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Acquisition of Resistance to Butyrate Enhances Survival After Stress and Induces Malignancy of Human Colon Carcinoma Cells

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Acquisition of Resistance to Butyrate Enhances Survival After Stress and Induces Malignancy of Human Colon Carcinoma Cells [CANCER RESEARCH 64, 4593–4600, July 1, 2004] Acquisition of Resistance to Butyrate Enhances Survival after Stress and Induces Malignancy of Human Colon Carcinoma Cells Isabel Lo´pez de Silanes,1,3 Nieves Olmo,1 Javier Turnay,1 Gonzalo Gonza´lez de Buitrago,2 Pablo Pe´rez-Ramos,1 Ana Guzma´n-Ara´nguez,1 Marta Garcı´a-Dı´ez,1 Emilio Lecona,1 Myriam Gorospe,3 and M. Antonia Lizarbe1 1Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Quı´micas, Universidad Complutense, Madrid, Spain; 2Departamento de Inmunologı´a y Oncologı´a, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain; and 3Laboratory of Cellular and Molecular Biology, National Institute on Aging–Intramural Research Program, National Institutes of Health, Baltimore, Maryland ABSTRACT proposed to be a consequence of histone hyperacetylation because such modifications facilitate endonuclease access to chromatin and Acquired resistance to apoptosis by tumor cells remains a major ob- induce or repress the expression of genes encoding proteins such as stacle for cancer treatment, and hence the analysis of resistance to apo- caspase-3 and Bcl-2 that are involved in cell death programs (7–9, 13, ptosis constitutes a major goal in the development of antitumoral drugs. We have established a butyrate-resistant human colon adenocarcinoma 14). Other butyrate-driven modifications altering gene expression cell line (BCS-TC2.BR2) from nontumorigenic BCS-TC2 cells to analyze include alterations in histone phosphorylation, DNA methylation, whether the acquisition of such phenotype confers resistance to apoptosis transcriptional activation of gene promoters bearing butyrate-response and stress. Although BCS-TC2.BR2 cells exhibited a more differentiated elements, and the expression of genes downstream of the ␤-catenin phenotype than the parental BCS-TC2 cells, higher butyrate concentra- and Tcf-signaling pathways (4, 15, 16). tions remained capable of additionally enhancing their differentiation Because of its cytostatic and cytotoxic effects on tumor cells, there without inducing apoptosis. Survival rates of BCS-TC2.BR2 cells after has been much interest in exploring the use of butyrate and its glucose deprivation and heat shock were higher than those of parental analogues as anticancer drugs (9, 17, 18). In addition, butyrate has cells, revealing a stress-resistant phenotype. These findings were accom- been used to sensitize multidrug-resistant tumor cell lines (19), al- panied by key differences between parental and butyrate-resistant cells in though in certain instances, treatment with butyrate might itself in- gene expression profiles and the acquisition of in vivo tumorigenicity. In conclusion, cells gaining resistance to an endogenous physiological mod- crease the multidrug-resistant phenotype (20, 21). ulator of growth, differentiation, and apoptosis concurrently acquired Increased proliferation and decreased apoptosis are key features of resistance to other agents that influence cell survival. neoplasia. In the normal colonic epithelium, butyrate is a major player in the regulation of these processes. However, some cells can escape from butyrate-triggered apoptosis and become resistant to this agent, INTRODUCTION as evidenced by the existance of butyrate-resistant cell lines (21–24). Therefore, the identification of the mechanisms responsible for the A growing number of epidemiological studies suggest that a fiber- acquisition of resistance to butyrate-triggered apoptosis is of great rich diet exerts a protective role against colorectal cancer develop- potential value because it could reveal additional targets for therapeu- ment, the third most common cancer and the second leading cause of tic intervention and new markers for colorectal cancer detection and cancer deaths. It is generally accepted that the end-products of colonic diagnosis. fiber fermentation, particularly butyrate, play an important role in In the present study, we have established butyrate-resistant cells protection against this disease. Butyrate is essential for maintaining from a poorly differentiated, nontumorigenic human colon adenocar- the physical and functional integrity of the intestinal mucosa through cinoma cell line (BCS-TC2) (25) to analyze their biological properties its stimulatory influence on cell proliferation, differentiation, cation and their resistance to chemical and environmental stress. The degree absorption, and colonic blood flow (1–4). Butyrate increases prolif- of induction of apoptosis after exposure to different types