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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 410-418
Copyright © 2004 by The Endocrine Society

Inhibition of Nuclear Factor-{kappa}B Cascade Potentiates the Effect of a Combination Treatment of Anaplastic Thyroid Cancer Cells

Dmitriy Starenki, Hiroyuki Namba, Vladimir Saenko, Akira Ohtsuru and Shunichi Yamashita

Departments of Molecular Medicine (D.S., H.N., S.Y.) and International Health and Radiation Research (V.S., S.Y.), Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, and Takashi Nagai Memorial International Hibakusha Medical Center (A.O., S.Y.), Nagasaki University Hospital, Nagasaki 852-8523, Japan

Address all correspondence and requests for reprints to: Hiroyuki Namba, M.D., Ph.D., Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: namba{at}net.nagasaki-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nuclear transcription factor-{kappa}B (NF-{kappa}B) is a transcriptional complex that is rapidly activated in the course of an immediate early response of cells after exposure to different stresses including ionizing radiation (IR). To overcome the limitation of radiation therapy for thyroid cancers, we studied the response of the NF-{kappa}B cascade to IR in cultured normal human thyroid cells and various thyroid cancer cell lines. Exposure to IR resulted in a dose-dependent increase of DNA-binding activity of p65 and p50 subunits in all types of thyroid cells. Specific inhibitors of NF-{kappa}B or phosphorylation deficient mutant inhibitory protein I{kappa}B{alpha} reduced thyroid cancer cell survival after exposure to IR and enhanced IR-induced cell death in a model undifferentiated thyroid cancer cell line. Tumors harboring mutant I{kappa}B{alpha} implanted into nude mice exhibited delayed growth rate and increased radiosensitivity. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and annexinV-propidium iodide staining revealed the increase of radiation-induced apoptosis in the cells with inhibited NF-{kappa}B signaling. Our results indicate that radiosensitivity of transformed thyroid cells is due in part to elevated basal activity and rapid induction of the active form of NF-{kappa}B. We therefore suggest that inhibition of NF-{kappa}B could be an effective modality for radiation therapy of advanced human thyroid cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDIFFERENTIATED OR ANAPLASTIC thyroid cancer (ATC) is a highly aggressive and fatal tumor. Various treatment modalities including radiation and chemotherapy have been tried in ATC. However, they are largely unsuccessful because of the high radio- or chemoresistance of ATCs that remains a critical obstacle in such treatment (1).

Recent studies have shown that radiation-induced responses can be mediated by a variety of pathways, including the DNA-damage- and p53-independent cascades (2). ATC cells usually lack functional p53 (3), and therefore p53-independent signaling may be important in the regulation of tumor radiosensitivity. In our previous studies, we demonstrated that thyroid-specific cell signaling involving several molecules downstream of p53, and protein kinase C{delta}-MKK7-c-Jun N-terminal kinase may contribute to the radiation response of ATC cells (4, 5).

Nuclear factor-{kappa}B (NF-{kappa}B) is a key regulator of genes involved in the control of cellular proliferation and apoptosis (6). In most cases, activation of NF-{kappa}B protects against cell death by up-regulation of expression of antiapoptotic genes, such as TRAF, c-IAP, p21, and Bcl-XL (7, 8).

Exposure to ionizing radiation (IR) leads to phosphorylation and subsequent proteasome-mediated degradation of the NF-{kappa}B cellular inhibitory protein I{kappa}B (I{kappa}B) (9). Subsequently, the released NF-{kappa}B translocates to the nucleus and activates the transcription of target genes. Thus, NF-{kappa}B is a radiation-responsive transcription factor (10), whose role in modulation of radiosensitivity of various cancers has been explored in several models (11, 12, 13).

The NF-{kappa}B signaling pathway is dysregulated in thyroid cancers through the mechanism of sustained activation, which contributes to the malignant potential of ATC cell lines (14). Therefore, the apoptosis permissive effects of NF-{kappa}B blockade may be regarded as a promising strategy for the treatment of ATC in combination with radiation therapy. The present study was designed to determine the role of the NF-{kappa}B signaling cascade in radioresistance of various thyroid cancer cell lines. For this purpose, we interrupted the radiation-induced activation of NF-{kappa}B in thyroid cells using a specific inhibitor of translocation of NF-{kappa}B to the nucleus, SN50, and expression of phosphorylation-deficient mutant I{kappa}B{alpha} (15), to determine whether this could lead to the modulation of radiation effects in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and exposure

Human anaplastic thyroid carcinoma cell lines FRO and ARO, papillary carcinoma cell lines NPA, TPC-1, and follicular carcinoma cell line WRO were initially provided by J. A. Fagin (University of Cincinnati College of Medicine). Papillary carcinoma cell line KTC-1 was provided by J. Kurebayashi (Kawasaki Medical School).

Cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% (wt/vol) penicillin/streptomycin (all reagents from Invitrogen Life Technologies, Paisley, UK) in a 5% CO2 humidified atmosphere at 37 C. Primary human thyroid cell culture was established as described previously (16) and maintained in DMEM:F12 (1:2) mixture supplemented with 3% FBS and 1% (wt/vol) penicillin/streptomycin. After 2-d incubation, when the culture reached about 80% confluence, cells were washed twice with PBS (pH 7.4) at 37 C and fresh medium containing 0.1% of FBS was added to each dish. Cells were incubated for an additional 24 h, exposed to X-radiation (EXS-300 X-irradiator; Toshiba, Tokyo, Japan; 200 kV, 15 mA, 0.85 Gy/min), and then collected at different time intervals.

Preparation of cell extracts

Cells were washed twice with ice-cold PBS, scraped, and collected in 1 ml PBS and centrifuged for 3 min at 1000 rpm. The pellet was then resuspended in 200 µl of lysis buffer (20 mM HEPES, pH 7.5; 0.35 M NaCl; 10% glycerol; 1% Nonidet P-40; 1 mM MgCl2 6H2O; 0.5 mM EDTA; and 0.1 mM EGTA) containing a protease inhibitor cocktail (Roche Diagnostics, Tokyo, Japan). After 15 min on ice, the lysate was centrifuged for 15 min at 15,000 rpm. The supernatant was stored at -80 C until use. Nuclear extracts were prepared as described previously (17). Cells pellets were resuspended in hypotonic buffer containing 10 mM HEPES (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, and 0.1% Nonidet P-40 and incubated on ice for 15 min. After centrifugation at 10,000 rpm, the supernatants were carefully removed to clean tubes and used as cytoplasmic fractions. The pellets were then resuspended in 40 µl of nuclear extract buffer (20 mM HEPES, pH 7.6; 420 mM NaCl; 1.5 mM MgCl2; 1 mM DTT; 0.2 mM EDTA; 0.5 mM phenylmethylsulfonylfluoride; 20% glycerol) and incubated on ice for 15 min. After centrifugation at 15,000 rpm for 10 min, the supernatants were transferred to clean tubes and used as nuclear extracts.

Western blotting

Total cell lysates were boiled in the sample buffer (100 mM Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 10% DTT) and separated by SDS-PAGE in 10% polyacrylamide gels. Proteins were transferred onto 0.2 mm polyvinyl difluoride membranes (Millipore Corp., Bedford, MA) by semidry blotting. Membranes were blocked with Tris-buffered saline/0.1% Tween 20 containing 5% nonfat dry milk and incubated with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. After washing three times with Tris-buffered saline/0.1% Tween 20, blots were incubated with horseradish peroxidase conjugates of appropriate secondary antibodies (Cell Signaling Technology, Beverly, MA) for 1 h at room temperature and then washed again. Complexes were visualized using enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL).

DNA-binding assay

The multiwell colorimetric assay for active NF-{kappa}B was performed as described previously (18), using a Trans-AM NF-{kappa}B p65 and p50 transcription factor assay kits (Active Motif North America, Carlsbad, CA), according to the manufacturer’s instructions. Briefly, nuclear extracts were incubated in 96-well plates coated with immobilized oligonucleotide, containing a NF-{kappa}B consensus binding site. NF-{kappa}B binding to the target oligonucleotide was detected by incubation of parallel samples with the primary antibodies specific for p65 and p50 subunits provided with the kits. For quantification, OD was read at 450 nm with a microplate reader (ImmunoMini NJ-2300, System Instruments, Tokyo, Japan). Background binding, obtained by incubation with mutant {kappa}B probe provided in the kit was subtracted from the obtained value.

Cell survival assay

Cultures were established in the 96-well flat-bottom microtiter plates (Nalge Nunc International, Tokyo, Japan). Cells were counted and resuspended in RPMI 1640 containing 5% FBS. Cell suspensions (100 µl, 2000 cells/well) were added to each well and incubated for 24 h before treatment.

