Butyrate-Induced Apoptosis in HCT116 Colorectal Cancer Cells

ARTICLE pubs.acs.org/jpr

Butyrate-Induced Apoptosis in HCT116 Colorectal Cancer Cells Includes Induction of a Cell Stress Response Kim Y. C. Fung,*,†,‡ Gemma V. Brierley,†,‡ Steve Henderson,†,‡ Peter Hoffmann,§ Shaun R. McColl,|| Trevor Lockett,†,‡ Richard Head,† and Leah Cosgrove†,‡ †

CSIRO Preventative Health Flagship, Australia CSIRO, Food and Nutritional Sciences, Adelaide and North Ryde, Australia § School of Molecular and Biomedical Science, Adelaide Proteomics Centre, University of Adelaide, Adelaide, Australia School of Molecular and Biomedical Science, Chemokine Biology Laboratory, University of Adelaide, Adelaide, Australia

)

‡

bS Supporting Information ABSTRACT: Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines. KEYWORDS: colorectal cancer, butyrate, butyrate insensitive, MAPK, cell stress

’ INTRODUCTION Colorectal cancer (CRC) is the second most prevalent cancer in the western world and is also regarded as one of the most preventable cancers.1 Short chain fatty acids (SCFA) such as butyrate, propionate, and acetate are produced in the gut and are believed to be protective against the development of colorectal cancer.2 As a result, the generation of butyrate from the microbial fermentation of dietary fiber components such as resistant starch is being investigated as a potential preventative measure in the development of CRC.3-7 In particular, butyrate is known to exhibit antitumorigenic properties both in vivo and in vitro. In vivo, butyrate is the primary energy source for colonocytes, plays a key role maintaining homeostasis of the colonic mucosa and has been demonstrated to inhibit proliferation and induce apoptosis and differentiation in numerous cell lines, including colorectal cancer cell lines.8 Several proteomic and gene expression studies have been conducted in an attempt to determine the mechanisms by which butyrate exerts its effects.9-15 Gene expression studies have r 2011 American Chemical Society

identified transcripts involved in apoptosis, proliferation and differentiation, cell cycle regulation, transcription factors, cell surface receptors and transporters as being differentially regulated by butyrate in both HT29 and HCT116 cell lines.9,11,15 Recent studies by Tan et al. utilized iTRAQ and cICAT technologies to conduct a temporal study comparing the protein expression profile between untreated HCT116 cells and HCT116 cells treated with butyrate over a 48 h time period. In their study, differentially expressed proteins were found to cluster into 4 primary categories: growth arrest, apoptosis, metabolism, metastasis and cytoskeletal-associated proteins.12 Even though the precise mechanism(s) by which butyrate induces apoptosis in colorectal cancer cells is not well understood, inhibition of histone deacetylase activity by butyrate has been linked to many of its antitumorigenic actions.16,17 In addition, butyrate is known to induce cell cycle arrest in numerous colorectal cancer cell lines where this can occur via both p53-dependent and -independent Received: November 4, 2010 Published: January 15, 2011 1860

dx.doi.org/10.1021/pr1011125 | J. Proteome Res. 2011, 10, 1860–1869

Journal of Proteome Research pathways.18 This has encouraged the development of butyrate, butyrate analogues and other histone deacetylase inhibitors as potential anticancer agents, including their use as therapeutic agents for the treatment of colorectal cancer.19-22 In addition to butyrate-induced apoptosis, our research efforts have also focused on the mechanism by which a subpopulation of colorectal cancer cells is able to overcome its apoptotic effects. We have previously shown that a number of important cellular responses are affected by butyrate in HT29 cells and their butyrate insensitive derivative HT29-BR cells.10 In the HT29 cell line, the development of butyrate insensitivity was characterized by remodelling of the actin cytoskeleton, a decrease in protein biosynthesis and potential activation of cell stress pathways. To further define the potential mechanisms involved in butyrate-induced apoptosis, we have used proteomics to compare the global protein expression between HCT116 cells treated with butyrate and HCT116-BR cells, which are less sensitive to butyrate’s apoptotic effects. Our analysis has revealed that butyrate elicits an endoplasmic reticulum (ER) stress response and that an adaptive cellular response to this stress may provide a mechanism for the development of resistance to butyrate-induced apoptosis in the HCT116-BR cells. We discuss our findings and the implications of activation of the classical mitogen activated protein kinase (p38 MAPK) cell stress pathway by butyrate and its potential role in cellular responses to butyrate, including apoptosis.

’ METHODS Cell Culture

HCT116 colorectal cancer cells (ATCC) were maintained in McCoy’s 5A Medium, 1% Pen-Strep and 10% FCS. Butyrate insensitive (HCT116-BR) cells were selected by sustained exposure of HCT116 cells to gradually increasing concentrations of sodium butyrate as described previously.10,23 Surviving cells were maintained in medium containing 5 mM sodium butyrate. For all experimental work, cells were seeded at the appropriate densities and maintained in fresh media for 48 h prior to any treatment. Apoptosis, Proliferation and Differentiation Assays

