Affiliations of authors: S. Ito, Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, Japan, and Third Department of Internal Medicine, Yokohama City University School of Medicine, Japan; T. Fukusato, Department of Pathology, Faculty of Medicine, Gunma University, Maebashi, Japan; T. Nemoto, Y. Seyama, S. Kubota, Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo; H. Sekihara, Third Department of Internal Medicine, Yokohama City University School of Medicine.
Correspondence to: Shunichiro Kubota, M.D., Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan (e-mail: kubota{at}bio.m.u-tokyo.ac.jp).
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ABSTRACT |
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INTRODUCTION |
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The process of metastasis consists of many steps, of which invasion through the basement membrane of blood vessels is one of the most important (15). MMPs are thought to be important in degrading basement membrane because their activity is associated with invasive and metastatic potential (16). These structurally related enzymes consist of at least 24 members (17,18) that are classified on the basis of their substrate specificities: collagenases, gelatinases, stromelysins, membrane-type MMPs, and unclassified MMPs (17,18). MMP activity is controlled at multiple levels. MMP genes are transcriptionally regulated in response to growth factors, cytokines, and oncogenes (18). In addition, MMPs are secreted as inactive precursors or zymogens that require proteolytic processing to release the active enzyme. This processing is achieved by proteinases such as plasmin or membrane-type MMP (18,19).
One MMP, MMP-2 (a 72-kd metalloproteinase) specifically cleaves type IV collagen, the major component of basement membranes (18). In human tumors, MMP-2 expression has been directly associated with cancer invasiveness and metastasis (2025). Because Glut-1 is overexpressed in cancer cells, and because this overexpression is associated with metastasis, we hypothesized that Glut-1 regulates the expression of proteins involved in basement membrane and extracellular matrix degradation, such as MMP-2. In this article, we report on our testing of this hypothesis by modulating the expression of Glut-1 and examining the effects on MMP-2 expression and invasiveness.
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METHODS |
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All cell lines used in this study were obtained from the Human Science Research Resources Bank (Osaka, Japan) and grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). We used the following cancer cell lines: RD (rhabdomyosarcoma), YKG1 (glioblastoma), T98G (glioblastoma), A172 (glioblastoma), A549 (lung carcinoma), MKN45 (gastric carcinoma), HT-1080 (fibrosarcoma), MCF7 (breast carcinoma), and MDA-MB-231 (breast carcinoma).
Generation of Glut-1 Complementary DNA, Glut-1 Expression Vectors, and Antisense Oligonucleotides
Total RNA was isolated from human MDA-MB-231 breast cancer cells as described (26). Reverse transcriptionpolymerase chain reaction (RTPCR) was used to generate Glut-1 complementary DNA (cDNA) from 800 ng of total RNA, according to the manufacturer's instructions (RNA PCR Kit; TaKaRa, Otsu, Japan). The sequences for the Glut-1 sense and antisense primers were 5`-TCAGAGTCGCAGTGGGAGTC-3` and 5`-ACTCACACTTGGGAATCAGC-3`. A 1599-bp product was amplified by PCR and cloned into the TA cloning site of the pTargeT expression vector (Promega, Tokyo, Japan). The sequence of the entire coding region of Glut-1 was confirmed by using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan) to make sure that Glut-1 was correctly amplified by PCR. The control vector consisted of a pTargeT expression vector without Glut-1 cDNA. Glut-1 phosphorothioate oligonucleotides (sense, 5`-CGGGCCAAGAGTGTGC TAAA-3` and antisense, 5`-TGACGATACCGGAGCCAATG 3`) were synthesized in an automated DNA synthesizer (model 380B; Applied Biosystems).
Transfection With Glut-1 Antisense Oligonucleotides or Glut-1 Expression Vector
RD cells (1 x 106) were plated into 100-mm dishes and allowed to adhere overnight. The next day, the cells were transiently transfected by exposure to a mixture containing oligonucleotides (20 µM) or containing Glut-1 cDNA (2 µg) or control DNA expression vectors and LipofectAMINE PLUS Reagent (Life Technologies, Inc., Tokyo, Japan) in 6.5 mL of DMEM for 3 hours at 37 °C in a 5% CO2 incubator, according to the manufacturer's instructions. After the transfection, the mixture was removed and replaced with DMEM containing 10% FCS.
