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Carcinogenesis Advance Access originally published online on June 14, 2006
Carcinogenesis 2006 27(11):2258-2268; doi:10.1093/carcin/bgl097
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

NF-{kappa}B inhibition increases chemosensitivity to trichostatin A-induced cell death of Ki-Ras-transformed human prostate epithelial cells

Osong Kwon, Kyong A Kim, Sun Ok Kim, Ryong Ha, Won Keun Oh, Min Soo Kim, Hee Sik Kim, Gun Do Kim1, Jong Wan Kim2, Mira Jung3, Cheorl Ho Kim4, Jong Seog Ahn and Bo Yeon Kim*

Laboratory of Cellular Signaling Modulators, Korea Research Institute of Bioscience and Biotechnology (KRIBB) Yuseong, Daejeon, 305-333, Korea
1 Department of Microbiology, College of Natural Sciences 599-1, Pukyong National University, Daeyeon3-Dong, Nam-Gu, Pusan 608-737, Korea
2 Department of Radiation, Dangook University School of Medicine Cheonan, Korea
3 Department of Radiation Medicine, Georgetown University School of Medicine Washington, District of Columbia 20057-1482, USA
4 Department of Biological Sciences, Sungkyunkwan University Chunchun-Dong 300, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Korea

*To whom correspondence should be addressed. Tel: +82 42 860 4297; +82 42 860 4595; Email: bykim{at}kribb.re.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemoresistance has been one of the major problems in anticancer therapy. In our effort to find a potential molecular target for overcoming the chemoresistance in prostate cancer, a promising anticancer drug trichostatin A (TSA) induced cell death was found to be compromised by enhanced NF-{kappa}B activation in 267B1/K-ras human prostate epithelial cancer cells. However, both the NF-{kappa}B activation and chemoresistance were reduced by pretreatment with proteasome inhibitor-I (ProI), accompanied by accumulations of both I{kappa}B{alpha} and p65/RelA (but not p50/NF-{kappa}B1) in the cytoplasm. Clonogenic cell survival and soft agar assays further confirmed the increased TSA chemosensitivity of 267B1/K-ras cells by ProI treatment. Moreover, dominant negative mutant of IKKß, I{kappa}B{alpha} and p65 enhanced the chemosensitization, too. Unexpectedly, using LY294002 and PD98059, phosphatidylinositol-3-kinase and mitogen-activated protein kinase were also implied in TSA chemoresistance through NF-{kappa}B activation, while these compounds had showed no effect on radiosensitization in the cells. On the other hand, together with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, activations of caspase-8 and caspase-3 by TSA and ProI were noticed, suggesting the involvement of apoptotic process in chemosensitization of 267B1/K-ras cells. Altogether, these results suggest that blocking the NF-{kappa}B activation pathway could be an efficient target for improving the TSA chemosensitization and applying to the development of anticancer therapeutics in Ki-Ras-overexpressing tumorigenic cells, including prostate cancer.

Abbreviations: DTT, dithiothreitol; EMSA, electromobility shift assay; HDAC, histone deacetylase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PMSF, phenylmethylsufonyl fluoride; ProI, proteasome inhibitor-I; TNF-{alpha}, tumor necrosis factor-{alpha}; TSA, trichostatin A; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
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 (1). Tumor cells usually express high levels of constitutive NF-{kappa}B activity (2). Exposure of these cancer cells to various cytotoxic agents increases NF-{kappa}B activity, resulting in cell growth and survival advantage and resistance to the therapeutic reagents (3,4). In many cases, chemotherapeutics result in the NF-{kappa}B-induced expression of anti-apoptotic genes, probably IEX-1L (5), IAP (6) and Gadd45ß (7). Bcl-xL was recently reported to be involved in chemoresistance in pancreatic cancer cells (8). Moreover, genes for tumor growth and survival, including cyclin D1 and c-Myc, are also regulated by NF-{kappa}B and contribute to the chemoresistance (9,10). Thus, NF-{kappa}B signaling pathway could be a potent target for improving the chemosensitivity of the tumor cells. In this regard, curcumin was reported to downregulate the constitutive activation of NF-{kappa}B, increasing the chemosensitivity to vincristine and melphlan in human multiple myeloma (MM) cells (11). Triptolide was also found to inhibit NF-{kappa}B and enhance the chemosensitivity to doxorubicin (12). In this respect, proteasome has emerged as an excellent target for cancer therapy (13,14). Very recently, a therapeutic implication of proteasome inhibition for differential regulation of noxa in normal melanocytes and melanoma cells was reported (15). The proteasome inhibitor PS-341 was also demonstrated to enhance chemosensitivity to CPT-11, a topoisomerase inhibitor (1). Moreover, I{kappa}B{alpha} super-repressor mutant greatly enhanced chemosensitivity of human pancreatic carcinoma cells (16).

