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Galectin-3-independent Down-regulation of GABABR1 due to Treatment with Korean Herbal Extract HAD-B Reduces Proliferation of Human Colon Cancer Cells
Galectin-3-independent Down-regulation of GABABR1 due to Treatment with Korean Herbal Extract HAD-B Reduces Proliferation of Human Colon Cancer Cells
Journal of Pharmacopuncture. 2012. Jul, 15(3): 19-30
Copyright ©2012, KOREAN PHARMACOPUNCTURE INSTITUTE
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : July 04, 2012
  • Accepted : September 10, 2012
  • Published : July 30, 2012
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About the Authors
Kyung-Hee Kim
Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea
Yong-Kyun Kwon
East-West Cancer Center, Dunsan Oriental Hospital of Daejeon University, Daejeon, Korea
Chong-Kwan Cho
East-West Cancer Center, Dunsan Oriental Hospital of Daejeon University, Daejeon, Korea
Yeon-Weol Lee
East-West Cancer Center, Dunsan Oriental Hospital of Daejeon University, Daejeon, Korea
So-Hyun Lee
Research Institute, National Cancer Center, Goyang, Korea
Sang-Geun Jang
Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea
Byong-Chul Yoo
Research Institute, National Cancer Center, Goyang, Korea
Hwa-Seong Yoo
East-West Cancer Center, Dunsan Oriental Hospital of Daejeon University, Daejeon, Korea

Abstract
Objectives:
Many efforts have shown multi-oncologic roles of galectin-3 for cell proliferation, angiogenesis, and apoptosis. However, the mechanisms by which galectin-3 is involved in cell proliferation are not yet fully understood, especially in human colon cancer cells.
Methods:
To cluster genes showing positively or negatively correlated expression with galectin-3, we employed human colon cancer cell lines, SNU-61, SNU-81, SNU-769B, SNU-C4 and SNU-C5 in high-throughput gene expression profiling. Gene and protein expression levels were determined by using real-time quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. The proliferation rate of human colon cancer cells was measured by using a 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
Results:
Expression of γ-aminobutyric acid B receptor 1 (GABABR1) showed a positive correlation with galectin-3 at both the transcriptional and the translational levels. Downregulation of galectin-3 decreased not only GABABR1 expression but also the proliferation rate of human colon cancer cells. However, Korean herbal extract, HangAmDan-B (HAD-B), decreased expression of GABABR1 without any expressional change of galectin-3, and offset γ-aminobutyric acid (GABA)-enhanced human colon cancer cell proliferation.
Conclusions:
Our present study confirmed that GABABR1 expression was regulated by galectin-3. HAD-B induced galectin-3-independent down-regulation of GABABR1, which resulted in a decreased proliferation of human colon cancer cells. The therapeutic effect of HAD-B for the treatment of human colon cancer needs to be further validated.
Keywords
1. Introduction
Galectin-3 is a member of the family of β-galactoside-binding proteins that bind to the carbohydrate portion of cellsurface glycoproteins and glycolipids [ 1 ]. Galectin-3 has a chimera-type structure consisting of three different structural domains: a short NH2-terminal domain of 12 amino acids that contains a serine phosphorylation site; a repeated collagen-like sequence that rich in glycine, tyrosine, and proline amino acid residues, which serves as a substrate for matrix metalloproteinases (MMPs); and a COOHterminal carbohydrate recognition domain [ 1 - 3 ]. Galectin-3 is a multifunctional oncogene [ 1 ], which regulates cell growth [ 4 ], adhesion [ 5 ], proliferation [ 6 ], angiogenesis [ 7 ], and apoptosis [ 8 ].
Many studies have shown that galectin-3 regulates cancer cell proliferation. Galectin-3-stimulated cell proliferation of IMR-90 human lung fibroblasts [ 6 ]; a decrease of galectin-3 expression in activated T lymphocytes paralleled a downregulation or even a blocking of proliferation [ 9 ]; and the introduction of galectin-3 cDNA caused human lymphoma Jurkat T cells to grow faster [ 10 ]. A recent report provided evidence that downregulation of galectin-3 led to diminished human colon cancer cell proliferation via modulation of the hete-rogeneous nuclear ribonucleoprotein Q (hnRNP Q) level [ 11 ].
