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Lovastatin, a 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitor, Induces Apoptosis and Differentiation in Human Anaplastic Thyroid Carcinoma Cells

Graduate Institute of Physiology, College of Medicine, National Taiwan University (C.Y.W., W.B.Z., Y.F.T.); and Department of Anatomy and Cell Biology (L.S.M.) and Division of Endocrinology (C.Y.W., T.C.C.), Department of Internal Medicine, Far-Eastern Memorial Hospital and National Taiwan University Hospital, College of Medicine, National Taiwan University, 10063, Taipei, Taiwan

Address all correspondence and requests for reprints to: Dr. Tien-Chun Chang, Division of Endocrinology, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan. E-mail: tcchang1{at}ms10.hinet.net.

	Abstract

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Although only 1% of differentiated thyroid cancers transform into anaplastic thyroid cancer, this disease is always fatal. Differentiation therapy may provide a new therapeutic approach to increasing the survival rate in such patients. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are reported to promote cellular apoptosis and differentiation in many cancer cells; these effects are unrelated to lipid reduction. Recently, we found that TNF induces cytomorphological differentiation in anaplastic thyroid cancer cells and increases thyroglobulin expression; however, TNF is cytotoxic for normal human tissue. The aim of this study was to determine whether lovastatin, an HMG-CoA reductase inhibitor, could induce apoptosis and differentiation in anaplastic thyroid cancer cells. Anaplastic thyroid cancer cells were treated with lovastatin, then examined for cellular apoptosis and cytomorphological differentiation by DNA fragmentation, phosphatidylserine externalization/flow cytometry, and electron microscopy. Thyroglobulin levels in the culture medium were also measured. Our results showed that at a higher dose (50 µM), lovastatin induced apoptosis of anaplastic thyroid cancer cells, whereas at a lower dose (25 µM), it promoted 3-dimensional cytomorphological differentiation. It also induced increased secretion of thyroglobulin by anaplastic cancer cells. Our results show that lovastatin not only induces apoptosis, but also promotes redifferentiation in anaplastic thyroid cancer cells, and suggest that it and other HMG-CoA reductase inhibitors merit further investigation as differentiation therapy for the treatment of anaplastic thyroid cancer.

	Introduction

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CELLULAR DEDIFFERENTIATION is very common in malignant transformation, occurring in up to one third of differentiated thyroid cancers (1). Although only 1% of differentiated thyroid cancers transform into anaplastic thyroid cancer, this disease is invariably fatal within a few months (2). Normal thyroid epithelial cells are usually uniform in size and shape, and microvilli can be easily observed in three-dimensional (3-D) cytomorphology by scanning electron microscopy (SEM) (3, 4, 5, 6). Nesland et al. (3) found the major difference between different types of thyroid carcinoma to be the abundance of microvilli. In neoplastic states, the density of microvilli steadily decreases on going from ordinary papillary thyroid carcinoma through follicular variants of papillary thyroid carcinoma to follicular carcinoma, and anaplastic carcinoma thyrocytes have few, or no, microvilli. Microvilli are, therefore, 3-D cytomorphological features of thyrocyte differentiation, with fewer being seen in less differentiated cancers (7).

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been widely used to reduce cardiovascular morbidity and mortality in various clinical studies (8, 9, 10). They have a marked beneficial action on the lipid profile, but also have effects unrelated to lipid reduction (11, 12, 13). Their inhibitory effects on the growth of cultured malignant cell depend on their ability to block the isoprenylation of proteins, including Ras, Rho, and Rac (14, 15, 16). Recently, lovastatin, an HMG-CoA reductase inhibitor, has been used to reduce proliferation and induce apoptosis of leukemia cells, colon cancer cells, and breast cancer cells (17, 18, 19). In addition, lovastatin and other HMG-CoA reductase inhibitors have been shown to promote cellular differentiation of human monocytic cells, neuroectodermal sarcoma cells, and osteoblastic cells (20, 21, 22).

