Histone deacetylases (HDAC) and histone acetyltransferases exert opposing enzymatic activities that modulate the degree of acetylation of histones and other intracellular molecular targets, thereby regulating gene expression, cellular differentiation, and survival. HDAC inhibition results in accumulation of acetylated histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bis-hydroxamide, on thyroid carcinoma cell lines, including lines originating from anaplastic and medullary carcinomas. In these models, both SAHA and m-carboxycinnamic acid bis-hydroxamide induced growth arrest and caspase-mediated apoptosis and increased p21 protein levels, retinoblastoma hypophosphorylation, BH3-interacting domain death agonist cleavage, Bax up-regulation, down-regulation of Bcl-2, A1, and Bcl-xL expression, and cleavage of poly(ADP-ribose) polymerase and caspase-8, -9, -3, -7, and -2. Transfection of Bcl-2 cDNA partially suppressed SAHA-induced cell death. SAHA down-regulated the expression of the apoptosis inhibitors FLIP and cIAP-2 and sensitized tumor cells to cytotoxic chemotherapy and death receptor activation. Our studies provide insight into the tumor type–specific mechanisms of antitumor effects of HDAC inhibitors and a framework for future clinical applications of HDAC inhibitors in patients with thyroid cancer, including histologic subtypes (e.g., anaplastic and medullary thyroid carcinomas) for which limited, if any, therapeutic options are available.

Thyroid cancer is the most prevalent endocrine neoplasia and is diagnosed each year in ∼22,000 new cases (∼16,300 in women and 5,700 in men) in the United States alone, and >1,500 patients are dying of thyroid cancer annually (1). For well-differentiated tumors, surgical resection and radioactive iodine can be an effective treatment. However, poorer prognosis is still associated with less differentiated histologic types, such as the tall-cell and the Huerthle variants. The even more undifferentiated anaplastic type carries an ominous prognosis (median overall survival of <6 months). Moreover, the histologically and phenotypically distinct medullary carcinomas, originating from the parafollicular C cells of the neural crest, also exhibit aggressive behavior and resist current therapeutic modalities.

A major mechanism controlling cellular differentiation and the biological behavior of cancer cells is via the regulation of acetylation of lysine residues on their amino-terminal tails of histones. Histone acetylation modulates nucleosome and chromatin structure and regulates transcription factor accessibility and function (26). The turnover of histone acetylation is regulated by the opposing activities of histone acetyltransferases and histone deacetylases (HDAC). In general, chromatin composed of nucleosomes with underacetylated histones is transcriptionally silent. Importantly, dysregulated histone acetyltransferase or HDAC activity has been found in certain human cancers (710). In this manner, tumor cells are unable to undergo the normal cellular differentiation programs, which contributes to their neoplastic transformation.

These findings suggest that the histone acetyltransferase/HDAC function balance is a potential therapeutic target for human malignancy. Several compounds, such as butyrates, the anticonvulsant valproic acid and the antifungal agent trichostatin A, have been shown to act as HDAC inhibitors (1114), but their clinical effectiveness has been limited by low potency, high toxicity, or poor stability. Depsipeptide (FR901228), a fermentation product isolated from Chromobacterium violaceum, has shown biological activity in a phase I trial in patients with refractory neoplasms (15). Recently, a class of novel synthetic hybrid polar compounds with potent inhibitory effect on HDAC activity has been described. The prototypes of this class of compounds, hydroxamic acid–based suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bis-hydroxamide (CBHA), cause accumulation of acetylated histones in cultured cells, induce differentiation and/or apoptosis of transformed cells in culture (25, 10, 16, 17), and inhibit the growth of tumors in animals (14, 17). SAHA binds directly to the HDAC catalytic site, potently inhibiting enzymatic activity (18), and selectively induces the expression of specific genes, such as p21WAF1/CIP1 cyclin-dependent kinase inhibitor (19, 20). Induction of p21 mRNA by SAHA involves changes in promoter-associated proteins, including acetylation of core proteins, increased DNase I sensitivity, marked decrease in HDAC1 and Myc, and an increase in RNA polymerase II binding to the proximal portion of the p21 promoter (21). Because the growth-suppressive and apoptotic activity of these agents seems restricted to transformed cells (10), these novel HDAC inhibitors represent promising anticancer agents.

In this study, we characterized the effect of SAHA and CBHA on thyroid carcinoma cell lines. These novel HDAC inhibitors had activity against all cell lines tested and induced accumulation of p21 and caspase-mediated apoptosis associated with cleavage of BH3-interacting domain death agonist (Bid). These studies provide the framework for the clinical evaluation of HDAC inhibitors to overcome clinical drug resistance and improve clinical outcome in patients with thyroid carcinoma. Ongoing clinical evaluation in patients with solid tumors has revealed that SAHA is biologically active, as evidenced by histone acetylation in vivo, and well tolerated (2224).

Cell lines. The SW579 cell line, derived from a poorly differentiated human thyroid adenocarcinoma (poorly differentiated carcinoma with nuclear features of papillary carcinoma and squamous differentiation), and the TT cell line, derived from a medullary thyroid carcinoma, were purchased from American Type Culture Collection (Manassas, VA). The anaplastic thyroid carcinoma cell lines FRO and ARO and the follicular carcinoma cell line WRO were a generous gift of Dr. James A. Fagin (University of Cincinnati School of Medicine, Cincinnati, OH; refs. 25, 26). All cells were grown in DMEM (BioWhittaker, Walkersville, MD) with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% FCS (Life Technologies, Gaithersburg, MD), unless stated otherwise.