Overexpression of galectin-3 has been reported in gastric cancer [ 12 ]. Positive galectin-3 expression was observed in 84% of gastric cancer cases. In enhanced cells of a cancerous lesion, 48% showed stronger nuclear immunoreactivity than a cytoplasmic one whereas adjacent epithelial cells showed little or weak nuclear immunoreactivity [ 12 ]. In addition, decreased galectin-3 expression was found in breast [ 13 ], ovary [ 14 ], prostate [ 15 ], epithelial skin cancer [ 16 ], and head-and-neck squamous cell carcinomas [ 17 ] than in corresponding normal tissue.
HangAmDan (HAD)-B consists of eight species of Korean medicinal plants and animals (Table 1 ), and is an upgraded version of HangAmDan (HAD) used traditionally for solid masses, which also shows anti-angiogenic activity [ 18 ]. A mixture of these plants has been shown to exert strong anticancer activity against solid tumors, including pancreatic, lung, colorectal, and stomach cancers. Additionally, anti-angiogenesis effects and inhibition of cancer cell proliferation and metastasis have been reported [ 19 ]. In particular, case reports observed with HAD have been selected as part of the National Cancer Institute’s Best Case Series Program [ 20 ]. HAD-B has shown efficacy in inhibiting migration and proliferation of human umbilical vein endothelial cells and in limiting the formation of capillary tube structures [ 21 ]. Furthermore, a safety evaluation of HAD-B has revealed no side-effects in both healthy subjects and cancer patients [ 22 ].
Even though a number of studies have reported the functions of galectin-3 in many types of cancer, the mechanisms by which galectin-3 is involved in cell proliferation are not yet fully understood, especially in human colon cancer cells. In the present study we report that γ-aminobutyric acid B receptor 1 (GABABR1) expression is linked to galectin-3 in human colon cancer cell line, and we discuss the effect of galectin-3- independent down-regulation of GABABR1 by treatment with Korean herbal extract HAD-B in human colon cancer cells.
2. Materials and methods
- 2.1. Human colon cancer cell lines
Human colon cancer cell lines, SNU-61, SNU-81, SNU-769B, SNU-C4 and SNU-C5, were obtained from the Korean Cell Line Bank (Seoul, Korea).
- 2.2. Preparation of water extract of HAD-B
Ingredients of HAD-B
Scientific name Relative amount (mg)
Panax notoginseng Radix 84.0
Cordyceps Militaris 64.0
Santsigu Tuber 64.0
Ginseng Radix 64.0
Bovis Calculus 64.0
Margarita 64.0
Bostaurus var.domesticus Gmelin 48.0
Commiphora myrrha 48.0
Total amount (1 capsule) 500.0
HAD-B was provided from the East-West Cancer Center of Dunsan Oriental Medical Hospital, Daejeon University, Daejeon, Korea (Table 1 ). The water extract of HAD-B was prepared by extracting HAD-B powder with 10-times (v/w) the amount of distilled water at room temperature for 24 hrs. The extract was centrifuged at 1000×g for 30 mins and was then filtered and lyophilized. The extract powder was dissolved directly in distilled water.
- 2.3. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
A colorimetric assay using tetrazolium salt, MTT, was used to assess cell proliferation after galectin-3 suppression. MTT assays were performed as described in a previous report [ 11 ]. Briefly, equal numbers of cells were incubated in each well in 0.18 ml of culture medium to which 0.02 ml of 10 × 5-FU (Choongwae Pharma Corporation), HAD-B, GABA or PBS (for untreated 100% survival control) had been added. After 4 days of culture, 0.1 mg of MTT was added to each well and incubated at 37°C for a further 4 hrs. Plates were centrifuged at 450 × g for 5 mins at room temperature, and the medium was removed. Dimethyl sulfoxide (0.15 ml) was added to each well to solubilize the crystals, and plates were immediately read at 540 nm by using a scanning multiwell spectrometer (Bio-Tek Instruments Inc., Winooski, VT). All experiments were performed three times, and the IC50 (μg/ml) values are presented as means ± standard deviations.
- 2.4. Western blot analysis
Western blot analyses were performed as described in a previous report [ 11 ]. Primary antibodies against galectin-3 (Abcam, Cambridge, UK), γ-aminobutyric acid B receptor 1 (GABABR1) (Abcam) and actin (Abcam) (1:1,000) were used.
