Kaurane diterpene compounds have been known to be cytotoxic against several cancer cells through inhibition of nuclear factor-κB (NF-κB) activity. Here, we showed that inflexinol, a novel kaurane diterpene compound, inhibited the activity of NF-κB and its target gene expression as well as cancer cell growth through induction of apoptotic cell death in vitro and in vivo. These inhibitory effects on NF-κB activity and on cancer cell growth were suppressed by the reducing agents DTT and glutathione and were abrogated in the cells transfected with mutant p50 (C62S). Sol-gel biochip and surface plasmon resonance analysis showed that inflexinol binds to the p50 subunit of NF-κB. These results suggest that inflexinol inhibits colon cancer cell growth via induction of apoptotic cell death through inactivation of NF-κB by a direct modification of cysteine residue in the p50 subunit of NF-κB. [Mol Cancer Ther 2009;8(6):1613–24]

Nuclear factor-κB (NF-κB) represents a family of eukaryotic transcription factors participating in the regulation of various cellular responses involved in the immediate early processes of the immune (1) and inflammatory responses (2), apoptosis (3), and cell proliferation (4) through induction of a large array of target genes. NF-κB is now considered as an important transcription factor in the tumorigenic process because it exerts strong antiapoptotic functions in cancer cells (5). NF-κB activation is associated with colorectal cancer development. Colon cancer cell lines and human tumor samples, as well as nuclei of stromal macrophages in sporadic adenomatous polyps, were found to have increased NF-κB activity (6, 7). NF-κB can lead to a proliferation of transformed cells through enhanced production of growth factors and cytokines (8). NF-κB acts as a cell survival factor through its regulatory role in the expression of an array of apoptotic (caspase-3 and Bax), antiapoptotic (Bcl-2 and IAP family), and cell proliferation genes (cyclooxygenase-2 and cyclins; refs. 9, 10). Thus, inactivation of NF-κB by chemotherapeutics is intended as a new strategy to eliminate cancerous cells through induction of apoptosis.

Several compounds inhibiting NF-κB have shown to be useful for inhibition of cancer cell growth. Nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, sulindac, phenylbutazone, naproxen, indomethacin, diclofenac, dexamethasone, celecoxib, tamoxifen, and sulfasalazine have been shown to suppress the activity of NF-κB, which eventually leads to the inhibition of cancer cell growth (1113). Many natural compounds, such as polyphenols (resveratrol, curcumin, epigallocatechin gallate, quercetin), lignans [manassantins, (+)-saucernetin, (−)-saucerneol methyl ether], sesquiterpenes (costunolide, parthenolide, celastrol, celaphanol A), diterpenes (excisanin, kamebakaurin), and triterpenes (avicin, oleandrin), have also been shown to suppress NF-κB activity; these compounds are also suggested to be useful for the inhibition of cancer cell growth (1416). However, only a few studies have shown the chemical molecular targets and anticancer activity of these compounds in vivo.

Whole-plant extracts of Isodon excisus (Max.) kudo (Labiatae) have been used in folk medicine in China, Korea, and Japan for treating tumors and inflammatory diseases (17). Several diterpenoids with C-20 nonoxygenated or oxygenated ent-kaurane, 7,6-seco-ent-kaurane, 8,9-seco-ent-kaurane, and ent-kaurane dimer have been isolated and characterized from the genus Isodon (18), and some kaurane compounds exhibit not only cytotoxic activity against various cancer cell lines but also inhibitory activity against NF-κB (19, 20). We recently isolated and characterized several kaurane diterpenoids from I. excisus (21), and found that this compound (named inflexinol) has strong NF-κB inhibitory effects on lipopolysaccharide (LPS)-treated macrophage RAW 264.7 cells and astrocytes (22). To investigate the antitumor activity of inflexinol in vitro and in vivo, as well as its interaction with target molecules, we studied the effect of inflexinol on NF-κB activity and its binding to NF-κB subunit, as well as its effect on NF-κB target gene expression, in colon cancer cells in vitro and in vivo in parallel with the effects of this compound on colon cancer cell growth and the induction of apoptosis.

Chemicals

Inflexinol was isolated from I. excisus (Labiatae) and characterized as described elsewhere (21). The structure of inflexinol was shown in Supplementary Fig. S1A.5

1Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org).

The expression plasmid encoding p50 (cysteine residue in the p50 is replaced with alanine) was obtained from Dr. Warner C. Greene (University of California, San Francisco, CA; ref. 23).

Cell Culture

SW620, HCT116 (p53+/+), and HCT116 (p53−/−) human colon cancer cells were obtained from the American Type Culture Collection and were grown in RPMI 1640 with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 humidified air. The human colon CCD 112-CoN cells were also obtained from the American Type Culture Collection and were grown in Eagle's MEM with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 humidified air.

Cell Viability Assay

SW620, HCT116 (p53+/+), and HCT116 (p53−/−) human colon cancer cells were plated at a density of 104 per well in 96-well plates per 100 μL medium. The cytotoxic effect was evaluated in the cells cultured for 12, 24, 36, and 48 h using the cell counting assay kit according to the manufacturer's instructions (CCK-8 kit, Dojindo, Maryland). Briefly, 10 μL of the CCK-8 solution were added to cells and cultured for an indicated time period. The mixture of cells and solution was further incubated for 1 to 4 h at 37°C. CCK-8 is reduced by dehydrogenase in the cells to give an orange-colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye (450-nm absorbance) generated by dehydrogenase in the cells is directly proportional to the number of cells. The absorbance was measured at 450 nm using a microplate absorbance reader (Sunrise, Tecan). To determine the cell number, colon cancer cells were plated onto 12-well plates (5 × 104 per well) and were trypsinized and pelleted by centrifugation for 5 min at 1,500 rpm, resuspended in 10 mL of PBS, and 0.1 mL of 0.2% trypan blue was added to the cancer cell suspension in each of the solutions (0.9 mL each). Subsequently, a drop of suspension was placed into a Neubauer chamber and the living cancer cells were counted. Cells that showed signs of staining were considered to be dead, whereas those that excluded trypan blue were considered viable. Each assay was carried out in triplicate.