Reagents. SAHA and CBHA were obtained from Aton Pharma, Inc. (Tarrytown, NY). Administration of SAHA as an i.v. infusion at a dose of 300 mg/m2 (which was determined to be the maximum tolerated dose for the hematologic patients) resulted in an average plasma SAHA Cmax of 2,638 ng/mL (9.99 μmol/L) in hematologic malignancies and as high as 3,298 ng/mL (12.46 μmol/L) in solid malignancies. Intravenous infusion of SAHA at 600 mg/m2 resulted in average Cmax as high as 10,815 ng/mL (40.96 μmol/L), yet some toxicity was observed at this dose (22). These data suggest that the in vitro use of SAHA concentrations in the range of 1 to 10 μmol/L is physiologically relevant and clinically achievable without prohibiting toxicity. Most of our studies were done with 1 to 5 μmol/L HDAC concentrations.

The anti-Fas antibody CH-11 was from Panvera (Madison, WI) and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)/Apo2L was from Biomol (Plymouth Meeting, PA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and doxorubicin were from Sigma Chemical Co. (St. Louis, MO).

Histone acetylation. FRO cells were treated with SAHA, CBHA, or vehicle as indicated. Histones were then isolated as described previously (17). Briefly, the cells were harvested in ice-cold PBS, centrifuged at 1,000 rpm for 5 minutes, resuspended in 1 mL histone lysis buffer [8.6% sucrose, 1% Triton X-100, 50 mmol/L sodium bisulfite, 10 mmol/L Tris-HCl (pH 6.5), 10 mmol/L MgCl2], and Dounce homogenized. Cell lysates were centrifuged at 700 rpm for 5 minutes. The nuclear pellet was then washed thrice with the lysis buffer and once with 10 mmol/L Tris-HCl (pH 7.4) and 13 mmol/L EDTA. Histones were acid extracted from the nuclear pellets as described previously (27). Acetylation of core histones in the cells or tumors was determined by Western blotting using rabbit polyclonal antibodies against acetylated histone H3 and H4 (Upstate Biotechnology, Lake Placid, NY) and visualized using enhanced chemiluminescence.

Propidium iodide staining. For cell cycle analysis, 1 × 106 cells were incubated with or without 1 μmol/L SAHA in 10% FCS for 24, 48, or 72 hours. The cells were then washed twice with PBS, permeabilized with 70% ethanol in PBS for 30 minutes at 4°C, incubated with 0.5 mL of a 50 μg/mL propidium iodide solution containing 20 units/mL RNase A (Boehringer Mannheim) for 30 minutes, and analyzed by flow cytometry.

Bromodeoxyuridine incorporation assay. Cell proliferation in cells treated with SAHA or CBHA (2.5, 5, and 10 μmol/L for 48 hours) was quantified by measuring the amount of bromodeoxyuridine incorporated into nuclear DNA using the bromodeoxyuridine incorporation assay (Oncogene Research, Cambridge, MA) according to the instructions of the manufacturer.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assay. Cell survival was examined using the MTT colorimetric assay as described previously (28). Dye absorbance (A) in viable cells was measured at 570 nm, with 630 nm as a reference wavelength. Cell viability was estimated as a percentage of the value of untreated controls. All experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments.

Colony formation assay. The potential of SAHA-treated thyroid carcinoma cells for long-term growth and colony formation was evaluated with a modification of the method of de Nigris et al. (29). Briefly, cells were treated with SAHA or vehicle in 24-well plates for 48 hours at the IC50 concentration for each cell line. Subsequently, the cells from each well were trypsinized, plated on a respective T75 cm2 flask, and allowed to grow in drug-free medium containing 10% fetal bovine serum. Fourteen days later (7 days for FRO due to very rapid cell growth rate), live cell colonies were stained with MTT and counted.

Lactate dehydrogenase release assay. FRO cells were preincubated with the pan-caspase inhibitor ZVAD-FMK, the caspase-8 inhibitor IETD-FMK, the caspase-9 inhibitor LEHD-FMK, or the caspase-3/7 inhibitor DEVD-FMK (all used at 20 μmol/L and all from Oncogene Research) for 1 hour before exposure to SAHA (5 μmol/L for 36 hours). Quantification of cell death was done by measuring the activity of lactate dehydrogenase released from the cytosol of damaged cells into the culture supernatant using the Cytotoxicity Detection kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the instructions of the manufacturer.

Immunoblotting analysis. Immunoblotting analysis was done as described previously (28). The antibodies used were as follows: mouse monoclonal antibodies for Bcl-2, Bcl-xL, A1, Bax, and tubulin and polyclonal antibodies for caspase-3 and -9 (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody for caspase-8 and polyclonal antibodies for FLIP and phospho-retinoblastoma (Rb; Upstate Biotechnology); monoclonal antibody for poly(ADP-ribose) polymerase (Biomol); polyclonal antiserum against cIAP-2 (R&D Systems, Inc., Minneapolis, MN); monoclonal antibody for p21 (Oncogene Research); polyclonal antiserum against Bid and caspase-2 and -7 (Cell Signaling, Beverly, MA); Complete mixture of proteinase inhibitors and SDS (Life Technologies); and enhanced chemiluminescence kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies (Amersham, Arlington Heights, IL).

Effect of suberoylanilide hydroxamic acid in thyroid carcinoma cells overexpressing Bcl-2. To evaluate the role of the antiapoptotic molecule Bcl-2 in SAHA-induced cell death, anaplastic carcinoma FRO cells were stably transfected with a vector carrying the Bcl-2 cDNA (Upstate Biotechnology) or the empty (neo) vector using LipofectAMINE 2000 according to the instructions of the manufacturer. Forty-eight hours later, the cells were incubated in growth medium containing G418 (500 μg/mL, Life Technologies) to select pools of stable clones that were subsequently treated with SAHA (2.5-10 μmol/L for 36 hours). The overexpression of Bcl-2 in transfected cells has been confirmed by immunoblotting.

Statistical analysis. Statistical significance was examined by two-way ANOVA followed by Duncan's post hoc test. In all analyses, P < 0.05 was considered statistically significant.

