Abstract
Colorectal cancer (CRC) is the third most common cancer worldwide. Chronic inflammation appears to enhance the risk of CRC. Emerging evidence has suggested that epigenetic mechanisms play an important role in CRC. Aspirin [acetylsalicylic acid (ASA)] has been shown to prevent CRC; however, the epigenetic mechanisms of its action remain unknown. This study investigated the protective role of ASA in azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted colitis-associated colon cancer (CAC) and examined the epigenetic effects, particularly on histone 3 lysine 27 acetylation (H3K27ac), underlying the preventive effect of ASA. CF-1 mice were fed with AIN-93M diet with or without 0.02% ASA from 1 week prior to AOM initiation until the mice were killed 20 weeks after AOM injection. Our results showed that AOM/DSS + ASA significantly suppressed inflammatory colitis symptoms and tumor multiplicity. AOM/DSS + ASA reduced AOM/DSS-induced protein expression and the activity of histone deacetylases (HDACs) and globally restored H3K27ac. Furthermore, AOM/DSS + ASA inhibited AOM/DSS-induced enrichment of H3K27ac in the promoters of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) that corresponded to the dramatic suppression of the messenger RNA (mRNA) and protein levels. Surprisingly, no significant changes in the H3K27ac abundance in the prostaglandin–endoperoxide synthase 2 (Cox-2) promoters or in the Cox-2 mRNA and protein expression were observed. Collectively, our results suggest that a potential novel epigenetic mechanism underlies the chemopreventive effects of ASA, and this mechanism attenuates CAC in AOM/DSS-induced CF-1 mice via the inhibition of HDACs and the modification of H3K27ac marks that suppress iNOS, TNF-α and IL-6.
Introduction
Colorectal cancer (CRC) is the third most common diagnosed cancer and the third most common cause of cancer death among men and women. It is estimated that 134490 new cases of CRC will be diagnosed in 2016 and that 49190 patients will die from this disease in 2016 (1). Inflammatory bowel disease (IBD) is one of the top high-risk conditions for CRC, together with hereditary familial adenomatous polyposis syndromes and hereditary nonpolyposis colon cancer syndrome (2). In fact, the risk of developing CRC among patients with IBD (including ulcerative colitis and Crohn’s disease) was approximately 2- to 3-fold greater than that in healthy adults (3), indicating a strong association between chronic inflammation and CRC. To investigate the molecular mechanisms that underlie colitis-accelerated colon carcinogenesis (CAC) and to develop effective chemoprevention strategies, azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted mouse models were established, and these models are widely used today. AOM is a classic chemical carcinogen used to induce aberrant crypt foci by causing DNA damage in the liver and colon (4), and DSS is an inflammatory stimulus that damages the epithelial lining of the colon and induces colitis (5). When AOM injection (initiation factor) is followed by DSS in drinking water (promotion factor), CAC is induced similar to the multistep carcinogenesis process in humans. This reliable, reproducible and clinically relevant animal model is a useful tool to simulate the pathogenesis observed in patients with inflammatory CRC and recapitulates many histopathological features of human CRC (6).
The development of CAC is probably multifaceted and involves the accumulation of both genetic and epigenetic alterations (7). Epigenetic modifications, the heritable transcription alterations that do not include changes in DNA sequence, have been implicated in the regulation of gene expression in both normal and cancerous tissue, thereby controlling the transformation from normal epithelium into adenocarcinoma in CRC (8). The covalent modifications of specific residues in the N-terminal tails of the histones dynamically regulate the transition between heterochromatin (a tightly packed structure with gene repression) and euchromatin (a loosely packed structure with gene activation) (9). Lysine acetylation generally opens the chromatin, increases the accessibility of transcriptional factors to chromatin and activates gene transcription, whereas deacetylated histone is often associated with gene repression (10). Several lines of evidence have observed differential patterns of histone acetylation in IBD animal models (11,12), and alterations of histone H3 lysine 27 acetylation (H3K27ac) were found in patients with sporadic colon cancer (13). However, the alteration of H3K27ac in the regulation of the inflammatory network during CAC has not yet been investigated.
