 8 µmol/L. *, P < 0.05.