Suberoylanilide hydroxamic acid induces accumulation of acetylated histones in thyroid carcinoma cells. We next investigated the effect of SAHA and CBHA on histone acetylation status in anaplastic thyroid carcinoma FRO cells. FRO cells treated with SAHA or CBHA for 8 hours exhibited increased acetylation of histones H3 and H4 compared with controls (Fig. 1A). Protein loading was monitored by Coomassie blue staining. A dose-response experiment for levels of acetylated H3 in FRO cells treated with SAHA (1-10 μmol/L) is shown in Fig. 1B.

Fig. 1.

SAHA induces accumulation of acetylated histones in thyroid carcinoma cells. A, FRO cells treated with SAHA or CBHA (5 μmol/L) for 8 hours exhibited significantly increased acetylation of histones H3 and H4 (AcH3 and AcH4, respectively) than controls. Protein loading was monitored by Coomassie blue. B, dose-response experiment for levels of acetylated H3 in FRO cells treated with SAHA (1-10 μmol/L) for 8 hours.

Fig. 1.

SAHA induces accumulation of acetylated histones in thyroid carcinoma cells. A, FRO cells treated with SAHA or CBHA (5 μmol/L) for 8 hours exhibited significantly increased acetylation of histones H3 and H4 (AcH3 and AcH4, respectively) than controls. Protein loading was monitored by Coomassie blue. B, dose-response experiment for levels of acetylated H3 in FRO cells treated with SAHA (1-10 μmol/L) for 8 hours.

Close modal

Suberoylanilide hydroxamic acid induces growth arrest and apoptosis in thyroid carcinoma cells. We first investigated the effect of SAHA and CBHA on growth and survival of thyroid carcinoma cells. Treatment of papillary (SW579), follicular (WRO), anaplastic (FRO and ARO), and medullary (TT) thyroid carcinoma cells with SAHA or CBHA (2.5, 5, and 10 μmol/L) for 48 hours potently suppressed cellular proliferation as quantified by bromodeoxyuridine incorporation (Fig. 2).

Fig. 2.

SAHA and CBHA suppress the growth of thyroid carcinoma cells. A and B, quantification of proliferation of thyroid carcinoma cells using the bromodeoxyuridine (BrdU) incorporation assay according to the instructions of the manufacturer. Papillary (SW579, ⧫), follicular (WRO, ×), anaplastic (FRO, ▪; ARO, ▴), and medullary (TT, •) thyroid carcinoma cells were treated with SAHA (A) or CBHA (B) or vehicle at the indicated concentrations in the presence of 10% fetal bovine serum for 48 hours. Points, percentages over those of vehicle-treated controls. Both HDAC inhibitors potently inhibited the proliferation of all cell lines tested.

Fig. 2.

SAHA and CBHA suppress the growth of thyroid carcinoma cells. A and B, quantification of proliferation of thyroid carcinoma cells using the bromodeoxyuridine (BrdU) incorporation assay according to the instructions of the manufacturer. Papillary (SW579, ⧫), follicular (WRO, ×), anaplastic (FRO, ▪; ARO, ▴), and medullary (TT, •) thyroid carcinoma cells were treated with SAHA (A) or CBHA (B) or vehicle at the indicated concentrations in the presence of 10% fetal bovine serum for 48 hours. Points, percentages over those of vehicle-treated controls. Both HDAC inhibitors potently inhibited the proliferation of all cell lines tested.

Close modal

Cell cycle analysis by propidium iodide of FRO cells treated with SAHA or CBHA (1 μmol/L for 24-72 hours) revealed inhibition of proliferation and induction of apoptosis (Fig. 3). Treatment of FRO, TT, and ARO cells with SAHA at the concentration determined to inhibit 50% of cell proliferation (bromodeoxyuridine incorporation in Fig. 2), which was 3.85, 1.7, and 2 μmol/L, respectively, resulted in 97 ± 0.5%, 85 ± 2%, and 55 ± 3% decrease in colony formation, respectively, suggesting that SAHA severely impairs the long-term growth potential of thyroid carcinoma cells.

Fig. 3.

SAHA and CBHA induce growth arrest and apoptosis in thyroid carcinoma cells. A-F, propidium iodide analysis of thyroid carcinoma cells that were treated with SAHA (1 μmol/L) in 2% FCS for 24 (B), 36 (C), 48 (D), 60 (E), and 72 (F) hours or left untreated (A) for 72 hours. The percentage of cells in the sub-G1 region indicates significant HDAC inhibitor–induced cell death. Similar results were obtained with treatment with CBHA (1 μmol/L).

Fig. 3.

SAHA and CBHA induce growth arrest and apoptosis in thyroid carcinoma cells. A-F, propidium iodide analysis of thyroid carcinoma cells that were treated with SAHA (1 μmol/L) in 2% FCS for 24 (B), 36 (C), 48 (D), 60 (E), and 72 (F) hours or left untreated (A) for 72 hours. The percentage of cells in the sub-G1 region indicates significant HDAC inhibitor–induced cell death. Similar results were obtained with treatment with CBHA (1 μmol/L).

Close modal

Suberoylanilide hydroxamic acid increases p21 protein levels and decreases phosphorylation of retinoblastoma. To detect the mediators of SAHA-induced growth arrest, we evaluated the levels of p21 and p53 by immunoblotting analysis. SAHA rapidly increased p21 protein levels in anaplastic thyroid carcinoma FRO cells (Fig. 4). Because p21 induces growth arrest by inhibiting the ability of the cyclin E/cyclin-dependent kinase2 complex to phosphorylate the cell cycle regulator Rb, we investigated the effect of SAHA on the phosphorylation status of Rb. We found that SAHA profoundly and rapidly decreased the phosphorylation of Rb. These data suggest that SAHA-induced growth arrest occurs via p21-mediated inhibition of Rb phosphorylation. In addition, SAHA transiently stimulated the expression of the proapoptotic Bax.

Fig. 4.

Growth arrest induced by SAHA (5 μmol/L) in FRO cells is associated with early up-regulation of p21, and decrease in phospho-Rb levels, as evidenced by immunoblotting. Bax expression is also transiently up-regulated.

