Constitutive nuclear factor-κB (NF-κB) activity plays a crucial role in the development and progression of lymphoma, leukemia, and some epithelial cancers. Given the contribution of NF-κB in carcinogenesis, a novel approach that interferes with its activity might have therapeutic potential against cancers that respond poorly to conventional treatments. Here, we have shown that a new IκB kinase β inhibitor, IMD-0354, suppressed the growth of human breast cancer cells, MDA-MB-231, HMC1-8, and MCF-7, by arresting cell cycle and inducing apoptosis. In an electrophoretic mobility shift assay and a reporter assay, IMD-0354 abolished the NF-κB activity in MDA-MB-231 cells in a dose-dependent manner. In the cells incubated with IMD-0354, cell cycle arrested at the G0-G1 phase and apoptotic cells were increased. The expression of some cell cycle regulatory molecules and antiapoptotic molecules was suppressed in cells treated with IMD-0354. On the other hand, cyclin-dependent kinase suppressor p27Kip1 was up-regulated by the addition of IMD-0354. Daily administration of IMD-0354 inhibited tumor expansion in immunodeficient mice into which MDA-MB-231 cells were transplanted. These results indicate that NF-κB may contribute to cell proliferation through up-regulation of cell cycle progression; accordingly, inhibition of NF-κB activity might have a therapeutic ability in the treatment of human breast cancers. (Cancer Res 2006; 66(1): 419-26)

Investigation of molecular mechanisms essential for tumor cell proliferation may provide information concerning novel molecular targets of anticancer treatment for tumors that respond poorly to standard chemotherapy and radiation therapy. Nuclear factor-κB (NF-κB) is comprised of inducible transcription factors of the Rel family, which usually resides in the cytosol in an inactive form, binding to its endogenous inhibitor, IκB family proteins (1, 2). IκB kinase (IKK) α and β phosphorylate serine residues in the NH2-terminus of IκB, resulting in NF-κB release and translocation to the nucleus (1, 2). The liberated NF-κB binds to specific DNA sequences of target genes and contributes to cell proliferation and functions (14). NF-κB activation induces the expression of D-type cyclins and promotes the transition of the cell cycle from the G0-G1 phase to the S phase (46). NF-κB also closely relates to the expression of antiapoptotic molecules including Bcl family proteins that contribute to the preservation of mitochondrial integrity resulting in the protection from apoptosis (7, 8). In the previous study, abnormal activation of NF-κB in tumor cells was reported (911), indicating that this transcription factor may play crucial roles in neoplastic cell growth. Constitutive activation of NF-κB has emerged as a hallmark for human leukemia, lymphoma, and solid tumors (911). Thus, NF-κB may become one of the important molecular targets of a new therapy against malignant tumors.

Cell proliferation requires a chain of processes essential for cell cycle progression. Various cellular molecules and transcription factors coordinately regulate cell cycle according to the cellular status. The retinoblastoma tumor suppressor protein (pRb) family including pRb, p107, and p130, associates with each member of the E2F family (12, 13). Complex formation between E2F and pRb families arrests cell cycle in the G1 phase and prevents neoplastic cell proliferation (12, 13). Cyclin-dependent kinases (CDK) binding with cyclins have the ability to phosphorylate pRb and free E2F (14, 15). However, the Cip/Kip family of CDK inhibitors including p21Cip1/WAF1 and p27Kip1 negatively regulates the activity of the CDK-cyclin complex (16, 17). Activation of the p53 pathway induces apoptosis at least in part through the up-regulation of p21Cip1/WAF1 and p27Kip1 (1823). The existence of p53 induces the expression of ubiquitin ligase murine double minute 2 (MDM2) that controls overactivation of p53 (2430). MDM2 facilitates cell cycle progression in a p53-dependent and p53-independent fashion (2430). Transformation leading to disruption of the normal networks of cell cycle regulation lies at the bottom of tumorigenesis.

Some breast cancers remain incurable despite surgical removal and conventional chemotherapies. NF-κB activation is essential during distinct steps of breast cancer progression and transforming growth factor-β (TGF-β) may play a critical role in the process (3134). Self-production of TGF-β has been shown in some breast cancer cells, which may associate with the invasive phenotype (35, 36). In some human breast cancer lines, tumor cells become resistant against TGF-β-induced apoptosis (37, 38). TGF-β functions as a cytokine that induces cell motility and invasiveness by autocrine and paracrine mechanisms (39, 40). MDM2 plays a p53-independent role in the acquisition of resistance to TGF-β-induced apoptosis (41). However, the involvement of NF-κB in the process has not been fully understood.