Histone acetylation is a reversible modification dynamically mediated by the epigenetic enzymes histone acetyltransferases and histone deacetylases (HDACs) that add or remove acetyl groups, respectively. Of all the epigenetic enzymes, HDACs are perhaps the most extensively characterized epigenetic proteins in chronic inflammatory diseases and CRC. Abnormalities of the expression and activity of HDACs can lead to histone hyperacetylation or histone hypoacetylation, thereby altering the expression of key genes in cancer and inflammation (14). Emerging evidence from in vitro cell lines and in vivo models of IBD and inflammation-driven tumorigenesis has suggested that the inhibition of HDAC represents a novel therapeutic strategy for CRC (11,15). Notably, several HDAC inhibitors, including vorinostat, romidepsin and panobinostat, have been approved by the US Food and Drug Administration to treat hematopoietic tumors. Moreover, an increasing number of HDAC inhibitors are currently being evaluated in clinical trials to treat various cancer types. However, those epigenetic agents are usually toxic. The discovery and development of more specific and safer agents to prevent CRC by targeting HDACs are needed.
Acetylsalicylic acid, also known as aspirin (ASA), is one of the most widely used drugs in the world, particularly for the prevention of cardiovascular diseases. Compelling findings from epidemiological studies, clinical trials and laboratory data have indicated that ASA can protect against CRC (16). Case–control studies revealed that regular ASA users have a statistically significant lower risk of developing sporadic CRC (odds ratio = 0.62) compared with nonusers (17). In addition to preventing sporadic CRC, the effect of ASA has been evaluated in hereditary CRC. It was found that long-term ASA use (600mg/day for a mean of 25 months) reduces cancer incidence in carriers of hereditary CRC (18). The use of ASA was also shown to be associated with lower risk of CRC in patients with chronic ulcerative colitis (odds ratio = 0.3) (19). Moreover, preclinical animal models have been used to investigate the mechanisms underlying chemopreventive effects of ASA. Recently, ASA at 200mg/kg for 80 days was found to induce apoptosis in AOM/DSS-induced Balb/c mice by inhibiting interleukin 6 (IL-6)-STAT3 pathway, although tumor multiplicity was not significantly changed by ASA treatment (20). Thus, we aim to determine whether longer exposure (20 weeks) of ASA would prevent the carcinogenesis in AOM/DSS-induced CF-1 mice.
The well-characterized mechanism of ASA’s action is the modification of the COX enzymes. It has been reported that COX-2-derived prostaglandins played an essential role in tumorigenesis in hereditary and sporadic CRC in which pro-inflammatory cytokines are not strongly expressed (21). However, studies showed that tumor formation in the AOM/DSS model may not be COX-2 dependent, suggesting that cyclooxygenase-derived prostanoids does not play a major role in inflammatory CRC (21). Notably, although clinical evidence has indicated that regular ASA use reduces the risk of CRC exclusively in individuals with overexpressed COX-2 (22), the effect of low-dose ASA on the activity of COX-2 is marginal (16). Thus, the elucidation of COX-2-independent pathways underlying the effect of ASA in preventing inflammatory CRC is needed. A few recent studies have suggested that epigenetic events are involved in the action of ASA and other nonsteroidal anti-inflammatory drugs with regard to cancer prevention (23). Hence, the present study investigated the epigenetic effects of ASA, particularly the modulation of HDACs and H3K27ac, regarding the suppression of inflammatory responses and the prevention of CAC using an AOM/DSS-induced mouse model.
Materials and methods
Animals, chemicals and diets
Male CF-1 mice were purchased from Charles River Laboratory (Wilmington, MA). AOM, hematoxylin and eosin were obtained from Sigma–Aldrich (St Louis, MO). DSS (molecular weight: 36000–50000) was purchased from MP Biomedicals (Solon, OH). ASA was purchased from Sigma–Aldrich and blended with AIN-93M rodent diet at the ratio of 0.02% (w/w) by Research Diet Inc. (New Brunswick, NJ).
Animal experimental procedure
All animal experiments were conducted in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Rutgers University. Upon arrival, the mice were housed in sterilized cages in a room held at a controlled temperature (20–22°C), with controlled relative humidity (45–55%) and 12-h light–12-h dark cycles at the Rutgers Animal Care Facility. All the animals had free access to water and diet throughout the experiment. The experimental protocol is summarized in Figure 1A. Briefly, after 1 week of acclimatization, mice were randomly assigned to three groups (n = 12) and started AIN-93M diet with or without ASA. One week later, the CF-1 mice (6 weeks old) were injected with AOM (10mg/kg body wt) or the saline (vehicle) subcutaneously at the lower flank. At 7 weeks, the drinking water for the mice in the AOM/DSS and AOM/DSS + ASA groups was replaced with 1.2% DSS (w/v) in distilled water for 7 days, after which the fluid was replaced with drinking water until the end of the experiment. The body weight and consumption of food and water were recorded weekly. The mice were humanely killed via CO2 asphyxiation 20 weeks after AOM injection. Blood samples were collected by cardiac puncture. At necropsy, the colons were removed, flushed with saline and opened longitudinally on filter paper. The number of tumors was counted, and the size of the tumors was measured using a caliper ruler. The tumor volume was estimated using the formula V = 0.5 × (length × width × width) as reported by Carlsson et al. (24). After the removal of the proximal end of the colon and tumors, the remaining tissue was cut into two halves along the main axis. The left portion of the colon was fixed in 10% buffered formalin for 24h, and the right half was snap frozen in dry ice and stored at −80°C for further analysis.