Solutions of SN50 and inactive control SN50M (Calbiochem, La Jolla, CA) were added to each well in 20 µl of medium at various concentrations, 6 wells for each concentration. After incubation for 1 h, plates were exposed to x-rays. After a 48-h incubation, 10 µl of CKK-8 solution (Dojin, Osaka, Japan) were added to each well and incubated for 1 h at 37 C. Optical density was read at 450 nm in a microplate reader.

Establishment of mutant I{kappa}B{alpha} transfectants

ARO cells were transfected with mutant I{kappa}B{alpha} plasmid (19) using Lipofectamine reagent (Invitrogen Life Technologies). Subclones stably expressing the mutant I{kappa}B{alpha} (32/36 AA) were selected in RPMI 1640 medium containing 400 µg/ml of G-418 (Promega, Madison, WI). Cell lysates from 20 individual clones were analyzed for the expression of FLAG-tagged mutant I{kappa}B{alpha} by Western blotting. The clone with maximum expression was referred to as mutARO and was used in further experiments. Empty linearized pcDNA3 vector was transfected as a control. All experiments with transfected cell lines were performed after three passages without G-418.

Annexin V/propidium iodide staining

Detection of apoptotic cells was performed by means of the annexin V-propidium iodide (PI) detection kit (Wako Chemicals, Osaka, Japan) according to the manufacturer’s instructions. Briefly, 106 cells were incubated with annexin V-fluorescein isothiocyanate and PI for 15 min and then analyzed by dual-color flow cytometry.

Nude mouse xenograft model

All animal experiments described in this study were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals of the Nagasaki University School of Medicine. ARO or mutARO cells (5 x 106) suspended in RPMI 1640 were injected sc into both flanks of 8-wk-old female BALB/c nu/nu mice (Charles River, Tokyo, Japan), five animals per group. Tumor sizes were measured each alternate day with calipers, and tumor volumes were calculated according to the formula: a2 x b x 0.4, where a is the smallest tumor diameter and b is the diameter perpendicular to a. Two weeks after injection, the left leg of each animal was exposed to 5 Gy x-ray. Tumor size was monitored for three more weeks. The body weight, feeding behavior, and motor activity of each animal were monitored as indicators of general health.

Histological estimation of apoptosis in the tumors

Tumors were dissected, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Apoptotic cells were detected in 5-µm sections with ApopTag peroxidase kit (Intergen Co., Burlington, MA). Positively stained cells were counted in four fields (x100) for each specimen. The apoptotic index was determined as the ratio of apoptotic cell number to total cell number.

Statistical analysis

All data were expressed as mean ± SD. Differences between groups were examined for statistical significance using ANOVA and/or Student’s t test where appropriate. P < 0.05 denoted the presence of a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Elevated basal level and enhanced induction of NF-{kappa}B DNA-binding activity after exposure to IR in human thyroid cancer cell lines

The DNA-binding activity of nuclear NF-{kappa}B was investigated in untreated primary thyroid cells and various human thyroid cancer cell lines. The basal activity level was markedly elevated in all cancer cell lines tested. The highest activity was observed in an anaplastic thyroid cancer cell line, FRO, and a papillary cancer cell line, TPC-1, which harbors the ret/PTC1 rearrangement. In a follicular carcinoma cell line, WRO, and papillary carcinoma cell line, KTC-1, only a 1.5-fold increase of basal activity was observed (Fig. 1Go).



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  		FIG. 1. Basal DNA-binding activity of NF-{kappa}B in thyroid cancer cell lines, compared with primary thyrocytes. Nuclear extracts were isolated from unstimulated cells cultured under conditions described in Materials and Methods. The binding assay was performed with 5 µg of nuclear protein per well. Subunits of NF-{kappa}B were detected with anti-p65 and anti-p50 antibodies. Data are representative of at least two separate experiments. Each bar represents mean ± SD value (n = 6).

 
IR induced an increase of DNA-binding activity of the p65 subunit, which has a transactivation domain, in all thyroid cell lines tested (Fig. 2AGo). The highest relative values of induced activity were found in the ATC cell line, ARO.