- 2.5. Immunoprecipitation
All procedures were performed at 4°C unless otherwise specified. Approximately 10 7 cells in 1 ml of cold 1 × radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche Diagnostics) were incubated on ice for 30 mins with occasional mixing. Cell lysates were centrifuged at 12,000 × g for 10 mins, and the supernatant was collected carefully without disturbing the pellet. The supernatant was mixed with primary antibody against either galectin-3 (Abcam) or GABABR1 (Abcam) and was incubated for 2 hrs on a rocking platform. Prepared protein G sepharose beads (GE Health Care Life Sciences) were added and further incubated on ice for 1 hrs on a rocking platform. The mixture was centrifuged at 10,000 g for 30 s, and the supernatant was removed completely. Protein G sepharose beads were washed 5 times with 1 ml of cold 1 × RIPA to minimize the background. Next, 100 μl of 2 × sodium dodecyl sulfate (SDS) sample buffer was added to the bead pellets and heated to 100°C for 10 mins. After boiling, immunoprecipitates were centrifuged at 10000 × g for 5 mins, and the supernatant was collected for the Western blot analysis.
- 2.6. Intracellular cAMP measurement
The intracellular cAMP for human colon cancer cells was determined by using a cAMP Direct Immunoassay Kit (Abcam), as recommended by the manufacturer.
- 2.7. RNA preparation and Affymetrix GeneChip hybridization
Total RNA was extracted using Trizol reagent (Life Technologies, Inc., Carlsbad, CA), according to the manufacturer’s instructions. Genes expressed in the chemosensitive and chemoresistant groups were analyzed on a high-density oligonucleotide microarray (HG-U133A; Affymetrix, Santa Clara, CA) containing 22,283 transcripts. Target preparation and microarray processing procedures were performed, following the Affymetrix GeneChip Expression Analysis Manual (Affymetrix). Briefly, total RNA extracted was purified with an RNeasy kit (Qiagen). Double-stranded cDNA was synthesized from total RNA (20 μg) with SuperScript II reverse transcriptase (Life Technologies, Inc. Rockville, MD) and a T7-(dT)24 primer (Metabion, Germany). Biotinylated cRNA was synthesized from double-stranded cDNA by using a RNA Transcript Labeling kit (Enzo Life Sciences, Farmingdale, NY), purified, and fragmented. Fragmented cRNA was hybridized to the oligonucleotide microarray, which was washed and stained with streptavidinphycoerythrin. Scanning was performed with an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA).
- 2.8. Affymetrix GeneChip data analysis
A GeneChip analysis was performed based on the Affymetrix GeneChip Manual (Affymetrix) with Data Mining Tool (DMT) 2.0 and Microarray Database software. All genes represented on the GeneChip were globally normalized and scaled to a signal intensity of 500. Fold changes were calculated by comparing transcripts between the cell lines tested. The DMT 2.0 software employed changed calls (increased or decreased) to analyze the expression of a particular transcript statistically and to determine whether it had been relatively increased, decreased or remained unchanged. After filtration through a "present" call ( p 〈0.05), a transcript was considered differentially expressed at a fold change of greater than 2.0.
- 2.9. Real-time quantitative reverse transcription polymerase chain reaction
Four genes (ELF3, AXIN2, ENO2 and SACS) were selected for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for validation of the microarray data. Using the SuperScript Pre-amplification System for first strand cDNA synthesis, 5 mg of total RNA was used for creation of singlestranded cDNA (Life Technologies). The cDNA was diluted and quantitatively equalized for PCR amplification. For real-time qRT-PCR, the ABI Prism 7900 sequence detection system (Applied Biosystems) was used. AccuPower GreenStar PCR Master Mix (Bioneer Corporation, Daejeon, Korea) was used for each PCR reaction, and the GAPDH gene was simultaneously run as a control and was used for normalization. Non-template-control wells without cDNA were included as negative controls. Each test sample was run in triplicate. The primer sets for PCR amplification were designed as follows: ELF3-F: 5’-TGAGCTGCTGGAGAAGGATG- 3’, ELF3-R: 5’-CCCTTCTTGCAGTCACGAAA- 3’, AXIN2-F: 5’-AATCATTCGGCCACTGTTCA-3’, AXIN2-R: 5’- CACAGGCAAACTCATCGCTT-3’, ENO2-F: 5’-CTGATGCTGGAGTTGGATGG- 3’, ENO2-R: 5’-CCATTGATCACGTTGAAGGC-3’, SACS-F: 5’-CCATTTGTTGGCATTTTTGG-3’, and SACS-R: 5’- CGCTCATGTTTCAGTGCCTT-3’. Following the standard curve method, the expressed quantities of the examined genes were determined using the standard curves and the CT values and were normalized using the GAPDH expression quantities.