The dietary administration of ASA inhibits CAC in AOM/DSS-induced CF-1 mice. (A) The experimental protocol for a chemoprevention study with ASA using the AOM/DSS model. (B) AOM/DSS + ASA suppressed the elevated DAI starting at day 3 of DSS administration, and the suppression was significant at day 7. (C) The administration of ASA did not cause significant weight loss. (D) AOM/DSS + ASA decreased tumor incidence and tumor multiplicity. (E) The effect of ASA on tumor volume. *P < 0.05 versus AOM/DSS.
Clinical scoring of colitis
To monitor the general clinical symptoms of acute colitis, the disease activity index (DAI) was calculated by scoring the percent of weight loss, stool consistency and bleeding during the administration of DSS as described previously (25). Briefly, each parameter was scored on a scale of 0–4, and the summed score of the three parameters was recorded as the DAI of each mouse. The percent of weight loss was determined as follows: 0 = weight loss <1%; 1 = 1% ≤ weight loss <5%; 2 = 5% ≤ weight loss <10%; 3 = 10% ≤ weight loss <20%; and 4 = weight loss ≥20%. The stool consistency parameter scores were determined as follows: 0 = well-formed stools; 2 = pasty and loose stools; and 4 = diarrhea (liquid form that adheres to the anus). Bleeding scores were determined as follows: 0 = no bleeding; 2 = blood present in the stool; 4 = gross bleeding.
Histopathological analysis
After fixation in the 10% buffered formalin for 24h, the left half of the colon was dehydrated, embedded in paraffin, sectioned (4 µm) and mounted on glass slides. The sections were stained with hematoxylin and eosin and evaluated by the histopathologist Dr Guangxun Li. Colonic neoplasms and dysplasia were classified into three categories: (i) dysplasia; (ii) adenoma; and (iii) adenocarcinoma, according to the criteria previously described (26).
Immunohistochemical analysis
Immunohistochemistry staining was performed as previously described (27) using a primary antibody against H3K27ac (Abcam, Cambridge, MA) at a 1:500 dilution. The results of the immunohistochemistry staining were acquired using an Aperio Scanscope scanner (Aperio Technologies, Inc., Vista, CA). The percentage of positive nuclei staining was analyzed using ImageScope software (Aperio Technologies).
RNA isolation and quantitative PCR
Total RNA was extracted from snap-frozen precancerous colonic mucosa using the AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA). First-strand complementary DNA was synthesized from 1 µg of RNA using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Carlsbad, CA). qPCR analysis was performed in an ABI7900HT system (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) using complementary DNA as the template. The primer sequences for tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), prostaglandin–endoperoxide synthase 2 (Cox-2) and interleukin 6 (IL-6) are provided in Supplementary Table 1, available at Carcinogenesis Online.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation assay (ChIP) assay was performed using the MAGnifyTM Chromatin Immunoprecipitation System (ThermoFisher Scientific, Waltham, MA) following the manufacturer’s instructions. Briefly, chromatin sample was prepared from approximately 50mg of snap-frozen noncancerous colonic tissue and sheared to an average length of 200–500bp via sonication at 4°C using a Bioruptor sonicator (Diagenode Inc., Sparta, NJ). The diluted chromatin solution was immunoprecipitated with 2 µg of anti-H3K27ac antibody (Abcam) or mouse immunoglobulin G. After washing, cross-link reversal, DNA elution and DNA purification, the relative amount of immunoprecipitated DNA was quantified via qPCR using the primers listed in Supplementary Table 2, available at Carcinogenesis Online. The enrichment of the precipitated DNA was calibrated using the standard curve from the serial dilution of the inputs, and the data were presented as the fold changes in the signal-to-input ratio normalized to the control.
