The role of butyrate in surgical and oncological outcomes in colorectal cancer

Abstract

Butyrate is a short-chain fatty acid produced by colonic gut bacteria as a result of fermentation of dietary fibers. In the colon, butyrate is a major energy substrate and contributes to the nutritional support and proliferation of a healthy mucosa. It also promotes the intestinal barrier function by enhancing mucus production and tight junctions. In addition to its pro-proliferative effect in healthy colonocytes, butyrate inhibits the proliferation of cancer cells. The antineoplastic effect of butyrate is associated with the inhibitory effect of butyrate on histone deacetylase (HDAC) enzymes, which promote carcinogenesis. Due to the metabolic shift of cancer cells toward glycolysis, unused butyrate accumulates and inhibits procarcinogenic HDACs. In addition, recent studies suggest that butyrate may improve the healing of colonic tissue after surgery in animal models, specifically at the site of reconnection of colonic ends, anastomosis, after surgical resection. Here, we review current evidence on the impact of butyrate on epithelial integrity and colorectal cancer and present current knowledge on data that support its potential applications in surgical practice.

INTRODUCTION

Short-chain fatty acids (SCFAs) are metabolites produced in the colon by gut bacteria (1). SCFAs are key players in intestinal homeostasis and contribute to the modulation of local and systemic immune and inflammatory processes (2–4). In the colon, SCFAs are the primary energy source for epithelial cells, contributing to their energy requirements (5). Propionate, acetate, and butyrate constitute the most abundant SCFAs (6, 7). Of these, butyrate has drawn significant attention in recent years due to accumulating evidence on its beneficial effects in the preservation of the colonic epithelial layer, modulation of inflammation, and maintenance of the intestinal barrier function (8).

Among its beneficial roles in health, butyrate has demonstrated a potential protective effect against colorectal cancer (CRC) (8). As one of the most prevalent cancers worldwide, CRC ranks fourth in terms of incidence and third for cancer mortality (9). Interestingly, butyrate is reported to concomitantly promote the proliferation of noncancerous colonocytes and suppress the proliferation of cancer cells (10). Data suggest that butyrate may improve epithelial healing after surgery and may prevent some major postoperative complications in the colon, specifically anastomotic leak (AL) after a colorectal resection. AL, a major complication in colorectal surgery, is defined as “a defect of the intestinal wall at the anastomotic site leading to a communication between the intra- and extraluminal compartments” (11). AL is associated with higher mortality rates and possibly increased CRC recurrence (12). Despite significant efforts to reduce its occurrence in colorectal surgery, AL remains unpredictable. Preventive measures such as butyrate supplementation thus become of interest. Given its potential anticancer activity, butyrate provides a particularly promising option for CRC patients undergoing colonic surgery.

This mini-review aims to provide an overview of the current evidence on the impact of butyrate on epithelial preservation and colorectal carcinogenesis and report the current knowledge on data that support its potential applications in surgical practice.

ROLE OF BUTYRATE IN MAINTAINING HEALTHY COLONIC PHYSIOLOGY

Butyrate is produced in the large intestine as a result of bacterial fermentation of dietary fibers and contributes to 70% of the energy requirements of colonic epithelial cells, which are the main cells that metabolize this SCFA (13–18). In healthy colonocytes, butyrate is usually taken up by a monocarboxylase transporter and metabolized via mitochondrial β-oxidation (7, 19). As an energy substrate, butyrate promotes cell proliferation and epithelial growth (20, 21). Paucity of SCFAs in the colonic lumen has been shown to induce epithelial atrophy, to decrease nutrient absorption, and to increase inflammation (22–24). In this respect, the proliferative effect of butyrate results in an enhanced mucosal thickness as measured by an increase in the depth of villi height and crypts (7), and hence reinforces the gut barrier. In addition, butyrate may promote the release of stimulatory growth factors and gastrointestinal peptides, including gastrin (25) and insulin-like growth factor-2 (IGF-2) (26), known to stimulate the proliferation and survival of intestinal cells (7, 27–29). Moreover, butyrate has been found to inhibit apoptosis by increasing the expression of antiapoptotic proteins (7). In summary, under normal physiological conditions, the beneficial effects of butyrate on the proliferation of colonic epithelial cells and thickness of the mucosal layer are well established.

ROLE OF BUTYRATE IN MAINTAINING THE INTESTINAL BARRIER

A dysfunctional intestinal barrier is a common feature of CRC, enabling cancer dissemination and metastasis (30). A healthy intestinal barrier is fundamental for the prevention of mucosal inflammation, bacterial translocation, and potential ensuing sepsis (31). Essential components for barrier integrity of the intestinal epithelium include the mucus layer and tight junctions controlling paracellular transport (31).