Transfection and Assay of Luciferase Activity

SW620, HCT116 (p53+/+), and HCT116 (p53−/−) human colon cancer cells (1 × 105 per well) were plated in 24-well plates and transiently transfected with pNF-κB-Luc plasmid (5× NF-κB; Stratagene) or p50 (C62S) mutant plasmid using a mixture of plasmid and LipofectAMINE PLUS in OPTI-MEN according to the manufacturer's specification (Invitrogen). The transfected cells were treated with different concentrations (10–40 μmol/L) of inflexinol in the absence or presence of tumor necrosis factor α (TNF-α; 10 ng/mL) for 8 h. Luciferase activity was measured by using the luciferase assay kit (Promega) according to the manufacturer's instructions (Perkin-Elmer Life and Analytical Sciences, Inc.).

Gel Electromobility Shift and Supershift Assays

Gel electromobility shift assay (EMSA) was done as described previously (24). The relative density of the protein bands was scanned by densitometry using MyImage and quantified by Labworks 4.0 software (UVP, Inc.).

Western Blot Analysis

Western blot analysis was done as described previously (24). The membrane was incubated for 5 h at room temperature with specific antibodies: mouse polyclonal antibodies against p65 and p50 (1:500 dilution; Santa Cruz Biotechnology, Inc.) and Ki-67 (1:500 dilution; Dakocytomation) and rabbit polyclonal for Bax and Bcl-2 (1:500 dilution; Santa Cruz Biotechnology), caspase-3, cleaved caspase-3, cleaved caspase-9, poly(ADP-ribose) polymerase, antihuman proliferating cell nuclear antigen (PCNA), X-chromosome linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 (cIAP1; 1:1,000 dilution; Cell Signaling Technology, Inc.). The blot was then incubated with the corresponding conjugated antirabbit and antimouse immunoglobulin G-horseradish peroxidase (1:2,000 dilution; Santa Cruz Biotechnology). Immunoreactive proteins were detected with the enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was scanned by densitometry using MyImage and quantified by Labworks 4.0 software.

Detection of Apoptosis

Detection of apoptosis was done as described elsewhere (24). In short, cells were cultured on eight-chamber slides. After treatment with inflexinol (10–40 μmol/L) for 24 h, the cells were washed twice with PBS and fixed by incubation in 4% paraformaldehyde in PBS for 1 h at room temperature. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays were done by using the In situ Cell Death Detection Kit (Roche Diagonostics GmbH) according to the manufacturer's instructions. Total number of cells in a given area was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. The apoptotic index was determined as the number of TUNEL-positive stained cells divided by the total cell number counted ×100.

Sol-Gel Biochip and Surface Plasmon Resonance Analysis

Sol-gel biochip analysis was done as described elsewhere (25). In brief, inflexinols (7.46 and 746 pmol) were aliquoted individually onto sol-gel spots in a polymethylmethacrylate-coated 96-well plate using an OmniGrid Accent Microarrayer (DIGI LAB) and immobilized by gelation. For the binding assay, p50 (10 ng/μL), anti-p50 (10 ng/μL), and Cy3-labeled goat secondary antibody (2 ng/μL) were sequentially incubated for 1 h in the wells. After washing, the spots were analyzed with a Multi-Image Analyzer (FUJIFILM). We also included on each plate a negative control (without inflexinol) and a positive control (with labeled antibodies and p50 protein). We also analyzed the p50 and inflexinol binding using plasmon resonance analysis, as described elsewhere (26), using Biacore system and CM5 sensor chip (both supplied by Biacore AB).

Antitumor Activity Study in In vivo Xenograft Animal Model

Six-week-old male BALB/c athymic nude mice were purchased from Japan SLC. The mice were housed and maintained under sterile conditions in facilities accredited by the American Association for Accreditation of Laboratory Animal Care and in accordance with the current regulations and standards of the Korea Food and Drug Administration. Human colon cancer SW620 cells were injected s.c. (1 × 107 tumor cells/0.1 mL PBS per animal) with a 27-gauge needle into the right lower flanks of carrier mice. After 20 d, when the tumors had reached an average volume of 300 to 400 mm3, the tumor-bearing nude mice were i.p. injected with inflexinol (12 and 36 mg/kg dissolved in 0.01% DMSO) twice per week for 3 wk. Docetaxel (10 mg/kg dissolved in 0.6% Tween 80 and 0.3% ethanol, i.p.) was also injected once a week as a positive control. The group treated with 0.01 mol/L DMSO was used as the control. The weight and tumor volume of the animals were monitored twice per week. The tumor volumes were measured with vernier calipers and calculated by the following formula: (A × B2)/2, where A and B are the larger and smaller dimensions, respectively. At the end of the experiment, the animals were sacrificed with cervical dislocation. The tumors were separated from the surrounding muscles and dermis, excised, and weighed.

Immunohistochemistry

All specimens were fixed in formalin and embedded in paraffin for examination. Sections, 5 μm thick, were stained with H&E and analyzed by immunohistochemistry. Paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval treatment. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen peroxide in methanol for 15 min, then cleared in PBS for 5 min. The sections were blocked for 30 min with 3% normal horse serum diluted in PBS; the sections were then blotted and incubated with primary mouse PCNA and Ki-67 monoclonal antibodies (1:200 dilution) at the appropriate dilution in blocking serum for 4 h at room temperature, or primary mouse anti-human p65 antibody (1:100 dilution) or primary rabbit anti-human p50 and cleaved caspase-3 polyclonal antibody (1:100 dilution) at the appropriate dilution in blocking serum overnight at 4°C. The next day, slides were washed thrice for 5 min each in PBS and incubated with biotinylated rabbit anti-mouse antibody for 2 h. The slides were washed in PBS, followed by the avidin-biotin-peroxidase complex (Vector Laboratories, Inc.). The slides were washed and the peroxidase reaction developed with diaminobenzidine and peroxide, then counterstained with hematoxylin, mounted in Aquamount, and evaluated with light microscopy (×200; Olympus). A negative control was done in all cases by omitting the primary antibody. All slides were counterstained with hematoxylin. For quantification, 200 cells at three randomly selected areas were assessed, and cells positively staining for PCNA, Ki-67, p65, p50, and cleaved caspase-3 were counted and expressed as percentage of stained cells. For detection of apoptotic cell death in tumor tissue, the paraffin-embedded sections were then incubated in the mixture of labeling solution (450 μL) and enzyme solution (50 μL) for 1 h at 37°C and washed thrice in 0.1 mol/L PBS for 5 min each, according to the manufacturer's instructions. Next, the sections were incubated with DAPI for 15 min at 37°C. Finally, the sections were rinsed, mounted on slides, and coverslipped for fluorescence microscopy (DAS microscope). Positive TUNEL stains were recorded by counting the number of positively stained DAPI in the definite area.

