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BRIEF COMMUNICATION |
Dipartimenti di Biologia e Patologia Cellulare e Molecolare (S.L., I.I., A.Fu., G.P.) and Endocrinologia e Oncologia Molecolare e Clinica (M.V.), and Cattedra di Patologia Clinica (G.P.), Università di Napoli "Federico II," 80131 Napoli, Italy; Naples Oncogenomic Center-Centro Ingegneria Genetica (A.Fe., A.Fu.), Biotecnologie Avanzate, 80145 Naples, Italy; and Dipartimento di Scienze Farmaceutiche (M.B.), Università degli Studi di Salerno, 84084 Naples, Italy
Address all correspondence and requests for reprints to: Giuseppe Portella, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, via S. Pansini 5, 80131 Napoli, Italy. E-mail: portella{at}unina.it.
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Abstract |
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Introduction |
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Anaplastic carcinoma (ATC) constitutes 1–7% of all thyroid cancer cases. It arises from thyroid follicular cells showing morphological features of a highly malignant, undifferentiated neoplasm with multiple atypical mitotic figures; it reveals no follicular structures, colloid formation, or other features of thyroid differentiation (2). p53 and β-catenin mutations are the genetic alterations most frequently found in poorly differentiated and anaplastic carcinomas (3).
ATC represents one of the most aggressive human malignancies because it invariably has a fatal prognosis. In fact, the absence of active therapies is the major impediment to the successful control of the disease (2, 3, 4). Therefore, novel therapeutic approaches are required.
Selective replication oncolytic viruses represent a novel therapeutic approach; adenovirus and other viruses have been engineered for selective replication within neoplastic cells. The most common approach is the deletion of viral gene whose product is necessary for replication in normal cells but expendable in cancer cells. Unfortunately, due to the multifunctional nature of many viral proteins, the gene deletion approach for viral selective replication frequently results in a reduced replication and therapeutic potency (5).
The first replication-competent adenoviral mutant described, dl1520 (Onyx-015), contains a deletion of E1B-55K, which inhibits p53 and prevents apoptosis, allowing dl1520 replication in cancer cells lacking functional p53 pathway but not in normal cells. dl1520 was expected to replicate selectively in a high percentage of human cancers being the p53 pathway nonfunctional in about 50% of human neoplasia. However, the role of p53 in determining dl1520 selectivity has remained controversial for years because it was shown that dl1520 can replicate in tumor cell lines retaining wild-type p53 sequences, and its replication in primary cells is not restricted by p53.
Recently it has been shown that E1B-55K mediates late-viral RNA transport, and dl1520 tumor-selective replication is determined by a unique property of tumor cells to efficiently export late viral RNA in the absence of E1B-55K. Thus, loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, is the major determinant of dl1520 selective replication in neoplastic cells (5).
The use of dl1520 oncolytic virus as monotherapy has demonstrated limitations in efficacy, making more research necessary for the design of strategies that will increase its tumor-eradicating potential (6).
dl1520 has been used in conjunction with chemotherapeutic agents in clinical trials with evidence for potential synergistic antitumor activity. The use of dl1520 in conjunction with cisplatin and 5-fluorouracil has been examined in a phase II clinical trial involving head and neck carcinoma patients. dl1520 has been used in combination with leucovorin and 5-fluorouracil in a phase II trial in patients with gastrointestinal carcinoma metastatic to the liver and with gemcitabine in a phase I/II trial in patients with unresectable pancreatic carcinoma (6, 7). All these studies have shown that combined treatment with dl1520 and chemotherapy increases the therapeutic effects.
We previously demonstrated an antineoplastic activity of dl1520 against ATC cell lines; however, high multiplicity of infections (MOIs) were used (8). Synergistic cell killing effects of dl1520 with doxorubicin or paclitaxel were observed in ATC cell lines, with the IC50 drug values reduced 1.75- to 11-fold in the presence of dl1520 (8). We have also shown increased efficacy of dl1520 followed by ionizing radiations, compared with either treatment alone (9). These data, previously obtained by us, suggest that dl1520 may be a valid tool in the treatment of anaplastic thyroid carcinoma in combination with other agents.
