Identification of Potential Pathways Involved in Induction of Apoptosis

Article pubs.acs.org/jpr

Identification of Potential Pathways Involved in Induction of Apoptosis by Butyrate and 4‑Benzoylbutyrate in HT29 Colorectal Cancer Cells Kim Y. C. Fung,*,†,‡ Cheng Cheng Ooi,†,‡,§ Tanya Lewanowitsch,†,‡ Sandra Tan,∥ Hwee Tong Tan,# Teck Kwang Lim,∥ Qingsong Lin,∥ Desmond B. Williams,§ Trevor J. Lockett,†,‡ Leah J. Cosgrove,†,‡ Maxey C. M. Chung,∥,# and Richard J. Head†,‡ †

CSIRO Preventative Health National Research Flagship, Adelaide, Australia CSIRO Animal, Food and Health Sciences, Adelaide and North Ryde, Australia § School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia, Australia ∥ Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore # Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore ‡

S Supporting Information *

ABSTRACT: Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure−activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects. KEYWORDS: butyrate, 4-benzoylbutyrate, 3-hydroxybutyrate, apoptosis, proteomics

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INTRODUCTION Epidemiological and experimental studies suggest that dietary fiber is protective against the development of colon carcinoma. This is attributed in part to the production of short chain fatty acids, in particular butyrate.1 Butyrate is a pleiotropic molecule performing a range of roles in the gastrointestinal tract, including acting as an energy source for colonocytes and maintaining homeostasis in the gut mucosa. Furthermore, butyrate has also been demonstrated to induce apoptosis and differentiation in cancer cells in vitro, including in colorectal cancer cell lines.2−8 Butyrate has long been known to act as a histone deacetylase (HDAC) inhibitor,9 and structurally unrelated HDAC inhibitors have gained increasing popularity in current cancer research as chemotherapeutic agents.10 Despite its potent apoptotic activity in cancer cells, butyrate © 2012 American Chemical Society

is not a suitable drug candidate due to its rapid metabolism and short half-life in the peripheral circulation. As a result, much attention has been given to the development of butyrate analogues for cancer therapy with 4-phenylbutyrate and the butyrate prodrug, tributyrin, being the most promising.11−13 Although the biological effects of butyrate are in part correlated with inhibition of HDAC activity,14 more recent studies have demonstrated that such effects can also be mediated via butyrate binding to and activating G-protein coupled receptors (GPCR).15−17 Using iTRAQ and ICAT technology, we have previously demonstrated that butyratemediated changes in protein expression in HCT116 colorectal Received: July 31, 2012 Published: October 11, 2012 6019

dx.doi.org/10.1021/pr3007107 | J. Proteome Res. 2012, 11, 6019−6029

Journal of Proteome Research

Article

using trypsin, and cell pellets were washed thrice with ice-cold PBS and stored at −80 °C until required.

cancer cells could be ascribed into clusters of biological function including cell cycle progression, apoptosis, metabolism and metastasis.18 These findings were confirmed and extended in HT29 colorectal cancer cells using 2D-DIGE and mass spectrometry where, in addition to an influence on proliferation, apoptosis and differentiation, butyrate-mediated changes in energy production, protein biosynthesis and folding, DNA transcription and translation and the expression of structural proteins were also observed.4 Our previous structure activity relationship (SAR) studies demonstrated that there was no simple direct correlation between the inhibition of cell proliferation, induction of apoptosis and inhibition of HDAC activity.19,20 These SAR studies showed that at selected concentrations, only one of the tested butyrate analogues, namely, 4-benzoylbutyrate, had an influence comparable to that of butyrate on cell proliferation and apoptosis in both HT29 and HCT116 colorectal cancer cell lines. The predominant naturally occurring metabolite of butyrate, 3-hydroxybutyrate, had minimal to no effect on these parameters and therefore offers the potential to discriminate between effects that are associated with apoptosis and/or proliferation and those that are less specific. Using iTRAQ labeling and 2D LC−MS/MS, this study aims to identify the proteins that are differentially expressed because of the effects of butyrate and 4-benzoylbutyrate on the growth of HT29 cells. These proteome changes may lead us to better understand the mechanisms underpinning the apoptotic responses to butyrate analogues and their potential utility as chemotherapeutic agents.