Data Analysis

Data were analyzed using GraphPad Prism 4 software (version 4.03, GraphPad software, Inc.). Data are presented as mean ± SE. Homogeneity of variances was assessed using Bartlett's test. If variances were homogeneous, differences between groups and treatment were assessed by one-way ANOVA. If the P value in the ANOVA test was significant, the differences between pair of means were assessed by Dunnett's test. P < 0.05 was considered to be statistically significant.

Inflexinol Inhibited NF-κB Activation In vitro in SW620, HCT116 (p53+/+), and HCT116 (p53−/−) Human Colon Cancer Cells

NF-κB has been known to be an implicated factor in apoptotic cell death of several cancer cells. To determine whether inflexinol inhibits the activation of NF-κB in colon cancer cells, we first determined the DNA binding activity of NF-κB by an EMSA. We found that a high level of DNA binding activity of NF-κB (constitutive activation) in the untreated SW620, HCT116 (p53+/+), and HCT116 (p53−/−) human colon cancer cells lasted for up to 24 h in the culture. Treatment of inflexinol for 1 hour inhibited the constitutively activated DNA binding activity of NF-κB in a concentration-dependent manner (10–40 μmol/L; Fig. 1A). We also found that 1-hour exposure to inflexinol inhibited the DNA binding activity of NF-κB, which was induced by TNF-α (10 ng/mL), 12-O-tetradecanoylphorbol-13-acetate (50 nmol/L), LPS (1 μg/mL), and H2O2 (1 mmol/L) in cultured colon cancer cells in a concentration-dependent manner (Fig. 1B). This DNA binding activity of NF-κB was confirmed by a competition assay using excessive [γ-32P]ATP–labeled oligonucleotides of NF-κB consensus (Fig. 1C, lanes 2 and 3). In the presence of a p65 antibody, the DNA binding activity of NF-κB was supershifted (Fig. 1C, lanes 6–8), but in the presence of the p50 antibody, the DNA binding activity of NF-κB was decreased (Fig. 1C, lanes 4–5 and 8). Along with the inhibitory effect on NF-κB, we also found that inflexinol concentration-dependently inhibited the translocation of p50 and p65 into the nucleus through inhibition of the phosphorylation of IκB (Fig. 1D).

Figure 1.

Effect of inflexinol on NF-κB activation in colon cancer cells. A and B, nuclear extract from colon cancer cells treated with inflexinol (10–40 μmol/L) for 1 h was incubated in binding interaction with 32P-end-labeled oligonucleotide containing the κB sequence. The activation of NF-κB was investigated using EMSA as described in Materials and Methods. C, for competition assays, nuclear extracts from SW620 colon cancer cells treated with inflexinol (10 μmol/L) were incubated for 1 h before EMSA with unlabeled NF-κB oligonucleotide or labeled NF-κB oligonucleotide. For supershift assays, nuclear extracts from SW620 colon cancer cells treated with 10 μmol/L inflexinol were incubated for 1 h before EMSA with specific antibodies against the p50 and p65 NF-κB isoforms. SS, supershift band. Quantification of band intensities from three independent experimental results was determined by densitometry (Imaging System), and the values (A) under the band indicate fold difference (average) from untreated control group. D, the cells were treated with different concentrations (10–40 μmol/L) of inflexinol at 37°C for 1 h. Nuclear or cytosolic proteins (50 μg) extracted after treatment were subjected to 12% SDS-PAGE. The expression of p50, p65, IκB-α, and p-IκB-α proteins was detected by Western blotting using specific antibodies. β-Actin and histone-H1 (NE) proteins were used as internal controls. NE, nuclear extract; CE, cytosolic extract. E, colon cancer cells were transfected with pNF-κB-Luc plasmid (5× NF-κB) and then activated with TNF-α (10 ng/mL) alone or TNF-α plus different concentrations (10–40 μmol/L) of inflexinol at 37°C, and then the luciferase activity was determined. Columns, mean of three independent experiments done in triplicate; bars, SD. RLU is relative to luciferase activity–transfected unstimulated cells. *, P < 0.05, versus the TNF-α–treated group.

Figure 1.

Effect of inflexinol on NF-κB activation in colon cancer cells. A and B, nuclear extract from colon cancer cells treated with inflexinol (10–40 μmol/L) for 1 h was incubated in binding interaction with 32P-end-labeled oligonucleotide containing the κB sequence. The activation of NF-κB was investigated using EMSA as described in Materials and Methods. C, for competition assays, nuclear extracts from SW620 colon cancer cells treated with inflexinol (10 μmol/L) were incubated for 1 h before EMSA with unlabeled NF-κB oligonucleotide or labeled NF-κB oligonucleotide. For supershift assays, nuclear extracts from SW620 colon cancer cells treated with 10 μmol/L inflexinol were incubated for 1 h before EMSA with specific antibodies against the p50 and p65 NF-κB isoforms. SS, supershift band. Quantification of band intensities from three independent experimental results was determined by densitometry (Imaging System), and the values (A) under the band indicate fold difference (average) from untreated control group. D, the cells were treated with different concentrations (10–40 μmol/L) of inflexinol at 37°C for 1 h. Nuclear or cytosolic proteins (50 μg) extracted after treatment were subjected to 12% SDS-PAGE. The expression of p50, p65, IκB-α, and p-IκB-α proteins was detected by Western blotting using specific antibodies. β-Actin and histone-H1 (NE) proteins were used as internal controls. NE, nuclear extract; CE, cytosolic extract. E, colon cancer cells were transfected with pNF-κB-Luc plasmid (5× NF-κB) and then activated with TNF-α (10 ng/mL) alone or TNF-α plus different concentrations (10–40 μmol/L) of inflexinol at 37°C, and then the luciferase activity was determined. Columns, mean of three independent experiments done in triplicate; bars, SD. RLU is relative to luciferase activity–transfected unstimulated cells. *, P < 0.05, versus the TNF-α–treated group.