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  		FIG. 2. Induction of the NF-{kappa}B binding activity in thyroid cells by IR. A, Fold increase of p65 DNA-binding activity over basal level in normal thyrocytes and cancer cells after irradiation. Cell extracts were isolated from cultured cells 2 h after exposure to 10 Gy of x-rays. The binding assay was performed as described in the legend to Fig. 1Go. B and C, Dose effects of ionizing radiation on NF-{kappa}B DNA-binding activity in primary thyrocytes (B) and ATC cells ARO (C). Cell extracts were collected 2 h after cell exposure to varying doses of ionizing radiation. Extracts from cells treated with 5 ng/ml TNF{alpha} for 1 h were used as positive controls. In A, B, and C, data are mean ± SD. D, Degradation of cytoplasmic I{kappa}B{alpha} and translocation of NF-{kappa}B subunits into the nucleus 2 h after exposure of ARO cells to IR.

 
Because our specific aim was to elucidate the effects of modulation of NF-{kappa}B activity in ATC, we concentrated further on the ATC cell line, ARO. Activity of NF-{kappa}B after exposure increased in a dose-dependent manner in both normal thyroid cells and the anaplastic ARO cancer cell line (Fig. 2Go, B and C, respectively). There was no significant change in the p65:p50 subunit ratio after the IR-induced activation, which can suggest that the active DNA-binding complex was represented predominantly by the p65/p50 heterodimer.

To confirm the mechanism of NF-{kappa}B activation in thyroid cells after IR exposure, we examined changes in the I{kappa}B{alpha} level in cytoplasmic protein extracts and translocation of active NF-{kappa}B to the nucleus using Western blotting of nuclear protein extracts with subunit-specific antibodies. IR induced a decrease of I{kappa}B{alpha} protein levels in a dose-dependent manner in ARO cell line, suggestive of proteasomal degradation of the cytoplasmic pool of I{kappa}B{alpha} (Fig. 2DGo). In additional experiments we observed that a proteasome inhibitor, ALLN, abrogated postradiational I{kappa}B{alpha} depletion (data not shown). Levels of nuclear p65 and p50 increased concordantly with radiation dose within short time after exposure. These findings indicate that the classical pathway of NF-{kappa}B activation is functional and occurs in thyroid cells after exposure to IR.

We next studied the kinetics of NF-{kappa}B induction by IR in ARO cultures. The cells were exposed to 5 Gy of IR, and cell extracts were collected at different time points between 0 and 8 h after IR exposure. The results showed a rise from the basal level to maximum activity between 1.5 and 2 h after irradiation and a rapid decline to the basal level 8 h later (Fig. 3Go).



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  		FIG. 3. Kinetics of the NF-{kappa}B response to IR. ARO cells were exposed to 5 Gy of x-rays. At the indicated time, cell extracts were collected and DNA binding activity was determined by multiwell assay. Data are mean ± SD.

 
Enhanced cell-killing effect of the NF-{kappa}B inhibitor, SN50, by IR

We examined the effect of SN50, a synthetic peptide designed to inhibit nuclear translocation of the p50 subunit of NF-{kappa}B (15), in thyroid cancer cells at the time of exposure to IR. Treatment of ARO cells with SN50 resulted in the inhibition of basal and induced p65/p50 DNA-binding (Fig. 4AGo). Incubation with SN50 alone for 48 h led to a concentration-dependent inhibition of ARO cell growth (Fig. 4BGo). The mutated control peptide SN50M that lost NF-{kappa}B inhibitory activity produced no effect on ARO cell proliferation. We next evaluated the effect of SN50 on ATC cell survival after exposure to IR. Cells pretreated with a SN50 showed significantly lower survival after exposure to IR than untreated control cells or cells treated with inactive SN50M (Fig. 4CGo).



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  		FIG. 4. Effect of SN50 and IR on the NF-{kappa}B DNA binding and cell proliferation. A, Inhibition of basal and induced NF-{kappa}B activity by SN50 in ARO cells. Cells were pretreated with varying doses of SN50 during 1 h before exposure to IR or 5 ng/ml TNF{alpha}. After 2 h incubation subcellular extracts were collected. DNA binding assay was performed with 5 µg of nuclear protein. B, Growth inhibition of ARO cells by SN50. C, Effect of combined treatment with SN50 and IR. Cells in a 96-well plate (2000 cells/well) were treated with SN50 or with the inactive control SN50M and then exposed to 10 Gy of x-rays. Cell number was estimated by water-soluble tetrazolium salt assay after 48 h incubation. Data are mean ± SD.