Western blotting
Protein sample was isolated from snap-frozen noncancerous colonic mucosa using radioimmunoprecipitation assay buffer (Cell Signaling Technology, Boston, MA). After homogenization by passing through the syringe with a 21G needle 10 times, resting and sonication at 4°C, the lysates were cleared via centrifugation at 14,000g for 10min at 4°C. The supernatants were collected and quantified using the Pierce™ bicinchoninic acid protein assay kit (ThermoFisher Scientific). Next, 20 µg of total protein was diluted with Laemmli’s sodium dodecyl sulfate sample buffer (Boston Bioproducts, Ashland, MA) and denatured at 90°C for 5min. Then, western blotting was performed as described previously (28). The antibodies were obtained from Cell Signaling Technology (HDAC 1, 2, 3 and 4), Santa Cruz Biotechnology (Santa Cruz, CA; iNOS, β-ACTIN, and all of the secondary antibodies) and Abcam (COX-2 and HDAC5). The protein bands were visualized using the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) with the Gel Documentation 2000 system (Bio-Rad, Hercules, CA). The relative protein expression was semiquantitated via densitometry using ImageJ (Version 1.48d; NIH) and presented as fold changes by calculating the density of each sample compared with the control sample and then normalized to the intensity of β-ACTIN.
Enzyme-linked immunosorbent assay
The level of TNF-α and IL-6 was determined using the protein lysate as prepared in western blotting and plasma using the Mouse TNF-α and IL-6 enzyme-linked immunosorbent assay kit (ThermoFisher Scientific), respectively, according to the manufacturer’s protocol. The level of TNF-α (pg/mg) was calculated by dividing the concentration of the total protein (mg/mL) by the concentration of TNF-α (pg/mL).
HDAC activity assay
The HDAC activity in noncancerous colonic tissue was determined using an Epigenase HDAC Activity/Inhibition Direct Assay Kit (Epigentek, Farmingdale, NY) following the manufacturer’s instruction. The nuclear extract was prepared from snap-frozen colonic mucosa using NE-PER™ Nuclear and Cytoplasmic Protein Extraction Reagents (ThermoFisher Scientific) and quantitated. The relative HDAC activity was calculated as the ratio of the HDAC activity of the AOM/DSS group or AOM/DSS + ASA group compared with that of the control group.
Statistical analysis
The data are presented as mean ± SEM. One-way analysis of variance with Tukey’s multiple comparison tests were used to test comparisons among multiple groups, and two-tailed Student’s t-tests were employed for simple comparisons between two groups. A P-value of <0.05 was regarded as significant.
Results
ASA at the dose of 0.02% attenuates AOM/DSS-induced acute colitis and colon tumorigenesis in CF-1 mice
DSS treatment after AOM injection was used to induce acute colitis. The clinical severity of colitis was estimated by assessing the DAI from day 0 to day 7 during the DSS treatment. The DAI has been widely used as an estimator of colitis severity and is associated with the presence of erosions and inflammation (29). As shown in Figure 1B, the DAI score in the AOM/DSS group increased gradually, but AOM/DSS + ASA slightly attenuated the increase of DAI starting at day 3, and the inhibition was significant at day 7. This result suggests that ASA protects against DSS-induced acute colitis. The effect of ASA in AOM/DSS-induced colon tumorigenesis was examined 20 weeks after AOM injection. As shown in Figure 1D, 7 of the 12 animals in the AOM/DSS group showed tumor growth in the colon, and the tumor multiplicity was 6.5±2.7 tumors per mouse. This finding is comparable with that of previous publication with a similar experimental design in CF-1 mice (26). The dietary supplementation of 0.02% ASA for 21 weeks resulted in only 2 of the 12 mice showing tumor growth and a significantly lower tumor multiplicity (0.2±0.1 tumors per mouse; Figure 1D). However, the tumor volume was only slightly reduced by AOM/DSS + ASA (13.7±1.6mm3 in the AOM/DSS group and 9.3±8.2mm3 in AOM/DSS + ASA group; P > 0.05; Figure 1E), possibly because of the large variability in the tumors in the AOM/DSS + ASA group due to the limited number of tumors. Weekly monitoring throughout the animal experiments showed no noticeable body weight loss in mice fed with diet supplemented with 0.02% ASA compared with the mice fed the control diet (Figure 1C).