Cellular proliferation and apoptosis in HCT116 and HCT116-BR cells were measured 48 h following the addition of butyrate (concentration range 0-20 mM) using the CellTiterBlue Assay Kit and Apo-ONE homogeneous Caspase 3/7 Assay Kit respectively according to manufacturer protocols (Promega). Cellular differentiation in HCT116 and HCT116-BR cells was measured 48hrs following the addition of 5 mM butyrate. Differentiation was determined using the Alkaline Phosphatase Detection Kit (Sigma Aldrich) according to manufacturer specifications, except fluorescence was recorded at 355 nm/460 nm (Wallac Victor3 1420 multilabel counter, Perkin-Elmer). All experiments were repeated at least 3 times with all measurements performed in triplicate. Representative data are shown and statistical analysis of the data was performed using Prism 4.0 Software (Graph Pad, San Diego, CA). Protein Isolation and 2D Electrophoresis

Experiments were performed in duplicate where untreated HCT116 cells, HCT116 cells treated with 5 mM butyrate and HCT116-BR cells were compared. Cells were seeded at a density of approximately 11.5 106 cells/mL in T175 flasks and maintained for 24 h prior to any treatment with butyrate. After 48 h, cells were harvested by trypsinisation, washed three times with

ARTICLE

PBS to remove excess media and then lysed on ice (150 mM NaCl, 1% IGEPAL, 1 mM Na3VO4, 10 mM NaF, 20 mM β-glycerophosphate 50 mM Tris, pH 7.5, protease inhibitors (Roche)). Following this, proteins were isolated by CH3OH:CHCl3 precipitation (4:3; 30 min; 4 °C) and the resulting pellet washed in cold acetone to remove remaining solvent and contaminants. The protein sample was then resolublised in rehydration buffer (7 M urea, 3 M thiourea, 2% CHAPS buffer, 30 mM Tris) and the protein concentration determined using the EZQ protein assay (Invitrogen). An aliquot of each protein sample (corresponding to 100 μg of protein) was then labeled with either Cy3 or Cy5 dye (200 pmol) according to manufacturer protocols (GE Healthcare). The internal standard was prepared by combining equal amounts (50 μg) of each respective sample and labeled with Cy2 dye (200 pmol) according to manufacturer protocols. Two dimensional gel electrophoresis (2DE) was performed as previously described.10 Proteins were separated in the first dimension using 24 cm pH 3-11 IEF strips. Strips were rehydrated (450 μL, 7 M urea; 3 M thiourea; 2% CHAPS buffer; 1 M DTT; ampholytes and 0.1% bromophenol blue) and then focused for approximately 70,000 V.Hrs (hold at 300 V for 2 h, hold at 500 V for 2 h, hold at 1000 V for 2 h, ramp to 8000 V for 5 h, hold at 8000 V for 6 h) (IPGphor II system, GE Healthcare). Focused strips were then reduced and alkylated using manufacturer protocols (ETC Elektrophorese-Technik GmbH, Germany) followed by second dimension separation using precast 2DGel DALT NF 12.5% gels (ETC Elektrophorese-Technik GmbH, Germany) under the following conditions: constant temperature of 20 °C, initially at 10 mA/gel for 1 h followed by 30 mA/gel for at least 16 h or until the dye front had reached the bottom of the gel. Gels were scanned using the Ettan DIGE Imager (GE Healthcare) and image analysis performed using the Decyder software (v6.5, GE Healthcare). Protein spots differentially expressed between any of the three groups being compared (ANOVA, p < 0.05) were excised (Ettan Spot Handling Workstation, GE Healthcare) for identification by mass spectrometry. Mass Spectrometry and Protein Identification

Excised gel spots were initially dehydrated in CH3CN (200 μL), prior to the addition of trypsin (0.1 μg in 25 mM NH4HCO3; 37 °C, overnight; Promega, Madison, WI). Following in-gel digestion, peptides were extracted by sequential washes of each gel piece in CH3CN/0.1% CF3COOH (1:1). The samples were then dried under vacuum (SpeedVac) and the peptides resolubilised (0.1% CF3COOH, 10 μL) prior to identification by mass spectrometry. Mass spectra were collected using an Ultraflex III MALDI TOF/TOF mass spectrometer (Bruker Daltonics) operated in the positive ion mode. Peptide mass maps and fragmentation spectra were collected using Rcyano-4-hydroxycinnamic acid as the matrix (Bruker Daltonics). Proteins were identified using the MASCOT search engine (MatrixScience, London, U.K.) and the search was performed against the IPI human protein database (v3.76) and Swiss-Prot database (human subset, v2010-09). Database search parameters included: maximum of two missed tryptic cleavage sites, carboxyamidomethylation of cysteine residues (fixed modification), methionine oxidation (variable modification) and mass tolerance of (50 ppm for peptide mass fingerprinting and (0.5 Da for fragmentation spectra. Western Blot Analysis

