NF-{kappa}B inhibition radiosensitizes Ki-Ras-transformed cells to ionizing radiation

Bo Yeon Kim 1, {dagger}, Kyung A. Kim 1, {dagger}, Osong Kwon 1, Sun Ok Kim 1, Min Soo Kim 1, Beom Seok Kim 1, Won Keun Oh 1, Gun Do Kim 2, Mira Jung 3 and Jong Seog Ahn 1, *

1 Laboratory of Cellular Signaling Modulators, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, 305-333, Korea, 2 Department of Microbiology, College of Natural Sciences, 599-1, Pukyong National University, Daeyeon3-Dong, Nam-Gu, Pusan 608-737, Korea and 3 Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC 20057-1482, USA

* To whom correspondence should be addressed. Tel: +82 42 860 4312; Fax: +82 42 860 4595; Email: jsahn{at}kribb.re.kr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most cancer cells show resistance to ionizing radiation (IR)-induced cell death. Recently, Ki-Ras was reported to be responsible for the increased radioresistance. We report here that inhibition of IR-induced activaton of nuclear transcription factor kappa B (NF-{kappa}B) but not of either Akt or MAPK kinase (MEK), increased the radiosensitization of Ki-Ras transformed human prostate epithelial 267B1/K-ras cells. Proteosome inhibitor-1 (Pro1) reduced NF-{kappa}B activation, and this inhibition was accompanied by increased levels of cytoplasmic I{kappa}B{alpha} and p65/RelA. However, translocation of p50/NF-{kappa}B1 did not occur on exposure to IR, suggesting the cell-specific involvement of p50 in radiation signaling. Clonogenic cell survival and soft agar assays further confirmed the increased radiosensitivity of 267B1/K-ras cells by proteosome inhibition. In addition, proteosome inhibition enhanced the IR-induced degradation of apoptotic protein caspases 8 and 3, with the level of antiapoptotic protein Bcl-2 being unaffected, suggesting the involvement of an apoptotic process in IR-induced cell death of 267B1/K-ras cells. LY294002 and PD98059, specific inhibitors of phosphatidylinositol-3-kinase (PI3K) and MEK, respectively however, did not affect the radiosensitization. All these results suggest an application of blocking NF-{kappa}B activation pathway to the development of anticancer therapeutics in IR-induced radiotherapy of Ki-Ras-transformed cancer cells.

Abbreviations: DTT, dithiothreitol; EMSA, electromobility shift assay; FT1, farnesyltransferase inhibitor-I; IR, ionizing radiation; MAPK, mitogen-activated protein kinase; NK-{kappa}B, nuclear transcription factor kappa B; PI3K, phosphatidylinositol-3-kinase; PMSF, phenylmethylsulfonyl fluoride; Pro1, proteosome inhibitor-1; SS, sulindac sulfide; TNF{alpha}, tumor necrosis factor {alpha}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exposure of eukaryotic cells to multiple forms of environmental stress, including ionizing radiation (IR), gives rise to a variety of cellular responses, such as DNA repair, gene induction, cell cycle arrest apoptosis and lethality (1,2). These responses are involved in both resistance to or delay of apoptosis depending on the cell type and on the stresses administered (3). Although IR is one of the most commonly used cancer treatments, its therapeutic efficacy decreases when cancer cells develop resistance to radiation. A partial explanation for this resistance comes from the observation that radiotherapy promoting cell death also activates the nuclear transcription factor kappa B (NF-{kappa}B), enhancing the expression of anti-apoptotic proteins (4).

NF-{kappa}B has been shown to play a critical role in blocking apoptosis induced by a variety of stimuli, including tumor necrosis factor {alpha} (TNF{alpha}), chemotherapeutic compounds and {gamma} radiation (5). Tumor cells usually express high levels of constitutive NF-{kappa}B activity (6). In addition, exposure of these cells to various cytotoxic agents including IR increases NF-{kappa}B activity (6,7), resulting in cell growth and survival advantage and resistance to IR and a variety of cytotoxic agents (8,9). NF-{kappa}B activation was also revealed to play an antiapoptotic role in human leukemic K562 cells exposed to IR (10). In addition, the relationship of constitutive NF-{kappa}B activity, basal apoptosis and radiosensitivity was reported in head and neck carcinoma cell lines (11). Similar findings were also observed in several other types of human cancer cells, including human breast and colon cancer cells, providing evidence for the role of NF-{kappa}B in oncogenesis and resistance to cell death.