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To determine the effects of inflexinol on the NF-κB–dependent reporter gene activity, we transiently transfected the cells with a NF-κB–regulated luciferase reporter construct, and then stimulated the transfected cells with TNF-α or with a combination of TNF-α and inflexinol. We treated the cells with TNF-α because NF-κB–regulated luciferase activitieswere not detected without stimulation. Consistent with the inhibitory effects on the NF-κB DNA binding activity, inflexinol (10–40 μmol/L) concentration-dependently inhibited TNF-α–induced NF-κB luciferase activity (Fig. 1E). We also found that inflexinol inhibited 12-O-tetradecanoylphorbol-13-acetate–induced NF-κB transcriptional activity (data not shown).

Inflexinol Inhibited Human Colon Cancer Cell Growth

To investigate whether the inhibition of the NF-κB activation is related to cancer cell growth, the inhibitory effect of inflexinol on SW620, HCT116 (p53+/+), and HCT116 (p53−/−) human colon cancer cell growth was analyzed by a CCK-8 assay kit and direct cell counting. Morphologic observation showed that the cells were gradually reduced in size and changed into a small round single cell shape with the treatment of inflexinol (Fig. 2A). Cell growth inhibition by inflexinol was also confirmed by the trypan blue dye exclusion method (Fig. 2B). Inflexinol (10–40 μmol/L) treatment resulted in significant concentration- and time-dependent inhibition of cell growth with IC50 values of 29 μmol/L in SW620, 30 μmol/L in HCT116 (p53+/+), and 34 μmol/L in HCT116 (p53−/−; Fig. 2C). In the presence of TNF-α or 12-O-tetradecanoylphorbol-13-acetate, the susceptibility of cells to inflexinol was greater than that after treatment with inflexinol alone. The IC50 values (24 and 48 hours) were 27 and 25 μmol/L in SW620, 28 and 22 μmol/L in HCT116 (p53+/+), and 32 and 24 μmol/L in HCT116 (p53−/−), respectively (Supplementary Fig. S1B).5 However, inflexinol was not cytotoxic in the normal CCD-112 CoN cells in the tested concentration (Supplementary Fig. S1C).5

Figure 2.

Morphologic changes and cell viability of colon cancer cells by inflexinol. A, morphologic changes were observed under a microscope. B, colon cancer cells were incubated with inflexinol at concentrations of 10 to 40 μmol/L for up to 48 h. At 12, 24, 36, and 48 h, cells were harvested by trypsinization and stained with 0.2% trypan blue. Relative cell survival rate was determined by counting live and dead cells. C, cell viability was determined by CCK-8 assay as described in Materials and Methods. Points, mean of three experiments each done in triplicate; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

Figure 2.

Morphologic changes and cell viability of colon cancer cells by inflexinol. A, morphologic changes were observed under a microscope. B, colon cancer cells were incubated with inflexinol at concentrations of 10 to 40 μmol/L for up to 48 h. At 12, 24, 36, and 48 h, cells were harvested by trypsinization and stained with 0.2% trypan blue. Relative cell survival rate was determined by counting live and dead cells. C, cell viability was determined by CCK-8 assay as described in Materials and Methods. Points, mean of three experiments each done in triplicate; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

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Inflexinol Induced Apoptotic Cell Death

To delineate whether the inhibition of cell growth by inflexinol was due to induction of apoptotic cell death, we evaluated the changes in the chromatin morphology of cells by using DAPI staining. To further characterize the apoptotic cell death induced by inflexinol, we performed TUNEL staining assays, and then the labeled cells were analyzed with a fluorescence microscope. The number of TUNEL-labeledcells was increased and the fluorescence intensity was also increased in treated cells (Fig. 3A). Apoptotic cells numbers (DAPI-positive TUNEL-stained cells) were increased to 0 ± 3%, 3 ± 5%, 5 ± 3%, 38 ± 13%, and 82 ± 8% in SW620 colon cancer cells; 0 ± 2%, 2 ± 4%, 44 ± 18%, 81 ± 5%, and 86 ± 7% in HCT116 (p53+/+) colon cancer cells; and 0 ± 5%, 2 ± 6%, 50 ± 20%, 89 ± 6%, and 93 ± 2% in HCT116 (p53−/−) colon cancer cells by 10 to 40 μmol/L inflexinol treatment (Fig. 3A, bottom graphs).

Figure 3.

Apoptotic cell death and expression of apoptosis-related proteins of colon cancer cells by inflexinol. A, colon cancer cells were treated with several concentration of inflexinol for 24 h; cell morphologic changes were observed under a microscope (top); and apoptotic cells were examined with a fluorescence microscope after TUNEL staining (bottom). Total number of cells in a given area was determined by DAPI nuclear staining (fluorescent microscope; middle). The apoptotic index was determined as the number of DAPI-stained TUNEL-positive cells counted. Columns, mean of three experiments each done in triplicate; bars, SD. *, P < 0.05, versus the untreated group. B, the cells were treated with different concentrations (10–40 μmol/L) of inflexinol at 37°C for 24 h. Equal amounts of total proteins (50 μg/lane) were subjected to 12% SDS-PAGE. Expression of Bax, cleaved caspase-3, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP), cyclin D1 (CyD1), Bcl-2, XIAP, cIAP1/2, and β-actin were detected by Western blotting using specific antibodies. β-Actin protein was used as an internal control. Each blot is representative of three independent experimental results. Bar, 100 μm.

Figure 3.

