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Keywords:

  • thyroid cancer;
  • roscovitine;
  • TRAIL;
  • apoptosis;
  • NF-κB

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Among thyroid carcinomas, highly aggressive undifferentiated or anaplastic carcinomas still await effective therapeutic strategies. R-roscovitine is a novel cyclin-dependent kinase inhibitor in clinical trials as anti-cancer agent. We have investigated the effects of R-roscovitine on proliferation and apoptosis of 4 thyroid cancer cell lines with different degrees of malignancy. R-roscovitine induced cell cycle arrest in G2/M phase in all cells analyzed possibly by inhibiting the CDK1-cyclin B1 complex. However, the compound was unable to induce a significant cell apoptosis. R-roscovitine has been shown to sensitize cancer cells to TRAIL-induced apoptosis. We report that R-roscovitine sensitized thyroid cell lines to TRAIL-induced apoptosis with the highest degree of synergism observed in the most undifferentiated cancer cells. Apoptosis was associated with the activation of caspases. In thyroid cancers, NF-κB is constitutively activated contributing to the proliferation of malignant cells. Accordingly, we observed that R-roscovitine inhibited p65 expression and nuclear translocation. Moreover, IKKβ over-expression inhibited R-roscovitine- and TRAIL-induced apoptosis. The combined treatment also caused down-regulation of anti-apoptotic proteins transcriptionally regulated by NF-κB. Finally, R-roscovitine up-regulated expression of DR5 TRAIL receptors. These results demonstrate that undifferentiated thyroid carcinoma cells can be effectively killed by a combination treatment of subtoxic doses of R-roscovitine and TRAIL. R-roscovitine sensitization of TRAIL-induced apoptosis appears to be mediated by the inhibition of the IKK/NF-KB pathway leading to down-regulation of anti-apoptotic genes and up-regulation of TRAIL death receptors. The combination of R-roscovitine and TRAIL may represent a novel approach to the treatment of anaplastic thyroid carcinomas resistant to conventional chemotherapy. © 2009 Wiley-Liss, Inc.

Thyroid carcinoma is the most common malignancy of the endocrine system. Four types of thyroid cancer, classified according to origin and grade of differentiation, comprise more than 98% of all thyroid tumors: papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and undifferentiated anaplastic thyroid carcinoma (ATC).1, 2 For well differentiated tumors, surgical resection and radioactive iodine can be an effective treatment. Undifferentiated or anaplastic thyroid carcinomas are highly aggressive, metastatic and fatal tumors. Various treatment modalities including radiation and combined chemotherapy have been tried in ATC with poor results as radio- or chemo- resistance remains a critical obstacle for such therapies. Effective treatment for ATC is not established until now and consequently there is need for new therapeutics for treatment of these tumors.3

The loss of regulatory control of the cell cycle is a hallmark of cancer, leading to unstrained tumor cell proliferation. The cell cycle is governed by the activities of cyclin-dependent kinases (CDK) and their regulatory cyclin partners.4 Abnormalities in CDK activity and regulation in cancers5 have proposed CDK functions as an attractive target for cancer therapy, and several CDK inhibitors are currently entering into clinical trials.6 The R-stereoisomer of roscovitine, a 2, 6, 9-substituited purine analogue, is one of the most frequently studied and used CDK inhibitors. Also referred to as CYC202 or Seliciclib, R-roscovitine has been tested in animals7, 8 and humans9 and has entered clinical trials against B-cell malignancies, lung and breast cancers.10 More recently, Rossi et al.11 have reported that R-roscovitine enhanced the resolution of inflammation by promoting inflammatory cell apoptosis suggesting that CDK inhibitors may have a potential in the treatment of inflammatory disorders.

Selective induction of apoptosis in malignant cells may represent an attractive mechanism to control neoplastic cell proliferation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of proteins including FasL, and TNF-α. TRAIL induces apoptosis by interacting with 2 cell-surface receptors, death receptor DR4 (TRAIL-R1) and DR5 (TRAIL-R2).12 Because TRAIL is able to induce apoptosis in many transformed and malignant cells, but not in normal cells,13 it has raised strong interest as a new promising anti-cancer agent. TRAIL receptors are broadly expressed in thyroid carcinomas and thyroid carcinoma cell lines.14 Interestingly, follicular undifferentiated thyroid carcinoma cells are very responsive to TRAIL-induced apoptosis, whilst the anaplastic thyroid carcinoma cells show very low sensitivity to TRAIL effects.14

