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Original Studies |
University of California, San Francisco, School of Medicine; University of California, San Francisco/Mount Zion Medical Center, Department of Surgery (E.K., M.G.W., A.E.S., O.H.C.); and San Francisco Veterans Affairs Medical Center, Surgical Services (Q.-Y.D.)
Address all correspondence and requests for reprints to: Orlo H. Clark, University of California, San Francisco/Mount Zion Medical Center, Department of Surgery, 1600 Divisadero Street, San Francisco, California 94143-1674. E-mail: clarkaa{at}mzsurgery.his.ucsf.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30%) and mortality
(40%) (2, 3). Cytotoxic drugs and external radiation have been, at
best, palliative in patients with thyroid cancer (3). Cellular dedifferentiation is a common event in malignant transformation and/or progression, and it occurs in up to one-third of differentiated thyroid cancers (DTC) (4). About 1% of DTCs transform into anaplastic thyroid cancer, which is almost uniformly lethal (5). In DTC, iodine uptake and metabolism, TSH receptor expression, thyroglobulin synthesis and secretion, and thyroid peroxidase activity are markers of thyrocyte differentiation (6, 7, 8). Patients with DTCs that have decreased or no radioactive iodine uptake often have more aggressive tumors and are difficult to treat. Several antiproliferative and differentiating agents (such as tamoxifen, retinoic acid, and Octreotide) have been evaluated in thyroid cancer experimental models leading to clinical evaluation in patients who did not benefit from conventional therapy (9, 10, 11, 12, 13).
Phenylacetate (an aromatic fatty acid) has been reported to have a potent antiproliferative and differentiating effect in hematologic malignancies and multiple solid tumors at nontoxic concentrations (14). It is present in low concentration (2–4 µmol/L) in human serum as a product of phenylalanine metabolism and is conjugated with glutamine in the liver by phenylacetyl coenzyme A to form phenylacetylglutamine (15). Based on this, it has been used safely to treat children with inborn errors of urea synthesis and also in patients with hyperammonemia (16, 17, 18). In patients with hormone refractory prostate cancer and high-grade glioma, phase I trials of phenylacetate showed minimal toxicity; and a partial response was observed at a serum concentration of 2–10 mmol/L (19, 20). Increasing evidence points to phenylacetate having multiple effects on gene expression and regulatory proteins responsible for its antineoplastic effects. In this study, we evaluated the antiproliferative and differentiating effect of phenylacetate in well-characterized human thyroid cancer cell lines of follicular cell origin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following reagents and materials were used: DMEM:F12 media, L-glutamine, 1x Trypsin/EDTA solution, 1x PBS from Cellgro Mediatech (Newark, DE); penicillin-streptomycin, FBS and fungizone from Irvine Scientific (Santa Ana, CA); RNAzol from Tel-Test B Inc. (Friendswood, TX); 125I, 32P, Rediprime II random prime labeling system, Hybond-N transfer membrane from Amersham Pharmacia Biotech (Piscataway, NJ); EcoRI, ClaI, and SstI restriction enzymes from Promega Corp. (Madison, WI); ExpressHyb hybridization solution from CLONTECH Laboratories, Inc. (Palo Alto, CA); quantitative protein assay from Bio-Rad Laboratories, Inc. (Richmond, CA); human vascular endothelial growth factor (VEGF) VEGF enzyme-linked immunosorbent assay (ELISA) Quantikine immunoassay from R & D (Minneapolis, MN) and all other materials from Sigma Chemical Co. (St. Louis, MO).
Cell lines and culture conditions
All three follicular thyroid cancer (FTC) cell lines were established from a single patient and were kindly provided by Dr. Peter Goretzki (Germany). FTC-133 was derived from a primary thyroid tumor, FTC-236 from a lymph node metastasis, and FTC-238 from a lung metastasis (21). A papillary thyroid cancer cell line (TPC-1) was kindly provided by Dr. Nabuo Satoh (Japan), and the Hürthle cell carcinoma cell line (XTC-UC1) was established in our laboratory from a metastasis to the breast (22).
The cell lines were maintained in DME:12 supplemented with 10% FBS, penicillin (10,000 U/mL), streptomycin (10,000 U/mL), fungizone (250 mg/mL), TSH (10 mU/mL), glutamine (12.5 mg/L), and insulin (5 mg/mL) in a standard humidified incubator at 37 C in a 5% CO2-95% O2 atmosphere. All experiments were performed in a serum-free environment using DME:12 media supplemented with four hormones (H5); transferrin (5 µg/mL), SRIF (10 ng/mL), glycl-L-histidyl acetate (2 ng/mL), and hydrocortisone (0.36 ng/mL), a modified Ambesi-Impiombato method (23). The culture media were changed to H5 media, 48 h before conducting experiments.
