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Ann Thorac Surg 2006;81:1034-1042
© 2006 The Society of Thoracic Surgeons

Original article: General thoracic

Effectiveness of Trichostatin A as a Potential Candidate for Anticancer Therapy in Non–Small-Cell Lung Cancer

Nishit K. Mukhopadhyay, PhD a , * , Ellen Weisberg, PhD b , David Gilchrist, BS a , Raphael Bueno, MD a , David J. Sugarbaker, MD a , Michael T. Jaklitsch, MD a

a Division of Thoracic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
b Department of Adult Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts

Accepted for publication June 22, 2005.

* Address correspondence to Dr Mukhopadhyay, Division of Urology, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115 (Email: nmukhopadhyay{at}partners.org).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: A well-known histone deacetylase inhibitor, trichostatin A, was applied to non–small-cell lung cancer cells to determine whether inhibition of histone deacetylase leads to the production of proteins that either arrest tumor cell growth or lead to tumor cell death.

METHODS: Trichostatin A (0.01 to 1.0 µmol/L) was applied to one normal lung fibroblast and four non–small-cell lung cancer lines, and its effect was determined by flow cytometry, annexin-V staining, immunoprecipitation, and Western blot analysis.

RESULTS: Trichostatin A demonstrated tenfold greater growth inhibition in all four non–small-cell lung cancer lines compared with normal controls, with a concentration producing 50% inhibition ranging from 0.01 to 0.04 µmol/L for the tumor cell lines and 0.7 µmol/L for the normal lung fibroblast line. Trichostatin A treatment reduced the percentage of cells in S phase (10% to 23%) and increased G1 populations (10% to 40%) as determined by flow cytometry. Both annexin-V binding assay and upregulation of the protein, gelsolin (threefold to tenfold), demonstrated that the tumor cells were apoptotic, whereas normal cells were predominantly in cell cycle arrest. Trichostatin A increased histone H4 acetylation and expression of p21 twofold to 15-fold without significant effect on p16, p27, CDK2, and cyclin D1.

CONCLUSIONS: Collectively, these data suggest that inhibition of histone deacetylation may provide a valuable approach for lung cancer treatment. We evaluated trichostatin A as a potential candidate for anticancer therapy in non–small-cell lung cancer.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
The nucleosomal complexes formed by the histone octamer and associated DNA are the fundamental organizational unit of eukaryotic chromatin [1, 2]. The reversible acetylation of the {Sigma}-amino groups of lysine residues within the NH2-terminal tails of core histones is important in the modulation of chromatin topology and regulation of gene transcription. Histone acetylation contributes to the formation of a transcriptionally competent environment by opening chromatin and allowing general transcription factors to gain access to the DNA template [3, 4]. On the other hand, histone deacetylation maintains chromatin in a transcriptionally silent state [5].

In normal cells, a balance exists between histone acetyltransferase and histone deacetylase (HDAC) activity that leads to a cell-specific pattern of gene expression. Ten structurally related HDAC enzymes have been identified in mammalian cells to date [3, 6]. Several transcriptional adaptor proteins, including p300/CBP, PCA, hTAFII250, TFIIIC, activator of thyroid and retinoic acid receptors, and steroid receptor coactivator 1, possess histone acetyltransferase activity [7–9]. These enzymes appear to be recruited by, and act as cofactors for, a large number of DNA sequence–specific transcription factors.

Inhibition of HDAC activity causes transcriptional activation of certain genes (p21 waf1/cip1 and LDL receptor) [10], but repression of others including p51kip2 [11], c-Myc [12], cyclin D1 [13], and BCL-XL [14]. Treatment of cells with commercially available HDAC inhibitors such as butyrate, trichostatin A (TSA), and trapoxin A [10, 15, 16] results in transcription of target genes.