The mucus layer serves as a barrier that limits the contact and subsequent deleterious effects of luminal bacteria on the colonic epithelium. The outer mucus layer is in contact with the gut microbiome, whereas the impenetrable inner layer is in contact with epithelial cells (32). Both the inner and outer mucus layers allow access of metabolites to epithelial cells (32). The colonic barrier is also reinforced by the intercellular apparatus of tight junctions that regulate the paracellular flux of ions and water (32). Major components of tight junctions include transmembrane proteins called claudins, tight-junction-associated marvel proteins (TAMPs) such as occludins, junctional adhesion molecules (JAMs), and cytosolic proteins such as zonula occludens (33). These cellular tight junction proteins prevent the translocation of luminal contents beyond the epithelial layer and into the systemic circulation (31, 32). Occludins, zonula occludens 1 (ZO-1), claudin-1, claudin-3, claudin-4, and claudin-5 have been shown to promote barrier integrity, whereas claudin-2, claudin-7, claudin-10, and claudin-12 have the opposite effect (31, 34–36).

Butyrate is consistently associated with the preservation of the intestinal barrier function and integrity via regulation of these tight junction proteins and maintenance of the mucus layer. In vitro, butyrate was associated with an increase in claudin-1, claudin-3, and claudin-4, and a concomitant reduction of claudin-2, resulting in augmented tight junctions (34–36). In mice, butyrate has been shown to enhance tight junctions, decrease inflammation, and prevent bacterial translocation (37–39). Expression of mucin-2 (MUC2), a major component of the colonic mucosal protection system at the surface of the gastrointestinal tract (40), was also shown to be elevated by butyrate (37, 39).

In addition to benefits conferred by enteral butyrate on intestinal barrier preservation, parenteral administration of butyrate may also exert a protective effect against gut-derived sepsis (41). Intravenous butyrate administered to rats undergoing cecal ligation and puncture improved survival and associated mucosal injury (41). The mechanisms by which parenteral butyrate improved the barrier were similar to those related to enteral butyrate, via increased expression of tight junction proteins claudin-1 and ZO-1 (41). Further work corroborated these findings in a rat model dependent on parenteral nutrition, where adding intravenous butyrate resulted in increased expression of tight junction proteins and mucins, and a decrease in intestinal permeability (42).

The protective effect of butyrate on the intestinal barrier integrity in in vitro and in vivo experiments, strongly implicates butyrate as an important factor for mucosal proliferation, barrier integrity, and surgical healing. These effects are of relevance to CRC surgery.

BUTYRATE AND TISSUE REPAIR AFTER SURGERY

The proliferative effect that butyrate exerts on the colonic epithelium suggests that it is associated with tissue repair and mucosal continuity and may prevent leaks at surgical sites. In rat models of anastomotic leakage, rectal administration of butyrate was associated with better anastomotic healing (43–45). One of the potential underlying factors promoting colonic healing is the promotion of tissue repair at the anastomosis site via higher synthesis and maturation of collagen (43). However, this beneficial increase in collagen does not seem to be a sine qua non mechanism since intravenous butyrate administered to rats undergoing anastomosis enhanced anastomotic strength without measurable effects on collagen (46).

Although this favorable effect of butyrate on colonic anastomotic healing has not been translated into clinical studies so far, the usefulness of this SCFA in surgical patients was tested in the context of intestinal diversion. Colorectal diversion involves an evacuation of the intestinal fecal content into an ostomy bag before it reaches some segments of the colon or rectum (47, 48). This procedure, common in rectal cancer surgery, diverts the digesta transit away from the colon to avoid stress on the confectioned, fragile rectal anastomosis and to prevent overt contamination if leakage occurs at the anastomosis site. In this situation, the defunctionalized colorectal mucosa of the unused colonic segment is prone to inflammation and bleeding due to the absence of transit and luminal nutrients, particularly SCFAs (22). Although this diversion is necessary in certain circumstances, the resulting SCFA depletion may affect epithelial integrity and harbor underestimated deleterious effects. Irrigation with a mix of the SCFAs butyrate, acetate, and propionate was shown to decrease mucosal inflammation while providing essential nutrients to the diverted mucosa (47). Butyrate enemas were also evaluated on the prevention of atrophy and inflammation of the colorectal mucosa after diverting ostomy and have proven to be efficient in restoring mucosal integrity and epithelial health (22). More specifically, butyrate promoted mucosal proliferation and was associated with an upregulation of transforming growth factor (TGF)-β3 and insulin-like growth factor (IGF) among others, which facilitate migration of epithelial cells toward the wounded epithelium (13, 22, 49). In patients with ulcerative colitis, butyrate diminished blood discharge, decreased the frequency of diarrhea, and improved mucosal inflammation as assessed by endoscopic examination (50).