To develop novel therapeutic strategies based on replication selective adenoviruses, it is important to identify drugs able to enhance the oncolytic activity. In the present study, we evaluated the effects of lovastatin in combination with dl1520 against ATC cells.
Lovastatin (3-hydroxy-methylglutaryl-CoA reductase inhibitor), a drug used for the treatment of hypercholesterolemia, has been reported to exert growth-inhibitory activity in vitro and in vivo. Farnesyl- and geranylgeranylpyrophosphate, intermediates in the cholesterol synthetic pathway, are needed for isoprenylation, a crucial step for membrane attachment of cellular proteins like Ras, Rho, Cdc42, Rac, etc. (10, 11). By inhibiting protein isoprenylation, lovastatin induces a reduction of cell proliferation and apoptosis (10, 11). Moreover, it has been demonstrated that low doses of lovastatin induce apoptosis of ATC cells (12, 13).
We have observed that lovastatin significantly enhanced the antineoplastic activity of dl1520 against ATC cells and its replication. Furthermore, a significant increase in viral gene expression was induced by lovastatin in ATC cells. Finally, the combined treatment induced a significant reduction of the growth of ATC tumor xenografts.
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Materials and Methods |
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ARO and FRO human thyroid anaplastic carcinoma cell lines were established by Dr. G. J. Juillard (Department of Radiation Oncology, University of California, Los Angeles, Los Angeles, CA), and ARO and FRO cell lines were kindly provided by Professor J. A. Fagin (University of Cincinnati College of Medicine, Cincinnati, OH). KAT-4 cells were obtained from Dr. Ain (University of Kentucky, Lexington, KY). Cal 62 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), German Collection of Microorganisms and Cell Cultures.
The NPA line derives from poorly differentiated papillary carcinoma and was obtained by Dr. G. J. Juillard. The FB1 cell line derives from a follicular carcinoma (14). Human embryonic kidney (HEK)-293 cells were obtained from American Type Culture Collection (Manassas, VA).
KAT-4 cells harbor a mutated p53 gene, (273 Arg->His), whereas FRO cells express very low levels of p53, but no p53 gene mutation was observed (8).
Cells were grown in DMEM supplemented with 10% fetal calf serum, glutamine, and ampicillin/streptomycin.
dl1520 (ONYX-015), a gift from Dr. A. Balmain (Cancer Research Institute, University of California, San Francisco, CA) and Dr. I. Ganly (Memorial Sloan-Kettering Cancer Center, New York, NY), is a chimeric human group C adenovirus (Ad2 and Ad5) that has a deletion between nucleotides 2496 and 3323 in the E1B region that encodes the 55-kDa protein. In addition, there is a C to T transition at position 2022 in region E1B that generates a stop codon at the third codon of the protein. Viral stocks were expanded in the HEK-293 cell line and purified, as previously reported (8).
Stocks were stored at –70 C after the addition of glycerol to a concentration of 50% (vol/vol). Virus titer was determined by plaque-forming units (pfu) on the HEK-293 cells.
Adenovirus infection
Cells were detached, counted, and plated in 6-well plate at 70% cell density. After 24 h, cells were infected with a green fluorescent protein (GFP)-transducing adenovirus (AdGFP), a nonreplicating E1 and E2-deleted adenovirus encoding GFP, diluted in growth medium at different MOIs; medium was replaced after 2 h. Cells were washed 24 h after infection and then trypsinized, washed, and resuspended in 300 µl PBS and analyzed for GFP expression on a fluorescence-activated cell sorter (FACS) cytometer (Dako Cytomation, Carpinteria, CA) and Summit version 4.3 software (Dako, Carpinteria, CA).
