Article pubs.acs.org/jnp
Antitumor Activity of 2‑Hydroxycinnamaldehyde for Human Colon Cancer Cells through Suppression of β‑Catenin Signaling Min Ai Lee,† Hyen Joo Park,† Hwa-Jin Chung,† Won Kyung Kim,† and Sang Kook Lee*,† †
College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742, Korea ABSTRACT: The antiproliferative and antitumor activities of 2-hydroxycinnamaldehyde (1), a phenylpropanoid isolated from the bark of Cinnamomum cassia, were investigated using human colorectal cancer cells. Compound 1 exhibited antiproliferative effects in HCT116 colon cancer cells, accompanied by modulation of the Wnt/β-catenin cell signaling pathway. This substance was found also to inhibit β-catenin/T-cell factor (TCF) transcriptional activity in HEK293 cells and HCT116 colon cancer cells. Further mechanistic investigations in human colon cancer cells with aberrantly activated Wnt/β-catenin signaling showed that 1 significantly suppressed the binding of β-catenin/TCF complexes to their specific genomic targets in the nucleus and led to the down-regulation of Wnt target genes such as c-myc and cyclin D1. In an in vivo xenograft model, the intraperitoneal administration of 1 (10 or 20 mg/kg body weight, three times/week) for four weeks suppressed tumor growth in athymic nude mice implanted with HCT116 colon cancer cells significantly, without any apparent toxicity. In an ex vivo biochemical analysis of the tumors, compound 1 was also found to suppress Wnt target genes associated with tumor growth including β-catenin, c-myc, cyclin D1, and survivin. The suppression of the Wnt/β-catenin signaling pathway is a plausible mechanism of action underlying the antiproliferative and antitumor activity of 1 in human colorectal cancer cells.
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into the nucleus, which complexes with members of the T-cell transcription factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors and induces the transcription of downstream target genes.16−18 These target genes include cell growth-regulating genes and genes that are associated with cancer cell invasion and metastasis such as cmyc,19 cyclin D1,20 MMPs,21 and uPA.22 Dysregulation of the Wnt/β-catenin signaling pathway has been associated with a number of diseases in humans, including a wide range of malignancies such as lung, breast, skin, and colorectal cancers.17,23,24 Aberrant Wnt signaling is particularly important in the carcinogenesis and progression of colorectal cancer; APC mutations are found in 80−90% of cases of sporadic colorectal cancer.17,25 β-Catenin mutations are also found in a majority of cases without APC mutations, highlighting the importance of understanding the function of Wnt/β-catenin signaling in colorectal cancer.26,27 Therefore, the identification of Wnt/βcatenin signaling inhibitors is considered an attractive strategy toward developing targeted therapies for the treatment of colon cancer. 2-Hydroxycinnamaldehyde (1) is a phenylpropanoid that can be isolated from the cortex of Cinnamomum cassia Blume (Lauraceae).28 This is a medicinal plant that is widely used in alternative medicine and is used also as a spice worldwide.29 As one of the active constituents of C. cassia, 1 is known to have many biological activities, such as antiangiogenic, anti-inflam-
he Wnt signaling pathway is an evolutionarily conserved pathway that is involved in various biological processes, including axis formation, stem cell maintenance, cell motility, and cell-cycle regulation.1−3 Among the Wnt signaling pathways, the canonical pathway involving β-catenin as a key mediator is by far the best characterized. Other Wnt signaling pathways (noncanonical pathways) include the planar cell polarity pathway and the Wnt/Ca2+ pathway.4−6 β-Catenin performs dual roles; its membrane-bound form interacts with the cytoplasmic domain of E-cadherin and contributes to cell−cell adhesion, while the cytosolic form participates predominantly in signal transduction.7,8 In the absence of Wnt ligands, unbound β-catenin is sequestered in a multiprotein destruction complex that consists of adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3β (GSK3β), and casein kinase 1α (CK1α). The formation of this complex induces the phosphorylation of βcatenin at Ser45 by CK1α, which, in turn, initiates β-catenin phosphorylation at Thr41, Ser37, and Ser33 by GSK3β.9,10 Phosphorylated β-catenin is specifically recognized and polyubiquitinated by the F-box protein β-transducin-repeat-containing protein (β-TrCP), an E3 ligase, and subsequently undergoes proteasomal degradation.11,12 Wnt signaling is activated when Wnt proteins bind to a receptor complex comprised of the Frizzled receptor and the low-density lipoprotein receptorrelated protein 5/6 (LRP5/6) coreceptor.13,14 This induces the phosphorylation of dishevelled (dvl), which leads to the inhibition of GSK3β, resulting in the dissociation of the destruction complex and the accumulation of unphosphorylated β-catenin.15 As a consequence, “free” β-catenin is translocated © 2013 American Chemical Society and American Society of Pharmacognosy
Received: March 15, 2013 Published: July 15, 2013 1278
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matory, and antiplatelet aggregative activities.30,31 This compound has also been reported to have antiproliferative or proapoptotic activities in various cancer cell lines, such as skin, ovarian, colon, and oral cancer cells.32,33 However, the precise mechanism of action of 1 in the inhibition of human colorectal cancer cell growth remains to be clarified.