As anaplastic thyroid cancer is fatal and refractory to conventional chemotherapy, differentiation therapy combined with radioactive iodine (¹³¹I) therapy after surgical intervention may provide a new therapeutic approach. In a recent study we found that TNF induces the 3-D cytomorphological differentiation of microvilli in anaplastic thyroid cancer cells and increases thyroglobulin expression (23). However, TNF might not be used to treat patients clinically for its cytotoxicity. The effects of the differentiating agents, retinoic acid and somatostatin, have been tested on slowly growing, well differentiated, thyroid cancer lines (24, 25), but none has been tested on the rapidly developing and fatal anaplastic thyroid carcinomas. In the present study we showed that an HMG-CoA reductase inhibitor was able to induce not only apoptosis, but also differentiation, of anaplastic thyroid cancer cells. In addition, as the secretion of thyroglobulin, a glycoprotein produced only by normal or well differentiated neoplastic thyroid follicular cells, is a differentiation marker of thyrocytes (26, 27), we measured thyroglobulin levels in culture supernatants from untreated and lovastatin-treated anaplastic thyroid cancer cells as a further parameter of thyrocyte differentiation and found a large increase in the lovastatin-treated cultures.

	Materials and Methods

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Cell culture and reagents

Lovastatin was a gift from the Standard Chemical and Pharmaceutical Co. (Taiwan). Human ARO anaplastic thyroid cancer cells, provided by Dr. S. D.Chen (Chang-Gung Memorial Hospital, Taiwan), were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 µg/ml streptomycin. For differentiation, 1 x 10⁵ cell/ml were incubated in 10-cm cell culture plates for 24 h in the above medium, then for 24 or 48 h in the same medium containing 25 µM lovastatin, and 3-D cytomorphology was examined. Mevalonate, geranylgeraniol (GGOH), and farnesol (FOH) were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Cytotoxicity assay

To investigate the cytotoxic effects of lovastatin, ARO cells (2 x 10⁵ cells/ml) were incubated with different concentrations of lovastatin for 24–96 h, then viable cell numbers were determined using a hemocytometer and the Trypan Blue dye exclusion method.

DNA fragmentation analysis by agarose gel electrophoresis

ARO cells (2 x 10⁵ cells) were treated with different concentrations of lovastatin for various times, harvested, and suspended in lysis buffer [10 mM Tris-HCl (pH 8.0), 20 mM NaCl, 25 mM EDTA, 1% sodium dodecyl sulfate, and 1 mg/ml proteinase K] for 24 h in a 55 C water bath. A phenol/chloroform/isoamyl alcohol solution (25:24:1) was used to remove protein and extract nucleic acid, then the extracted nucleic acid was digested with ribonuclease A (200 µg/ml) for 12 h at 37 C, and DNA was electrophoresed on a 2% agarose gel at 50 V for 45 min and visualized with ethidium bromide under UV illumination.

Analysis of apoptotic cells by phosphatidylserine externalization

After treatment with lovastatin (50 µM), 1 x 10⁶ cells were suspended in 100 µl binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 5 mM CaCl₂], then annexin V-fluorescein isothiocyanate (Boehringer Ingelheim GmbH, Vienna, Austria) and propidium iodide (PI) were added (final concentration of both, 5 µg/ml). The cells were then incubated for 15 min at room temperature in the dark and diluted with 300 µl binding buffer for flow cytometric analysis. Bivariate analysis was performed on a FACSCaliber (BD Biosciences, Mountain View, CA) equipped with a 488-nm argon laser for excitation. Bandpass 530/30-nm and 585/42-nm emission filters were used, respectively, for fluorescein isothiocyanate and PI. Data analysis was performed using standard CellQuest software (BD Biosciences). Annexin V-positive/PI-negative cells were defined as apoptotic cells.