Analysis of the Expression of Glut-1, MMP-2, -actin, and Membrane Type-MMP by RTPCR and Northern Blotting
Thirty-six hours after transfection with Glut-1 sense or antisense oligonucleotides or Glut-1 expression vector, total RNA from RD cells was isolated using the acid guanidiniumphenolchloroform method. mRNA expression of Glut-1, MMP-2, -actin, and membrane type (MT)-MMP was measured by RTPCR starting with 800 ng of total RNA and specific primers, according to the manufacturer's instructions (RNA PCR kit, version 2.1; TaKaRa). We used the sense and antisense primers MMP-2, 5`-TTCAAGGACCGGTTCATTTGGCGGACTGTG-3` and 5`-TTCCAAACTTCACGCTCTTCAGACTTTGGTT-3`; MT-MMP, 5`-AACAACCAGAAGCTGAAGGTA-3` and 5`-ACCTTGTC CAGCAGGGAACG-3`;
-actin, 5`-CGTGGGGCGCCCCAG GCACCA-3` and 5`-TTGGCCTTGGGGTTCAGGGGG-3`; and Glut-1, 5`-CGGGCCAAGAGTGTGCTAAA-3` and 5`-TGACGAT ACCGGAGCCAATG-3`. PCR was carried out in a Gene Amp PCR Thermal Cycler 9600 (PerkinElmer, Fremont, CA) for 23 cycles, with each cycle consisting of denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. The PCR products were subjected to electrophoresis through a 2% agarose gel, which was then stained with ethidium bromide to visualize the bands. The sequence of the amplified PCR products was verified to make sure that PCR was correctly performed. NIH Image software (version 1.61; National Institutes of Health, Bethesda, MD) was used to quantify the intensities of the bands. To compare differences between samples, the relative intensity of each band was normalized to the intensity of the
-actin band amplified from the same sample.
Northern blotting was performed using 10 µg of total RNA isolated from RD cells, RD cells transfected with Glut-1 sense or antisense oligonucleotides, and RD cells transfected with Glut-1 or control DNA expression vectors. RNAs were separated by electrophoresis through a 1% formaldehyde/agarose/MOPS gel and transferred to a nylon membrane. The northern blot was hybridized with riboprobes for Glut-1 and MMP-2, as described (26). To generate the probes, cDNAs for Glut-1, MMP-2, or -actin were cloned into the SK multiple cloning site of the pBluescript II vector (TOYOBO, Tokyo, Japan). Glut-1 cDNA contained the sequence from 62 to 1660 base positions of the gene (GenBank accession number K03195), MMP-2 cDNA contained the sequence from 1415 to 1907 base positions of the gene (GenBank accession number J03210), and
-actin cDNA contained the sequence from 112 to 354 base positions of the gene (GenBank accession number AB004047). Glut-1, MMP-2, and
-actin riboprobes were generated by in vitro transcription using the T7 polymerase binding site of each cloning vector and an in vitro transcription kit (Roche Japan, Tokyo). Specific riboprobes were labeled with digoxigenindeoxyuridine triphosphate (dUTP) by random priming (Roche Japan). The mRNA bands on the northern blot were quantified using NIH Image software, and the intensities of the MMP-2 and Glut-1 mRNA bands were normalized to the intensities of the control
-actin bands from the same samples.
Gelatin Zymography
Sixty hours after transfection with Glut-1 or control DNA expression vectors, RD cells were detached with trypsin and plated on 12-well cluster dishes at a concentration of 5 x 105 cells/well. The cells were adhered in the presence of serum, which was then replaced with serum-free medium. After 24 hours, the serum-free conditioned media were collected, and aliquots (45-µL) were assayed for MMP-2 and MMP-9 activities by gelatin zymography, as described (27), and the data were quantified using NIH Image software. An MMP-1 enzyme assay was performed using an enzyme-linked immunosorbent assay (ELISA) kit (Amersham Biosciences, Tokyo, Japan), according to the manufacturer's recommended protocol.
Western Blotting
Cells were cultured in complete growth medium until they were approximately 80%90% confluent. The cells were washed twice with phosphate-buffered saline (PBS), collected with cell scrapers, homogenized, and extracted with 50 mM TrisHCl buffer (pH 7.5) containing proteinase inhibitors (5.0 µg/mL phenylmethylsulfonyl fluoride, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin). The protein concentration of the extracts was measured using a Bradford assay kit (Bio-Rad, Hercules, CA). Extracted proteins (100 µg) were separated by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with specific rabbit polyclonal antibodies against human Glut-1 and MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA), as described (28). Protein bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences). Band intensities were quantified using NIH Image software.