Previously, trichostatin A (TSA), the most potent inhibitor of histone deacetylase (HDAC), was found to strongly suppress growth of pancreatic adenocarcinoma cells (17). However, there also appeared observations in regard to the TSA-induced NF-{kappa}B activation. TSA not only induced caspase-3 activation and neuronal apoptosis but also activated NF-{kappa}B (18). NF-{kappa}B-mediated interleukin-6 (IL-6) gene promoter activity was exclusively potentiated by TSA (19). Furthermore, chromatin remodeling and p65-mediated transcriptional initiation and elongation by TSA were reported (20). A more direct evidence for the involvement of NF-{kappa}B in TSA-induced gene expression comes from an observation demonstrating that HDAC1 and HDAC2 associate with p65 and TSA interrupted this interaction, leading to the enhanced IL-8 gene expression (21). Thus, although TSA is one of the promising agents useful for cancer treatment, its therapeutic efficacy decreases when the compound also activates the transcription factor NF-{kappa}B, enhancing the expression of anti-apoptotic proteins (22).

It was recently reported that Ras is responsible for the resistance to Fas-induced apoptosis (23). 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 (70–90%), colon (50%) and lung adenocarcinomas (25–50%) (24–26). RelA (p65/NF-{kappa}B) is constitutively activated in pancreatic adenocarcinoma and pancreatic tumor cells expressing a mutant Ki-Ras; however, disruption of K-ras was shown to result in downregulation of cancer-prone activities in the invasive colon cancer cell line HCT116 (27). 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 (28), and further demonstrated that I{kappa}B{alpha} inhibition enhanced the radiosensitization (29).

Given that 267B1/K-ras cells were refractory to TSA-induced cell death, in this study, we have explored the possibility that inhibition of NF-{kappa}B activation could increase the TSA sensitivity of the cells. Our data showed that NF-{kappa}B inhibition by a proteasome inhibitor enhanced the chemosensitivity of 267B1/K-ras cells. This TSA-induced cell death is dependent on I{kappa}B{alpha} degradation and p65/RelA translocation but not on p50/NF-{kappa}B1. Akt and mitogen-activated protein kinase (MAPK) signaling pathways are also suggested to be involved in this process.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Lipofectamine Plus, RPMI1640 and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Proteasome inhibitor-1 (ProI) and antibodies to caspase-8 and caspase-3 were obtained from Calbiochem (San Diego, CA). Antibodies to IkBa, Akt1, actin, p50, p52, p65, Rel-B, c-Rel and IKKa/b were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p53 and Bcl-2 were purchased from Oncogene (Darmstadt, Germany). Antibody to phospho-IkBa was obtained from Cell Signaling (Beverley, MA). The luciferase assay kit was from Promega (Madison, WI), Sephadex G-25 columns and enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech (Amersham, NJ). The pNF-kB-Luc plasmid was obtained from Stratagene (La Jolla, CA), TUNEL assay kit was from Roche (Mannheim, Germany) and [{gamma}-32P]ATP was obtained from NEN, Dupont (Boston, MA). Wild-type p65 cDNA was cloned and sequenced as described elsewhere (30). Trichostatin A (TSA) and all the other reagents were obtained from Sigma (St Louis, MO).

Cell culture, transfection and luciferase reporter gene assay
The preparation of both 267B1 and 267B1/K-ras cell lines was described previously (31). The human neonatal prostate cell line, 267B1, was established by transfecting primary human neonatal prostate epithelial cells with a plasmid containing SV40 early region genes. These cells became immortalized but retained the essential characteristics of primary human prostate epithelial cells: an epithelial morphology, expression of cytokeratins specific for epithelial cells, an antigenic profile similar to adult prostatic epithelial cells and non-tumorigenicity in nude mice. The 267B1 cells were infected with Ki-MSV containing an activated K-ras oncogene, subcultured every 7–10 days, and the morphology, growth pattern, p21 expression and tumorigenicity study in nude mice were observed for selection of transformed 267B1/K-ras cell line.

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, cells (2 x 105/ml) were incubated in six-well plates for 24 h: with the use of Lipofectamine Plus, they were then co-transfected 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 of NIK, IKK{alpha} or IKKß. In some cases, cells were treated with TSA in the presence or absence of varying concentrations of ProI (1 hr) 3 h post-transfection with pNF-{kappa}B-Luc. After 24 h, luciferase activity was measured with a detection kit.

Immunoblot analysis
Cells were grown to semiconfluence in 100 or 150 mm dishes. They were washed four times with ice-cold phosphate-buffered saline (PBS) and then scraped on ice into, and maintained for 15 min in, a solution containing 10 mM HEPES–KOH (pH 7.9), 10 mM KCl, 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM NaF, 0.4 mM phenylmethylsufonyl fluoride (PMSF), leupeptin (10 µg/ml), aprotinin (10 µg/ml), 0.1 mM Na3VO4 and 1 mM dithiothreitol (DTT). 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.

