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Istituto di Medicina Interna e di Malattie Endocrine e del Metabolismo (V.V., R.M., F.F., G.P., R.V.), Cattedra di Endocrinologia, University of Catania, Ospedale Garibaldi, 95123 Catania, Italy; Dipartimento di Biomorfologia e Biotecnologia (E.M.), University of Messina, Torre Biologica, Policlinico Universitario, 98122 Messina, Italy; and Dipartimento di Medicina Sperimentale e Clinica (A.B.), Cattedra di Endocrinologia, University of Catanzaro, Policlinico Mater Domini, 88100 Catanzaro, Italy
Address all correspondence and requests for reprints to: Antonino Belfiore, M.D., Dipartimento di Medicina Sperimentale e Clinica, Cattedra di Endocrinologia, Università di Catanzaro, Policlinico Mater Domini, via Tommaso Campanella, 115-88100 Catanzaro, Italy. E-mail: belfiore{at}unicz.it.
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Abstract |
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), IL-4 has antiproliferative and apoptotic effects in many malignancies. Its effect in thyroid cancer is unknown.
We found that surgical specimens of thyroid carcinomas express both IL-4R and IL-4 in the majority of cases. Thyroid glands affected by Graves’ disease also express IL-4. We also studied a panel of eight thyroid cancer cell lines from different histotypes and found that thyroid cancer cells express high levels of IL-4R although they do not express IL-4.
We then compared the biological effects of IL-4 in TPC-1, a thyroid cancer cell line, and in MCF-7 breast cancer cells. IL-4 very weakly stimulated thyroid cancer cell proliferation, but it was very effective in protecting thyroid cancer cells from apoptosis induced by staurosporin. The protective effect of IL-4 was similar in magnitude to that of IGF-I and was associated with up-regulation of the antiapoptotic molecule Bcl-2 and weak down-regulation of the proapoptotic molecule Bax. Moreover, IL-4 slightly potentiated the survival effect of IGF-I. In contrast, IL-4 reduced growth and induced apoptosis in MCF-7 cells.
Taken together, these findings suggest that thyroid cancer cells receive significant protection from apoptosis by IL-4 produced in the thyroid gland by activated T lymphocytes when concomitant Graves’ disease is present.
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Introduction |
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IL-4, a pleiotropic cytokine produced by Th2 cells, elicits major regulatory effects in the immune response by inducing Th2 cell differentiation, proliferation, and apoptosis inhibition in both B and T cells. It also induces IgE and IgG1 class switching and major histocompatibility complex class II molecule expression (9). In addition, IL-4 has effects on nonimmune cells, i.e. it is mitogenic for fibroblasts and endothelial cells and favors adhesiveness of endothelial cells for T lymphocytes and eosinophils rather than for granulocytes (10).
IL-4 has raised the interest of oncologists because cells from a variety of epithelial malignancies express IL-4 receptors (IL-4R). In these malignant cells, including melanoma, colon, renal, and breast cancer cells, IL-4 has antiproliferative and/or apoptotic effects that markedly contrast with its growth-stimulating effects in T cells (11, 12, 13). In breast cancer cells, IL-4 antagonizes the antiapoptotic effect of IGF-I (13). These data have suggested that IL-4, or agonist IL-4 analogs, could be used in antitumor therapy.
The data on IL-4 antiproliferative and/or proapoptotic effects in epithelial malignancies may appear in contrast with the observation that thyroid carcinomas concurrent with Graves’ disease are usually aggressive.
To investigate this issue, we studied the presence of IL-4R and the biological effects of IL-4 in a panel of thyroid cancer cells. By comparison, we studied MCF-7 breast cancer cells, where IL-4 is known to be antimitogenic and proapoptotic (13).
We found that thyroid cancer specimens express both IL-4R and IL-4 in the majority of cases, whereas thyroid cancer cell lines express variable levels of IL-4R but not IL-4. IL-4 protected thyroid cancer cells from apoptosis induced by exposure to staurosporin with a potency similar to that of IGF-I. In contrast, IL-4 induced apoptosis and antagonized the survival effect of IGF-I in breast cancer MCF-7 cells.
The survival effect of IL-4 in thyroid cancer cells was associated with up-regulation of the antiapoptotic molecule Bcl-2 and down-regulation of the proapoptotic molecule Bax. These data suggest, therefore, that IL-4 produced by Th2 lymphocytes may contribute to thyroid cancer growth and aggressiveness in Graves’ patients and that antagonists to these cytokines or to their receptors may be helpful in the treatment of aggressive thyroid papillary carcinomas.