SEM and transmission electron microscopy (TEM)

For SEM, ARO cells on albumin-coated coverglasses were fixed for 1 h with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), then the slide was washed three times for 5 min each time with cacodylate buffer (pH 7.4) (CD buffer), fixed for 30 min with 1% osmium tetroxide in CD buffer, and washed three times for 5 min each time with CD buffer. The specimen was dehydrated successively with 70%, 85%, and 95% alcohol (5 min each), then three times for 5 min each time with 100% alcohol. Critical point drying was performed using an HCP2 critical point drier (Hitachi, Tokyo, Japan), then the specimen was coated with Au-Pd using a JEC-1100 ion spatter (Japan Electron Optical Laboratory, Tokyo, Japan) and observed using a JSM-T330A scanning microscope (Japan Electron Optical Laboratory).

For TEM, ARO cells, treated with lovastatin, were fixed for 1 h in 4% glutaraldehyde, washed three times for 10 min each time with CD buffer, fixed for 30 min with 1% osmium tetroxide in CD buffer, and again washed three times for 10 min each time with CD buffer. The specimen was dehydrated successively with 70%, 85%, and 95% alcohol (10 min each), then three times for 10 min each time with 100% alcohol. The dehydrated specimen was immersed in propylene oxide twice for 10 min each time, then in a propylene oxide/Epon mixture (1:1) for 1 h, a propylene oxide:Epon mixture (1:3) for 2 h, and pure Epon overnight before being embedded in a commercially available capsule and polymerized in a 60 C oven for 48 h. Ultrathin sections (1 µm thick) were cut using an Ultracut E ultramicrotome (Reichert-Jung, Vienna, Austria) and mounted on copper grids. The sections were subjected to electron staining with uranium acetate and lead citrate for 5 min each, then observed using a JEM-2000EXII electron microscope (Japan Electron Optical Laboratory).

Thyroglobulin RIA

Triplicate samples of culture medium were collected from 10⁵ untreated ARO cells and cells treated with lovastatin (25 µM) for 24, 48, and 72 h, and assayed for thyroglobulin using an RIA kit (Double Antibody Thyroglobulin, EURO/DPC Ltd. Glyn Rhonwy, United Kingdom). The sample was first preincubated with the antithyroglobulin antiserum, then ¹²⁵I-labled thyroglobulin was added. After incubation for a fixed time, bound and free label were separated by the double-antibody method, and the antibody-bound fraction was precipitated and counted. Sample concentrations are read from a calibration curve. The tracer has a high specific activity, with total counts of 35,000 cpm at iodination. Maximal binding was approximately 40%, and nonspecific binding was negligible.

Western blot analysis for farnesylation and geranylgeranylation

Lovastatin (25 µM)-treated ARO cells were cotreated with mevalonate, FOH, and GGOH, respectively. Thirty-microgram samples of cytoplasmic proteins were electrophoresed on 10% polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblotting was carried out with specific antibodies in PBS with 0.2% Tween 20 (Sigma-Aldrich Corp.) and 5% BSA (Sigma-Aldrich Corp.). FOH and GGOH proteins were visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Arlington Heights, IL).

Apoptotic rate in farnesylation and geranylgeranylation

Triplicate samples of culture medium were collected from 10⁵ untreated ARO cells and cells treated with lovastatin (50 µM) for 24 h. Apoptosis was assessed according to the percentage of cells with hypodiploid DNA, using the PI-staining technique. Lovastatin (50 µg/ml)-treated ARO cells were cotreated with mevalonate, FOH, and GGOH. Then cells were washed in PBS, dispersed in 70% ethanol, and stored overnight at -20 C. Then cells were incubated at 37 C for 30 min in PBS containing 1 mg/ml ribonuclease A and 50 µM PI at room temperature in the dark for 1 h. The samples were detected using a FACSCaliber flow cytometer (BD Biosciences) and were analyzed using CellQuest software.

Statistical analysis

Thyroglobulin levels in untreated and treated ARO cell culture supernatants were compared using paired t test; P < 0.05 was considered statistically significant.

	Results

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Lovastatin inhibits survival of ARO cells

When different concentrations of lovastatin (10–75 µM) were added to ARO cell cultures for up to 96 h, a time- and dose-dependent inhibition of cellular survival was seen (Fig. 1).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Effect of lovastatin on ARO cell survival rate.