Fig. 4.

Growth arrest induced by SAHA (5 μmol/L) in FRO cells is associated with early up-regulation of p21, and decrease in phospho-Rb levels, as evidenced by immunoblotting. Bax expression is also transiently up-regulated.

Close modal

Involvement of caspases, but not p53, in suberoylanilide hydroxamic acid–induced apoptosis. We subsequently assessed the functional role of caspases in SAHA-induced apoptosis in thyroid carcinoma cells. We found by immunoblotting that SAHA induced cleavage of caspase-8 and -9 followed by caspase-3, -7, and -2 in our model (Fig. 5A). We also found cleavage of poly(ADP-ribose) polymerase, a protein known to be enzymatically cleaved during apoptosis, resulting in the classic 85-kDa poly(ADP-ribose) polymerase fragment. Of note, p53 expression was not significantly modified by SAHA treatment.

Fig. 5.

Involvement of caspases, but not p53, in SAHA-induced apoptosis in thyroid carcinoma cells. A, immunoblotting for p53 and caspase-8, -9, and -3 in FRO cells treated with SAHA (5 μmol/L) detected cleavage of caspase-8, -9, and -3 as well as poly(ADP-ribose) polymerase (PARP). p53 expression remained practically unchanged. It should be noted that FRO cells express minimal amounts of p53 (26), necessitating very prolonged film exposure to obtain signal. B, quantification of cell death (average ± SD) induced in FRO cells by SAHA (5 μmol/L for 36 hours), in the presence of specific caspase inhibitors (1 hour pretreatment), using the lactate dehydrogenase release assay. The pan-caspase inhibitor ZVAD-FMK (20 μmol/L) completely abrogated SAHA-induced apoptosis. Moreover, the caspase-8 inhibitor IETD-FMK, the caspase-9 inhibitor LEHD-FMK, and the caspase-3 inhibitor DEVD-FMK (all used at 20 μmol/L) also had a protective effect.

Fig. 5.

Involvement of caspases, but not p53, in SAHA-induced apoptosis in thyroid carcinoma cells. A, immunoblotting for p53 and caspase-8, -9, and -3 in FRO cells treated with SAHA (5 μmol/L) detected cleavage of caspase-8, -9, and -3 as well as poly(ADP-ribose) polymerase (PARP). p53 expression remained practically unchanged. It should be noted that FRO cells express minimal amounts of p53 (26), necessitating very prolonged film exposure to obtain signal. B, quantification of cell death (average ± SD) induced in FRO cells by SAHA (5 μmol/L for 36 hours), in the presence of specific caspase inhibitors (1 hour pretreatment), using the lactate dehydrogenase release assay. The pan-caspase inhibitor ZVAD-FMK (20 μmol/L) completely abrogated SAHA-induced apoptosis. Moreover, the caspase-8 inhibitor IETD-FMK, the caspase-9 inhibitor LEHD-FMK, and the caspase-3 inhibitor DEVD-FMK (all used at 20 μmol/L) also had a protective effect.

Close modal

We next further evaluated the functional role of caspases in our model. We found that the pan-caspase inhibitor ZVAD-FMK completely abrogated SAHA-induced cell death in FRO cells (Fig. 5B), establishing a role for caspases in this model. Moreover, the caspase-8 inhibitor IETD-FMK, the caspase-9 inhibitor LEHD-FMK, and the caspase-3/7 inhibitor DEVD-FMK also exerted protective effect.

Suberoylanilide hydroxamic acid–induced cell death is regulated by members of the Bcl-2 family. Subsequently, we investigated the involvement of members of the Bcl-2 family in SAHA-induced cell death. SAHA treatment promotes cleavage of the Bcl-2 family member Bid (Fig. 6A). Cleavage of Bid results in a truncated form, which translocates to the mitochondria and results in an allosteric activation of Bak and Bax, inducing their intramembranous oligomerization that leads to mitochondrial dysfunction (30). These events are counteracted by the antiapoptotic members of the Bcl-2 family, such as Bcl-2, A1, and Bcl-xL. We also found that SAHA down-regulated the expression of Bcl-2, A1, and Bcl-xL, thus shifting the balance toward the proapoptotic members of the Bcl-2 family. Therefore, we hypothesized that inhibition of Bid-induced mitochondrial events would protect from SAHA-induced cell death. Indeed, overexpression of Bcl-2 in thyroid carcinoma cells partially protected them from SAHA-induced cell death (Fig. 6B). These data suggest a role for mitochondria and the Bcl-2 family members in SAHA-induced apoptotic signaling.

Fig. 6.

Involvement of Bcl-2 family members in SAHA-induced cell death in thyroid carcinoma cells. A, SAHA (5 μmol/L) induced cleavage of Bid in FRO cells, a member of the Bcl-2 family that, on cleavage, translocates to the mitochondria to promote apoptosis. In addition, SAHA down-regulated the expression of the antiapoptotic Bcl-2 family members Bcl-2, A1, and Bcl-xL. B, overexpression of Bcl-2, which stabilizes the mitochondria and antagonizes the effects of Bid, partially protected FRO cells from SAHA-induced cell death (36 hours; ▪, Bcl-2-transfected FRO cells; ⧫, FRO cells transfected with the empty vector). Percent cell viability (mean ± SD) was quantified by MTT. Experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. Inset, expression of Bcl-2, as evidenced by immunoblotting, in FRO cells transfected with the empty vector or the Bcl-2 cDNA.

Fig. 6.

Involvement of Bcl-2 family members in SAHA-induced cell death in thyroid carcinoma cells. A, SAHA (5 μmol/L) induced cleavage of Bid in FRO cells, a member of the Bcl-2 family that, on cleavage, translocates to the mitochondria to promote apoptosis. In addition, SAHA down-regulated the expression of the antiapoptotic Bcl-2 family members Bcl-2, A1, and Bcl-xL. B, overexpression of Bcl-2, which stabilizes the mitochondria and antagonizes the effects of Bid, partially protected FRO cells from SAHA-induced cell death (36 hours; ▪, Bcl-2-transfected FRO cells; ⧫, FRO cells transfected with the empty vector). Percent cell viability (mean ± SD) was quantified by MTT. Experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. Inset, expression of Bcl-2, as evidenced by immunoblotting, in FRO cells transfected with the empty vector or the Bcl-2 cDNA.