Different treatment conditions were used for the evaluation of lovastatin effects: antineoplastic effects were evaluated in the presence of lovastatin, whereas experiments designed to evaluate the effects of the drug on the early phases of viral infection were performed with a short treatment time.
For the evaluation of lovastatin effects on adenoviral entry and GFP expression, the cells were treated for 16 h with 10 µM lovastatin and then infected for 24 h with AdGFP. Cells were harvested and prepared for FACS analysis as previously described.
For the evaluation of the cytoxic effects of the dl1520 virus, 1 x 103 cells were seeded in 96-well plates, and 24 h later lovastatin was added to the incubation medium. After an additional 16 h, medium was replaced with a medium containing or not lovastatin plus dl1520 at different MOIs. After 14 d the cells were fixed with 10% trichloroacetic acid and stained with 0.4% sulforhodamine B in 1% acetic acid (15). The bound dye was solubilized in 200 µl of 10 mM unbuffered Tris solution, and the optical density was determined at 490 nm in a microplate reader (Bio-Rad Laboratories, München, Germany). The percent of survival rates of treated cells were calculated by assuming the survival rate of untreated cells to be 100%.
Quantitative PCR of dl1520
To quantify the amount of dl1520 virus genome on lovastatin treatment, ATC cells were treated with 10 µM lovastatin for 16 h and then media replaced with a medium containing different MOIs of dl1520 alone or with lovastatin. After 24 h of infection, cell media were collected and viral DNA extracted using a QIAamp DNA minikit (QIAGEN, Valencia, CA). Viral DNA was then quantified by real-time PCR using assay-specific primer and probe. A real-time-based assay was developed using the following primers: 5'-GCCACCGAGACGTACTTCAGCCTG-3' (upstream primer) and 5'-TTG TAC GAG TAC GC G GTA TCCT-3' (downstream primer) for the amplification of 143 bp sequence of the viral hexon gene (from bp 99 to 242 bp). For quantification, a standard curve was constructed by assaying serial dilutions of dl1520 virus ranging from 0.1 to 100 pfu.
To quantify the amount of dl1520 virus genome in tumor xenografts, total DNA was extracted from 20 mg of each sample using a DNA purification system (Promega Corp., Madison, WI). DNA was resuspended in 200 µl of water and 2 µl used for the real-time PCR-based assay. For each experiment the DNA was extracted from three different samples of each treatment group.
Quantitative RT-PCR of dl1520 gene expression
Cells were infected with 10 or 100 pfu of dl1520, in the presence or in the absence of 10 µM of lovastatin, and harvested at 24 h after infection. Cells were dissolved in 1 ml Trizol (Invitrogen, Carlsbad, CA). RNA quality was evaluated by agarose gel electrophoresis; DNase treatment was performed. One microgram of total RNA was reverse transcribed and the expression of E1A 13 S and Penton genes monitored using real-time PCR with the following specific primers: E1A 13 S forward, 5'-AATGGCCGCCAGTCTTTT-3' and reverse, 5'-ACACAGGACTGTAGACAA-3'; Penton forward, 5'-TAACCAGTCACAGTCGCAAG-3' and reverse, 5'-CCCGCGCCTTAAACTTATT-3' (16). To calculate the relative expression levels, we used the 2–[
][]cycle threshold (Ct) method. Negative controls, samples without RT-PCR, or CDNA template were included in every PCR run, always resulting negative (data not shown).
Detection of cell surface coxsackievirus-adenovirus receptor (CAR) receptor
Cells were grown in six-well plates. After 48 h cells were detached in PBS-EDTA 10 mM, washed with PBS, and then incubated 1.5 h in FACS buffer with a mouse anti-CAR monoclonal antibody RmcB (1:250) (17). After washing, the cells were incubated in FACS buffer containing the secondary antibody conjugated to fluorescein isothiocyanate (FITC) (1:100; Sigma, St. Louis, MO) and analyzed for CAR expression on a Cyan cytometer (Dako Cytomation) and Summit version 4.3 software (Dako).