Thyroid tissue culture
Fresh normal thyroid tissues adjacent to thyroid neoplasms were obtained immediately upon surgical removal and maintained on ice. All experiments and tissue procurement were approved by the Committee on Human Research at the University of California, San Francisco. The tissues were cut into 1- to 5-mm pieces in 1x PBS, then pelleted and treated with collagenase for 45 min and 1x trypsin/EDTA solution for 15 min. The digested solution was pelleted, resuspended in maintenance media, and incubated in a 125-cm2 flask in a humidified incubator. Geneticin (10 ng/mL) was used to eliminate fibroblast growth, if present, in the thyroid monolayer cultures. Primary culture thyroid cells were harvested at a 100% confluency to confirm the accuracy of 125I-uptake assay studies.
Proliferation assay
Growth experiments were done in a 96-well plate in triplicate. Cells at 100% confluency were harvested with 1x Trypsin/EDTA solution and seeded into a 96-well plate at 5 x 103 cells per well and maintained in 200 µL H5 medium in a humidified incubator. Phenylacetate salt was reconstituted in H5 media, to a pH of 7.4, using NaOH. After 24 h, the cells were incubated with 0, 2.5, 5.0, 7.5, 10, and 50 mmol/L phenylacetate with the media changed daily. Colorometric MTT (dimethylthiazol-diphenyltetrazolium bromide) proliferation assays were performed at 0, 24, 48, 72, and 96 h. MTT (400 µg/mL) was added to each well and incubated for 3 h. It was solubilized with 0.04 N HCL/Isopropanol/3% SDS and incubated for 1 h. The optical densities in the 96-well plates were determined using an ELISA microplate reader (Molecular Devices) at 595 nm/620 nm (1-reference).
Flow cytometry analysis
The thyroid cancer cell lines (FTC-236, TPC-1, and XTC-UC1) were incubated, with and without phenylacetate (10 mmol/L) for 24 h, to assess the effect of phenylacetate on cell cycle progression. Subconfluent cells were harvested, pelleted, alcohol fixed, and resuspended in 1x PBS to a concentration of 1 x 106 cells/mL. To eliminate staining artifacts from double-stranded RNA binding propidium iodide, ribonuclease (deoxyribonuclease-free @ 100 µg/mL) was added to each cell suspension and incubated for 1 h at 4 C.
Flow cytometric analysis was performed on a Becton Dickinson and Co. FACScan. Data files were generated for 20,000 events (cells) or more per sample using the CELLQuest software. The ModFit LT cell cycle analysis software from Verity Software House, Inc. was used to analyze the data files.
TSH growth response
The cells were maintained in H5 medium, with phenylacetate treatment (10 mmol/L) or without (control), for 72 h, then harvested and seeded into a 96-well plate at 1 x 104 cells/well in 200 µL H5 medium with 10 mU/mL TSH. As described earlier, proliferation MTT assays were performed 24 h after TSH stimulation.
Iodine-uptake studies
Cells were maintained in H5 media, with (7.5 mmol/L) and without phenylacetate, for 72 h, then harvested using a cell scraper, and pelleted. The total cell number of each sample was determined by hemocytometry, and 2 x 106 cells were resuspended in HBSS with 0.5 mmol/L sodium iodine and 2 µCi of 125I for an activity of 20 mCi/mmol. The solution was then incubated at 37 C for 2 h, then pelleted, and washed with 1 mL ice-cold HBSS three times.
To determine the 125I associated with the cells, the pellet was resuspended in 1 mL 95% alcohol and incubated for 20 min at room temperature. Triplicates (200 µL) of each sample were immediately counted in a ß counter (COBRA Auto-gamma) for 5 min. Blank background samples (without cells) showed less than 5.5% of sample radioactivity. The 125I uptake in the cell lines, using 10% trichloroacetic acid instead of alcohol, gave similar results. Before sample incubation with 125I and uptake measurement, the cell viability was greater than 96%, by trypan blue dye exclusion. The 125I uptake is reported as cpm/100,000 cells.