Close modal

Suberoylanilide hydroxamic acid sensitizes thyroid carcinoma cells to death receptor–induced cell death. We also investigated the effect of SAHA on cell death induced by cell surface death receptors. SAHA sensitized thyroid carcinoma cells to cell death mediated by cross-linking Fas with the CH-11 antibody and to cell death induced by TRAIL/Apo2L (Fig. 7A). This sensitizing effect was associated with decreased expression of the antiapoptotic proteins FLIP and cIAP-2 (Fig. 7B).

Fig. 7.

Sensitizing effect of SAHA to death receptor–induced apoptosis in thyroid carcinoma cells. A, FRO cells were pretreated with SAHA (5 μmol/L) for 6 hours and then treated with the Fas activating antibody CH-11 (200 ng/mL) or a low concentration of TRAIL/Apo2L (10 ng/mL) for additional 18 hours. SAHA potently sensitized the anaplastic thyroid carcinoma cells to both Fas and TRAIL death receptor pathways. Percent cell survival (mean ± SD) was quantified by MTT. All experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. B, SAHA down-regulated the antiapoptotic proteins FLIP and cIAP-2, which inhibit death receptor–mediated apoptotic signaling. SAHA also up-regulated TRAIL-R1 and TRAIL-R2 and, to a lesser extent, Fas expression.

Fig. 7.

Sensitizing effect of SAHA to death receptor–induced apoptosis in thyroid carcinoma cells. A, FRO cells were pretreated with SAHA (5 μmol/L) for 6 hours and then treated with the Fas activating antibody CH-11 (200 ng/mL) or a low concentration of TRAIL/Apo2L (10 ng/mL) for additional 18 hours. SAHA potently sensitized the anaplastic thyroid carcinoma cells to both Fas and TRAIL death receptor pathways. Percent cell survival (mean ± SD) was quantified by MTT. All experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. B, SAHA down-regulated the antiapoptotic proteins FLIP and cIAP-2, which inhibit death receptor–mediated apoptotic signaling. SAHA also up-regulated TRAIL-R1 and TRAIL-R2 and, to a lesser extent, Fas expression.

Close modal

Suberoylanilide hydroxamic acid sensitizes thyroid carcinoma cells to cytotoxic chemotherapy. Finally, we studied the effect of SAHA on the sensitivity of thyroid carcinoma cells to DNA damage using as a model the chemotherapeutic agent doxorubicin. We treated SW579, FRO, and TT cells concurrently with doxorubicin (0.25 μg/mL) and SAHA (at indicated concentrations) for 48 hours. Thyroid carcinoma cells are relatively resistant to conventional chemotherapy, but cotreatment with SAHA had a strong synergistic effect (Fig. 8). The LD50 for SAHA alone in this experiment was 7.66, 2.38, and 0.72 μmol/L for SW579, FRO, and TT cells, respectively, compared with 1.68, 0.39, and 0.38 μmol/L, respectively, for treatment with SAHA in the presence of doxorubicin.

Fig. 8.

Sensitizing effect of SAHA to conventional cytotoxic chemotherapy in thyroid carcinoma cells. SW579 (A), FRO (B), and TT (C) cells were concurrently treated with doxorubicin (0.25 μg/mL) and SAHA (1-5, 0.25-0.5, and 0.5-1 μmol/L, respectively) for 48 hours. Percent cell survival (mean ± SD) was quantified by MTT. All experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. Thyroid carcinoma cells are relatively resistant to cytotoxic chemotherapy, but cotreatment with SAHA had a strong sensitizing effect on doxorubicin-induced cell death.

Fig. 8.

Sensitizing effect of SAHA to conventional cytotoxic chemotherapy in thyroid carcinoma cells. SW579 (A), FRO (B), and TT (C) cells were concurrently treated with doxorubicin (0.25 μg/mL) and SAHA (1-5, 0.25-0.5, and 0.5-1 μmol/L, respectively) for 48 hours. Percent cell survival (mean ± SD) was quantified by MTT. All experiments were repeated at least thrice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Columns, average values of representative experiments; bars, SD. Thyroid carcinoma cells are relatively resistant to cytotoxic chemotherapy, but cotreatment with SAHA had a strong sensitizing effect on doxorubicin-induced cell death.

Close modal

From a clinical and biological standpoint, thyroid cancers represent a broad spectrum of neoplastic disorders that include histologic subtypes, such as poorly differentiated, anaplastic and medullary thyroid carcinomas, which are refractory to most conventional systemic anticancer therapeutic strategies, highlighting the need for novel therapeutic strategies. In this study, we evaluated the effects of the novel, potent HDAC inhibitors SAHA and CBHA in a panel of thyroid carcinoma cell lines and found that they potently suppress proliferation and induce apoptosis. HDAC inhibitor–induced apoptosis in this tumor model involved cleavage of Bid, down-regulation of Bcl-2, A1, and Bcl-xL, and activation of caspases. Furthermore, SAHA increased the sensitivity of thyroid carcinoma cells to death receptor–induced apoptosis and cytotoxic chemotherapy.