Given the considerable contribution of NF-κB to the proliferation of many breast cancers (2, 7, 9), inhibition of NF-κB may have a therapeutic potential for the control of tumor progression. We have already reported that a novel IKKβ inhibitor IMD-0354 suppresses the proliferation of neoplastic mast cells by inhibiting cell cycle progression (42). In the current study, we have clearly shown that IMD-0354 prevented the proliferation of estrogen receptor–positive (MCF-7) and –negative (MDA-MB-231 and HMC1-8) human breast cancers. Cell cycle progression was inhibited in a dose- and time-dependent manner when those cells were incubated with IMD-0354. The effect of IMD-0354 was manifested through down-regulation of D-type cyclins. IMD-0354 also diminished phosphorylation of pRb family proteins and expression of antiapoptotic Bcl family proteins and MDM2. On the other hand, IMD-0354 up-regulated the expression of p27Kip1 and the mRNA levels of TGF-β in MDA-MB-231 cells. Furthermore, administration of IMD-0354 abrogated tumor expansion in immunodeficient mice that were transplanted with MDA-MB-231 cells. These results indicate that NF-κB is essential for breast cancer progression; thus, the reagent that interferes with NF-κB signaling may be useful for antitumor therapies against breast cancers.

Cell culture. Human breast cancer cell line MDA-MB-231 was purchased from the American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% FCS and antibiotics. MCF-7 cells were provided from Japan Health Science Foundation (Osaka, Japan) and cultured in DMEM supplemented with nonessential amino acids, 1 mmol/L sodium pyruvate, 10 μg/mL insulin, 10% FCS, and antibiotics. HMC1-8 cells were also obtained from Japan Health Science Foundation and cultured in RPMI 1640 supplemented with 10% FCS and antibiotics.

Reagents. Rabbit anti-phospho-IκBα antibody, rabbit anti-IκBα antibody, and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG antibody were obtained from Cell Signaling Technology (Beverly, MA). Anti-pRb monoclonal antibody (clone G3-245) and anti-cyclin D1/D2 antibody were purchased from PharMingen (San Diego, CA) and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. Anti-cyclin D3 antibody, anti-cyclin E antibody, anti-p21Cip1/WAF1 antibody, anti-p27Kip1 antibody, anti-p57Kip2, anti-Bcl-2 antibody, anti-Bcl-xL antibody, anti-p107 antibody, and anti-p130 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-p53 antibody and anti-MDM2 antibody were provided from Sigma Chemicals (St. Louis, MO). IMD-0354, a novel inhibitor of IKKβ, was molecularly designed, synthesized, and provided from the Institute of Medicinal Molecular Design, Inc. (Tokyo, Japan; ref. 42). Unless otherwise indicated, all chemicals used in this report were obtained from Sigma Chemicals.

Electrophoretic mobility shift assay. After incubation with the indicated concentrations of IMD-0354 or each signal inhibitor for 24 hours, nuclear extractions were prepared from 107 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's instructions. A biotin-labeled double-stranded DNA probe containing the consensus DNA binding sequence for NF-κB was synthesized by incubating sense and antisense oligonucleotides (sense, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; antisense, 5′-GCCTGGGAAAGTCCCCTCAACT-3′, Promega Corp., Madison, WI) in Tris-EDTA buffer for 2 minutes at 85°C, for 15 minutes at 65°C, for 15 minutes at 37°C, for 15 minutes at room temperature, and for 15 minutes on ice. With a LightShift chemiluminescent electrophoretic mobility shift assay kit (Pierce), 0.02 pmol of the biotin-labeled DNA probe was incubated with 5 μg of the nuclear extraction for 20 minutes at room temperature. The conjugate was mixed with 5× loading buffer and 20 μL of the mixture containing 4 μg nuclear protein was applied onto each lane of 6% DNA-PAGE mini gel (Tefco, Tokyo, Japan). Electrophoresis was done in Tris–boric acid–EDTA buffer, and the separated proteins were transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ). After UV cross-linking, the membrane was blocked, incubated with LightShift stabilized streptavidin-horseradish peroxidase conjugate (Pierce) for 60 minutes. Positive reactions were visualized by incubating the membrane in LightShift luminol/enhancer solution (Pierce). All procedures were done according to the manufacturer's instructions except where indicated. For competition assays, unlabeled NF-κB consensus oligonucleotides and mutant oligonucleotides with a single base substitution (Santa Cruz) were used. For supershift assays, 4 μg of anti-p65, anti-cRel, or anti-p50 subunit antibodies (Santa Cruz) in each reaction was added.

Luciferase assay. Using an Effectene transfection reagent kit (Qiagen, Hilden, Germany), 200 ng of pNF-κB-TA-Luc plasmid (BD Bioscience Clontech, Palo Alto, CA) was introduced into MDA-MB-231 cells according to the manufacturer's instructions. We used β-galactosidase expression vector (pCMVβ) to control transfection efficiency. Twenty-four hours later, cells were washed and treated with various concentrations of IMD-0354 in DMEM containing 10% FCS and further incubated for 24 hours. Luciferase activity in supernatants of cell lysates was measured with a Bright-Glo luciferase assay system (Promega Corp.) as a substrate. Results were normalized to the luciferase activity of mock TA-Luc plasmid.