as a potent inhibitor in this assay system. Therefore, in the present study, the plausible mechanisms of action underlying the antiproliferative activity and the modulation of the Wnt/βcatenin signaling pathway in human colon cancer cells by 1 were investigated. Compound 1 effectively inhibited the TOPflash reporter gene in a concentration-dependent manner (Figure 1A). In addition, this compound was found to have a far more potent inhibitory effect on TOPflash reporter activity than did 2methoxycinnamaldehyde (2) and cinnamaldehyde (3), which are other phenylpropanoids isolated from C. cassia (Figure 1A). To further confirm the specificity of 1 to Wnt/β-catenin signaling, the TOPflash and FOPflash reporter activity was evaluated in HEK293 cells. Although compound 1 inhibited the TOPflash activity in a concentration-dependent manner, this substance did not affect the activity of FOPflash, which contains mutations in its TCF binding sites. These results suggest that 1 is a specific inhibitor of Wnt/β-catenin/TCF-responsive transcriptional activity (Figure 1B). Since Wnt signaling is known to be activated aberrantly in a majority of colon cancer cells,1,12,27 the effects of 1 were studied on Wnt signaling in HCT116 colon cancer cells. HCT116 cells have a heterozygous deletion of codon 45 of the β-catenin gene, resulting in a loss of the serine residue. As this serine residue is essential for the phosphorylation and degradation of β-catenin, the deletion of serine 45 inhibits the degradation of β-catenin. This leads to the accumulation of β-catenin and consequently evokes sustained activation of the Wnt signaling pathway.35 To investigate the suppressive effects of 1 on Wnt/β-cateninresponsive transcriptional activity in colon cancer cells, HCT116 cells were transfected with either the TOPflash or FOPflash plasmid and incubated with 1 for 24 h. HCT116 cells transfected with the TOPflash reporter gene showed the highest transcriptional activity, and treatment with 1 decreased the luciferase activity in a concentration-dependent manner (Figure 2A). These findings suggest that 1 inhibits the endogenously activated Wnt/β-catenin signaling in HCT116 colon cancer cells. To further elucidate whether the suppressive effect of 1 on the transcriptional activity of β-catenin/TCF is associated with the
In the present study, reported are the antitumor activity of 1 and its mechanisms of action in human colon cancer cells, which involve the Wnt/β-catenin signaling pathway, both in cultured cells and in the nude mouse xenograft model used.