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Materials and Methods |
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Human thyroid cancer cell lines BC-PAP (papillary), FRO (follicular), WRO (follicular), ARO (anaplastic), and TPC-1 (papillary) provided by Dr. A. Fusco and M. Santoro (Naples, Italy); FF-1 (anaplastic) established in our laboratory; and 8505-C (papillary) purchased from DMSZ (Braunschweig, Germany) were grown in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), 100 µg/ml penicillin, and streptomycin. The human thyroid papillary cancer cell line PAP was provided by Prof. Edgar Selzer (Vienna, Austria) and grown in RPMI 1640 (Sigma) and DMEM (Sigma) (1:1) supplemented with 2 mM glutamine, 10% FBS, 100 µg/ml penicillin, and streptomycin. Normal thyroid cells in primary culture were obtained from surgical specimens after treatment with 1 mg/ml collagenase IV (Sigma). The human MCF-7 cell line was cultured in MEM (Sigma) plus 10% FBS, 100 µg/ml penicillin, and streptomycin. The human acute T cell leukemia cell line (JURKAT) was cultured in RPMI 1640 (Sigma) supplemented with 10% FBS, 2 mM glutamine, 100 µg/ml penicillin, and streptomycin. In all cell cultures, medium was routinely changed every 2 d.
Human thyroid cancer specimens were obtained at surgery and stored in liquid nitrogen until processing. Informed consent was obtained from patients.
Immunohistochemistry
Immunohistochemical staining was performed on 7-µm-thick sections of unfixed tissue specimens. Sections were cut with a cryostat at –30 C, fixed with acetone at –20 C for 10 min, and hydrated with PBS at room temperature for 45 min. After blocking in 2% normal serum for 20 min, sections were incubated overnight with the anti-IL-4 or anti-IL-4R polyclonal antibodies (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). Specific labeling was detected with biotin-conjugated antimouse/antirabbit IgG and avidin-biotin peroxidase complex. Sections were counterstained with either hematoxylin QS or Nuclear Fast Red (Bio-Optica, Milano, Italy), examined, and photographed using an Olympus BH-2 microscope (Olympus, Orangeburg, NY). In every experiment, sections were incubated with secondary antibody alone to further verify the specificity of the reaction. Both the intensity and the proportion of stained cells were determined by examining the entire slide and scored as: + (low staining intensity in less than 10% cells in the section); ++ (moderate staining intensity in 10–40% cells in the section); or +++ (high and diffuse staining intensity in more than 50% cells in the section).
Immunoblot analysis
Cell lysates were prepared in RIPA buffer containing 0.1% sodium dodecyl sulfate (SDS) and protease inhibitor cocktail (Roche Biochemical Inc, Basel, Switzerland). Samples were resuspended in loading buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% milk-Tris-buffered saline with Tween 20 and then immunoblotted with primary antibodies (1 µg/ml). Appropriate horseradish peroxidase-conjugated secondary antibodies were added at 1:2000 (Amersham Biosciences, Little Chalfont, UK), and proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).
The following antibodies were used for Western blotting: anti-IL-4R polyclonal antibody (Santa Cruz Biotechnology); anti-p27 monoclonal antibody (Exalpha Biologicals, Inc., Boston, MA); anti-p21 polyclonal antibody (Santa Cruz Biotechnology); anti-Bax monoclonal antibody (Trevigen Inc., Gaithersburg, MA); anti-Bcl-2 monoclonal antibody and anti-Bcl-XL monoclonal antibody (Santa Cruz Biotechnology); anti-FLIP polyclonal antibody (Calbiochem, La Jolla, CA); and anti-ß-actin monoclonal antibody (Sigma).
