FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1982057613> ?p ?o ?g. }

• W1982057613 endingPage "39223" @default.
• W1982057613 startingPage "39211" @default.
• W1982057613 abstract "The short chain fatty acid (SCFA) buyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive. The short chain fatty acid (SCFA) buyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive. Short chain fatty acids are the final product of bacterial fermentation of dietary fibers (1Bugaut M. Comp. Biochem. Physiol. B. 1987; 86: 439-472Crossref PubMed Scopus (16) Google Scholar). Butyrate, a 4-carbon fatty acid, is the main energy source for normal colonocytes (2Roediger W.E. Gastroenterology. 1982; 83: 424-429Abstract Full Text PDF PubMed Scopus (891) Google Scholar, 3Wong J.M. de Souza R. Kendall C.W. Emam A. Jenkins D.J. J. Clin. Gastroenterol. 2006; 40: 235-243Crossref PubMed Scopus (1853) Google Scholar), which are found in high concentrations in the colonic lumen. It is also thought to be responsible for colorectal cancer prevention, as butyrate can also function as a histone deacetylase (HDAC) 4The abbreviations used are: HDAChistone deacetylasePEPCKphosphoenolpyruvate carboxykinasePKLRpyruvate kinaseMCT1monocarboxylates transporter 1EMTepithelial to mesenchymal transitionRQrelative quantifyingMMPmatrix metalloproteinasesHEXIIhexokinase II. inhibitor to inhibit cell proliferation and induce apoptosis of cancer cells (4Leng S.L. Leeding K.S. Gibson P.R. Bach L.A. Carcinogenesis. 2001; 22: 1625-1631Crossref PubMed Google Scholar, 5Hinnebusch B.F. Meng S. Wu J.T. Archer S.Y. Hodin R.A. J. Nutr. 2002; 132: 1012-1017Crossref PubMed Scopus (399) Google Scholar, 6Shi S.L. Wang Y.Y. Liang Y. Li Q.F. World J. Gastroenterol. 2006; 12: 1694-1698Crossref PubMed Scopus (37) Google Scholar); probably because of this, cancer cells are normally unable to metabolize butyrate. histone deacetylase phosphoenolpyruvate carboxykinase pyruvate kinase monocarboxylates transporter 1 epithelial to mesenchymal transition relative quantifying matrix metalloproteinases hexokinase II. The anti-tumor effects of butyrate were described in studies using colorectal cancer cell lines, in which butyrate inhibits growth, and induces differentiation and apoptosis (7Heerdt B.G. Houston M.A. Augenlicht L.H. Cancer Res. 1994; 54: 3288-3293PubMed Google Scholar). In other studies butyrate was able to inhibit tumor growth in vivo in murine models (8Mariadason J.M. Velcich A. Wilson A.J. Augenlicht L.H. Gibson P.R. Gastroenterology. 2001; 120: 889-899Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 9Medina V. Afonso J.J. Alvarez-Arguelles H. Hernández C. González F. J. Parenter. Enteral. Nutr. 1998; 22: 14-17Crossref PubMed Scopus (83) Google Scholar). Despite these findings, there is an unresolved paradox concerning the putative protective role of butyrate in colon cancer, colorectal cancers still develop and grow despite the high concentrations of butyrate in the colon. Furthermore, several studies have now shown that butyrate-resistant cells may be selected and give rise to more aggressive cancers (8Mariadason J.M. Velcich A. Wilson A.J. Augenlicht L.H. Gibson P.R. Gastroenterology. 2001; 120: 889-899Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Chai F. Evdokiou A. Young G.P. Zalewski P.D. Carcinogenesis. 2000; 21: 7-14Crossref PubMed Scopus (80) Google Scholar, 11López de Silanes I. Olmo N. Turnay J. González de Buitrago G. Pérez-Ramos P. Guzmán-Aránguez A. García-Díez M. Lecona E. Gorospe M. Lizarbe M.A. Cancer Res. 2004; 64: 4593-4600Crossref PubMed Scopus (29) Google Scholar). The mechanisms by which cancer cells develop resistance to butyrate and progress remain unknown. Here, we demonstrate that chronic exposure of colon cancer cells to butyrate may result in the selection of more aggressive clones. Our data provides evidence showing that butyrate-resistant colon cancer cells acquire the ability