of stresses eration of nondifferentiated colonocytes from the crypt base, where was found to be markedly elevated in the parental cells, suggesting the butyrate concentration is low, whereas it induces differentiation and existence of apoptosis-resistant mechanisms in the butyrate-resistant apoptosis of cells at the neck and top of the crypts, exposed to the cells. Importantly, this increase in apoptosis resistance was accompa- higher butyrate concentrations of the intestinal lumen (5). Alterations nied by the acquisition of a malignant phenotype in vivo. in butyrate metabolism can lead to inflammatory bowel disease, atrophic colonic epithelium, and colorectal cancer. Butyrate inhibits cell proliferation and induces differentiation and MATERIALS AND METHODS first apoptosis in transformed cells from colon and other tissues (5–7). It first imposes cell cycle arrest (8, 9), afterward triggering a differ- Cell Culture, Proliferation, and Treatment. Human colon adenocarci- entiation program reflected in changes in specific markers such as noma cells were cultured in DMEM (Sigma, Alcobendas, Spain) supplemented alkaline phosphatase (ALP) activity, which can subsequently induce with 5% FCS (Bio-Whittaker, Verviers, Belgium). Cell proliferation and cells to undergo apoptosis. Butyrate strongly influences transcrip- viability were measured as described previously (26). Spheroids were obtained by seeding the cells on nonadherent dishes coated with 1% (w/v) agar (Difco, tional activity, causing extensive modifications in gene expression Detroit, MI), their growth monitored using an ocular with a grid (Olimpus, patterns (10, 11). Although the precise mechanisms underlying such Melville, NY), and their size calculated according to (a ϫ b)1/2, where a and alterations have not been fully elucidated, the inhibitory effect of b are the largest diameter and its perpendicular diameter, respectively. Heat butyrate on histone deacetylase activity appears to be a major influ- shock was performed at 42°C for the indicated times. Glucose-free DMEM encing factor (3, 12). Butyrate-triggered apoptosis has also been was from Biochrom AG (Berlin, Germany). Butyrate (Sigma) was prepared in standard medium and used at the indicated final concentrations. After estab- Received 2/26/04; revised 4/30/04; accepted 5/13/04. lishment of the butyrate-resistant cell line BCS-TC2.BR2, cultures were rou- Grant support: Direccio´n General de Ensen˜anza Superior Grant PM98-0083 and tinely maintained in standard growth medium in the presence of 2 mM butyrate. Direccio´n General de Investigacio´n Grant BMC2002-01407. Unless otherwise specified, all experiments comparing parental and butyrate- The costs of publication of this article were defrayed in part by the payment of page resistant cells were performed after trypsinization and reseeding of the cells charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. without butyrate. Plating densities were chosen to achieve exponential cell Requests for reprints: Maria Antonia Lizarbe. E-mail: [email protected]. growth by 2–3 days in culture, whereupon experiments were performed. 4593 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. ANTIAPOPTOTIC PHENOTYPE OF BUTYRATE-RESISTANT CELLS Preparation of Cell Lysates and Western Blot Analysis. Cell lysates viability, causing the detachment of cells from the monolayer, and were prepared as previously described (27) and protein content determined induced profound morphological changes, as evidenced by the pres- using the DC Protein Assay (Bio-Rad, Madrid, Spain). For Western blotting, ence of multinuclear cells and by increases in cell volume and vacu- monoclonal antibodies recognizing the 70-kDa constitutive (HSC70) and in- olization (Fig. 1A). The decrease in cell viability was a consequence ducible (HSP70) heat-shock proteins (Stressgen, Victoria, British Columbia, of the induction of apoptosis, as revealed by the detection of internu- Canada), p21 (Neomarkers, Fremont, CA), or vinculin (Sigma) were used. cleosomal DNA fragmentation (Fig. 1B), the exposure of phosphati- Detection was performed using horseradish peroxidase-labeled secondary an- tibodies and enhanced chemiluminescence (Amersham-Pharmacia-Biotech, dylserine (Fig. 1C), and the dispersion and loss of mitochondrial Buckinghamshire, United Kingdom). Vinculin was used as loading control for membrane potential (Fig. 1D). All of these changes documented a Western blotting data, and densitometric analysis was performed using a time-dependent induction of apoptosis in BCS-TC2 cells. However, photodocumentation system (UVItec, Cambridge, United Kingdom) and the after treatment with butyrate for 30 days (Fig. 1A), the number of cells UVIBand V.97 software (28). released from the monolayer began to decrease, and the remaining Flow Cytometry, DNA Fragmentation,
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