3. Results
- 3.1. Galectin-3 expression related to 5-FU susceptibility in human colon cancer cells
To confirm the correlation between galectin-3 expression and 5-FU susceptibility in human colon cancer cells, we performed Western blot and MTT analyses on three human colon cancer cell lines, SNU-769B, SNU-C4 and SNU-C5. 5-FU susceptibility showed a decreasing tendency that depended on both the transcriptional (Fig 1 A) and the translational (Fig 1 Ba) levels of galectin-3. To cluster the genes showing positively or negatively correlated expression with galectin-3, we employed SNU-61, which had almost the same 5-FU susceptibility as SNU-769B, in a high-throughput gene expression profiling experiment (Fig. 1 B & Tables 2 , 3 ). Figure 1 Ba shows an example of 19 genes clustered in a galectin-3 expression pattern, which was confirmed by real-time PCR (Fig 1 Bb). The top 50 down- and up-regulated genes in SNU-C5, compared to SNU-769B, are listed in Tables 2 , 3 respectively.
PPT Slide
Lager Image
Galectin-3 expression correlated with 5-FU susceptibility in human colon cancer cell lines and gene expression profiling linked to galectin-3.

(A) Galectin-3 protein expression correlated with 5-FU susceptibility in three human colon cancer cell lines, SNU-769B, SNU-C4 and SNU-C5. Whole proteomes obtained from the human colon cancer cell lines employed were subjected to SDS-PAGE and were electro-transferred to PVDF membranes for western blot analysis. When galectin-3 expression was higher, human colon cancer cell lines showed more 5-FU susceptibility. (B) Gene expression profiling liked to galectin-3. To satisfy minimum clustering sample size, we added SNU-61, which has almost the same 5-FU susceptibility as SNU-769B, and as shown in the enlarged yellow box, genes linked to galectin-3 expression were selected (a). The expressional profiling was further confirmed by using real-time PCR as shown in panel (b). All genes showing positive and negative expressional correlations with galectin-3 are listed in Tables , respectively.

- 3.2. Galectin-3-dependent γ-aminobutyric acid B receptor 1 (GABABR1) expression in human colon cancer cells
Although the genes listed in Tables 2 , 3 contain γ-aminobutyric acid B receptor 1 (GABABR1), its expression was positi-vely correlated with galectin-3 as previously reported (Fig 2 A) [ 11 ]. GABABR1 expression at the translational level was highest in SNU-769B among the three human colon cancer cell lines tested (Fig 2 B). To validate the interaction between galectin-3 and GABABR1, we performed reverse immunoprecipitation: however, galectin-3 did not form a complex with GABABR1 (Fig 2 C).
PPT Slide
Lager Image
Gene and protein expressions of GABABR1 positively linked to galectin-3 expression.

(A) GABABR1 in the list of the genes showing positive expressional correlation with galectin-3. (B) Protein expression of GABABR1 in the three human colon cancer cell lines tested. GABABR1 protein expression also showed positive correlation with galectin-3 expression. (C) Reverse immunoprecipitation using anti-galectin and GABABR1 antibody. Results demonstrated that two proteins did not interact to form a complex in SNU-C4 with modest expression of galectin-3 and GABABR1.

- 3.3. Galectin-3-independent down-regulation of GABABR1 protein by HAD-B in human colon cancer cells
To check the effect of HAD-B treatment on the expression level of galectin-3 and GABABR1, we cultured SNU-C4 with modest expression of galectin-3 and GABABR1 in the presence of HADB, and we performed a Western blot analysis. At 96 hrs after treatment with 1 mg/ml HAD-B, expression of GABABR1 was reduced, but galectin-3 did not show any expressional change (Fig 3 A).