Since the majority of reported studies have demonstrated that NF-{kappa}B plays an important role in the development of tumor resistance to radiotherapy and chemotherapy, inhibition of NF-{kappa}B targeted at the molecular level has been actively pursued as a potential and novel adjuvant treatment for cancer in conjunction with radiotherapy and chemotherapy. IR-induced NF-{kappa}B activation was repressed by the expression of an undegradable form of I{kappa}B{alpha}, consequently leading to the killing of HT1080 fibrosarcoma cells (12). Recently, indomethacin was shown to lower the temperature and heating times required to inhibit the activation of NF-{kappa}B and induce significant hyperthermic radiosensitization (13). Enhanced radiosensitization was also achieved in NF-{kappa}B-suppressed human oral cancer cells (14).

It was reported that ras oncogene enhanced the resistance of breast cancer cells to radiotherapy (15). Of the three genes in the ras family (K-ras, N-ras and Ha-ras), K-ras appears to be mutated most frequently in human tumors, including pancreatic, colon and lung adenocarcinomas (16,17). RelA (p65/NF-{kappa}B) is constitutively activated in pancreatic adenocarcinoma and pancreatic tumor cells expressing a mutant Ki-Ras (18). Disruption of K-ras, on the other hand, was shown to result in downregulation of cancer-prone activities in the invasive colon cancer cell line HCT116 (19). Recently, we reported that IKKß and I{kappa}B{alpha} are responsible for Ki-Ras-induced NF-{kappa}B activation in 267B1/K-ras human prostate epithelial cells (20). Our further study showed that tumorigenic growth of Ki-Ras-transformed cells was also pronounced when compared with normal cells. These results suggested that NF-{kappa}B activation by Ki-Ras might be responsible for the enhanced growth rate, tumorigenesis and radioresistance of Ki-Ras-transformed 267B1/K-ras cells.

In this study, we have explored the possibility that inhibition of NF-{kappa}B activation could increase the sensitivity of Ki-Ras-transformed cells to IR. Our data showed that NF-{kappa}B inhibition by a proteosome inhibitor enhanced the radiosensitivity of 267B1/K-ras cells. This radiation-induced cell death is dependent on I{kappa}B{alpha} degradation and p65/RelA translocation but not on p50/NF-{kappa}B1, Akt- or mitogen activated protein kinase (MAPK)-signaling pathways.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Lipofectamine Plus, RPMI1640 and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Proteosome inhibitor-1 (Pro1), farnesyltransferase inhibitor-I (FTI), sulindac sulfide (SS) and antibodies to caspases 8 and 3 were obtained from Calbiochem (San Diego, CA). Antibodies to I{kappa}B{alpha}, Akt1, actin, p50, p52, p65, Rel-B and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p53 and Bcl-2 were purchased from Oncogene (Darmstadt, Germany). A specific antibody to PARP was obtained from Pharmingen (San Jose, CA). Antibody to phosphor-I{kappa}B{alpha} and phospho-Akt1 were from Cell Signaling (Beverley, MA). The luciferase assay kit was purchased from Promega (Madison, WI), Sephadex G-25 columns and enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Amersham, NJ). The pNF-{kappa}B-Luc plasmid was from Stratagene (La Jolla, CA) and [{gamma}-32P]ATP was purchased from NEN, Dupont (Boston, MA). Wild-type p65 cDNA was cloned and sequenced as described elsewhere (21). All the other reagents were obtained from Sigma (St Louis, MO).

Cell culture, transfection and luciferase reporter gene assay
267B1 and 267B1/K-ras cells were maintained in RPMI1640 medium supplemented with 2 mM L-glutamine, hydrocortisone (0.5 µg/ml) and 10% heat-inactivated fetal bovine serum and were cultured in a humidified CO2 incubator at 37°C. For luciferase reporter assays, the cells (2 x 105/ml) were incubated in 6-well plates for 24 h, with the use of Lipofectamine Plus, followed by cotransfection with 0.5 µg of pNF-{kappa}B-Luc, 0.5 µg of pCMV/ß-galactosidase plasmid and 10 ng of a plasmid encoding the wild type NIK, IKK{alpha} or IKKß. In some cases, the cells were treated with Pro1 at varying concentrations 3 h post-transfection with pNF-{kappa}B-Luc. After 24 h, luciferase activity was measured with a detection kit.

MTT assay
Working solution of MTT was prepared by 1:5 dilution from a stock solution [5 mg/ml, in phosphate buffered saline (PBS)] with prewarmed medium just prior to assay. An aliquot of 50 µl of this MTT working solution was added to each well of microculture plate. After 4 h incubation, the cells were centrifuged at 1200 r.p.m. for 5 min, and the plate was reversed on paper towels to remove the medium. Following thorough formazan solubilization by adding 150 µl of dimethyl sulfoxide to each well, the absorbance of each well was measured using a microplate reader (Dynatech MR700) at 540 nm (single wavelength, calibration factor = 1.00). For larger size of culture dishes, the amount of MTT solution was increased. The cells were collected into microculture plate and proceeded for measurement of absorbance.