Apoptotic cell death and expression of apoptosis-related proteins of colon cancer cells by inflexinol. A, colon cancer cells were treated with several concentration of inflexinol for 24 h; cell morphologic changes were observed under a microscope (top); and apoptotic cells were examined with a fluorescence microscope after TUNEL staining (bottom). Total number of cells in a given area was determined by DAPI nuclear staining (fluorescent microscope; middle). The apoptotic index was determined as the number of DAPI-stained TUNEL-positive cells counted. Columns, mean of three experiments each done in triplicate; bars, SD. *, P < 0.05, versus the untreated group. B, the cells were treated with different concentrations (10–40 μmol/L) of inflexinol at 37°C for 24 h. Equal amounts of total proteins (50 μg/lane) were subjected to 12% SDS-PAGE. Expression of Bax, cleaved caspase-3, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP), cyclin D1 (CyD1), Bcl-2, XIAP, cIAP1/2, and β-actin were detected by Western blotting using specific antibodies. β-Actin protein was used as an internal control. Each blot is representative of three independent experimental results. Bar, 100 μm.

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Inflexinol Induced the Expression of Apoptotic Regulatory Protein Expression

NF-κB activation in cancer cells correlates well with the resistance to apoptosis and expression of apoptotic regulatory proteins. To figure out the relationship between the induction of apoptosis and the expression of their regulatory protein by inflexinol, expression of apoptosis-related proteins was investigated. In the expression of proapoptotic proteins, the expression of antiapoptotic protein Bcl-2, XIAP, and cIAP1/2 was decreased. However, the expression of caspase-3 and caspase-9 was decreased, but that of cleaved caspase-3, cleaved caspase-9, and poly(ADP-ribose) polymerase was increased, by treatment of inflexinol (Fig. 3B). Moreover, the ratio of Bax/Bcl-2 significantly increased after inflexinol treatment in a concentration-dependent manner. It was also found that inflexinol inhibited the growth regulatory NF-κB target genes cyclin D1 and Bcl-2 (Fig. 3B).

Suppression of Inflexinol-Induced Inhibition of the DNA Binding Activity of NF-κB and Cell Growth by Thiol-Reducing Agents and in the Cells Transfected with Mutant p50

It is well known that the cyclopentanone moiety of a compound is bound to cysteine residue of molecules such as p50, and reducing agents can block this binding activity. To determine whether inflexinol reacts with p50 and whether it inhibits NF-κB, we exposed cells to inflexinol both in the absence and presence DTT and glutathione for 1 hour, and then examined the DNA binding activity of NF-κB. We found that these reducing agents significantly suppressed the inhibitory effects of inflexinol on the DNA binding activity of NF-κB (Fig. 4A). We also examined the suppressive effect of DTT and glutathione on the inhibitory effect of inflexinol in cancer cell growth. In agreement with the suppressive effect on the DNA binding activity of NF-κB, DTT and glutathione suppressed the inhibitory effect of inflexinol in colon cancer cell growth (Fig. 4B and C). Both the supershift assay of the DNA binding activity, showing the decreased DNA binding activity of NF-κB in the presence of p50 antibody, and the suppressive effect of DTT and glutathione on the inhibitory effects of inflexinol on the DNA binding activity of NF-κB prompted us to assume that sulfhydryl residue in p50 might be the target of inflexinol. To further investigate this possibility, we studied the DNA binding activity and cell growth inhibitory effects of inflexinol in the SW620 cells transfected with the p50 mutant (C62S), in which a cysteine residue at 62 of p50 is replaced by alanine. As expected, the inhibitory effect of inflexinol was much greatly reduced in the DNA binding activity of NF-κB (Fig. 4D) and in the cell growth of these transfected cells (Fig. 4E). These results clearly suggested that inflexinol mediates its effects through modulation of cysteine residues of the p50 subunit of NF-κB.

Figure 4.

Abolition of the inhibitory effect of inflexinol by DTT and reduced glutathione (GSH) and in the cells harboring mutant p50 on DNA binding activation of NF-κB and colon cancer cell growth and binding between p50 and inflexinol. A, colon cancer cells grown in six-well plates were cotreated with the indicated concentrations of DTT (10 μmol/L) or glutathione (100 μmol/L) with inflexinol (30 μmol/L) for 1 h. Nuclear extracts were then prepared and examined by EMSA as described in Materials and Methods. B and C, effect of reducing agents on cancer cell growth. Colon cancer cells grown in 96-well plates were cotreated with the indicated concentrations of DTT (5–20 μmol/L) or glutathione (1–100 μmol/L) with inflexinol (30 μmol/L) for 24 h. D and E, colon cancer cells were transiently transfected with wild or mutant types of p50 for 24 h as described in Materials and Methods, and then the cells were treated with inflexinol for 1 h to determine DNA binding activity of NF-κB (D) or for 24 h to determine cell growth (E), as described above and in Materials and Methods.

Figure 4.

Abolition of the inhibitory effect of inflexinol by DTT and reduced glutathione (GSH) and in the cells harboring mutant p50 on DNA binding activation of NF-κB and colon cancer cell growth and binding between p50 and inflexinol. A, colon cancer cells grown in six-well plates were cotreated with the indicated concentrations of DTT (10 μmol/L) or glutathione (100 μmol/L) with inflexinol (30 μmol/L) for 1 h. Nuclear extracts were then prepared and examined by EMSA as described in Materials and Methods. B and C, effect of reducing agents on cancer cell growth. Colon cancer cells grown in 96-well plates were cotreated with the indicated concentrations of DTT (5–20 μmol/L) or glutathione (1–100 μmol/L) with inflexinol (30 μmol/L) for 24 h. D and E, colon cancer cells were transiently transfected with wild or mutant types of p50 for 24 h as described in Materials and Methods, and then the cells were treated with inflexinol for 1 h to determine DNA binding activity of NF-κB (D) or for 24 h to determine cell growth (E), as described above and in Materials and Methods.

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Binding between Inflexinol and p50 of NF-κB

To determine the direct binding of p50 with inflexinol, sol-gel biochip and surface plasmon resonance analysis were done. Sol-gel biochip analysis showed clear binding signal between p50 and inflexinol. However, the binding signal in negative control (without inflexinol) was not observed, whereas in the positive control (binding between labeled p50 protein and its antibody) it was strong (Fig. 5A). We also found binding between p50 and inflexinol, determined by surface plasmon resonance analysis, indicating that inflexinol binds to p50, and the binding activity is about 1.6 × 10−7 mol/L (Kd; Fig. 5B).