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RESULTS AND DISCUSSION To identify novel small molecule regulators of the Wnt/βcatenin signaling pathway, several natural products were evaluated using an HEK293 cell-based TOPflash reporter gene assay. T-cell factor (TCF) reporter plasmid contains two sets (with the second set in the reverse orientation) of three copies of the binding site (wild type) upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame (TOPflash, pGL3-OT). However, FOPflash (pGL3-OF) contains mutated TCF binding sites and is, thus, used as a negative control. TCFs comprise a family of DNA-binding transcriptional activators that are essential for lymphoid cell development. These transcription factors are activated by the Wnt pathway. In particular, TCF-4 is mainly localized in the cytoplasm and is translocated into the nucleus bound to β-catenin in a cooperative manner. In a previous study, it was found that several classes of natural compounds such as lignans,34 phenylpropanoids, and curcuminoids have the potential to inhibit TOPflash activity. Among the active compounds, 2-hydroxycinnamaldehyde (1) was identified
Figure 1. The inhibitory effect of 2-hydroxycinnamaldehyde (1) on β-catenin/TCF transcriptional activity in HEK293 cells. (A) HEK293 cells were transfected transiently with β-catenin, TCF4, TOPflash, and the Renilla gene and then incubated for 24 h. The luciferase activity was determined after treatment with compounds 1−3 for 24 h. (B) The TOPflash or FOPflash activity was determined in the presence of compound 1 in HEK293 cells that were transiently transfected with the TOPflash or FOPflash reporter gene. The TOPflash or FOPflash activity was normalized to Renilla activity and expressed as relative units. The results indicate the means ± SD (n = 3) and are representative of the findings from four separate determinations (*p < 0.05 or **p < 0.01 compared to control). 1279
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Figure 2. The inhibitory effect of 2-hydroxycinnamaldehyde (1) on the β-catenin/TCF transcriptional activity in HCT116 colon cancer cells. (A) HCT116 cells were cotransfected with TOPflash (or FOPflash) and the Renilla gene for 24 h and then treated with 1 for an additional 24 h. The luciferase activity for TOPflash or FOPflash was determined. The results indicate the means ± SD (n = 3) and are representative of the findings from four separate determinations (*p < 0.05, **p < 0.01). (B) The inhibitory effects of 1 on the binding of TCF complexes to DNA. HCT116 cells were treated with 5 or 10 μM 1 for 30 min, and then nuclear extracts were isolated. EMSA was performed with nuclear extracts from 1-treated or vehicle-treated control cells.
Figure 3. The suppressive effect of 2-hydroxycinnamaldehyde (1) on the expression of Wnt target genes in HCT116 colon cancer cells. (A) The inhibitory effects of 1 on the mRNA levels of Wnt target genes. HCT116 cells were treated with the indicated concentrations of 1 for 12 h, and the mRNA levels of Wnt target genes were examined using real-time PCR. The results are presented as a relative expression compared to control cells and normalized to β-actin. (B) The suppressive effects of 1 on the levels of proteins encoded by Wnt target genes. HCT116 cells were treated with the indicated concentrations of 1 for 24 or 48 h, and the protein expressions of β-catenin, c-myc, and cyclin D1 were determined by Western blot analysis. βActin was used as an internal standard. The data shown are the means ± SD of four determinations (*p < 0.05, **p < 0.01).