Electromobility shift assay (EMSA) analysis
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 PMSF, leupeptin (10 µg/ml), 1 mM 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 1 x kinase buffer (supplied with the kinase). The labeling 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 before 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.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Working solution of MTT was prepared by 1 : 5 dilution from a stock solution (5 mg/ml in PBS) with pre-warmed medium just before assay. Fifty microliters of this MTT working solution was added to each well of microculture plate. After 4 h incubation, 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 DMSO 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. Cells were collected into microculture plate and the absorbance was measured.

Staining for determination of cell proliferation
Cells treated with TSA 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 ~40°C. Cells pre-exposed to TSA or chemicals in 60 mm dishes were trypsinized, counted and poured into the pre-made soft agarose medium (200 cells/well) in six-well plates. Five days later, cells were photographed under inverted microscope and the number of colonies were counted. Three independent experiments were performed in triplicates.

Clonogenic assay
Semiconfluent growth of cells in 60 mm dishes were exposed to TSA or ProI at appropriate concentrations and immediately trypsinized to be transferred to six-well plates at 1 x 103 cells/well in complete RPMI1640 medium. After 7–10 days' 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.

TUNEL assay
All the procedure using the TUNEL assay kit was performed according to the suggestion by the manufacturer. In brief, confluent cells were serum-starved 24–48 h in the presence of appropriate test samples in a humidified CO2 incubator and then washed with PBS buffer for the TUNEL assay. Cells were fixed (4% paraformaldehyde in PBS, pH 7.4) at 15–25°C for 1 h, washed again with PBS and blocked (3% H2O2 in MeOH) for 10 min. After cell permeabilization (0.1% Triton X-100 in 0.1% sodium citrate) on ice for 2 min and drying out, TUNEL reaction mixture was added into the wells for 1 h in the dark. Cells were stained by adding the DAB substrate following POD solution treatment as supplied in the kit and photographed in the visible light.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Resistance of Ki-Ras-transformed cells to TSA-induced cell death
It has been reported that TSA strongly suppressed the growth of pancreatic adenocarcinoma cells (17). To evaluate the effect of TSA on the proliferation of Ki-Ras-transformed cells, both 267B1 and 267B1/K-ras cells (normal and Ki-Ras-transformed, respectively) were treated with varying doses of TSA for 1 h and allowed to grow further after removal of the compound. An MTT assay after three days of incubation showed a dose-dependent significant decline in 267B1 cell proliferation (black diamond) while only a small fraction of 267B1/K-ras cells were dead by TSA treatment (black circle) (Figure 1A), as supported by a cell-staining experiment (Figure 1B). In addition, a time-course experiment revealed that the normal 267B1 cells were noticeably dead by the second or third day of TSA treatment (diamonds), whereas the Ki-Ras-transformed cells showed significant resistance to TSA cytotoxicity (circles) (Figure 1C). Correspondingly, a clonogenic survival assay further confirmed the increased TSA resistance of Ki-Ras-transformed cells (Figure 1D). After treatment with TSA for 1 h, cells were trypsinized, seeded into six-well plates, incubated for 7 days and the colonies having >50 cells were counted. As expected, 267B1/K-ras cells showed only a slight decrease in cell survival while most of the normal cells were dead by TSA treatment. These results suggested that the TSA resistance might be due to the enhanced NF-{kappa}B activity in the 267B1/K-ras transformed cells, given that Ki-Ras induced NF-{kappa}B activation in these cells (28).


Figure 1
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  		Fig. 1 TSA-induced cell death resistance by Ki-Ras overexpression. (A) 267B1/K-ras cells grown in complete medium in 12-well plates were exposed to varying concentrations of TSA, washed twice with PBS, refreshed with a new PRMI1640 complete medium, incubated for 3 days and the cell number was measured by MTT assay. (B) Cells treated with 0.2 µg/ml of TSA were processed as in (A) and stained with Eosin-Y dye for visualization as described in Materials and methods. (C) Cells were treated with 0.2 µg/ml of TSA, processed as in (A), and incubated for the indicated days for measurement of cell proliferation by MTT assay. (D) Clonogenic assay; cells grown in 60 mm dishes were treated with 0.2 µg/ml of TSA, trypsinized, seeded into six-well plates (1 x 103 cells/well) and incubated for 7 days. Following colony formation, cells were stained and counted as described in Materials and methods. Two independent experiments were performed in triplicate and all the points are means ± SE from a representative triplicate experiment.

 
NF-{kappa}B is constitutively activated in Ki-Ras-transformed cells
A luciferase reporter gene assay showed that there was ~5- to 6-fold increase in Ki-Ras-induced NF-{kappa}B transcriptional transactivation (Figure 2A). An EMSA using the nuclear lysates from both cells identified the activated NF-{kappa}B complex consisting of a p65/p50 heterodimer and a p50/50 homodimer (Figure 2B). The phosphorylation and subsequent degradation of I{kappa}B{alpha} was found to occur in accordance with the NF-{kappa}B activation in Ki-Ras-transformed cells (Figure 2C). Hence, all these results together with those in Figure 1 suggested the contribution of NF-{kappa}B activation by Ki-Ras to cell death resistance to TSA treatment.


