5,6-Dichloro-ribifuranosylbenzimidazole- and apigenin-induced sensitization of colon cancer cells to TNF-{alpha}-mediated apoptosis

Myriam Farah, Kuljit Parhar, Maryam Moussavi, Sharlene Eivemark, and Baljinder Salh

The Jack Bell Research Centre, Vancouver, British Columbia, Canada V6H 3Z6

Submitted 6 May 2003 ; accepted in final form 24 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) is a multifunctional cytokine involved in the expression of many genes integral to the inflammatory response. In addition, it activates both apoptotic and survival pathways, the latter being mediated through the activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B). Protein kinase CK2, a serine-threonine kinase that is universally upregulated in human malignancies, may be involved at multiple levels in this process. However, its role in mediating a survival response within colon cancer cells remains incompletely understood. Here we report that inhibition of CK2 in HCT-116 and HT-29 cells with the use of two specific CK2 inhibitors, 5,6-dichloro-ribifuranosylbenzimidazole (DRB) and apigenin, effected a synergistic reduction in cell survival when used in conjunction with TNF-{alpha}. Furthermore, there was a demonstrable synergistic reduction in colony formation in soft agar with the use of the same combinations. Western blot analysis showed that poly-ADP ribose polymerase and procaspase-3 cleavage complemented the fluorescence-activated cell sorter analysis findings of significantly increased subdiploid DNA-containing cell populations using these conditions. Remarkably, these events occurred in the absence of any reduction in the expression of the Bcl-2 family members Bcl-2, Mcl-1, and Bcl-xL or any change in the proapoptotic molecules Bad or Bax. One-hybrid NF-{kappa}B promoter assays utilizing a Gal4-p65 transactivation domain construct revealed that the TNF-induced transactivation was inhibited by both DRB and apigenin. This was associated with a concomitant reduction in the expression of a recognized anti-apoptotic NF-{kappa}B target, manganese superoxide dismutase, demonstrated by Q-PCR. Our findings indicate a potentially novel strategy for the treatment of colon cancer, one that targets CK2 simultaneous with TNF-{alpha} administration.

CK2; tumor necrosis factor-{alpha}; apoptosis; nuclear factor-{kappa}B; manganese superoxide dismutase


TUMOR NECROSIS FACTOR-{alpha} (TNF-{alpha}) is a key component of the immune response, which effects the regulation of a diverse array of proinflammatory molecules (see Refs. 2 and 3 for review) and which in addition has been shown to possess tumoricidal activity (7, 33). TNF-{alpha} sends signals through the two cell surface receptors TNFR1 (p55) and TNFR2 (p75) to activate transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B) and activator protein-1 (AP-1). Whereas most biological activities are elicited via signaling through either receptor, apoptosis occurs predominantly through TNFR1 signaling (4). After binding of TNF-{alpha} to TNFR1, the TNFR1-associated death domain protein, receptor-interacting protein, and Fas-associated death domain protein are recruited, which leads to interaction with other players, such as TNF receptor-associated factors 1 and 2 (TRAF1 and -2). Exposure of the death domain of Fas protein leads to interaction with caspase-8 with the consequent activation of the caspase cascade and cell death. In many cell types, the simultaneous activation of NF-{kappa}B prevents cell death, and its inhibition counteracts this phenomenon (5, 37, 40). The fact that this phenomenon also occurs with chemotherapy underscores its importance as a mechanism involved in cancer cell resistance (9). The mechanism is purported to involve the expression of antiapoptotic molecules, including Bcl-xL and Bfl-1/A1, as well as TRAF and and inhibitors of apoptosis proteins (8, 39, 41, 45).

Protein kinase CK2 is a serine-threonine kinase that is dysregulated in various types of human cancer (1, 16, 2527, 35). Transgenic models have implicated its involvement in the pathogenesis of both lymphoma and breast cancer (19, 20). Further relevance for its role in cancer is indicated by its mediation of resistance to chemotherapy-induced apoptosis (17). More recently, enhanced activities were correlated with activation of NF-{kappa}B in human breast cancer (28). In the context of colon cancer, it has been implicated in Wnt signaling, occurring in complexes with dishevelled and {beta}-catenin (32).