binding of β-catenin/TCF complexes to DNA promoter regions, an electrophoretic mobility shift assay was performed to investigate the potential change in DNA-TCF complex binding in HCT116 cells. A 30 min treatment with compound 1 effectively inhibited the binding of DNA to TCF through the TCF binding site (Figure 2B). These findings suggest that 1
suppresses β-catenin/TCF transcriptional activity in HCT116 cells by inhibiting the binding between DNA and β-catenin/TCF complexes. Since compound 1 inhibited β-catenin/TCF responsive transcriptional activity in colon cancer cells, the effects of 1 were assessed on the expression of endogenous Wnt target genes 1280
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colorectal cancer,46 was used as a reference compound. The tumor volumes in the vehicle-treated control group were approximately 1300 mm3 on day 35 after inoculation with the HCT116 cells. The tumor growth was inhibited significantly in mice treated with 10 or 20 mg/kg 1 compared with control mice (p < 0.05, Figure 5A). The tumor volumes in the treatment
using real-time PCR. The mRNA levels of Wnt target genes such as CCND1 (cyclin D1) and CMYC (c-myc), which are also essential components of the cell cycle progression and cell proliferation signaling pathways,36−38 were down-regulated by 1 in HCT116 cells (Figure 3A). Furthermore, treatment with 1 decreased the mRNA expression levels of MMP7 (mmp7 gene) and PLAU (urokinase plasminogen activator gene) (Figure 3A), which are Wnt target genes known to be linked to cancer cell invasiveness and motility.39,40 The mRNA levels of AXIN2, NKD1, and DKK1, all of which are known to be Wnt target genes,41−44 were also suppressed (Figure 3A). These results demonstrated that compound 1 is able to inhibit cancer cell proliferation and invasiveness in Wnt-activated colon cancer cells by inhibiting the Wnt/β-catenin signaling pathway. In addition, Western blot analysis revealed that the down-regulation of CCND1 and CMYC by 1 suppressed the protein expression of cyclin D1 and c-myc (Figure 3B). The levels of total β-catenin protein were also down-regulated by 1 (Figure 3B), implying that the Wnt inhibitory effects of 1 might be associated with the down-regulation of β-catenin. Since sustained Wnt signaling and the upregulation of βcatenin play essential roles for cancer cell survival and proliferation,45 and because compound 1 suppressed the expression of cyclin D1 and c-myc, which are major factors in cancer cell proliferation, it was investigated if compound 1 affects the proliferation of cancer cells through the inhibition of Wnt signaling in colon cancer cells. The antiproliferative activity of 1 was assessed using a sulforhodamine B (SRB) assay in cultured HCT116 colon cancer cells. Compound 1 exhibited time- and concentration-dependent antiproliferative effects in vitro (Figure 4).
Figure 5. The antitumor activity of 2-hydroxycinnamaldehyde (1) in a tumor xenograft model. (A) HCT116 cells (2 × 106 cells/mouse) were injected subcutaneously into the flanks of nude mice. Treatment with the test compounds was initiated when tumor volumes reached ∼100 mm3. Compound 1 (10 or 20 mg/kg body weight) was administered intraperitoneally three times per week in a volume of 200 μL. The control group was treated with an equal volume of vehicle (normal saline containing 0.5% Tween 80). Irinotecan was used as a reference compound and administered twice a week (12.5 mg/kg body weight, ip). Tumor volumes were measured with a caliper every 2−3 days (**p < 0.01 was considered statistically significant). (B) Changes in the body weights of the mice were monitored during the experiments.
Figure 4. The antiproliferative effect of 2-hydroxycinnamaldehyde (1) in HCT116 human colon cancer cells. HCT116 cells were treated with various concentrations of 1 for 24, 48, or 72 h. Cell proliferation was measured using the SRB assay. IC50 values were calculated by nonlinear regression analysis using TableCurve 2D v5.01 (Systat). The data shown are the means ± SD of four determinations (*p < 0.05, **p < 0.01).
groups by 2-hydroxycinnamaldehyde were 52% and 69% of the control volume (100%) for 10 and 20 mg/kg 1, respectively (Figure 5A). No overt toxicity or change in body weight was observed in the treatment groups as compared to the control group (Figure 5B). In addition, biochemical analysis of the tumors was conducted to assess the relationship between the antitumor effect of 1 and its Wnt inhibitory effect. The mRNA levels of Wnt target genes CCND1, CMYC, MMP7, PLAU, and NKD1 were downregulated in the tumors of the 1-treated groups compared to those of the vehicle-treated control groups (Figure 6A).