Human thyroid tissue samples and real time RT-PCR
Normal and neoplastic human thyroid tissues were obtained from surgical specimens and immediately frozen in liquid nitrogen. Thyroid tumors were collected at the Laboratoire d’Histologie et de Cytologie, Centre Hospitalier (Lyon Sud, France) and the Laboratoire d’Anatomie Pathologique, Hospital de L’Antiquaille (Lyon, France).
Total RNA was isolated and DNase digested using the RNeasy minikit (QIAGEN) according to the manufacturer’s recommendations. One microgram of total RNA from each sample was reverse transcribed using random hexamers as primers and Moloney leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) to yield cDNA.
To design a quantitative PCR assay, a human ProbeLibray system (Exiqon, Vedbaek, Denmark) was used. Probe and primers pair to amplify a fragment for real-time PCR of Cxadr mRNA were selected entering its accession number (NM001338.3) on the assay design page of the ProbeFinder software. ProbeFinder generated an intron-spanning assay identifying the exon-exon boundaries within submitted transcript. An amplicon of 93 nucleotides scattered among sixth and seventh exon was selected. The primer sequences were: Cxadr forward, 5'-ATGAAAAGGAAGTTCATCAACGTA-3', Cxadr reverse, 5'-AATGATTACTGCCGATGTAGCTT-3'. The same procedure was used to select probe and primes for housekeeping gene G6PDH, accession no. X03674. The primer sequences were: G6PDH forward, 5'-ACAGAGTGAGCCCTTCTTCAA-3', G6PDH reverse, 5'-GGAGGCTGCATCATCGTACT-3'. All fluorigenic probes were dual labeled with FAM at 5'end and with a black quencer at the 3'end.
Relative quantitative TaqMan RT-PCR was performed in a Chromo4 detector (MJ Research, Waltham, MA) in 96-well plates using a final volume of 25 µl. The conditions used for PCR were 10 min at 95 C and then 45 cycles of 20 sec at 95 C and 1 min at 60 C. Each reaction was performed in duplicate. To calculate the relative expression levels, we used the 2–[][]Ct method, where [][]Ct = []Ct,sample – []Ct,reference.
Written informed consent was obtained from all participants. The study protocol was approved by the ethics committees of the Centre Hospitalier Lyon Sud and the University Federico II, Napoli, and conducted in accordance with the principles of the Declaration of Helsinki as revised in 2000.
Protein extraction and Western blot analysis
In all experiments 70% confluent cells were used. Cells were homogenized directly into lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.5 mM sodium orthovanadate, 20 mM sodium pyrophosphate). The lysates were clarified by 20 min centrifugation at 14,000 x g. Protein concentrations were estimated by a Bio-Rad assay, and then proteins were boiled in Laemmli buffer [Tris-HCl (pH 6.8) 0.125 M, sodium dodecyl sulfate 4%, glycerol 20%, 2-mercaptoethanol 10%, bromophenol blue 0.002%] for 5 min before electrophoresis. Proteins were subjected to SDS-PAGE (10% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membranes (Immobilon; Millipore Corp., Billerica, MA); complete transfer was assessed using prestained protein standards (Bio-Rad, Hercules, CA). After blocking with Tris-buffered saline-BSA [25 mM Tris (pH 7.4), 200 mM NaCl, 5% BSA], the membrane was incubated with the primary polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) against CAR receptor SC-15–405 (1:200) for 1 h (at room temperature) or with the monoclonal antibody against against β-actin (Sigma). Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) for 45 min (at room temperature), and the reaction was detected with an ECL system (Amersham Life Science, Buckinghamshire, UK).