Nuclear factor-κB (NF-κB) is a key regulator of genes involved in the control of cellular proliferation15 and apoptosis.16 NF-κB is the major anti-apoptosis transcription factor that regulates the expression of a number of anti-apoptotic genes, such as cellular inhibitors of apoptosis (c-IAPs), Bcl-2, Bcl-XL, c-FLIP and TRAF.17, 18 Activation of NF-κB has been implicated in resistance to different chemotherapeutic agents and to cytokines, such as TNF-α, TRAIL and Fas ligand.19, 20 Basal NF-κB activity is often increased in various types of human hematopoietic and solid tumors.21 In thyroid cancers NF-κB signaling pathways are constitutively activated contributing to the malignant potential of anaplastic thyroid carcinoma cells.22, 23 Indeed, inhibition of the NF-κB cascade potentiates the effect of a combination treatment of anaplastic thyroid cancer cells.24 It has been recently shown that R-roscovitine is able to sensitize tumor cells to apoptosis induced by protein agonists of the TNF family. It potentiates TNF-α-induced apoptosis in adenocarcinoma and melanoma cell lines via p53 activation and NF-κB inhibition.25 Moreover, R-roscovitine sensitizes glioma cells26 and breast cancer cells27 to TRAIL-induced apoptosis via several mechanisms including regulation of the expression of apoptotic genes like survivin, XIAP, FLIP, and Mcl-1.

Given the interest of R-roscovitine use in cancer, the effects of the drug alone or in combination with TRAIL in several thyroid cancer cell lines have been investigated. We report that R-roscovitine is able to sensitize anaplastic thyroid cancer cells to TRAIL-induced apoptosis via caspase activation and inhibition of the IKK/NF-κB pathway.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Drugs

R-roscovitine (Alexis Corporation, Vinci-Biochem, Italy) was dissolved in DMSO to obtain a stock solution of 40 mM, stored at -20°C, which was then diluted in culture medium to obtain the desired concentration. Soluble human recombinant SuperKiller TRAIL, SN50 NF-κB peptide blocker and SN50M control inactive peptide were purchased from Alexis Corporation. The General Caspase Inhibitor Z-VAD-FMK was from BD Pharmingen (BD Bioscience, Bedford, USA).

Cell cultures

Human papillary (NPA), follicular (WRO) and undifferentiated follicular (FRO) thyroid carcinoma cell lines were cultured in DMEM medium supplemented with 2-mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (all from Cambrex Bioscience, Verviers, Belgium). Undifferentiated thyroid carcinoma cells (ARO) were cultured in RPMI 1640 supplemented with 2-mM L-glutamine, 10% FBS, 1% penicillin/streptomycin at 37°C in an atmosphere of 95% O2 and 5% CO2 as previously described.28 Cells were plated at a density of 1 × 105 cells/well (Falcon, BD Bioscience, Bedford, USA) the day before treatment. At the end of the incubation period the cells were processed for Western blotting and FACS analyses. The cells were used up to a maximum of 10 passages.

Analysis of apoptosis

Hypodiploid DNA was analysed using the method of propidium iodide (PI) staining and flow cytometry as described.29 Briefly, cells were washed in phosphate-buffered saline (PBS) and resuspended in 500 μl of a solution containing 0.1% sodium citrate, 0.1% Triton X-100 and 50 μg/ml propidium iodide (Sigma-Aldrich, Italy). After incubation at 4°C for 30 minutes in the dark, cell nuclei were analyzed with Becton Dickinson FACScan flow cytometer using the Cells Quest program. Cellular debris was excluded from analysis by raising the forward scatter threshold, and the DNA content of the nuclei was registered on logarithmic scale. The percentage of the cells in the hypodiploid region was calculated.

Cell cycle analysis

Cells were plated at 1 × 105 in 60-mm dish and exposed to increasing concentrations of R-roscovitine (1–40 μM). After the incubation period cells were harvested and fixed in cold 70% ethanol at −20°C. Cell cycle profiles were evaluated by DNA staining with 2.5 mg/ml propidium iodide in phosphate buffered saline (PBS) supplemented with 100U/ml ribonucleases A, for 30 min at room temperature. Samples were analyzed with a FACScan flow cytometer (Becton Dickinson, CA) using the Cells Quest evaluation program. Distribution of cells in distinct cell cycle phases was determinate using ModFit LT cell cycle analysis software as described.30