Electromobility shift assay (EMSA) analysis
EMSA analysis was performed as described by Janssen and Sen (22). In brief, cells (5 x 106 in 10 ml) grown in 100 mm dishes were lysed on ice for 15 min in a hypotonic solution containing 10 mM HEPES–KOH (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA (sodium salt), 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (10 µg/ml), 1 mM dithiothreitol (DTT) and 0.15% NP-40. The lysate was centrifuged at 16 000 r.p.m. for 1 min at 4°C and the resulting nuclear pellet was resuspended in ice-cold extraction buffer [50 mM HEPES–KOH (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM PMSF, 1 mM DTT and 10% glycerol] and incubated for 30 min at 4°C with occasional vortex. The nuclear lysate was then centrifuged at 16 000 r.p.m. for 30 min at 4°C and the resulting supernatant was stored at –80°C or immediately subjected to EMSA analysis. An oligonucleotide containing NF-{kappa}B binding site (3.5 pmol) (Santa Cruz) was incubated for 10 min at 37°C in 10 µl containing 10 µCi of [{gamma}-32P]ATP, 5 U of T4 polynucleotide kinase and 1x kinase buffer (supplied with the kinase). The labelling reaction was terminated by the addition of 100 mM EDTA, after which the reaction mixture was centrifuged through a Sephadex G-25 column to remove unincorporated 32P. The 32P-labeled oligonucleotide was then stored at –80°C until use. For EMSA assay, nuclear protein extract (10 µg) was incubated for 30 min at room temperature in a final volume of 10 µl containing 0.03 pmol of 32P-end-labeled oligonucleotide, 40 mM HEPES–KOH (pH 7.8), 10% glycerol, 1 mM MgCl2, 0.1 mM DTT and 1 µg of poly(dI-dC). For supershift analysis, the nuclear extract was incubated with specific antibodies (2 µg) for 30 min at room temperature prior to the addition of the labeled oligonucleotide. The binding reaction was terminated by the addition of electrophoresis sample buffer, and the samples were fractionated on 5% non-denaturing polyacrylamide gels in 0.5x Tris–boric acid–EDTA (TBE) buffer. The gels were then subjected to autoradiography.

Immunoblot and image analysis
Cells were grown to subconfluency in 100 or 150 mm dishes. They were washed four times with ice-cold PBS and then scraped on ice into a solution containing 10 mM HEPES–KOH (pH 7.9), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.2 mM NaF, 0.4 mM PMSF, leupeptin (10 µg/ml), aprotinin (10 µg/ml), 0.1 mM Na3VO4 and 1 mM DTT, and maintained for 15 min on ice. After the addition of NP-40 to a final concentration of 0.15%, the lysate was vigorously mixed for 15 s and then centrifuged at 16 000 r.p.m. for 1 min at 4°C. The resulting supernatant was stored at –80°C as the cytoplasmic extract, and the nuclear pellet was resuspended in a solution containing 50 mM HEPES–KOH (pH 7.9), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.2 mM NaF, leupeptin (10 µg/ml), aprotinin (10 µg/ml), 0.4 mM PMSF, 0.1 mM Na3VO4, 1 mM DTT and 10% glycerol. The resulting suspension was incubated for 30 min on ice with occasional vortex and then centrifuged at 16 000 r.p.m. for 30 min at 4°C for withdrawal of the supernatant as a nuclear fraction. For preparation of total cell lysate, cells washed with PBS buffer were scraped and collected as described above. The cells were gently resuspended with a pipette in a lysis buffer containing 10 mM HEPES–KOH (pH 7.9), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.2 mM NaF, 0.4 mM PMSF, leupeptin (10 µg/ml), aprotinin (10 µg/ml), 0.1 mM Na3VO4, 1 mM DTT and 1% CHAPS. The mixture was placed on ice for 30 min and centrifuged at 19 000 r.p.m. at 4°C for 30 min. The supernatant was collected and preserved at –80°C until use. Equal amounts (50 µg) of cytoplasmic or nuclear extract after determination of protein concentration by Bradford method were subsequently applied to SDS–PAGE and subjected to immunoblot analysis with specific antibodies (1:1000 diluted). Immune complexes were detected with enhanced chemiluminescence reagents. Images of the bands were scanned and quantified using DNR Bio-Imaging System.

Staining for determination of cell proliferation
Cells treated with IR and chemicals were washed twice with PBS buffer to remove the dead cells detached from the well and were fixed in methanol for 1 min followed by dipping into 0.5% Eosin Y solution in 90% ethanol containing several drops of glacial acetic acid for another 1 min and photographed under inverted microscope.