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Proliferation Coupled Apoptosis Assays

Cell proliferation and apoptosis of the treated cells were determined after 48 h using the CellTiter-Blue coupled with Apo-ONE Homogeneous Caspase-3/7 assay (Promega, Australia) following the manufacturer’s protocol as previously described.4 For both proliferation and apoptosis assay of butyrate and its analogues treatment, the dose−response curves of fluorescence vs concentration were obtained. The fluorescence data for individual test solutions were expressed as a percentage of the values (for proliferation assays) or fold change (for apoptosis assays) relative to the respective control. HDAC Activity Assays

For the HDAC activity assays, cells were seeded into T75 flasks and collected for lysis when they reached approximately 70% confluence. The nuclear fractions of HT29 and HCT116 cells were first obtained using the BioVision Nuclear/cytosol fractionation kit following the manufacturer’s protocol (BioVision, USA). HDAC activity was measured using the HDAC inhibitory assay kit (BioVision, USA) following the manufacturer’s protocol. The values obtained for individual test solutions were expressed as a percentage of decrease relative to the respective control (expressed as 100%). Histone Extraction and Western Blotting

Acid extraction of histone components from cell pellets was performed according to Kiefer et al. (2006).21 In brief, cell pellets (approximately 8 × 106 cells) were resuspended twice in 1 mL lysis buffer (1 mM Tris, pH 6.5; 35 mM Na2SO4; 10 mM MgCl2; 250 mM sucrose; 1 mM Pefabloc; 1% Triton X-100; 10 mM dithiothreitol). After centrifugation (1 × 103 g; 10 min, 4 °C), the supernatant (cytosol) was discarded and the nuclear pellets were solubilized in 200 μL histone extraction buffer (30 mM Tris, pH 7.4; 40 mM Na2EDTA; 1 mM Pefabloc; l0 mM dithiothreitol; 1.1% concentrated H2SO4). After centrifugation (1.1 × 104 g; 10 min; 4 °C), the supernatant was collected for acetone precipitation (overnight, 4 °C). The mixture was again centrifuged (1.1 × 104 g; 20 min; 4 °C) to obtain pellets containing histones. The pellets were resolubilised in Milli-Q water and stored at −20 °C until required. Protein concentration was estimated using the bicinchoninic acid (BCA) assay. Histone extracts (2 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidenefluoride (PVDF) membrane using the iBlot dry transfer system (Invitrogen). After incubation with blocking buffer (Tris-buffered saline0.05% Tween 20 [TBST] containing 3% ECL blocking agent [GE Healthcare, Australia]), the membrane was probed for 30 min at room temperature with agitation with primary antibodies to histone H4 rabbit monoclonal antibody (1:30 000) (Upstate Biotechnology, USA) or acetyl H4 rabbit polyclonal antibody (1:4000) (Upstate Biotechnology, USA) specific for acetylation at lysine residues 5, 8, 12, and 16 of the histone H4 protein. After washing with TBST, the blots were incubated with goat antirabbit horseradish peroxidise-conjugated secondary antibody (Dako Deutschland GmbH, Germany) for 0.5 h with shaking at room temperature. Signals were detected with Amersham ECL Advance Western blotting detection kit (GE Life Sciences, Australia).

MATERIALS AND METHODS

Chemicals and Reagents

When not specified, all chemicals and reagents were purchased from Sigma-Aldrich (Australia). Stock solutions of 1 M butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were prepared in a solution consisting of equal volumes of 40% ethanol/60% propylene glycol, 1× phosphate buffered saline and dimethyl sulfoxide (EP/PBS/DMSO). Cell Culture

HT29 and HCT116 human colorectal adenocarcinoma cells were obtained from the American Tissue Culture Collection (ATCC, USA). HT29 cells were maintained in a 1:1 ratio of Dulbecco modified Eagle’s medium (DMEM) (Gibco, Invitrogen, Australia) and Hams F12 medium (Gibco, Invitrogen) containing 5% fetal calf serum (FCS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2. HCT116 cells were maintained in McCoy’s 5A medium (Gibco, Invitrogen) containing 10% FCS, penicillin (100 U/ mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2. For the proliferation coupled apoptosis assays, cells were seeded at 5 × 104 cells/well into black 96-well plates for 24 h and then treated with butyrate or its analogues for 48 h. Test solutions were made up to final concentrations (0−20 mM) in DMEM/ F12 or McCoy’s 5A medium supplemented with 3% FCS and 40 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES) and adjusted to pH 6.8. For iTRAQ analysis and Western blots, 2 × 107 cells were seeded into T175 flasks for 24 h and treated with EP/PBS/ DMSO (control), butyrate (5 mM), 3-hydroxybutyrate (5 mM) or 4-benzoylbutyrate (10 mM) for 24 h (as well as 48 h for Western blotting). Cells were then detached from flasks 6020



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Protein Digestion and iTRAQ Labeling

GPS Explorer was also used to assemble and report the iTRAQ data. The false discovery rate (FDR) was determined as previously described in Tan et al. (2008),18 except that a 2% FDR was used. All peptides with significant ion scores (p < 0.05) were included in the analysis to identify proteins that were differentially regulated. Proteins with ratios above 1.5 and under 0.67 when compared to the control group (untreated HT29 cells labeled with iTRAQ reagent 114, Student’s t test, p < 0.05) were determined to be differentially regulated. Proteins showing similar patterns of changing abundance in cells treated with butyrate and 4-benzoylbutyrate but not with 3hydroxybutyrate when compared to mock treated cells were further validated with Western blotting.