A histopathologist subsequently examined and characterized the histological alterations using hematoxylin and eosin staining. As shown in Figure 2, AOM/DSS treatment induces severe crypt dysplasia, adenomas and adenocarcinomas in the colon. The adenomas observed were associated with inflammation, increased nucleus-to-cytoplasm ratio, nuclear crowding, mitosis and nuclear hyperchromasia (Figure 2C displays a representative image). Inflammation, leukocyte infiltration into the lumen, nuclear hyperchromasia, nuclear mitosis and the loss of nuclear polarity with respect to the basement membrane were observed in the adenocarcinomas of AOM/DSS-treated mice (Figure 2D). Administering ASA attenuated inflammation severity and cancer lesions. As shown in Figure 2E and F, mice in the AOM/DSS + ASA group exhibited normal colon morphology or dysplasia with inflammation.
Histologic characterization of colonic tumors and lesions in AOM/DSS-treated CF-1 mice. Normal mucosa was observed in the mice in the control groups (A). AOM/DSS resulted in crypt dysplasia (black star) with the loss of goblet cells in the crypts (B); adenomas with inflammation, increased nucleus-to-cytoplasm ratios, nuclear crowding (solid arrow) and mitosis (arrow head) (C); adenocarcinoma with inflammation; leukocytes infiltrated into the diluted lumen (triangle) composed of a pattern of cribriform glands, nucleus hyperchromasia (dotted arrow) and mitosis (D). The colon from AOM/DSS + ASA-treated mice showed attenuated inflammation severity and cancer lesions, normal mucosa (E) or dysplasia with inflammation (F). The average number of histologically characterized alterations, including dysplasia, adenoma and adenocarcinoma, per mouse are presented (G).
Together these results demonstrate that dietary feeding of ASA at 0.02% effectively suppresses acute colitis and tumor growth in AOM/DSS-induced CF-1 mice without affecting body weight.
ASA at the dose of 0.02% suppresses AOM/DSS-induced HDAC activity
Emerging evidence has suggested that HDACs are important modulators of the inflammatory response and colon cancer progression (14), and HDAC inhibitors might be a promising therapeutic option for IBD and colon cancer (30,31). As shown in Figure 3A and B, the protein expression levels of HDAC 1, 2, 3, 4 and 5 are significantly elevated in precancerous colonic tissue from AOM/DSS-treated mice. Accordingly, the HDAC activity was significantly higher in the colonic mucosa of AOM/DSS-treated mice (Figure 3C). AOM/DSS + ASA significantly suppressed the protein expression of HDACs (subtypes 2–5) and HDAC activity compared with the AOM/DSS group (Figure 3A–C). These findings implicated that the activation of the HDACs may be involved in the CAC induced by AOM/DSS and ASA treatment at the dose of 0.02% could effectively inhibit HDACs.
The effect of ASA on the protein expression of HDAC 1–5 and HDAC activity. Total proteins were extracted and examined via western blotting. AOM/DSS treatment significantly increased the protein expression of HDAC 1–5, whereas AOM/DSS + ASA effectively inhibited the protein expression of HDAC 2–5. Representative bands are shown (A), and the bar graph presents the fold change of the blot density determined by ImageJ (B). Nuclear proteins were extracted and assayed for HDAC activity. AOM/DSS + ASA strongly suppressed significantly elevated HDAC activity in AOM/DSS-treated mice (C). *P < 0.05 and **P < 0.01.
AOM/DSS and ASA alter the level H3K27ac
The activity and expression of HDAC regulate histone acetylation. Immunohistochemical staining was performed to investigate the level of H3K27ac in formalin-fixed paraffin-embedded tissue. Although the tissue from all three groups revealed pronounced positive nuclear staining for H3K27ac, the percentage of H3K27ac-positive cells analyzed using ImageScope software was dramatically lower in the AOM/DSS-treated mice (Figure 4A–D). AOM/DSS + ASA significantly increased H3K27ac staining compared with AOM/DSS, and the percentage of H3K27ac-positive cells was similar to that of the mice in the control group (Figure 4A–D). These results suggest that the exposure of AOM/DSS diminishes the overall H3K27ac level in the colon, whereas AOM/DSS + ASA restores H3K27ac expression.
AOM/DSS + ASA counters the AOM/DSS-induced alteration of H3K27ac expression. The expression of H3K27ac was examined by immunohistochemical staining. Representative images of the control (A), AOM/DSS (B) and AOM/DSS + ASA (C) are presented. The percentage of H3K27ac-positive cells was analyzed using ImageScope software, indicating that AOM/DSS significantly suppress nuclear staining, whereas AOM/DSS + ASA restores reduced H3K27ac staining (D). The enrichment of the H3K27ac mark in the promoter regions of selective pro-inflammatory genes was determined using a ChIP-qPCR assay. AOM/DSS + ASA significantly suppressed the increased relative abundance of H3K27ac following AOM/DSS in the promoter regions of iNOS (E), TNF-α (F) and IL-6 (G). No significant changes in the H3K27ac level in the Cox-2 promoter were observed (H). *P < 0.05, **P < 0.01 and ***P < 0.005.