The in vivo antitumor activity of 1 was evaluated in a nude mouse xenograft model implanted with HCT116 human colon cancer cells. HCT116 cells (2 × 106 cells/mouse) were injected sc into the right flank region of each nude mouse. When the tumor volume reached approximately 100 mm3, compound 1 (10 or 20 mg/kg body weight) was administered intraperitoneally three times a week for approximately four weeks. Irinotecan, a chemotherapeutic agent that is widely used to treat 1281
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Figure 6. The suppressive effect of 2-hydroxycinnamaldehyde (1) on the expression of Wnt target genes in vivo in tumor tissues. (A) mRNA was extracted from frozen xenograft tumor tissue samples, and the mRNA levels of Wnt target genes were determined using real-time PCR. The results are presented as a relative expression compared to vehicle-treated controls and normalized to β-actin. (B) Small portions of tumors from each group were thawed on ice and homogenized in Complete Lysis Buffer (Active Motif). The expression levels of the β-catenin, c-myc, cyclin D1, and survivin proteins were determined by Western blot analysis. β-Actin was used as an internal standard. (C) Immunohistochemical analysis of β-catenin, cyclin D1, and Ki67 in tumor tissue sections. Formalin fixed, paraffin-embedded tumor sections were blocked, probed with the indicated antibodies, and detected using the LSAB+ System-HRP Kit (Dako). Sections were counterstained with hematoxylin and photographed with an inverted phase-contrast microscope (200× magnification).
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Furthermore, Western blot analysis showed that protein levels of cyclin D1, c-myc, and survivin, all of which are Wnt targets associated with cell proliferation and cell survival,36,38,47,48 were suppressed in the tumors of the 1-treated groups (Figure 6B). An immunohistochemical analysis of the tumors also indicated that 1 suppressed the expression of β-catenin and cyclin D1 (Figure 6C). Compound 1 also inhibited the expression of the proliferation biomarker Ki-67 in tumor cells (Figure 6C). These results confirm that 1 effectively inhibits the in vivo tumor growth of colon cancer cells, and the antitumor activity of 1 might be associated with the inhibition of the Wnt signaling pathway. In summary, the present study demonstrates that 2hydroxycinnamaldehyde (1) might be a candidate for the development of small-molecule inhibitors of Wnt signaling and that the antitumor activity of 1 is associated with the downregulation of Wnt signaling in colon cancer cells. These findings indicate that this phenylpropanoid is a promising candidate natural anticancer agent for the management of human colorectal cancer.
EXPERIMENTAL SECTION
General Experimental Procedures. Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), antibiotics-antimycotics solution, TRI reagent, and Lipofectamine 2000 were purchased from Invitrogen (Grand Island, NY). Bovine serum albumin (BSA), trichloroacetic acid (TCA), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. pTOPFlash and pFOPFlash reporter plasmids were obtained from Upstate Biotechnology (Lake Placid, NY). Mouse anti-β-catenin, anticyclin D1, and anti-c-myc antibodies were purchased from BD Biosciences (San Diego, CA). Mouse antisurvivin, anti-TCF4, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A complete protease inhibitor cocktail was purchased from Roche Applied Science (Penzberg, Germany). Gene-specific primers for real-time PCR were synthesized by Bioneer (Daejeon, Korea). The Reverse Transcription Kit and the Dual Luciferase Reporter Assay System were purchased from Promega (Madison, MA). The TCF-electrophoretic mobility shift assay kit was purchased from Panomics (Redwood City, CA). 2-Hydroxycinnamaldehyde (1; purity > 98%), 2-methoxycinnamaldehyde (2; purity > 98%), and cinnamaldehyde (3; purity > 98%), were provided by Korea Food & Drug Administration (KFDA). 1282