Cell pellets from control and treated HT29 cells were resuspended in lysis buffer (0.5 M triethyl ammonium bicarbonate [TEAB], pH 8.5 and 1% SDS) and boiled for 10 min prior to centrifugation at 18800g for 1h at 23 °C.18 The protein concentration of the supernatant was estimated with Coomassie Plus Protein Assay kit (Pierce Biotechnology, USA). Proteins (100 μg) from each treatment were reduced, alkylated, digested with trypsin and labeled with iTRAQ 4-plex reagent following the manufacturer’s protocol (AB Sciex, USA). The samples were labeled as follows: (i) HT29 control lysate was labeled with iTRAQ reagent 114, (ii) 5 mM butyrate lysate with iTRAQ reagent 115, (iii) 5 mM 3-hydroxybutyrate lysate with iTRAQ reagent 116, and (iv) 10 mM 4-benzoylbutyrate lysate with iTRAQ reagent 117. The iTRAQ results were obtained from three biological replicate samples with two technical runs for each (total of 6 analyses).

Western Blotting for iTRAQ Data Validation

Cell pellets were resuspended in 1 mL of cell lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 20 mM βglycerophosphate; 1 mM Na3VO4; 10 mM NaF; 1% IGEPAL) containing protease-inhibitor cocktail (Roche Diagnostics, Australia). Resuspension was performed on ice for 30 min with occasional vortex mixing to achieve complete cell disruption and protein solubilization. The sample was centrifuged (1.1 × 104 g; 4 °C; 15 min) and the supernatant was collected for protein quantitation using the BCA protein assay. Protein extracts (15 μg) were separated on SDS-PAGE and then transferred onto a nitrocellulose membrane using the iBlot dry transfer system. After incubation with blocking buffer (phosphate buffered saline−0.05% Tween 20 [PBST] containing 3% ECL blocking agent), the membrane was probed (1 h at room temperature with agitation) with primary antibodies to either ERO1α (ERO1L) rabbit polyclonal antibody (1:2000) (Novus Biologicals, USA), ProTα (PTMA) mouse monoclonal antibody (1:3000) (Alexis Biochemicals, Switzerland) or βtubulin mouse monoclonal antibody (1:2000) (Sigma, USA). After washing with PBST, the blots were incubated with goat antirabbit or goat antimouse horseradish peroxidise-conjugated secondary antibody, respectively (Dako Deutschland GmbH), for 30 min with agitation at room temperature. Signals were detected with ECL Plus or ECL Advance Western blotting detection kits (GE Healthcare).

Fractionation of iTRAQ Peptides

Tryptic peptides were separated using an Ultimate dualgradient LC system (Dionex LC Packings, USA). The combined labeled mixture was resuspended in 30 μL of buffer A (2% CH3CN; 0.05% TFA), and 25 μL was injected into a 0.3 × 150 mm strong cation exchange (SCX) column (Dionex LC Packings). The column was allowed to equilibrate for 20 min in mobile phase A (5 mM KH2PO4; pH 3; 5% CH3CN) before a gradient was initiated. Nine fractions were separated by step gradients of mobile phase B (5 mM KH2PO4; pH 3; 5% CH3CN; 500 mM KCl) at a 6 μL/min flow rate. The fractions were captured alternatively onto two 0.3 × 1 mm trap columns (Dionex LC Packings) and washed with 0.05% TFA followed by gradient elution in a 0.2 × 50 mm reverse-phase column (Dionex LC Packings). The peptides were eluted from the reverse-phase column using buffer A (2% CH3CN; 0.05% TFA) and B (80% CH3CN; 0.04% TFA) in a 60 min gradient step of 0−60% mobile phase B at a flow rate of 2.7 μL/min. These LC fractions were mixed with MALDI matrix solution (7 mg/mL α-cyano-4-hydroxycinnamic acid; 13 μg/mL ammonium citrate in 75% CH3CN) using a 25 nL mixing tee (Upchurch Scientific, USA) at a flow rate of 5.4 μL/min prior to spotting onto 192-well stainless steel MALDI target plates (AB Sciex) using a Probot Micro Fraction collector (Dionex LC Packings) at a speed of 5 s/well. Mass Spectrometry (MS) Analysis

Densitometry

The fractionated iTRAQ-labeled peptides were then analyzed using the MALDI-TOF/TOF 4700 Proteomics Analyzer (Applied Biosystems). One thousand shots were accumulated for each MS spectrum. MS/MS analyses were performed using nitrogen, at a collision energy of 1 kV and collision gas pressure of ∼2 × 10−6 Torr. Six thousand shots were combined for each precursor ion with signal-to-noise ratio (S/N) greater or equal to 100, and for precursors with S/N between 50 and 100, 1 × 104 shots were acquired. Data files were processed using the GPS Explorer software version 3.5 (Applied Biosystems). The MASCOT search engine (version 2.1; Matrix Science) was used for peptide and protein identifications, using the IPI human database (version 3.85) restricted to tryptic peptides. N-terminal iTRAQ labeling and iTRAQ labeled-lysine were selected as fixed modifications and methionine oxidation was set as a variable modification. One missed cleavage was allowed. Precursor error tolerance was set to 100 ppm and MS/MS fragment error tolerance to 0.4 Da. The maximum peptide rank was set at 2.