Trichostatin A is an antifungal antibiotic and a potent noncompetitive reversible inhibitor of HDAC activity. It is stable and can modulate gene transcription at micromolar concentrations in vivo in adult mice without major toxicity and does not perturb mouse embryonic or postnatal development [17]. Trichostatin A has been shown to induce differentiation in acute myeloid leukemia [18] and displays potent antitumor activity against prostate [19] and breast cancer cells in vitro and in vivo [15]. The gene expression profile of a TSA-treated lung adenocarcinoma cell line, LT23, has also been reported [10]. Although the antitumor activity of TSA has been tested in lung cancer cells in combination with DNA methyl transferase inhibitor 5-aza-2'-deoxycidine [20], the study is limited to two lung cancer cell lines and is without a normal control. Moreover, Zhu and colleagues [20] did not study the details of TSA action, nor did they identify any potential marker for TSA-induced apoptosis. We also came across a recent paper by Maxhimer and associates [21] in which they have shown the effect of TSA on two lung cancer cell lines with variable effects. We have investigated the antiproliferative activity of TSA in four human non–small-cell lung cancer (NSCLC) lines and in one normal lung fibroblast (NLF) line to explore its effectiveness as a drug candidate and its mechanism of action in these cells.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Reagents
Dulbecco's modified Eagle's medium, RPMI 1640 medium, fetal bovine serum, trypsin, phosphate-buffered saline, penicillin-streptomycin, and glutamate were purchased from Invitrogen Life Technologies (Carlsbad, CA). Trichostatin A was purchased from Calbiochem (San Diego, CA). Bovine serum albumin and soybean trypsin inhibitor were from Sigma Chemical Company (St. Louis, MO). Proto-gel was from National Diagnostic (Atlanta, GA). Anti-acetylated histone H4 was from Upstate Biotechnology (Lake Placid, NY). Gelsolin, p21, p27, p16, and CDK2 antibodies were from Transduction Laboratories (San Diego, CA). Phosphotyrosine monoclonal antibody 4G10 was a kind gift from Professor Thomas Roberts, PhD, of the Dana-Farber Cancer Institute (Boston, MA). Monoclonal anti-ß-actin antibody was from Sigma Chemical Company.

Cell Culture and Cell Survival Assay
The human NSCLC lines used in these studies (Calu-1, NCI-H520, NCI-H23, and NCI-H441) are available through American Type Culture Collection (Rockville, MD; www.atcc.org). The NLF line mrc-9 was a kind gift from Dr LanBo Chen, PhD, of the Dana Farber Cancer Institute, but is also available from American Type Culture Collection. All the above cell lines were grown in either Dulbecco's modified Eagle's medium or RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 2% glutamine at 37°C and 5% CO2. Cells were passaged for not more than eight generations and then replaced from frozen stocks of earlier passaged cells.

The cell survival assay began with seeding six-well microtiter plates with 1 x 105 cells/well in 3 mL of growth medium. Either TSA (in dimethylsulfoxide [DMSO]) or DMSO (vehicle control) at final concentrations ranging from 10 nmol/L to 10 µmol/L was added to the cell culture medium after 3 to 4 hours of subculturing. The cells were incubated at 37°C, 5% CO2, for specific periods, and the viable cells were counted in triplicate during 24-hour intervals by trypan blue assay. Cells staining black were considered as dead cells and unstained cells as viable cells.

Immunodetection of Acetylated Histone H4
Non–small-cell lung cancer lines (1 x 105) were treated with 1 µmol/L TSA in DMSO or with DMSO as a vehicle control for 24 hours at 37°C. Cells were lysed in triple detergent lysis buffer containing 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% NP-40, 1% Triton X-100, and 0.01% SDS, and a cocktail of protease inhibitors including 1 mmol/L phenylmethylsulfonyl fluoride, 2 mg/mL leupeptin, and 5 mg/mL aprotinin. Whole cell extract proteins (50 µg) were then separated by 15% sodium dodecylsulfate polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes, blocked with 5% milk in Tris–borate–T-20 buffer (Boston Bioproducts, Inc, Boston, MA) and then probed with anti-acetylated histone H4 antibody (Upstate Biotechnology; 1:1,000 dilution in 3% milk) overnight at 4°C. Membranes were washed briefly with Tris–borate–T-20 buffer and incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature (1:2,500 dilution). After extensive washing, immunoreactive bands were visualized by chemiluminescence (ECL reagent, New England Nuclear, Boston, MA).