 
Influence of mutant I{kappa}B{alpha} on cell survival after exposure to IR

To assess the role of stress-inducible NF-{kappa}B activation in cell survival after exposure to IR, we established a cell line stably transfected with a mutant form of an I{kappa}B{alpha}-expressing plasmid (mutARO). The mutant I{kappa}B{alpha}, in which Ser32 and 36 were substituted to Ala, cannot be phosphorylated by I{kappa}B kinases, resulting in resistance to ubiquitination and degradation by the proteasome, thus inhibiting signal-dependent NF-{kappa}B activation (20). Mutant cells expressed both normal and mutant I{kappa}B{alpha}. The expression level of the NF-{kappa}B subunits, p50 and p65, did not change (Fig. 5AGo). We observed no change in basal NF-{kappa}B DNA binding in the mutARO cells (data not shown). However, the IR-induced activation of DNA-binding was abrogated in this cell line (Fig. 5BGo). The growth rate of the mutI{kappa}B{alpha} transfectants in culture did not differ significantly from that of control ARO cells (Fig. 5CGo). We next assessed the radioresistance of the parental ARO cells and mutI{kappa}B{alpha} transfectants. In a clonogenic assay, mutARO cells exhibited decreased radioresistance, compared with control cells (Fig. 5DGo).



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  		FIG. 5. A, Expression of I{kappa}B{alpha}, p65, and p50 in the clone of ARO cells stably transfected with control vector (cARO) or mutI{kappa}B{alpha} (mutARO). FLAG-tagged mutI{kappa}B{alpha} appears as a band above the endogenous I{kappa}B{alpha}. B, Inhibition of IR-induced NF-{kappa}B binding in mutARO cells. Cell extracts were collected 2 h after exposure. DNA binding assay was performed with 5 µg of nuclear protein. C, Growth rate of control ARO and mutARO cell lines in culture by water-soluble tetrazolium salt assay. D, Repression of radioresistance in mutI{kappa}B{alpha} transfectants. Two hundred ARO cells stably transfected with control vector or mutI{kappa}B{alpha} plasmids were plated in 60-mm dishes and irradiated with a range of doses. Cells were cultured for 14 d, and colonies with more than 50 cells were counted to determine clonogenic survival. Three dishes were used for each dose. Data are mean ± SD. *, P < 0.001.

 
Inhibition of NF-{kappa}B enhances radiation-induced apoptosis

We analyzed whether the modulation of NF-{kappa}B signaling could induce an apoptotic response in thyroid cancer cells. The annexin V/PI double-labeling revealed that ARO cells were resistant to apoptosis induced by IR or SN50 alone (Fig. 6AGo). In contrast, irradiation of ARO cells in the presence of SN50 did evoke the apoptotic response 24 h after exposure. After the combined treatment, we detected a simultaneous increase of the annexin V+/PI- fraction (early apoptotic) and annexin V+/PI+ fraction that usually is referred to as necrosis. To confirm the programed cell death in treated cells, we analyzed the cleavage of poly-(ADP-ribose) polymerase (PARP), a major substrate of the effector caspase-3 during apoptosis (21). As demonstrated in Fig. 6BGo, only the combined treatment of control ARO cells with SN50 and IR or irradiation of mutARO cells yielded the products of PARP cleavage.



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  		FIG. 6. Effect of the NF-{kappa}B inhibition on induction of apoptosis in ARO cells. A, AnnexinV/PI staining was performed to quantify phosphatidylserine externalization and advanced stage of cell death in ARO cells treated with SN50 (25 µg/ml) and exposed to 5 Gy of x-rays. B, Immunoblotting analysis of PARP cleavage was performed in lysates of ARO and mutARO cells treated with SN50 (25 µg/ml) and/or exposed to IR. C, Levels of Bcl-2 family proteins in the mutARO clone at different time points after exposure to 5 Gy. D, Levels of IAP family proteins in control ARO and mutARO cells at different time points after exposure to 5 Gy.