Band intensities were determined using Adobe Photoshop version 7.0.1 (Adobe Systems Inc., USA). For comparing effects of butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate treatments, the resulting signals of all antibodies were normalized relative to the control in the respective cell lines (equal to 1). For histone Western blotting, histone H4 served as the loading control while for iTRAQ data validation, βtubulin was the loading control. Statistics

For cell based assays, all data presented are mean values from three independent experiments ± SEM. Dose−response curves were generated using a nonlinear regression sigmoidal dose− response curve (variable slope) using GraphPad Prism 4.0 (GraphPad Software Inc., USA). Where indicated, one-way analysis of variance (ANOVA) was used to compare means. P values used for significance are indicated in figure legends. 6021



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Figure 1. Proliferation and apoptosis in HT29 cells (A and B) and HCT116 cells (C and D) treated with butyrate or its analogues for 48 h at pH 6.8. For the proliferation assay, the values shown are expressed as % of the baseline responses (untreated control, taken as 100%). For the apoptosis assay, values are expressed as fold change from baseline response (untreated control equal to 1). Results reflect the mean ± SEM for three independent experiments in triplicate.

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RESULTS

HT29 and HCT116 cell lines did not appear to alter with butyrate, 3-hydroxybutyrate or 4-benzoylbutyrate treatment at the concentrations tested (Figure 2B), only butyrate treatment increased H4 acetylation in both cell lines in comparison to the control group. 4-Benzoylbutyrate did not alter histone H4 acetylation in the HT29 cells, but in the HCT116 cell line, a 1.5 fold increase in H4 acetylation was observed. However, this increase was less than that observed for butyrate (4.5 fold increase). Since 4-benzoylbutyrate had no detectable acetylation effect in HT29 cells, this suggested that 4-benzoylbutyrate induced apoptosis and inhibited proliferation in HT29 cells independently of H4 acetylation. Treatment of either cell lines with 3-hydroxybutyrate did not alter the acetylation status of the histone H4 protein.

Proliferation and Apoptosis in HT29 and HCT116 Cells in Response to Butyrate and Its Analogues

In order to maximize the specificity of our proteomic profiles for the discovery of apoptotic and cell proliferative signatures, we first determined the minimum concentrations of butyrate and its analogues required to induce maximal responses in these parameters. HT29 and HCT116 cells displayed different responses to cell proliferation and apoptosis when treated with butyrate or 4-benzoylbutyrate (Figure 1). The minimum concentration that produced the maximal responses for butyrate-induced apoptosis and proliferation inhibition was 5 mM in both cell lines. For 4-benzoylbutyrate, these concentrations were 10 mM in HT29 cells and 5 mM in HCT116 cells. Figure 1 also illustrates that the naturally occurring metabolite of butyrate, 3-hydroxybutyrate, had minimal influence on cell proliferation and apoptosis at any of the concentrations tested. These results are consistent with those we published previously.19,20 Proteomic analysis was therefore performed on HT29 cells treated with 5 mM butyrate, 5 mM 3-hydroxybutyrate and 10 mM 4-benzoylbutyrate.

Proteomic Analysis

In the HT29 cells, proteomic analysis identified 4508 peptides that could be mapped to 627 unique gene products. Of these, 111 proteins (18%) were determined to be regulated by butyrate, 3-hydroxybutyrate and/or 4-benzoylbutyrate when compared with the control HT29 cells. Of interest are those proteins with iTRAQ ratios that are relatively high for butyrate or 4-benzoylbutyrate, but with corresponding low values for 3hydroxybutyrate (Figure 3). An arbitrary iTRAQ ratio of ≥1.5 was used to identify proteins that were up-regulated relative to control, and proteins with iTRAQ ratios ≤0.67 were determined to be down-regulated. From the results, butyrate and 4-benzoylbutyrate were found to regulate the expression of 60 (24 up-regulated and 36 down-regulated) and 58 (23 upregulated and 35 down-regulated) proteins, respectively. Interestingly, despite 3-hydroxybutyrate exhibiting no significant effects on HDAC activity, H4 acetylation, cell proliferation, or apoptosis, it did induce differential expression of 35

HDAC Activity and Histone H4 Acetylation

HDAC activity in both HT29 and HCT116 cells was significantly reduced (p < 0.001) by butyrate and 4benzoylbutyrate treatment at the concentrations tested (Figure 2A). 3-Hydroxybutyrate did not have an observable effect on HDAC activity in either of these cell lines. Although the above results are consistent with those we have published previously, they do not correlate with the observed H4 acetylation results. While the expression level of histone H4 protein in both the 6022



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Figure 2. (A) HDAC activity of HT29 and HCT116 cell lysates upon 24 h treatment with butyrate or its analogues. (B) Acetylation of histone H4 in HT29 cells upon 24 h treatment with butyrate or its analogues. (C) Acetylation of histone H4 in HCT116 cells upon 24 h treatment with butyrate or its analogues. The data are expressed as (A) % of the respective baseline HDAC activity (untreated control, taken as 100%); (B) and (C) relative intensity of the respective untreated control (equal to 1). Results reflect the mean ± SEM for three independent experiments in triplicate. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the respective untreated control. Bu, butyrate; 3-OHBu, 3-hydroxybutyrate; 4-BzBu, 4benzoylbutyrate.