Aberrant modifications of H3K27ac at specific genetic regions have been reported in DSS-induced colitis (32). We performed a ChIP-qPCR analysis to investigate the level of H3K27ac in the promoter regions of selective pro-inflammatory genes in noncancerous colonic tissue. In contrast to reducing the H3K27ac level globally, AOM/DSS treatment increased the enrichment of H3K27ac at the promoters of iNOS, TNF-α and IL-6 by 2.99±0.32, 1.20±0.11 and 1.91±0.18-fold, respectively, compared with the control (Figure 4E–G). AOM/DSS + ASA led to 33.4%, 26.7% and 39.8% decrease in the H3K27ac level at the promoter regions of iNOS, TNF-α and IL-6, respectively, compared with AOM/DSS (Figure 4E–G). However, the level of H3K27ac at the Cox-2 promoter was not significantly affected by AOM/DSS with or without ASA (Figure 4H).
Our results suggest that AOM/DSS exposure introduces differential patterns of alterations at the global H3K27ac level as well as H3K27ac enrichment at specific loci, whereas AOM/DSS + ASA resulted in the opposite modification of the H3K27ac level compared with AOM/DSS and exhibited similar H3K27ac levels with the control group.
ASA suppresses the AOM/DSS-induced expression of pro-inflammatory genes
The H3K27ac mark is associated with an open chromatin configuration and transcriptional activation (10). Because AOM/DSS treatment in CF-1 mice led to local hyperacetylation at H3K27 in the promoters of iNOS, TNF-α and IL-6 and because AOM/DSS + ASA suppressed this hyperacetylation, we examined whether this modification at the H3K27ac mark influenced the transcription activity of iNOS, TNF-α and IL-6. A qPCR analysis revealed that the mRNA expressions of iNOS, TNF-α and IL-6 were dramatically increased in noncancerous colonic tissue exposed to AOM/DSS by 55.10±19.06, 3.92±0.22 and 5.59±1.25-fold, respectively, compared with the control (Figure 5A–C). The mRNA expressions of iNOS, TNF-α and IL-6 from AOM/DSS + ASA group were only 1.96±0.61, 1.53±0.29 and 1.77±0.86-fold, respectively, compared with the control, leading to 96.4, 61.0, and 68.3% decreases of those markers in the AOM/DSS-treated mice, respectively (Figure 5A–C). We also examined the protein expressions of iNOS, TNF-α and IL-6 via western blotting and enzyme-linked immunosorbent assay, respectively. Figure 5E showed that the blotting of iNOS was only detectable in the protein extracted from the AOM/DSS group, demonstrating that AOM/DSS + ASA reduced the dramatically elevated level of iNOS protein expression. AOM/DSS + ASA significantly decreased the induction of the TNF-α protein level in the AOM/DSS group by 59.9% (Figure 5F). Since the IL-6 protein level is below the detection limit in protein lysate (data now shown), we examined the IL-6 level in plasma samples instead. As shown in Figure 5G, the dramatically elevated IL-6 concentration in AOM/DSS group was significantly decreased in AOM/DSS + ASA group by 80.8%. In accordance with the unchanged H3K27ac abundance in the Cox-2 promoter, both AOM/DSS and AOM/DSS + ASA failed to significantly influence the mRNA and protein levels of Cox-2 (Figure 5D and E). These data demonstrate that AOM/DSS + ASA significantly attenuated the AOM/DSS-induced mRNA and protein expressions of iNOS, TNF-α and IL-6.
The effect of ASA on the expressions of iNOS, TNF-α, IL-6 and Cox-2. The mRNA expression of pro-inflammatory genes was examined using qPCR. AOM/DSS + ASA strikingly suppressed the AOM/DSS-induced mRNA expressions of iNOS (A), TNF-α (B) and IL-6 (C). However, the mRNA expression of Cox-2 was not changed in any treatment group (D). The protein expressions of COX-2 and iNOS were determined using western blotting, and the representative blots are presented. AOM/DSS + ASA strongly inhibited the protein expression of iNOS, but the COX-2 protein was not inhibited (E). The protein expression of TNF-α and IL-6 was quantified using ELISA, and the results demonstrated that AOM/DSS + ASA effectively inhibits the protein concentration of TNF-α (F) and IL-6 (G), respectively. *P < 0.05, **P < 0.01 and ***P < 0.005.