For validation of proteomic targets, Western blot analysis was performed using two independent biological replicates. Untreated HCT116 cells, HCT116 cells treated with butyrate, 1861

dx.doi.org/10.1021/pr1011125 |J. Proteome Res. 2011, 10, 1860–1869

Journal of Proteome Research

ARTICLE

Figure 1. Apoptosis, proliferation and differentiation in HCT116 and HCT116-BR cells. HCT116 and HCT116-BR cells were treated with butyrate for 48 h prior to measuring apoptosis, proliferation and differentiation. Error bars are the standard deviation of triplicate measurements from a single representative experiment. (A) Apoptosis in each cell population was determined by measuring caspase 3 and 7 activity. Butyrate induced apoptosis in each cell population, but the HCT116-BR cell population was less responsive to the effects of butyrate. A significant degree of apoptosis was observed in each cell population with the addition of 2.5 mM butyrate (p < 0.001). Lower levels of apoptosis were observed in the HCT116-BR cell line at all concentrations of butyrate used indicating that this cell population is less responsive to the apoptotic effects of butyrate. (B) Butyrate was found to inhibit proliferation in both the HCT116 and HCT116-BR cell lines. Significant levels of inhibition were seen with the addition of 2.5 mM butyrate in each cell population in comparison to the 0 mM control population (p < 0.001). (C) Butyrate was found to induce differentiation in HCT116 and HCT116-BR cells as measured by alkaline phosphatase activity. After 48hrs, significantly higher levels of alkaline phosphatase activity was seen in the HCT116-BR cells in comparison with both the control HCT116 cell population and following treatment of these cells with 5 mM butyrate (p < 0.001). ### p < 0.001 when compared to 0 mM control, 2-way ANOVA with Bonferroni post test. *** p < 0.001, ANOVA with Tukey’s multiple comparison test.

and HCT116-BR cells were harvested 48 h following the addition of 5 mM butyrate. Cells were harvested and protein lysates were obtained as described above. Lysates (equivalent to 12 μg protein) were separated using 4-12% gradient Bis-tris mini-gels according to manufacturer protocols (Invitrogen). Following electrophoresis, proteins were transferred onto Hybond-ECL membrane (GE Healthcare) and the membranes blocked using skim milk powder in TBS-T prior to overnight incubation with goat antitriosephosphate isomerase (1:50 000; Abnova, Taiwan) or rabbit antiperoxiredoxin 1 (1:10 000; Upstate, NY). Rabbit anti-β-actin antibody (1:5000; AbCam,

Cambridge, UK) was used as loading control. HRP-R-mouse IgG1 (1:10 000; Dako), HRP-R-goat IgG (1:5000; Dako) and HRP-R-rabbit IgG (1:5,000; Dako) were used as secondary antibodies. Membranes were then visualized using enhanced chemiluminescence (ECL Plus, GE Healthcare) and quantitative analysis performed using ImageQuant TL software 7.0 (GE Healthcare). For p38 MAPK and HSP27 phosphorylation, HCT116 and HCT116-BR cells were seeded into 10 cm dishes at 3106 cells/ dish and maintained for 24 h prior to treatment. Cells were then cultured in the presence of 5 mM butyrate for up to 72hrs. At 1862



ARTICLE

Table 1. Summary of the 142 Differentially Expressed Proteins Identified by 2D-DIGE in HCT116 and HCT116-BR Cells Exposed to Butyrate general effect with butyrate exposure cellular function

# proteins

HCT116 cells

HCT116-BR cells

Protein biosynthesis

14

inhibitory

inhibitory

Chaperones, protein folding

20

inhibits protein folding

inhibits protein folding

Protein trafficking, endocytosis Protein degradation, proteolysis

3 2

up regulated expression inhibitory

up regulated expression inhibitory

DNA repair

2

potentially enhanced

potentially enhanced

Transcriptional regulation

5

negative regulation, activation of gene transcription negative regulation, activation of gene transcription

RNA splicing and mRNA processing

11

negative regulation

negative regulation

Amino acid, purine/nucleotide biosynthesis

4

inhibitory

enhanced

Stress response, redox regulation

6

enhanced

enhanced

Metabolism

21

enhanced glycolysis

enhanced glycolysis

Cellular respiration, energy production Structural proteins

7 26

enhanced up regulated expression

enhanced up regulated expression

Heme biosynthesis

2

negative regulation

negative regulation

Immunoregulation

3

enhanced immune response

enhanced immune response

Apoptosis

2

inhibitory

inhibitory

Nuclear lamina proteins

4

down regulated expression

down regulated expression

Inflammation

2

anti-inflammatory

anti-inflammatory

Angiogenesis

1

potentially promotes

potentially promotes

Unknown function/miscellaneous

7

N/A

N/A

appropriate time points, cells were washed in PBS (3 times) to remove excess culture medium and then lysed on ice for 30 min (50 mM HEPES, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM Na4P2O7, 2 mM Na3VO4, 10 mM NaF, and protease inhibitor cocktail (Roche), pH 7.5). Lysates were then centrifuged (10 000 g, 15 min, and 4 °C) to remove cell debris, transferred to clean tubes and stored at -80 °C until analysis. The protein concentration was determined using the bicinchoninic acid protein assay (Sigma) and electrophoresis performed as described above. Following electrophoresis, proteins were transferred onto nitrocellulose membranes and the membranes blocked with either skim milk powder (5% w/v, HSP27 antibodies) or BSA (5% w/v, p38 antibodies) in 1xPBST prior to overnight incubation with either rabbit anti-p38 thr180/tyr182 (1:1000, CST), rabbit anti-Hsp27ser15 (1:500, Stressgen), rabbit anti-Hsp27ser78 (1:1000, Stressgen), or rabbit anti-Hsp27ser82 (1:1000, Stressgen). Membranes were stripped and reprobed using rabbit anti-p38 (1:1000, CST) or rabbit anti-Hsp27 (1:1000, Stressgen) for detection of total p38 or HSP27 respectively. HRP-R-rabbit IgG (1:5000; Dako) was used as the secondary antibody. Bound antibodies were then visualized using enhanced chemiluminescence (Western Lightning Plus-ECL, Perkin-Elmer) and densitometry analysis performed using ImageJ software 1.41o (NIH). Data presented is representative of three independent experiments.