(12 up-regulated and 23 down-regulated) proteins. Eight proteins were found to be regulated by both butyrate and its two analogues. The identities of the proteins regulated by each analogue and details of their fold changes are provided in Supporting Information Tables S1 and S2.

factor receptor-bound protein 2 (GRB2), calpain small subunit 1 (CAPNS1), serine/threonine-protein phosphatase 2A catalytic subunit beta isoform (PPP2CB)), including activation of tyrosine kinase receptors (RTK) such as the epidermal growth factor (EGF) receptor.

Comparison of Proteins Regulated by Both Butyrate and 4-Benzoylbutyrate but Not 3-Hydroxybutyrate

Proteins Regulated by 4-Benzoylbutyrate Only

Twenty-three proteins were found to be regulated by 4benzoylbutyrate uniquely, and these are listed in Table 2. Sixteen proteins were found to be down-regulated, while seven proteins were up-regulated. Of note, several nuclear proteins were identified, and they include two proteins, nucleosome assembly protein 1-like 1 (NAP1L1) and nucleosome assembly protein 1-like 4 (NAP1L4) belonging to the nucleosome assembly protein 1 (NAP1) family, transcriptional regulators (i.e., E3 ubiquitin-protein ligase (HUWE1) and ATP-dependent RNA helicase A (DHX9)) and nucleoporin 93 kDa (NUP93), a component of the nuclear pore complex. Two additional proteins including heat shock 70 kDa protein 9 (HSPA9) and cell division cycle 5-like protein (CDCL5) are believed to be involved in cell cycle progression.

The expression of 20 proteins was altered when HT29 cells were treated with either butyrate or 4-benzoylbutyrate but not by 3-hydroxybutyrate (see Table 1). They also showed similar patterns of up- or down-regulation (6 and 13 proteins, respectively) by both compounds with the exception of eukaryotic translation initiation factor 3 subunit E (EIF3E, up-regulated by butyrate and down-regulated by 4-benzoylbutyrate). Of note were c-Myc transcriptional targets such as prothymosin alpha (PTMA), nucleolar protein 16 (NOP16) and basic leucine zipper and W2 domain-containing protein 2 (BZW2) and proteins involved in cellular energy metabolism (beta-hexosaminidase subunit beta (HEXB), beta-glucuronidase (GUSB), hydroxyacyl-coenzyme A dehydrogenase (HADH), acyl-protein thioesterase 1 (LYPLA1)). Also identified were proteins involved in regulation of signal transduction pathways (integrin alpha-V (ITGAV1), growth

Western Blot Validation of Selected Proteins

The differential expressions of two proteins, PTMA and ERO1like protein alpha (ERO1L) were validated by Western blotting 6023



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Figure 3. iTRAQ ratio for proteins identified as being differentially regulated by 3-hydroxybutyrate (x-axis) and (A) butyrate or (B) 4benzoylbutyrate plotted on the y-axis when compared to control untreated cells. Dashed lines indicate iTRAQ ratios of 1.5 and 0.67 relative to control untreated cells. The shaded region indicates those proteins regulated by either butyrate or 4-benzoylbutyrate, but not 3-hydroxybutyrate. Unshaded regions indicate proteins that were also regulated by 3-hydroxybutyrate and are therefore not specific to apoptosis induction by either butyrate or 4-benzoylbutyrate. A subset of proteins regulated by both butyrate and 4-benzoylbutyrate are indicated. The differential regulations of ERO1L and PTMA proteins were validated by Western blot analysis.

point, although butyrate appeared to down-regulate the expression of PTMA further. The expression of ERO1L in response to these analogues, on the other hand, remained constant from 24 to 48 h of treatment of the HT29 cells. 3Hydroxybutyrate did not alter the expression of these proteins in this cell line after 24 h, which is consistent with our proteomic findings. However, a slight downward trend in the expression of PTMA was observed after 48 h. In addition, the expression of the same two proteins was determined in the HCT116 cell line after 24 and 48 h exposure to each analogue. In the HCT116 cells, butyrate and 4-

in the HT29 and HCT116 cells. The results in Figure 4 show their trends in response to each of the analogues at 24 and 48 h treatment time points, and for the HT29 cells, these are in agreement with the iTRAQ data; i.e., butyrate and 4benzoylbutyrate up-regulated the expression of ERO1L and down-regulated the expression of PTMA. The expression of these proteins in response to each analogue was also determined after 48 h treatment, i.e., the time point when cell proliferation and apoptosis were measured. After 48 h treatment, both butyrate and 4-benzoylbutyrate down-regulated PTMA to a similar extent when compared to the 24 h time 6024