Immunodetection of p21, p16, and p27 Protein
Immunodetection of cell cycle inhibitors was performed as described above with the following modifications: Cells (1 x 105) were lysed at 4°C in the lysis buffer containing 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% NP-40, 200 µmol/L sodium orthovanadate, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mg/mL leupeptin, and 5 mg/mL aprotinin. The lysates were centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatants (approximately 80 µg of protein) were transferred using Western blotting technique to a nitrocellulose membrane after a 12% sodium dodecylsulfate polyacrylamide gel electrophoresis. The membrane was first probed with primary antibody against p21 at a dilution of 1:2,500 in 5% milk overnight at 4°C. After probing with secondary antibody (1:2,500 dilution) and chemiluminescence, as described earlier, the membrane was stripped in 62.5 mmol/L Tris-HCl (pH 6.8), 2% sodium dodecylsulfate, and 1.0 mmol/L ß-mercaptoethanol for 30 minutes at 50°C. The same membrane was again blotted with p27, p16, and ß-actin antibodies sequentially with the same dilution of primary and secondary antibodies by subsequent stripping and probing with the specific antibodies.

Immunodetection of Gelsolin, CDK2, and Cyclin D1
Gelsolin, CDK2, and cyclin D1 were immunodetected in the same blot sequentially as described earlier with the following modifications: 80 µg of protein was separated in a 10% sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred using Western blotting technique to a nitrocellulose membrane. The dilution of primary antibody for gelsolin and CDK2 was 1:2,000 in 5% milk. For cyclin D1, the primary antibody was used at a dilution of 1:2,500 in 3% milk. All the blots were probed at room temperature for 1 hour with a secondary antibody at a dilution of 1:5,000. Washing with chemiluminescence reagent was performed as described earlier. The gelsolin blot was probed first, followed by CDK2, ß-actin, and cyclin D1.

Annexin-V Staining and Cell Cycle Analysis
Apoptosis of HDAC inhibitor-treated cells was measured using the Annexin-V-Flues Staining Kit (Boehringer Mannheim, Indianapolis, IN). Approximately 0.5 to 1.0 x 106 cells cultured in the presence or absence of TSA were washed once with phosphate-buffered saline and centrifuged at 1,500 rpm for 5 minutes. Cell pellets were resuspended in 100 µL of 20% annexin-V–fluorescein labeling reagent and 20% propidium iodide in HEPES (N-[2-hydroxyethyl]piperazine-N'-[4-butanesulfonic acid]) buffer. Cells were incubated at room temperature for 15 minutes, diluted with 0.8 mL of HEPES buffer, and analyzed by flow cytometry. Control cells were incubated for 15 minutes with annexin-V–fluorescein labeling reagent alone, propidium iodide alone, or HEPES buffer alone. All controls were then diluted with 0.8 mL of HEPES buffer and analyzed by flow cytometry.

Cell cycle analysis was performed using approximately 500,000 cells per treatment. Trichostatin A–treated and untreated cells were centrifuged for 5 minutes at 1,500 rpm and washed in phosphate-buffered saline. Cell pellets were then resuspended in 500 µL of propidium iodide solution (50 µg/mL propidium iodide, 0.1% NP-40, 0.1% sodium citrate). The mixture was incubated in the dark at 4°C for a minimum of 15 minutes, before analysis with flow cytometry.