Previous work has revealed that CK2 may be involved in various steps of the NF-{kappa}B activation process. First, it has been shown to phosphorylate I{kappa}B{alpha} not only on Ser32 and Ser36 but also on COOH-terminal proline-glutamic acid-serine-threonine residues (22, 23, 30, 31, 36). Furthermore, it has been implicated in the phosphorylation of the Rel A on Ser529, and this has a direct effect on the amplitude of transactivation (42). However, to our knowledge, the role of this molecule in mediating TNF-mediated cell survival has not been examined.

Several laboratories, including ours, (29, 38) have previously shown that TNF-{alpha} activates CK2. In this report, by using two specific inhibitors of CK2, we present evidence that this enigmatic molecule is involved in the survival response of HCT-116 cells after treatment with TNF-{alpha} through activation of NF-{kappa}B.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. HCT-116 cells were kindly donated by B. Vogelstein and maintained in McCoy's 5A media (Life Technologies, Burlington, ON, Canada) containing 10% FBS (Hyclone, Logan, UT), supplemented with penicillin and streptomycin (Life Technologies). HCT-116 I{kappa}B mutant cells were kindly donated by J. Piette and maintained in 10% McCoy's 5A media supplemented with penicillin, streptomycin, and 250 µg/ml G418 (Life Technologies). HT-29 cells were obtained from ATCC (Rockville, MD) and maintained in medium 199 supplemented with 10% FBS and gentamicin (5 mg/100 ml medium).

3-(4,5-Dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt cell viability assay. HCT-116 (or HT-29) cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were grown for 24 h before being serum-starved in 0.1% FBS-containing medium for 24 h. Cells were then preincubated with the inhibitors apigenin and 5,6-dichloro-ribofuranolsylbenzimadole (DRB; Calbiochem, San Diego, CA) for 2 h before being stimulated with TNF-{alpha} (Calbiochem) at the appropriate concentration for 24 h. A 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt (MTS) solution containing phenazine methosulfate (PMS; Promega, Madison, WI) was added at a final concentration of 250 µg/ml MTS and 20 µM PMS for 2 h, before absorbance at 490 nm was recorded with an ELISA plate reader. Each condition was plated in quintuplicate.

Colony formation assay. Six-well plates were precoated with 2.8 ml of an agarose mix consisting of 0.5% agarose (Bio-Rad, Missisauga, ON, Canada), McCoy's 5A medium, and 20% FBS and left to solidify. Cell suspension (500 µl) containing 150,000 cells was mixed with 1.2 ml of agarose mix and added onto each precoated well. After an overnight incubation at 37°C, each well was covered with 1 ml of 20% McCoy's 5A medium containing apigenin and DRB at the appropriate concentrations for 2 h, before TNF-{alpha} was added at the appropriate concentration. The medium was replenished every 4 days. Fourteen days after being seeded, the cells were stained with 0.1% methylene blue dye (Sigma, Oakville, ON, Canada) containing 50% methanol for 30 min. The cells were destained in distilled water at room temperature and dried overnight at 37°C before being photographed.

Fluorescence-activated cell sorter analysis. Cells were seeded in 6-well plates and grown to confluence. After overnight stimulation in 1% FBS-containing medium, the cells were preincubated with the inhibitors apigenin and DRB for 2 h before being stimulated with TNF-{alpha} for 24 h. Cells were harvested using trypsin-EDTA (Life Technologies), centrifuged, and resuspended in hypotonic lysis buffer, 25 µg/ml RNAse (Qiagen, Toronto, ON, Canada) 0.1% sodium citrate, 50 µg/ml propidium iodide (Sigma), and 0.1% Triton X-100. After 24 h incubation at 4°C, fluorescence was measured with the use of a fluorescence-activated cell sorter (Beckman Coulter Epics XL-MCL). At least 104 cellular events were counted.

Nuclear extracts. Cells were seeded onto 60-mm plates and grown to confluence. Cells were serum starved in 1% FBS-containing medium overnight and then preincubated with the appropriate inhibitor for 2 h before being stimulated with TNF-{alpha} for 30 min. Cells were washed once with ice-cold PBS and scraped into 1 ml of PBS. Cells were centrifuged at 14,000 revolutions/min and resuspended in 200 µl buffer A, which was composed of (in mM) 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.2 EGTA, 1 DTT, and 0.5 PMSF for 15 min, before 13 µl of 10% Nonidet P-40 was added. Cells were then vortexed for 10 s before being centrifuged again at 14,000 revolutions/min for 30 s. The supernatant was removed, and the nuclear pellet was resuspended in 30 µl of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, and 1 mM PMSF) and shaken for 15 min before being centrifuged for 5 min at 14,000 revolutions/min. The supernatant was retained, and the protein concentration was determined by the Bradford (6) assay (Bio-Rad). Samples were stored at -80°C until use.