Western blot analysis. After washing in PBS, 2 × 106 cells were lysed in 100 μL of a CelLytic-M reagent supplemented with a protease inhibitor cocktail (Sigma Chemicals). Supernatants were collected by centrifugation, mixed with the same volume of 2× sample buffer [20% glycerol, 10% 2-ME, 4% SDS, 100 mmol/L Tris-HCl (pH 6.8)], and boiled for 5 minutes. Each sample was applied to SDS-PAGE with 12.5% gels (Bio-Rad Laboratories, Hercules, CA). Separated proteins were transferred onto Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocked in 5% nonfat dry milk and blotted with primary antibodies diluted in a blocking solution. After washing, the membrane was incubated with HRP-conjugated second antibodies as indicated in the manufacturer's instructions. Positive reactions were visualized with an enhanced chemiluminescence plus Western blotting detection system (Amersham Bioscience, Buckinghamshire, England). As positive controls, we used 3T3 cell lysate (Upstate Biotechnology), MOLT-4 cell lysate (PharMingen), or lysate from HeLa cells stimulated with or without tumor necrosis factor-α as indicated in each figure. Images were analyzed by Basic Quantifier (Genomic Solutions, Tokyo, Japan).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells (2 × 105 cells/mL) were incubated in phenol red–free DMEM containing 10% FCS and antibiotics with or without various concentrations of IMD-0354. One hundred microliters of the cell suspension was applied to each well of 96-well culture plates and incubated for 24, 48, and 72 hours. Four hours prior to the end of the culture period, 10 μL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dissolved in PBS was added to each well. The reaction was stopped by adding 100 μL of 10% SDS in 0.01 N HCl. The absorbance was measured at 577 nm with ImmunoMini NJ-2300 (Nalge Nunc International K.K., Tokyo, Japan).

Cell cycle analysis. For a cell cycle analysis, MDA-MB-231 cells (2 × 105 cells/mL) were incubated in the presence or absence of various doses of each IMD-0354. After 24 hours, cells were washed in ice-cold PBS, resuspended in 100 μL of PBS, and fixed in 70% ethanol for 30 minutes on ice. Cells were recovered by centrifugation and treated with 0.5 mg/mL RNase A for 20 minutes at 37°C to avoid nonspecific propidium iodide (PI) binding. DNA was stained by incubating cells in 50 μg/mL PI dissolved in 0.1% sodium citrate for 10 minutes on ice. To remove clots, cell suspensions were passed through a nylon mesh. DNA contents were analyzed by an EPICS XL flow cytometer (Coulter, Hialeah, FL) with MacCycle AV software (Phoenix, San Diego, CA). We analyzed DNA histograms in the G0-G1, S, and G2-M phases in cells after incubation with IMD-0354 and calculated the ratio of cells in each phase (42).

Annexin V analysis. The apoptotic status was analyzed by using a TACS annexin V-FITC kit (R&D Systems, Minneapolis, MN). Briefly, cells (1 × 106 cells/mL) were incubated for 48 hours in the presence or absence of indicated doses of IMD-0354. After washing in ice-cold PBS, cells were collected by EDTA treatment and incubated with a mixture of annexin V-FITC and PI for 15 minutes at room temperature according to the manufacturer's instruction. Early apoptotic (only annexin V-positive) cells were distinguished from late apoptotic or necrotic (annexin V and PI double-positive) cells by a flow cytometric analysis.

Real-time PCR. Total RNA was recovered from MDA-MB-231 cells using Isogen (Nippongene, Tokyo, Japan) according to the manufacturer's instruction. Two to five micrograms of total RNA obtained from each sample was reverse-transcribed into cDNA with Oligo(dT)12-18 primers using a Superscript II (Invitrogen Corp., Carlsbad, CA). Reaction mixtures were amplified with a SYBR Green PCR Master Mix (PE Applied Biosystems, Tokyo, Japan) in the presence of 0.2 μmol/L each of the sense and antisense primers for TGF-β (5′-CCTGAGCAGGATGGAGAATTACA-3′ and 5′-TCCAGAACATGCCGCAGAG-3′) or with primers for glyceraldehyde-3-phosphate dehydrogenase (5′-TGGTCGTACCACAGGCATTG-3′ and 5′-TGATGTCACGCACGATTTCC-3′) by the following thermal cycling programs: stage 1, 50°C for 2 minutes; stage 2, 95°C for 10 minutes; stage 3, 35 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fluorescence intensity was measured in real-time during extension steps for a SYBR Green assay by using the ABI Prism 7000 Sequence Detector (PE Applied Biosystems). Relative expression levels of the target gene was normalized using glyceraldehyde-3-phosphate dehydrogenase as an endogenous reference using the comparative CT (threshold cycle) method. The amount of gene expression that was normalized to endogenous reference is given by: 2−ΔΔCT.