Densitometry and Statistical Analysis
The concentration of TSA resulting in a 50% decrease in proliferation (IC50) was determined graphically in each case using nonlinear regression analysis to fit inhibition data to the appropriate dose–response curve using Graph Pad Prism v.3.2 (Graph Pad Software, San Diego, CA). The same software was also used to assess quantified differences in the number of adherent cells using a two-tailed Student's (parametric) t test. All differences were determined to be statistically significant if p was less than 0.05. Quantitation of the Western blots was accomplished with a densitometric-based analysis performed on scanned fluorograms using Scion Image Beta 4.02 software from Scion Corporation (Frederick, MD).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Trichostatin A Possesses Antiproliferative Activity Against Non–Small-Cell Lung Cancer Cells
Proliferation in all four NSCLC cell lines was inhibited by TSA in a dose-dependent fashion (Table 1). We determined that effective growth inhibition by TSA occurred within 48 hours. After 48 hours of incubation, the IC50 of the two squamous lines (Calu-1 and H520) was 0.034 and 0.046 µmol/L, respectively. The IC50 of the two adenocarcinoma lines (H441 and H23) was 0.012 and 0.054 µmol/L, respectively. The mean IC50 for all four NSCLC lines was 0.036 ± 0.019 µmol/L. In contrast to the tumor cell lines, the NLF line (mrc-9) was more than a log less sensitive to TSA (IC50 = 0.76 µmol/L). Figure 1 represents a comparison of all five cell lines at 0.1 µmol/L TSA, further indicating that NLF cells were about tenfold more resistant to TSA treatment.


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  		Table 1. Antiproliferative Effect of Trichostatin A on Non–Small-Cell Lung Cancer Cells Compared With Normal Lung Fibroblast Cell Line a
 

Figure 1
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  		Fig 1. Antiproliferative effect of trichostatin A. Non–small-cell lung cancer cells (NCI-H441, NCI-H23, NCI-H520, and Calu-1) and normal lung fibroblast cell line (mrc-9) were treated with 100 nmol/L trichostatin A. Data are plotted in reference to the dimethylsulfoxide control, and viability assays were performed at 48 hours. Error bars represent the standard deviation of at least three experiments.

 
Trichostatin A Treatment Induced Acetylation of Histone H4
To establish a link between the cellular effects of TSA and its presumed mechanism of action, the levels of histone H4 were examined after 24 hours of 1 µmol/L TSA treatment (Fig 2). An increased level of acetylated histone H4 was observed in all tumor cells treated with TSA in immunodetection assays using antibodies specific for acetylated histone H4. The acetylation level is higher for H520 and H23 compared with Calu-1 and H441. ß-actin levels were measured to verify equal loading.


Figure 2
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  		Fig 2. Increased acetylation of histone H4 by trichostatin A treatment. All four non–small-cell lung cancer cell lines were treated with either 1 µmol/L trichostatin A or dimethylsulfoxide (vehicle control) for 24 hours. Cells were disrupted with lysis buffer, and 50 µg of protein was separated by 15% sodium dodecylsulfate polyacrylamide gel electrophoresis. After transfer, the blot was probed with anti-acetylated histone H4 antibody. The same blot was stripped in buffer (1 mol/L Tris at pH 6.8, 10% sodium dodecylsulfate, and 0.67% ß-mercaptoethanol) for 30 minutes at 50°C and reprobed with ß-actin antibody as loading control. The results have been verified in two independent experiments.

 
Trichostatin A–Induced Cell Cycle Profiles
The effect of TSA treatment on cell cycle progression was analyzed in all four NSCLC lines and in the NLF line, mrc-9. Cells were treated with different doses for 48 hours, as described in Table 2. Cell cycle profiles were compared with DMSO-treated controls. G1 arrest was observed in Calu-1, H441, and H23 cells treated with 0.1 and 1.0 µmol/L TSA. The squamous line, H520, showed G2 arrest at a lower dose, but S phase arrest at 1.0 µmol/L TSA concentrations. Representative flow cytometry patterns for H23 cells treated with 1 µmol/L TSA for 48 hours are shown in Figure 3. Trichostatin A treatment also markedly increased the sub-G1 populations in H23 cells after 48 hours (Fig 3) and in other tumor lines (data not shown), indicating that these cells were undergoing apoptosis. Compared with most of the NSCLC lines, mrc-9 cells showed arrest in S phase at higher TSA concentrations (Table 2).