Electromobility shift assay. A synthetic {kappa}B oligonucleotide was cloned into the cloning vector pBS (Stratagene, La Jolla, CA) with the use of the EcoRI and HindIII sites to create pBS-electrophoretic mobility shift assay (EMSA)-{kappa}b. To radiolabel the probe, the probe was excised from pBS-EMSA{kappa}b with the use of EcoRI and HindIII and labeled with [{gamma}-32P]dCTP (Amersham, Montreal, QC, Canada) and the Klenow fragment of DNA polymerase (New England Biolabs, Missisauga, ON, Canada). The probe was purified by being run on a 5% nondenaturing gel, by cutting out the fragment, and by incubating the gel slice in elution buffer (0.6 M ammonium acetate, 1 mM EDTA, 0.1% SDS) overnight. Nuclear extracts (10 µg) were preincubated in binding buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 10% glycerol, 1 mM DTT) and 1 µg of poly(dI-dC) (Amersham) for 15 min. A probe (20,000 counts/min) was then added, and the reaction mixture was incubated at room temperature for 30 min and then resolved on a 5% nondenaturing polyacrylamide gel in 0.25x Tris-borate EDTA at 200 V for 1.5 h. The gel was subsequently dried for 45 min before phosphorimaging analysis with the use of a Bio-Rad molecular imager FX (or alternatively exposed to film overnight at -80°C and then developed). For supershift or cold competitor reactions, the nuclear extract was preincubated with 1 µg of anti p65 antibody (Calbiochem) or 100-fold excess of a cold probe with binding buffer and poly(dI-dC) for 30 min before the addition of a radiolabeled probe. The sequence of probe was the following: 5'-AATTCGGTTACAAGGGACTTTCCGCTGA-3' and 3'-GCCAATGTTCCCTGAAAGGCGACTTCGA-5'.

Western blot analysis. Cells were washed once with ice-cold PBS, resuspended in homogenization buffer (20 mM MOPS, 50 mM {beta}-glycerophosphate, 5 mM EGTA, 50 NaF, 1 mM DTT, 1 mM sodium vanadate, 1% Nonidet P-40, and 1 mM PMSF) for 30 min, sonicated for 15 s, and centrifuged at 14,000 revolutions/min for 15 min. The protein concentration in the supernatant was determined by the Bradford assay. Protein (50 µg) from each sample was resolved with the use of 10% SDS-PAGE before being transferred to nitrocellulose membranes (Bio-Rad). The blots were blocked in 5% skim milk in 20 mM Tris · HCl, pH 7.4, 250 mM NaCl, 0.05% Tween 20 (TBST) for 1 h before being probed for 2–4 h with the use of the appropriate primary antibody. The blots were washed with TBST for 10 min three times before being incubated with the appropriate secondary antibody for 1.5 h. After three more washes in TBST, the blots were developed with the use of an enhanced chemiluminescence detection system (Amersham). The blots were probed with antibodies to caspase-3, Bad, Bax, and Bcl-2 (Stressgen Biotechnologies, Victoria, BC, Canada), Mcl-1, Bid (Santa Cruz Biotechnology, Santa Cruz, CA), BAK (Upstate Biotechnology, Lake Placid, NY), Bcl-xL (Transduction Laboratories, San Diego, CA), phospho-MAPKs, I{kappa}B{alpha} (New England Biolabs), and poly-ADP ribose polymerase (PARP; Oncogene, San Diego, CA).

Transient transfections. Transient transfections were performed with the use of the Effectene reagent (Qiagen) according to the manufacturer's instructions. Cells were seeded and transfected on reaching 90% confluency. For one 35-mm well, 0.2 µg of DNA was transfected using a DNA-Effectene ratio of 1:25. Transfections were carried out overnight before being replaced with fresh medium for 24 h before the experiments were conducted.

Reporter gene assays. One-hybrid p65/RelA reporter assays were carried out using Gal4-p65TAD (kindly provided by Dr. Bryan Cullen, Duke University) and Gal4-luciferase constructs, which were cotransfected with a LacZ plasmid (kindly donated by William Jia, University of British Columbia) using Effectene (Qiagen). Cells were pretreated for 2 h with the appropriate inhibitor and then stimulated with TNF-{alpha} for 6 h before being harvested. Luciferase and {beta}-galactosidase activities were measured according to the manufacturer's instructions (Promega, Madison, WI). Light emission was measured using a luminometer, and the results were normalized using {beta}-galactosidase.