Total, cytosolic and nuclear proteins extraction

Total intracellular proteins were extracted from the cells by freeze/thawing in lysis buffer 50 mM Tris-HCl containing protease and phosphatase inhibitors (1-mM PMSF, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 μg/ml aprotinin, 1 μM Na3VO4, 1 μM NaF; all from Sigma Sigma-Aldrich, Gallarate, Italy). Protein content was estimated according to Biorad protein assay (BIO-RAD, Milan, Italy) and the samples either analysed immediately or stored at −80°C. For cytosolic and nuclear extracts ARO cells were plated in 10-cm plates and incubated with or without R-roscovitine at different times. After incubation period cells were collected in PBS and centrifuged for 5 min at 1,200 rpm. The pellets were resuspended in 100-μl lysis buffer A (HEPES 10 mM pH 7.9, EDTA 1mM pH 8.0, KCl 60 mM, N-P40 0.2%, DTT 1 mM, PMSF 2 mM containing cocktails protease inhibitors) (all from Sigma-Aldrich, Italy) and then incubated on ice for 5 min. Supernatant obtained after centrifugation were separated from nuclei by addition of 50-ml lysis buffer C (TRIS-HCl 250 mM pH 7.8, KCl 60 mM, DTT 1 mM, PMSF 2 mM, 20% glycerol) and then centrifuged for 15 min at 9,500 rpm. Cytosolic protein extracts were stored at −80 °C until use. Nuclear pellets were suspended in 100-μl lysis buffer B (HEPES 10 mM pH 7.9, EDTA 1mM pH 8.0, KCl 60 mM, DTT 1 mM, PMSF 2 mM) and centrifuged for 5 min at 2,500 rpm. Pellets were suspended in 50-μl buffer C and nuclear proteins were obtained by 3 freeze/thawing cycles and after centrifugation at 9,500 rpm for 15 min. Total, cytosolic, and nuclear extracts were then analysed by Western blotting.

Western blotting analysis

Samples (30-μg protein) were loaded onto 10–12% acrylamide gels and separated by SDS-PAGE in denaturating conditions at 50 V. The separated proteins were then transferred electrophoretically (100 mA per blot 90 min; Trans Blot Semi-Dry, BIO-RAD) to nitrocellulose paper (Immobilon-NC, Millipore, Bedford, USA) soaked in transfer buffer (25 mM Tris, 192 mM glycine, Sigma-Aldrich) and 20% methanol vol/vol (Carlo Erba, Milan, Italy). Non specific binding was blocked by incubation of the blots in 5% no fat dry-milk powder (BIO-RAD) in TBS/0.1%Tween (25 mM Tris; 150 mM NaCl; 0.1% Tween vol/vol, Sigma-Aldrich) for 60 min. After washing, the blots were incubated overnight at 4°C with the primary following antibodies: rabbit polyclonal anti-p65 (diluted 1:1,000), rabbit polyclonal anti-caspase-3 (diluted 1:1,000), rabbit polyclonal IKKβ (diluted 1:500), mouse monoclonal anti-PARP (diluted 1:1,000), mouse monoclonal Bcl-XL (diluted 1:500) (all from Santa-Cruz Biotechnology, D.B.A. ITALIA s.r.l, Milan, Italy), rabbit polyclonal anti-histone H4 (diluted 1:500, Upstate Biotechnology, Lake Placid, NY), mouse monoclonal anti-cyclin B1 (diluted 1:1,000), mouse monoclonal anti-XIAP and survivin (diluted 1:1,000) (all from Stressgen, USA), rabbit polyclonal anti-p-ERK (diluted 1:1,000), mouse monoclonal anti-caspase-8 (diluted 1:1,000) (both from Cell Signaling Technology, Denvers, MA) and mouse monoclonal anti β-actin (Sigma-Aldrich, Italy). After incubation with the primary antibodies and washing in TBS/0.1% Tween, the appropriate secondary antibody, either anti-mouse (diluted 1:5,000), or anti-rabbit (diluted 1:5,000) (both from Sigma-Aldrich, Italy) was added for 1 hr at room temperature. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemiluminescence reagents (ECL) and exposed to Hyperfilm (both from Amersham Biosciences, Italy). The blots were then scanned and analysed (Gel-Doc 2000, BIO-RAD)

Cell transfection

ARO cells were plated in 10-cm cell culture plates in media containing 10% FBS. After 48 hr, cells were washed once with PBS, harvested with Trypsin/EDTA (Cambrex Bioscience, Verviers, Belgium), counted and centrifuged at 900 rpm for 5 min. Cell pellets were resuspended in room temperature Nucleofector Solution V (“Nucleofector® Solution,” Amaxa, USA) to a final concentration of 1 × 105 cells/100 μl for each sample. Hundred microliter of cell suspension were then mixed or not with 3 μg of IKβ kinase (IKKβ) DNA plasmid or pCMV plasmid (kindly provided by Dr. M.C. Turco, Dep. Pharmaceutical Sciences, University of Salerno) transferred in cuvettes, and electroporated by appropriate Nucleofector program according to the manufacturer's instructions. Cell suspension was then added to a 6-well plate containing prewarmed medium and incubated at 37°C, 5% CO2 in a humidified atmosphere. After 48 hr, cells were incubated with R-roscovitine and TRAIL for further 16 hr. At the end of the incubation period the cells were processed for Western Blot analysis and flow cytometry.