 
The ratio between the amounts of proapoptotic and antiapoptotic proteins of the Bcl-2 family is an important determinant of cell viability. Proapoptotic Bax promotes the release of cytochrome c from mitochondria, and antiapoptotic Bcl-2 and Bcl-XL prevent this process (22). IR can change the Bax/Bcl-2 ratio, inducing apoptosis in radiosensitive cells (23). Previously, we have shown that exposure to IR did not change the levels of the apoptosis inhibitors Bcl-2, Bcl-xL, and proapoptotic Bax protein in thyroid carcinoma cells, including the ARO cell line (4). Here we evaluated whether cells with impaired NF-{kappa}B signaling had altered expression of these proteins. Similar to our earlier findings, no change in Bcl-2, Bcl-xL, and Bax levels was observed in the mutI{kappa}B{alpha} transfectants at different time points after 5Gy exposure (Fig. 6CGo).

Other mechanisms of preventing apoptosis in cells include inhibition of the caspase cascade by proteins of the inhibitor of apoptosis (IAP) family (24). cIAP-1 and -2 were identified as NF-{kappa}B-inducible gene products (7) and can be regulated by different activators and inhibitors of the NF-{kappa}B pathway (25). These members of the IAP family can prevent apoptosis by inhibiting the downstream caspases-3 and -7 (26). We therefore examined the levels of proteins of the IAP family, cIAP-1 and c-IAP-2, after exposure to IR. In the control ARO cells, we observed that exposure to 5Gy induced a sustained increase of expression level of IAPs. Deregulation of NF-{kappa}B signaling by mutant I{kappa}B{alpha} abolished the induction of these apoptotic inhibitors (Fig. 6DGo).

These data suggest that in thyroid cancer cells, exposure to IR activates the antiapoptotic pathway via induction of NF-{kappa}B and up-regulation of IAPs. Inhibition of NF-{kappa}B at the time of exposure can change the balance toward the proapoptotic effects of radiation.

In vivo effect of IR on mutARO tumors

To examine the effects of IR in model thyroid anaplastic tumors with an impaired NF-{kappa}B cascade in vivo, we implanted ARO and mutARO cells into athymic mice and exposed the growing tumors to x-rays. Because the tumor volume is critical for the formation of radiation response, exposure to IR was performed when tumors reached nearly same size. As shown in Fig. 7Go, in vivo growth of mutARO tumors was significantly slower than that of control ARO tumors (P = 0.038; ANOVA test). After exposure to IR, we observed a profound delay of tumor progression in mutARO neoplasms (Fig. 7BGo). In contrast, radiation exposure did not produce observable effects in ARO tumors (Fig. 7AGo).



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  		FIG. 7. Effect of IR in control ARO and mutARO cells implanted into athymic mice. Animals from each group (n = 5) were exposed to 5 Gy of x-rays after developing tumors from injected cell suspension. The graphs show the dynamics of tumor growth of cARO (A) and mutARO cells (B) and response to irradiation. Data are mean ± SEM.

 
In separate experiments, we confirmed the expression of the mutant form of I{kappa}B{alpha} in mutARO tumors (Fig. 8AGo) and estimated the level of apoptosis in control and mutant tumors 48 h after exposure to 5 Gy (Fig. 8BGo). In the ARO tumors, the basal level of apoptotic cells was less than 1%. After exposure to IR, we observed an increase of apoptosis to 2.9%. The basal level of apoptotic cells was higher in the tumors of mutARO cells (7.5%) and increased 48 h after irradiation to 22% (Fig. 8CGo). This difference was significant.



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  		FIG. 8. Characteristics of implanted tumors. A, Expression of I{kappa}B{alpha} and mutI{kappa}B{alpha} in tumors grown in athymic mice. Protein extracts from tumor homogenates were separated by SDS-PAGE and immunoblotted with antibody to I{kappa}B{alpha}. Each lane in the control and mutARO groups correspond to different animals. B, Detection of apoptotic cells in the tumors. Mice with control and mutARO tumors were exposed to 5 Gy IR. Tissues were dissected 48 h after irradiation. Magnification, x100. C, Apoptotic index in the tumors. Data are mean ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NF-{kappa}B is one of the most critical transcription factors to regulate gene expression of antiapoptotic proteins and cytokines such as IL-1, IL-2, IL-6, IL-8, TNF-{alpha}, and interferon-{gamma} (27, 28). It is generally assumed that the NF-{kappa}B-induced factors promote apoptotic resistance, transformation, cell growth, metastasis, and angiogenesis in neoplastic tumors (29). Basal NF-{kappa}B activity is often increased in various types of cancer by various activated oncogenes such as ras and raf (30). Therefore, the inhibition of NF-{kappa}B activity appeals as an approach to cancer therapy, and indeed it has been attempted in many types of cancer (31). The results, unfortunately, have not always been promising. Inhibition of NF-{kappa}B alone does not dramatically induce cell death in most solid cancers, perhaps because many types of tumors can up-regulate NF-{kappa}B-independent antiapoptotic pathways. However, survival of some cancer cells, such as several hematological malignancies and prostate cancer, clearly depend on NF-{kappa}B (32, 33, 34).