’ RESULTS Apoptosis, Proliferation and Differentiation in HCT116 and HCT116-BR Cells

Figure 1 shows representative data comparing the apoptotic, proliferative and differentiation response of HCT116 and HCT116-BR cells to butyrate. We have shown that butyrate induces apoptosis and inhibits the proliferation of both HCT116 and HCT116-BR cells at concentrations of 2.5 mM and higher

(p < 0.001). The HCT116 cell line showed a greater apoptotic response in comparison to the butyrate insensitive HCT116-BR cells whereas a similar antiproliferative response was observed in both cell lines (Figures 1A and B). Figure 1C shows the level of differentiation in these cell lines by measuring alkaline phosphatase activity. After 48 h, butyrate induced differentiation in both the HCT116 and HCT116-BR cells. Although the HCT116 cells showed a slight increase in alkaline phosphatase activity in response to butyrate, this was not statistically significant. The HCT116-BR cells showed significantly higher levels of alkaline phosphatase activity (p < 0.001) in comparison to the untreated HCT116 cell population and HCT116 cells treated with butyrate. Proteomic and Biological Analysis

Proteomics analysis identified 154 protein spots as differentially abundant (ANOVA, p < 0.05) when comparing HCT116, HCT116 cells treated with butyrate and HCT116-BR cells. An example of an annotated 2D DIGE gel image is shown in Supporting Information 1 and the proteins identified are listed in Supporting Information 2. In total, 142 (92%) differentially regulated spots were identified by mass spectrometry and database searching, and this corresponded to 102 (72%) unique gene products with the remaining 40 being isoforms or posttranslationally modified forms of these. Western blot analysis was performed to confirm the expression of two proteins identified as being differentially expressed by butyrate and this is shown in Supporting Information 3. Table 1 summarizes the proteins identified grouped according to their cellular function based on gene ontology annotation. In addition to proliferation, apoptosis and differentiation, this analysis shows that butyrate induces changes in a range of cellular processes including cell metabolism and energy production (20% of proteins identified), protein biosynthesis and folding (28% of proteins identified), DNA transcription and translation (14% of proteins of identified) and also regulates the expression of structural proteins (18% of proteins 1863


Journal of Proteome Research identified). In addition to these, we also identified proteins involved in inflammation, immunoregulation, angiogenesis and regulators of the apoptotic response. The proteins determined to be involved in immunoregulation (four isoforms of FKBP4) and angiogenesis (ANXA2) were identified in one replicate experiment only. We have also identified proteins involved in chromatin remodelling and histone modification, thus influencing gene transcription. This includes proteins such as cold shock domain-containing protein E1 (CSDE1), PRP19/PSO4 pre-mRNA processing factor 19 S. cerevisiae homologue (PRPF19), tripartite motif-containing 28 (TRIM28) and protein arginine N-methyltransferase 5 (PRMT5). In this study, we also provide the first evidence that butyrate mediates cellular heme biosynthesis and the expression of nuclear lamina proteins. The nuclear lamina proteins (LMNA, LMB1 and LMNB2) were identified in only one replicate experiment. Of particular note, we also identified proteins involved in the cellular response to endoplasmic reticulum (ER) stress, including three well characterized protein markers of ER stress. PDIA3 was found to be up-regulated in the butyrate treated HCT116 cell line (1.6 fold change) and HCT116-BR (2.2 fold change) cell line. The expression of HSPA5 was up-regulated by butyrate (1.5 fold) in HCT116 cells, but its expression remained stable in the HCT116BR cells after 48 h. The expression of ERP29 decreased in response to butyrate (-1.29 fold change) in the HCT116 cells whereas an increase in its expression was detected in the HCT116-BR cell line (1.3 fold change). In addition to these proteins, butyrate also modulated the expression of several chaperones involved in protein folding. HSP27 and P38 MAPK Activation

Analysis by 2D-DIGE revealed that HSP27 is down-regulated in HCT116 cells following 48 h exposure to butyrate whereas in HCT116-BR cells, its expression remains relatively stable. Western blot analysis using a more detailed kinetic analysis confirmed the results obtained by 2D-DIGE (See Figure 2). Furthermore, we have also demonstrated that butyrate induces phosphorylation of this protein at residues ser-15, -78, and -82 in a timedependent manner in both cell lines. Constitutive levels of phosphorylation were also detectable in the HCT116-BR cell population (See Figure 2). In both the HCT116 and HCT116BR cell lines, ser-78 and -82 appeared to be the preferred phosphorylation sites in response to butyrate, and lower levels of phosphorylation were detected at ser-15. Maximum levels of phosphorylation were reached within 5 min in each of the cell lines, and phosphorylation was sustained over the 72 h time period investigated with the exception of ser-15. Overall, there were no apparent differences in either the timing or magnitude of HSP27 phosphorylation between the two cell lines. We used Western blot analysis to determine if the p38 MAPK pathway was activated in response to butyrate and was therefore responsible for the phosphorylation of HSP27. Using densitometry, we show that the level of total p38 protein does not appear to change significantly in response to butyrate over time in either the HCT116 or HCT116-BR cell lines (See Figure 3). However, the pattern of p38 phosphorylation differed markedly between these two cell lines under the same experimental conditions. In the HCT116 cell population, phosphorylated p38 was detected at 48 and 72 h following butyrate treatment (Figure 3A), whereas in the HCT116-BR cell population there appeared to be constitutive activation of the phosphorylated p38 protein following butyrate treatment, but this did not appear to alter over time

ARTICLE

(Figure 3B). These data indicate that the p38 MAPK pathway is not solely responsible for the activation and phosphorylation of HSP27 in the cell lines examined in this study. Further experiments are currently being conducted to further characterize both the role of p38 activation by butyrate and the pathways involved in HSP27 phosphorylation.