to metabolize butyrate, resembling normal colonocytes. In butyrate-preselected cells we also found genes involved in EMT, cell proliferation, tumor angiogenesis, and metastasis to be up-regulated compared with the unselected cells. Human colon cancer cell lines (HCT15-CCL-225 and SW480-CCL-228, ATCC) were maintained in DMEM (Invitrogen 41965 with glucose 4.5 g/liter and l-glutamine) at 37 °C in a humidified 5% CO2 incubator. For butyric acid chronic exposure, cells were cultured in DMEM supplemented with 1 mm butyric acid (Sigma) for 5 days. For metabolic assays using NMR, cells grew in DMEM with 1 mm [U-13C]butyrate sodium salt (Sigma). For assaying if butyrate is directly incorporated into membranes or first transformed to acetate, HCT15 cells were grown in a total butyrate concentration of 1 mm: 20% [U-13C]butyrate and 80% butyric acid. For 13C NMR analysis of ethanol extracts HCT15 cells were grown overnight in DMEM with 10 mm [U-13C]butyrate sodium salt (Sigma). Control and butyrate-preselected HCT15 cells were collected and the lipidic fractions were extracted with 15 ml of chloroform/methanol/HCl 12 m (2:1:0.01, v/v/v) and 3.8 ml of KCl (66 mm). After centrifugation, the organic phase was evaporated under nitrogen, and lipids were suspended in chloroform d (deuterium) for NMR analysis. For analysis of the intracellular content, the cells were extracted with ice-cold ethanol (80%) and the supernatants were freeze-dried and suspended in D2O for NMR analyses. Proton-decoupled 13C NMR spectra of cellular extracts were acquired in a Bruker AVANCE III 500 (Bruker, Rheinstetten, Germany) at 125.77 MHz, using a 5-mm 13C selective probe head. Proton decoupling was applied during the acquisition time only. Spectra were recorded at a temperature of 300 K. The chemical shifts in aqueous sample were referred to (trimethylsilyl) propanesulfonic acid, whereas the samples in chloroform d (deuterium) were referred to the solvent signal designated at 77.0 ppm. Assignments were made by comparison with chemical shifts found in the literature for metabolic intermediates. The 13C/13C-correlation spectrum (COSY) was acquired with a standard Bruker Pulse sequence using a 13C 90° flip angle under continuous 1H decoupling. The amount of glucose in culture supernatants was determined by using D_Glucose UV-method (Roche Applied Science/R-Biopharm). Xenografted subcutaneous tumors were induced by inoculation of 5 × 106 HCT15 cells, butyric acid preselected and controls, into the subcutaneous region of 6-week-old male Balb/SCID mice. Animals were sacrificed at day 20. For the VEGF-KDR autocrine loop assay, the animals were injected intra-peritoneally every 3 days with 500 ng of IMC-11C1 (ImClone Systems, New York) or PBS. Tumors were fixed in 2% paraformaldehyde and embedded in gelatin. The antibodies used were anti-CD31 and anti-phospho-Histone3 (Table 1). Secondary antibodies were, respectively, anti-rat and rabbit FITC/phycoerythrin-coupled IgG (Alexa Fluor 488/594, Molecular Probes). Cells were examined in microscopy (Axioplan Microscope, Zeiss, Germany). Microvessel density was evaluated through CD31 immunoreactivity of the five most vascular areas (×200 field). Proliferation was evaluated through phospho-Histone3 immunoreactivity of the five most proliferative areas (×200 field).TABLE 1Primary antibodies used in immunofluorescense and Western blottingAntigenAntibodyDilutionCommercialTechniqueaIF, immunofluorescense; IH, immunohistochemistry; WB, Western blotting.