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  		Table 2. Trichostatin A Induced Cell Cycle Arrest in Non–Small-Cell Lung Cancer Cells and in Normal Lung Fibroblast Lines a
 

Figure 3
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  		Fig 3. Trichostatin A induction of cell cycle arrest in H23 cells. H23 cells were treated with vehicle (dimethylsulfoxide) or 1 µmol/L trichostatin A for 48 hours and analyzed for cell cycle inhibition by propidium iodide staining and flow cytometry as described in Material and Methods.

 
Trichostatin A–Induced Apoptosis in Lung Cancer Cells
There was a statistically significant increase in apoptotic fractions within cell populations of Calu-1, H23, H520, and H441 cells after 48 hours of treatment with 1 µmol/L TSA compared with mrc-9 cells (Table 3). A representative annexin–propidium iodide staining scatter plot of H23 cell populations treated with 1 µmol/L TSA or vehicle (DMSO) is shown in Figure 4.


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  		Table 3. Trichostatin A Induction of Apoptosis in Human Lung Cancer Cell Lines and Normal Human Fibroblast Cells a
 

Figure 4
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  		Fig 4. Trichostatin A induction of apoptosis in H23 cells. H23 cells were treated with vehicle (dimethylsulfoxide) or 1 µmol/L trichostatin A for 48 hours and analyzed for apoptotic cell death by annexin/propidium iodide (PI) staining. (FITC = fluorescein isothiocyanate.)

 
To determine the extent to which TSA induces apoptosis in NLF as compared with NSCLC cells for an earlier time point, mrc-9 cells and H441 cells were treated for 24 hours with 1 µmol/L TSA and then analyzed by annexin–propidium iodide staining. A 17% increase was observed in the percentage of apoptotic and necrotic H441 cells treated with 1 µmol/L TSA after 24 hours, as compared with a 7% increase in the percentage of apoptotic and necrotic mrc-9 cells treated with 1 µmol/L TSA after 24 hours (Table 2). In an independent experiment directly comparing mrc-9 cells and H23 cells treated for 48 hours with 1 µmol/L TSA, a 21% increase in the percentage of apoptotic and necrotic H23 cells was observed (compared to control cells), and a 12.9% increase in the percentage of apoptotic and necrotic mrc-9 cells was observed (data not shown).

Trichostatin A Treatment Induced Gelsolin Expression
Gelsolin, a multifunctional actin-binding protein, is used as a marker of apoptotic activity. Treatment with 1 µmol/L TSA for 24 hours increased gelsolin expression in all NSCLC lines (threefold to tenfold) as observed by the gelsolin-specific antibody (Fig 5). The baseline gelsolin protein level was lower in both adenocarcinoma lines (H441 and H23) compared with the two squamous lines (Calu-1 and H520).


Figure 5
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  		Fig 5. Increased gelsolin expression after trichostatin A treatment. Non–small-cell lung cancer cells were treated with 1 µmol/L trichostatin A for 24 hours, and equal amounts of protein (80 µg) from vehicle (dimethylsulfoxide) and trichostatin A–treated cell lysates were transferred to a nitrocellulose membrane using Western blotting technique. The figure represents the chemiluminescence band after antibody probing. The results in the figure represent one of the three independent experiments with similar results. The same blot was stripped and reprobed with ß-actin as loading control.

 
Expression of p21, p16, and p27 After Trichostatin A Treatment
Three NSCLC lines showed higher p21 expression (twofold to 15-fold) after 1 µmol/L TSA treatment for 24 hours (Fig 6A). A small level of p21 is detectable in H520 only after TSA treatment. The levels of two other cell cycle inhibitors, namely p16 and p27, were not significantly changed, indicating that p21 is an important mediator by which histone deacetylase inhibitors may arrest cell proliferation. However, the level of CDK2 and cyclin D1 was unaltered in these NSCLC lines after TSA treatment (Fig 6B).