’ DISCUSSION Butyrate is a well established endogenous inhibitor of histone deacetylase activity in the colon and is also known to induce cell cycle arrest, apoptosis and differentiation in tumorigenic cell lines. Moreover, aspects of structure-activity relationships for butyrate and its analogues have been described.24 Proteomic analysis identified 154 proteins that were differentially regulated between the HCT116 and HCT116-BR cell lines and these proteins were primarily involved with protein biosynthesis and turnover, transcriptional regulation, cellular respiration and metabolism, or were structural proteins (See Table 1). These processes were also identified as being regulated by butyrate in a previous study we conducted utilizing HT29 cells and its butyrate-resistant derivative.10 However, in this current study, we also identified novel proteins modulated by butyrate including enzymes involved in heme biosynthesis, and also differential expression of proteins comprising the nuclear lamina. Heme is a cofactor for a number of proteins (hemoproteins) such as P450 enzymes, catalases, peroxidases, guanylate cyclases, nitric oxide synthases, and also the cytochrome C proteins involved in cellular respiration in the mitochondria.25,26 The biosynthesis of heme is therefore essential for a diverse range of cellular processes, including cellular respiration and metabolism, oxygen sensing, cell growth and differentiation.25,26 In the HCT116 and HCT116-BR cell lines, butyrate was found to reduce the expression of two enzymes involved in heme biosynthesis, coproporhyrinogen III oxidase and uroporphyrinogen decarboxlyase. The expression of these enzymes was reduced approx 1.2 fold in the HCT116 cells but their reduction in expression is even greater in the HCT116-BR cells (approx 2.3 fold). The breadth of cellular processes involving heme underscores the importance of this cofactor in maintaining cell function, and the effects of disrupting this biosynthetic pathway by butyrate needs further investigation. Butyrate Induces a Cell Stress Response in HCT116 and HCT116-BR Cells

Figure 4 outlines possible mechanisms involved in stressinduced apoptosis in HCT116 cells in response to butyrate, and the potential pro-survival adaptive responses elicited in the HCT116-BR cells based on our proteomic data. Proteomic analysis identified several proteins involved in the induction of both a cell stress and an ER stress response. This includes mitochondrial and endoplasmic reticulum (ER) chaperone proteins indicative of an ER stress response, oxidative-stress related proteins and heat shock protein 27 (HSP27), an antiapoptotic protein and a well characterized marker of cellular stress. HSP27 is a well characterized down stream target of the MAPK pathway, and in particular p38 MAPK is recognized as the primary arm of the MAPK pathway responsible for its activation. The MAPK signaling pathway is activated in response to stress and growth factors and modulates the transcription of specific genes involved in proliferation and migration.27 In this study, p38 activation was evident 48 h following butyrate exposure in the HCT116 cells whereas constitutive p38 phosphorylation was observed in the HCT116-BR cells (See Figure 3). We also determined that the 1864


Figure 2. Representative Western blot for HSP27 protein expression and phosphorylation at residues ser-15, ser-78 and ser-82 in HCT116 and HCT116-BR cells in response to butyrate. The data shown is representative of two independent biological replicates. Densitometry was performed using ImageJ software. (A) Phosphorylation of HSP27 in HCT116 cells treated with butyrate. The expression of total HSP27 protein was found to decrease with butyrate treatment in the HCT116 cells after 24hrs. This is in agreement with our 2D-DIGE analysis (Supporting Information 2). Butyrate induced phosphorylation at all three serine residues within 5 min of butyrate treatment. Phosphorylation at residues ser-78 and -82 was sustained over the 72 h time period. After 6 h, phosphorylation at ser-15 declined and was not detectable after 24 h. (B) Phosphorylation of HSP27 in HCT116-BR cells treated with butyrate. In the HCT116-BR cells, the expression of HSP27 remained stable over the time period measured. In this cell line, constitutive levels of HSP27 phosphorylation were detectable at all three serine residues and higher levels of phosphorylation was induced within minutes of butyrate exposure. As with the HCT116 cell population, phosphorylation at residues ser-78 and -82 was sustained over the 72 h time period. After 6 h, phosphorylation at ser-15 declined and was not detectable after 24 h.

Journal of Proteome Research ARTICLE

1865


Figure 3. Representative Western blot and densitometry for p38 and phosphorylated p38 in HCT116 and HCT116-BR cells in response to butyrate. The expression of the p38 protein was found to be stable in both the (A) HCT116 and (B) HCT116-BR cell lines in response to butyrate over time. Phosphorylation of the p38 protein was not detectable in the HCT116 cells until 24 h following butyrate exposure. At the 48 and 72 h time points, high levels of phosphorylated p38 were observed. In the HCT116-BR cells, p38 appeared to be constitutively phosphorylated over the 72 h time period. Data are representative of three independent biological replicates.