Thyroglobulin measurement
To assess the effect of phenylacetate on thyroglobulin synthesis and secretion in the thyroid cancer cell lines, each cell line (2 x 106 cells) was maintained in H5 media, with (10 mmol/L) and without phenylacetate, in a 25-cm2 flask. To determine thyroglobulin secretion, the culture media was collected at 12, 24, and 48 h; and the thyroglobulin levels were measured commercially (Quest Diagnostics, Inc., Nicholas Institute, Clinical Studies Center, San Juan Capistrano, CA). To measure the effect of phenylacetate on thyroglobulin synthesis, the intracellular thyroglobulin levels, after incubation with and without phenylacetate (10 mmol/L) at 12, 24 and 48 h, were determined after protein lysate preparation. The cells were harvested, pelleted, and washed with 1 mL of 1x PBS and homogenized in 1x PBS/1% IGEPAL CA-630/0.5% SDS for 1 h. The protein concentration of the lysates was quantified using the Bio-Rad Laboratories, Inc. protein assay (based on the Bradford method), as described by the manufacturer. The protein lysate samples were diluted 1:10 in H5 media, and the thyroglobulin levels were measured.
VEGF ELISA
VEGF secretion in the thyroid cancer cell lines was measured in 96-well plates (5 x 103 cells/well) triplicates maintained in 250 µL H5 media after 12 h incubation with and without phenylacetate (10 mmol/L). The medium from each well was collected to determine VEGF secretion. VEGF ELISAs were done using the Quantikine immunoassay kit, as described in the manufacturer’s protocol. The optical density in the 96-well plates was determined using an ELISA microplate reader at 450 nm/595 nm (1-reference). Protein lysates were prepared and quantified as described above under the same culture conditions. To measure the intracellular VEGF level, the VEGF ELISA system was also used after a 1:4 dilution of the protein lysate samples.
RNA extraction and Northern blot analysis
Total RNA was extracted by the single-step acid guanidium thiocyanate-phenol-chloroform method using the RNAzol solution. Ten micrograms of total RNA was electrophorized on a 1.2% agarose/1.9% formaldehyde gel, transferred onto a nylon membrane, and cross-linked by ultraviolet irradiation. VEGF complementary DNA (cDNA) probes (160-bp fragment) were excised from 2.1 kb Bluescript II KS plasmid with ClaI and SstI, and ß-actin cDNA probes (1.1-kb fragment) from a Bluescript SK plasmid with EcoRI. The probes were radiolabeled with [32P] deoxycycidine triphosphate by random priming. The nylon membranes were prehybridized at 65 C and hybridized at 65 C with labeled cDNA probes. The membranes were washed to a stringency of 0.1% SSPE and 0.1% SDS at 65 C and exposed to Kodak XAR-5 x-ray films for 24–48 h at -80 C. The resulting autoradiograph band intensities were quantified using an autoradiography densitometry imaging analyzer. The VEGF messenger RNA (mRNA) signals were normalized to the ß-actin mRNA signals in the same lanes to control for RNA loading and transfer. For rehybridization, the membranes were stripped for 10 min at 100 C using a 0.05% XSSPE, 0.1% SDS, and 20 mmol/L EDTA solution.
Statistical analysis
The student’s t test for paired data and ANOVA for multiple group (control plus multiple phenylacetate treatment concentration) comparison were used with a statistically significant result defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found growth inhibition, with phenylacetate treatment, in all
the thyroid cancer cell lines studied at nontoxic concentrations
(2.5–10 mmol/L). The antiproliferative effect of phenylacetate was
time- and dose-dependent in all the cell lines (Fig. 1
, A and B). The antiproliferative effect
of phenylacetate does not seem to be a cytotoxic effect, because the
cell viability was greater than 96% with exposure to 10 mmol/L of
phenylacetate. Growth inhibition, with phenylacetate (10 mmol/L),
occurred as early as 24 h after exposure in all the cell lines
ranging, from 12–37% (P < 0.05). FACS
analysis of subconfluent cell lines (FTC-236, TPC-1, and XTC-UC1)
exposed to phenylacetate, compared with unexposed (control) cell lines,
showed a decrease (5–14%, P 
0.03) in the percent
of S-phase cells, with a corresponding increase (2–7%,
P 0.03) in the percent of G0–1-phase
cells. It has been shown that phenylacetate can bind glutamine and
deplete serum circulating levels in humans. Because we used a
glutamine-free culture experimental model, we also conducted
experiments to see whether the presence of glutamine reversed the
antiproliferative effect of phenylacetate observed. Glutamine
supplementation of two times the physiologic level did not reverse the
antiproliferative effect of phenylacetate (data not shown). Morphologic
changes in the thyrocyte monolayer culture system occurred late with
phenylacetate treatment and consisted of cell detachment from the
flask, rounded-up cells, and decreased cellular cytoplasm with
prominent nuclei formation (Fig. 2).
