Microbiota-derived metabolites have a profound effect on host physiology, and it has been estimated that approximately 50% of all metabolites in the plasma have a bacterial origin [
38]. With an estimated number of 10
14 bacteria [
39], the human colon harbors the densest and metabolically active microbial community in the body. Over the past decades, several catogries of gut microbial specific metabolites have been identified, including SCFAs, secondary bile acids, polyamines, indoles, methylamines, polyphenolics, vitamines and others [
40]. Accumulating evidence indicates microbiota-derived metabolites exert an important influence on host physiology and diseases development. In the following parts, we will mainly summarize SCFAs, secondary bile acids, polyamine metabolism and their underlying molecular functions in colorectal carcinogenesis [
41] ().
1.1. Short-Chain Fatty Acids (SCFAs)
SCFAs are fatty acids with fewer than six carbon atoms, and they consist principally of acetate (C2), propionate (C3) and butyrate (C4). They are mostly produced from bacterial anaerobic fermentation of undigested dietary carbohydrates and endogenous epithelial-derived mucus [
42] () in the colon. The most abundant acetate is produced by many gut commensal bacteria primarily through the fermentation of organic substrates, some acetogenic bacteria also generate acetate through the Wood-Ljundahl pathway [
43,
44]. Propionate is mainly produced by Bacteroidetes and some Firmicutes through the succinate pathway, while the other two pathways including the acrylate pathway and propanediol pathway also contribute to the formation of relatively smaller amounts of propionate [
45]. Butyrate is formed by the most SCFA-productive microbial species in
Clostridium and
Bifidobacterium, dominantly using acetate as substate via butyryl-CoA:acetyl-CoA transferase activity [
46,
47]. The less commonly employed metabolic pathway is the butyrate kinase pathway [
48]. Some bacterial species also undergo fermentation of protein and amino acids to generate butyrate through the lysine degradation pathway [
49].
Acetate, propionate and butyrate typically have a combined concentration of 10–130 mM in the colon with a 3:1:1 ratio, which can be influenced by the gut microbiota composition, diet and other environmental factors. The concentration of SCFAs varies along the intestinal tract, with the highest levels in the cecum and proximal colon, and its levels decrease in the distal colon due to absorption by colonic epithelial cells. SCFAs are rapidly absorbed by colonocytes. In fact, butyrate is one of the chief energy sources for local colonic epithelial cells, while the majority of acetate and propionate enter the circulation to exert systemic effects, which influence various pathological conditions including obesity, fatty liver disease and metabolic syndrome [
50,
51]. Due to extensive absorption, only a small amount of unabsorbed SCFAs (about 5–10% of the total) are detected in fecal samples [
52]. Hence, SCFAs could come directly into contact with the colon epithelium, and such interaction has received increasing attention due to the putative roles of SCFAs in colorectal tumorigenesis.
Butyrate is the most widely studied SCFA and it has been suggested that it plays a protective role in CRC [
53]. Several clinical studies have reported the depletion of butyrate-producing bacteria species and diminished fecal butyrate levels are associated with colon tumorigenesis, suggesting that SCFAs may confer protective effects against carcinogenesis [
54,
55]. In homeostasis, butyrate helps to control the gut barrier function by providing colonocytes with energy, decreasing activity of type IV collagen to stimulate epithelial cell attachment and deterring the colonization of pathogens [
46]. In CRC, butyrate inhibits tumorigenesis via directly inhibition of histone deacetylases (HDACs) activity to modulate translation of tumor suppressor genes. It also mediates its effect via alternative pathways such as metabolic rewiring of cancer cells, activation of G protein-coupled receptors (GPCRs) signaling pathways, which culminates in cancer cells apoptosis and anti-inflammatory responses [
56].
Butyrate has a major impact on the epigenetic landscape in cancer cells, by virtue of its inhibitory effect on HDACs. In CRC, the dysregulation of HDACs creates a non-permissive chromatin conformation that prevents the transcription of tumor suppressive genes. HDACs inhibitors have shown great potential in anticancer therapy by reversing this process [
57]. A growing body of work reported butyrate stimulates apoptosis of cancer cells through HDACs inhibition. In a gnotobiotic mice model treated with a butyrate-producing bacterium and high-fiber diet, the increased level of butyrate was associated with a lower tumor burden when compared with normal controls [
58]. Furthermore, in CRC cell lines, butyrate was shown to induced expression of cell-cycle regulators such as p21 and p27 and pro-apoptotic genes such as FAS through inducing histone acetylation, thus to inhibit proliferation and promote apoptosis [
59]. Butyrate also promotes an anti-inflammatory microenvironment, which is crucial for preventing tumorigenesis. By enhancing histone H3 acetylation and inhibiting the NF-κB signaling pathway, butyrate attenuated the production of inflammatory cytokines such as TNF-α, IL-6 and IL-12 to relieve colitis in mice model [
60]. In addition, butyrate promotes regulatory T
reg cell differentiation [
61] and inhibits macrophage pro-inflammatory function [
62] through enhancing histone acetylation, thereby contributing to an anti-inflammatory microenvironment.
Apart from epigenetic modifications, butyrate also suppresses CRC development by modulating tumor metabolism. It has been well documented that butyrate serves as an energy source for normal colonic epithelial cells to sustain cell proliferation [
63]. In contrast, butyrate induces metabolic rewiring in cancer cells to inhibit proliferation and induce apoptosis. For example, a study reported that butyrate could directly bind to PKM2, the M2 isoform of a pyruvate kinase, to inhibit aerobic glycolysis and reverse Warburg effects, thereby compromising the growth of cancer cells which are frequently dependent on glycolysis while normal cells are spared [
64].