Journal of Proteome Research ARTICLE

1866



ARTICLE

Figure 4. Schematic diagram outlining a possible intracellular pathway involved in butyrate-induced apoptosis in HCT116 cells and the adaptive response in HCT116-BR cells to promote cell survival. This model is based on the interpretation of our proteomics data. Butyrate induces an acute stress response as determined by HSP27 phosphorylation. This is independent of p38 MAPK signaling. The cellular stress response induced by butyrate may lead to activation of the ASK1 signaling pathway. Activation (phosphorylation) of p38 in response to ASK1 induces apoptosis in the HCT116 cells. It is possible that protection from oxidant-induced stress in the HCT116-BR cells, for example by PRDX1, may influence ASK1 and p38 signaling to promote cell survival. Butyrate also elicits an ER stress response which leads to the regulation of protein biosynthesis and mRNA translation, potentially inducing apoptosis in HCT116 cells. An adaptive response to ER stress in the HCT116-BR cell population promotes cell survival.

phosphorylation of HSP27 occurs independently of p38 MAPK in these cell lines, indicating that butyrate initiates an alternate signaling pathway which leads to the activation of HSP27. p38 MAPK Activation and Cell Stress Response

The expression of peroxiredoxin 1 (PRDX1) was found to be upregulated in response to butyrate in both the HCT116 cells (approx 1.3 fold increase) and HCT116-BR (approx 1.9 fold increase) cell lines and this was confirmed by Western blot analysis. This protein is an antioxidant enzyme responsible for cell redox homeostasis and has been reported to be overexpressed in colorectal cancer tissue.28 Increased expression of PRDX1 has been linked to resistance to apoptosis under conditions of oxidative stress29,30 and this is most likely mediated through the apoptosis signal-regulated kinase-1 (ASK-1) signaling pathway.31 Binding of PRDX1 to ASK-1 inhibits its activation, influencing the downstream activation of p38 MAPK and JNK signaling cascades.31 The role of PRDX1 and its potential to influence the ASK1 and MAPK signaling cascades in both the HCT116 and HCT116-BR cell lines needs to be further investigated. ASK-1 signaling is also activated by ER stress and in this study we have identified specific proteins modulated by butyrate which are indicative of an ER stress response. This includes well

characterized markers such as PDIA3, HSPA5 (GRP78, BiP) and ERp29 as well as the expression of several other ER chaperones. Recently, Bambang et al (2009) have shown that cytokeratin 19 (CK19) modulates ER stress signaling and p38 signaling in breast cancer cells.32 The expression of CK19 is up regulated by butyrate and similarly, may also influence the cellular response to ER stress in HCT116 cells and HCT116BR cells. In the HCT116 cells, a 2-fold increase in CK19 expression was observed in comparison to untreated cells, and this correlated with activation of p38 MAPK signaling, reduced ERP29 expression and increased HSPA5 expression, potentially enhancing cell survival. In the HCT116-BR cells, increased ERP29 expression and reduced HSPA5 expression indicates that this cell population has potentially adapted to ER stress. HSP27 and the Inflammatory Response

In this study, we also determined that butyrate altered the expression of HSP27. Butyrate is known to regulate HSP27 expression in vitro,13,33,34 but this is the first evidence of its regulation and phosphorylation kinetics in HCT116 cells. We have determined that butyrate induces an acute cellular stress response resulting in the activation of HSP27. Butyrate is known 1867


Journal of Proteome Research to activate cellular signaling pathways via GPCR receptors to influence the inflammatory response, glucose and fatty acid homeostasis as well as the cell stress response.35-37 Thus it is possible that the acute activation of HSP27 is a result of GPCR activation. The protective effect of HSP27 and other inducible heat shock proteins (in particular HSP72 and 90) in the inflammatory and immune response is highly complex and thought to involve the interplay between both the MAPK and NFκb pathways.38,39 These pathways regulate the expression of pro-inflammatory cytokines, and it has been shown that elevated expression of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-R) inhibits the translation of inducible heat shock proteins such as HSP27 in intestinal epithelial cells.40 Further investigations are required to understand the nature of the stress induced during this early phase and the role of HSP27 activation in these cell lines.

’ CONCLUSION In this study we have identified 154 gene products whose expression is modulated by butyrate in HCT116 cells and its butyrate insensitive derivative (HCT116-BR cells). Figure 4 is a schematic diagram outlining our proposed intracellular mechanism involved in butyrate-induced apoptosis in HCT116 cells, and the adaptive response in the HCT116-BR derivative which enables cells to overcome butyrate’s antitumorgenic effects. Proteomic analysis has revealed that butyrate elicits a stress response characterized by HSP27 activation, in addition to oxidative stress and ER stress in the HCT116 cells, activating the p38 MAPK pathway leading to stressinduced cell death. In the HCT116-BR cells, p38 activation did not alter over time. Since this did not result in complete inhibition of apoptosis in the HCT116-BR cells, butyrate must induce apoptosis via additional alternate mechanisms. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Kim Fung, PhD CSIRO, Food and Nutritional Sciences, PO Box 10041, Adelaide BC, South Australia, 5000 Australia (Email) [email protected] (Tel) þ61 8 8303 8840, (Fax) þ61 8 8303 8899.