To determine whether lovastatin could induce apoptosis in ARO cells, DNA extracts from control cells and cells treated with different concentrations of lovastatin were separated by agarose gel electrophoresis. DNA fragmentation was seen in cells treated for 24 h with lovastatin at concentrations of 50 µM or higher (Fig. 2). In addition, the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane in apoptotic cells was examined using the FITC-annexin V binding assay. Figure 3 shows that the percentage of annexin V-positive/PI-negative cells (apoptotic cells) increased in a time-dependent manner after treatment with 50 µM lovastatin.

View larger version (97K): [in this window] [in a new window]   FIG. 2. Agarose gel electrophoresis of DNA from ARO cells treated with different concentrations of lovastatin for 24 h.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Percentage of annexin V-positive/PI-negative cells after treatment of ARO cells with lovastatin (50 µM) for different times.

Using SEM, untreated ARO cells showed a disorganized cellular arrangement with a smooth cellular surface and an absence of microvilli (Fig. 4, A and B), whereas after 24- or 48-h treatment with 25 µM lovastatin, a more uniform cell population with distinct borders and an abundance of microvilli was seen (Fig. 5, A-1 and A-2). Using TEM, microvilli and cytoplasmic dense-core secretory granules could be identified after 24-h treatment with 25 µM lovastatin (Fig. 5B).

View larger version (117K): [in this window] [in a new window]   FIG. 4. SEM of untreated ARO cells (magnification: A, x2000; B, x3500). SEM showed a disorganized cellular arrangement with a smooth cellular surface and the absence of microvilli.

View larger version (151K): [in this window] [in a new window]   FIG. 5. SEM and TEM after treatment with 25 µM lovastatin for 24 h. A, SEM showed a more uniform cell population with distinct borders and an abundance of microvilli (magnification: A-1, x2000; A-2, x3500). B, TEM identified microvilli (arrow), a 3-D cytomorphological feature of thyroid follicular cell differentiation.

Although both untreated and treated ARO cells secreted thyroglobulin into the medium (Fig. 6), the amount secreted by lovastatin-treated cells was much greater and increased with time (Fig. 6, B, D, and F; 24, 48, and 72 h, respectively).

View larger version (13K): [in this window] [in a new window]   FIG. 6. Thyroglobulin levels in culture medium from lovastatin-treated and untreated ARO cells. A, C, and E, Corresponding untreated cells. B, D, and F, Cells treated with 25 µM lovastatin for 24 h (B), 48 h (D), or 72 h (F).

Ras protein cannot be inhibited by lovastatin, but Rho protein could be suppressed after treatment of lovastatin in ARO cells. Mevalonate and GGOH, the metabolites of HMG-CoA, restored Rho protein in lovastatin-treated ARO cells; however, FOH did not alter the suppression of Rho (Fig. 7). Meanwhile, lovastatin-related cytomorphological differentiation in ARO cells was not influenced by farnesylation and geranylgeranylation. FOH transferase and GGOH transferase inhibitors were used in lovastatin-treated ARO cells, and the features of cytomorphological differentiation still existed (data not shown). The isoprenylated proteins were closely related to apoptosis in lovastatin-treated ARO cells; especially Rho could reverse the apoptosis induced by lovastatin in ARO cells (Fig. 8).

View larger version (22K): [in this window] [in a new window]   FIG. 7. A, Ras protein had not inhibited by lovastatin, and Rho protein was suppressed by lovastatin. B, Mevalonate could reverse the suppression of Rho protein in lovastatin-treated ARO cells. C, FOH could not reverse the suppression of Rho, but GGOH could restore the expression of Rho (D).

View larger version (13K): [in this window] [in a new window]   FIG. 8. Mevalonate and GGOH could reserve the suppression of Rho protein after treatment with lovastatin, and Rho could protect against apoptosis induced by lovastatin in ARO cells.