α2 Integrin6110161:50BD BiosciencesIFα3 Integrinsc-70191:50Santa Cruz Biotechnology, Inc.IFα5 Integrinsc-13541:50Santa Cruz Biotechnology, Inc.IFα6 Integrin13781:50ChemiconIFαV Integrin99691:50Santa Cruz Biotechnology, Inc.IFβ3 Integrinsc-200581:50Santa Cruz Biotechnology, Inc.IFKDRV30031:25SigmaIFCytochrome oxidase-2sc-652391:50Santa Cruz Biotechnology, Inc.IFE-Cadherin4A2C71:50ZymedIFCK-19RCK1081:100DakoCytomationIHFoxO1C29H41:50Santa Cruz Biotechnology, Inc.IFFoxM1263C2a1:50Santa Cruz Biotechnology, Inc.IFCD315533701:100BD PharmigenIFpH306–5701:100Cell Signaling SolutionsIFSCADABVAP696Y1:1000AbnovaWBMCAD100–11211:1000Novus BiologicalsWBβ-ActinA54411:1000SigmaWBa IF, immunofluorescense; IH, immunohistochemistry; WB, Western blotting. Open table in a new tab “Metastasis” was induced by tail vein injection of 1 × 106 HCT15 cells, butyric acid-preselected and controls, in 6-week-old male Balb/SCID mice. Animals were sacrificed 4 weeks after injection and lung, liver, and spleen were collected, fixed in 10% formalin, and embedded in paraffin. Tissues were serially sectioned (3 μm), deparaffinized, and stained with hematoxylin and eosin (H&E). Xenografted orthotopic tumors were induced by inoculation of 1 × 106 HCT15 cells, butyric acid selected and controls, into the visceral cecal wall of 6-week-old male Balb/SCID mice. Animals were sacrificed at day 60 and cecum, lung, liver, kidney, and spleen were collected, fixed in 10% formalin, and embedded in paraffin. Tissues were serially sectioned (3 μm), deparaffinized, and stained with hematoxylin and eosin (H&E), as well as used in immunohistochemistry for CK-19 detection and in immunofluorescense for CK-19 and E-cadherin detection. Antibodies used are presented in Table 1. Cells were fixed in 2% paraformaldehyde for 15 min at 4 °C. Antibodies used are presented in Table 1. Secondary antibodies used were anti-rat/goat/rabbit FITC/phycoerythrin-coupled IgG (Alexa Fluor 488/594, Molecular Probes). Cells were examined using Axioplan microscope (Zeiss) and confocal microscopy using a LSM510 META microscope (Zeiss). Cell extracts were performed with RIPA buffer + 1 mm Na2VO3 + 1× proteinase inhibitors (Roche Applied Science), on ice for 30 min. Mitochondria extracts were performed according to the Mitochondria Isolation Kit (MITOISO1, Sigma). Primary antibodies used are shown in Table 1. Secondary antibodies were conjugated with HRP (horseradish peroxidase) and reactivity was developed using ECL Western blotting detection kit (Amersham Biosciences). Bands were quantified using Image J software (rsb.info.nih.gov/ij). Media supernatants of HCT15 cells treated with 1 mm butyric acid were loaded in a 12% polyacrylamide gel (30% acryamide/bis solution, 29:1 (3.3% C), Bio-Rad) with 0.1% gelatin and electrophoresis was performed in 1× TGS buffer (Bio-Rad) during 6 h at 150 V. The gel was incubated in renaturating buffer (25% Triton X-100, v/v) in agitation at room temperature, after which the gel was incubated overnight at 37 °C in developing buffer (50 mm Tris base, 200 mm NaCl, 5 mm CaCl2, 0.02% Brij 35). Staining was performed with 0.5% (w/v) Coomassie Blue R-250 for 30 min and destaining with methanol/acetic acid/water (50:10:40). Bands were quantified using ImageJ software. HCT15 cells (1 × 105) were seeded in the upper well ( 5 μm) in 100 μl of DMEM, 10% FBS and in the lower well in 500 μl of DMEM, 10% FBS, cells were added in the presence or absence of EGF (20 ng/μl) and/or 1 mm butyric acid. Each condition was performed in triplicate and after 12 h the number of cells in the lower well was counted. Expression of VEGF was measured in medium from cultures of HCT15 control cells and HCT15 cells were treated with 1 mm butyric acid, using VEGF ELISA kit Human (Calbiochem) according to the manufacturer's instructions. RNA extraction (RNeasy mini kit, Qiagen), RT-PCR, and mRNA arrays (Oligo GEArray, SuperArray Bioscience corporation) were performed following standard protocols. RNA extraction (RNeasy mini kit, Qiagen) using RT-PCR was performed using SuperScript® II Reverse Transcriptase (Invitrogen) and RQ-PCR with SYBR® Green PCR Master Mix (Applied Biosystems) in a ABI PRISM® 7900HT (Applied Biosystems). The primers used to detect glucose transporters GLUT1 and SGLT1, monocarboxylates transporter 1 (MCT1), hexokinase II (HEXII), glucose-6-phosphate