IL-4R and IL-4 measurement
IL-4R and IL-4 transcripts were measured by RT-PCR. Total RNA (1 µg) was reverse transcribed with Superscript II (Invitrogen, Paisley, UK) and Random Examers. Two microliters of the synthesized cDNA were then combined in a PCR using primers 5'-AAC-CAG-AGT-GAG-TAT-GAG-GAT-3' (forward) and 5'-CCG-TTC-CAG-AGC-GAA-GTG-CTT-3' (reverse) specific for IL-4R (fragment size, 309 bp). The IL-4 transcript was detected using primers 5'-TCA-CCT-CCC-AAC-TGC-TTC-CC-' (forward) and 5'-TGT-CGA-GCC-GTT-TCA-GGA-AT-3' (reverse) (fragment size, 327 bp) for first PCR and primers 5'-CAC-GGA-CAC-AAG-TGC-GAT-AT-3' (forward) and 5'-GCA-GCG-AGT-GTC-CTT-CTC-AT-3' (reverse) for nested PCR. ELE-1 amplification (ubiquitous gene) was performed using the following primers: 5'-ATT-GAA-GAA-ATT-GCA-GGC-TC-3' (forward) and 5'-TGG-AGA-AGA-GGA-GCT-GTA-TCT-3' (reverse) (fragment size, 280 bp).
Viability assay
Cell viability was measured by the methyl thiazolyl tetrazolium (MTT) test (MTT, Amersham Biosciences), which measures the ability of viable mitochondria to reduce soluble tetrazolium salts to insoluble formazan (14). MTT dye conversion is, therefore, proportional to the number of viable cells. Cells (1 x 103) were seeded in 96-well plates. After 24 h, medium was removed and replaced with BSA (0.1%). After an additional 24 h, 10 nM IGF-I or 50 ng/ml IL-4 was added in fresh medium. MTT conversion was measured 1, 3, 5, and 7 d later by dissolving formazan in dimethylsulfoxide and reading the absorbance at 405 nm. For dose response experiments, increasing IL-4 concentrations were used (10, 25, 50, 70, and 100 ng/ml). Cell viability was measured 4 d later, as described above.
Cell growth studies
Cell growth was measured by [3H]thymidine incorporation: cells (3 x 104/well) were plated in 24-well tissue culture plates and grown in their regular medium for 48 h. Medium was then replaced with fresh medium containing 0.1% BSA. Twenty-four hours later, either 50 ng/ml IL-4 or 10 nM IGF-I was added to each well. After 24 h, 18.5 kBq/well of [3H]thymidine was added for 4 h. Cell monolayers were then washed twice with cold buffer, incubated with cold 10% TCA solution for 30 min, solubilized with 0.1 N NaOH, and counted by liquid scintillation.
Invasion assay
Cell ability to invade collagen-coated filters was measured with the Boyden’s chamber technique. Approximately 105 cells, resuspended in 200 µl medium, were placed on 6.5-mm diameter polycarbonate filters (8-µm pore size, Corning Costar Corporation, Camdridge, MA) coated at the upper and lower side with 1.0 mg/ml collagen (invasion assay). Either IL-4 (50 ng/ml) or IGF-I (10 nM) in 1 ml medium was added to the lower compartment. The plates were incubated at 37 C with 5% CO2 overnight, and then the collagen at the upper side of the filter was removed with a cotton swab. Cells that had migrated to the lower side of the filter were fixed with 11% glutheraldeyde for 15 min at room temperature and stained with 0.1% crystal violet in 20% methanol for 20 min. After three washes with water and complete drying, the crystal violet was solubilized by immersion of the filters in 10% acetic acid. The concentration of the solubilized crystal violet was evaluated as absorbance at 590 nm.
Apoptosis assay
Apoptosis was determined with Cell Death Detection ELISA PLUS kit (Roche Diagnostic, Mannheim, Germany), a photometric enzyme immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone-associated DNA fragments after induced cell death.
Briefly, cells (2 x 104/well) were plated in 12-well tissue culture plates and grown in their regular medium for 24 h. Medium was then replaced with fresh medium containing IL-4 (50 ng/ml) or IGF-I (10 nM) in the presence or absence of staurosporin (Sigma) (2 nM for TPC-1 cell line and 40 nM for MCF-7 cells) for 24 h. At the end of incubation, the absorbance was read at 405 nm.
Statistical analysis
Differences between means were evaluated using the Student’s paired t test, as indicated in Results. The statistical program Statview 5.01 for Macintosh (SAS Institute, Inc., Cary, NC) was used.
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Results |
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IL-4 and IL-4R expression was first examined in a panel of eight thyroid cancer cell lines (four papillary, two follicular, and two anaplastic) and in one primary cell culture from normal thyroid by RT-PCR. MCF-7 human breast cancer cells were also examined. Phytoemoagglutinin-stimulated peripheral blood leukocytes were used as positive control for IL-4 expression; JURKAT cells were used as positive controls for IL-4R.