Tumorigenicity assay
Experiments were performed with 6-wk-old female athymic mice (Charles River, Calco, Lecco, Italy). FRO cells (4 x 106) were injected into the right flank of 80 athymic mice. After 20 d, when tumors were clearly detectable, the animals were divided into four groups (20 animals/group), and tumor volume was evaluated. Two groups received lovastatin in the drinking water (40 mg/1000 ml) and after 6 d dl1520 (5 x 107 pfu) was injected in the peritumoral area in a group treated with lovastatin and in an untreated group.
Tumor diameters were measured with calipers every second day by two blind and neutral observers until the animals were killed. No mouse showed signs of wasting or other indications of toxicity. Tumor volumes were calculated by the formula of rotational ellipsoid: Tumor volume = A x B2/2, where A is the axial diameter and B is the rotational diameter.
All mice were maintained at the Dipartimento di Biologia e Patologia Cellulare e Molecolare Animal Facility. The animal experimentations described herein were conducted in accordance with accepted standards of animal care and in accordance with the Italian regulations for the welfare of animals used in studies of experimental neoplasia, and the study was approved by our institutional committee on animal care.
The amount of lovastatin administered was chosen considering that the average daily water intake for each mouse was 5–7.5 ml and 10 mg/kg/d was the best dose of lovastatin for obtaining biological effects in mice.
To evaluate the genome equivalent copies of dl1520 in tumor xenografts, three animals bearing FRO xenografts received lovastatin in the drinking water (40 mg per 1000 ml). After 6 d, dl1520 (5 x 107 pfu) was injected in the peritumoral area of the mice treated with lovastatin and in three untreated mice.
After 48 h animals were killed, tumors excised, DNA extracted, and viral replication evaluated by real-time PCR. DNA quality was analyzed by real-time PCR of the β-actin gene.
Statistical analysis
Comparisons among different treatment groups in the experiments in vivo were made by the ANOVA method and the Bonferroni post hoc test using a commercial software (Prism 4; GraphPad, San Diego, CA). Assessment of differences among rate of tumor growth in mice was made for each time point of the observation period; treatment groups were control, dl1520, lovastatin, and lovastatin plus dl1520.
The analysis of the cell-killing effect in vitro was also made by the ANOVA method and the Bonferroni post hoc test. For all the other experiments, comparisons among groups were made by the ANOVA method and t test.
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Results |
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Because adenoviral infection occurs via the attachment to the CAR (18), we analyzed CAR expression by Western blot; two CAR-specific bands of 44 and 46 kDa, respectively, were detected in all the samples (Fig. 1B
). However, certain differences were observed in their levels; in ATC cell lines, variable levels ranging from very low to intermediate were observed; the papillary thyroid carcinoma-derived NPA cell line, the follicular carcinoma-derived FB-1 cell lines, and the ATC-derived KAT-4 cell line express the highest CAR levels. A cytofluorimetric analysis was performed to evaluate the presence of CAR on the cell membrane (Fig. 1C), showing the absence of CAR on the cell surface in FRO, NPA, Cal 62, and FB1 cells. This observation suggests a CAR-independent entry of AdGFP in these cell lines. The cell lines ARO and KAT-4 showed the presence of CAR on the cell membrane despite being the less susceptible to adenoviral infection.
To confirm that anaplastic thyroid carcinomas express low levels of CAR, we evaluated the CAR mRNA levels in human thyroid carcinoma tissues by RT-PCR. Significantly lower levels (**, P < 0.01) in CAR gene expression were observed in papillary carcinomas, follicular carcinomas, and anaplastic thyroid carcinomas with respect to normal thyroid samples (Fig. 2).
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In both cell lines, the combined treatment enhanced the cell-killing activity of dl1520. In FRO cells treated with 10 pfu/cell of dl1520, an additive and significant (2.5 µM) and highly significant (5 µM) increase in cell killing was observed. In KAT-4 cells a highly significant increase in cell killing was induced by lovastatin treatment at all MOIs and lovastatin concentrations used (Fig. 3A).