Figure 2
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  		Fig. 2 NF-{kappa}B activation in Ki-Ras-transformed cells. (A) Luciferase reporter gene assay: cells co-transfected with 0.5 µg pNF-{kappa}B-Luc and 0.5 µg pCMV/ß-galactosidase plasmids for 24 h in six-well plates 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. (B) EMSA analysis: nuclear fraction (10 µg) was applied to EMSA with or without the indicated antibodies (1 µg); non-specific binding (ns), supershifts (SS). (C) Western blot analysis. Cytosolic fraction (50 µg) obtained as in (B) was immunoblotted with a specific antibody to phospho-I{kappa}B{alpha} or I{kappa}B{alpha}. The membrane was stripped off and reblotted with a ß-actin antibody.

 
TSA enhances the NF-{kappa}B activation in 267B1/K-ras cells
There have been controversial reports concerning the effect of HDAC inhibitors on NF-{kappa}B activation. The interaction of p65 subunit with HDAC1 and HDAC2 could be reversed by TSA treatment, leading to the increased IL-8 gene expression (21). In addition, TNF-{alpha}-induced NF-{kappa}B activation was found to be enhanced by TSA or sodium butyrate at multiple levels (32). On the other hand, the luminal short-chain fatty acid butyrate inhibited the TNF-{alpha}-induced NF-{kappa}B activation as well as the nuclear localization of p50 (33). In order to determine whether TSA induces NF-{kappa}B activation in the prostate cells, cells were treated with TSA at 0.2 µg/ml for 24 h after transfection with a pNF-{kappa}B-Luc reporter plasmid. The result showed that TSA induced NF-{kappa}B activation in both 267B1 and 267B1/K-ras cells, with the increase more pronounced in the transformed cells (Figure 3A). Moreover, NF-{kappa}B DNA binding was significantly increased by TSA treatment in 267B1/K-ras cells while there was little increase in 267B1 cells when determined by EMSA analysis (Figure 3B). Thus, it was possible from all these results that NF-{kappa}B activation by TSA as well as by Ki-Ras could be responsible for the increased resistance of 267B1/K-ras cells to TSA-induced cell death.


Figure 3
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  		Fig. 3 TSA induces NF-{kappa}B activation in 267B1/K-ras cells. (A) Luciferase reporter gene assay; cells in six-well plate were transfected with 0.5 µg of pNF-{kappa}B-Luc for 3 h and then refreshed with a new serum-free medium containing 0.2 µg/ml TSA. After 24 h incubation, cells were lysed and equal amount of proteins were used for luciferase measurement. The bars represent mean ± SE from a representative triplicate experiment of two independent experiments. (B) EMSA analysis; serum-starved cells were treated with 0.2 µg/ml TSA, lysed at appropriate time points and the nuclear fractions (10 µg) were applied to EMSA analysis; non-specific (ns) and free oligonucleotide bands are marked.

 
TSA-induced cell death is enhanced by proteasome inhibition
After I{kappa}B{alpha} is phosphorylated by an upstream I{kappa}B{alpha} kinase complex, it is degraded by proteasome, releasing NF-{kappa}B into the nucleus (34). Previously, we reported that ProI reduced the Ki-Ras-induced NF-{kappa}B activation, leading to the increased radiosensitization of the cells (29). Thus, we explored whether the inhibition of I{kappa}B{alpha} degradation could enhance the TSA-triggered cell death. The 267B1/K-ras cells were treated with ProI for 1 h before the exposure to TSA and lysed for western blot analysis. It was found that TSA increased the degradation of I{kappa}B{alpha} in the cytosol (Figure 4A), corresponding with the results showing the upregulation of NF-{kappa}B activation by TSA (Figure 3). ProI pretreatment, however, dose-dependently restored the level of I{kappa}B{alpha} (Figure 4A, top panel). As of NF-{kappa}B, the nuclear translocation of p65 by TSA was blocked by ProI pretreatment (Figure 4A, second and third panel), all these results indicating the ProI inhibition of TSA-induced NF-{kappa}B activation. In addition, ProI showed no effect on the level of IKK{alpha}/ß, supporting the result that ProI affected NF-{kappa}B activation by inhibiting the I{kappa}B{alpha} degradation. Unexpectedly, however, translocation of the p50 component of NF-{kappa}B could not be seen, regardless of TSA treatment to the cells (Figure 4A, fourth and fifth panel). A further study with pNF-{kappa}B-Luc reporter gene assay clearly supported the inhibitory effect of ProI on TSA-induced NF-{kappa}B activation (Figure 4B), corresponding with the results of EMSA analysis (Figure 4C). Interestingly, however, use of the antibodies to p65 and p50 (Figure 4C, lanes 7 and 8) could make their respective supershift bands. Hence, although more detailed study for the identification of the complex should be needed, these results suggested that the TSA-induced NF-{kappa}B complex consists of p65/p65 homodimer, with the p50 : DNA complex possibly due to the Ki-Ras effect itself since Ki-Ras increased the nuclear translocation and DNA binding activity of p50 (Figure 2B).