Soft agar assay
A 0.3% soft agar medium was prepared by adding equal volume of autoclaved low melting temperature (LMT) agarose to RPMI1640 medium to make the temperature around 40°C. Cells pre-exposed to IR or chemicals in 60 mm dishes were trypsinized, counted and poured into the premade soft agarose medium (200 cells/well) in 6-well plates. After 5 days, the cells were photographed under inverted microscope and the number of colonies counted. Three independent experiments were performed in triplicates.

Clonogenic assay
Semiconfluent growth of cells in 60 mm dishes were exposed to IR or ProI at appropriate doses and immediately trypsinized to be transferred to 6-well plates at 1 x 103 cells/well in complete RPMI1640 medium. After 7–10 days of incubation, the medium was removed and 200 µl of 0.3% crystal violet solution (dissolved in 1:1 mixture of methanol and H2O) was added into each of the wells for 2 min. The cells were then washed once with PBS buffer, air dried, counted and photographed. Two independent experiments were conducted in triplicates.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Radioresistance of K-Ras-transformed cells
In order to compare the radiosensitivity of the 267B1 and 267B1/K-ras cells (normal and Ki-Ras transformed, respectively), cells were exposed to IR at various doses. After 3 days of incubation, both of the cell types not exposed to IR were almost intact and alive when measured by MTT assay. Upon exposure to IR, however, most of the normal cells were dead while the transformed cells showed only a slight decrease in cell number (Figure 1A). Time-dependent pattern of cell death also showed that 267B1/K-ras cells are more resistant to IR-induced cell death than the normal 267B1 cells, with the latter cells being almost dead by the third day post-irradiation (Figure 1B). To further confirm the increased radioresistance of Ki-Ras transformed cells, a clonogenic survival assay was performed. Cells were exposed to IR at 10 Gy, trypsinized immediately, seeded into 6-well plates, incubated for 7–10 days and the colonies having >50 cells were counted. As shown in Figure 1C, there was only a slight decrease in 267B1/K-ras cells survival while most of the normal cells were dead by IR exposure, supporting the above observations. Since Ki-Ras was reported to induce NF-{kappa}B activation in these cells (20), it could be suggested that the radioresistance displayed by 267B1/K-ras cells might be ascribed to the enhanced NF-{kappa}B activity in the transformed cells.



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Fig. 1. Ki-Ras overexpression confers resistance to IR-induced cell death. (A) Cells grown in complete medium in 12-well plate were exposed to varying doses of IR, incubated for 3 days and the cell number measured by MTT assay. (B) Cells grown as in (A) were exposed to 10 Gy of IR and incubated for the indicated days for measurement of cell proliferation by MTT assay. (C) Clonogenic assay; cells grown in 60-mm dishes were irradiated at 10 Gy, trypsinized, seeded into 6-well plates (1 x 103 cells/well) and incubated for 7–10 days. Following colony formation, cells were stained and counted as described in Materials and methods. Two independent experiments were performed in triplicates and all the points are mean ± SE from a representative triplicate experiment.

 
Constitutive activation of NF-{kappa}B in Ki-Ras-transformed cells
The nuclear lysates from both the normal and the transformed cells were applied to EMSA to determine the NF-{kappa}B activation in the cells. It was found that 267B1/K-ras cells showed much higher level of NF-{kappa}B DNA binding activity compared with 267B1 cells, the complex consisting of a p65/p50 heterodimer and p50/p50 homodimer (Figure 2A). A luciferase reporter gene assay further supported the result demonstrating that there was ~6-fold increase in NF-{kappa}B transcriptional transactivation by Ki-Ras (Figure 2B). In addition, it was found that the Ki-Ras-induced NF-{kappa}B activation could be through the enhanced phosphorylation and degradation of I{kappa}B{alpha} (Figure 2C). These results suggested that the increased level of constitutive NF-{kappa}B activation in Ki-Ras-transformed cells lead to the radioresistance as shown in Figure 1.



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Fig. 2. NF-{kappa}B activation in Ki-Ras-transformed cells. (A) EMSA analysis: nuclear fraction (10 µg) was applied to EMSA with or without the indicated antibodies (2 µg); non-specific binding (NS), supershifts (SS). (B) Luciferase reporter gene assay: cells cotransfected with 0.5 µg pNF-{kappa}B-Luc and 0.5 µg pCMV/ß-galactosidase plasmids for 24 h were lysed and the luciferase activity was measured and expressed as fold increase relative to that of 267B1 cells normalized on the basis of ß-galactosidase activity. (C) Western blot analysis. Cytosolic fraction (50 µg) obtained as in (A) was immunoblotted with a specific antibody to phospho-I{kappa}B{alpha}. The membrane was stripped off and reblotted with a ß-actin antibody. The results are shown in duplicates.