The reason for the high NF-{kappa}B basal activity in thyroid cancer cells may be in part due to mutations characteristic in this type of malignancy. Our earlier study has shown that activated mutations of the ras oncogene were found in about 30% of thyroid tumors (35). Oncogenic Ras signaling has been shown to up-regulate NF-{kappa}B RelA/p65 subunit transcriptional activity in transformed NIH3T3 cells (36). More recently we and others (37, 38) also found activated b-raf gene mutations in about 30% of advanced human thyroid cancers. Induction of NF-{kappa}B by activated RAF is relayed by MEKK1, which phosphorylates I{kappa}B kinase ß protein, finally resulting in NF-{kappa}B activation (39). Also, in papillary thyroid cancer, RET activation by gene rearrangement is found in nearly 30–40% of tumors (40). Although, to our knowledge, direct NF-{kappa}B activation by rearranged RET has not been demonstrated so far, one can draw some parallels with point-mutated RET characteristic for medullary thyroid cancer. Such oncogenes have been shown to stimulate NF-{kappa}B activity in a manner similar to activated RAF (41).

One more possible explanation of higher NF-{kappa}B DNA-binding activity in anaplastic thyroid cancer cells can be an alteration of the PTEN gene. The tumor suppressor PTEN has been shown to play an important role in the regulation of NF-{kappa}B activity. For instance, it has been demonstrated that PTEN can down-regulate the phosphatidylinositol 3-kinase/Akt signaling, which prosurvival effect, at least in part, is associated with the activation of NF-{kappa}B transcription factor (42, 43). PTEN has been found to be deleted and/or mutated in several tumor types, including thyroid cancers (44, 45). Recently it has been shown that the PTEN gene is often silenced to a greater or lesser extent in ATCs due to complex epigenetic and/or structural mechanisms (46). Thus, PTEN deficiency may contribute to the constitutive activation of phosphatidylinositol 3-kinase/Akt pathway (47) resulting in higher basal NF-{kappa}B activity in ATC cells. Perhaps usage of additional cell lines lacking PTEN, such as FTC-133, would allow to address this question in larger details.

NF-{kappa}B has been shown to play an important role in maintenance of malignant phenotype of ATC. Indeed, blockade of p65 NF-{kappa}B protein synthesis with specific antisense oligonucleotides has been demonstrated to greatly reduce the ability of two ATC cell lines to form colonies in agar and inhibited their growth rate in vitro (14). Because the most common therapeutic approach to this type of cancer is radical surgery followed by combination chemotherapy and radiation therapy, it is important to take into account that NF-{kappa}B is activated in cancer cells by the latter. In our experiments we observed that IR exposure induced stronger enhancement of NF-{kappa}B activity in thyroid cancer cells than in normal thyroid cells. It was shown previously that transformation or carcinogenetic events may sensitize cells to induction of NF-{kappa}B by IR (48). Our results also suggest that thyroid cancer cells are more sensitive to IR for activation of NF-{kappa}B, compared with normal thyrocytes. Our previous study demonstrated the resistance to IR of cell lines derived from different types of thyroid cancer (49). Considered together, these data suggest that NF-{kappa}B may play an important role in determining the intrinsic radiosensitivity of thyroid cells. Thus, for instance, the enhanced response of NF-{kappa}B cascade may block the ability of radiation therapy to induce cell death. Consistently, inhibition of NF-{kappa}B during the course of chemotherapy or radiation therapy may strongly potentiate the cell-killing effect of the treatment. For example, HT1080 fibrosarcoma cells exposed to IR or to daunorubicin exhibited enhanced activation of NF-{kappa}B. Inhibition of NF-{kappa}B under these experimental conditions led to a dramatically increased apoptotic response to IR or to the drug as compared with the control cells (50, 51). In our experiments, we used the cell-permeable peptide SN50, which acts as an inhibitor of NF-{kappa}B nuclear translocation to suppress NF-{kappa}B activity in a thyroid cancer cell line. The treatment markedly inhibited both basal and IR-induced NF-{kappa}B activity and promoted cell death in vitro in combination with IR exposure. Interestingly, no increase of cell killing but only inhibition of cell growth was observed in the cells treated with NF-{kappa}B inhibitor alone. This finding suggests that additional proapoptotic signal is required to initiate the process. As we have shown, in the case of ATC, the exposure to IR can play a proapoptotic role in the cells with inhibited NF-{kappa}B.