Flow cytometry of TRAIL receptors

ARO cells were plated in 6-cm wells and then treated with R-roscovitine at different times (0–24 hr). Cells were washed with PBS and collected in PBS-EDTA 1 mM. Pellets were incubated with 100 μl PBS with monoclonal antibodies anti-DR4, anti-DR5, anti-DcR1 and anti-DcR2 (diluted 1:100) (Alexis Corporation, Vinci-Biochem, Italy) on ice for 1 hr. After incubation, cells were washed twice and incubated in 100 μl PBS with secondary FITC-conjugated monoclonal antibody (diluted 1:100) (Alexis Corporation, Vinci-Biochem, Italy) on ice for 1 hr. After washing with PBS the expression of death receptors was analysed by flow cytometry.

Statistical analysis

All results are mean ± SEM of 3 experiments performed in triplicate. The optical density of the protein bands detected by Western blotting was normalized on β-actin levels. Statistical comparison between groups were made using Bonferroni parametric test. Differences were considered significant if p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

R-roscovitine induces cell cycle arrest in G2/M phase in thyroid carcinoma cell lines

To determine the effects of R-roscovitine on cell cycle of thyroid cancer, the 4 cell lines ARO, FRO, WRO and NPA were treated with increasing concentrations of R-roscovitine (1–40 μM) for 24 hr. Figure 1 shows that the drug induced a concentration- dependent accumulation of cells in G2/M phase that was significant (p < 0.001) at 20 μM in all thyroid carcinoma cells analyzed. The observed values for the different cells at 20-μM R-roscovitine were: ARO (30.1%, Fig. 1a), FRO (47.3% Fig. 1b), WRO (42.9%, Fig. 1c) and NPA cells (34.9%, Fig. 1d) compared with controls. These effects are maintained at 48-hr incubation with the drug (data not shown). Cell cycle progression phases are regulated through the binding of specific regulatory cyclins with cyclin- dependent kinases. To elucidate the role of specific regulatory proteins in the anti-proliferative effects of R-roscovitine, the expression of cyclin B1 in ARO cells incubated for different times (0–24 h) with 20-μM R-roscovitine was analyzed by Western blotting. R-roscovitine induced a significant down-regulation of cyclin B1 after 8–24 hr (Fig. 1e) suggesting that cell cycle arrest is due to inactivation of CDK1-cyclin B1 complex by R-roscovitine. Figure 1f also shows that 20-μM R-roscovitine inhibited ERK1/2 phosphorylation at 8–24 hr incubation in ARO cells. Similar results were obtained in the other thyroid cancer cells (data not shown).

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Figure 1. Effects of R-roscovitine in cell cycle progression and cell cycle regulatory proteins in thyroid carcinoma cell lines. ARO (a), FRO(b), WRO (c) and NPA (d) cells were treated with R-roscovitine (10–40 μM) for 24 hr. Cells were then collected and percentage of G1, S and G2/M cells was analyzed by flow cytometry. Cell cycle analysis and relative hystograms of thyroid cancer cells show accumulation of cells in G2/M phase after R-roscovitine treatment (***p < 0.001 vs. control cells). Data are expressed as mean ± SEM percentage of cell cycle distribution (n = 3). (e) Levels of cyclin B1 protein expression after 20-μM R-roscovitine were assessed at different times by Western blotting analysis and normalized on total β-actin levels. (f) Levels of phosphorylated ERK1/2 levels after 20-μM R-roscovitine were assessed at different times by Western blotting analysis and normalized on total ERK1/2 levels. Immunoblots show one representative experiment performed in ARO cells (n = 3). Similar results were obtained in FRO, WRO and NPA cells.

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R-roscovitine sensitizes thyroid carcinoma cells to TRAIL-induced apoptosis

To investigate the effect of R-roscovitine on apoptosis, ARO, FRO, WRO, and NPA cells were incubated with different concentration (1–40 μM) of the drug for 24 hr and apoptosis was measured by PI staining of hypodiploid nuclei as described in Material and Methods. Figure 2a shows that R-roscovitine was unable to induce significant apoptotic effects in ARO, NPA, and WRO thyroid cancer cells. High concentration (40 μM) caused 20% apoptosis in follicular undifferentiated FRO cells after 24-hr incubation. As R-roscovitine has been shown to sensitize cancer cells26, 27 to TRAIL-induced apoptosis we decided to investigate the effect of the combination of R-roscovitine and TRAIL on the apoptosis of the thyroid cancer cells. ARO, FRO, WRO and NPA cells were incubated with R-roscovitine (20 μM) and TRAIL (0.5 ng/ml) alone or in combination for 24 hr and apoptosis analyzed by PI staining. Figure 2b shows that R-roscovitine sensitized thyroid cell lines to TRAIL-induced apoptosis to a different extent. Interestingly, the highest degree of synergism of the 2 treatments was observed in the ARO and FRO cells that correspond to the more undifferentiated thyroid tumors that are associated to poorer prognosis because of the lack of effective treatment.