Invasion Assay
The invasiveness of RD, YKG1, and T98G cells (2 x 105) transfected with Glut-1 sense or antisense oligonucleotides was assayed 60 hours after transfection by using a Boyden chamber as described (27). Invasiveness was measured by counting the number of cells that penetrated the polycarbonate filters (Neuro Probe, Inc., Gaithersburg, MD). The invasiveness of each cell line was expressed as a percentage of the control (cells transfected with sense oligonucleotides). Transfected cells were assayed in triplicate for invasiveness, and the experiments were repeated twice.
In some experiments, antibodies were added to neutralize MMP-2 activity. In these experiments, 60 hours after transfection with the Glut-1 cDNA or control DNA expression vectors, RD cells were incubated with anti-MMP-2 antibody (456 µg/mL) and then tested in the invasion assay, as described (27). This antibody concentration effectively neutralizes MMP-2 activity (27). In control experiments, we substituted immunoglobulin G (IgG; 456 µg/mL) of the same species and isotype as the anti-MMP-2 antibody.
Cloning of the Human MMP-2 Promoter
We identified and cloned the human MMP-2 promoter from human placental DNA by using RTPCR with specific primers for human MMP-2 promoter, as described (29). The PCR product was cloned into the pGL3 vector (Promega), which was then sequenced. The constructs used for the experiments were pGL3a, containing the full-length MMP-2 promoter (from positions 1659 to +57); pGL3b, containing a deletion mutant starting at position 1079 upstream of the transcription start site (from positions 1079 to +57); and pGL3e, containing no MMP-2 promoter.
Luciferase Assay
RD cells were detached with trypsin, plated on 12-well cluster dishes at a concentration of 5 x 105 cells/well, and cultured for 12 hours in complete medium. One microgram of pGL3a, pGL3b, or pGL3e was cotransfected with 0.1 µg of pRL-CMV vector, which has a cytomegalovirus promoter in front of a renilla luciferase gene. After 48 hours, luciferase activity was measured in lysates from the transfected cells, according to the manufacturer's recommended protocol (Dual-Luciferase Reporter Assay System; Promega). The activity of renilla luciferase was used to normalize any variation in transfection efficiency. After the firefly and luciferase activities in cell lysates were sequentially quantified, the promoter activity of each plasmid construct was calculated as the fireflyrenilla luciferase activity ratio. The transfection efficiency of RD cells using the pCMV vector (Clontech, Tokyo, Japan), which contains the
-galactosidase gene, was 75.0% ± 5.8%.
Preparation of Nuclear Extracts and Gel Mobility Shift Assays
Forty-eight hours after transfection, nuclear extracts were prepared, as described (28), from untreated control RD cells, from transfection control RD cells (those treated with LipofectAMINE PLUS Reagent or with control vector), or from RD cells transfected with Glut-1 cDNA or Glut-1 sense or antisense oligonucleotides. The protein concentration in the nuclear extracts was measured using the Bradford assay kit (Bio-Rad). For each gelmobility shift assay, 2 µg of each nuclear extract was used.
Consensus or mutant p53 oligonucleotides and consensus or mutant Ets-1 family oligonucleotides (Santa Cruz Biotechnology) were used as probes and labeled with DIG-112`,3`-dideoxyribonucleoside uridine 5`-triphosphate (DIG-11-ddUTP) by using the terminal deoxynucleotidyl transferase method, according to the manufacturer's recommended protocol (Roche Japan). Binding reaction mixtures (20 µL containing 2 µg of nuclear extract, 0.4 ng of labeled probe, 20 mM HEPES [pH 7.6], 5 mM EDTA, 10 mM (NH4)2SO4, 1 mM dithiothreitol, 0.2% [w/v] Tween 20, 30 mM KCl, and 1 µg of poly [dI-dC]) were mixed on ice and incubated for 15 minutes at room temperature. For the competition experiments, 125- or 250-fold molar excess of the unlabeled probe was added to the mixture before incubation.
The proteinDNA complexes were resolved by electrophoresis through a 6% polyacrylamide gel in 0.25x TrisborateEDTA buffer (22.25 mM Tris, 22.25 mM borate, 0.5 mM EDTA [pH 8.0]) for 2 hours at 4 °C. The proteinDNA complexes were transferred to a nylon membrane (Roche Japan) by electroblotting, and the proteinDNA complexes were detected with a chemiluminescence detection kit (Roche Japan).