Discussion
Old, even abandoned drugs might hold promise for cancer therapy by targeting epigenetic mechanisms. For example, azacitidine was originally developed as a cytotoxic agent that was rejected by the US FDA more than 25 years ago. The elucidation of epigenetic modifications in cancer have prompted its reevaluation and led to its approval as an epigenetic drug (Vidaza) for myelodysplastic syndromes in 2004 (33). The investigation of ASA in the context of epigenetic modifications might reveal novel insights into its mechanisms and provide useful information regarding the dosage regimen when used as an epigenetic modulator in cancer chemoprevention. In the present study, ASA was administered in mouse diets at a dose of 0.02% for 21 weeks (equivalent to a dose of approximately 110mg/day in humans). At this dosing regimen, ASA remarkably reduced tumor multiplicity and strongly suppressed HDAC activity and the enrichment of H3K27ac in the promoter regions of iNOS, TNF-α and IL-6 in CF-1 mice (Figures 1D, 3A–C and 4E–G). A recently published study found that ASA at a similar dose for a shorter duration (less than 12 weeks) failed to significantly inhibit the tumor number in AOM/DSS-induced Balb/c mice (20), possibly due to different variability to DSS in these two mouse strains and different cycles of DSS were used in these two studies. Although clinical trials and observational studies have suggested that the long-term use of ASA at both low (81–160mg/day) and high doses (300–325mg/day) can reduce cancer risk (34), the optimal dose and duration of ASA needed to prevent CAC have not yet been established. Our present study provides useful information suggesting that the chronic use of ASA at a low dose (~110mg/day) might be a reasonable starting point for investigating ASA’s role in modulating histone acetylation to prevent CAC in humans.
In the current study, the normal-appearing colonic mucosa was used in all the molecular assays to elucidate the epigenetic effects of ASA partly due to the limited quantity of tumor samples in this study, especially in AOM/DSS + ASA group (Figure 1D). The use of precancerous mucosa (rather than tumor samples) might explain the relatively unchanged COX-2 expression observed in our study since upregulated COX-2 was predominantly located in the tumor tissue but not in the adjacent normal tissue (35). Cancer epigenomic studies have indicated that DNA methylation abnormalities in malignant tumors are already accumulated in the precancerous stages in the kidney, liver, lung, urinary tract, pancreas and gastric mucosa obtained from patients with carcinomas (36,37). These data have suggested that abnormal epigenetic patterns may have already established in precancerous tissue and further determine the tumor development and patient outcomes. Thus, the modification of HDACs and H3K27ac we observed in precancerous tissue in the current study may reveal the early epigenetic events during colon carcinogenesis in AOM/DSS-induced CF-1 mice.
Recently, evidence has suggested that epigenetic modifications are involved in the chemopreventive actions of ASA. The chronic use of ASA is associated with the reduced prevalence of E-cadherin (CDH1) promoter methylation in human gastric mucosa, suggesting that ASA protects against promoter DNA methylation (38). In the context of histone acetylation, both the induction and inhibition of HDACs by ASA have been reported. Kamble et al. showed that ASA induces Sirtuin 1 (SIRT1, a class III HDAC) in liver cells (39). A total of 33 cellular proteins (including histones) were identified as targets of ASA-mediated acetylation in colon cancer HCT-116 cells, implying that histone acetylation plays a role in the action of ASA in colon cancer (40). Another study that investigated the mechanisms of ASA in atherosclerosis found that a low concentration of ASA inhibited HDAC activities and increased the expression of acetylated H3, thereby promoting the transcription of netrin-1 in TNF-α-treated cells (41). The present study provides the first evidence suggesting that the inhibition of the protein expression and the activity of HDACs are involved in the prevention of CAC in AOM/DSS-induced CF-1 mice by ASA at the dose of 0.02% (Figure 3A–C). Additional investigations are needed to determine whether the inhibition of HDACs by ASA is in a dose-dependent manner and to delineate the mechanisms that underlie the actin of ASA in modifying HDAC activity and histone acetylation.