CK2 kinase activity assay. Ten microliters of lysate (corresponding to 10 µg) were incubated with the specific substrate RRREDEESDDEE (150 µM) and GTP for 10 min at 30°C. Subsequently, 20 µl of the reaction mixture was spotted onto p81 filter paper and washed extensively for 2 h in 0.01% phosphoric acid. After the addition of 250 µl scintillation fluid, the samples were counted in a Wallac scintillation counter. Assays were done in triplicate on at least two separate occasions.

IKK activity assay. IKK complexes were immunoprecipitated with IKK{gamma} antibody (Santa Cruz Biotechnology) from cell lysates (prepared as described under Western blot analysis, above) by adding 5 µl of antibody to 400 µg cell lysate. The tubes were placed on a Labquake shaker overnight at 4°C. Thirty microliters of 1:1 slurry of protein A-Sepharose beads were then added, and the rotator continued for 1 h further at 4°C. After this procedure, the beads were washed twice with homogenization buffer and twice with KII buffer composed of (in mM) 12.5 {beta}-glycerol phosphate, 20 MOPS, pH 7.2, 5 EGTA, 7.5 MgCl2, 50 NaF, and 0.25 DTT. The beads were then resuspended and subjected to an immune complex kinase assay by adding 5 µl of the ATP cocktail (250 mM ATP, 10 µCi [{gamma}-32P]ATP) and by using glutathione-S-transferase-I{kappa}B{alpha} (500 ng; Santa Cruz Biotechnology) as the substrate. After a 20-min reaction, 30 µl of 2x sample buffer were added to the beads, and after boiling for 5 min, the sample was resolved by 11% SDS-PAGE and subjected to autoradiography.

Isolation of RNA and RT-PCR. Cells were pretreated for 2 h with the appropriate inhibitor and stimulated with TNF-{alpha}. RNA was isolated with the use of the TRIzol method (Life Technologies). RNA (1 µg) was reverse transcribed with the use of 0.5 µg oligo(dT)12–18 (Amersham), 1 µl 10 mM dNTPs, 2 µl 0.1 M DTT, 40 units of RNA guard (Amersham) in 1x first-strand buffer (Life Technologies) using 200 units of Moloney murine leukemia virus reverse transcriptase, by incubating the reaction mixture for 50 min at 37°C. cDNA (2 µl) was used in each subsequent PCR reaction. For each 50-µl PCR reaction, 2 units of Taq (PE Biosystems, Branchburg, NJ), 1x PCR buffer (PE Biosystems), 10 pmol of each primer, 1 µl of 10 mM dNTPs, and 3 µl of 25 mM MgCl2 were used. The PCR temperatures used were 94°C denaturing for 45 s, 55°C annealing for 1 min, and 72°C extension for 1 min. Ten-microliter aliquots of the reaction were electrophoresed on a 2% agarose gel containing ethidium bromide. Negative controls for cDNA synthesis were run without template and also without RT. The linearity of PCR reactions was determined between 20 and 40 cycles. Densitometry was performed with the use of Bio-Rad Quantity One software. The sizes of the PCR products for manganese superoxide dismutase (MnSOD) and actin were 306 and 235 bp, respectively. The primers were the following: MnSOD forward 5'-GAGTTGCTGGAAGCCATCAAACGT-3', MnSOD reverse 5'-GTATCTTTCAGTTACATTCTCCCA-3', {beta}-actin forward 5'-CCAACCGCGAGAAGATGACC-3', and {beta}-actin reverse 5'-GATCTTCATGAGGTAGTCAGT-3'.