Overall, these studies point toward an advantageous use of butyrate on a wide spectrum of adverse postoperative outcomes, including leakage at the colonic anastomosis site. However, although butyrate may be especially useful in surgical CRC patients, it requires further evaluation on its effect on carcinogenesis and CRC recurrence.

EFFECT OF BUTYRATE ON COLORECTAL CANCER

Butyrate promotes the epithelial proliferation of noncancerous colonocytes. In contrast, butyrate also inhibits the proliferation of cancer cells, a phenomenon described as the “butyrate paradox” (10) that may be partially explained by the differential regulation of the Wnt/β-catenin (canonical Wnt) signaling pathway (51). As Wnt signaling is fundamental to the normal function of the colorectal epithelium, deregulation of the Wnt pathway is linked to CRC, mostly involving activating mutations in components of the pathway (51). Several studies have investigated the effect of butyrate on the Wnt pathway by assessing the expression levels of pathway components. In vitro, butyrate was found to induce apoptosis of colon carcinoma cells by increasing the activity of the transcription factor T-cell factor (TCF) (14) as well as the levels of activated β-catenin and the formation of β-catenin-TCF complexes (52, 53), which resulted in the transcription of target oncogenes (51).

Most studies in vitro and in vivo show that, upon hyperactivation of Wnt signaling, butyrate triggers apoptosis through the induction of both intrinsic and extrinsic apoptotic pathways. The antiproliferative effect of butyrate in the human colon carcinoma HCT116 cell line was associated with induction of apoptosis and was mediated by the tumor suppressor protein p21 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (15). ERK1/2 play a crucial role in cell cycle progression, cell survival, and cell proliferation (15, 54,55), and ERK1/2 inhibitors display excellent antitumor activity (56, 57). Importantly, butyrate decreases the phosphorylation of ERK1/2 and consequently, the proliferation of human HCT 116 and HT 29 colon cancer cells (15, 58). Butyrate can also inhibit tumor growth by decreasing the expression of silent mating-type information-regulation 2 homolog (SIRT)1 (59), a protein with histone deacetylase (HDAC) function that promotes tumor growth (59). The mammalian target of rapamycin (mTOR), a serine/threonine kinase, induces phosphorylation of ribosomal protein S6 kinase β-1 (S6K1), a process promoted by SIRT1 and which normally induces cell proliferation (59). However, butyrate in HCT116 cells was shown to downregulate SIRT1 and prevented SIRT1-dependent proliferation (59). In both HCT116 cells and LoVo human colon adenocarcinoma cells, butyrate appears to downregulate Bmi-1, a proto-oncogene whose expression induces cell proliferation (60). Similarly, the proliferation and cell viability of the human CRC cell lines WiDr and LS1034 decreased in the presence of butyrate through enhanced apoptosis driven by an increase in the ratio of proapoptotic protein Bcl-2-associated X (Bax) to antiapoptotic protein B cell lymphoma 2 (Bcl-2) (61). Interestingly, even when cancer cells were resistant to proapoptotic triggers such as deoxycholic acid, butyrate was still able to exert an antiproliferative effect (62).

The differences between the effects of butyrate on the proliferation of healthy cells compared with cancer cells have been suggested to be due to the sensitivity and responsiveness of the cells to butyrate (63). In healthy cells with relatively moderate levels of Wnt activity, butyrate contributes to normal regulation of proliferation and controlled self-renewal, whereas in CRC cells with hyperactive Wnt pathway, butyrate further enhances Wnt signaling and triggers apoptosis.

Oxidation of butyrate is required for energy in normal epithelial cells (64) but is decreased in CRC cells (20, 64). A shift toward glycolysis in cancer cells is known as the “Warburg effect” (20), reducing their use of butyrate, which accumulates and in turn, inhibits HDAC function (20) and cell proliferation (10, 64). Histone acetylation and deacetylation play important roles in the epigenetic modulation of gene expression or repression (20, 65). As HDAC enzymes may also contribute to cancer development via the silencing of tumor suppressor genes (65), HDAC inhibition has been proposed as a promising option for anticancer therapy (20). Accordingly, the accumulation of unused butyrate in cancer cells exerts an HDAC inhibitor effect, which essentially affects chromatin modulation and gene expression, resulting ultimately in cell cycle arrest and cell death (10, 20, 66).