dehydrogenase, pyruvate kinase (PKLR), phosphoenolpyruvate carboxykinase (PEPCK), and SNAIL are presented in Table 2.TABLE 2Primers used in relative quantifying PCRGenePrimerSequence (5′–3′)GLUT1FORCACGGCCTTCACTGTCGTGREVGGACATCCAGGGTAGCTGCSGLT1FORCACGGGTCTCTGGTCTGCREVCATGATGAACATGGGCATCAGMCT1FORGGACTCTTTGCACCTTTGGTGTREVCAACAAGGTCCATCAATGTTTCAAHEXIIFORGCATGAGTTTGACCAGGAGREVCAAAGAGCAGCTCCTCCTTGG6PDFORGGCAACAGATACAAGAACGTGAREVGCAGAAGACGTCCAGGATGAGPKLRFORAGGCGTGAAGAGGTTTGATGAGREVCCCCCGTGCCACCATPEPCKFORGCGGATCATGACGCGGATGREVGAGCGTCAGCTCCGGGTTGSNAILFORCTCTTTCCTCGTCAGGAAGCREVGGCTGCTGGAAGGTAAACTC Open table in a new tab SCAD, MCAD, VEGF, and KDR promoters were analyzed for transcription factor binding site prediction using the TFBlast data base. Detection fragments corresponding to promoter regions were amplified by PCR using primers described in Table 3. For transient transfection assays, 2.5 × 105 HCT15 cells/well were seeded overnight in 24-well plates. Transfections were performed using Lipofectamine 2000 (Invitrogen), according to the standard protocol, with 0.5 μg of the Renilla luciferase expressing vector (transfection control) and 0.5 μg of the pGL3 construct of interest. After transfection, some cells were maintained in DMEM, 10% FBS and others in DMEM, 10% FBS, and 1 mm butyric acid, and all conditions were tested in triplicate. Total cell extracts were prepared after 48 h according to the Dual Luciferase reporter gene assay system protocol (Promega). The luciferase activity of test plasmids was expressed as -fold of induction compared with the activity of the corresponding empty vector (pGL3 basic, Promega), after correction for transfection efficiency with the levels of R. luciferase activity.TABLE 3Primers used for SCAD, MCAD, VEGF, and KDR promoters constructsPromoter genePrimerSequenceaMluI and HindIII restriction sites (in bold) were included, respectively, in forward and reverse primers. (5′–3′)SCADFOR1ATGACGCGTCCTGGCCGCG AGCGCACCTCSCADFOR2ATGACGCGTGGCCCTGATAGACAAGGCAGGSCADREVATGAAGCTTCCAGGCTTCGCGACCTCCCGMCADFOR1ATGACGCGTGTTGGCTACCCGGCGCCGGGMCADFOR2ATGACGCGTGGGCCAGAGGTGGAAACGCAGMCADFOR3ATGACGCGTCCCCACCGTTCAGCGCAACCMCADREVATGAAGCTTGTTGGCTCCCGTTCCGGCCACVEGFFOR1ATGACGCGTGGGTGCTAGAGGCGCACVEGFFOR2ATGACGCGTCCAGATGAGGGCTCCAGTGGVEGFFOR3ATGACGCGTCCCCGCGGGCGCGTGTTCVEGFFOR4ATGACGCGTCCCGGCGGGGCGGAGCCAVEGFREVATGAAGCTTCCCCCAGCGCCACGACCKDRFOR1ATGACGCGTGGAGATCGCCGCCGGGTACCKDRFOR2ATGACGCGTCCAGCTTGCACCCGGCATACKDRREVATGAAGCTTCGGGCGAAATGCCAGAACTCGa MluI and HindIII restriction sites (in bold) were included, respectively, in forward and reverse primers. Open table in a new tab Results are expressed as mean ± S.D. Data were analyzed using the unpaired two-tailed Student's t test or the Tukeys test one-way analysis of variance. p values of <0.05 were considered significant. Under the culture conditions defined under “Experimental Procedures” (in the presence of 1 mm butyrate, for up to 5 days), the HCT15 colon cell line thrives and shows growth patterns similar to untreated cells, whereas SW480 grew much slower when exposed to butyrate (supplemental Fig. 1A) and the levels of cell death were 2-fold higher in the SW480 cell line compared with HCT15 after 5 days of exposure to butyrate (supplemental Fig. 1B). Therefore, we used these culture conditions to study the effects of butyrate in metabolic profiling and the acquisition of phenotypic changes by HCT15 cells. The 13C NMR spectra of lipid extracts of butyrate-treated HCT15 cells showed no substantial alterations in lipid contents, compared with untreated cells (Fig. 1A). The major difference observed was an intensity increase of the resonances in the spectral region characteristic of the olephinic carbons (127 to 131 ppm), indicating an increase in lipid unsaturation. In the 13C NMR spectra of HCT15 cells treated with [U-13C]butyrate extracts, it was possible to observe the incorporation of the labeled carbons from [U-13C]butyrate in the lipid fraction (Fig. 1A). The peaks assigned to ω-1 (14 ppm), ω-2 (22 ppm), ω-3 (32 ppm), C1 (174 ppm), C2 (34 ppm), C3 (24–25 ppm), and the elongation carbons (27–30 ppm) of long chain fatty acids indicated the existence of carbons originating from the [U-13C]butyrate and from other sources. The incorporation of labeled carbons in glycerol was not detected (data not shown), indicating that in triacylglycerol molecules, carbons from butyrate were only incorporated in the fatty acid chain. For the SW480 cell line, there was no alteration in the spectra from the lipidic fraction with and without [U-13C]butyrate, suggesting that butyrate is not metabolized by these cells. In the 13C-13C COSY spectra of lipid extracts from HCT15 cells grown in 20% [U-13C]butyrate and 80% of butyric acid, each signal shows a single COSY correlation, i.e. ω-2 correlated only with ω-1 and not with ω-3, which in turn presented a correlation with an elongation carbon (Fig. 1C), meaning that carbon is incorporated in two atom blocks. Hence, 13C-13C COSY spectra showed that butyrate enters in β-oxidation and is catabolized to acetate prior to incorporation into fatty acids. The 13C NMR spectra of ethanol extracts of HCT15 cells treated overnight with 10 mm [U-13C]butyrate showed that the labeled carbons were incorporated in position 5 of proline and in position 4 of glutamate (Fig. 1B). The J13C-13C coupling constant present in carbon 4 of glutamate indicated that carbon 5 is also labeled. These results suggest that carbons from butyrate enter the oxidative pathway of the Kreb's cycle as acetyl-CoA, being converted to glutamate through oxaloacetate. These data are in agreement with increased expression of SCAD and MCAD (Fig. 2A), acyldehydrogenases that can catalyze the first step of butyrate β-oxidation. Luciferase reporter gene assays showed that the SCAD and MCAD promoters were activated in HCT15 cells previously exposed to butyrate (Fig. 2B). We also found that butyrate-preselected cells express more resistin (RETN) (Fig. 3), which is a hormone that represses the uptake of glucose. This indicates that like normal colonocytes, these cells preferred to metabolize butyrate rather than glucose. Taken together, these data showed that at least a subset of HCT15 cells can fully metabolize butyrate.FIGURE 3HCT15 cells preselected with butyrate express more FoxM1 and membrane and metastases-associated proteins. SuperArray analysis for cell membrane and metastasis markers revealed that HCT15 cells preselected with butyric acid express higher levels of genes related to inhibition of glucose uptake, low differentiation, cell cycle and proliferation, and a more invasive (metastatic) behavior. These data were obtained from three independent experiments, with consistent results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RQ-PCR showed decreased mRNA expression of glucose transporters GLUT1 and SGLT1, whereas MCT1 was increased in the butyrate-preselected HCT15 cells, indicating that HCT15 cells uptake butyrate rather than glucose, because MCT1 is a transporter for butyrate (Fig. 2C). Despite the fact that HEXII is not the unique hexokinase encoding gene, its decreased expression is in agreement with the lower rates of glucose uptake observed in HCT15 cells cultured in butyrate-supplemented medium, which is about 20% less than control cells (Fig. 2D). The expression of PKLR was increased, indicative of glycolysis. The decreased expression of glucose-6-phosphate dehydrogenase indicates that the pentose-phosphate pathway was partially affected by the lack of glucose, however, the increased expression of PEPCK indicates that gluconeogenesis was activated (Fig. 2C). In SW480, the expression of GLUT1 was slightly increased in butyrate-preselected cells, whereas SGLT1 was expressed at the same levels as in control cells. MCT1 expression was also increased in SW480 butyrate-preselected