Figure 6
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  		Fig 6. Expression of cell cycle proteins after trichostatin A treatment. (A) Western blot analysis of 80 µg of whole cell extract protein with specific antibodies as indicated. The blot was first probed with p21 followed by p27, p16, and ß-actin antibody after stripping. Vehicle (dimethylsulfoxide)-treated control and trichostatin A treatment are indicated by – and + signs, respectively. (B) Immunodetection of cell cycle protein CDK2 and Cyclin D1 in all four NSCLC cells after dimethylsulfoxide or 1 µmol/L trichostatin A treatment for 24 hours. The blot was first probed with CDK2 antibody followed by Cyclin D1 and ß-actin after stripping. Similar results are obtained in three independent experiments.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The antiproliferative properties of TSA were characterized in four NSCLC lines and in one NLF line. Differential effects in normal versus tumor cells were observed because certain concentrations of TSA produced significant cell death in NSCLC cells compared with normal controls, suggesting the possibility of a therapeutic window. The IC50, as determined on cell growth inhibition assay, is well below the physiologic dose. However, the significance of our observation needs to be further evaluated in vivo. Antiproliferative activity of TSA against acute myeloid leukemia, breast, and prostate cancer cells has been documented either alone or in combination with retinoic acid [22] and androgen receptors [15]. It is important to note that the antiproliferative activity of TSA depends on the cell line used and the duration of exposure. Maxhimer and coworkers [21] recently reported that two NSCLC lines, namely H322 and H460, need a relatively higher dose (>5 µmol/L) for TSA-induced apoptosis.

To elucidate the mechanism of TSA-induced cell death in NSCLC lines, we performed cell cycle analysis and annexin V staining, and monitored the expression level of gelsolin, an apoptotic marker. As expected, there was a substantial G1 arrest in three of four cell lines, including an increase in sub-G1 populations, indicating apoptosis. In contrast, the squamous NCI-H520 line showed substantial arrest in S phase with nonsignificant sub-G1 populations, suggesting a different mechanism of TSA activity in this cell line. To confirm further the apoptotic nature of cell death in NSCLC lines, we did independent experiments, studying the TSA-treated and vehicle (DMSO) -treated cells by annexin V staining. The flow cytometric analysis of stained cells confirmed the presence of TSA-induced apoptotic cell death in NSCLC lines. The NLF cells also showed apoptosis after 48 hours of treatment, but did not show any significant apoptotic staining after 24 hours of treatment. Our data suggest that mrc-9 cells had predominantly undergone cell death by cell cycle arrest after TSA treatment.

As further confirmation of the presence of apoptotic cell death, we investigated the overexpression of gelsolin in all four NSCLC lines after TSA treatment, which also supported the presence of apoptosis after compound treatment. Submicromolar concentrations of TSA have been reported to upregulate gelsolin expression and growth inhibition in human colon, cervical, and bladder cancer cells [23, 24]. Gelsolin has also been shown to inhibit apoptosis in Jarkat cells, promote apoptosis in neutrophils, and increase the sensitivity of Hela cells to tumor necrosis factor-1 (TNF1)-induced apoptosis, indicating the existence of multiple pathways for its recruitment into the process [25].

All of the above findings, in addition to the deformed morphology of the TSA-treated NSCLC cells (data not shown), are consistent with the process of apoptosis. This, taken together with viability counts for untreated and TSA-treated mrc-9 cells and untreated and treated NSCLC cells, suggests that NLF cells are less sensitive to TSA treatment than NSCLC lines in terms of drug-induced apoptotic cell death.

Increase in the acetylation status of histone H3 and H4 acts as a surrogate marker of HDAC inhibitor treatment. To investigate whether the effect of TSA correlates with modifications of histone acetylation, we evaluated the status of histone H4 acetylation in NSCLC cells. Increased histone acetylation after exposure to TSA has been observed in all tumor cells, suggesting that acetylated histone can be a surrogate marker in NSCLC lines after TSA treatment. The relationship between the effects of TSA on histone deacetylation and antiproliferative actions, however, remains to be determined.