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). KAT-4 cells kept in the presence of lovastatin also showed a highly significant increase in viral replication (**, P < 0.01), whereas 16 h treatment did not modify viral replication.
Lovastatin enhances the expression of viral genes
Because ATC cells infected in the presence of lovastatin show an increase in viral replication, we decided to evaluate the infectivity of ATC cells in the presence of lovastatin. KAT-4 and FRO cells were pretreated with a noncytotoxic concentration of lovastatin (10 µM) for 16 h and then infected with AdGFP at different MOIs. FACS analysis of treated or untreated cells showed that lovastatin treatment induced a highly statistically significant (**, P < 0.01) increase in the number positive cells ranging from 3 up to 10% (Fig. 4A).
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Pharmacological inhibitors of the Raf-MAPK kinase (MEK)-ERK pathway up-regulates CAR expression on the cell surface of cancer cells (19); lovastatin is a well-known inhibitor of p21 Ras isoprenylation and activity. Therefore, we evaluated the effects of lovastatin on CAR expression in ATC cells. CAR levels on cell surface, evaluated by FACS analysis (Fig. 4B), were not modified by lovastatin treatment.
Because lovastatin-treated cells showed an increased in fluorescence to cell ratio, we hypothesized that lovastatin treatment could enhance viral gene expression. To monitor the expression of adenoviral genes in lovastatin-treated ATC cells we selected two adenoviral genes, whose expression is correlated with different stages of the adenoviral life-cycle: E1A 13 S representing genes transcribed from the immediate early region and Penton representing genes expressed from the late region.
FRO and KAT-4 cells were infected respectively with a MOI of 100 and 10 pfu of dl1520 in the presence or in the absence of lovastatin (10 µM). The expression levels of E1A 13 S and Penton gene were measured by real-time RT-PCR at 24 h. Cotreatment with lovastatin significantly (*, P < 0.05) enhanced the expression levels of E1A 13 S and Penton gene in both cell lines (Fig. 5).
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FRO cells were injected into the right flank of athymic mice, and after 20 d, when tumors were clearly detectable (T = 0), animals were randomized into four groups and lovastatin was added in the drinking water of two groups. The animals received lovastatin until the end of the experiment. After 6 d dl1520 was injected within the tumor (T = 6), twice per week. In Fig. 6A, tumor growth is expressed as a percentage of growth relative to the volume observed at T = 0.
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Finally, we evaluated by real-time PCR (Fig. 6B) the genome equivalent copies of dl1520 in animals bearing FRO xenografts and treated with dl1520 and dl1520 plus lovastatin. Lovastatin treatment was able to induce a 10-fold increase in viral replication.
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Discussion |
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Replication-selective oncolytic viruses (virotherapy) are being developed as a novel, targeted form of anticancer treatment. dl1520 (Onyx-015) was the first virus that had been genetically engineered for replication-selectivity for treatment in cancer patients. Recent results from a phase III clinical trial have confirmed the ability of an oncolytic adenovirus (H101) bearing an E1B-55kD gene deletion similar to that present in dl1520 to increase the response rate of nasopharyngeal carcinoma in combination with cisplatin (20).
Selective oncolytic replicating adenoviruses have been tested in ATC cell lines and tumor xenografts showing promising results (8, 9); however, high MOIs of dl1520 were required to efficiently kill ATC cells.
The efficacy of oncolytic adenoviruses as therapeutic agents relies on the capability of target neoplastic cells to bind, internalize, and sustain the replication of adenoviruses. We analyzed the infectivity of a panel of human thyroid carcinoma cell lines, showing a low infectivity in most ATC cell lines. Conversely, papillary or follicular thyroid carcinoma cells display a higher infectivity. In a previous study, we evaluated the infectivity of ATC cell lines ARO, KAT-4, and FRO infecting the cells for 48 h with a lacZ-transducing adenovirus and evaluating the percentage of infected cells counting stained cells in randomly selected fields. The different results obtained in the two studies can be explained with the use of different reporter genes, infection times, and detection techniques.