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Figure 2. Effects of R-roscovitine on apoptosis of thyroid cancer cell lines. (a) ARO, FRO, WRO and NPA cells apoptosis after treatment with different concentration of R-roscovitine (1–40 μM) for 24 hr. (b) Analysis of percentage of cells in apoptosis after combination treatment of R-roscovitine and TRAIL. ARO, FRO, WRO and NPA cells were co-treated with 20-μM R-roscovitine and 0.5 ng/ml TRAIL for 24 hr. Analysis of apoptosis as percentage of hypodiploid nuclei was performed by PI staining using flow cytometry. Results are shown as mean ± SEM of 3 experiments performed in triplicate. **p< 0.01 vs. control FRO cells; ***p < 0.001 vs. ARO, FRO, WRO and NPA control cells.

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Combined treatment with R-roscovitine and TRAIL induces caspase activation in ARO cells

In the light of the interesting results obtained with the combination treatment in ARO cells, we decided to further investigate apoptotic pathways in these cells. Thus, ARO cells were treated with different concentration of R-roscovitine (1–40 μM) and TRAIL (0.5–1 ng/ml) alone or in combination for 24 hr. Figure 3a shows that the effect of the combination treatment was dependent of the concentration of R-roscovitine. The role of caspase activation was also investigated. ARO cells were incubated with the caspase inhibitor Z-VAD-FMK (50 μM) 30 min before adding TRAIL (0.5 ng/ml) and R-roscovitine (20 μM) for further 16 hr. Z-VAD-FMK efficiently inhibited apoptosis induced by the combination of R-roscovitine and TRAIL (Fig. 3b). Caspase involvement was confirmed by Western blotting analysis of caspase-3, caspase-8 and PARP expression. Figure 3c shows that the combination treatment of R-roscovitine (20 μM) and TRAIL (0.5 ng/ml) for 16 hr induced activation of caspase-3, caspase-8, and cleavage of PARP, a substrate of caspase-3 in ARO cells.

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Figure 3. (a) Effects of the combined treatment with R-roscovitine and TRAIL at different concentrations in ARO cells. ARO cells were treated with R-roscovitine (1–40 μM) alone or in combination with TRAIL (0.5–1 ng/ml) for 24 hr. Analysis of apoptosis was performed by PI staining using flow cytometry. Results are showed as mean ± SEM of 3 experiments performed in triplicate.***p < 0.001 vs. control cells. (b) Effects of caspase inhibitor Z-VAD-FMK on R-roscovitine- and TRAIL-induced apoptosis. Z-VAD-FMK (50 μM) was administered 30 min before incubation with 20-μM R-roscovitine and 0.5 ng/ml TRAIL for 16 hr and percentage of apoptotic cells was evaluated by flow cytometry (mean ± SEM of 3 experiments performed in triplicate) ***p < 0.001 vs. control cells, °°°p < 0.001 vs. R-roscovitine and TRAIL. (c) Western blotting analysis of caspase-3, caspase-8 and PARP expression after 16-hr incubation with 20-μM R-roscovitine alone or in combination with 0.5 ng/ml TRAIL. Blots are representative of 3 different experiments.

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R-roscovitine inhibits NF-kB nuclear translocation in ARO cells

In anaplastic thyroid cancer sustained basal activation of NF-κB contributes to malignancy of cell lines.22, 23 Hence the effect of R-roscovitine on NF-κB expression in ARO cells was evaluated. ARO cells were incubated with 20-μM R-roscovitine for different times. At the end of incubations expression of subunit p65 of NF-κB was assessed by Western blotting in whole cell, cytosol and nuclear extracts using β-actin as control for whole cell and cytosol extracts and histone H4 for nuclear extracts. After 16-hr incubation, R-roscovitine clearly reduced p65 expression in whole cell and almost abolished nuclear expression of the subunit suggesting a likely inhibition of NF-κB nuclear translocation (Fig. 4a). Figure 4b shows that R-roscovitine (20 μM) inhibited p65 expression in whole cell extracts. This inhibitory effect was potentiated by the combined treatment of R-roscovitine (20 μM) and TRAIL (0.5 ng/ml).