Induction of p21, a key mediator of G1 arrest, is observed when tumor cells are treated with HDAC inhibitors, including TSA [10]. Overexpression of p21 is observed in all four NSCLC lines with a lesser effect observed in the H520 line. To establish a correlation between cell death, apoptosis, and p21 expression, we used the same dose and time of TSA treatment. Our data support the premise that TSA-induced NSCLC cell death leads to overexpression of a cell cycle regulator gene, one possible mechanism for how TSA may affect cell proliferation in NSCLC cells. Overexpression of p21 after TSA treatment has also been reported in myeloid cells [18], megakaryocytes [26], and other cell lines, with the exception of prostate cells, in which p21 expression is unaltered after TSA treatment [19]. Overexpression of inhibitor of cycline-dependent kinase 4 A (p16INK4A) has also been reported for other cell lines after TSA treatment [27]. Surprisingly, p16 protein level did not change in NSCLC cells, as did the level of p27 (twofold), another cell cycle inhibitor of CDK2. Trichostatin A inhibits cyclin D1 expression in a nuclear factor kappa-B–dependent manner in mouse JB6 cells [28], but the level of cyclin D1 and CDK2 were unaltered in these NSCLC cells after TSA treatment.

The mechanism of TSA-induced apoptosis in NSCLC cells is far from clear. Our results are consistent with the reports that the effect of TSA on NSCLC lines is to induce specific gene expression (eg, p21), which is associated with G1 arrest in many different cell types. The cdk inhibitor p21 is classically known to mediate cell arrest, and several reports have indicated the upregulation of p21 in different cell types including NSCLC cells [22]. However, the expression of p21 after TSA treatment differs for individual cell types. Murine embryo fibroblasts deficient in both p21 and p27 are also sensitive to TSA-induced growth arrest [29]. Similar observations were made in p21WAF/Cip–/– HCT116 cells [30]. Besides that, TSA inhibits proliferation of prostate cancer cell lines LNCaP and PC-3 without affecting p21 expression [19]. Of the four NSCLC cell lines we have tested, only the H520 line did not show significant overexpression of p21 after TSA treatment. Moreover, the relationship between p21 induction and antiproliferative activity of various lung cancer cell lines needs to be determined.

Our data regarding the overexpression of gelsolin in TSA-treated NSCLC cells are of particular interest. In recent years, studies of gelsolin displayed association with carcinogenesis and tumor prognosis. In human lung, breast, colon, prostate, and bladder carcinomas, downregulation of the gelsolin gene has been commonly observed [23–25, 31, 32]. Moreover, introduction of human wild-type complementary DNA of the gelsolin gene into murine and human cancer cell lines abrogates the in vivo tumorigenicity and colony-forming ability [31]. These phenomena indicate that gelsolin can suppress tumor growth. If gelsolin has tumor suppressor potential, it is plausible that patients with high levels of gelsolin should demonstrate improved survival compared with patients with diminished gelsolin. Various studies indicate the multifunctional and variable properties of gelsolin in different circumstances depending on the tumor type. Increased expression of gelsolin can increase cellular motility and help tumor cells to invade different organs faster to interfere with patient survival [33]. On the other hand, deficiency of gelsolin can lead to a variety of physiologic defects such as retarded remodeling of the actin cytoskeleton, impaired neutrophil migration, prolonged bleeding time, delayed blebbing, and DNA fragmentation after induction of apoptosis in gelsolin knockout mice [34, 35]. Our reports regarding the involvement of gelsolin during apoptosis probably imply that gelsolin downregulation may be one mechanism through which NSCLC cells evade apoptotic signaling pathways but become apoptotic after TSA treatment. The mechanism of overexpression of gelsolin after TSA treatment is far from clear. To our knowledge, this is probably the first example of TSA-induced overexpression of gelsolin in NSCLC cells, although TSA-induced gelsolin overexpression has been reported in breast, colon, gastric, and bladder cancers. It is also possible that the deformed morphology of NSCLC cells after TSA treatment is associated with the overexpression of gelsolin.