Figure 4
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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 TSA; 267B1/K-ras cells were pretreated with ProI at the indicated concentrations for 1 h before the TSA treatment. One hour later of challenging with TSA at 0.2 µg/ml, 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, p50, IKK{alpha}/ß and actin. (B) Luciferase reporter gene assay; cells were pretreated with varying concentrations of ProI 1 h before the TSA treatment (0.2 µg/ml) following transfection with pNF-{kappa}B-Luc for 3 h. After 24 h, cells were lysed and the NF-{kappa}B activity was measured as described above. All the bars are means ± SE from a representative triplicate experiment. (C) EMSA analysis; nuclear lysates (10 µg) from 267B1/K-ras cells pretreated with ProI at the indicated concentrations for 1 h before the TSA treatment at 0.2 µg/ml were subjected to EMSA analysis with or without an antibody (2 µg) to p50 or p65 for supershift as described in Materials and methods. (D) 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} or actin. (E) Confluent cells were treated with ProI at 5 µM for 1 h, lysed and the nuclear fractions (10 µg) were analyzed for EMSA analysis. (F) Semiconfluent 267B1/K-ras cells in 12-well plate were treated with ProI (0.4 µM), TSA (0.2 µg/ml) or both and incubated for 3 days. Cells were stained with Eosin-Y as described in Materials and methods.

 
Next, given that the TSA-induced NF-{kappa}B activation was inhibited by ProI, we determined whether the Ki-Ras-induced NF-{kappa}B activation itself could also be affected by ProI. As described in Figure 2C, I{kappa}B{alpha} degradation occurred in 267B1/K-ras cells. ProI treatment, however, restored the cytosolic I{kappa}B{alpha} to a significant level in 267B1/K-ras cells while there was no change in the normal 267B1 cells (Figure 4D). Correspondingly, an EMSA analysis revealed a reduction in Ki-Ras-induced NF-{kappa}B DNA binding activity when the cells were treated with ProI (Figure 4E). In addition, a cell proliferation assay 3 days after TSA treatment in the presence or absence of ProI showed that ProI treatment drastically enhanced the sensitivity of 267B1/K-ras cells to TSA-induced cell death, whereas neither of the compounds when treated alone showed a noticeable effect (Figure 4F). Hence, all these results indicate that NF-{kappa}B inhibition could enhance the chemosensitivity of the Ki-Ras-transformed cells to TSA.

Involvement of IKKß, I{kappa}B{alpha} and p65 in the TSA sensitization of 267B1/K-ras cells
For convincing the enhanced TSA sensitization by NF-{kappa}B inhibition, 267B1/K-ras cells were dose-dependently pretreated with ProI for 1 hour and then exposed to TSA for another 1 h. After removal of the compounds, the cells were applied to clonogenic survival assay. It was found that ProI drastically enhanced the TSA-induced cell death (Figure 5A), as supported by a soft agar assay demonstrating that inhibition of I{kappa}B{alpha} degradation by ProI enhanced the TSA sensitivity of 267B1/K-ras cells (Figure 5B). Results from cell proliferation assay by MTT also showed the ProI enhancement of TSA sensitization (Figure 5C). Moreover, when 267B1/K-ras cells were transfected with a vector containing wild-type p65, the TSA-induced cell death was significantly blocked (the lipofectamine reagent itself showed some cytotoxicity), with the DNA binding activity of p65 increased (Figure 5D), implying that I{kappa}B{alpha} degradation and p65 translocation are closely associated with chemoresistance of the cells to TSA.


Figure 5
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  		Fig. 5 Enhanced chemosensitization by ProI treatment. (A) Clonogenic assay; cells pretreated with varying doses of ProI for 1 h and then exposed to TSA at 0.2 µg/ml in 60 mm dishes were immediately trypsinized, seeded into six-well plates (1 x 103 cells/well) and incubated for 7 days for survival colony counting. Two independent experiments were conducted in triplicates and all the points are means ± 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 six-well plates and colonies of >50 cells were counted. Three independent experiments were done in triplicates and all the points are means ± SE from a representative triplicate experiment. (C) 267B1/K-ras cells in 12-well plates were treated with 0.5 µM of ProI followed by immediate exposure to TSA at 0.2 µg/ml. Cells were incubated for the indicated days and subjected to MTT assay for the determination of cell proliferation. (D) Cells as above were transfected with 0.5 mg of wild-type p65 plasmid for 3 h and then treated with TSA. One hour later, cells were washed once with PBS buffer, refreshed with a new medium and incubated for the indicated days for MTT cell proliferation analysis. For the insert in (D), nuclear lysate from 267B1/K-ras cells transfected with wild-type p65 for 48 h was applied to an EMSA analysis.