Figure 5.

Binding of inflexinol to p50 of NF-κB. A, sol-gel biochip analysis was done as described in Materials and Methods. The representative spots were analyzed with a Multi-Image Analyzer (FUJIFILM). The figure also showed a negative control (without inflexinol) and a positive control (p50 protein with labeled antibody). Similar results were obtained from three separate experiments. Ifl H, high dose of inflexinol (746 pmol); Infl L, low dose of inflexinol (7.46 pmol); N, negative control; p50, positive control with anti-p50. B, binding affinity between p50 and inflexinol analyzed by surface plasmon resonance analysis as described in Materials and Methods using Biacore system and CM5 sensor chip (both supplied by Biacore). All values represent means ± SD of three independent experiments done in triplicate. *, P < 0.05, versus the control group.

Figure 5.

Binding of inflexinol to p50 of NF-κB. A, sol-gel biochip analysis was done as described in Materials and Methods. The representative spots were analyzed with a Multi-Image Analyzer (FUJIFILM). The figure also showed a negative control (without inflexinol) and a positive control (p50 protein with labeled antibody). Similar results were obtained from three separate experiments. Ifl H, high dose of inflexinol (746 pmol); Infl L, low dose of inflexinol (7.46 pmol); N, negative control; p50, positive control with anti-p50. B, binding affinity between p50 and inflexinol analyzed by surface plasmon resonance analysis as described in Materials and Methods using Biacore system and CM5 sensor chip (both supplied by Biacore). All values represent means ± SD of three independent experiments done in triplicate. *, P < 0.05, versus the control group.

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Inflexinol Inhibited the Growth of Colon Cancer Cells in In vivo Xenograft Model

To elucidate the anticancer effects of inflexinol in vivo, the tumor growth in colon cancer xenograft–bearing nude mice following inflexinol treatments was investigated. In SW620 xenograft studies, inflexinol was administrated i.p. twice per week for 3 weeks to mice with tumors ranging from 100 to 300 mm3 in volume. The mice were weighed twice per week. The changes in body weights between the control and the inflexinol-treated mice (n = 10) were not remarkably different during the experiment (data not shown). Figure 6A represents the relative tumor growth delay measured after treatment of inflexinol in comparison with the control group. On day 21, the final tumor weights were recorded. Tumor volumes in mice treated with inflexinol at 12 and 36 mg/kg doses and docetaxel at 10 mg/kg dose were 64.5%, 47.8%, and 18.5% of control group, respectively. Tumor weights in mice treated with inflexinol at 12 and 36 mg/kg and docetaxel at 10 mg/kg were 75.9%, 58.0%, and 48.1% over the control group, respectively (Fig. 6B). The immunohistochemical analysis of tumor sections by H&E and by proliferation antigens against PCNA and Ki-67 staining revealed that inflexinol dose-dependently inhibited tumor cell growth (Fig. 6C). In agreement with the in vitro results, these results suggested that inflexinol suppressed colon cancer cell growth in vivo.

Figure 6.

Effect of inflexinol on tumor growth in SW620 xenograft in vivo model. Growth inhibition (as assessed by tumor volume) of s.c. transplanted SW620 xenografts in mice treated with inflexinol (12 or 36 mg/kg twice a week) for 3 wk. Xenografted mice (n = 10) were i.p. given saline (1 mL/kg) or inflexinol (12 or 36 mg/kg) or docetaxel (10 mg/kg). A, photographs of nude mice and tumors at day 21. B, tumor volume was measured at 3- and 4-d intervals starting from the first day of treatment. Tumor weights were measured at study termination on day 21. Points and columns, mean estimates of tumor growth and weight, respectively, from 10 mice in each treatment; bars, SD. C, immunohistochemistry was used to determine expression levels of H&E, PCNA, and Ki-67 in nude mouse xenograft tissues by the different treatments as described in Materials and Methods. Columns, mean from five animal tumor sections; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

Figure 6.

Effect of inflexinol on tumor growth in SW620 xenograft in vivo model. Growth inhibition (as assessed by tumor volume) of s.c. transplanted SW620 xenografts in mice treated with inflexinol (12 or 36 mg/kg twice a week) for 3 wk. Xenografted mice (n = 10) were i.p. given saline (1 mL/kg) or inflexinol (12 or 36 mg/kg) or docetaxel (10 mg/kg). A, photographs of nude mice and tumors at day 21. B, tumor volume was measured at 3- and 4-d intervals starting from the first day of treatment. Tumor weights were measured at study termination on day 21. Points and columns, mean estimates of tumor growth and weight, respectively, from 10 mice in each treatment; bars, SD. C, immunohistochemistry was used to determine expression levels of H&E, PCNA, and Ki-67 in nude mouse xenograft tissues by the different treatments as described in Materials and Methods. Columns, mean from five animal tumor sections; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

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Effect of Inflexinol on the DNA Binding Activity of NF-κB and on the Expression of Proapoptotic and Antiapoptotic Genes In vivo

We also determined the effect of inflexinol on NF-κB activity in vivo. Similar to the inhibitory effects in vitro, inflexinol dose-dependently inhibited the DNA binding activity of NF-κB in tumor tissue (Fig. 7A). Western blotting analysis also showed that treatments of inflexinol decreased p65 and p50 translocation into the nucleus as well as IκB phosphorylation in the cytosol (Fig. 7B). We also characterized the immunohistochemical staining pattern of the NF-κB subunits p65 and p50 in tumor tissue. There was a trend toward decreased intensity of nuclear staining of p65 and p50 in inflexinol-treated tumor tissue (Fig. 7C). These data agree with the inhibitory effects of inflexinol on the DNA binding activity of NF-κB in vivo and in vitro, and indicate that NF-κB is the target of inflexinol. Inflexinol also increased Bax and cleaved caspase-3, but deceased the expression of Bcl-2, XIAP, and cIAP in tumor tissue (Fig. 7D). Immunohistochemical analysis also showed considerable increase in the expression of cleaved caspase-3–positive cells in the inflexinol-treated tumor tissue compared with those of the control group. However, Bcl-2–positive cells were significantly decreased (Fig. 7E). Apoptotic cell death was also significantly reduced in the inflexinol-treated tumor tissue sample (Fig. 7F).