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Table 1. Proteins Identified As Being Up-Regulated (Average Ratio ≥ 1.5) or Down-Regulated (Average Ratio ≤ 0.67) by Butyrate (Bu) and 4-Benzoylbutyrate (4-BzBu) in HT29 Cells Relative to the Untreated Control (p < 0.05)a accession no.

gene ID

protein name

IPI00012585 IPI00892827 IPI00555991

HEXB XPO1 ITGAV

Beta-hexosaminidase subunit beta Exportin 1 Integrin alpha-V

IPI00429689

PPP2CB

IPI00966602 IPI00017454 IPI00549891 IPI00025084 IPI00924436 IPI00398727 IPI00465016 IPI00024911 IPI00455510 IPI00973922

CASP6 TUBA4B HS2ST1 CAPNS1 HSPB1 LYPLA1 QSOX1 ERP29 PTMA EIF3E

Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform Caspase 6 Putative tubulin-like protein alpha-4B Heparan sulfate 2-O-sulfotransferase 1 Calpain small subunit 1 Heat shock protein beta-1 Acyl-protein thioesterase 1 Sulfhydryl oxidase 1 Endoplasmic reticulum resident protein 29 Prothymosin alpha Uncharacterized protein

IPI00032849 IPI00807524

NOP16 BZW2

IPI00298406

HADH

IPI00021327 IPI00219516 IPI00386755

GRB2 GUSB ERO1L

cell location

Nucleolar protein 16 Basic leucine zipper and W2 domain-containing protein 2 Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial Growth factor receptor-bound protein 2 Beta-glucuronidase ERO1-like protein alpha

protein function

Bu Av ratio (SD)

4-BzBu avg ratio (SD)

# peptides quantified

cytoplasm nucleus plasma membrane cytoplasm

enzyme transporter not classified

0.55 0.55 0.56 (0.13)

0.45 0.56 0.63 (0.05)

1 1 2

phosphatase

0.57 (0.13)

0.63 (0.13)

4

cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm nucleus cytoplasm

0.58 0.58 0.59 0.60 0.63 0.65 0.65 0.66 0.66 1.66

0.61 0.58 0.49 0.64 0.35 0.67 0.53 0.60 0.54 0.65

1 3 1 1 3 3 2 2 1 2

cytoplasm

peptidase not classified enzyme peptidase not classified enzyme enzyme transporter not classified translation regulator not classified translation regulator enzyme

cytoplasm cytoplasm cytoplasm

not classified enzyme enzyme

nucleus cytoplasm

(0.22)

(0.10) (0.07) (0.15) (0.07) (0.14)

(0.32)

(0.03) (0.07) (0.05) (0.03) (0.13)

1.66 1.75

2.07 1.89

1 1

1.75

1.56

1

1.76 2.06 (0.53) 3.08

1.59 1.70 (0.09) 2.01

1 2 1

a

These proteins were not regulated by 3-hydroxybutyrate. The average ratio and standard deviation (SD) are based on iTRAQ ratios for each peptide identified. The number of peptides quantified for each protein is also included. The details for single peptide matches have been provided in the Supporting Information. The cell location and function for each protein are based on Gene Ontology classifications.

documented that HDAC inhibitors can modify the acetylation status of other protein classes. In the present study, we have further characterized the effect of butyrate and 4-benzoylbutyrate on human colorectal cancer cells by comparing the biological properties and proteomic profiles of the two analogues. On the basis of our observations that 3-hydroxybutyrate, the predominant natural metabolite of butyrate, had minimal influence on apoptosis, cell proliferation and HDAC activity, it follows that a comparison of the proteome profiles of either butyrate- or 4-benzoylbutyrate- with 3-hydroxybutyrate- treated cells offers the potential to identify proteins that are likely to be associated with apoptosis and proliferation. From the iTRAQ analysis of butyrate and 4benzoylbutyrate treated HT29 cells, 111 proteins were found to be differentially regulated, and 20 of these proteins were regulated commonly by both analogues. 4-Benzoylbutyrate regulated an additional 23 proteins uniquely indicating that this analogue may also influence cellular processes via mechanisms distinct from butyrate. Although a direct role for these proteins in apoptosis and cellular proliferation is not known, two of these proteins including heat shock 70 kDa protein 9 (HSPA9) and E3 ubiquitin-protein ligase (HUWE1) have been implicated in colorectal tumorigenesis.22,23 Each of these proteins were down-regulated by 4-benzoylbutyrate. These proteins are reportedly overexpressed in colorectal adenocarcinomas and suppress the activity of the p53 tumor suppressor protein. Those proteins commonly regulated by both analogues have been implicated in apoptosis or cell proliferation. Figure 5 outlines the potential pathways involved in the induction of apoptosis by these compounds, including the oxidative stress

benzoylbutyrate reduced the expression of PTMA after 24 and 48 h, whereas the expression of ERO1L was found to increase only after exposure to each of these analogues for 48 h. Furthermore, exposure to 3-hydroxybutyrate did not appear to alter the expression of these proteins in HCT116 cells. With the exception of ERO1L, these findings are consistent with those observed in the HT29 cell line.