	Discussion

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The inhibitory effects of HMG-CoA reductase inhibitors on the growth of cultured malignant cells depend on their ability to block isoprenylation of proteins, including Ras, Rho, and Rac (14, 15, 16), by inhibiting the biosynthesis of FOH and GGOh pyrophosphate (30, 31). Ras is involved in cellular apoptosis (16), and Rho is involved in actin cytoskeletal reorganization and regulation of cell adhesion, morphology, motility, and invasion (32, 33), playing a pivotal role in cancer cell growth, invasion, and metastasis. In our study we used mevalonate, FOH, and GGOH to treat ARO cells with lovastatin, and surprisingly, we found that Rho protein is the pivotal element for cellular survival of ARO cells after treatment with lovastatin. We also used FOH and GGOH transferases inhibitors to evaluate the role of isoprenylated proteins in cytomorphological differentiation. However, we found the isoprenylated proteins cannot influence the expression of microvilli. Recently, Zhong et al. (34) found an increment in caspase-2 and caspase-3 activities after treatment with 50 µM lovastatin for 48 h, and the hypodiploid cell percentage was noted in a dose-dependent manner. In addition, lovastatin can elicit the cytochrome c accumulation into cytoplasm fraction from mitochondria in 12 h.

In this study we found that lovastatin treatment could result in either apoptosis or differentiation of anaplastic thyroid cancer cells. Lovastatin caused a time- and dose-dependent inhibition of cellular survival as well as differentiation at a dose of 25 µM and apoptosis at a dose of 50 µM. HMG-CoA reductase inhibitors have been used to induce cancer cellular apoptosis (12, 17, 18, 19), but there are few reports of their use in cellular differentiation (20, 21, 22). Because anaplastic thyroid cancer is fatal and refractory to conventional therapies, including surgery, radioactive iodine, and chemotherapy, differentiation therapy combined with conventional strategies might provide the means to treat such patients.

We previously reported cytomorphological and biochemical differentiation of thyroid anaplastic cancer cells after treatment with TNF (23). These cytomorphological features could provide early information on differentiation and thus allow the use of radioactive iodine, but as TNF is highly cytotoxic for normal human tissue, its use in the treatment of anaplastic thyroid cancer is not practical. In contrast, HMG-CoA reductase inhibitors have been widely used to treat hypercholesterolemia for more than 10 yr, and high dose lovastatin has been used in a phase II study in patients with advanced gastric carcinoma (35). On the basis of these investigations, the safety of HMG-CoA reductase inhibitors is good, and they may therefore prove useful in differentiation therapy. In a pilot study we have shown that lovastatin treatment of an anaplastic thyroid cancer patient resulted in an increase in serum thyroglobulin levels from undetectable to greater than 300 ng/ml and in increase radioactive iodine uptake at metastatic lung lesions (unpublished data).

Recently, Zelvyte et al. (36) reported that increased peroxisome proliferator-activated receptor- (PPAR) levels in statin-treated monocytes. Statins added to monocytes can significantly inhibit the generation of inflammatory matrix metalloproteinases, monocyte chemotactic protein-1, and TNF. On the other hand, activation of PPAR was reported to inhibit cellular growth in anaplastic thyroid cancer cells in a dose-dependent manner (37). However, PPAR generally plays a critical physiological role in differentiation of fat cells and regulates lipid metabolism (38). The pleiotropic role of statins may suggest the involvement of PPAR in the modulation of cancer cell differentiation. Further studies will be needed to survey the possible relationship between PPAR and cytomorphological differentiation of anaplastic thyroid cancer cells.

In conclusion, our data show that lovastatin treatment of anaplastic thyroid cancer cells induces thyrocyte apoptosis at higher doses and differentiation at lower doses, as shown by DNA fragmentation, flow cytometry, 3-D cytomorphological changes (microvilli), and increased thyroglobulin secretion. These results suggest that the use of lovastatin and other HMG-CoA reductase inhibitors as both apoptotic and differentiation therapy for the treatment of anaplastic thyroid cancer merits further investigation.

	Footnotes

 
Abbreviations: CD buffer, Cacodylate buffer; 3-D, three-dimensional; FOH, farnesol; GGOH, geranylgeraniol; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; PI, propidium iodide; PPAR, peroxisome proliferator-activated receptor-; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Received November 22, 2002.

Accepted March 19, 2003.

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