- 3.4. GABABR1-mediated proliferation of human colon cancer cells suppressed by HAD-B treatment
Treatment with γ-aminobutyric acid (GABA) in the culture medium promoted proliferation of the human colon cancer cell line SNU-C4 (Fig 3 B). At 48 hrs after treatment with GABA, cell proliferation was increased up to ~50% compared to nonetreated controls, but rate of increase of proliferation was not maintained (Fig 3 B). HAD-B significantly decreased cell proliferation at 48 hrs after treatment compared to the control, but the suppressed proliferation had recovered at 96 hrs (Fig 3 B). Cells co-treated with GABA and HAD-B showed almost the same pattern of proliferation as that of the control (Fig 3 B). Either GABA or HAD-B treatment slightly increased the intracellular cAMP in SNU-C4 compared to that in the nontreated control (Fig 3 C).
PPT Slide
Lager Image
Reduced GABABR1 expression and suppressed cell proliferation of SNU-C4 by treatment with HAD-B.

(A) Decreased GABABR1 expression by treatment with HAD-B. At 96 hrs after treatment of with 1 mg/ml HAD-B, protein expression of GABABR1 was decreased in SNU-C4. (B) Suppressed cell proliferation by treatment with HAD-B. GABA treatment recovered the rate of proliferation of SNU-C4 that had been suppressed by HAD-B treatment. (C) Increase in the intracellular cAMP by either GABA or HAD-B treatment. Treatment with GABA or HAD-B increased the basal level of intracellular cAMP.

Top 50 down-regulated genes in SNU-C5 compared to SNU-769B
Probe Set IDGene SymbolSNU- 769ASNU- C4SNU- C5FCGO Biological Process TermGO Cellular Component TermGO Molecular Function Term
8101893 ADH1C 2263 1125 14 -7.3 alcohol metabolic process cytoplasm alcohol dehydrogenase activity, zinc-dependent
7989501 CA12 2428 789 26 -6.5 one-carbon compound metabolic process membrane carbonate dehydratase activity
8022692 DSC3 904 35 10 -6.5 cell adhesion membrane fraction calcium ion binding
7979658 GPX2 2129 1408 24 -6.5 response to oxidative stress cytoplasm glutathione peroxidase activity
7919055 HMGCS2 2091 1229 28 -6.2 acetyl-CoA metabolic process mitochondrion hydroxymethylglutaryl-CoA synthase activity
8036591 LGALS4 5079 4762 69 -6.2 cell adhesion cytosol sugar binding
7928770 PCDH21 1522 291 21 -6.2 homophilic cell adhesion membrane calcium ion binding
7953200 CCND2 2210 60 37 -5.9 regulation of progression through cell cycle nucleus protein binding
7928766 C10orf99 1970 493 33 -5.9      
8138392 AGR3 292 174 6 -5.7      
7919984 SELENBP1 2409 1499 54 -5.5     selenium binding
8174654 KLHL13 650 164 16 -5.3     protein binding
7967107 C12orf27 367 229 9 -5.3      
8161884 PRUNE2 396 230 12 -5.1      
8106354 IQGAP2 704 95 22 -5.0 signal transduction intracellular actin binding
8134339 PEG10 658 28 21 -5.0 negative regulation of transforming growth factor beta receptor signaling pathway cytoplasm nucleic acid binding
8135378 PRKAR2B 543 85 18 -4.9 protein amino acid phosphorylation cAMP-dependent protein kinase complex nucleotide binding
8091283 PLOD2 358 98 13 -4.8 protein modification process endoplasmic reticulum iron ion binding
8128123 RRAGD 311 58 12 -4.7   nucleus nucleotide binding
7983606 EID1 759 568 30 -4.7 negative regulation of transcription from RNA polymerase II promoter cellular component protein binding
8100734 UGT2B17 125 6 5 -4.6 metabolic process membrane fraction glucuronosyltransferase activity
8080964 PPP4R2 293 130 12 -4.6 protein modification process centrosome protein binding
8151592 CA1 256 23 11 -4.6 one-carbon compound metabolic process cytoplasm carbonate dehydratase activity
8101757 GPRIN3 711 256 31 -4.5      
7926545 PLXDC2 499 87 22 -4.5 multicellular organismal development membrane receptor activity
7916185 ZCCHC11 266 262 12 -4.5   intracellular nucleic acid binding
8008172 B4GALNT2 693 38 30 -4.5 UDP-N-acetylgalactosamine metabolic process membrane acetylgalactosaminyltransferase activity
8040374 FAM84A 998 492 44 -4.5      
8168589 ZNF711 378 106 19 -4.4 regulation of transcription, DNA-dependent intracellular DNA binding