To test the specificity of the proteinDNA binding reaction, nuclear extracts were preincubated with 25 µg of mouse monoclonal anti-p53 antibody (BD Biosciences, Tokyo, Japan), 25 µg of mouse monoclonal anti-Ets-1 antibody (BD Biosciences), or 25 µg of control mouse IgG (Cosmo Bio, Tokyo, Japan) for 1 hour before the addition of the DNA probe.
Immunohistochemical and Fluorescence Analyses
Tumor tissues of various organs from 80 Japanese patients were examined. The tumors had been surgically resected at the Second Department of Surgery, Gunma University Hospital, and its affiliated hospitals from 1992 through 2000. The mean age of the patients was 58.1 years (range = 589 years). Thirty-four patients were male and 46 were female. Tumors were histologically classified according to the World Health Organization International Histological Classification of Tumors (30). The study protocol conformed to the ethical guidelines of the Gunma University School of Medicine.
Archival formalin-fixed, paraffin-embedded tissue sections (3-µm thick) were deparaffinized and immunostained by following a standard protocol. Briefly, after inhibiting endogenous peroxidase activity with a 3% aqueous hydrogen peroxidemethanol solution for 20 minutes and blocking the section with 10% normal rabbit serum, we incubated the tissue sections overnight with primary antibodies (polyclonal goat anti-MMP-2 and anti-Glut-1 antibodies, 1 : 500 dilution, Santa Cruz Biotechnology) at 4 °C. The sections were processed by the streptavidinbiotinperoxidase method according to the manufacturer's recommended protocol (Histofine SAB kit; Nichirei, Japan) that used diaminobenzidine as the chromogen. Nuclei were lightly counterstained with hematoxylin. To confirm the specificity of the MMP-2 or Glut-1 immunostaining, nonimmune serum was substituted for the primary antibodies and used to stain additional tissue sections.
For the purpose of this study, tissue sections were considered negative for MMP-2 or Glut-1 expression if 100% of the cells were Glut-1 or MMP-2 negative by immunohistochemistry. If any expression was detected, tissue sections were considered Glut-1 or MMP-2 positive.
Immunofluorescence staining was performed on breast cancer tissue sections by using a combination of rabbit polyclonal anti-Glut-1 (1 : 500 dilution; Santa Cruz Biotechnology) and mouse monoclonal anti-MMP-2 (1 : 500 dilution; Fuji, Toyama, Japan) antibodies. After the tissue sections were incubated with the combination of primary antibodies for 1 hour at room temperature, they were rinsed extensively in PBS and then incubated with rhodamine-conjugated goat anti-rabbit IgG (1 : 1000 dilution; Santa Cruz Biotechnology) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1 : 1000; Santa Cruz Biotechnology) for 1 hour at room temperature. After extensive rinsing in PBS, the sections were examined under a fluorescence microscope (IX70; Olympus, Tokyo, Japan).
Effect of Glucose on Glut-1 and MMP-2 mRNA Expression
To determine the time-dependent and concentration-dependent effects on Glut-1 and MMP-2 mRNA expression, RD cells (1 x 106 cells/dish) were plated in 100-mm dishes and allowed to adhere for 2 hours. Next, the medium was changed to DMEM containing 2% FCS in which the glucose concentrations were adjusted to very low (2.7 mM), low (5.6 mM), or high (25 mM). RD cells were incubated for various periods of time before RNA was extracted and examined by northern blot analysis for expression of Glut-1, MMP-2, and -actin mRNAs. The mRNA bands on the northern blot were quantified by using NIH Image software, and the fold of intensities of the MMP-2 and Glut-1 mRNA bands were normalized to the intensities of the control
-actin bands from the same samples. After image analysis, the data from three samples per group were expressed as fold induction relative to
-actin.
Statistical Analysis
The data were provided as means of three experiments with 95% confidence intervals (CIs). All experiments were performed three times, unless stated otherwise. All statistical analyses were performed by using Statview 4.5 software (Abacus Concepts, Inc., Berkeley, CA). The statistical significance of differences between treatment groups was determined by the use of analysis of variance (ANOVA) followed by Fisher's partial least-squares difference test. All P values were two-sided, and P values of less than .05 were considered statistically significant.