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DISCUSSION Our previous SAR studies demonstrated that treatment of HT29 and HCT116 cells with 4-benzoylbutyrate exhibited comparable in vitro effects to treatment with butyrate.19,20 The present study confirmed these findings on the basis of our results obtained from cell proliferation and apoptosis assays as well as HDAC activity in these cell lines (Figures 1 and 2). We also confirmed that 3-hydroxybutyrate had no or only minimal influence on these biological properties. Because inhibition of HDAC activity by butyrate leads to hyperacetylation of histone proteins, primarily histones 3 (H3) and 4 (H4), we extended our analysis to include H4 acetylation to determine if this mechanism was also involved in 4-benzoylbutyrate-induced apoptosis. Our results here show that 4-benzoylbutyrate failed to induce H4 acetylation in both the HT29 and HCT116 cell lines, despite being effective in the inhibition of HDAC activity. In the absence of a full kinetic analysis, it remains possible that under the appropriate experimental conditions (e.g., appropriate dose and time course), this analogue may be effective in inhibiting HDAC activity and inducing significant acetylation of histone H4. Alternatively, HDAC activity may lead to hyperacetylation of other histone proteins (e.g., histone H3) that were not examined in this study. It has also been 6025



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Table 2. Proteins Identified as Being Differentially Regulated by 4-Benzoylbutyrate (4-BzBu) in HT29 Cells Relative to the Untreated Control (Average Ratio ≤ 0.67 and ≥ 1.5, p < 0.05)a protein name

4-BzBu avg ratio (SD)

# peptides quantified

0.63 0.55 (0.04) 1.77 0.50 0.63 0.56 (0.02) 0.60 (0.22)

1 2 1 1 1 3 2

plasma membrane nucleus cytoplasm cytoplasm cytoplasm

not classified enzyme transporter not classified not classified not classified transcription regulator transmembrane receptor not classified enzyme enzyme not classified

0.58

1

0.52 (0.10) 0.50 0.57 (0.02) 0.54

16 1 12 1

cytoplasm cytoplasm

transporter peptidase

0.56 (0.00) 0.45 (0.08)

2 11

nucleus cytoplasm nucleus nucleus cytoplasm

enzyme not classified not classified enzyme enzyme

1.66 (1.38) 0.66 1.54 1.55 2.42

20 1 1 1 1

nucleus cytoplasm

not classified not classified

1.67 0.67

1 1

nucleus unknown

not classified not classified

0.67 1.50 (0.06)

1 2

accession no.

gene ID

cell location

IPI00966238 IPI00843910 IPI00930710 IPI00965004 IPI00465294 IPI00072044 IPI00445401

HSPA9 FUCA1 SLC25A13 TTC37 CDC5L C11orf54 HUWE1

heat shock 70 kDa protein 9 fucosidase, alpha-L- 1, tissue solute carrier family 25, member 13 tetratricopeptide repeat domain 37 cell division cycle 5-like protein ester hydrolase C11orf54 E3 ubiquitin-protein ligase HUWE1

cytoplasm cytoplasm cytoplasm unknown nucleus nucleus nucleus

IPI00943111

FCGRT

Fc fragment of IgG, receptor, transporter, alpha

IPI00797545 IPI00477231 IPI00027223 IPI00946039

NAP1L1 MGEA5 IDH1 RG9MTD1

IPI00185503 IPI00943181

ABCD3 PSME2

IPI00844578 IPI00294242 IPI00915022 IPI01012856 IPI00793381

DHX9 MRPS31 THOC2 SNRNP200 PSMD6

IPI00644506 IPI00552652

NUP93 MAPRE2

IPI00798071 IPI00217852

NAP1L4 PM20D2

nucleosome assembly protein 1-like 1 bifunctional protein ncoat isocitrate dehydrogenase [NADP] cytoplasmic RNA (guanine-9-) methyltransferase domain containing 1 ATP-binding cassette subfamily D member 3 proteasome (prosome, macropain) activator subunit 2 ATP-dependent RNA helicase A 28S ribosomal protein S31, mitochondrial THO complex subunit 2 small nuclear ribonucleoprotein 200 kDa proteasome (prosome, macropain) 26S subunit, non-ATPase, 6 nucleoporin 93 kDa microtubule-associated protein, RP/EB family, member 2 nucleosome assembly protein 1-like 4 peptidase M20 domain-containing protein 2

protein function

a

These proteins were not regulated by butyrate or 3-hydroxybutyrate. The average ratio and standard deviation (SD) are based on iTRAQ ratios for each peptide identified. The number of peptides quantified for each protein is also included. The details for single peptide matches have been provided in the Supporting Information. The cell location and function for each protein are based on Gene Ontology classifications.