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Our previous studies show that all the thyroid cancer cell lines
have increased growth with TSH (10 mU/mL) stimulation (22, 24).
Therefore, we were interested in determining whether phenylacetate
exposure could potentiate this response (i.e. have a
redifferentiating effect). Phenylacetate treatment decreased the growth
response to TSH stimulation, compared with unexposed cell lines (Fig. 3). This suggests a primarily
antiproliferative effect of phenylacetate in the thyroid cancer cell
lines evaluated. Phenylacetate treatment in the TPC-1 and XTC-UC1 cell
lines increased 125I uptake by 82% and 108%,
respectively, compared with unexposed cell lines (Table 1). This effect was not observed in the
more invasive FTC cell lines (FTC-133, FTC-236, and FTC-238) (24). Only
the XTC-UC1 cell line had an appreciable thyroglobulin level secreted
in the condition media collected. When exposed to phenylacetate, we
found a time-dependent inhibition of thyroglobulin secretion 20% (8 h)
and 69% (24 h) (P < 0.05). To differentiate whether
this was an effect on the secretion or synthesis of thyroglobulin, we
determined the intracellular thyroglobulin levels. Phenylacetate
treatment in the XTC-UC1 cell line had no effect up to 24 h; but
by 48 h, there was an increased intracellular thyroglobulin level,
compared with control (250% increase, P = 0.03). This
suggests that phenylacetate inhibits the secretion of thyroglobulin,
causing intracellular accumulation.
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Because of our finding that phenylacetate inhibited secretion of
thyroglobulin (a glycoprotein like VEGF) and another report that
suggests that phenylacetate may inhibit angiogenesis, we reasoned that
phenylacetate might also inhibit the secretion of VEGF (a glycoprotein
known to be dependent on N-linked glycosylation for efficient
secretion) (25). Phenylacetate inhibited the secretion of VEGF, in all
the cell lines, 38–68% at 12 h [P < 0.05 for
all the cell lines, except TPC-1 (P = 0.06)]. We
performed Northern blot analysis to determine whether phenylacetate
exposure might have decreased VEGF secretion by down-regulating VEGF
mRNA expression, but we found no difference between exposed and
unexposed cell lines (Fig. 4).
Phenylacetate treatment did increase intracellular VEGF levels in the
TPC-1 and XTC-UC1 cell lines (78% and 70%, respectively, compared
with untreated cell lines at 12 h, P < 0.05).
This also suggests that phenylacetate inhibits the efficient secretion
of VEGF in human thyroid carcinoma cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phenylacetate has been shown to have an antiproliferative effect in
prostate carcinoma (27), melanoma (28), rhabdomyosarcoma (29), breast
cancer (30), pancreatic adenocarcinoma (31), chronic lymphocytic
leukemia (32), ovarian carcinoma (33), and medulloblastoma and
astrocytoma cell lines (34). Consistent with other studies, we also
found that growth inhibition occurred at concentrations of
phenylacetate (2.5–10 mmol/L) clinically achievable. Presently, the
mechanism of action of phenylacetate is not clearly understood.
Although glutamine depletion has been suggested as a possible effect of
phenylacetate, glutamine supplementation (up to 2 times the
physiologic level) did not reverse the antiproliferative effects in the
thyroid cancer cell lines in vitro, consistent with the
findings of Ferrandina et al. (33) and Samid et
al. (27). There are other potential actions of phenylacetate that
may mediate its antineoplastic effects. First, it inhibits protein
prenylation and glycosylation, which might affect the synthesis of
proteins involved in cell cycle control and signal transduction (35).
Indeed, phenylacetate has been shown to down-regulate Bcl-2 and
up-regulate bax/p21 apoptosis-related genes in ovarian carcinoma cells
and up-regulate p21 in K-ras mutant MCF-7ras breast cancer cell lines
(33, 36). Second, phenylacetate activates the human peroxisome
proliferator-activated receptors (PPAR) (37). The PPAR belong to the
superfamily of nuclear steroid receptors (such as retinoids, vitamin D,
and thyroid hormone receptors), all important regulators of cell growth
and differentiation in thyroid cells (38, 39). Last, phenylacetate may
lead to DNA hypomethylation (40).