Cell death in solid tumors after treatment with IR or chemotherapeutic drug can proceed through different processes, including apoptosis, mitotic catastrophe, and necrosis. Our results suggest that at least a portion of ATC cells in our model undergo apoptosis after irradiation provided NF-{kappa}B activation is suppressed. However, the detected increase of necrosis can also be a consequence of the treatment. Most likely, the overall cell killing effect in vitro is due to a combination of all three processes. Additional study is necessary to clarify the significance of these different components of radiosensitization by NF-{kappa}B inhibition. In the in vivo model, however, our results suggest that namely apoptosis may be the major point of cell death once again attesting for the difference of biological effects between in vitro and in vivo systems.

SN50 is a synthetic peptide consisting of 26 amino acids. Its associated immune response and high cost render it impractical for an in vivo study. To overcome these obstacles, in the in vivo models, we used an ATC cell line stably transfected with the Ser32/36 mutant I{kappa}B{alpha} (mutI{kappa}B{alpha}), which has been reported as one of the methods of selective inhibition the NF-{kappa}B cascade (52). The responses to IR were then compared between the tumors derived from the transfectants and control cells and were found to be significantly different between two groups. To mention, in contrast to the in vitro characteristics, mutI{kappa}B{alpha} tumors implanted into nude mice exhibited a slower growth rate and higher basal apoptosis index than tumors originating from control cells. This effect, perhaps, could partially be attributed to the less favorable conditions for cell growth in vivo, compared with in vitro culture. Similarly the in vitro results of clonogenic assay, tumors with mutI{kappa}B{alpha} responded to radiation therapy better then controls.

Recently, several low-molecular-weight inhibitors of NF-{kappa}B have been designed as anticancer, immunosuppressant, and antiinflammatory agents. Most NF-{kappa}B inhibitors, such as panepoxydone, are designed to target factors upstream of I{kappa}B (53). One such compound, dehydroxymethylepoxyquinomicin (DHMEQ), has been derived from the structure of the antibiotic epoxyquinomicin C and tested in vivo (54). DHMEQ was found to inhibit TNF{alpha}-induced activation of NF-{kappa}B and was effective in suppressing rheumatoid arthritis and hormone-refractory prostate cancer in an in vivo model without any toxicity (34). Application of this kind of low-molecular-weight NF-{kappa}B inhibitor may be a promising molecular target therapy for advanced thyroid cancers.

In conclusion, our results demonstrate the critical role of NF-{kappa}B signaling in human thyroid cancer cells as antiapoptotic intrinsic factor. The inhibition of stress-induced signaling via the NF-{kappa}B pathway in an ATC cell line can potentiate the therapeutic effect of ionizing radiation. Our study, therefore, implicates the usefulness of targeted NF-{kappa}B inhibition as an adjuvant approach in combination with radiation therapy for treatment of human advanced thyroid cancers.


   Acknowledgments
 
We thank Dr. Jun-ichiro Inoue (Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan) for the generous gift of the mutant I{kappa}B{alpha} (32/36 AA) plasmid.


   Footnotes
 
This work was supported by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to H.N., Grant 13671158; to V.S., Grant 14380256; to S.Y., Grant 12576020).

Abbreviations: ATC, Anaplastic thyroid cancer; DTT, dithiothreitol; FBS, fetal bovine serum; IAP, inhibitor of apoptosis; I{kappa}B, inhibitory protein {kappa}B; IR, ionizing radiation; mutI{kappa}B{alpha}, mutant I{kappa}B{alpha}; NF-{kappa}B, nuclear transcription factor-{kappa}B; PARP, poly-(ADP-ribose) polymerase; PI, propidium iodide.

Received July 15, 2003.

Accepted October 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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