IL-4 transcripts were detected only in phytoemoagglutinin-stimulated peripheral blood leukocytes but neither in thyroid cells nor in MCF-7 cells (data not shown). In contrast, IL-4R transcripts were expressed in all thyroid cancer cell lines as well as in normal thyroid cells (Fig. 1A
). IL-4R protein expression was evaluated by Western blot and results expressed in all cells studied, with highest values in follicular and anaplastic cancer cells (Fig. 1B).
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was evaluated in surgical specimens by both RT-PCR and immunohistochemistry. We obtained specimens from: 1) normal thyroids (n = 8); 2) thyroids affected by Graves’ disease (n = 10); and 3) thyroid carcinomas of different histotypes (n = 24). No IL-4 or IL-4R were found in normal thyroid specimens. In Graves’ glands, IL-4R mRNA was found in all cases, although IL-4R protein was undetectable by immunohistochemistry; IL-4 mRNA was found in 80% of cases and IL-4 protein in 50% (data not shown). In cancer specimens, IL-4R mRNA was found in all cases studied and IL-4R protein in approximately 70% of cases. IL-4 was detected in 14 of 16 cases by RT-PCR and in 17 of 24 cases by immunohistochemistry. IL-4 immunostaining was localized at the level of the malignant thyrocytes in 11 cases, whereas it was localized at both the thyrocytes and the stromal cells in six cases (Table 1 and Fig. 2). In one case of papillary cancer concurrent with Graves’ disease, both IL-4 and IL-4R were found in both the neoplastic and the nonneoplastic tissues (Table 1). In papillary carcinomas, there was an association between lymphocyte infiltration and IL-4 positivity: in seven cases, both features were present; in three cases, only IL-4 was found; and in two cases, only lymphocyte infiltration was found. No apparent association between lymphocyte infiltration and IL-4 was present in follicular or anaplastic carcinomas (Table 1).
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We then evaluated the effect of IL-4 on cell viability in thyroid cancer cells maintained in serum-free medium in monolayer culture. IGF-I (10 nM), previously described as a relevant mitogenic and antiapoptotic factor for cultured thyroid cancer cells, was used as a control growth factor. Human breast cancer cells MCF-7, known to be growth-inhibited by IL-4 and growth-stimulated by IGF-I (13), were studied in parallel experiments. IL-4 was used at 50 ng/ml, a dose able to elicit maximal effects in most model systems, and cell viability was evaluated by MTT staining 4 d later.
IL-4 increased cell viability (up to 171% of unstimulated) in all papillary and anaplastic thyroid cell lines; although in one papillary cell line (8505C), this increase was very modest and not significant. IL-4 did not increase cell viability in follicular cancer cell lines, rather decreased it in one cell line (WRO) (Table 2). As expected, IL-4 reduced cell viability in MCF-7 cells (77% of unstimulated). IGF-I increased cell viability in all thyroid cancer cell lines; although in one of them (8505C), this increase was not significant. MCF-7 cell viability increased markedly in response to IGF-I (Table 2).
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). Maximal IL-4 effect was observed after 5–7 d and with a dose of 50–100 ng/ml.
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To evaluate whether IL-4 increased cell viability in TPC-1 cells by either increasing cell proliferation or decreasing the apoptosis rate, [3H]-thymidine incorporation was carried out after cell exposure to either IL-4 or IGF-I. Parallel experiments were carried out in MCF-7 cells. Exposure to IL-4 slightly increased [3H]-thymidine incorporation in TPC-1 cells, although not significantly. In contrast, IL-4 significantly reduced growth in MCF-7 cells (P = 0.03) (Fig. 4). IGF-I significantly increased [3H]-thymidine incorporation in both cell lines (P = 0.007 and P = 0.004 in TPC-1 and MCF-7, respectively) (Fig. 4).
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). In contrast, IL-4 increased p21Cip1/Waf1 levels at 24 h in MCF-7 cells (150% of unstimulated cells) and reduced p27Kip1 levels to 88% of unstimulated after 24 h (Fig. 5).
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We evaluated the ability of IL-4 to induce chemoinvasion in TPC-1 cells. Chemoinvasion was measured as the cell ability to cross a collagen barrier in Boyden chambers. Both IL-4 and IGF-I similarly stimulated chemoinvasion in TPC-1 thyroid cancer cells. This stimulation was weak for both ligands but consistent: 123 ± 6% for IL-4 (P = 0.0013) and 123 ± 4.7% for IGF-I (P = 0.0012). In MCF-7 cells, only IGF-I (129 ± 8%, P = 0.028) but not IL-4 (110 ± 12%, P = 0.45) stimulated chemoinvasion.