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RESULTS |
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We first examined the expression of MMP-2 and Glut-1 in eight cancer cell lines by western blotting. All of the cell lines coexpressed MMP-2 and Glut-1 proteins (Fig. 1, A). The relative band intensities of four cell lines (A549, T98G, YKG1, and RD) were comparable for MMP-2 and Glut-1 expression levels (lower panel). The relative band intensities in three cell lines (A172, MCF7, and MDA-MB-231) were higher for MMP-2 expression than for Glut-1 expression. The relative band intensities for MKN45 cells were higher for Glut-1 expression than for MMP-2 expression. The results suggest that MMP-2 and Glut-1 were coexpressed at the protein level in various cancer cell lines.
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We next used RTPCR analysis to evaluate the effect of altering Glut-1 on mRNA expression levels of MMP-2. To inhibit Glut-1 expression, we transfected RD cells, which constitutively express Glut-1, with Glut-1 antisense oligonucleotides. Transfection with specific Glut-1 antisense oligonucleotides decreased mRNA levels of Glut-1 and MMP-2 but not of -actin (Fig 1, B
). Compared with control cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 68.8% (95% CI = 68.0% to 69.6%; P<.001), and the level of MMP-2 mRNA decreased by 71.5% (95% CI = 71.1% to 71.9%; P<.001) in cells transfected with Glut-1 antisense oligonucleotides.
Next, we examined the effect of overexpressing Glut-1 mRNA in RD cells by transfecting the cells with a Glut-1 expression vector. Compared with cells transfected with control vector, the level of Glut-1 mRNA expression increased by 5.4-fold (95% CI = 4.6-fold to 6.2-fold; P<.001) and the level of MMP-2 mRNA increased by 4.3-fold (95% CI = 3.7-fold to 4.9-fold; P<.001) (Fig. 1, C). Overexpression of Glut-1 mRNA did not have a statistically significant effect on the level of MT-MMP mRNA expression (Fig. 1, D
). These results suggest that MMP-2 but not MT-MMP expression is associated with Glut-1 expression in RD cells.
To determine whether the association between Glut-1 and MMP-2 was unique to RD cells, we transfected other cancer cell lines with Glut-1 antisense oligonucleotides and examined the effect on Glut-1 and MMP-2 mRNAs. Decreased expression of Glut-1 and MMP-2 mRNAs was observed in the human glioblastoma cell lines YKG1 and T98G after transfection with Glut-1 antisense oligonucleotides (Fig. 2). Compared with control cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 68.8% (95% CI = 67.4% to 70.2%; P<.001), and the level of MMP-2 mRNA decreased by 71.5% (95% CI = 67.1% to 75.9%; P<.001) in YKG1 cells transfected with Glut-1 antisense oligonucleotides (Fig. 2, A
). Similar results were obtained with T98G cells. Compared with T98G cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 74.1% (95% CI = 73.8% to 74.4%; P<.001), and the level of MMP-2 mRNA decreased by 65.9% (95% CI = 65.7% to 66.1%; P<.001) in T98G cells transfected with Glut-1 antisense oligonucleotides (Fig. 2, B
). The results suggest that the association between the expression of Glut-1 and MMP-2 is not unique to RD cells.
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Effect of Glut-1 Antisense Oligonucleotides and Glut-1 cDNA on MMP Protein Expression
Next, to assess the effect of Glut-1 on MMP-2 protein activity, which is a direct reflection of MMP-2 protein expression, we measured the gelatinolytic activity in the supernatants of Glut-1-overexpressing RD cells by zymography. RD cells secrete the latent, inactive form of MMP-2 (27). Neither activated MMP-2 nor the related molecule MMP-9 was detected in culture supernatants from control RD cells (Fig. 3, lane 1). RD cells that overexpressed Glut-1 secreted 4.0-fold (95% CI = 3.96-fold to 4.06-fold) more active MMP-2 than did untransfected cells (P<.001). By contrast, RD cells transfected with Glut-1 antisense oligonucleotides secreted only 29.4% (95% CI = 27.5% to 31.3%) of the level of active MMP-2 secreted by untreated cells (P<.001). Thus, MMP-2 expression was regulated at the mRNA level, and this corresponded to changes at the protein level.