8043981 IL1R2 663 54 33 -4.3 immune response membrane receptor activity
7923578 FMOD 359 74 18 -4.3 transforming growth factor beta receptor complex assembly proteinaceous extracellular matrix protein binding
8138553 FAM126A 210 69 11 -4.3 biological process cellular component signal transducer activity
8077323 CNTN4 168 11 10 -4.1 cell adhesion plasma membrane protein binding
7999553 FLJ11151 337 207 20 -4.1     hydrolase activity
7940565 FADS2 502 380 31 -4.0 lipid metabolic process membrane fraction iron ion binding
7951554 RDX 259 67 16 -4.0 cytoskeletal anchoring cytoplasm actin binding
8044212 SULT1C2 215 39 13 -4.0 amine metabolic process cytoplasm sulfotransferase activity
7903742 GSTM2 946 183 59 -4.0 metabolic process   glutathione transferase activity
7937335 IFITM1 402 343 25 -4.0 regulation of progression through cell cycle plasma membrane receptor signaling protein activity
8041383 LTBP1 470 213 30 -4.0 biological process proteinaceous extracellular matrix transforming growth factor beta receptor activity
8142171 SLC26A3 185 18 12 -4.0 transport membrane fraction transcription factor activity
7951789 FAM55D 318 209 21 -3.9      
8078544 MLH1 155 121 10 -3.9 mismatch repair nucleus single-stranded DNA binding
8111772 DAB2 344 72 23 -3.9 cellular morphogenesis during differentiation coated pit protein C-terminus binding
8094988 FLJ21511 270 75 18 -3.9      
7918223 C1orf59 122 101 8 -3.9      
8095110 KIT 160 29 11 -3.9 protein amino acid dephosphorylation external side of plasma membrane nucleotide binding
8125149 SLC44A4 1306 1163 88 -3.9   membrane  
8178653 NEU1 1306 1163 88 -3.9 metabolic process lysosome exo-alpha-sialidase activity
8179861 NEU1 1306 1163 88 -3.9 metabolic process lysosome exo-alpha-sialidase activity
FC: Fold-change was calculated from the signal Log ratio value.
Top 50 up-regulated genes in SNU-C5, compared to SNU-769B
Probe Set IDGene SymbolSNU- 769ASNU- C4SNU- C5FCGO Biological Process TermGO Cellular Component TermGO Molecular Function Term
7954330 SLCO1B3 6 551 921 7.3 ion transport integral to plasma membrane transporter activity
7959856 PIWIL1 11 17 834 6.3 multicellular organismal development cytoplasm single-stranded RNA binding
7954090 EMP1 23 51 1597 6.1 multicellular organismal development membrane fraction  
7954344 LST-3TM12 14 236 875 6.0 transport membrane transporter activity
8026490 LOC729642 29 34 1815 5.9      
8108217 TGFBI 36 105 2089 5.9 cell adhesion proteinaceous extracellular matrix integrin binding
8176026 FLNA 67 699 2852 5.4 cell motility nucleus actin binding
7954356 SLCO1B1 10 55 327 5.1 ion transport membrane fraction transporter activity
8124437 HIST1H3F 45 49 1482 5.0 nucleosome assembly nucleosome DNA binding
7997139 CALB2 33 35 1020 5.0     calcium ion binding
8102950 INPP4B 25 131 786 5.0 signal transduction   phosphatidylinositol-3, 4-bisphosphate 4-phosphatase activity
8095728 EREG 62 230 1896 4.9 regulation of progression through cell cycle extracellular space epidermal growth factor receptor binding
8155849 ANXA1 65 980 1942 4.9 lipid metabolic process cornified envelope phospholipase inhibitor activity
8089082 DCBLD2 131 308 3841 4.9 cell adhesion integral to plasma membrane protein binding
8140668 SEMA3A 36 190 1067 4.9 multicellular organismal development extracellular region chemorepellant activity
7920128 S100A11 41 806 1209 4.9 signal transduction ruffle calcium ion binding
8098470 WWC2 13 20 358 4.8      
8067233 TMEPAI 50 115 1311 4.7 androgen receptor signaling pathway membrane molecular function
7909789 TGFB2 18 22 440 4.6 cell morphogenesis extracellular region beta-amyloid binding
8015016 TNS4 42 240 1027 4.6 apoptosis cytoskeleton actin binding
8095744 AREG 44 240 1080 4.6 cell-cell signaling extracellular space cytokine activity
8021442 ZNF532 28 73 673 4.6   intracellular nucleic acid binding
7933312 LOC653110 27 29 648 4.6      
7981514 AHNAK2 22 42 532 4.6     protein binding
8027778 FXYD5 65 433 1459 4.5 ion transport membrane actin binding