IL-4 protects thyroid cancer cells from apoptosis
Because IL-4 did not elicit a consistent proliferative effect in TPC-1, we interpreted the increased cell viability as mostly due to protection from apoptosis. To better study this effect, we then evaluated the possible protective effect of IL-4 on apoptosis induced by exposure to staurosporin, a potent apoptotic agent. TPC-1 or MCF-7 cells were plated in monolayers in the presence or the absence of 2 nM staurosporin, and protection from staurosporin-induced apoptosis was then evaluated by adding either IL-4 (50 ng/ml) or IGF-I (10 nM) or both compounds. Apoptosis was evaluated by measuring histone-associated-DNA-fragments (mono- and oligonucleosomes), as described in Materials and Methods. In TPC-1 cells, neither IL-4 nor IGF-I affected basal apoptosis whereas both of them effectively reduced staurosporin-induced apoptosis in a similar manner. The simultaneous treatment with the two compounds had an additive effect on apoptosis reduction (Fig. 6A). In MCF-7 cells, IL-4 did not significantly affect staurosporin-induced apoptosis, whereas IGF-I was highly protective and almost completely inhibited the effect of staurosporin. The combined effect of the two compounds was similar to that of IGF-I alone (Fig. 6A).
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Staurosporin reduced cell survival by approximately 30–40% in both cell lines. In agreement with results obtained measuring histone-associated-DNA-fragments, both IL-4 and IGF-I were equipotent in protecting TPC-1 cells from the effect of staurosporin. The simultaneous exposure to both ligands completely blocked the effect of staurosporin (Fig. 6B
). Different results were obtained in MCF-7 cells. IL-4 slightly potentiated the effect of staurosporin, whereas IGF-I was protective. The simultaneous addition of IL-4 reduced IGF-I protection (Fig. 6B).
IL-10, another Th2 cytokine present in Graves’ disease, was also tested in parallel experiments and produced results very similar to those of IL-4: it protected thyroid cancer cells from staurosporin-induced apoptosis with a potency similar to IL-4, whereas it potentiated the effect of staurosporin in MCF-7 cells (data not shown).
IL-4 differentially affects apoptosis regulatory molecules in MCF-7 and TPC-1 cells
To gain insight into the mechanisms underlying the differential effect of IL-4 on cell survival, we analyzed the effect of IL-4 on a panel of molecules involved in apoptosis regulation.
Bcl-2, Bcl-XL, and Bax belong to the same family and are major regulators of the mitochondrial arm of apoptosis pathway; Bcl-2 and Bcl-XL are antiapoptotic, whereas Bax is proapoptotic.
After 12–24 h exposure to IL-4, Bcl-2 expression increased by approximately 4-fold in TPC-1, whereas it remained unchanged in MCF-7 cells (Fig. 7A). Bcl-XL expression was increased in TPC-1 cells by 30% after 12 h and reduced by 20% after 24 h. In MCF-7 cells, Bcl-XL expression was reduced by 24% and 40%, after 12 and 24 h exposure to IL-4, respectively. Proapoptotic protein Bax expression was slightly affected by IL-4 treatment, changing in an opposite way in the two cell types (–18% and –16% in TPC-1 cells vs. +12% and +12% in MCF-7 cells after 12 h and 24 h, respectively) (Fig. 7A).
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). In addition to reducing antiapoptotic molecules Bcl-2 and Bcl-XL, staurosporin increased the levels of proapoptotic molecule Bax (to 140% and 280% of untreated cells in TPC-1 and in MCF-7, respectively). The addition of IL-4 not only blocked the staurosporin-mediated Bax increase in TPC-1 cells but also reduced Bax levels to approximately 60% of the level of untreated cells. By contrast, in MCF-7 cells, IL-4 did not affect staurosporin-mediated Bax increase.
To evaluate the potential role of IL-4 on the caspase arm of apoptosis, caspase 3 activation was assessed by Western blot by an antibody that detects both procaspase and the cleaved caspase fragment. We also measured cellular FLICE-inhibitory protein (cFLIP), an antagonist of the effector caspases, by Western blot. No significant effect of IL-4 on caspase-3 processing or cFLIP expression was found in both TPC-1 and MCF-7 cells (data not shown). Exposure to staurosporin reduced cFLIP levels by approximately 30% in both cell lines. Neither IL-4 nor IGF-I significantly affected these changes (data not shown).