Prior studies have shown that the HDAC inhibitor depsipeptide enhances apoptotic killing by p53 gene therapy in thyroid carcinoma cells (31) and increases expression of the Na+/I symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells (32). Moreover, the HDAC inhibitors sodium butyrate and trichostatin A induce cell cycle arrest and promote apoptosis in anaplastic thyroid cancer cells (33). The novel synthetic hybrid polar compounds, such as SAHA and CBHA, potently inhibit HDAC activity and induce accumulation of acetylated histones, differentiation, and/or apoptosis of transformed cells in vitro (25, 10, 16, 17). SAHA suppresses the growth of prostate carcinoma cell lines at micromolar concentrations (2.5-7.5 μmol/L) in vitro (17) and induces accumulation of acetylated histones in tumor tissue from xenografts of human prostate carcinomas and neuroblastomas (34) in nude mice. In this study, we show potent activity of SAHA and CBHA against a panel of thyroid carcinoma cell lines that includes lines originating from papillary, follicular, anaplastic, and medullary carcinomas. The activity of these novel agents against medullary carcinoma cells is particularly impressive, because this type of tumor resists current chemotherapeutic approaches.

SAHA and CBHA induced accumulation of acetylated histones in our model, early up-regulation of the cyclin-dependent kinase inhibitor p21, decrease in phosphorylation of the cyclin-dependent kinase substrate Rb, followed by growth arrest and apoptosis. It should be noted that the SW579, ARO, and WRO cell lines carry mutant p53 (26, 35) and that the FRO line expresses very low levels of p53 mRNA (26), which did not increase with SAHA treatment. Therefore, p21 up-regulation seems to be p53 independent, consistent with studies by Richon et al. (19), Huang et al. (20), and Vrana et al. (36), showing that SAHA-induced up-regulation of p21 is mediated by Sp1 sites in the p21 promoter and is p53 independent. Therefore, HDAC inhibitors, such as SAHA and CBHA, are expected to be active even against malignant cells with defects in the p53 pathway. This is of particular clinical importance because p53 mutations are among the most common genetic aberration in human cancer in general and because they are also very frequent in poorly differentiated and anaplastic thyroid cancer (26).

We then investigated the mechanism of SAHA-induced apoptosis in our model and detected cleavage of caspase-8 and -9 and (later) caspase-3, -7, and -2. Moreover, poly(ADP-ribose) polymerase was cleaved into an 85-kDa fragment, indicating caspase activation. In support, the pan-caspase inhibitor ZVAD-FMK completely abolished SAHA-induced apoptosis in FRO cells, and the specific caspase-8, -9, and -3/2 inhibitors, respectively, exerted a significant protective effect. Therefore, in this model, SAHA-induced apoptosis seems to be caspase mediated. The role of caspases in HDAC inhibitor–induced apoptosis seems to be tumor type or tissue specific. We and others have reported cleavage of Bid and caspase-independent cell death in SAHA-treated malignant cells of hematopoietic origin (37, 38), whereas, in solid tumor models, HDAC inhibitor–induced cell death is caspase mediated (39, 40).

SAHA treatment of multiple myeloma cells irreversibly commits them to apoptosis within 8 hours as well, as documented by experiments where the drug was washed away after brief exposures and the cells were further incubated in drug-free medium to detect the resulting apoptosis (41). In agreement, SAHA treatment of thyroid carcinoma cells in the present study resulted in evidence of caspase cleavage as early as within 8 hours of incubation, suggesting early commitment to apoptosis.

The induction by SAHA of a dual apoptotic cascade mediated by activation of both caspase-8 and -9 prompted us to further investigate the involvement of death receptor–mediated and mitochondrial regulators of apoptosis. Activation of caspase-8 is the initiator of death receptor (e.g., Fas or TRAIL receptor)–mediated apoptosis (42, 43), whereas caspase-9 activation is a nodal point for mitochondrial-induced apoptotic signaling (44). There is also cross-talk between the two apoptotic pathways, because caspase-8 can cleave the Bcl-2 family member Bid, which then translocates to the mitochondria and results in an allosteric activation of Bak and Bax, inducing their intramembranous oligomerization that leads to mitochondrial dysfunction (30). Cleavage of Bid was also detected in our model together with transient up-regulation of Bax on SAHA treatment. Moreover, SAHA down-regulated the levels of Bcl-2, A1, and Bcl-xL, suggesting that SAHA-induced apoptosis can be related to a shift in the balance between proapoptotic and antiapoptotic members of the Bcl-2 family toward the proapoptotic side. In support of these findings, overexpression of Bcl-2 in thyroid carcinoma cells partially attenuated SAHA-induced cell death. The degree of antiapoptotic protective effect of Bcl-2 overexpression varies between experimental models and proapoptotic stimuli (45, 46), ranging between complete suppression of apoptosis and no effect. In experimental models where the proapoptotic agent efficiently activates caspase-8, apoptosis proceeds in a mitochondrial-independent manner and overexpression of Bcl-2 has no protective effect (47). On the contrary, overexpression of Bcl-2 completely abrogates apoptosis in models where caspase-8 is inefficiently activated and the apoptotic pathway requires mitochondrial involvement (47). Our data suggest that SAHA-induced apoptosis in thyroid carcinoma cells is at least partially mediated by the mitochondria. Multiple myeloma cells, on the other hand, are completely protected against SAHA-induced cell death by overexpression of Bcl-2 (37), suggesting a greater importance of mitochondrial-dependent signaling in that model.