Figure 7.

NF-κB activity and expression of NF-κB and apoptosis-related proteins as well as induction of apoptosis by inflexinol in in vivo xenograft animal model. A, DNA binding activity of NF-κB was determined by EMSA in nuclear extract from xenograft tumor samples (five samples per group) as described in Materials and Methods. B, expression of NF-κB proteins was detected by Western blotting using specific antibodies in nuclear extract from xenograft tumor samples (three samples per group) as described in Materials and Methods. C, immunohistochemistry was used to determine the nuclear p65 and p50 reactive cell number in nude mouse xenograft tissues by the different treatments as described in Materials and Methods. A representative sample from each group was stained in the picture. D, Western blot analysis of cleaved caspase-3, Bax, Bcl-2, IAP1/2, and XIAP expression. E, immunohistochemical analysis of cleaved caspase-3. F, apoptotic cell death by inflexinol in vivo. Columns, mean from five animal tumor sections; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

Figure 7.

NF-κB activity and expression of NF-κB and apoptosis-related proteins as well as induction of apoptosis by inflexinol in in vivo xenograft animal model. A, DNA binding activity of NF-κB was determined by EMSA in nuclear extract from xenograft tumor samples (five samples per group) as described in Materials and Methods. B, expression of NF-κB proteins was detected by Western blotting using specific antibodies in nuclear extract from xenograft tumor samples (three samples per group) as described in Materials and Methods. C, immunohistochemistry was used to determine the nuclear p65 and p50 reactive cell number in nude mouse xenograft tissues by the different treatments as described in Materials and Methods. A representative sample from each group was stained in the picture. D, Western blot analysis of cleaved caspase-3, Bax, Bcl-2, IAP1/2, and XIAP expression. E, immunohistochemical analysis of cleaved caspase-3. F, apoptotic cell death by inflexinol in vivo. Columns, mean from five animal tumor sections; bars, SD. *, P < 0.05, versus the control group. Bar, 100 μm.

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Recent evidence indicates that NF-κB signaling pathways are significantly involved in tumor development (27, 28). The constitutively activated NF-κB transcription factor has been associated with several aspects of tumorigenesis such as tumor cell growth, antiapoptosis, metastasis, angiogenesis, resistance against chemotherapeutics, and tumor promotion in many cancer cells including colon cancer (8). The NF-κB transcription factor is constitutively activated in human colorectal carcinoma tissue and colon cancer cells (29, 30). Therefore, activation of NF-κB may give favorable circumstances for colon cancer cell growth. From this knowledge, the transcription factor NF-κB can be specifically targeted to prevent colon cancer cell growth. We, in the present study, found that inflexinol, a kaurane diterpene compound, inhibited constitutively activated NF-κB in colon cancer cells. Inflexinol also prevented NF-κB target antiapoptosis gene expressions (but increased apoptosis gene expression) as well as colon cancer cell growth in in vitro cell cultures and in in vivo xenograft animal model. In further chemical target identification studies, we found that inflexinol modifies a cysteine residue of p50 in NF-κB. The present data, therefore, indicated that inflexinol, a diterpenoid compound isolated from genus Isodon, effectively inhibited colon cancer cell growth through inactivation of NF-κB by directly acting on cysteine residue of p50.

The genus Isodon (also called Rabdosia) is a rich source of diterpenoids, especially the highly oxidized kaurane diterpenes. It was previously found that kaurane diterpenoid compounds have a number of biological properties such as the induction of apoptosis (19, 31) and cytotoxicity against cancer cells, as well as anti-inflammatory effects through inactivation of NF-κB (20, 21). Kamebakaurin, isolated from Isodon japonicus, suppressed the TNF-α–induced expression of antiapoptotic proteins via inactivation of NF-κB DNA-binding activity in human breast cancer cells (MCF-7; ref. 19). Kamebanin and kamebacetal A, which are isolated from I. japonicus, also inhibited the LPS-induced nitric oxide and prostaglandin E2 production through suppression of NF-κB activation in RAW 264.7 cells (21). Similarly, oridonin and ponicidin, isolated from the leaves of Rabdosia rubescens, and ent-kaurane diterpenoid compounds significantly inhibited the cell growth through inactivation of NF-κB in human breast carcinoma cells (MCF-7; ref. 32). We also previously showed that inflexinol inhibited the NF-κB activity in LPS-treated macrophage RAW 264.7 cells (33) and astrocytes (22). Together, these data indicated that inflexinol, in agreement with other kaurane diterpenoid compounds, inhibits NF-κB in colon cancer cells, and this effect was in conjunction with the effect on cell growth inhibition and on the induction of apoptosis. Several studies have shown that the naturally occurring anticancer drugs inhibit cancer cell growth through inhibition of NF-κB. Curcumin inhibited the growth of human myeloid leukemia cells (U937) through inhibition of NF-κB (34). Similar effects were found with indole-3-carbinol in human T-cell leukemia cells (Jurkat; ref. 16), and with epigallocatechin 3-gallate and capsaicin in other cancer cells (35, 36). Trichostatin A and eriocalyxin B (37) induced apoptosis through inactivation of NF-κB in tongue carcinoma cells as well. We also recently reported that 2′-hydroxycinnamaldehyde and obovatol inhibit colon cancer cell growth through inhibition of TNF-α–induced activation of NF-κB in human colon cancer cells (38, 39).