 
Both 267B1 and 267B1/K-ras cells were shown to express equal amounts of IKK{alpha} and IKKß (Figure 6A). To determine the association of the proteins upstream of I{kappa}B{alpha} with the TSA resistance of the 267B1/K-ras cells, dominant negative forms of the plasmids IKK{alpha}, IKKß, p65 and I{kappa}B{alpha} were transfected into the cells and the cell proliferation was measured by an MTT assay (Figure 6B). TSA alone did not show much effect on the cell death after 3 days of treatment and only a marginal effect by IKK{alpha} mutant could be seen. The IKKß mutant, however, most profoundly enhanced the TSA-induced cell death. As expected, the mutants of p65 and the non-degradable I{kappa}B{alpha} showed significant inhibition in cell proliferation. Without TSA, however, all the mutant plasmids showed little, if any, effect on the cell proliferation (data not shown), suggesting the much more significant contribution of NF-{kappa}B activation by TSA rather than by Ki-Ras to the chemoresistance of the cells. Thus, when a reporter gene assay was performed without TSA treatment, it was revealed that the mutant forms of IKKß and NIK reduced while their wild-types increased the Ki-Ras-induced NF-{kappa}B transcriptional transactivation. However, IKK{alpha}, regardless of wild-type or mutant, showed little effect on NF-{kappa}B activation (Figure 6C). These results, showing the proper working of all the plasmids in the intact cells, further supported the suggestion that NF-{kappa}B activation by TSA rather than by Ki-Ras could contribute more strongly to the cell death resistance In addition, in a determination of whether other signaling pathways could also be involved in TSA-induced cell death, 267B1/K-ras cells were pretreated for 1 h with LY294002 and PD98059, the specific inhibitors of phosphatidylinositol-3-kinase (PI3K) and MAPK, respectively. Unexpectedly, the cell proliferation assay showed that both compounds were effective in enhancing the cell death by TSA (Figure 6D), even though we previously noticed that IR-induced cell death could not be increased by the compounds (29). Treatment of the inhibitors without TSA did not show significant cell death (data not shown). Hence, it is suggested that chemosensitization by TSA might be different from radiosensitization in its signaling pathways for inducing cell death. On the other hand, in connection with the cell death-enhancing activity of the compounds with NF-{kappa}B regulation, it was revealed that LY294002 reduced the NF-{kappa}B activation in response to TSA as well as to Ki-Ras itself. However, PD98059 significantly decreased only the TSA-induced NF-{kappa}B activation (Figure 6E). These results suggest that TSA-induced NF-{kappa}B activation has a close relation with cell proliferation and that PI3K could be mediating the NF-{kappa}B activation in response to both TSA and Ki-Ras. However, MAPK only seems to be involved in TSA-induced signaling.


Figure 6
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  		Fig. 6 Association of IKKß, Akt1 and MAPK in TSA-induced cell death. (A) Expression of IKK{alpha} and IKKß. Equal amounts of cell lysates prepared as described in Materials and methods were subjected to western blot analysis with an antibody to IKK{alpha}/ß. (B) Effect of dominant negative mutants of IKKs, I{kappa}B{alpha} and p65 on cell proliferation; 267B1/K-ras cells in 12-well plates were transfected for 3 h with each of the dominant mutant IKK{alpha}, IKKß, I{kappa}B{alpha} or p65 (1 µg) followed by 1 h incubation in the presence of TSA (0.2 µg/ml). The cells were washed once with PBS buffer and processed for the determination of cell proliferation by MTT. All the bars are means ± SE from a representative duplicate experiment. (C) NIK and IKKß regulate the Ki-Ras-induced NF-{kappa}B activation; 267B1/K-ras cells in six-well plates were co-transfected with 0.5 µg pNF-{kappa}B-Luc and wild-type or dominant negative form of 10 ng NIK, IKK{alpha}, IKKß, I{kappa}B{alpha} or p65. Three hours later, the cells were washed, refreshed with a new medium and further incubated for 24 h for measurement of luciferase activity. The bars show means ± SE from a triplicate experiment. (D) Enhancement of TSA chemosensitization by LY294002 and PD98059. 267B1/K-ras cells in 12-well plates were pretreated with LY294002 (10 µM) or PD98059 (5 µM) for 1 h before TSA exposure at 0.2 µg/ml for the indicated days. MTT assay was done as in Materials and methods. All the bars show means ± SE from a triplicate experiment. (E) Effect of LY294002 and PD98059 on NF-{kappa}B activation in response to Ki-Ras or TSA. 267B1/K-ras cells in six-well plates were transfected with 0.5 µg pNF-{kappa}B-Luc for 3 h, washed once with PBS buffer, refreshed for 1 h with a new medium containing LY294002 (10 µM) or PD98059 (5 µM) and further incubated for 24 h in the presence or absence of TSA (0.2 µg/ml) for measurement of luciferase activity. The bars show means ± SE from a triplicate experiment.