’ ACKNOWLEDGMENT We would also like to thank Dr. Tanya Lewanowitsch and Ms. Ilka Priebe for their assistance with cell culture work. The Adelaide Proteomics Centre is supported by a grant from the Australian Cancer Research Foundation. ’ ABBREVIATIONS SCFA, short chain fatty acid; Bu, butyrate; CRC, colorectal cancer; HCT116-BR, butyrate insensitive HCT116 colorectal cancer cells

ARTICLE

’ REFERENCES (1) U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999-2005 Incidence and Mortality Web-based Report; U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute: Atlanta, 2009. (2) Topping, D. L.; Clifton, P. M. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol. Rev. 2001, 81 (3), 1031–64. (3) Bajka, B. H.; Clarke, J. M.; Cobiac, L.; Topping, D. L. Butyrylated starch protects colonocyte DNA against dietary protein-induced damage in rats. Carcinogenesis 2008, 29 (11), 2169–74. (4) Clarke, J. M.; Topping, D. L.; Bird, A. R.; Young, G. P.; Cobiac, L. Effects of high-amylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats. Carcinogenesis 2008, 29 (11), 2190–4. (5) Corfe, B. M.; Williams, E. A.; Bury, J. P.; Riley, S. A.; Croucher, L. J.; Lai, D. Y.; Evans, C. A. A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study. BMC Cancer 2009, 9, 332. (6) Dronamraju, S. S.; Coxhead, J. M.; Kelly, S. B.; Burn, J.; Mathers, J. C. Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch: a randomised controlled trial. Gut 2009, 58 (3), 413–20. (7) Le Leu, R. K.; Hu, Y.; Brown, I. L.; Young, G. P. Effect of high amylose maize starches on colonic fermentation and apoptotic response to DNA-damage in the colon of rats. Nutr. Metab. (London) 2009, 6, 11. (8) Hamer, H. M.; Jonkers, D.; Venema, K.; Vanhoutvin, S.; Troost, F. J.; Brummer, R. J. Review article: the role of butyrate on colonic function. Aliment Pharmacol. Ther. 2008, 27 (2), 104–19. (9) Daly, K.; Shirazi-Beechey, S. P. Microarray analysis of butyrate regulated genes in colonic epithelial cells. DNA Cell Biol. 2006, 25 (1), 49–62. (10) Fung, K. Y.; Lewanowitsch, T.; Henderson, S. T.; Priebe, I.; Hoffmann, P.; McColl, S. R.; Lockett, T.; Head, R.; Cosgrove, L. J. Proteomic analysis of butyrate effects and loss of butyrate sensitivity in HT29 colorectal cancer cells. J. Proteome Res. 2009, 8 (3), 1220–7. (11) Iacomino, G.; Tecce, M. F.; Grimaldi, C.; Tosto, M.; Russo, G. L. Transcriptional response of a human colon adenocarcinoma cell line to sodium butyrate. Biochem. Biophys. Res. Commun. 2001, 285 (5), 1280–9. (12) Tan, H. T.; Tan, S.; Lin, Q.; Lim, T. K.; Hew, C. L.; Chung, M. C. Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells. Mol. Cell. Proteomics 2008, 7 (6), 1174–85. (13) Tan, S.; Seow, T. K.; Liang, R. C.; Koh, S.; Lee, C. P.; Chung, M. C.; Hooi, S. C. Proteome analysis of butyrate-treated human colon cancer cells (HT-29). Int. J. Cancer 2002, 98 (4), 523–31. (14) Vanhoutvin, S. A.; Troost, F. J.; Hamer, H. M.; Lindsey, P. J.; Koek, G. H.; Jonkers, D. M.; Kodde, A.; Venema, K.; Brummer, R. J. Butyrate-induced transcriptional changes in human colonic mucosa. PLoS One 2009, 4 (8), e6759. (15) Williams, E. A.; Coxhead, J. M.; Mathers, J. C. Anti-cancer effects of butyrate: use of micro-array technology to investigate mechanisms. Proc. Nutr. Soc. 2003, 62 (1), 107–15. (16) Davie, J. R. Inhibition of histone deacetylase activity by butyrate. J. Nutr. 2003, 133 (7 Suppl), 2485S–93S. (17) Wilson, A. J.; Byun, D. S.; Popova, N.; Murray, L. B.; L’Italien, K.; Sowa, Y.; Arango, D.; Velcich, A.; Augenlicht, L. H.; Mariadason, J. M. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J. Biol. Chem. 2006, 281 (19), 13548–58. (18) Archer, S. Y.; Meng, S.; Shei, A.; Hodin, R. A. p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (12), 6791–6. (19) Frew, A. J.; Johnstone, R. W.; Bolden, J. E. Enhancing the apoptotic and therapeutic effects of HDAC inhibitors. Cancer Lett. 2009, 280 (2), 125–33. 1868