DIETARY COMPONENTS, BUTYRATE, AND CRC

Several studies have enhanced our understanding of the influence of dietary components on the production of SCFAs, including butyrate, and their effect on cancer cells. Dietary carbohydrates such as pectin and starch were shown to increase the gut concentrations of SCFAs (67). In contrast, high-fat diets seem to decrease butyrate production and increase inflammation (67, 68). However, addition of fibers, such as guar gum, were found to reverse this effect (67). Protein diets are associated with less SCFA production compared with fiber diets (67). Whether this effect may be replicated locally in the gut to prevent CRC development is yet to be investigated.

Much less is known about the potential interactions between butyrate and other metabolic byproducts of intestinal bacteria to modulate colorectal carcinogenesis. Deoxycholic acid for instance is produced from the deconjugation of primary bile acids by gut bacteria (69) and is increased by high-fat diets (70). The effects of this secondary bile acid byproduct are known to promote colorectal proliferation and tumorigenesis but are also inhibited by butyrate (71, 72). On the other hand, dietary components may also directly interact with butyrate and potentiate its effects. For example, polyunsaturated fatty acids were shown to enhance butyrate-induced apoptosis of HT29 adenocarcinoma cells (73). How other dietary components may influence the effects of butyrate on normal and cancerous colonic epithelia remains to be investigated.

BUTYRATE AND CANCER CELL INVASION

Butyrate has also been shown to decrease the invasion capacity of human CRC cell lines HCT116, HT29, LoVo, and HCT8 (52) by decreasing cell motility via reduced phosphorylation of Akt1 and ERK1/2 (52). Akt1 is a serine/threonine protein kinase involved in multiple cell processes, including growth, proliferation, survival, and angiogenesis, and Akt1 mutations are found in many cancer types (74). Its aberrant activation in cancer cells contributes to the modulation of cytoskeleton components that increases the motility and the invasiveness of metastatic cells (74, 75).

In a mouse xenograft model using WiDr colon cancer cells, adding butyrate to the chemotherapeutic agent irinotecan was associated with reduced tumor growth (61). Tian et al. (76) have shown that oral delivery of a mix of SCFAs containing acetate, butyrate, and propionate decreased the development of colonic tumors in mice induced by azoxymethane (AOM)/dextran sodium sulfate (DSS) administration when compared with control mice that did not received SCFAs. Finally, further studies have outlined a possible butyrate effect on distant cancer dissemination (77). In BALB/c mice subjected to an intrasplenic injection of mouse CT26 colon cancer cells, supplementation with butyrate was associated with fewer liver metastases compared with untreated mice (77).

Despite the antineoplastic role ascribed to butyrate, previous studies have shown that cancer cells may acquire resistance to its antineoplastic effect. The human colon adenocarcinoma BCS-TC2 cell line was shown to undergo apoptosis when cultured with butyrate (16). However, after 30 days of exposure to butyrate, cancer cells maintained their viability and resumed proliferation, suggesting the development of resistance to prolonged butyrate exposure (16). Similar resistance was also found upon sustained exposure of HT29 and HCT116 cells to butyrate (17, 78). Butyrate-resistant HCT116 cells were established after exposing them to the SCFA for several months and was accompanied with enhanced chemoresistance to paclitaxel, 5-fluorouracil, doxorubicin, and trichostatin A (78). These cells also did not display the same pattern of apoptosis as nonresistant cells (78). The underlying mechanisms related to this resistant phenotype were associated with decreased expression of proapoptotic proteins, including Bax and Bim, and an increased expression of antiapoptotic counterparts, such as Bcl-xL (78). However, butyrate resistance did not confer a higher invasive potential to the cancer cells (78). In this respect, the prospects of using butyrate perioperatively to improve surgical outcomes in CRC and prevent anastomotic leakage would likely not be problematic due to the short-term use of this metabolite before and shortly after surgery.