Tumor transplantation and in vivo experiments. MDA-MB-231 cells were suspended in PBS (5 × 106 cells/100 μL mouse) and s.c. injected to the back of female BALB/c nude mice (purchased from Charles River Japan, Inc., Yokohama, Japan) at the age of 4 to 5 weeks. After growth, the tumor was removed surgically and 100 mg of each established tumor was transplanted to the back of other female nude mice at the age of 4 weeks under ether anesthesia. IMD-0354 was suspended in saline and 5 mg/kg body weight IMD-0354 (suspended in 100 μL/mouse) was given to each mouse by i.p. injection once a day for 28 days after the implantation. Saline was injected in nude mice as a control. Estimated tumor volume (mm3) and tumor weight (mg) were given as the following formulas: tumor volume = [(width)2 × length] / 2 (43), tumor weight = (longest diameter (mm) × [shortest diameter (mm)]2) / 2 (44). All mice were maintained in an air-filtered specific pathogen–free condition and tumor sizes were measured weekly. All experiments with animals complied with the standards in the guidelines of the University Animal Care and Use Committee of the Tokyo University of Agriculture and Technology.

Statistical analysis. Two-tailed Student's t test was done for statistical analysis of the data, and P < 0.01 was taken as the level of significance.

Constitutive activation of NF-κB in breast cancer cells inhibited by IMD-0354. Inhibitory effect of IMD-0354 on NF-κB activity was examined by a reporter assay using MDA-MB-231 cells transfected with pNF-κB-TA-Luc plasmid as previously described (42). Luciferase activity, which represents the transcriptional activation of NF-κB, was reduced dose-dependently by treatment with IMD-0354 for 24 hours (Fig. 1A). When MDA-MB-231 cells were incubated with IMD-0354 for 24 hours, translocation of NF-κB to the nucleus was completely abolished (Fig. 1B). In a Western blot analysis, we showed that IMD-0354 suppressed phosphorylation of IκB after 4 hours in a dose-dependent manner (Fig. 1C and D). Because the Akt signaling pathway plays a major role in the activation of NF-κB and in the subsequent expression of antiapoptotic molecules (6, 7), we examined the effect of IMD-0354 on the phosphorylation of Akt. As shown in Fig. 1C and D, incubation of cells with various concentrations of IMD-0354 for 24 hours completely failed to alter the phosphorylation level of Akt.

Figure 1.

NF-κB activity suppressed by IMD-0354 without affecting Akt phosphorylation (A-D). The inhibitory effect of IMD-0354 on NF-κB activity was examined by a reporter assay using MDA-MB-231 cells transfected with pNF-κB-TA-Luc plasmid (A). Luciferase activity was reduced in a dose-dependent manner by treatment with IMD-0354 for 24 hours. Mock TA-Luc plasmid was used for controls and the results obtained were normalized to control values. Points, mean values at each time point of three different experiments with duplication; bars, ± SE; *, P < 0.01 as compared with medium alone. NF-κB binding activity in nuclear extracts obtained from MDA-MB-231 cells was detected by an electrophoretic mobility shift assay (B). Treatment with IMD-0354 for 24 hours decreased the translocation of NF-κB to the nucleus in a dose-dependent manner. Lanes 1 and 2, nuclear extract and probe, respectively. IκB phosphorylation was also suppressed 4 hours after IMD-0354 addition, whereas Akt phosphorylation level was not affected (representative of three different experiments, C). Relative intensities of phospho-IκB (pIκB) and phospho-Akt (pAkt) were normalized to total protein expression of IκB and Akt, respectively (D). Each value was standardized to a value without IMD-0354 as 1.

Figure 1.

NF-κB activity suppressed by IMD-0354 without affecting Akt phosphorylation (A-D). The inhibitory effect of IMD-0354 on NF-κB activity was examined by a reporter assay using MDA-MB-231 cells transfected with pNF-κB-TA-Luc plasmid (A). Luciferase activity was reduced in a dose-dependent manner by treatment with IMD-0354 for 24 hours. Mock TA-Luc plasmid was used for controls and the results obtained were normalized to control values. Points, mean values at each time point of three different experiments with duplication; bars, ± SE; *, P < 0.01 as compared with medium alone. NF-κB binding activity in nuclear extracts obtained from MDA-MB-231 cells was detected by an electrophoretic mobility shift assay (B). Treatment with IMD-0354 for 24 hours decreased the translocation of NF-κB to the nucleus in a dose-dependent manner. Lanes 1 and 2, nuclear extract and probe, respectively. IκB phosphorylation was also suppressed 4 hours after IMD-0354 addition, whereas Akt phosphorylation level was not affected (representative of three different experiments, C). Relative intensities of phospho-IκB (pIκB) and phospho-Akt (pAkt) were normalized to total protein expression of IκB and Akt, respectively (D). Each value was standardized to a value without IMD-0354 as 1.