Q-PCR was performed as described (28a) with the use of an ABI Prism 5700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Briefly, this relies on the SYBR Green I dye binding to the dsDNA directly in the reaction tube. The software detects the threshold cycle number when signals reach 10-fold the standard deviation of the baseline. Each reaction contained 25 µlof2x SYBR Green Master Mix (containing 200 µM deoxyadenosine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate; 400 µM deoxyuridine triphosphate; 2.5 mmol/l MgCl2; and 0.625 units of AmpliTaq Gold DNA polymerase). Reactions were incubated at 50°C for 2 min, followed by 95°C for 15 min. The PCR parameters used were 40 cycles of 15-s denaturation at 95°C, followed by 1-min annealing at 65°C and 1 min at 72°C. All reactions were performed in triplicate. Data analysis used sequence detection system software provided by the manufacturer.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
CK2 inhibition leads to enhanced TNF-{alpha}-mediated reduction in cell viability. HCT-116 colon cancer cells were either treated with the two CK2-specific inhibitors DRB (43) and apigenin alone (32) or with TNF-{alpha} for 24 h. The data indicates (Fig. 1A) that between doses of 10–80 ng/ml of TNF there is no significant difference/reduction in cell viability. This implicates the possible simultaneous activation of a survival pathway. We initially established that DRB at a concentration of 20 µM also had an insignificant effect (<20% reduction) on cell viability. On combined treatment with TNF-{alpha}, however, it is apparent that there is significantly reduced cell viability. This indicates that at this concentration, DRB affects the survival response mediated by TNF-{alpha}. We repeated the experiment with apigenin at 7.5 µM and saw a similar effect (Fig. 1B). Above this concentration, as with DRB, there was a concentration-dependent reduction in cellular viability (data not shown). To substantiate these findings the experiment was repeated in HT-29 cells, where a similar phenomenon was observed (Fig. 1C). The data indicate that the two inhibitors when used in two different colon cancer cell lines exposed to TNF-{alpha} have the capacity to induce a significant reduction in cell viability.



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Fig. 1. Pretreatment of HCT-116 (A and B) or HT-29 (C) colon cancer cells with apigenin or 5,6-dichloro-ribofuranosyl benzimidazole (DRB) causes decreased viability in response to tumor necrosis factor-{alpha} (TNF-{alpha}) stimulation. Cells were plated out at a density of 10,000 cells per well in a 96-well plate for 24 h before being serum-starved in 0.1% FBS-containing media for 24 h. Cells were then preincubated with DRB (A) or apigenin (B) at the indicated concentrations for 2 h before stimulation with TNF-{alpha} at the appropriate concentration for 24 h. Cell viability was determined by the [3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt] (MTS) formazan method, as described in MATERIALS AND METHODS. This assay is based on the cellular bioreduction of MTS by mitochondrial dehydrogenase enzymes in metabolically active cells. The quantity of formazan product formed was measured by the amount of 490-nm absorbance and is directly proportional to the number of viable cells in culture. Readings were taken in quintuplicate, and data are representative of 4 independent experiments. Data are presented as means ± SD; *P < 0.01, **P < 0.001.

 

CK2 inhibition leads to enhanced TNF-{alpha}-mediated subdiploid population of HCT-116 cells. To characterize the cellular changes further, we performed fluorescence-activated cell sorter analysis on cells treated in the same way by using propidium iodide staining to determine the subdiploid (apoptotic) cell fractions. The data indicate that the combined treatment led to a synergistic effect in mediating cell death (Fig. 2, AF). With either inhibitor alone the apoptotic fraction comprised ~5% of the cells. Not surprisingly, with the use of TNF-{alpha} alone a slightly higher frequency (15%) was observed. However, when this is compared with the combined treatments, there is a significantly increased population of apoptotic cells. The magnitude of change was two- and threefold for DRB and apigenin, respectively. To confirm that NF-{kappa}B was involved in this response, it can be seen that cells that were stably transfected with I{kappa}B{alpha}, in which Ser32 and Ser36 were mutated to alanine, exhibited significantly enhanced apoptosis with TNF alone (Fig. 2, G and H).



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Fig. 2. Pretreatment of HCT-116 colon cancer cells with apigenin or DRB enhances hypoploidy in response to TNF-{alpha} stimulation (AH). Cells were plated in 6-well plates and grown to confluency. After overnight starvation in 1% FBS-containing media, cells were preincubated with DRB or apigenin at the indicated concentrations for 2 h before being stimulated with TNF-{alpha} at the indicated concentration for 24 h. Total cells were harvested and stained with propidium iodide as described in MATERIALS AND METHODS. At least 104 cellular events were counted to determine the subdiploid cell population. Data shown are representative of 3 independent experiments. A: control; B: apigenin; C: DRB; D: TNF; E: TNF + apigenin; F: TNF + DRB; G: I{kappa}B mut; H: I{kappa}B mut + TNF. Pretreatment of HCT-116 colon cancer cells with apigenin or DRB induces apoptosis through activation of caspase-3 (I) and poly-ADP ribose polymerase (PARP) cleavage (J) in response to TNF-{alpha} stimulation. Western immunoblotting was carried out as described in MATERIALS AND METHODS, and data are representative of the experiment performed on 3 separate occasions.