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Discussion |
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First we observed that human thyroid cells in culture, both normal and malignant, express IL-4R mRNA and protein. When thyroid surgical specimens were examined by immunohistochemistry, IL-4R was found in most malignant specimens but not in normal thyroid specimens. These observations indicate that most thyroid carcinomas express high IL-4R levels, independently of the histotype. The expression of the ligand, IL-4, was not found in established thyroid cancer cell lines but was found in approximately 70% of fresh specimens from thyroid carcinomas. In 17 of 24 cases, IL-4 immunoreactivity appears to be localized in the malignant thyrocytes. Moreover, IL-4 was present in over 80% of specimens from thyroid glands affected by Graves’ disease. In one case of papillary thyroid cancer concomitant with Graves’ disease, IL-4 was found both in the malignant and in the nonmalignant tissue. IL-4 was not present in normal thyroid specimens. These observations are in agreement with previous data indicating that IL-4, together with other Th2 cytokines (e.g. IL-10), is expressed by lymphocytes infiltrating thyroid glands affected by Graves’ disease (7, 17). While this paper was being reviewed, Stassi et al. (18) reported that primary cultures from thyroid carcinomas from different histotypes, and apparently free of lymphocyte infiltration, express IL-4. They concluded that malignant thyrocytes may express IL-4 in an autocrine manner (18). Taken together, these findings indicate that thyroid cancer cells overexpress IL-4R and are exposed to IL-4, locally produced either by thyrocytes themselves or by infiltrating lymphocytes. IL-4 production may be especially important in tumors concomitant with Graves’ disease.
Second, we found that IL-4 exerted a potent antiapoptotic effect and protected cells from apoptosis induced by staurosporin. This effect was comparable with that of IGF-I, a major survival factor in thyroid cancer (19). In contrast with observations in thyroid cancer cells and in agreement with previous observations (13), we found that IL-4 induced apoptosis in MCF-7 breast cancer cells and did not protect those cells from staurosporin-induced apoptosis.
IL-4 also elicited opposite effects on cell growth of TPC-1 vs. MCF-7 cells: it weakly stimulated thymidine incorporation in thyroid cancer cells, whereas it inhibited growth in MCF-7 cells. This IL-4 effect on MCF-7 growth inhibition is small probably as a consequence of our procedure (cells are cultured in the absence of serum), because IL-4 mainly inhibits MCF-7 cell growth induced by FCS and estrogen (13).
Finally, IL-4 had a stimulatory effect on TPC-1 cell chemo-invasion, a property related to in vivo cancer invasion and metastatic potential. The degree of chemo-invasion stimulated by IL-4 was rather weak, but it was comparable with that observed in response to IGF-I, a well-known stimulator of cell motogenesis (20). The observed dissociation between the proliferative and the chemoinvasion effect in response to IL-4 in malignant thyrocytes is not surprising. A similar dissociation has been observed in SKUT-1 leiomiosarcoma cells in response to insulin (21) and in the estrogen receptor-negative breast cancer cells MDA-MB231 in response to IGF-I (22). It appears, therefore, that mitogenic and motogenic pathways stimulated by IL-4 in thyroid cancer cells may be dissociated; likewise, it may happen in response to other growth factors. These non-growth-related motogenic effects, nevertheless, may be relevant to the metastatic process.
To gain insight into the molecular mechanisms underlying the different effects on apoptosis and proliferation in TPC-1 and in MCF-7 cells, we studied the effects of IL-4 on a panel of apoptosis regulatory molecules. In particular, members of the Bcl-2 family have a key role in regulating cytochrome c efflux from the mitochondria into the cytosol and consequent activation of IL-1ß-converting enzyme family of proapoptotic cysteine proteases (23). Because these mitochondrial proteins may heterodimerize, the net effect on apoptosis protection results from the balance between antiapoptotic (Bcl-2, Bcl-XL) and apoptotic proteins (Bax, Bad) (24). We found that IL-4 markedly increases Bcl-2 expression and slightly decreases Bax in TPC-1 cells. In contrast, IL-4 decreased Bcl-XL and slightly increased Bax in MCF-7 cells. These findings may help explain the antiapoptotic effect in TPC-1 and the proapoptotic effect in MCF-7 cells. These data confirm previous findings indicating that Bax is less modulated by cytokines than Bcl-2 or Bcl-XL (25).