 
Ionizing radiation induced NF-{kappa}B activation
It has been reported that IR causes cell death after deleterious damage to DNA (1,2). However, a cellular defense mechanism is also triggered to prevent the cells from the radiation-induced death. NF-{kappa}B is reported to be activated by IR, consequently leading to the increased radiation resistance (10). To determine whether the irradiation induces NF-{kappa}B activation in the cells, both 267B1 and 267B/K-ras cells were exposed to IR at 10 Gy. The luciferase reporter gene assay showed that IR induced NF-{kappa}B activation in both cell types, with more pronounced effect on the transformed cells (Figure 3A). In addition, EMSA analysis confirmed that there is a time-dependent increase in NF-{kappa}B DNA binding by irradiation in 267B1/K-ras cells (Figure 3B). In this case, the amount of proteins applied to the EMSA analysis was reduced (5 µg) for the clear detection of radiation effect. Hence, these results raised a possibility that the NF-{kappa}B activation by Ki-Ras as well as by IR could be responsible for the increased radioresistance of 267B1/K-ras cells.



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Fig. 3. IR-induced NF-{kappa}B activation. (A) Luciferase reporter gene assay; cells in 6-well plate were transfected with 0.5 µg of pNF-{kappa}B-Luc for 3 h and then refreshed with a new medium without serum. After 24 h incubation, cells were irradiated at 10 Gy for another 24 h and lysed. Equal amounts of proteins were used for luciferase measurement. The bars represent mean ± SE from a triplicate experiment. (B) EMSA analysis; serum-starved cells were irradiated at 10 Gy, lysed at appropriate time points and the nuclear fractions (10 µg) applied to EMSA analysis.

 
Proteosome inhibition decreases NF-{kappa}B activation but increases radiation-induced cell death
To determine whether inhibition of NF-{kappa}B activity could increase the radiosensitivity of 267B1/K-ras cells, the cells were treated with ProI for 1 h before exposure to IR. Westernblot analysis showed that the cytosolic degradation of I{kappa}B{alpha} by IR was inhibited by Pro1 treatment (Figure 4A, top panel). In accordance with this, the level of cytosolic p65 was increased while the nuclear translocation of p65 by radiation exposure was slightly diminished by ProI treatment (Figure 4A, second and third panel, respectively), which clearly indicated the inhibition of radiation-induced NF-{kappa}B activation by ProI. Unexpectedly, however, the translocation of p50 component of NF-{kappa}B did not occur regardless of the radiation exposure to the cells (Figure 4A, fourth and fifth panel). In agreement with the protein analysis, ProI significantly reduced the DNA binding activity of NF-{kappa}B in response to IR (Figure 4B, lanes 3–5). In addition, when an antibody specific to either p65 or p50 was preincubated with the EMSA reaction mixture before the addition of the labeled NF-{kappa}B oligonucleotides, there appeared a clear supershift of p65:DNA or p50:DNA complex (Figure 4B, right panel). Hence, it could be suggested from these results that the IR-induced NF-{kappa}B complex consists of p65/p65 homodimer, with the p50:DNA complex being just the result of Ki-Ras stimulation since Ki-Ras itself increased the nuclear translocation and DNA binding activity of p50 (18). However, a more detailed study of the identification of the complex is needed.



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Fig. 4. Inhibition of NF-{kappa}B activation by ProI. (A) Western blot analysis for the determination of I{kappa}B{alpha} degradation and NF-{kappa}B translocation in response to IR; 267B1/K-ras cells were pretreated with ProI at the indicated concentrations for 1 h prior to the IR exposure at 10 Gy. After 1 h of irradiation, cells were lysed and equal amounts (50 µg) of cytosolic (C) or nuclear (N) fractions were applied to western blot analysis with specific antibodies to I{kappa}B{alpha}, p65 and p50. The fold changes in the expressions of nuclear p65 with respect to control (without any treatment) are shown below the p65 (N) panel. (B) EMSA analysis; nuclear lysates (10 µg) from 267B1/K-ras cells pretreated with ProI at the indicated concentrations for 1 h prior to the IR exposure at 10 Gy were subjected to EMSA analysis with or without an antibody (2 µg) to p50, p65 or RelB for supershift as described in Materials and methods. (C) Western blot for I{kappa}B{alpha} accumulation by ProI treatment; cells treated with ProI for 1 h at the indicated concentrations were lysed and the equal amounts of the cytosolic fractions (30 µg) were subjected to western blot analysis with an antibody to I{kappa}B{alpha}. The membrane was stripped off and reblotted with an antibody to Akt1. (D) Confluent growth of cells were treated with ProI at 5 µM for 1 h, lysed and the nuclear fractions (10 µg) were analysed for EMSA analysis. (E) Semiconfluent growth of 267B1/K-ras cells in 12-well plates were treated with ProI (0.4 µM), IR (10 Gy) or both and incubated for 3 days. Cells were stained with Eosin Y as described in Materials and methods.