 
Apoptotic processes are involved in TSA-induced cell death
The 267B1/K-ras cells exposed to ionizing radiation showed activation of apoptotic proteins, caspase-8 and caspase-3, with a little bit enhanced p53 expression (29). As shown in Figure 7A, the levels of the activated caspase-8 and the large fragment of the two degradation products (20 kD, 18 kD) of caspase-3 increased upon treatment with either TSA or ProI, and their appearance corresponded with the pattern of TSA-induced NF-{kappa}B activation (Figure 3B), supporting the close connection of NF-{kappa}B activation with chemosensitization in 267B1/K-ras cells. However, no additive effect by the co-treatment of the two compounds could be seen, possibly reflecting the maximum limit of caspase activation in 267B1/K-ras cells. On the other hand, the caspase fragments activated by TSA disappeared at later time (>48 h). The levels of p53 and bcl-2 did not change. Supporting data for the apoptotic process in response to TSA and ProI were obtained by a modified TUNEL assay in which the color of the apoptotic cells turns to brown when exposed to visible light. As shown in Figure 7B, compared with the transformed cells, the normal cells exhibited a little bit but noticeable apoptotic pattern in serum-free condition even without any treatment. Apoptotic death of 267B1/K-ras cells was more pronounced by ProI than by TSA. Furthermore, combination of both the compounds drastically increased the apoptosis even at 24 h post-treatment, and a large part of the cells began to die out by 48 h.


Figure 7
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  		Fig. 7 Apoptotic proteins are involved in chemosensitization of 267B1/K-ras cells to TSA. (A) Semiconfluent growth of 267B/K-ras cells were treated with TSA (0.2 µg/ml), ProI (0.5 µM) or combination of both. At the indicated time points, whole cell lysates were prepared and subjected to western blotting analysis (50 µg) with antibodies to Bcl-2, p53, caspase-8 and caspase-3. (B) TUNEL assay; 7 x 104 cells/well in 48-well plates were grown to confluence overnight and the medium was changed for 1 or 2 days in the presence of TSA (0.2 µg/ml) and ProI (5 µM) alone or in combination. The remaining procedure for the detection of apoptotic cells is as described in Materials and methods. The brown color represents the cells undergoing apoptosis.

 

   Discussion
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 Materials and methods
 Results
 Discussion
References
 
Several groups have reported the potentiation of NF-{kappa}B-mediated gene induction by TSA or sodium butyrate (21,35,36). Conversely, inhibition of p50 nuclear localization and NF-{kappa}B activation was also reported (37). In our study, TSA was shown to significantly enhance the Ki-Ras-induced NF-{kappa}B activation (Figure 3), consequently leading to the increased cell death resistance (Figure 1). However, proteasome inhibition by ProI reduced the TSA-induced NF-{kappa}B activation and enhanced the chemosensitivity of the cells (Figures 4 and 5). Since proteasome-mediated I{kappa}B{alpha} degradation has emerged as a promising target for anticancer therapeutics (13–15,38) and the peptide boronic acid bortezomib (also known as PS-341/Velcade) showing selective and reversible binding to proteasome has been approved for the treatment of MM (14), our data further reemphasize the critical role of NF-{kappa}B in chemosensitization in Ki-Ras-overexpressing tumorigenic cells.

NF-{kappa}B activation in 267B1/K-ras cells appeared to peak at around 1 h post-TSA treatment (Figure 3B), with the concomitant change of I{kappa}B{alpha} level (Figure 4A). As of the NF-{kappa}B complex in response to TSA treatment, it seems to be a homodimer of p65 although a specific antibody to p50 could also make a supershift (Figure 4B). Hence, reminding of a report demonstrating that p65 can directly bind to and be acetylated by CBP/p300 histone acetyltransferase (HAT) after phosphorylation at serine 276 by the catalytic subunit of PKA (39), it is likely that p65, as a main TSA-induced NF-{kappa}B subunit, could associate with one of the acetyltransferases in 267B1/K-ras cells and that the p50 supershift is simply due to the Ki-Ras-induced p50 translocation. However, it could not be excluded that the p50 could be induced to translocate into the nucleus by TSA but the level is beyond the limit of our detection with the EMSA or western blot analysis since p50 was also reported to be acetylated by p300/CBP in vivo and in vitro (40,41). Thus, a more detailed study should be required for the identification of the TSA-induced NF-{kappa}B complex.

NF-{kappa}B activation has been reported to be regulated by acetyltransferases at multiple steps; IKK{alpha}, IKK{gamma} and p65 were found to competitively bind to CBP/p300 (37,42), and I{kappa}B{alpha} was revealed to associate with HDAC1 and HDAC3 (43). In 267B1/K-ras cells, TSA induced the proteasome-mediated degradation of I{kappa}B{alpha} and subsequent nuclear translocation of p65 (Figure 4A). Given that TSA prolonged the TNF-{alpha}-induced IKK activity, thus causing a persistent proteasome-mediated degradation of neo-synthesized I{kappa}B{alpha} (35), and that the dominant negative mutant IKKß downregulated the cell proliferation of TSA-treated 267B1/K-ras cells (Figure 6B), there is a possibility that TSA enhanced the IKK activity, subsequently leading to the increased I{kappa}B{alpha} degradation and NF-{kappa}B activation in 267B1/K-ras cells. At present, however, the detailed mechanism and the target of TSA in the Ki-Ras-transformed cells remain to be resolved.