Journal of Proteome Research (20) Schrump, D. S. Cytotoxicity mediated by histone deacetylase inhibitors in cancer cells: mechanisms and potential clinical implications. Clin. Cancer Res. 2009, 15 (12), 3947–57. (21) Tarasenko, N.; Nudelman, A.; Tarasenko, I.; Entin-Meer, M.; Hass-Kogan, D.; Inbal, A.; Rephaeli, A. Histone deacetylase inhibitors: the anticancer, antimetastatic and antiangiogenic activities of AN-7 are superior to those of the clinically tested AN-9 (Pivanex). Clin. Exp. Metastasis 2008, 25 (7), 703–16. (22) Villar-Garea, A.; Esteller, M. Histone deacetylase inhibitors: understanding a new wave of anticancer agents. Int. J. Cancer 2004, 112 (2), 171–8. (23) Chalkley, R.; Shires, A. The isolation of HTC variant cells which can replicate in butyrate. Changes in histone acetylation and tyrosine aminotransferase induction. J. Biol. Chem. 1985, 260 (12), 7698–704. (24) Cheng Ooi, C.; Good, N. M.; Williams, D. B.; Lewanowitsch, T.; Cosgrove, L. J.; Lockett, T. J.; Head, R. J. Structure-activity relationship of butyrate analogues on apoptosis, proliferation and histone deacetylase activity in HCT-116 human colorectal cancer cells. Clin. Exp. Pharmacol. Physiol. 2010. (25) Tsiftsoglou, A. S.; Tsamadou, A. I.; Papadopoulou, L. C. Heme as key regulator of major mammalian cellular functions: molecular, cellular, and pharmacological aspects. Pharmacol. Ther. 2006, 111 (2), 327–45. (26) Mense, S. M.; Zhang, L. Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res. 2006, 16 (8), 681–92. (27) Wagner, E. F.; Nebreda, A. R. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat. Rev. Cancer 2009, 9 (8), 537–49. (28) Rho, J. H.; Qin, S.; Wang, J. Y.; Roehrl, M. H. Proteomic expression analysis of surgical human colorectal cancer tissues: upregulation of PSB7, PRDX1, and SRP9 and hypoxic adaptation in cancer. J. Proteome Res. 2008, 7 (7), 2959–72. (29) Berggren, M. I.; Husbeck, B.; Samulitis, B.; Baker, A. F.; Gallegos, A.; Powis, G. Thioredoxin peroxidase-1 (peroxiredoxin-1) is increased in thioredoxin-1 transfected cells and results in enhanced protection against apoptosis caused by hydrogen peroxide but not by other agents including dexamethasone, etoposide, and doxorubicin. Arch. Biochem. Biophys. 2001, 392 (1), 103–9. (30) Chang, R.; Wang, E. Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress. J. Cell Biochem. 2007, 100 (2), 267–78. (31) Kim, S. Y.; Kim, T. J.; Lee, K. Y. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway. FEBS Lett. 2008, 582 (13), 1913–8. (32) Bambang, I. F.; Lu, D.; Li, H.; Chiu, L. L.; Lau, Q. C.; Koay, E.; Zhang, D. Cytokeratin 19 regulates endoplasmic reticulum stress and inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in breast cancer cells. Exp. Cell Res. 2009, 315 (11), 1964–74. (33) Wang, H. P.; Hanlon, J. G.; Rainbow, A. J.; Espiritu, M.; Singh, G. Up-regulation of Hsp27 plays a role in the resistance of human colon carcinoma HT29 cells to photooxidative stress. Photochem. Photobiol. 2002, 76 (1), 98–104. (34) Yang, Y. X.; Sun, X. F.; Cheng, A. L.; Zhang, G. Y.; Yi, H.; Sun, Y.; Hu, H. D.; Hu, P.; Ye, F.; Chen, Z. C.; Xiao, Z. Q. Increased expression of HSP27 linked to vincristine resistance in human gastric cancer cell line. J. Cancer Res. Clin. Oncol. 2009, 135 (2), 181–9. (35) Maslowski, K. M.; Vieira, A. T.; Ng, A.; Kranich, J.; Sierro, F.; Yu, D.; Schilter, H. C.; Rolph, M. S.; Mackay, F.; Artis, D.; Xavier, R. J.; Teixeira, M. M.; Mackay, C. R. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009, 461 (7268), 1282–6. (36) Tiwari, A. GPR43: an emerging target for the potential treatment of type 2 diabetes, obesity and insulin resistance. Curr. Opin. Investig. Drugs 2010, 11 (4), 385–93. (37) Yonezawa, T.; Kobayashi, Y.; Obara, Y. Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein

ARTICLE

kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line. Cell Signal. 2007, 19 (1), 185–93. (38) Alford, K. A.; Glennie, S.; Turrell, B. R.; Rawlinson, L.; Saklatvala, J.; Dean, J. L. Heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (TAK1)-mediated signaling. J. Biol. Chem. 2007, 282 (9), 6232–41. (39) Malago, J. J.; Koninkx, J. F.; van Dijk, J. E. The heat shock response and cytoprotection of the intestinal epithelium. Cell Stress Chaperones 2002, 7 (2), 191–9. (40) Hu, S.; Ciancio, M. J.; Lahav, M.; Fujiya, M.; Lichtenstein, L.; Anant, S.; Musch, M. W.; Chang, E. B. Translational inhibition of colonic epithelial heat shock proteins by IFN-gamma and TNF-alpha in intestinal inflammation. Gastroenterology 2007, 133 (6), 1893–904.

1869


Butyrate-Induced Apoptosis in HCT116 Colorectal Cancer Cells

Recommend Documents