The effects of phenylacetate, as a differentiating agent, varied among the cell lines we studied. This may be attributable to the FTC cell lines being too dedifferentiated, because they have no detectable TSH receptor, exhibit a biphasic growth response to TSH, and secrete a minimal amount of thyroglobulin (24). Several in vitro studies have shown that TSH is an important promoter of malignant thyroid cell growth, invasion, and angiogenesis; it also inhibits apoptosis in malignant thyroid cell lines (24, 41, 42). The decreased TSH growth response of the thyroid cancer cell lines, with phenylacetate treatment, may be attributable to the effect of phenylacetate on intracellular regulatory protein function or synthesis by inhibiting protein prenylation and glycosylation. One of the markers of DTCs is their ability to synthesize and secrete thyroglobulin, which is clinically useful in identifying patients who have persistent or recurrent thyroid cancer (43). Because efficient glycoprotein secretion requires N-linked glycosylation, and phenylacetate inhibits protein glycosylation, we expected phenylacetate to decrease thyroglobulin secretion in the XTC-UC1 cell line (31, 44, 45). Furthermore, the intracellular accumulation of thyroglobulin, with phenylacetate treatment of the XTC-UC1 cell line, supports this mechanism. Although there was increased radioactive iodine uptake in two (TPC-1 and XTC-UC1) cell lines, no differences were observed in the FTC cell lines. The FTC cell lines that failed to have increased radioactive iodine uptake with phenylacetate treatment are perhaps more dedifferentiated than the TPC-1 and XTC-UC1 cell lines. Similar findings have been observed with retinoic acid in human FTC cell lines; a redifferentiation effect, with respect to radioactive iodine uptake, was observed in some, but not all, cases (10, 11, 46). In fact, Schmutzler et al. (46) have reported that the FTC cell lines (FTC-133, FTC-236, and FTC-238) did not have 125I uptake in their experimental model but yet reported that these cells express the Na+/I- symporter. These apparent differences in results could also be caused by subtle differences in experimental methods. In addition to the reproducible result with our model, we conducted 125I-uptake assays in primary thyroid tissue culture cells and with TSH stimulation to further confirm the accuracy of our experimental model. As would be expected, we found a three- to six-times higher 125I uptake in normal thyroid cells and an increase in 125I uptake (60% in the XTC-UC1 cell line) after TSH stimulation. This further validates our system for measuring 125I uptake, which is similar to that of van Herle et al. (10) who also reported 125I uptake in FTC cell lines. Because phenylacetate activates the PPAR, up-regulates the retinoic acid receptor ß, and has a synergistic effect with retinoic acid in inducing redifferentiation of neuroblastoma cells, the redifferentiation effect of phenylacetate (with respect to 125I uptake) may therefore act through a mechanism similar to that of retinoic acid (37, 47).
VEGF is a glycoprotein shown to be dependent on N-linked glycosylation for efficient cellular secretion (48). VEGF promotes tumor angiogenesis and growth and is overexpressed in thyroid cancers (49). We did find phenylacetate to inhibit VEGF secretion with intracellular accumulation in some of the cell lines, which has not been previously identified. This suggests a possible antiangiogenic effect mediated by the inefficient secretion of VEGF. Adams et al. (25) have shown decreased angiogenesis in phenylacetate-treated breast cancer (MCF-7ras) xenograft, compared with unexposed tumors, and this might have been attributable to lower VEGF secretion. Taken together, they suggest that the inhibition of VEGF secretion by phenylacetate may be an important aspect of the in vivo antiproliferative effect of phenylacetate.
We have shown that phenylacetate can inhibit the growth of human thyroid cancer cell lines with an early cell cycle arrest in the G0–1 phase and late morphologic changes. Phenylacetate may inhibit tumor angiogenesis by inhibiting VEGF secretion. Because radioiodine uptake may be increased with phenylacetate treatment, it may be useful in patients who have tumors that take up little or no radioiodine. Given that serum phenylacetate concentrations have been safely achieved in humans, it may be beneficial in patients with DTC who fail conventional therapy or as an adjuvant treatment. Its interaction with retinoic acid is also of interest.
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Footnotes |
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Received March 11, 1999.
Revised May 4, 1999.
Accepted May 10, 1999.
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