7908072 LAMC2 73 317 1591 4.4 cell adhesion basement membrane protein binding
8075310 LIF 54 56 1139 4.4 immune response extracellular region cytokine activity
8138466 7A5 61 316 1226 4.3      
7986446 ALDH1A3 88 114 1741 4.3 alcohol metabolic process   3-chloroallyl aldehyde dehydrogenase activity
8041179 CLIP4 18 20 356 4.3      
8124413 HIST1H4D 63 338 1234 4.3      
7924029 LAMB3 77 169 1473 4.3 electron transport proteinaceous extracellular matrix structural molecule activity
8129379 ECHDC1 38 685 717 4.3 metabolic process   catalytic activity
7945321 LOC89944 33 510 625 4.2 carbohydrate metabolic process beta-galactosidase complex catalytic activity
8179731 HLA-C 153 616 2862 4.2 ciliary or flagellar motility axonemal dynein complex microtubule motor activity
8064613 SLC4A11 72 239 1300 4.2 anion transport membrane inorganic anion exchanger activity
8167185 TIMP1 215 857 3816 4.1 multicellular organismal development extracellular region enzyme inhibitor activity
8120602 OGFRL1 28 141 438 4.0   membrane receptor activity
8178489 HLA-C 190 730 2990 4.0 ciliary or flagellar motility axonemal dynein complex microtubule motor activity
8060758 PRNP 108 486 1632 3.9 copper ion homeostasis cytoplasm copper ion binding
8178498 HLA-B 164 517 2463 3.9 antigen processing and presentation of peptide antigen via MHC class I cellular component molecular function
8124911 HLA-B 131 408 1955 3.9 antigen processing and presentation of peptide antigen via MHC class I cellular component molecular function
7973985 MIPOL1 9 92 134 3.9      
7944722 STS-1 32 58 473 3.9   nucleus  
8124901 HLA-C 205 724 2974 3.9 ciliary or flagellar motility axonemal dynein complex microtubule motor activity
8095736 LOC727738 43 170 615 3.8      
8091411 TM4SF1 38 220 538 3.8 biological process integral to plasma membrane molecular function
7917875 F3 78 87 1078 3.8 immune response plasma membrane transmembrane receptor activity
8092726 CLDN1 57 195 772 3.8 cell adhesion integral to plasma membrane structural molecule activity
8126820 GPR110 16 20 217 3.8 signal transduction membrane receptor activity
FC: Fold-change was calculated from the signal Log ratio value.
4. Discussion
Colon cancer causes almost a half million deaths every year [ 23 ]. In the past 3 decades, 5-fluorouracil (5-FU) chemotherapy and 5-FU-based chemotherapy have been the mainstream in adjuvant treatment of colon cancer [ 24 ]; however, partial or complete responses of colon cancer to 5-FU are generally followed by eventual tumor re-growth [ 25 ]. Numerous studies have focused on identifying the mechanisms and key molecules involved in natural or acquired 5-FU resistance. Nevertheless, conclusive and consistent results have not been obtained so far. A recent proteome approach identified galectin-3 as a protein affecting 5-FU resistance and the proliferation rate of human colon cancer cells [ 11 ]. Our present study confirmed the correlation between galectin-3 expression and 5-FU susceptibility in three human colon cancer cell lines. 5-FU susceptibility of human colon cancer cells was different depending on both the transcriptional and the translational levels of galectin-3 (Fig 1 A,B). Because the identification of genes showing positively or negatively correlated expression with galectin-3 can provide further information on how galectin-3 regulates proliferation of human colon cancer cells, a high-density oligonucleotide microarray was performed. From this transcriptional analysis, we were able to list the genes down- and up-regulated based on the level of galectin-3 expression (Fig. 1 B, Tables 2 , 3 ). Though γ-aminobutyric acid B receptor 1 (GABABR1) was not in the top 50 genes linked to galectin-3 (Table 2 and 3 ), interestingly we found that both the transcriptional and the translational levels of GABABR1 were positively correlated with galectin-3 (Fig 2 A,B). Even though the biological functions of each individual protein have been well studied, we could not find a report describing the relation between galectin-3 and GABABR1.