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Effect of Glut-1 on Transcriptional Activity of MMP-2
To elucidate the mechanism by which Glut-1 may regulate MMP-2 expression, we next evaluated whether Glut-1 could influence the MMP-2 gene promoter. We subcloned the full-length MMP-2 promoter region into pGL3e (pGL3a), transiently cotransfected the vector along with pRL-CMV into RD cells, and measured the resulting luciferase activity after 48 hours. We compared this luciferase activity with that from cells transfected with the control vector pGL3e (containing no MMP-2 promoter) and pRL-CMV. Fig. 4, A, shows the relative luciferase activity expressed as the ratio of firefly luciferase (from the pGL3 vector) to renilla luciferase (from the pRL-CMV vector) detected in cell lysates. Cells transfected with pGL3e had very low levels of the firefly luciferase activity. However, cells transfected with pGL3a containing the full-length MMP-2 promoter (from positions 1659 to +57) expressed statistically significantly more luciferase activity (2.1-fold; 95% CI = 1.6-fold to 2.6-fold; P = .002) than did control cells.
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Identification of Factors Associated With Glut-1-Mediated MMP-2 Transcriptional Activation
The region spanning from 1659 to 1079, which appeared to have a crucial role in Glut-1-mediated MMP-2 transcriptional activity, consists of one p53 binding site and one Ets-1 binding site (29). To determine whether either transcription factor is important for Glut-1-mediated MMP-2 promoter activity, we performed gel mobility shift assays by using oligonucleotide probes containing Ets-1 or p53 binding consensus sequences. Nuclear extracts from untreated RD cells contain p53 binding factors that interact with specific p53 oligonucleotide consensus sites, generating two specific bands (Fig. 4, B). The intensity of the bands increased with increasing endogenous MMP-2 transcriptional activity (Fig. 4, B
). There was no substantial difference in the intensity of the bands among untreated RD cells, control transfection reagent-treated RD cells, control-transfected RD cells, and Glut-1 sense oligonucleotide-transfected RD cells. By contrast, there was an increase in the intensity of the bands derived from Glut-1-transfected RD cells and a decrease in the intensity of the bands derived from Glut-1 antisense oligonucleotide-transfected RD cells.
Nuclear extracts from untreated RD cells contain an Ets-1 binding factor that interacts with a specific Ets-1 oligonucleotide consensus site, generating a single specific band (Fig. 4, C). There was no difference in the intensity of the Ets-1 bands among untreated RD cells, control reagent-treated RD cells, control transfected RD cells, Glut-1-transfected RD cells, or among Glut-1 sense or antisense oligonucleotide-transfected RD cells.
The p53 and Ets-1 proteinDNA complexes were specific because their formation was completely inhibited by 125-fold or 250-fold molar excesses of unlabeled oligonucleotide probe or 250-fold molar excess of unlabeled mutated oligonucleotide probe and partially inhibited by a 125-fold molar excess of unlabeled mutated oligonucleotide probe (Fig. 4, B and C). When this mutated oligonucleotide was used as a probe, fewer low-mobility complexes were formed relative to those formed in the presence of the nonmutated oligonucleotide, and these were completely inhibited by a 125-fold molar excess of unlabeled oligonucleotide probe (Fig. 4, B and C
, lanes 1015). Furthermore, we confirmed the identity of the bands by preincubating the nuclear extracts with antibodies to p53 or Ets-1 for 1 hour at room temperature. This incubation prevented the interaction between the protein and the DNA probe, as evidenced by a lack of slower mobility bands. Preincubation with nonimmune IgG had no effect on the formation of p53 and Ets-1 proteinDNA complexes (Fig. 4, B and C
, lane 13).
Effect of Glut-1 Expression on Cell Invasion
Because MMP-2 is involved in cell invasion, we next examined the functional consequences of suppressing Glut-1 expression in RD, T98G, and YKG1 cells in a Boyden chamber invasion assay (27). The invasion of RD, T98G, and YKG1 cells transfected with Glut-1 antisense oligonucleotides was suppressed by 53.0% (95% CI = 47.5% to 58.5%; P = .007), 55.1% (95% CI = 47.5% to 62.7%; P = .01), and 52.4% (95% CI = 47.6% to 57.2%; P = .001), respectively, compared with cells transfected with Glut-1 sense oligonucleotides (Fig. 5, A). The magnitude of the suppression of invasion was similar to MMP-2 mRNA suppression seen in RD, T98G, and YKG1 cells68.8%, 71.5%, and 65.9%, respectively (Fig. 2
).