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Figure 4. Effects of R-roscovitine on p65 protein expression in whole cell, cytosol and nuclei lysates of ARO cells. (a) ARO cells were treated with 20-μM R-roscovitine for different times and whole, cytosolic and nuclear expression of p65 was analyzed by Western blotting analysis. (b) Effects of R-roscovitine and TRAIL on p65 expression. ARO cell were treated for 16 hr with 20-μM R-roscovitine alone or in combination with 0.5 ng/ml TRAIL. Whole cell levels of p65 were assessed by Western blotting analysis. Blots are representative of three experiments. ***p < 0.001 vs. control cells.

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NF-κB inhibition sensitizes cells to TRAIL-induced apoptosis

To confirm the role of NF-κB inhibition on TRAIL-induced apoptosis ARO cells were treated with increasing concentrations (25–100 μg/ml) of either SN50, a specific NF-κB inhibitor peptide,31 or SN50M, a control inactive peptide, 1 hr before TRAIL treatment (0.5 ng/ml). After 16-hr incubation, apoptosis was measured by PI staining of hypodiploid nuclei as described in Material and Methods. Results in Figure 5 show that SN50 sensitized ARO cells to TRAIL-induced apoptosis in a concentration-dependent manner. The control peptide had no effect.

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Figure 5. Effects of SN50 and SN50M on TRAIL-induced apoptosis in ARO cells. Cells were pretreated with increasing concentrations (25–100 μg/ml) of SN50 or inactive control peptide SN50M for 1 hr before TRAIL treatment (0.5 ng/ml). After 16-hr incubation, percentage of apoptotic cells was evaluated by flow cytometry analysis. Results are expressed as mean ± SEM of 3 experiments performed in triplicate. *p < 0.05, ***p < 0.001 vs. TRAIL-treated cells.

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Overexpression of IKβ kinase inhibits apoptosis induced by combined treatment of R-roscovitine and TRAIL in ARO cells

Next we investigated the mechanisms of NF-κB regulation by R-roscovitine. ARO cells were transfected in transient for 48 hr with a plasmid to over-express IKβ kinase (IKKβ) or with CMV as control plasmid, as described in Material and Methods. Western blotting analysis in Figure 6a shows a marked IKKβ overexpression in ARO cells transfected with pIKKβ plasmid, whereas in CMV transfected cells kinase expression levels were similar to control cells. After 48 hr post-transfection, cells were incubated or not with R-roscovitine (20 μM) and TRAIL (0.5 ng/ml) for 16 hr. At the end of incubations percentage of apoptotic cells was analyzed by flow cytometry. Results in Figure 6b show that in ARO cells overexpressing IKKβ apoptosis induced by R-roscovitine in combination with TRAIL were significantly inhibited. These data suggest that inhibition of IKKβ expression/activity may be instrumental in the mechanism of R-roscovitine sensitization of ARO cells to TRAIL-induced apoptosis.

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Figure 6. Effects of IKKβ over-expression on apoptosis induced by R-roscovitine and TRAIL in ARO cells. (a) ARO cells were transfected for 48 hr with a plasmid overexpressing IKKβ or with an empty CMV plasmid as control using Nucleofection protocols as described in Material and Methods. Analysis of expression of IKKβ in transfected cells was evaluated by Western blotting and normalized on total β-actin levels. (b) After transfection, control, CMV- and IKKβ−treated cells were incubated with 20-μM R-roscovitine and 0.5 ng/ml TRAIL for 16 hr and analysis of apoptosis was performed by flow cytometry. Results are expressed as mean ± SEM of 3 experiments performed in triplicate. °°°P < 0.001 vs. control cells, §§§p < 0.001 vs. CMV-treated cells; ***p < 0.001 vs. R-roscovitine/TRAIL-treated cells.

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Effects of R-roscovitine on the expression of anti-apoptotic proteins

Alteration in the expression of members of the inhibitor of apoptosis (IAP) family contributes to chemotherapy resistance in thyroid cancer cells.32 As discussed above NF-kB inhibits apoptosis via its ability to regulate expression of a variety of anti-apoptotic proteins as Bcl-2, Bcl-XL, and IAPs family.17, 18 Hence we evaluated by Western blotting analysis the expression of XIAP, survivin and Bcl-XL in ARO cells treated with R-roscovitine and TRAIL. Figure 7 shows that TRAIL alone (0.5 ng/ml) had no effect on protein expression, whereas R-roscovitine alone (20 μM) inhibited XIAP expression after 16-hr incubation. R-roscovitine in combination with TRAIL (0.5 ng/ml) almost abolished the expression levels of XIAP, survivin, and Bcl-XL after 16-hr incubation.