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Proliferation of breast cancer cells inhibited by IMD-0354. Next, we evaluated the effect of IMD-0354 on the proliferation of estrogen receptor–positive MCF-7 cells and estrogen receptor–negative MDA-MB-231 cells and HMC1-8 cells. Those cells were incubated with increasing concentrations of IMD-0354 for 24, 48, and 72 hours, and their viability was determined by a MTT assay. As shown in Fig. 2A, IMD-0354 suppressed the proliferation of those three cell lines in a dose-dependent manner after 48 hours. IC50 of IMD-0354 on inhibition of cell proliferation of those three cell lines were calculated by a trypan blue dye exclusion test as follows: 0.8 μmol/L for MDA-MB-231 cells, 1.6 μmol/L for HMC1-8 cells, and 22.4 μmol/L for MCF-7 cells.

Figure 2.

Inhibitory effect of IMD-0354 on the proliferation of breast cancer cells. MDA-MB-231 cells, HMC1-8 cells, or MCF-7 cells were incubated in the presence of increasing concentrations of IMD-0354 for 48 hours, and the viability of cells were determined by a trypan blue dye exclusion test and MTT assay (A). Columns, means of five to six different experiments; bars, ± SE. *, P < 0.05 compared with medium alone. MDA-MB-231 cells were incubated with IMD-0354 for 24 hours, and the cell cycle was analyzed by PI uptake (B). The percentage of cells in each phase was measured; cells in hypodiploid range were 5.5 ± 0.3% and 29.4 ± 2.1% in each condition (C). MDA-MB-231 cells, HMC1-8 cells, or MCF-7 cells were incubated with IMD-0354 (0 or 10 μmol/L) and an annexin V-binding assay was done (D). Percentages of negative (viable) cells, annexin V-FITC–positive (early apoptotic) cells, PI-positive (necrotic) cells, or annexin V-FITC and PI–positive (late apoptotic) cells were shown (mean of three different experiments).

Figure 2.

Inhibitory effect of IMD-0354 on the proliferation of breast cancer cells. MDA-MB-231 cells, HMC1-8 cells, or MCF-7 cells were incubated in the presence of increasing concentrations of IMD-0354 for 48 hours, and the viability of cells were determined by a trypan blue dye exclusion test and MTT assay (A). Columns, means of five to six different experiments; bars, ± SE. *, P < 0.05 compared with medium alone. MDA-MB-231 cells were incubated with IMD-0354 for 24 hours, and the cell cycle was analyzed by PI uptake (B). The percentage of cells in each phase was measured; cells in hypodiploid range were 5.5 ± 0.3% and 29.4 ± 2.1% in each condition (C). MDA-MB-231 cells, HMC1-8 cells, or MCF-7 cells were incubated with IMD-0354 (0 or 10 μmol/L) and an annexin V-binding assay was done (D). Percentages of negative (viable) cells, annexin V-FITC–positive (early apoptotic) cells, PI-positive (necrotic) cells, or annexin V-FITC and PI–positive (late apoptotic) cells were shown (mean of three different experiments).

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Effect of IMD-0354 on the cell cycle of MDA-MB-231 cells. Because NF-κB was constitutively activated in most human breast cancer cells, we attempted to determine the effect of IMD-0354 on cell cycle regulation. MDA-MB-231 cells were incubated with IMD-0354 for 24 hours, and the cell cycle analysis was done by PI uptake. As shown in Fig. 2B, cell cycle was arrested at the G0-G1 phase in cells treated with 10 μmol/L IMD-0354. The number of cells with hypodiploid DNA contents was increased 24 hours after addition of 10 μmol/L IMD-0354 (Fig. 2B). We also analyzed cell cycle in MCF-7 cells and HMC1-8 cells and found that the percentages of cells arrested at the G0-G1 phase were increased by treatment with 10 μmol/L IMD-0354 after 24 hours as well [medium alone versus IMD-0354, 10 μmol/L (%); MCF-7 cells, 59.2 ± 1.2% versus 71.2 ± 0.8%; HMC1-8 cells, 58.6 ±1.5% versus 69.9 ± 1.6%]. The ratio of cells in S and G2-M phases was significantly decreased and cells in hypodiploid range were increased in a dose-dependent manner when MDA-MB-231 cells were treated with IMD-0354 for 24 hours (Fig. 2C). In addition, an annexin V-binding assay showed that IMD-0354 treatment for 48 hours induced apoptosis but not necrosis to breast cancer cells (Fig. 2D).