 

To characterize the mode of cell death, we examined two well-described targets of apoptosis, PARP and procaspase-3, with Western immunoblotting. Not suprisingly, we found that PARP was cleaved to its 84-kDa form when cells were treated with the TNF and CK2 inhibitors or when the I{kappa}B{alpha}-mutant HCT-116 (S32A, S36A) cells were treated with TNF alone. These changes corresponded to reductions in the levels of procaspase-3 (Fig. 2, I and J). The data for TNF-{alpha} alone reveal a partial response in both the reduction in procaspase-3 as well as an increase in the subdiploid cell population. Taken together, these complementary assays indicated that CK2 was involved in the survival response consequent on TNF-{alpha} treatment of HCT-116 cells.

CK2 inhibitors abolish anchorage-independent growth. To our knowledge, the involvement of CK2 in anchorage-independent growth has not been addressed. Therefore, using the same conditions, we wondered whether either agent alone or in combination with TNF-{alpha} would affect this property. As shown in Fig. 3, none of the agents used in isolation significantly affected colony formation in soft agar. However, in conjunction with our MTS assay results, we observed a dramatic reduction in this parameter when the agents were used together. The quantitative differences between the findings from this experiment and the MTS assay likely reflect the duration of this assay (14 days) versus the MTS assay (24 h).



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Fig. 3. Pretreatment of HCT-116 colon cancer cells with apigenin or DRB inhibits growth on soft agar in response to TNF-{alpha} stimulation. The cells (150,000) were seeded in soft agar as described in MATERIALS AND METHODS. Cells were preincubated in media containing apigenin or DRB for 2 h and then stimulated with TNF-{alpha} as described. Colony formation was observed over 14 days, during which medium was replenished every 3 to 4 days, before colonies were stained with methylene blue dye and photographed (A). After colonies were counted in five different fields of view, the mean number of colonies was calculated and expressed as a percentage of control (B). Results are indicative of 3 independent experiments.

 

Inhibition of NF-{kappa}B transactivation. The next objective was to determine whether or not NF-{kappa}B was inhibited in this system. EMSA assays were performed to see whether an effect on DNA binding could be established. However, this was not the case in HCT-116 cells (Fig. 4A) at the inhibitor concentrations used in this study. With the use of a one-hybrid Gal4-p65TAD construct cotransfected with a Gal4-luciferase construct, we were able to show an activation of the NF-{kappa}B promoter with TNF-{alpha}. This activation could be blunted by preincubation of the cells with both apigenin and DRB (Fig. 4B). With the use of a 4x {kappa}-luc reporter a fourfold activation of promoter activity was found with TNF-{alpha}, and this was completely attenuated in the presence of the two inhibitors (data not shown).



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Fig. 4. A: TNF-{alpha}-induced nuclear factor-{kappa}B (NF-{kappa}B) transactivation is reduced by apigenin and DRB. Electrophoretic mobility shift assays (EMSA) were performed on nuclear extracts obtained from HCT-116 cells as described in MATERIALS AND METHODS. B: TNF-{alpha}-induced transactivation of NF-{kappa}B is inhibited by CK2 inhibitors. HCT-116 cells were transfected with a one-hybrid construct composed of a Gal4-RelATAD + Gal4-luciferase reporter and grown to confluence. C: cells were then prepretreated with DRB or apigenin (APIG) at the indicated concentration for 2 h and then stimulated with TNF-{alpha} at the indicated concentration for 6 h. Cells were harvested, and luciferase activity was determined as described in MATERIALS AND METHODS. Results were normalized for transfection efficiency by using LacZ activity. Results are representative of at least 3 independent experiments. *P < 0.05; **P < 0.001. RLU, relative light units; cpm, counts per million.

 

We confirmed that CK2 was enzymatically activated on TNF-{alpha} stimulation in HCT-116 cells by using a specific substrate and GTP as the phospho donor. There was a reliable inhibition of its activation, which coincided very well with the promoter activities observed above, using either DRB or apigenin at the concentrations used in the functional studies (Fig. 4C).