cells, indicating the uptake of butyrate. The expression levels of HEXII, PKLR, and PEPCK were similar expression levels in SW480 control and butyrate-preselected cells, whereas glucose-6-phosphate dehydrogenase is up-regulated in butyrate-preselected cells. In addition, butyrate-preselected cells formed mitochondria aggregates in their cytoplasm, whereas the mitochondria were dispersed in the controls as well as the SW480 control and butyrate-exposed cells (Fig. 2E). Having shown that HCT15 butyrate-preselected cells fully metabolize butyrate, we reasoned that these cells might exhibit different cell phenotypes and behavior in vivo and in vitro compared with control HCT15 cells. Accordingly, butyrate pre-treatment of HCT15 cells prior to subcutaneous inoculation into Balb/SCID mice generated tumors that were significantly more aggressive than those established from control cells. In particular, the tumor sizes (Fig. 4A) and proliferation rates (Fig. 4B) of butyrate-preselected tumors were significantly higher than those established from control cells. Moreover, the microvessel density of butyrate-preselected tumors was also increased (Fig. 4C). In addition to the significant effects in primary tumor growth, butyrate treatment also promoted the formation of lung homing spots. Butyrate-preselected HCT15 cells formed a significant number of lung metastases shortly after inoculation, whereas control cells formed significantly fewer metastases (Fig. 4D). In both groups of animals the liver demonstrated no alterations or metastasis (data not shown). Taken together, these data show that butyrate-preselected cells have increased tumorigenic and metastatic potential. These cancer cells were also implanted orthotopically into the cecal wall. Sixty days after implantation, mice injected with control HCT15 cells showed gross lesions only in the cecum, whereas those injected with butyrate-preselected HCT15 cells showed gross lesions in the peritoneum, liver, pancreas, kidney, lung, and mediastinum (Fig. 5A). Histological examination showed that tumors derived from the control HCT15 cells were multifocal to diffuse, with severe infiltration of the muscle layer, submucosa, and lamina propria. The tumor patterns were mainly nested, with areas of solid growth and moderate lymphatic invasion (Fig. 5B). Primary tumors from butyrate-preselected HCT15 cells were multifocal, with much smaller foci confined to the submucosa. These tumors were rarely detected in the muscle layer, with no infiltration of the lamina propria; but lymphatic invasion was severe for these tumors (Fig. 5B). Notably, metastasis to the liver and lung occurred through vascular dissemination but not by diffusion/infiltration from the serosal surfaces, as observed in the spleen, kidney, and pancreas (Fig. 5C). Next, we explored the molecular basis for the phenotypic features seen in tumors from butyrate-preselected HCT15 cells. First, as determined by zymography, butyrate-preselected HCT15 cells secreted significantly higher levels of active MMP9 and MMP2 (Fig. 6A) compared with control cells, suggestive of increased invasive behavior. Moreover, butyrate-preselected HCT15 cells showed increased migratory capacity to EGF gradient (Fig. 6B), and increased VEGF production into the culture supernatants (Fig. 7A), consistent with the increased angiogenesis seen in vivo (Fig. 4C), and the increased KDR expression (Fig. 7B).FIGURE 7HCT15 cells preselected with butyrate have a more angiogenic phenotype. A, as determined by ELISA, butyrate-preselected HCT15 cells express more VEGF than control cells (p = 0.03); B, as determined by immunofluorescence, butyrate-preselected HCT15 cells express more KDR than control cells. These data were obtained from three independent experiments, with consistent results; C, luciferase reporter gene assay showed that the VEGF promoter sequence −131 to −1 is significantly more active (p < 0.02) than the other tested sequences in HCT15 cells, and deletion constructs pVEGF1, pVEGF2, and pVEGF4 are significantly stimulated in cells preselected by butyric acid (p = 0.041 for pVEGF1, p = 0.0073 for pVEGF2 and p = 0.0098 for pVEGF4); D, luciferase reporter gene assay showed that the KDR promoter sequence −446 to −1 is significantly more active (p = 0.046) than the sequence −796 to −1 in HCT15 cells, and both deletion constructs of KDR promoter are significantly stimulated in cells preselected by butyric acid (p = 0.0205 for pKDR1 and p = 0.0477 for pKDR2; E, as shown by immunofluorescence, butyrate-preselected HCT15 cells show low levels of FoxO1 and increased expression of FoxM1 transcription factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We exploited further the regulation of VEGF and KDR expression in butyrate-preselected HCT15 cells. As shown using luciferase reporter gene assays, both VEGF and KDR promoters exhibited higher activities in butyrate-preselected cells (Fig. 7, C and D). The regulatory elements responsible for the induction of promoter activity in the butyrate-preselected cells appeared to locate between −131 and −66 upstream of the ATG translational start site, whereas for the KDR promoter the responsive region was mapped between −446 and the ATG. We next analyzed the involvement of the forkhead Fox transcription factors (previously shown to regulate VEGF expression (12Wang Z. Banerjee S. Kong D. Li Y. Sarkar F.H. Cancer Res. 2007; 67: 8293-8300Crossref PubMed Scopus (237) Google Scholar, 13Furuyama T. Kitayama K. Shimoda Y. Ogawa M. Sone K. Yoshida-Araki K. Hisatsune H. Nishikawa S. Nakayama K. Nakayama K. Ikeda K. Motoyama N. Mori N. J. Biol. Chem. 2004; 279: 34741-34749Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar)) in the establishment of butyrate-preselected HCT15 cells. As shown in Fig. 7E, there were no detectable differences in FoxO1 expression between the control and butyrate-preselected cells, whereas FoxM1 expression was increased in the butyrate-preselected cells compared with the controls. Taken together, these data strongly suggest that butyrate-preselected cells have higher levels of activated VEGF and KDR, and this induction is mediated at the transcriptional level; in the case of VEGF, it may involve the transcription factor FoxM1 (14Zhang Y. Zhang N. Dai B. Liu M. Sawaya R. Xie K. Huang S. Cancer Res. 2008; 68: 8733-8742Crossref PubMed Scopus (166) Google Scholar, 15Dormond O. Madsen J.C. Briscoe D.M. J. Biol. Chem. 2007; 282: 23679-23686Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Because butyrate-selected HCT15 cells showed increased expression of VEGF and KDR, we reasoned it might result in the generation of a functional VEGF:KDR autocrine loop, as shown in other tumor models (16Santos S.C. Dias S. Blood. 2004; 103: 3883-3889Crossref PubMed Scopus" @default.
• W1982057613 created "2016-06-24" @default.
• W1982057613 creator A5002815845 @default.
• W1982057613 creator A5023037813 @default.
• W1982057613 creator A5027082180 @default.
• W1982057613 creator A5035579330 @default.
• W1982057613 creator A5041150382 @default.
• W1982057613 creator A5044261553 @default.
• W1982057613 creator A5047987801 @default.
• W1982057613 creator A5055347634 @default.
• W1982057613 creator A5065286374 @default.
• W1982057613 creator A5077594914 @default.
• W1982057613 creator A5080331399 @default.
• W1982057613 date "2010-12-01" @default.
• W1982057613 modified "2023-10-16" @default.
• W1982057613 title "Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells" @default.
• W1982057613 cites W1612925887 @default.
• W1982057613 cites W1849320643 @default.
• W1982057613 cites W1965985248 @default.
• W1982057613 cites W1986574885 @default.
• W1982057613 cites W1990624032 @default.
• W1982057613 cites W2015867957 @default.
• W1982057613 cites W2016592775 @default.
• W1982057613 cites W2024813257 @default.