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Figure 7. Effects of R-roscovitine and TRAIL on expression of anti-apoptotic proteins in ARO cells. (a) ARO cells were treated with 20-μM R-roscovitine and 0.5 ng/ml TRAIL alone or in combination for 16 hr. Expression of XIAP, survivin and Bcl-XL was then evaluated by Western blotting analysis. (b) Densitometric analysis of the effects on XIAP expression of R-roscovitine (20 μM) and TRAIL (0.5 ng/ml) alone or in combination for 16 hr. Results are expressed as mean ± SEM of 3 experiments performed in triplicate. ***p < 0.001 vs. control cells.

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Effects of R-roscovitine on TRAIL receptor expression

We next investigated the expression of TRAIL receptors after R-roscovitine (20 μM) treatment for different times by immunofluorescence flow cytometry analysis. Results in Figure 8 show up-regulation of DR5 expression after 24-hr R-roscovitine treatment. However, no significant changes of DR4 and decoy TRAIL receptors were observed after treatment with R-roscovitine.

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Figure 8. Effects of R-roscovitine on TRAIL receptor expression. ARO cells were treated with 20-μM R-roscovitine for 24 hr. Immunofluorescence of DR5-, DR4-, DcR1-, and DcR2-TRAIL receptors was measured by flow cytometry analysis as described in Material and Methods. Results are representative of 3 experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Anaplastic thyroid carcinomas are highly aggressive, metastatic and fatal tumors. Prognosis is very poor, with a median survival of only 6 months from the original symptom.3 As current treatments yield poor results because of radio- or chemo-resistance there is a clear need for new therapeutics in the treatment of these tumors. The loss of regulatory control of the cell cycle is a hallmark of cancer, leading to unstrained tumor cell proliferation. In thyroid tumors defects in transcriptional and post-transcriptional regulation of cell-cycle control elements seem to affect tumor progression.33

In the first part of the present study we have evaluated the effects of R-roscovitine, a CDK inhibitor in clinical trials as anti-cancer molecule, on cell proliferation and apoptosis in human thyroid carcinoma cells. R-roscovitine inhibited cell proliferation by induction of cell cycle arrest at G2/M phase in ARO, FRO, WRO and NPA thyroid cancer cells. Cell cycle arrest at G2/M phase was associated with the reduction of cyclin B1 expression levels suggesting a possible direct inhibition of CDK1/cyclinB1 binding complex. Moreover R-roscovitine was able to inhibit ERK1/2 phosphorylation, an important regulator of cell G2/M transition and mitosis, in a time-dependent manner in all thyroid cancer cells analyzed.

At variance with its anti-proliferative effects, R-roscovitine induced about 20% apoptosis in FRO cells at the highest concentration tested, whereas it was unable to induce cell death in the other cell lines analyzed. Previous data have shown that R-roscovitine is able to sensitize cancer cells to the apoptotic effects of TRAIL, a new potential anti-cancer agent of the TNF family of cytokines.26, 27 It is also known that FRO cells are very responsive to TRAIL-induced apoptosis, whilst the anaplastic thyroid carcinoma ARO cells show very low sensitivity to TRAIL effects.14 Hence, we decided to investigate the effects of the combined treatment of R-roscovitine and TRAIL on apoptosis of the thyroid cancer cell lines. The combined treatment of subtoxic doses of R-roscovitine and TRAIL induced apoptosis to a different extent in all 4 cell lines with the highest synergistic effects observed in undifferentiated thyroid carcinoma cells (ARO) that are the cell counterpart of the anaplastic thyroid carcinomas. On the basis of these results the mechanisms of the sensitizing effects of R-roscovitine on TRAIL-induced apoptosis was investigated in ARO cells. The synergistic effect on apoptosis of ARO cells was dependent on the concentration of R-roscovitine administered in combination with TRAIL (Fig. 3a) and on activation of caspases. Indeed, apoptosis was almost abolished by incubation of cells with Z-VAD-FMK, a caspase inhibitor. The combined treatment also induced activation of caspase-3, caspase-8, and cleavage of PARP, a substrate of caspase-3 in ARO cells.

In thyroid cancers NF-κB signaling pathways are constitutively activated contributing to the malignant potential of anaplastic thyroid carcinoma cells.22, 23 It has been recently reported that R-roscovitine causes potentiation of TNF-α−induced apoptosis adenocarcinoma and melanoma cell lines by suppression of the NF-κB pathway.25 Thus the effect of R-roscovitine on NF-κB activation was investigated. In ARO cells R-roscovitine inhibited the nuclear translocation of subunit p65 of NF-κB and p65 whole cell expression. This inhibition was potentiated by the combined treatment of R-roscovitine and TRAIL. These results suggest that NF-κB may be a target of R-roscovitine action in anaplastic thyroid cancer and that this negative regulation may be important in sensitizing ARO cells to TRAIL-induced apoptosis. The role of NF-κB inhibition in the sensitization of ARO cells to TRAIL-induced apoptosis was confirmed by the effect of SN50, a specific NF-κB peptide blocker. Pretreatment of ARO cells with SN50 was able to sensitize the cells to TRAIL-induced apoptosis in a concentration-dependent manner.