The expression of Akt1 is reported to be regulated by NF-{kappa}B (44). Furthermore, there has been accumulating evidences that Akt1 is involved in the chemotherapy resistance (45). However, our study showed that the expression of Akt1 did not change upon time-dependent exposure of the cells to both TSA and ProI (data not shown), indicating that Akt1 is not downstream of NF-{kappa}B activation in our system. However, we observed that LY294002 enhanced the TSA-induced cell death (Figure 6D) and inhibited the NF-{kappa}B transcriptional transactivation (Figure 6E) without effect on the NF-{kappa}B DNA binding activity (data not shown). Furthermore, the chemosensitivity of the cells was increased by PD98059 (Figure 6D), although this compound showed no inhibition against NF-{kappa}B activation in response to Ki-Ras itself (Figure 6E). Since neither LY294002 nor PD98059 enhanced the radiosensitivity of the cells (29), these results suggest that the apoptotic signaling mechanism by TSA might be different from the one used by irradiation and that both Akt and MAPK could partly contribute to the chemoresistance of the 267B1/K-ras cells to TSA. In addition, it could be that MAPK might play an important role in TSA-induced cell signaling.

Bcl-2 and p53, concerned in anti-apoptosis and pro-apoptosis, respectively, are directly and indirectly regulated by NF-{kappa}B (46,47). 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 might be one mechanism for tumor resistance (48). In our study, however, the levels of neither Bcl-2 nor p53 did change upon exposure to TSA and Pro1 (Figure 7A), although there was some change in p53 in response to irradiation (29). In comparison, the activation of caspase-8 and caspase-3 could easily be detected (Figure 7A). Unexpectedly, however, the TSA-induced activation of caspase-8 could not be seen at later times (>48 h), and this correlated with the disappearance of the larger fragment of the two degradation products of pro-caspase-3, suggesting that TSA-induced apoptotic signaling pathway and the functional significance might be different from those by ProI. Furthermore, it could be that the levels of the degradation of pro-caspase-8 and pro-caspase-3 by the compounds were maximum, thus making it difficult to observe the synergistic effect even when the cells were co-treated with both the compounds together. At any rate, all these results strongly suggest the apoptotic process in chemosensitization of 267B1/K-ras cells to TSA as supported by the TUNEL assay (Figure 7B), and the apoptosis could be further exploited with the aid of other compounds, such as curcumin (11) and others, specifically inhibiting particular enzymes concerned in NF-{kappa}B activation.

In our study, TSA seems to have functions both cytoplasmic and nuclear (Figure 8). With the observations of the effects of LY294002 and PD98059 on NF-{kappa}B activation and cell death by TSA (Figure 6D and E), it could be that TSA might induce NF-{kappa}B activation through several pathways. Moreover, given that CBP/p300 can interact with IKK{alpha}, IKK{gamma} and p65 (37,42), the nuclear interference of HDAC activity by TSA could enhance the NF-{kappa}B activity. Hence, all these combined effects could contribute to the blocking of the caspases-mediated apoptosis in response to TSA.


Figure 8
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  		Fig. 8 A proposed signaling pathway of TSA-induced NF-{kappa}B activation and apoptosis. Caspase-mediated apoptosis in response to TSA can be antagonized by NF-{kappa}B activation through several pathways. TSA seems to trigger I{kappa}B{alpha} degradation by either Ki-Ras-dependent or Ki-Ras-independent mechanism. MAPK is also implied in NF-{kappa}B transcriptional activation in response to TSA independently of Ki-Ras. PI3K, downstream of Ki-Ras, is suggested to mediate the TSA-induced NF-{kappa}B transcriptional activity. The combined effects of these signaling pathways, converging on NF-{kappa}B, may contribute to the chemoresistance to TSA.

 
In conclusion, our data suggest that p65/RelA activation by TSA contributes to chemoresistance. Hence, inhibition of NF-{kappa}B can lead to the increased cell death upon exposure of Ki-Ras-overexpressing cells to TSA. Our results provide a potential means for developing a target-oriented anticancer therapeutics, at least in curing of pancreatic, prostate and colon cancers since they overexpress constitutively activated Ki-Ras oncoprotein.


   Acknowledgments
 
This work was supported by Nuclear Research Program from the Ministry of Science and Technology (Grant NAM0080611), Pharmacogenomics Program from the Ministry of Health and Welfare (Grant BGW0200311) and KRIBB Research Initiative Program.

Conflict of Interest Statement: None declared.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
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