Upregulated HDACs might be associated with abnormal histone acetylation, which can lead to the massive deregulation of gene transcription during the course of cancer. Reduced histone acetylation marks, including H3ac, H4ac, H4K16ac, H3K18ac and H3K9/14ac, are implicated in CRC (42). In addition, aberrant histone acetylation, particularly H4K8ac and H4K12ac, was observed in the inflamed mucosa of a murine colitis model (12). However, the alteration of H3K27ac in CAC has not yet been determined. Recently, Karczmarski et al. showed that the level of H3K27ac is increased in patients with sporadic colon cancer (13). Contrary to this finding, our results showed that the expression of H3K27ac in the colons of CF-1 mice was significantly reduced in AOM/DSS group (Figure 4B and D). Importantly, AOM/DSS + ASA significantly restored the reduction of H3K27ac, possibly through the inhibition of HDACs (Figures 3 and 4C and D). Nevertheless, the involvement of other epigenetic enzymes (e.g. histone acetyltransferases) in the control of the H3K27ac mark by ASA should be investigated in the future. We postulated that the reduction of the H3K27ac mark predisposes the epigenetic trait in the epithelium in favor of tumor growth in CAC, and the preventive effect of ASA might be associated with the restoration of H3K27ac expression.
Experimental results showed that the abundance of histone acetylation marks are altered in the promoter regions of pro-inflammatory genes, including Cox-2, iNOS, TNF-α and IL-6 under inflammatory conditions, thereby influencing the expression of these genes (43–46). It would be important to examine whether AOM/DSS or AOM/DSS + ASA influences the enrichment of H3K27ac in the promoters of these genes. Interestingly, although AOM/DSS diminished global H3K27ac expression, the relative abundance of H3K27ac was increased in the promoters of iNOS, TNF-α and IL-6. AOM/DSS + ASA significantly countered the effect of AOM/DSS on the H3K27ac mark, both globally and locally (Figure 4). The H3K27ac mark is frequently associated with active transcriptional enhancers and chromatin-accessible transcription factor binding regions; moreover, it predicts active transcription (47). Notably, AOM/DSS + ASA dramatically suppressed the abnormally high levels of the transcription and protein expressions of iNOS, TNF-α and IL-6 in the noncancerous colonic tissue from AOM/DSS-induced mice, possibly as a result of the modifications in the abundance of the H3K27ac mark in the promoter regions (Figures 4E–G and 5A–C and E–G). Given the critical roles that iNOS, TNF-α and IL-6 play in the CAC (48,49), the suppressive effect of ASA in AOM/DSS-induced colon cancer might be partially attributed to the inhibitions of iNOS, TNF-α and IL-6 via histone modification. Future research should determine whether other histone marks, such as H3K27me1, 2 and 3, as well as H3K9ac, are associated with CAC, plus whether the epigenetic effects of ASA involves the modifications of these histone marks. In addition, ChIP coupled with next-generation sequencing (ChIP-seq) might identify the potential genetic locus regulated by histone modification upon ASA use. Moreover, ASA was administered prior to the AOM injection in the current study. Thus, whether ASA interferes with the metabolism of AOM and thereby inhibits tumor initiation should be investigated by future experiments.
In conclusion, we used an AOM/DSS-induced CAC model to demonstrate the preventive effect of the chronic use of low-dose ASA, suggesting that the in vivo mechanism of ASA appear to involve epigenetic modifications. Specifically, ASA at the dose of 0.02% inhibited the protein expression and activity of HDACs, as well as restoring the global H3K27ac level. In addition, ASA dramatically reduced the expressions of iNOS, TNF-α and IL-6, activities that might be associated with the suppression of the local enrichment of the H3K27ac mark in promoter regions. These findings provide novel insights for the future development of old drug ASA as a potential epigenetic modulator in the prevention of inflammatory CRC.
Supplementary material
Supplementary Tables 1 and Supplementary Data can be found at Supplementary Data
Funding
R01AT007065 from the National Center for Complementary and Alternative Medicines (NCCAM) and the Office of Dietary Supplements (ODS).
Abbreviations
- AOM
azoxymethane
- ASA
aspirin (acetylsalicylic acid)
- CAC
colitis-accelerated colon carcinogenesis
- CRC
colorectal cancer
- DAI
disease activity index
- DSS
dextran sulfate sodium
- HDAC
histone deacetylase
- H3K27ac
histone H3 lysine 27 acetylation
- iNOS
inducible nitric oxide synthase
- IBD
inflammatory bowel disease
Acknowledgements
We thank Dr Guangxun Li for his assistance with the histology evaluation. We also thank all the members of A.-N.T.K. laboratory for their helpful discussions and the preparation of this manuscript.
Conflict of Interest Statement: None declared.
References