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Expression of Glut-1 and MMP-2 in Tissue Sections
Because all the cancer cell lines we tested coexpressed Glut-1 and MMP-2 in vitro, we next examined whether human cancers expressed MMP-2 and Glut-1 in the same cells by immunohistochemistry and immunofluorescence. In Fig. 6, A, the upper and lower panels show the typical staining patterns for MMP-2 and Glut-1, respectively, in a human lung squamous cell carcinoma. By immunofluorescence, we detected expression of MMP-2 and Glut-1 in the same cells (Fig. 6, B
). The results of MMP-2 and Glut-1 expression analyses in 80 human cancer specimens analyzed by immunoperoxidase staining by using anti-MMP-2 and Glut-1 antibodies are summarized in Table 1
. Of the 80 cancer specimens, 45 coexpressed Glut-1 and MMP-2, 19 expressed neither Glut-1 nor MMP-2, 14 expressed Glut-1 but not MMP-2, and two expressed MMP-2 but not Glut-1. The relationship between the expression of Glut-1 and MMP-2 was statistically significant (P<.001).
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To assess the effect of glucose on Glut-1 and MMP-2 mRNA expression, RD cells were incubated with varying concentrations of glucose (2.725 mM) for 0, 36, and 60 hours, and total RNA was extracted. Glut-1 and MMP-2 mRNA expression was analyzed by northern blotting. As shown in Fig. 7, glucose did not affect Glut-1 and MMP-2 mRNA expression at any of the tested concentrations or incubation times.
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DISCUSSION |
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Glut-1 gene expression can be induced in cancer cells by oncogenes such as activated Ras and Src, autocrine growth factor loops, and local hypoxia (9,35,36). Glut-1 expression appears to reflect the biologic behavior of cancer cells because the expression of Glut-1 is regulated by conditions that are related to cell proliferation, differentiation, and nutritional starvation. The results of overexpressing Glut-1 are consistent with increased glucose uptake, increased adaptive changes to glycolytic metabolism, and increased cellular proliferation of cancer cells. For example, overexpression of Glut-1 was associated with increased uptake of 2-fluorodeoxyglucose as measured by positron emission tomography of pancreatic cancer cells (37). Younes et al. (38) reported that Glut-1 expression is increased in breast carcinomas with higher histologic grades and higher proliferative activity. However, the direct mechanism by which Glut-1 expression is associated with poor prognosis has not been clearly understood.
In this study, we analyzed expression of Glut-1 and MMP-2 in eight cancer cell lines and 80 cancer specimens. All eight cancer cell lines coexpressed Glut-1 and MMP-2, albeit to various levels. Although the reason for the variability in expression levels is not clear, it may be a result of the difference in the sensitivity of the MMP-2 regulatory machinery induced by Glut-1 in various cancer cells. However, we cannot exclude the possibility that other mechanisms in addition to Glut-1 regulation are also involved in the regulation of MMP-2 expression in A172, MCF7, MDA-MB-231, and MKN45 cells. As summarized in Table 1, there was a statistically significantly frequent coexpression of Glut-1 and MMP-2 in 80 human tumor tissues.
To our knowledge, this is the first report to show a statistically significant association between Glut-1 and MMP-2 expression. It is important to note that the relationship between Glut-1 and MMP-2 is independent of available glucose because three different concentrations of glucose did not alter Glut-1 or MMP-2 mRNA expression (Fig. 7). The overexpression of MMP-2 that results from overexpression of Glut-1 may contribute to neoplastic invasiveness. This finding is consistent with the fact that Glut-1 overexpression is strongly associated with increased tumor cell invasion, increased metastasis, and poor clinical prognosis (914). Thus, the data further support our idea that Glut-1 modulates MMP-2 expression, which could promote tumor cell invasion. Because MMP-2 is closely associated with neoplastic invasiveness, regulation of MMP-2 expression or activity may be a possible target for therapeutic intervention. In this study, loss of Glut-1 expression suppressed MMP-2 expression in invasive human rhabdomyosarcoma and glioblastoma cell lines, with a concomitant suppression of cell invasion. Because a high frequency of human cancers coexpressed Glut-1 and MMP-2 (Table 1
), suppression of Glut-1 expression may be a rational approach to suppressing the metastasis of various human cancers that overexpress Glut-1.
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NOTES |
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We are grateful to Satoko Yamashita for her excellent technical assistance.
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Manuscript received December 4, 2001; revised May 3, 2002; accepted May 22, 2002.
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