Adenovirus internalization relies on the presence of cell surface of the CAR; a Western blot analysis has been performed on total cell lysates showing discrete or high expression levels of CAR. However, cytofluorimetric assay for CAR expression on cell surface showed a low or absent expression of CAR, suggesting a CAR-independent adenoviral entry in thyroid carcinoma cells lines. Furthermore, a significant reduction in CAR gene expression was observed in anaplastic thyroid carcinomas samples, compared with normal thyroid tissues.
Our observations are in agreement with previous studies and confirm a low susceptibility of ATC cells to therapeutic approaches based on adenoviral vectors (21).
It is worth noting that CAR expression is negatively regulated by TGF-β during the switch from epithelial to mesenchymal phenotype, and human pancreatic carcinoma cells show loss of epithelial differentiation and CAR surface localization on TGF-β treatment (22). In ATC cell lines, we observed discrete or high total expression levels of CAR and loss of surface localization of CAR. Our results indicate that the loss of thyroid differentiation, a typical feature of ATC, causes an intracellular localization of CAR receptor, probably by increasing the recycling of CAR receptors or shifting them to the cytosol.
Because ATC cells display a low infectivity and a high MOI of dl1520 is required to efficiently kill these cells, we decided to enhance the oncolytic activity by using lovastatin in combination with dl1520.
Lovastatin, a drug used for the treatment of hypercholesterolemia, has previously been reported to exert growth-inhibitory activity in vitro and in vivo. Farnesyl- and geranylgeranylpyrophosphate, intermediates in the cholesterol synthetic pathway, are needed for isoprenylation a crucial step for membrane attachment of cellular proteins like of p21 Ras, Rho, Cdc42, Rac, etc. (10). By inhibiting protein isoprenylation, lovastatin exerts its cellular effects, including a reduction of cell proliferation and induction of apoptosis (10). It is worth noting that low doses of lovastatin induce apoptosis or differentiation of ATC cells (11, 12).
Our study demonstrates that the treatment of ATC cell lines KAT-4 and FRO with lovastatin significantly increased the effects of dl1520.
A significant increase in viral replication was observed in lovastatin-treated cells. In FRO cells a significant increase was observed infecting lovastatin-treated cells with 10 pfu/cell, whereas at 1 pfu/cell, no significant differences were observed. In KAT-4 cells kept in the presence of lovastatin and infected with 5 or 10 pfu/cell, significant increase was observed; however, the pretreatment with lovastatin for 16 h was not able to increase viral replication.
These results suggest that the increase in viral replication observed treating ATC cells with lovastatin is viral dose or lovastatin treatment time dependent. Therefore, it is possible that higher MOIs of dl1520 in combination with a 16-h lovastatin pretreatment could result in enhanced viral replication in KAT-4 cells.
Increased infection efficacy could account for the increased oncolytic activity after lovastatin treatment; moreover, lovastatin inhibits the isoprenylation and the activity of p21 ras, blocking MEK activity, and it has been reported that MEK inhibitors, U0126 and PD184352, increase the expression of CAR protein and adenoviral entry in cancer cells (19). Therefore, we evaluated the adenoviral entry and CAR expression levels in ATC cell lines treated with lovastatin. We observed a modest, albeit significant, increase in adenoviral entry (3–10%), which that it is not sufficient to explain the effects of lovastatin on viral replication and cell survival.
CAR expression levels either total (data not shown) or on the cell surface were not modified by lovastatin treatment.
Cholesterol deprivation of FRO and KAT-4 cells did not modify adenoviral entry (data not shown), suggesting that the effects of lovastatin were not mediated by the inhibition of cholesterol synthesis.