We next investigated the effect of SAHA on apoptosis induced by the cell surface death ligands FasL and TRAIL via their respective receptors Fas and TRAIL-R1(DR4)/TRAIL-R2(DR5) (ref. 48). FasL and TRAIL are death ligands expressed by activated immune effector cells and participate in cell-mediated cytotoxicity and antitumor surveillance (48). Modulation of cancer cell sensitivity to the Fas and TRAIL apoptotic pathways could affect their response to immune-based therapeutic approaches. This is of particular importance in thyroid carcinomas that are frequently associated with an intense inflammatory reaction (“peritumoral thyroiditis”; ref. 49). Thyroid carcinoma cells are sensitive to apoptosis induced by TRAIL and resistant to FasL (50). We now found that SAHA sensitized thyroid carcinoma cells to the Fas-activating monoclonal antibody CH-11 and to a low, subtoxic concentration of recombinant TRAIL. Therefore, HDAC inhibitors may modulate immune responsiveness of tumor cells and could be useful to overcome refractoriness to immune-based therapies. Furthermore, these data indicate that HDAC inhibitors can influence the interaction of the tumor cells with their local microenvironment. This sensitizing effect was associated with decreased expression of the antiapoptotic proteins FLIP and cIAP-2 as well as up-regulation of TRAIL-R1 and TRAIL-R2 and, to a lesser extent, Fas expression. FLIP inhibits death receptor–induced apoptosis in thyroid carcinomas (51) and the protein synthesis inhibitor cycloheximide, the protein kinase C inhibitor bisindolylmaleimide, and a FLIP antisense oligonucleotide sensitize thyroid carcinoma cells to death receptor–mediated apoptosis by down-regulating FLIP (51). However, none of these reagents represents a currently available clinical approach for the treatment of aggressive thyroid cancer. Our study identifies SAHA as the first clinically applicable pharmacologic agent that can down-regulate FLIP and increase sensitivity to TRAIL and, more importantly, restore sensitivity to Fas-mediated apoptosis.

Thyroid carcinomas are generally poorly responsive to cytotoxic chemotherapy, which could be attributed to the presence of intracellular inhibitors of the apoptotic signaling cascade. Having shown the ability of HDAC inhibitors to down-regulate the expression of antiapoptotic proteins, such as Bcl-2, Bcl-xL, cIAP-2, and FLIP, and to up-regulate expression of proapoptotic Bax, we investigated its effect on doxorubicin-induced cell death and found that HDAC inhibition confers to thyroid cancer cells a potent chemosensitizing effect. This finding suggests that novel therapies combining SAHA with conventional chemotherapy could improve the outcome in aggressive thyroid cancer.

The clinical applicability of SAHA administration in cancer patients is highlighted by the early experience generated from phase I clinical trials, where SAHA, despite a relatively short plasma half-life after i.v. infusion, was found to be biologically active (inducing accumulation of acetylated histones in vivo for at least 4 hours after infusion), with an acceptable safety profile (22). There was also early evidence of antitumor activity (2224), including reduction in measurable disease in refractory papillary thyroid cancer (52). Early clinical studies of an oral SAHA formulation are currently under way (52).

In conclusion, we have evaluated the effects of HDAC inhibition in a panel of thyroid carcinoma cell lines using the novel HDAC inhibitors SAHA and CBHA. We found that these HDAC inhibitors suppressed growth in all cell lines tested, including those with defects in the p53 pathway. SAHA up-regulated p21 expression, promoted cleavage of Bid, down-regulated Bcl-2, A1, and Bcl-xL levels, and induced caspase-mediated apoptosis. SAHA sensitized thyroid carcinoma cells to apoptosis mediated by death receptors and cytotoxic chemotherapy. These findings provide the preclinical rationale for clinical studies of SAHA, either as a monotherapy or in combination with other anticancer therapies, in an effort to improve the clinical outcome of patients with aggressive cases of thyroid cancer.

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: The Memorial Sloan-Kettering Cancer Center and Columbia University jointly hold patents on the hydroxamic acid–based hybrid polar compounds, including SAHA, which are exclusively licensed to Aton Pharma, Inc. (Tarrytown, NY), a subsidiary of Merck & Co., Inc. (Rahway, NJ). Both institutions and founder have an equity position in Aton Pharma. P.A. Marks is a founder and member of the Board of Directors of Aton Pharma.