ANASTOMOTIC LEAK AND LOCAL CANCER RECURRENCE

Despite improvements in technique and perioperative care, AL remains a major and unpredictable complication in colorectal surgery and has been shown above all to increase CRC recurrence rates (79). The mechanisms by which poor anastomotic healing and AL are associated with higher local CRC recurrence are not fully elucidated. However, AL has been suggested as an independent predictor of recurrence when taking into consideration cancer stage (80). It is worth noting that local recurrences occur mainly at the anastomosis site and surrounding tissue in contrast with only a minority occurring in the bowel lumen (81, 82). Furthermore, approximately one in five patients will develop metastatic disease after surgical treatment (83). One of the potential explanations for this event is the leak and implantation of luminal floating cancer cells on the bowel wall and pericolonic tissue at the anastomotic site during and soon after surgery (81). In fact, it has been shown that healthy colonic and anastomotic tissue during surgery harbors microscopic tumoral cells that can potentially induce tumorigenesis (84, 85). A leak at the anastomotic location can induce a significant local inflammatory state, with a potential shift of the local anastomotic microbiota toward a pathogenic profile that promotes collagenase activity, extracellular matrix disruption, and seeding of exfoliated leaking tumoral cells at the anastomotic site and surrounding tissue (18, 81). These events potentially allow for easier fixation of cancer cells and promotion of recurrence (81). Therefore, prevention of AL may decrease the occurrence of local and metastatic cancer recurrence.

FUTURE PERSPECTIVES

Despite compelling evidence on the beneficial effects of butyrate on the proliferation and health of the colonic mucosa, very few studies have explored its use in digestive surgery to improve postoperative intestinal healing (Fig. 1). Moreover, the protective anticancer role of butyrate in respect to concentrations, exposure time, and type of cancer requires further characterization to properly assess its anticancer effect.

Figure 1.

Butyrate-mediated effects on colonocytes and potential mechanisms of action in the colon. Indigestible dietary fibers are fermented by microbes in the large intestine. Butyrate, a major product of dietary fiber fermentation by bacteria, is actively metabolized and used as an energy source by colonocytes. The beneficial effects of butyrate are related, in part, to the genetic and epigenetic regulation of gene expression. In addition, butyrate exhibits several antitumor effects that suppress colorectal cancer (CRC) development. Strategies to enhance butyrate production, such as dietary prebiotic supplementation, may be beneficial in CRC surgery to enhance tissue repair after an invasive colorectal procedure and prevent potential anastomotic leakage (AL) and dissemination of cancer cells. ERK, extracellular signal-regulated kinase; HDAC, histone deacetylase; IGF-2, insulin-like growth factor 2; MUC2, mucin 2; Wnt, Wingless and INT-1.

The use of butyrate in surgical animal models has not evolved into clinical practices, which may be due to the reluctance of surgeons to administer rectal enemas to patients who underwent recent surgery with fragile, fresh anastomoses. An alternative to this limitation would be to apply dietary interventions such as supplementation with prebiotics, which are “substrates selectively utilized by microorganisms that confer a health benefit” (86) and increase butyrate production by shifting the colonic microbiota toward a butyrogenic profile. Considering that the gut microbiota in patients with CRC displays a decrease in butyrate-producing bacteria (87), these interventions would not only help to inhibit cancer cell proliferation, but would also facilitate postoperative healing and prevent AL. However, one should keep in mind that bowel preparations and oral antibiotics given presurgery (88–90) may deplete butyrate-producing bacteria. Hence, supplementation with prebiotics to stimulate the growth of butyrate-producing bacteria (91) should be initiated weeks in advance before surgery to promote mucosal health and strengthen the barrier function, and may need to be resumed immediately after surgery. Alternately, direct oral butyrate supplementation may be an attractive option to bypass the effect of antibiotics on the gut microbiota, as it has been found to improve the intestinal barrier in mice (92) and to potentially reduce inflammation in humans (93). Further investigations using animal models and human trials are needed to evaluate the potential benefit of such interventions in enhancing anastomotic healing and preventing AL as well as to unravel the mechanisms involved.

In summary, the growing importance of butyrate in colonic health and disease requires further studies using surgical models of CRC to corroborate and consolidate these emerging findings. Further research is required to confirm the consistency of beneficial clinical findings and to validate the real clinical impact of butyrate on colonic healing and prevention to allow translational applications in colorectal surgery.

GRANTS

This work was supported by Canadian Institutes of Health Research (CIHR) Grant FRN-159775, Cancer Research Society (CRS) Grant 25262, and Natural Sciences and Engineering Research Council of Canada (NSERC) Grant RGPIN-2018-06442 (to M. M. Santos). R. Hajjar is the recipient of a scholarship from the Fonds de recherche du Québec – Santé (FRQ-S)/Ministère de la Santé et des Services sociaux (MSSS; Resident Physician Health Research Career Training Program).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.H., C.S.R., and M.M.S. drafted manuscript; R.H., C.S.R., and M.M.S. edited and revised manuscript; R.H., C.S.R., and M.M.S. approved final version of manuscript.