• W1982057613 cites W2033540301 @default.
• W1982057613 cites W2039090342 @default.
• W1982057613 cites W2050395200 @default.
• W1982057613 cites W2051477844 @default.
• W1982057613 cites W2052223356 @default.
• W1982057613 cites W2053865741 @default.
• W1982057613 cites W2057082948 @default.
• W1982057613 cites W2057946453 @default.
• W1982057613 cites W2077560715 @default.
• W1982057613 cites W2086107224 @default.
• W1982057613 cites W2094433942 @default.
• W1982057613 cites W2114927063 @default.
• W1982057613 cites W2121982452 @default.
• W1982057613 cites W2134176478 @default.
• W1982057613 cites W2136498065 @default.
• W1982057613 cites W2138373575 @default.
• W1982057613 cites W2145693683 @default.
• W1982057613 cites W2151148306 @default.
• W1982057613 cites W2151309632 @default.
• W1982057613 cites W2156122059 @default.
• W1982057613 cites W2156870300 @default.
• W1982057613 cites W2156892844 @default.
• W1982057613 cites W2164265402 @default.
• W1982057613 doi "https://doi.org/10.1074/jbc.m110.156026" @default.
• W1982057613 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2998102" @default.
• W1982057613 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20926374" @default.
• W1982057613 hasPublicationYear "2010" @default.
• W1982057613 type Work @default.
• W1982057613 sameAs 1982057613 @default.
• W1982057613 citedByCount "70" @default.
• W1982057613 countsByYear W19820576132012 @default.
• W1982057613 countsByYear W19820576132013 @default.
• W1982057613 countsByYear W19820576132014 @default.
• W1982057613 countsByYear W19820576132015 @default.
• W1982057613 countsByYear W19820576132016 @default.
• W1982057613 countsByYear W19820576132017 @default.
• W1982057613 countsByYear W19820576132018 @default.
• W1982057613 countsByYear W19820576132019 @default.
• W1982057613 countsByYear W19820576132020 @default.
• W1982057613 countsByYear W19820576132021 @default.
• W1982057613 countsByYear W19820576132022 @default.
• W1982057613 countsByYear W19820576132023 @default.
• W1982057613 crossrefType "journal-article" @default.
• W1982057613 hasAuthorship W1982057613A5002815845 @default.
• W1982057613 hasAuthorship W1982057613A5023037813 @default.
• W1982057613 hasAuthorship W1982057613A5027082180 @default.
• W1982057613 hasAuthorship W1982057613A5035579330 @default.
• W1982057613 hasAuthorship W1982057613A5041150382 @default.
• W1982057613 hasAuthorship W1982057613A5044261553 @default.
• W1982057613 hasAuthorship W1982057613A5047987801 @default.
• W1982057613 hasAuthorship W1982057613A5055347634 @default.
• W1982057613 hasAuthorship W1982057613A5065286374 @default.
• W1982057613 hasAuthorship W1982057613A5077594914 @default.
• W1982057613 hasAuthorship W1982057613A5080331399 @default.
• W1982057613 hasBestOaLocation W19820576131 @default.
• W1982057613 hasConcept C100544194 @default.
• W1982057613 hasConcept C154945302 @default.
• W1982057613 hasConcept C185592680 @default.
• W1982057613 hasConcept C2776107976 @default.
• W1982057613 hasConcept C2776979534 @default.
• W1982057613 hasConcept C3020616263 @default.
• W1982057613 hasConcept C41008148 @default.
• W1982057613 hasConcept C502942594 @default.
• W1982057613 hasConcept C55493867 @default.
• W1982057613 hasConcept C70721500 @default.
• W1982057613 hasConcept C81917197 @default.
• W1982057613 hasConcept C86803240 @default.
• W1982057613 hasConcept C89423630 @default.
• W1982057613 hasConcept C95444343 @default.
• W1982057613 hasConceptScore W1982057613C100544194 @default.
• W1982057613 hasConceptScore W1982057613C154945302 @default.
• W1982057613 hasConceptScore W1982057613C185592680 @default.
• W1982057613 hasConceptScore W1982057613C2776107976 @default.
• W1982057613 hasConceptScore W1982057613C2776979534 @default.

• next