Effect of CK2 inhibitors on TNF-{alpha}-induced IKK and MAPK responses. Because previous work indicates activation of the MAPKs by TNF-{alpha} (4), as well as a role for JNK downregulation by NF-{kappa}B (11, 34), we were interested in pursuing their activation under the conditions of this experiment. We also wanted to evaluate IKK activity and I{kappa}B{alpha} degradation at the same time. In confirmation of our findings in Fig. 4, we observed no effects of apigenin or DRB on TNF-{alpha}-induced I{kappa}B{alpha} degradation or on IKK activity (Fig. 5, A and B). When these findings are compared with the changes in MAPK activation, there is surprisingly an absence of p42/44 MAPK activation under any condition (Fig. 5D); however, the responses of the stress-activated kinases are decidedly different. Whereas the p38 MAPK exhibits a mild activation with the apigenin alone, and at least an additive one with the TNF-{alpha}, this is not the case for DRB (Fig. 5C). For the JNKs, there is no response to either inhibitor alone and no obvious additive response with the addition of TNF-{alpha} (Fig. 5E). These findings indicate that apigenin and DRB do not target the p42/44 MAPKs directly in a manner correlative with the subsequent induction of cell death.



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Fig. 5. Pretreatment with CK2 inhibitors does not impact on TNF-{alpha}-induced IKK activity or p42/44 MAPK activity. IKK activity was determined as described in MATERIALS AND METHODS, and Western blot analysis with the use of the indicated antibodies was performed on whole cell lysates resolved on 10% SDS-PAGE. The data represented were reproduced on 2 other occasions. GST, gluthathione S-transferase.

 

Evaluation of Bcl-2 family members. Several NF-{kappa}B-induced genes are involved in the anti-apoptotic response to chemotherapeutics, and the Bcl-2 family members are an important group among these (14, 18). Furthermore, an alteration in the ratio of Bax to Bcl-xL has been proposed to play an important role in nonsteroidal anti-inflammatory drug-induced cell death (44). We therefore examined the relative amounts of these and were unable to detect changes in Bcl-2, Bcl-xL, Mcl-1, Bad, or Bax (Fig. 6, A and B). This data appeared to indicate that the absolute amounts of these molecules did not significantly change during the apoptotic response.



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Fig. 6. A: cells grown to confluence were serum starved overnight before being preincubated with apigenin or DRB at the indicated concentration for 2 h and then stimulated with TNF-{alpha} at the indicated concentration for 24 h. After total cells were harvested, proteins were extracted and resolved using 10% SDS-PAGE as described in MATERIALS AND METHODS and probed with the antibodies indicated. B: relative amounts of protein were measured by scanning the film with the Bio-Rad gel-doc apparatus into a TIFF-format file, and data are presented as means ± SD. Open bars, Bax; gray bars, Bad; solid bars, Bcl-2; hatched bars, Bcl-xL.

 

Effects of apigenin and DRB on TNF-{alpha}-induced MnSOD expression. To investigate a potential alternate mechanism whereby apigenin and DRB mediate their effects, we examined the expression of MnSOD, which has been shown to offer partial protection to TNF-{alpha}-induced apoptosis (10). The data indicate that there is an ~2.5-fold induction of MnSOD with TNF-{alpha} treatment. This is reliably attenuated to below control values using either apigenin or DRB (Fig. 7). We subsequently performed Q-PCR to validate this observation, and this revealed the threshold cycle number values of 27.7 ± 0.4, 25.2 ± 0.1, 27.8 ± 0.03, and 26.5 ± 0.1 for control, TNF-{alpha}-stimulated, and samples pretreated with apigenin and DRB before TNF-{alpha} stimulation, respectively. (These figures show the means ± SD and were repeated on two separate occasions with similar findings).



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Fig. 7. Treatment of HCT-116 colon cancer cells with apigenin or DRB attenuates manganese superoxide dismutase (MnSOD) expression in response to TNF-{alpha} stimulation. Cells were stimulated with TNF-{alpha} for 3 h, and total RNA was extracted with the use of TRIzol. After cDNA synthesis, semiquantitative RT-PCR was carried out using primers indicated in MATERIALS AND METHODS. The data were normalized for the expression of actin (densitometry was performed using Bio-Rad gel-doc), and the findings depicted are representative of 3 independent experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The mechanism of TNF-{alpha}-mediated cell survival has attracted considerable attention over the past few years, and significant progress has been made in delineating the mechanisms involved. The simultaneous activation of both apoptotic and antiapoptotic pathways implies that, depending on the balance of signaling within a given cell system, either effect may be accomplished. This has especial relevance for cancer biology, where several reports now indicate that using proteosomal inhibitors or the stable expression of I{kappa}B{alpha} to inhibit activation of NF-{kappa}B can have a markedly enhanced cell killing effect with antitumor modalities (14, 18). In this report we propose that CK2 is involved in the TNF-{alpha}-activated survival response, as indicated by the finding that treatment with two discrete CK2 inhibitors led to a synergistic reduction in the viability of the colon cancer cell lines HCT-116 and HT-29. It should be noted that the concentrations of inhibitors used were those at which there is a more specific effect on CK2. We also showed that the inhibitors used have an inhibitory effect on NF-{kappa}B transactivation, thus excluding a more proximal site of action.