1
Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003.
CA Cancer J Clin
2003
;
53
:
5
–26.
2
Cheung P, Allis CD, Sassone-Corsi P. Signaling to chromatin through histone modifications.
Cell
2000
;
103
:
263
–71.
3
Jenuwein T, Allis CD. Translating the histone code.
Science
2001
;
293
:
1074
–80.
4
Wolffe AP, Pruss D. Deviant nucleosomes: the functional specialization of chromatin.
Trends Genet
1996
;
12
:
58
–62.
5
Turner BM. Histone acetylation and an epigenetic code.
Bioessays
2000
;
22
:
836
–45.
6
Strahl BD, Allis CD. The language of covalent histone modifications.
Nature
2000
;
403
:
41
–5.
7
Urnov FD, Wolffe AP. Chromatin organisation and human disease.
Emerg Ther Targets
2000
;
4
:
665
–85.
8
Mahlknecht U, Hoelzer D. Histone acetylation modifiers in the pathogenesis of malignant disease.
Mol Med
2000
;
6
:
623
–44.
9
Cress WD, Seto E. Histone deacetylases, transcriptional control, and cancer.
J Cell Physiol
2000
;
184
:
1
–16.
10
Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. Histone deacetylases and cancer: causes and therapies.
Nat Rev Cancer
2001
;
1
:
194
–202.
11
Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.
J Natl Cancer Inst
2000
;
92
:
1210
–6.
12
Marks PA, Richon VM, Breslow R, Rifkind RA. Histone deacetylase inhibitors as new cancer drugs.
Curr Opin Oncol
2001
;
13
:
477
–83.
13
Richon VM, Zhou X, Rifkind RA, Marks PA. Histone deacetylase inhibitors: development of suberoylanilide hydroxamic acid (SAHA) for the treatment of cancers.
Blood Cells Mol Dis
2001
;
27
:
260
–4.
14
Richon VM, Emiliani S, Verdin E, et al. A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.
Proc Natl Acad Sci U S A
1998
;
95
:
3003
–7.
15
Sandor V, Bakke S, Robey RW, et al. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms.
Clin Cancer Res
2002
;
8
:
718
–28.
16
Glick RD, Swendeman SL, Coffey DC, et al. Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma.
Cancer Res
1999
;
59
:
4392
–9.
17
Butler LM, Agus DB, Scher HI, et al. Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo.
Cancer Res
2000
;
60
:
5165
–70.
18
Finnin MS, Donigian JR, Cohen A, et al. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.
Nature
1999
;
401
:
188
–93.
19
Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.
Proc Natl Acad Sci U S A
2000
;
97
:
10014
–9.
20
Huang L, Sowa Y, Sakai T, Pardee AB. Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites.
Oncogene
2000
;
19
:
5712
–9.
21
Gui CY, Ngo L, Xu WS, Richon VM, Marks PA. Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1.
Proc Natl Acad Sci U S A
2004
;
101
:
1241
–6.
22
Kelly WK, Richon VM, O'Connor O, et al. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously.
Clin Cancer Res
2003
;
9
:
3578
–88.
23
Kelly WK, Richon VM, Curley T, et al. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), orally administered has good bioavailability and biologic activity. In: American Society of Clinical Oncology Annual Meeting Proceedings; 2002. p. 6b.
24
Kelly WK, Richon VM, Troso-Sandoval T, et al. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor: biologic activity without toxicity. In: American Society of Clinical Oncology Annual Meeting Proceedings; 2001. p. 11a.
25
Gonsky R, Knauf JA, Elisei R, Wang JW, Su S, Fagin JA. Identification of rapid turnover transcripts overexpressed in thyroid tumors and thyroid cancer cell lines: use of a targeted differential RNA display method to select for mRNA subsets.
Nucleic Acids Res
1997
;
25
:
3823
–31.
26
Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP. High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas.
J Clin Invest
1993
;
91
:
179
–84.
27
Yoshida M, Kijima M, Akita M, Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A.
J Biol Chem
1990
;
265
:
17174
–9.
28
Mitsiades CS, Treon SP, Mitsiades N, et al. TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma: therapeutic applications.
Blood
2001
;
98
:
795
–804.
29
de Nigris F, Mega T, Berger N, et al. Induction of ETS-1 and ETS-2 transcription factors is required for thyroid cell transformation.
Cancer Res
2001
;
61
:
2267
–75.
30
Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c.
Cell Death Differ
2000
;
7
:
1166
–73.
31
Imanishi R, Ohtsuru A, Iwamatsu M, et al. A histone deacetylase inhibitor enhances killing of undifferentiated thyroid carcinoma cells by p53 gene therapy.
J Clin Endocrinol Metab
2002
;
87
:
4821
–4.
32
Kitazono M, Robey R, Zhan Z, et al. Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na(+)/I(−) symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells.
J Clin Endocrinol Metab
2001
;
86
:
3430
–5.
33
Greenberg VL, Williams JM, Cogswell JP, Mendenhall M, Zimmer SG. Histone deacetylase inhibitors promote apoptosis and differential cell cycle arrest in anaplastic thyroid cancer cells.
Thyroid
2001
;
11
:
315
–25.
34
Coffey DC, Kutko MC, Glick RD, et al. The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid.
Cancer Res
2001
;
61
:
3591
–4.
35
Yoshimoto K, Iwahana H, Fukuda A, Sano T, Saito S, Itakura M. Role of p53 mutations in endocrine tumorigenesis: mutation detection by polymerase chain reaction-single strand conformation polymorphism.
Cancer Res
1992
;
52
:
5061
–4.
36
Vrana JA, Decker RH, Johnson CR, et al. Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-xL, c-Jun, and p21CIP1, but independent of p53.
Oncogene
1999
;
18
:
7016
–25.
37
Mitsiades N, Mitsiades CS, Richardson PG, et al. Molecular sequelae of histone deacetylase inhibition in human malignant B cells. Blood 2003;101:4055–62.
38
Ruefli AA, Ausserlechner MJ, Bernhard D, et al. The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species.
Proc Natl Acad Sci U S A
2001
;
98
:
10833
–8.
39
Henderson C, Mizzau M, Paroni G, Maestro R, Schneider C, Brancolini C. Role of caspases, bid and p53 in the apoptotic response triggered by histone deacetylase inhibitors TSA and SAHA.
J Biol Chem
2003
;
278
:
12579
–89.
40
Huang L, Pardee AB. Suberoylanilide hydroxamic acid as a potential therapeutic agent for human breast cancer treatment.
Mol Med
2000
;
6
:
849
–66.
41
Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Transcriptional signature of histone deacetylase inhibition in multiple myeloma: biological and clinical implications.
Proc Natl Acad Sci U S A
2004
;
101
:
540
–5.
42
Ashkenazi A, Dixit VM. Apoptosis control by death and decoy receptors.
Curr Opin Cell Biol
1999
;
11
:
255
–60.
43
Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors.
Eur J Biochem
1998
;
254
:
439
–59.
44
Green DR, Reed JC. Mitochondria and apoptosis.
Science
1998
;
281
:
1309
–12.
45
Zhang J, Alter N, Reed JC, Borner C, Obeid LM, Hannun YA. Bcl-2 interrupts the ceramide-mediated pathway of cell death.
Proc Natl Acad Sci U S A
1996
;
93
:
5325
–8.
46
Zhong LT, Sarafian T, Kane DJ, et al. Bcl-2 inhibits death of central neural cells induced by multiple agents.
Proc Natl Acad Sci U S A
1993
;
90
:
4533
–7.
47
Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways.
EMBO J
1998
;
17
:
1675
–87.
48
Mitsiades CS, Poulaki V, Mitsiades N. The role of apoptosis-inducing receptors of the tumor necrosis factor family in thyroid cancer.
J Endocrinol
2003
;
178
:
205
–16.
49
Baker JR Jr. The immune response to papillary thyroid cancer.
J Clin Endocrinol Metab
1995
;
80
:
3419
–20.
50
Mitsiades N, Poulaki V, Tseleni-Balafouta S, Koutras DA, Stamenkovic I. Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand.
Cancer Res
2000
;
60
:
4122
–9.
51
Poulaki V, Mitsiades CS, Kotoula V, et al. Regulation of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in thyroid carcinoma cells.
Am J Pathol
2002
;
161
:
643
–54.
52
Kelly W, O'Connor O, Richon VM, et al. A phase I clinical trial of an oral formulation of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA).
Eur J Cancer
2002
;
38
:
88
.