 
Since ProI was found to inhibit the radiation-induced NF-{kappa}B activation, it was necessary to determine whether the Ki-Ras-induced NF-{kappa}B activation could also be affected by the compound. The Ki-Ras-transformed 267B1/K-ras cells showed an elevated level of I{kappa}B{alpha} degradation compared with the normal cells as described earlier (Figure 2C). There was, however, a dose-dependent restoration of cytosolic I{kappa}B{alpha} to a significant level when treated with ProI although Akt1, another key enzyme involved in cell signaling, showed no change in its level (Figure 4C). In addition, ProI treatment also reduced the Ki-Ras-induced NF-{kappa}B activation as revealed by an EMSA analysis (Figure 4D). When cell proliferation was measured 3 days after IR exposure in the presence of ProI, it was found that ProI treatment drastically increased the radiosensitivity of 267B1/K-ras cells, whereas cells treated with ProI alone showed only a slight effect (Figure 4E). All these results indicate that inhibition of NF-{kappa}B activation could increase the radiosensitivity of the Ki-Ras transformed cells.

I{kappa}B{alpha}, but not Akt1 or MAPK, is responsible for the radioresistance of 267B1/K-ras cells
To test further the enhanced radiosensitization by NF-{kappa}B inhibition, 267B1/K-ras cells pretreated with ProI for 1 h were exposed to IR at 10 Gy, trypsinized, seeded into 6-well plates and incubated for 7 days. As mentioned above, ProI pretreatment drastically increased the radiosensitivity of the Ki-Ras transformed cells, with an almost complete inhibition of cell growth at 1 µM concentration (Figure 5A). Even a lower concentration of ProI (0.4 µM) enhanced the IR-induced 267B1/K-ras cell death when compared with cells treated with ProI alone. This result was in accord with that of a soft agar assay in which cells were treated either with ProI alone or with a combination of both ProI and IR as done above (Figure 5B). The soft agar assay showed a greater synergistic effect by ProI treatment, possibly due to a different condition of the culture medium. A supporting result could also be obtained from a cell proliferation assay in which 267B1/K-ras cells pretreated with ProI were irradiated and the cell survival was measured by MTT assay at appropriate time points, showing that ProI enhances the radiosensitivity of the transformed cells (Figure 5C).



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Fig. 5. Enhancement of radiosensitivity by ProI treatment. (A) Clonogenic assay; cells pretreated with varying doses of ProI for 1 h and exposed to 10 Gy of IR in 60-mm dishes were immediately trypsinized, seeded into 6-well plates (1 x 103 cells/well) and incubated for 7–10 days for survival colony counting. Two independent experiments were conducted in triplicates and all the points are mean ± SE from a representative triplicate experiment. (B) Soft agar assay; cells treated as in (A) were trypsinized, added into premixed RPMI1640 medium (200 cells/well) of 0.3% soft agarose for 5 days in 6-well plates and colonies of >50 cells were counted. Three independent experiments were done in triplicates and all the points are mean ± SE from a representative triplicate experiment. (C and D) 267B1/K-ras cells in 12-well plates were treated with 0.4 µM of ProI, 10 µM LY294002 or 5 µM PD98059 followed by immediate exposure to IR at 10 Gy. Cells were incubated for the indicated days and subjected to MTT assay for the determination of cell proliferation. For the insert in (D), whole cell lysate from 267B1/K-ras cells with or without LY294002 at 10 µM for 24 h was immunoblotted with an antibody to phospho-Akt1, the PVDF membrane stripped off and reblotted with an antibody to Akt1. All the bars represent mean ± SE from a representative triplicate experiment.

 
Since IR induces many kinds of enzymes and proteins, including MAPK, 267B1/K-ras cells were treated with LY294002 and PD98059, an inhibitor of PI3K and MAPK, respectively and analysed for cell proliferation by MTT assay. Interestingly, it was found that neither of the compounds increased the radiosensitivity of the cells although they are effective in the cells; LY294002 and PD98059 reduced the Ki-Ras-induced phosphorylation of Akt1 (Figure 5D, insert) and of p44/p42 MAPK (data not shown), respectively. These results clearly indicate that NF-{kappa}B activation through I{kappa}B{alpha} degradation confers the radiation resistance to the Ki-Ras-transformed cells, although neither Akt1 nor MAPK is required for the radioresistance.