In most cells, Rel/NF-κB subunits are sequestered in the cytoplasm as inactive homo- or heterodimers, bound to IκB inhibitory factors. The rapid and transient activation of NF-κB complex in response to a wide variety of stimuli generally involves phosphorylation of IκB by the IκB kinase complex (IKKα, IKKβ, NEMO). Phosphorylation targets IκB for degradation via proteasome and culminates in nuclear translocation of active Rel/NF-κB dimers, their binding to DNA and transcriptional activation of cellular genes involved in cell proliferation, apoptosis and inflammation.18 A recent report has shown that R-roscovitine suppresses NF-κB activation by inhibiting IKKβ kinase activity which leads to defective IκB phosphorylation, degradation and impaired nuclear function of NF-κB.25 To investigate the role of IKKβ in ARO cells, cells were transfected with a plasmid to over-express IKKβ. The over-expression of IKKβ blocks the effect of combined R-roscovitine and TRAIL-induced apoptosis supporting the hypothesis that R-roscovitine prevents the nuclear localization of p65 and thereby down-regulates NF-κB pathway by inhibiting IKK activity in anaplastic thyroid cancer cells.

Alteration in the expression of c-IAPs, survivin and Smac contributes to chemotherapy resistance in thyroid cancer cells.31 In several cancer cells, NF-κB protects against cell death by up-regulating anti-apoptotic factors as Bcl-2, Bcl-XL, and IAPs.17, 18 Moreover, R-roscovitine has been shown to sensitize glioma cells to TRAIL-induced apoptosis by down-regulation of survivin and XIAP.26 In ARO cells TRAIL alone had no effect on protein expression, whereas R-roscovitine alone inhibited XIAP expression after 16-hr incubation. R-roscovitine in combination with TRAIL almost abolished the expression levels of XIAP, survivin, and Bcl-XL after 16-hr incubation. These evidences suggest that R-roscovitine can sensitize anaplastic thyroid cancer cells to TRAIL-induced apoptosis by inhibiting the expression of anti-apoptotic genes transcriptionally regulated by NF-κB.

A study of the physiological role of the different subunits of NF-κB has shown that over-expression of p65/RelA subunit inhibits the expression of DR4 and DR5 receptors and enhances expression of cIAPs resulting in the inhibition of TRAIL-induced apoptosis of breast cancer cells. These results suggest that p65 subunit acts as a survival factor in TRAIL-induced apoptosis.34 According to this evidence, we observed that treatment with R-roscovitine of ARO cells caused an up-regulation of the expression of DR5 TRAIL receptors, whereas no changes in the expression of DR4 and decoy TRAIL receptors were noticed. Thus, DR5 up-regulation may be a consequence of the inhibition of p65 by R-roscovitine and may represent an additional mechanism of sensitization of ARO cells by R-roscovitine to TRAIL-induced apoptosis. Interestingly, a very recent report has shown that ABT-737, a novel compound targeting Bcl-2 family members, induces expression of DR5 and sensitizes renal, prostate, and lung cancer cells to TRAIL-induced apoptosis.35

In summary, our results show that undifferentiated thyroid carcinoma cells, that are usually resistant to chemotherapy, can be effectively killed by a combination treatment of subtoxic doses of R-roscovitine and TRAIL. R-roscovitine sensitization of TRAIL-induced apoptosis appears to be mediated by the inhibition of the IKK/NF-κB pathway leading to down-regulation of anti-apoptotic genes and up-regulation of TRAIL death receptors. Additional mechanisms for R-roscovitine sensitization cannot be ruled out, namely the observed inhibition of ERK1/2 phosphorylation that will be addressed by future experiments. Indeed, the role of ERK signal transduction pathway has been differently reported. It has been shown that IL-1β/TNF-α treatment sensitized human thyroid epithelial cells to TRAIL-mediated apoptosis through the inhibition of the ERK pathway.36 More recently, Kim et al. reported that in human prostate cancer cell lines quercetin enhanced TRAIL-induced apoptosis through ERK pathway activation.37 However, whatever the mechanisms, we propose that the combination of R-roscovitine and TRAIL may represent a novel approach to the treatment of anaplastic thyroid carcinomas resistant to conventional chemotherapy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors gratefully acknowledge the contribution of Dr. Adriano G. Rossi, University of Edinburgh Medical School, to the revised article.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References