It is possible that TSA regulates transcription through proteins other than histones. However, little is known about the nonhistone targets of HDAC inhibitors, such as TSA, or their role in gene regulation. Recent reports indicate that tumor suppressor gene p53 is regulated by the acetylation–deacetylation process [4]. Erythroid Kruppel-like factor, a transcription factor important for erythropoiesis, is also acetylated in an active form [36]. Our unpublished data indicate that TSA-induced apoptosis of NSCLC cells is probably independent of p53, which is consistent with the observation in melanoma cells [37] in which TSA-induced apoptosis was independent of wild-type p53. Moreover, of three classes of HDAC, which includes class I, class II, and class III HDAC [38], only classes I and II are affected by TSA. Class III HDAC is not affected by TSA or by other hydroxamate inhibitors. At this moment, we do not know which HDACs are affected by TSA in NSCLC cells.

The selection of a molecule for drug development requires a balance of biologic potency, safety, and pharmacokinetics. Therefore, it is of paramount importance to elucidate the detailed pharmacokinetics and toxicologic properties of TSA before it can be considered as a potential new drug. Currently, several hydroxamate-based inhibitors, including TSA, show a lot of promise, as they have been shown to selectively inhibit tumor growth in animals at low and apparently nontoxic doses [39–41]. Several data show their clinical potential for the treatment of leukemia and solid tumors [39, 41, 42]. Because hydroxamate-based inhibitors (SAHA, 3-benzoyl-PHA, oxamflatin, 3,4-dimethoxy-phenyl derivative, 4-Me 2N-BACAH, 4-biphenyl CAH, Scriptaid, and D9) [38] including TSA display great potential in the treatment of hyperproliferative diseases such as cancer, most research has been performed on tumor cell lines. It has been reported that TSA dose-dependently inhibits growth and induces apoptosis in a plethora of carcinoma cell lines in vitro. It was also found that TSA inhibits angiogenesis, which is important for the growth or metastasis of solid tumors, both in vivo and in vitro [30]. Trichostatin A could be useful for the topical treatment of epidermal malignancies as TSA can induce an irreversible growth arrest in normal human epidermal keratinocytes and in two keratinocyte-derived squamous cell carcinoma cell lines [43]. Trichostatin A also shows some potential for transcriptional or differentiation therapy in acute myeloid leukemia as it potentiates the effect of retinoic acid on the induction of endogenous retinoic acid target genes [44].

Whether TSA will be effective in vivo needs detailed investigation. It induces differentiation and shows chemotherapeutic activity against N-methylnitrosourea-induced rat mammary cancer without toxicity [15]. On the other hand, TSA failed to show antitumor activity in nude mice bearing xenografts of human melanoma cells [45]. The lack of activity in the melanoma model is probably a consequence of metabolic inactivation of TSA by the liver and kidneys. It might also be a problem of insolubility of TSA in the aqueous vehicle that was used for administration. Moreover, TSA does not disturb embryonic or postnatal development of mice and appears nontoxic in adult mice [17, 46]. It also attenuates airway inflammation in a mouse asthma model [47]. Maxhimer and colleagues [21] predicted from their experiments on two NSCLC cell lines that treatment with TSA might not be clinically achievable without significant systemic toxicity.

In summary, in this report we have demonstrated that TSA-induced death of NSCLC and NLF cells is dose dependent, indicating the possibility of a therapeutic window. We have identified apoptosis as the principal mechanism of death of NSCLC cells by TSA with a higher expression level of gelsolin and p21. Whether TSA, TSA analogs, or TSA in combination with other agents (eg, protein kinase C inhibitor) will be the proper choice of treatment needs further investigation.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors would like to thank Dr Gavin G. Gordon, Ann S. Adams, MS, and Angeline S. Ferdinand, BS, for critical reading and editing, and Debra H. Cole and Amy Y. M. Wong for secretarial support. This work was partially supported by grants from the Lowe Center for Thoracic Oncology of the Dana-Farber Cancer Institute and the Brigham and Women's Physician Organization.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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