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Cell Culture. HCT116 human colorectal cancer cells and HEK293 human embryonic kidney cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in DMEM (HEK293 cells) or RPMI1640 (HCT116 cells) supplemented with 10% FBS and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B) in a humidified incubator containing 5% CO2 at 37 °C. Transfection and Luciferase Reporter Gene Assays. Transient transfections were performed using Lipofectamine 2000 (Invitrogen). Cells were seeded in 48-well plates at a density of 1.5 × 104 cells per well. After 24 h of incubation, cells were transfected with 0.1 μg of the luciferase reporter plasmid (TOPflash or FOPflash) and 0.005 μg of the Renilla gene for normalization. HEK293 cells were also cotransfected with 0.02 μg pcDNA β-catenin and 0.004 μg of the TCF4 expression vector for Wnt activation. After 24 h of transfection, cells were treated with a test compound or solvent control. At 24 h, the cells were lysed and subjected to a dual luciferase activity assay using the Dual Luciferase Reporter Assay System (Promega), according to the manufacturer’s recommendations. All experiments were performed in triplicate. Luciferase activity was normalized to Renilla activity and is expressed relative to the vehicle control. Isolation of Nuclear Extracts. Cells (1 × 106 cells) were treated with the test compounds for 30 min. Harvested cells were washed with PBS, suspended in ice-cold lysis buffer (10 mM Tris-HCl [pH 8.0], 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1.5% NP-40, 50 mM sodium fluoride, 5 mM sodium orthovanadate, and protease inhibitor cocktail) on ice for 5 min. After centrifugation at 2 500 rpm for 4 min at 4 °C, the supernatant was decanted, and the pellets were washed twice with ice-cold lysis buffer without NP-40. The cells were resuspended in hypertonic nuclear extract buffer (20 mM Tris-HCl [pH 8.0], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 50 mM sodium fluoride, and protease inhibitor cocktail) on ice for 10 min and then centrifuged at 140 000 rpm for 15 min at 4 °C. This supernatant, which contained nuclear extracts, was collected and stored in aliquots at −70 °C. The protein content of the cell lysates was determined using the Bradford assay.49 Cell Proliferation Assay. Cells were seeded in 96-well plates (6 × 103 cells/well), incubated for 24 h, and fixed (for day zero controls) or treated with the test compounds for 24, 48, and 72 h. After incubation, cells were fixed with 10% trichloroacetic acid (TCA), dried, and stained with 0.4% SRB in 1% acetic acid. The unbound dye was washed out, and the stained cells were dried and resuspended in 10 mM Tris (pH 10.0). The absorbance at 515 nm was measured, and cell proliferation was determined as follows: cell proliferation (%) = (average absorbance compound − average absorbance day zero)/(average absorbance control − average absorbance day zero) × 100. IC50 values were calculated by nonlinear regression analysis using TableCurve 2D v5.01 (Systat Software Inc., Richmond, CA, USA). Western Blotting. Total cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The protein concentration was determined, and equal amounts of protein samples were subjected to 8−13% SDSPAGE. Separated proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA) and probed with the indicated antibodies. The blots were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, U.K.). RNA Extraction and Real-Time PCR. Total RNA was extracted from cells or tumor tissues using the TRI reagent (Invitrogen) and reverse-transcribed using the Reverse Transcription System (Promega), according to the manufacturer’s instructions. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions. The thermocycling conditions utilized were 20 s at 95 °C; 40 cycles of 20 s at 95 °C, 20 s at 56 °C, and 30 s at 72 °C; 1 min at 95 °C; and 1 min at 55 °C. All experiments were performed in quadruplicate. The sequences of the primers used are listed in Table 1. The threshold cycle (CT), indicating the fractional cycle number at which the amount of amplified target gene in each well reaches a fixed threshold, was determined using MJ Research Opticon Monitor software. Relative quantification, representing the difference in gene expression as measured by real-time quantitative PCR between 1 and
Table 1. Sequences of Target Gene-Specific Primers Used in Real-Time PCR target genes CCND1 (cyclin D1) CMYC
MMP7 PLAU
NKD1 DKK1
sequences sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense
AXIN2
sense antisense
ATCB (β-actin)
sense antisense