sion and accumulation of this protein in the nucleus is linked with cellular proliferation, and its expression is markedly enhanced in tumor tissues.30−32 In the cytosol, however, PTMA is reported to prevent apoptosis by inhibiting formation of the apoptosome, a complex comprised of cytochrome c and apoptotic protease activating factor 1 (APAF-1), and preventing the activation of procaspase-9 and caspase-3 in the intrinsic mitochondrial-dependent apoptotic pathway.33 More recently, this protein was reported to influence the cellular response to oxidative stress by up-regulating the transcription of stressprotective genes.34 Down-regulation of PTMA is consistent with our observed results, which indicate induction of apoptosis and inhibition of proliferation by both butyrate and 4benzoylbutyrate. Butyrate and 4-benzoylbutyrate also regulated proteins involved in signal transduction pathways including ITGAV, CAPNS1 and PPP2CB. Also of note, GRB2 is involved with activation of receptor tyrosine kinases (RTK). The GRB2 protein binds to activated RTK, such as the epidermal growth factor receptor (EGFR) and the Met receptor.35,36 Interaction of GRB2 with RTK leads to Ras activation and ultimately activation of downstream serine/threonine kinases such as the mitogen-activated protein kinase (MAPK) signaling pathway to enhance cell proliferation and tumor metastatic potential. In this study however, we observed inhibition of proliferation with butyrate and 4-benzoylbutyrate treatment and overexpression of GRB2. At least two mechanisms have been proposed for

and the ER stress signaling pathways. Dysregulated expression of endoplasmic reticulum resident protein 29 (ERP29, downregulated), EIF3E and ERO1L (up-regulated) also indicates that perturbation of ER homeostasis occurs with exposure to butyrate and 4-benzoylbutyrate. ERP29 expression is increased during the unfolded protein response (ER stress), potentially protecting the cell from cellular stress and inhibiting cell growth.24 EIF3E, a subunit of the eukaryotic translation initiation factor 3 (eIF3) translation initiation complex, has been implicated in the regulation of cell proliferation and apoptosis, and altered expression levels have been detected in human cancer tissues.25,26 ERO1L was found to show the largest increase in expression induced by butyrate and 4-benzoylbutyrate treatment. Of cellular significance is the reported observation that ERO1L is induced by C/EPB homologous protein (CHOP) under conditions of ER stress, stimulating ER calcium release into the cytosol and triggering apoptosis.27 Dysregulation of sulfhydryl oxidase 1 (QSOX1), heat shock protein beta-1 (HSPB1) and PTMA expression also indicates that these butyrate analogues influence the cellular response to oxidative stress-induced apoptosis. Up-regulated expression of HSPB1 protein can suppress cell death, and high constitutive expression of HSPB1 has also been detected in CRC tissue, indicating that this protein may play an important role in the early stage of colorectal tumorigenesis.28,29 Many oncogenic functions have been ascribed to PTMA, including dual roles in promoting proliferation and inhibiting apoptosis. Overexpres6026



Article

Figure 4. Representative Western blots for ERO1L and PTMA in HT29 and HCT116 cells following (A) 24 and (B) 48 h treatment with butyrate or its analogues. β-Tubulin was used as the control. Densitometry from Western blots for (C) HT29 cells and (D) HCT116 cells following 24 and 48 h treatment with each analogue relative to β-tubulin. The data are expressed as relative intensity of the respective untreated control (equal to 1). Results reflect the mean ± SEM for three independent experiments. Bu, butyrate; 3-OHBu, 3-hydroxybutyrate; 4-BzBu, 4-benzoylbutyrate.

tion.37 Lack of Gab2/GRB2 binding quenches downstream signaling cascades to potentially inhibit cellular proliferation.37 Kondo et al. (2008) have also proposed that enhanced coupling of GRB2-associated binding protein 1 (Gab1) and GRB2 is responsible for inhibition of cellular proliferation via enhanced extracellular signal-regulated kinase (ERK) signaling as a result of Met receptor activation.36 Further work is required to understand the signaling events involved in inhibition of cellular proliferation in response to butyrate and 4-benzoylbutyrate treatment.

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

Figure 5. Potential pathways involved in the induction of apoptosis by butyrate and 4-benzoylbutyrate. Proteomic analysis identified 20 proteins that were commonly regulated by both butyrate and 4benzoylbutyrate, but not 3-hydroxybutyrate, when compared to control untreated cells. These proteins are involved in oxidative stress and the cell stress response, ER homeostasis and modulation of tyrosine kinase receptor signaling.

*E-mail: [email protected]. Tel: +61 8 8303 8840. Fax: +61 8 8303 8899. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was funded by the CSIRO Preventative Health National Research Flagship. CCO was supported in part by University of South Australia Higher Degree by Research International Travel Grant.

these opposing results. Brummer et al. (2008) hypothesized that binding of specific 14-3-3 proteins to partner proteins such as GRB2-associated binding protein 2 (Gab2) is able to interfere with recruitment of GRB2 to signaling complexes, in particular signaling complexes associated with EGF stimula6027



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Identification of Potential Pathways Involved in Induction of Apoptosis

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