Ras inhibition increases the radiosensitivity of 267B1/K-ras cells
FTI and SS are inhibitors of Ras farnesylation and signaling, respectively (23,24). It was also reported that Ki-Ras was responsible for the radioresistance in tumor cells (25). To confirm that Ki-Ras induces NF-{kappa}B activation, leading to the increased radioresistance of the cells, to 267B1/K-ras cells transfected with wild-type of NIK, IKK{alpha} and IKKß plasmids were treated with these compounds (Figure 6A). FTI and SS by themselves significantly reduced the Ki-Ras-induced NF-{kappa}B activity (Figure 6A, c and g). However, this inhibition was reversed by NIK (Figure 6A, d and h) or IKKß (Figure 6A, f and j) transfection. However, the other hand, IKK{alpha} could not reverse the inhibitory effect of the compounds (Figure 6A, e and i). These results indicate the involvement of NIK and IKKß in Ki-Ras-induced NF-{kappa}B activation. To determine whether the Ras inhibitors could increase radiosensitivity, 267B1/K-ras cells were irradiated in the presence of the compounds. It was clearly shown that treatment with the Ras inhibitors potentiated the radiation-induced cell death (Figure 6B), implying that NF-{kappa}B inhibition by the compounds increased the radiation sensitivity.



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Fig. 6. Increased radiosensitivity by Ras inhibitors. (A) NIK and IKKß restore the reduced NF-{kappa}B activity by FTI and SS; 267B1/K-ras cells in 6-well plates were cotransfected with 0.5 µg pNF-{kappa}B-Luc and 10 ng NIK, IKK{alpha} or IKKß followed by treatment with 10 µM FTI or SS 3 h post-transfection and the luciferase activity was measured after 24 h incubation. For normalization, 0.5 µg of pCMV/ß-galactosidase plasmid was cotransfected. All the bars are mean ± SE from a triplicate experiment. (B) Effect of Ras inhibitors on cell proliferation; cells in 12-well plates were treated with 10 µM of FTI or SS followed by irradiation at 10 Gy. After 3 days of incubation, cell growth was measured by MTT assay. The bars show mean ± SE from a triplicate experiment.

 
Involvement of apoptotic proteins in radiation-induced cell death
Finally, the level of proteins concerned in apoptosis was evaluated by western blot analysis of the irradiated 267B1/K-ras cells. It was observed that degradation of the apoptotic proteins caspases 8 and 3 was enhanced by proteosome inhibition, accompanied by a slight increase in the level of p53 in 267B1/K-ras cells. However, the amount of antiapoptotic protein Bcl-2 did not change although the cells were exposed to IR and ProI for longer time periods (Figure 7). These results suggest the involvement of apoptotic process in radiation-induced cell death of Ki-Ras-transformed cells.



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Fig. 7. Apoptotic proteins are involved in IR-induced cell death. Semiconfluent growth of 267B/K-ras cells were irradiated at 10 Gy in the presence or absence of 1 µM ProI. At the indicated time points, whole cell lysates were prepared and subjected to western blotting analysis (50 µg) with antibodies to Bcl-2, p53, caspases 8 and 3. The fold changes in the level of p53 with respect to control (without any treatment) shown below the p53 panel.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Radiation-induced NF-{kappa}B activation has been reported in a variety of cancer cell types, leading to radioresistance and decreased apoptosis (26). In this respect, there was an observation demonstrating that inhibition of NF-{kappa}B might increase cellular radiosensitivity for some cancers and that expression of the super-repressor form of I{kappa}B leads to an improvement in the apoptotic killing of cells exposed to cytokines and other cytotoxic stimuli (27). In addition, in a preclinical study, the use of protease inhibitor increased radiation-induced apoptosis of lymphoma cells (28). Enhanced radiosensitivity by proteosome inhibition was also reported in Lewis lung or Lovo colorectal cancer cells (29). Our data with ProI are clearly in accordance with the results of others using another 26S proteosome inhibitor, PS-341, demonstrating the proteosome inhibition as a new target of anticancer radiotherapeutics (29,30). When ProI, as an alternative means to overcome the Ki-Ras-induced NF-{kappa}B activation, was applied to the cells, it restored the level of cytosolic I{kappa}B{alpha}, leading to the decreased DNA binding activity of NF-{kappa}B molecules in the nucleus of 267B1/K-ras cells (Figure 4C and D). Radiation-induced I{kappa}B{alpha} degradation and p65 translocation were also affected by ProI pretreatment, consequently leading to the NF-{kappa}B inhibition (Figure 4A and B). Furthermore, ProI treatment enhanced the radiation-induced cell death of the Ki-Ras-transformed cells (Figure 5A). These results imply the crucial role of NF-{kappa}B activation in radiotherapy resistance of Ki-Ras overexpressing tumor cells.