Cell exposure to staurosporin induced apoptosis and reduced Bcl-2 and Bcl-XL expression both in TPC-1 and MCF-7 cells. IL-4 antagonized, and almost completely blocked, Bcl-2 reduction in TPC-1 but not in MCF-7 cells. Furthermore, IL-4 slightly affected Bcl-XL levels in TPC-1, whereas it caused a more marked reduction in MCF-7 cells. IL-4 also blocked the staurosporin-mediated Bax increase in TPC-1 cells, whereas it did not affect staurosporin-mediated Bax increase in MCF-7 cells.
No clear effect of IL-4 was observed on caspase-3 cleavage and on the expression of cFLIP, a effectore caspase inhibitor (26).
In partial accordance with our present data, Stassi et al. (27) recently reported that both IL-4 and IL-10 protect Graves’ thyrocytes from CD95-mediated apoptosis. However, at variance with our data in thyroid cancer cells, these authors found that IL-4 up-regulates Bcl-XL and cFLIP in primary cultures from normal thyroid. However, in their last paper, the same authors reported that IL-4 protects malignant thyrocytes from chemotherapeutic drugs by up-regulating both Bcl-2 and Bcl-XL (18).
We found that IL-4 was weakly mitogenic in thyroid cancer cells, as assessed by thymidine incorporation. The progression in the cell cycle requires cyclin/CDK complexes that result in phosphorylation of the retinoblastoma protein. Upon phosphorylation, retinoblastoma releases the transcription factor E2F that activates transcription of genes involved in cell cycle progression (28). Cyclin/CDK complexes are negatively regulated by CDK inhibitors, such as p21Cip1/Waf1, p27Kip1, and others (29, 30). In particular, p27Kip1 has a key role in mediating G1 arrest by contact inhibition or serum deprivation and mainly targets cyclin D/CDK4 but also other cyclin/CDK complexes (31). We found that IL-4 elicits different effects on the expression of CDK inhibitors in the two cell lines. IL-4 down-regulates both p27Kip1 and p21Cip1/Waf1 in TPC-1 cells. The effect on p27Kip1 is, however, predominant. In contrast, IL-4 up-regulates p21Cip1/Waf1 and does not affect p27Kip1 in MCF-7 cells. These data indicate a positive effect of IL-4 on cell cycle progression in thyroid cancer cells but confirm a negative effect on cell cycle progression in MCF-7 cells.
Taken together, these findings suggest that, in patients with Graves’ disease and a concomitant thyroid papillary cancer, IL-4 and IL-10 produced by infiltrating lymphocytes may affect thyroid cancer biology and may potentiate the role of other antiapoptotic factors such as IGFs (19, 32). An impaired ability of the cell to undergo apoptosis in response to genotoxic insults may play a role in the accumulation of mutations that eventually leads to malignant transformation. Accordingly, others and we (2, 33) have reported that Graves’ patients have an increased rate of thyroid cancer. Moreover, the combined effect of increased survival, growth, and invasion in thyroid cells exposed to IL-4 may stimulate thyroid cancer aggressiveness and hematogenous spread. Interestingly, although the majority of Graves’-associated carcinomas are well differentiated and belong to the papillary histotype that is usually scarcely angioinvasive, these tumors may behave aggressively and show a higher rate of distant metastases and of persisting/relapsing disease than similar tumors occurring in euthyroid patients (3, 4).
These data suggest, therefore, that cytokines produced by Th2 lymphocytes may contribute to thyroid cancer aggressiveness in Graves’ patients and that antagonists to these cytokines or to their receptors may be helpful in the treatment of these aggressive thyroid papillary carcinomas.
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Footnotes |
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V.V. is the recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. F.F. is the recipient of a fellowship from the American Italian Cancer Foundation.
Abbreviations: CDK, Cyclin-dependent kinase; cFLIP, cellular FLICE-inhibitory protein; FBS, fetal bovine serum; IL-4R, IL-4 receptors; MTT, methyl thiazolyl tetrazolium; SDS, sodium dodecyl sulfate; TSAb, antibodies to the TSH receptor.
Received September 23, 2003.
Accepted March 7, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur J Immunol 26:294–299[Medline]
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