CK2 has been previously shown to be dysregulated in several malignancies, with some reports providing a correlation with tumor aggressivity (13, 24). However, a direct effect on the development of cancer has awaited the transgenic work linking overexpression of CK2 to the development of lymphoma and breast cancer (19, 20). However, its role in this process remains unclear. Our observation that transactivation of NF-{kappa}B was inhibited by both inhibitors indicates that it may provide a survival advantage to tumor cells by the facilitation of expression of anti-apoptotic molecules. However, our findings indicate that this is unlikely to involve Bcl-2, Bcl-xL, or Mcl-1.

Recent work (28) has examined the role of CK2 in the activation of NF-{kappa}B in breast cancer (28). With the use of apigenin and emodin as selective CK2 inhibitors, reduced NF-{kappa}B/Rel activity was observed in breast cancer cell lines exposed to these agents. Furthermore, it was reported that there existed a positive correlation between CK2 and IKK activities in those primary human cancers that displayed aberrant constitutive expression of NF-{kappa}B/Rel. In contrast to our findings, a direct effect on DNA binding (at doses higher than those used in our work) was demonstrated. We were not able to reproduce these data in HCT-116 cells. Thus it would appear that the response may be specific for tumors of different epithelial origin.

Interestingly, in a separate report (17) it was shown that overexpression of protein kinase CK2 could protect against drug-induced apoptosis in prostate cancer cells. The effect appeared to occur in association with a predominant nuclear matrix localization of the molecule. Taken together with the other findings, it would seem plausible that enhanced CK2 activity would be associated with aberrant NF-{kappa}B activation and a relative resistance to chemotherapy. Although our work has not specifically addressed either of these issues, it is the first to demonstrate that use of selective CK2 inhibitors may dramatically sensitize colon cancer cells to TNF-{alpha}-induced apoptosis. The findings from the colony formation assay were entirely supportive and perhaps even more compelling in that there was a dramatic attenuation of the growth of cells exposed to CK2 inhibitors and subsequently incubated with TNF-{alpha}.

The underlying mechanism for the phenomenon presented in this report is likely to be due at least in part to the expression of MnSOD but may also involve an alternate Bcl-2 family member. We have examined the expression of Bid, which has been reported to undergo phosphorylation by CK1 and CK2, thus rendering it more resistant to cleavage by caspase-8 (12), but this was not altered (data not shown). Another molecule that may be affected is ARC, a caspase-inhibiting protein, which is phosphorylated by CK2 at Thr149. This modification targets the protein to mitochondria, where it can bind and thus inhibit caspase-8 (21). An additional mechanism for the selective inhibitors DRB and apigenin could be that they directly target the interaction between CK2 and the TATA-binding protein component of the transcription apparatus (15).

In summary, by using two well-recognized pharmacological inhibitors of CK2, DRB and apigenin, we have shown a significant biological role for this serine-threonine kinase in protection from apoptosis when two colonic cancer cell lines were treated with TNF-{alpha}. That this effect coincided with a reduction in NF-{kappa}B transactivation but without an effect on DNA binding of the RelA subunit was also important. We also demonstrate for the first time that MnSOD can be regulated by both of the CK2 inhibitors used and thus provide a potential mechanism, which may in part explain our findings. Further work will help to clarify whether or not other Bcl-2 family members are involved in this response also. We conclude that the use of these inhibitors of CK2 may have therapeutic value when used with biological agents such as with TNF-{alpha} in colon cancer.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported in part by grants from the Crohn's and Colitis Foundation of Canada and from the Cancer Research Society. M. Farah was supported by a British Columbia Cancer Agency studentship, and K. Parhar was supported by a Michael Smith Graduate Studentship Award.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Salh, Division of Gastroenterology, Univ. of British Columbia, 100-2647 Willow St., Vancouver BC V5Z 3P1, Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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