Effect of IMD-0354 on the expression of cell cycle regulatory proteins in MDA-MB-231 cells. We have already shown that inhibition of NF-κB activity resulted in the down-regulation of cyclin D3 in neoplastic human mast cells (42). It becomes clear that NF-κB contributes to cell proliferation in various cell types through transcriptional regulation of cyclins (4, 5). MDA-MB-231 cells express cyclin D1 and D3 (45); therefore, we examined the role of NF-κB in the expression of several cyclins. Figure 3A shows spontaneous expression of cyclin D1, D3, and E in MDA-MB-231 cells. IMD-0354 had a minimal effect on expression of cyclin E in MDA-MB-231 cells (Fig. 3A). However, expression of cyclin D1 and D3 was dramatically decreased in a time-dependent manner by addition of 10 μmol/L IMD-0354 (Fig. 3A). IMD-0354 reduced the expression of cyclin D1 and D3 in a dose-dependent manner after 16 hours (Fig. 3B). D-type cyclins form a complex with CDK and phosphorylate pRb, thereby resulting in positive regulation of the G1 progression and transition to the S phase (1317). Compared with untreated cells, pRb phosphorylation was suppressed dose-dependently in cells incubated with IMD-0354 for 16 hours (Fig. 3C). We also detected other pRb family proteins p107 and p130 in MDA-MB-231 cells (Fig. 3D). IMD-0354 strongly inhibited phosphorylation of p130 (Fig. 3D). Although the effect on suppression of p107 was weaker than that on pRb and p130, IMD-0354 decreased phosphorylated p107 (Fig. 3D).

Figure 3.

Expression of cell cycle regulatory proteins in MDA-MB-231 cells down-regulated by treatment with IMD-0354. Expression of cyclin D1 and D3 was decreased by treatment with IMD-0354 (10 μmol/L). We used 3T3 cell lysates as positive controls (Control; A). Expression of cyclin D1 and D3 in MDA-MB-231 cells was suppressed by treatment with IMD-0354 for 16 hours in a dose-dependent manner (B). Relative intensities of cyclin D1 and D3 were standardized to a value without IMD-0354 as 1 (B). Spontaneously phosphorylated pRb (C) and pRb family proteins (p107 and p130; D) was down-regulated in MDA-MB-231 cells incubated with various concentrations of IMD-0354 for 16 hours (representative of three different experiments).

Figure 3.

Expression of cell cycle regulatory proteins in MDA-MB-231 cells down-regulated by treatment with IMD-0354. Expression of cyclin D1 and D3 was decreased by treatment with IMD-0354 (10 μmol/L). We used 3T3 cell lysates as positive controls (Control; A). Expression of cyclin D1 and D3 in MDA-MB-231 cells was suppressed by treatment with IMD-0354 for 16 hours in a dose-dependent manner (B). Relative intensities of cyclin D1 and D3 were standardized to a value without IMD-0354 as 1 (B). Spontaneously phosphorylated pRb (C) and pRb family proteins (p107 and p130; D) was down-regulated in MDA-MB-231 cells incubated with various concentrations of IMD-0354 for 16 hours (representative of three different experiments).

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Next, we analyzed the expression of Cip/Kip family proteins after treatment of cells with IMD-0354. We detected p21Cip1/WAF1 and p57Kip2 in MDA-MB-231 cells, but IMD-0354 did not affect the expression of those molecules (Fig. 4A). In contrast, treatment with IMD-0354 increased the protein level of p27Kip1 when >5 μmol/L IMD-0354 was applied (Fig. 4A).

Figure 4.

Effect of IMD-0354 on expression of the Cip/Kip family of CDK inhibitors (A). Treatment with IMD-0354 for 24 hours increased p27Kip1, but neither p21Cip1/WAF1 nor p57Kip2 was affected. Effect of IMD-0354 on p53 and MDM2 (B). Although IMD-0354 had no effect on p53 expression, MDM2 was decreased by treatment with IMD-0354. IMD-0354 also inhibited antiapoptotic proteins (Bcl-2 and Bcl-xL; photographs representative of three different experiments; C). Relative intensities of p27Kip1, MDM2, Bcl-2, and Bcl-xL are indicated (D). Each value, except p27Kip1, was standardized to values without IMD-0354 as 1. For p27Kip1 expression, each value was standardized to values with 10 μmol/L IMD-0354 as 1.

Figure 4.

Effect of IMD-0354 on expression of the Cip/Kip family of CDK inhibitors (A). Treatment with IMD-0354 for 24 hours increased p27Kip1, but neither p21Cip1/WAF1 nor p57Kip2 was affected. Effect of IMD-0354 on p53 and MDM2 (B). Although IMD-0354 had no effect on p53 expression, MDM2 was decreased by treatment with IMD-0354. IMD-0354 also inhibited antiapoptotic proteins (Bcl-2 and Bcl-xL; photographs representative of three different experiments; C). Relative intensities of p27Kip1, MDM2, Bcl-2, and Bcl-xL are indicated (D). Each value, except p27Kip1, was standardized to values without IMD-0354 as 1. For p27Kip1 expression, each value was standardized to values with 10 μmol/L IMD-0354 as 1.