5′-GAA GAT CGT CGC CAC CTG-3′ 5′-GAC CTC CTC CTC GCA CTT CT-3′ 5′-CAC CAG CAG CGA CTC TGA-3′ 5′-GAT CCA GAC TCT GAC CTT TTG3′ 5′-GAC ATC ATG ATT GGC TTT GC-3′ 5′-TCT CCT CCG AGA CCT GTC C-3′ 5′-TTG CTC ACC ACA ACG ACA TTG3′ 5′-GGC AGG CAG ATG GTC TGT AT-3′ 5′-GGG AAA CTT CAC TCC AAG CC-3′ 5′-CTC CCG ATC CAC TCC TCG AT-3′ 5′-CCT TGA ACT CGG TTC TCA ATT CC-3′ 5′-CAA TGG TCT GGT ACT TAT TCC CG-3′ 5′-CAA CAC CAG GCG GAA CGA A-3′ 5′-GCC CAA TAA GGA GTG TAA GGA CT-3′ 5′-AGC ACA ATG AAG ATC AAG AT-3′ 5′-TGT AAC GCA ACT AAG TCA TA-3′
the untreated control group, was calculated by the comparative CT method.50 The data were analyzed using the equation 2−△△CT, where △△CT = [CT of target gene − CT of housekeeping gene]treated group − [CT of target gene − CT of housekeeping gene]untreated control group. For the treated samples, 2−△△CT represents the fold change in gene expression, normalized to the housekeeping gene β-actin, relative to the untreated control. Electrophoretic Mobility Shift Assay. The binding of activated TCF and the sequence of the TCF response element were evaluated using the TCF-electrophoretic mobility shift assay kit. Activated TCF was applied as nuclear cell extracts from HCT116 cells. The binding reactions contained 8 μg of extracted protein and 1.5 pmol of biotinlabeled TCF binding probe and were incubated at 15 °C for 30 min. The products were separated on 6% nondenaturing polyacrylamide gels and visualized using an ECL detection system. In Vivo Tumor Xenograft Model. All animal use and care followed the guidelines approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC; permission number: SNU120102-2). Female nude mice (4−6 weeks old, BALB/c-nu) were purchased from Central Laboratory Animal, Inc. (Seoul, Korea) and housed in the animal care facility at Seoul National University under pathogen-free conditions with a 12 h light−dark schedule. After animals were acclimated for one week, HCT116 cells were injected subcutaneously into the flanks of mice (2 × 106 cells in 200 μL medium) using 27-gauge needles. The resulting tumors were allowed to develop for 8 days until they reached approximately 100 mm3. The mice were randomized into vehicle control and treatment groups of five animals per group. Compound 1 (10 or 20 mg/kg body weight) was dissolved in 200 μL vehicle solution (0.5% Tween 80 in normal saline) and administered intraperitoneally three times a week for 4 weeks. Irinotecan (12.5 mg/kg body weight), a positive reference sample, was administered two times a week. The control group was treated with an equal volume of vehicle. Tumor growth was measured using a digital slide caliper, and tumor volume was estimated according to the following formula: Tumor volume (mm3) = L × W × H/2, where L is the length, W is the width, and H is the height of the tumor. The experiment was terminated when the average tumor volume of the control group reached approximately 1300 mm3. The mice were euthanized, and the tumors were excised, weighed, and frozen for further biochemical analysis. Toxicity was assessed based on the lethality and body weight loss exhibited by the nude mice. 1283
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Immunohistochemistry of Tumors. Excised tumor tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Serial sections of the embedded specimens were deparaffinized, rehydrated, and subjected to antigen retrieval. The slides were incubated with antiKi67 antibody, which was detected using the LSAB+ System-HRP kit (Dako, Glostrup, Denmark) and counterstained with hematoxylin. Stained sections were observed and photographed under an inverted phase-contrast microscope. Ex Vivo Biochemical Analysis of Tumors. A portion of the frozen tumor excised from each of the nude mice was thawed on ice and homogenized using a hand-held homogenizer in Complete Lysis Buffer (Active Motif, Carlsbad, CA, USA). The protein concentrations of the tumor lysates were determined, and aliquots were stored at −80 °C. Statistical Analysis. The data are expressed as the means ± SD of the indicated number of independently performed experiments. Statistical significance (p < 0.05) was assessed using Student’s t-test or one-way analysis of variance (ANOVA) coupled with Dunnett’s t-test.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-2-880-2475. Fax: +82-2-762-8322. E-mail: sklee61@ snu.ac.kr.. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20120004939).
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dx.doi.org/10.1021/np400216m | J. Nat. Prod. 2013, 76, 1278−1284