Butyrate is also an agonist of several GPCRs and regulates downstream signaling pathways. GPR109A is a typical receptor of butyrate. Activation of GPR109A induced down-regulation of Bcl-2, Bcl-xL, and cyclin D1, and up-regulation of death receptor pathway to promote apoptosis in colon cancer cells, together with the inhibition of NK-κB signaling [
65]. In addition, GPR109A signaling induced by butyrate promotes the anti-inflammatory properties of macrophages and dendritic cells in colonic milieu to facilitate the differentiation of T
reg cells and IL-10-producing T cells [
66]. Butyrate is also recognized by GPR43. By interacting with GPR43, microbial-derived butyrate suppresses the Wnt/β-catenin signaling to inhibit intestinal tumor development [
67].
Although the majority of propionate is absorbed to enter the circulation and is metabolized in liver, it has also shown to have protective effects on colonic cells [
68]. Propionate inhibits colonic inflammation [
69,
70]. Much less is known with regards to the molecular mechanism of propionate in colon cancer; however, it may share a similar mechanism with butyrate through inhibition of HDACs [
71]. Acetate is also a potential chemopreventive agent against CRC by inhibiting cell proliferation and inducing apoptosis [
72]. However, others reported that acetate promoted plasma membrane relocalization of MCT-1 and triggered increased glucose consumption and lactate production, thus increasing the glycolytic phenotype in cancer cells [
73].
Based on current evidence, SCFAs are considered to exert preventive effects on CRC. More in-depth investigations will be required to understand its role in mediating colon epithelium and gut microbiota interactions in the context of tumorigenesis.
1.2. Bile Acids
Bile acids, which are primarily produced in the liver, are metabolized to secondary bile acids by the gut microbiota in the intestinal tract [
74]. Primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA), are produced from cholesterol in hepatocytes and are excreted through the bile duct after conjugation with glycine or taurine. The primary function of bile acid secretion is to emulsify lipids and to facilitate their absorption after meal ingestion. In the gastrointestinal tract, primary bile acids are mostly re-absorbed via enterohepatic circulation in the ileum. However, ~5% of bile acids are deconjugated and biotransformed to secondary bile acids via the action of gut microbiota in colon [
75]. CA and CDCA are dehydroxylated to generate deoxycholic acid (DCA) and lithocholic acid (LCA) respectively. CDCA can also be metabolized by bacterial hydroxysteroid dehydrogenase (HSDH) to generate ursodeoxycholic acid (UDCA). The main bacterial genera involved in secondary bile acids biosynthesis are
Bacteroides,
Clostridium,
Lactobacillus,
Bifidobacterium and
Eubacterium, they also serve key roles in regulating host fat metabolism [
76]. The most abundant bile acids in humans include the primary bile acid CA and CDCA and the secondary bile acid DCA ().
Metabolomics studies suggested a correlation between bile acid dysfunction and CRC in multiple cohorts. A multi-omics study combining metabolomics and microbiome analyses fecal/urinary metabolites and fecal microbiota revealed that the higher fecal concentrations of tumor-promoting DCA and increased levels of 7α-dehydroxylating bacteria were observed in CRC high risk population [
77]. Another study also showed bile acids such as DCA were increased in multiple polypoid adenomas and intramucosal carcinomas patients, and were positively associated with the abundance of Bilophila wadsworthia, whose growth is stimulated by bile acids [
78]. These studies indicate an extensive crosstalk between bile acids production and gut microbiota, which may form a positive feedback loop to drive CRC pathogenesis.
Secondary bile acids, especially DCA, are considered significant contributors to the development of CRC. DCA was first shown to be a carcinogen that promotes CRC development in mice in 1940 [
79]. In a recent study, administration of DCA induced colonic tumor formation particularly in the context of obesity [
80], as high-fat diet induced alterations in the gut microbiota contributed to increased intestinal DCA levels. Hydrophobic DCA exerts multiple pathogenic effects on the colon epithelium including the disruption of cell membrane, induction of oxidative damage to DNA, and activation of NF-κB to provoke inflammation. A study reports that DCA disrupted cell monolayer integrity and increased pro-inflammatory cytokine production in the intestine, leading to low grade inflammation and aggravation of intestinal tumorigenesis in
ApcMin/+ mice [
81]. In vitro experiments showed that DCA exposure induced single-strand breaks of DNA in CRC cells [
82]. DCA-induced mitochondrial oxidative stress can activate NF-κB signaling in CRC cells, leading to decreased apoptosis and tumor progression [
83]. Furthermore, bile acids could interact with bile acid receptors to promote CRC. DCA antagonizes intestinal farnesoid X receptor (FXR) function, which in turn, induces cell proliferation and Lgr
5+ cancer stem cell expansion [
84]. Collectively, DCA is a key secondary bile acid implicated in the promotion of colorectal tumorigenesis.
In contrast to DCA, UDCA and tauroursodeoxycholic acid (TUDCA) have been associated with suppression of colon tumor development. UDCA regulated intracellular reactive oxygen species (ROS) generation, suppressed cell cycle progression in colon cancer cells, and reduced the formation of colon cancer stem-like cells [
85]. On the other hand, TUDCA suppressed NF-κB signaling in CRC cells and ameliorated colitis-associated tumorigenesis in AOM/DSS-treated mice [
86]. The different biological and pathological effects of DCA and UDCA present are not been fully elucidated, one possible explanation for their different effects on cancer is the diverging effects on the same oncogenic signaling pathway [
87]. These results suggest that the mechanisms by which secondary bile acids affect the progression of CRC are complex and more research is needed to fully understand their roles in colorectal tumorigenesis.