GABABRs have been found to play a key role in regulating membrane excitability and synaptic transmission in the brain [ 26 ]. GABABRs are G-protein coupled receptors that associate with a subset of G-proteins that trigger cAMP cascades [ 26 ]. GABABR subtypes exist; two GABAB-receptor splice variants, GABABR1a and GABABR1b, have been cloned [ 27 ], and a new GABABR subtype, GABABR2, does not bind with available GABAB antagonists with measurable potency [ 28 ]. GABABR1a, GABABR1b and GABABR2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. GABABR2 and GABABR1a/b proteins immunoprecipitate and localize together at dendritic spines [ 28 ]. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABAB receptor [ 28 ]. As a previous report also showed that the transcriptional level of GABABR1 was decreased by transfection of galectin-3 small interfering RNA (siRNA) [ 11 ], expression of GABABR1 could be regulated by galectin-3. However, reverse immunoprecipitation to validate the interaction between two proteins revealed that galectin-3 did not affect the protein stability of GABABR1 because it formed a complex with GABABR1 (Fig 2 C).
Gamma-aminobutyric acid (GABA) has been reported to affect cancer development. For example, GABA can be a potential tumor suppressor for small airway-derived lung adenocarcinomas [ 29 ]. The GABA agonist nembutal has been reported to be a potent inhibitor of primary colon cancer and metastasis [ 30 ]. The GABABR agonist baclofen induced G(0)/G(1) phase arrest of human hepatocellular carcinomas (HCCs), which suggested the possibility of developing baclofen as a therapeutic drug for the treatment of HCCs [ 31 ]. Furthermore, stimulation of GABABR signaling has been suggested as a novel target for the treatment and the prevention of pancreatic cancer [ 32 ]. However, in our present study, treatment with GABA promoted proliferation of the human colon cancer cell line SNU-C4 (Fig 3 B). The Korean herbal extract HAD-B not only decreased GABABR1 expression but also reduced proliferation of human colon cancer cells without any expressional change of galectin-3 (Fig 3 A,B). GABABR activation can lead to down-regulation of the intracellular cAMP level in human cancer cells [ 30 , 32 ]. Downregulation of GABABR1 by HAD-B treatment increased the basal level of intracellular cAMP in SNU-C4 (Fig 3 C). However, such an increased cAMP was also observed after GABA treatment (Fig 3 C). The overall findings in the present study were inconsistent with those in previous reports describing the activation of GABABR1 to prevent the progression of a human carcinoma. Nevertheless, our present results showed a link between galectin-3 and GABABR1 in human colon cancer cell proliferation. Galectin-3 regulated GABABR1 expression [ 11 ]. Decreased galectin-3 expression reduced not only GABABR1 expression but also the proliferation rate of human colon cancer cells [ 11 ]. Even GABA promoted human colon cancer cell proliferation by activating GABABR1 signaling, and the increased proliferation was offset by HAD-B treatment because HAD-B led to galectin-3-independent down-regulation of GABABR1 (Fig. 3 ).
5. Conclusion
Our present study confirmed that GABABR1 expression was regulated by galectin-3. Korean herbal extract HAD-B induced galectin-3-independent down-regulation of GABABR1, which resulted in a decreased proliferation of human colon cancer cells. The therapeutic effect of HAD-B for the treatment of human colon cancer needs to be further validated.
Acknowledgements
This work was supported by a research grant (NCC-1010050) from the National Cancer Center, Korea.
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