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Furthermore, we investigated antiapoptotic molecules in MDA-MB-231 cells. Expression of p53 was obvious in untreated cells with IMD-0354 and was not affected by treatment with IMD-0354 (Fig. 4B). The p53 gene in MDA-MB-231 has a point mutation in codon 280 of exon 8, thereby resulting in a dysfunctional tumor suppressor (46, 47). Because expression of p53 induces anti-p53 molecule MDM2, we attempted to detect MDM2 and examined the effect of IMD-0354 on its level. As shown in Fig. 4B, MDA-MB-231 cells expressed MDM2 and IMD-0354 decreased its expression in a dose-dependent manner. IMD-0354 also inhibited protein levels of both Bcl-2 and Bcl-xL (Fig. 4C). Relative intensities revealed that IMD-0354 regulated the protein expression of p27Kip1, MDM2, Bcl-2, and Bcl-xL in a dose-dependent manner (Fig. 4D).

Effect of IMD-0354 on TGF-β expression in MDA-MB-231 cells. Because MDA-MB-231 cells have been reported to produce TGF-β (37), we analyzed the effect of IMD-0354 on TGF-β production by a real-time PCR. Twenty-four hours of incubation with IMD-0354 significantly increased mRNA levels of TGF-β in MDA-MB-231 cells in a dose-dependent manner (Fig. 5).

Figure 5.

Effect of IMD-0354 on mRNA expression of TGF-β in MDA-MB-231 cells. After 24 hours of treatment with various concentrations of IMD-0354. Total RNA was isolated and reverse-transcribed into cDNA. A real-time PCR revealed that TGF-β mRNA expression in MDA-MB-231 cells was increased by treatment with IMD-0354 in a dose-dependent manner. Columns, mean of three different experiments with duplication; bars, ± SE; *, P < 0.01 as compared with medium alone.

Figure 5.

Effect of IMD-0354 on mRNA expression of TGF-β in MDA-MB-231 cells. After 24 hours of treatment with various concentrations of IMD-0354. Total RNA was isolated and reverse-transcribed into cDNA. A real-time PCR revealed that TGF-β mRNA expression in MDA-MB-231 cells was increased by treatment with IMD-0354 in a dose-dependent manner. Columns, mean of three different experiments with duplication; bars, ± SE; *, P < 0.01 as compared with medium alone.

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Tumor progression suppressed by daily administration with IMD-0354. Finally, we examined the therapeutic advantages of IMD-0354 on breast cancer progression by in vivo experiments. Daily administration with 5 mg/kg IMD-0354 significantly suppressed tumor expansion in nude mice implanted with established MDA-MB-231 tumors (Fig. 6A). In mice treated with IMD-0354, tumor progression was restrained as indicated in Fig. 6B. There was no difference in blood cell counts (control versus IMD-0354; mean peripheral blood leukocyte numbers, 10,800 versus 11,200 cells/μL; packed cell volume, 44% versus 43%), and tests of blood biochemistry (control versus IMD-0354; alanine aminotransferase, 52.1 ± 3.2 versus 56.4 ± 9.8 units/L) between each group after the experiment.

Figure 6.

Effect of IMD-0354 on in vivo progression of human breast cancer. Established human breast cancer was transplanted to female BALB/c nude mice as described in Materials and Methods. IMD-0354 (5 mg/kg body weight) or saline alone was injected daily for 28 days and tumor weight and tumor volume were measured once a week. Administration of IMD-0354 suppressed tumor progression (A). Points, mean of two different experiments with eight mice in each group; bars, ± SE; *, P < 0.01 as compared with saline alone. Photographs of typical features of mice in each group (B).

Figure 6.

Effect of IMD-0354 on in vivo progression of human breast cancer. Established human breast cancer was transplanted to female BALB/c nude mice as described in Materials and Methods. IMD-0354 (5 mg/kg body weight) or saline alone was injected daily for 28 days and tumor weight and tumor volume were measured once a week. Administration of IMD-0354 suppressed tumor progression (A). Points, mean of two different experiments with eight mice in each group; bars, ± SE; *, P < 0.01 as compared with saline alone. Photographs of typical features of mice in each group (B).