The mechanism of the inhibitory effect on NF-κB by inflexinol is not clear. However, we found that the suppression of NF-κB activation by inflexinol was reversed by treatment of thiol-reducing agents, suggesting that inflexinol alters the thiol-redox status of the cells. This suppressive effect on NF-κB was parallel with the cell growth inhibitory effect of inflexinol. Moreover, the growth inhibition and DNA binding activity of NF-κB in colon cancer cells transfected a p50 mutant with a C62S mutation were not observed after treatment with inflexinol, further indicating the possibility that inflexinol may interact with cysteine residue of NF-κB. Thus, it is possible that inflexinol mediates its effects by modification of cysteine residue present in p50 of NF-κB. In fact, in the presence of the p50 antibody, the DNA binding activity was diminished in the supershift assay of NF-κB. It is noteworthy that inflexinol contains a α-methylenecyclopentanone moiety, which is known to interact with nucleophiles, especially cysteine sulfhydryl groups in proteins, by a Michael-type addition (40). In fact, direct interaction between p50 and inflexinol was found in the sol-gel biochip as well as surface plasmon resonance analysis. Very similar to our findings, kamebakaurin and kamebanin were found to be able to interact with both the p50 and p65 subunits of NF-κB (41). Similar inhibition mechanisms have been shown by other agents, such as N-ethylmaleimide, cyclopentenone prostaglandin, 2-hydroxycinnamaldehyde, snake venom toxin, and melittin, which showed anti-inflammatory and anticancer properties (24, 26, 38, 42). Cysteine residues in IκB kinases (IKK) have also been modified by same mechanism with several compounds such as arsenite (43), auranofin (44), and cyclopentenone prostanoids prostaglandin A1 and 15d-prostaglandin J2 and their derivatives (45). Inflexinol could act through inhibition of IKK activity by modification of cysteine residue of IKK. The inhibitory effect of inflexinol on IKKβ activity as well as phosphorylation of IKKα and IKKβ was also found. In addition, the inhibitory effect of inflexinol on cell growth was not observed in the cells transfected with IKKα (C178A) and IKKβ (C179A) mutants having cysteine residues replaced with alanine. Redox alteration of other molecules, such as glutathione having cysteine residues by inflexinol, could also be possibly seen with 13-hydroxy-15-oxo-zoapatlin, an ent-kaurane diterpene, which induced apoptosis in human leukemia cells (46), and with Melissoidesin G, a diterpenoid purified from Isodon melissoides, which induced leukemic cell apoptosis (47).

Many genes, including the antiapoptosis genes Bcl-XL, Bcl-2, IAP-1/2, XIAP, and TNF receptor–associated proteins 1/2 (TRAF 1/2), and the proapoptosis genes caspase-3, caspase-9, and Bax, were known to be regulated by NF-κB (8). It was also found that the naturally occurring anticancer drugs inhibit cancer cell growth through regulation of NF-κB target gene expressions. Curcumin regulated the expression of NF-κB–regulated gene products involved in cellular proliferation (cyclin D1, COX-2, and c-myc) and antiapoptosis (IAP1, IAP2, XIAP, Bcl-2, Bcl-xL, Bfl-1/A1, TRAF1, and cellular FLIP) protein in human myeloid leukemia cells (U937; ref. 30). Indole-3-carbinol also inhibited genes involved in cell proliferation (cyclin D1 and COX-2), antiapoptosis (survivin, cIAP1/2, XIAP, Bcl-2, Bfl-1/A1, TRAF1, and FLIP), and invasion (MMP-9) in human T-cell leukemia cells (Jurkat; ref. 16). Our previous study also showed that 2′-hydroxycinnamaldehyde inhibited genes involved in cell proliferation (cyclin D1) and antiapoptosis (Bcl-2) in human colon cancer cells (35). Moreover, kaurane diterpene and kamebakaurin also inhibited the antiapoptotic NF-κB target genes cIAP1 (hiap-2) and cIAP2 (hiap-1), members of the inhibitor of apoptosis family, and Bfl-1/A1, a prosurvival Bcl-2 homologue in cancer cells (19). Oridonin has also been found to down-regulate the levels of Mcl-1 and BCL-x(L), antiapoptotic Bcl-2 family members in lymphoid malignancies (48). Agreeing with these findings, our data showed that inflexinol clearly inhibited NF-κB–regulated gene products involved in cell proliferation (cyclin D1 and COX-2) and antiapoptosis (XIAP, IAP1/2, and Bcl-2) and increased proapoptosis genes (caspase-3, caspase-9, and Bax) in vitro as well as in vivo. Thus, it is possible that an alteration in the expression level of NF-κB target antiapoptotic and proapoptotic proteins is likely to influence apoptotic cell death by inflexinol.

Although many anticancer effects in vitro and other possible mechanisms have been shown by kaurane diterpenoid compounds, in vivo anticancer effects have not been shown. Here, we showed the inhibitory effect of inflexinol on tumor (SW620) growth in vivo using the colon tumor xenograft nude mouse model. We observed that inflexinol significantly inhibited the growth of colon tumors as determined by the decrease in tumor volume and weight accompanied by a decrease in the number of PCNA and Ki-67 reactive cells. In in vivo studies, we also found that inflexinol decreased the translocation of p65 and p50 into nucleus as determined by Western blotting or immunohistochemistry. The tumor growth inhibition (40% inhibition over untreated tumor bearing group) by inflexinol (36 mg/kg) treatment for 3 weeks is half of that seen with the conventional chemotherapeutic drug docetaxel (10 mg/kg, 75% inhibition). However, in contrast to docetaxel (40% weight loss), we did not observe weight loss, one indicator of side effects of chemotherapeutic drugs, or any cytotoxic effects in normal cultured cells. We also found that inflexinol did not show any toxicities in the 4-week repeated toxicity study, with 100 times higher dose of inflexinol than the dose used in antitumor study. We also found that inflexinol has good oral and intestinal absorption as determined by the Caco-2 and Madin-Darby canine kidney cell permeability assays, and was found to not pass through the brain-blood barrier in the absorption, distribution, metabolism, excretion, and toxicity (ADME/Toxicity) test using prediction program preADME (version 1.0.2). Inflexinol was also evaluated as having no rodent carcinogenicity (data not shown). In conclusion, the current study showed that inflexinol exerts its cell growth inhibitory effects through inhibition of NF-κB by binding to cysteine residue of p50 in human colon cancer cells, suggesting that inflexinol can be a useful agent for treatment of colon cancer cell growth.

No potential conflicts of interest were disclosed.

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