Radiation-induced NF-{kappa}B activation differs from the activation caused by proinflammatory cytokines. For example, NF-{kappa}B activation in response to IR occurs over a period of hours, rather than the immediate response seen within 15 min after tumor necrosis factor treatment (31). In our study, NF-{kappa}B activation in 267B1/K-ras cells persisted over 3 h after exposure to IR (Figure 3B), with concomitant change in the level of I{kappa}B{alpha} (Figure 4A). As of IR-induced NF-{kappa}B complex formation, it seems to be a homodimer of p65/p65 although a specific antibody to p50/NF-{kappa}B1 could make supershifts for both p65/p50 and p50/p50 complexes (Figure 4B). While it was reported that the NF-{kappa}B p50 subunit is a critical component of the NF-{kappa}B complex activated by IR in vivo (27), our study showed that p50 did not translocate into the nucleus on exposure to IR, raising the possibility that the p50:DNA complex as revealed by the p50 supershift is due to the Ki-Ras-induced p50 translocation. These results suggest that the involvement of p50 in IR-induced signaling could be cell type specific and that homodimeric form of p50/p50 might not be responsible for IR-induced NF-{kappa}B activation in 267B1/K-ras cells although the complex does exist in the cells (20). However, a more detailed study is required for the identification of the complex responsible for NF-{kappa}B activation in 267B1/K-ras cells exposed to IR.

Bcl-2, a potent inhibitor of apoptosis, is directly regulated by NF-{kappa}B (32). The finding that residual tumors at the completion of chemotherapy express increased levels of Bcl-2 compared with pretreatment specimens suggests that Bcl-2 expression may be one mechanism for tumor resistance (33). p53, a major apoptosis and cell cycle regulator, is also both directly and indirectly regulated by NF-{kappa}B (34). In our study, the level of Bcl-2 was unchanged upon exposure to IR and Pro1, while the amount of p53 was slightly increased (Figure 7). In addition, the IR-induced degradation of proapoptotic proteins, caspases 8 and 3, was enhanced by Pro1 treatment (Figure 7). However, the degradation product of poly (ADP-ribose) polymerase (PARP) could not be detected by either of the treatments (data not shown). These results strongly suggest the apoptotic process in radiosensitization of 267B1/K-ras cells, although the exact nature of the radiation-induced cell death of Ki-Ras-transformed cells is expected to be resolved using exogenous inhibitors of NF-{kappa}B, including curcumin (35), peptide inhibitors (36) and others.

The expression of Akt is reported to be regulated by NF-{kappa}B (37). Furthermore, there has been accumulating evidence that Akt is involved in resistance to chemotherapy (38). However, in our study the expression of Akt did not change on time-dependent exposure of the cells to both IR and ProI (data not shown), indicating that Akt is not downstream of NF-{kappa}B activation in our system. Moreover, Akt does not seem to be involved in radiation-induced cell death (Figure 5D). Recently, however, we observed that Akt1 is also involved in Ki-Ras-induced NF-{kappa}B activation (Kim, B.Y. et al., unpublished data). Thus, it is presumable that the effect of Akt1-mediated NF-{kappa}B activation on IR-induced cell death of Ki-Ras-transformed cells is only marginal compared with that of I{kappa}B{alpha}-mediated one. However, it cannot be excluded that downstream regulation of the expression of Akt by NF-{kappa}B is cell line- or agonist-specific and that Akt1 has other roles than the cell growth in Ki-Ras-overexpressing cells.

In conclusion, our data suggest a p65/RelA activation by IR and show that NF-{kappa}B activation, but not Akt or MAPK signaling, contributes to the radiation resistance. Hence, inhibition of NF-{kappa}B can lead to the increased cell death on exposure of Ki-Ras overexpressing cells to radiation. Our results provide a possibility of developing a target-oriented anticancer therapeutics, at least in curing of pancreatic, prostate and colon cancers since they overexpress constitutively activated Ki-Ras oncoprotein.


    Notes
 
{dagger} The first two authors equally contributed to this work. Back


    Acknowledgments
 
This work was supported by Pharmacogenomics Program from the Ministry of Health and Welfare (BGW0200311), Molecular and Cellular BioDiscovery Research Program from the Ministry of Science and Technology (Grant M1-0311-00-0023) and KRIBB Research Initiative Program.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 12, 2005; revised March 3, 2005; accepted March 22, 2005.





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