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In the present study, we clearly showed that constitutive activation of NF-κB up-regulated cell cycle progression and seriously contributed to the proliferation of human breast cancer cells. In MDA-MB-231 cells, NF-κB was spontaneously activated and inhibition of the activity with IMD-0354 led the cells to apoptosis. The inhibitory effect of IMD-0354 was also confirmed in other human breast cancer cell lines, MCF-7 and HMC1-8. However, the effect of IMD-0354 was weaker against estrogen receptor–positive MCF-7 cells than estrogen receptor–negative cell lines. Estrogen receptor–negative breast cancers are resistant to tamoxifen therapy and show extremely malignant features with the invasive and metastatic phenotype. Our results indicate that estrogen receptor–negative breast cancers may be more sensitive to NF-κB inhibition, so that IMD-0354 may be useful against recurrent or metastatic breast cancers with less sensitivity to tamoxifen. Because IMD-0354 completely suppressed expression of cyclin D1 and D3, NF-κB may be involved in cell cycle transition from the G1 phase to the S phase through regulation of those D-type cyclins. The cyclin D and CDK4/6 complex phosphorylates pRb family proteins and phosphorylated pRb proteins release E2F that plays a critical role in S phase entry (14, 16). Because IMD-0354 inhibited phosphorylation of pRb family proteins, activation of NF-κB may facilitate cyclin D expression resulting in phosphorylation of pRb members. p27Kip1 is one of the negative regulators of the cyclin D and CDK4/6 complex (14, 16, 24). Although we detected no signals of p27Kip1 in untreated MDA-MB-231 cells, expression of p27Kip1 was promoted in cells treated with IMD-0354. Positive reactions of weak p21Cip1/WAF1 and p57Kip2 were identified in MDA-MB-231 cells; however, inhibition of NF-κB activity by IMD-0354 had no effect. These results delineate that abnormal activation of NF-κB may suppress p27Kip1 expression and lead to up-regulation of the cyclin D and CDK4/6 complex activity resulting in pRb phosphorylation. Released E2F family proteins must strongly drive cell cycle progression in MDA-MB-231 cells. The antiproliferative role of p27Kip1 is frequently disrupted in human cancers. Because loss of p27Kip1 contributes to the progression of human breast cancers (48), up-regulation of p27Kip1 together with down-regulation of D-type cyclins by treatment with IMD-0354 may contribute to the inhibition of cell cycle progression. Although expression of p27Kip1 does not always bring a favorable prognosis in some breast cancer cases, our results suggest that induction of p27Kip1 may play a role in cell cycle arrest in MDA-MB-231 cells after treatment with IMD-0354.

IMD-0354 suppressed the expression of both Bcl-2 and Bcl-xL without affecting Akt activity, suggesting that NF-κB may play a key role in the downstream signaling pathway of Akt for the regulation of antiapoptotic molecules. As previously reported (46, 47), p53 was abnormally expressed in MDA-MB-231 cells. Although IMD-0354 had no effect on p53 expression, p53-repressor MDM2 was inhibited by treatment with IMD-0354. Independent from interaction with p53, MDM2 enhances phosphorylation of pRb and release of E2F (29). Functional inactivation of p53 and up-regulation of MDM2 may be closely associated with cell proliferation and tumor progression in breast cancers. In some tumor cells expressing mutant p53, MDM2 is sometimes stabilized by heat shock protein 90 (Hsp 90) that is accumulated by mutant p53 (49). Hsp 90 is involved in the NF-κB pathway by modulating kinase activity and biogenesis of IKK. Because IMD-0354 inhibited the expression of MDM2 without affecting the p53 level, a complicated crosstalk may be predicted between the Hsp 90 and the NF-κB pathway.

Moreover, IMD-0354 increased TGF-β production in MDA-MB-231 cells. TGF-β leads to apoptosis in various cell types. Although mutations in the p27Kip1 gene are rare (50), accelerated proteolysis causes a reduction in the p27Kip1 protein in many cancers, including breast cancers, and may contribute to TGF-β resistance (36, 45). MDM2 is another candidate that induces TGF-β resistance (41); MDA-MB-231 cells may manifest the resistance at least in part through induction of MDM2 and reduction of p27Kip1. Up-regulation of cyclin D, Bcl family members, and MDM2, together with down-regulation of p27Kip1 by activated NF-κB, may orchestrate an antiapoptotic phenotype in MDA-MB-231 cells. Our results emphasize that inhibition of NF-κB activity may correct disrupted cell cycle control in breast cancer cells. Thus, we speculate that IMD-0354 may recover the sensitivity to TGF-β by regulating its production and expression of cellular molecules that associate with TGF-β resistance.

IMD-0354 did not completely inhibit but suppressed growth of the established breast cancer in vivo without any serious side effects and toxicity. Thus, interference of NF-κB activity in breast cancer therapy may be considered as one of the candidates of combination chemotherapy in addition to standard methods. NF-κB must be an important molecular target, thus, IMD-0354 may bring a great advantage for anti–breast cancer therapy.

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

We thank Yoko Wakamatsu (Institute of Medicinal Molecular Design) for her technical support.

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