It has been shown that lovastatin inhibits the invasiveness of an ATC cell line (ARO) and focal adhesion kinase phosphorylation, a nonreceptor tyrosine kinase playing a crucial role in integrin-mediated cell adhesion (23); therefore, it is not possible to exclude that lovastatin induces changes in integrin pattern on cell surface. Because it has been suggested that integrins can act as a coreceptor for adenovirus types 2 and 5 (24), the modest increase in adenoviral entry observed by us could be due to changes in integrins pattern induced by lovastatin. Further studies are required to verify this hypothesis.
A high increase in the fluorescence to cell ratio of lovastatin-treated cells was observed. This observation suggested to us that the drug could enhance the expression of viral genes, thereby increasing viral replication. To confirm this hypothesis, the expression of two viral genes, representing the early and the late phases, was analyzed in ATC cells infected with dl1520. Lovastatin increased significantly the expression of early gene E1A 13 S and a late gene Penton.
It has been proposed that drugs able to affect the cell cycle could enhance viral oncolysis by modulating viral gene expression (25). Lovastatin together with other clinically used statins (fluvastatin and simvastatin) induces a G1 arrest of prostate cancer cells (26). A block of the cell cycle in ATC cells could enhance viral gene expression and increase the replication of dl1520. Further studies are required to clarify the mechanisms responsible for the effects of lovastatin on dl1520 gene expression and replication.
The administration of lovastatin per os to nude mice bearing ATC tumor xenografts enhances the antitumoral effects of dl1520 and increases the viral replication in vivo, confirming that this treatment could improve the clinical efficacy of replicating oncolytic adenoviruses. Interestingly, the injection of lovastatin and the virus within the tumor did not increase the effects of dl1520 (data not shown), indicating that a constant lovastatin blood concentration should be obtained to enhance the effects of dl1520.
The data presented here identify lovastatin as a promising agent for increasing the effects of oncolytic adenoviruses in ATC cells. It has been reported that FR901228 and trichostatin A, members of a new group of anticancer agents, the histone deacetylase inhibitors (27, 28, 29), or the MEK inhibitors U0126 and PD184352 (18) increase the antineoplastic effects of oncolytic adenoviruses. However, these agents have been used in a limited number of clinical trials, whereas 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have been widely used to reduce cardiovascular diseases without major adverse effects (10, 11). Lovastatin is generally well tolerated; the most important adverse effect is myotoxicity within the first 3 months of therapy (11). The risk of muscle dysfunction is increased by coadministration of other drugs able to interfere with lovastatin metabolism (11).
In the present study, in in vitro experiments, lovastatin was used at final concentrations ranging from 2.5 to 10 µM, and in in vivo, we used a lovastatin concentration of 10 mg/kg/d.
Although the therapeutic dose for the treatment of hypercholesterolemia is about 1 mg/kg/d, which yields serum levels of 0.1 µM, it has been shown that serum concentrations of 2–4 µM of lovastatin are well tolerated in animal models, and clinical trials of phase I and II have been performed using 25 mg/kg/d with encouraging results (11). All these data indicate that lovastatin at a dose of 10 mg/kg/d could probably be used in ATC patients in combination with dl1520 without major side effects.
In conclusion, we have identified lovastatin as a drug that increases adenoviral replication and enhances the effects of an oncolytic adenovirus in vitro and in vivo against ATC cells. Therefore, lovastatin could be useful in the context of gene therapy for anaplastic thyroid carcinoma. Further studies are required to confirm the feasibility of this approach in ATC patients.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online August 9, 2007
Abbreviations: AdGFP, GFP-transducing adenovirus; ATC, anaplastic thyroid carcinoma; CAR, coxsackievirus-adenovirus receptor; Ct, cycle threshold; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HEK, human embryonic kidney; MEK, MAPK kinase; MOI, multiplicity of infection; pfu, plaque-forming unit.
Received June 8, 2007.
Accepted for publication August 2, 2007.
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References |
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(v)β(1) is an adenovirus coreceptor. J Virol 75:5405–5409
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