SCFAs are organic carboxylic acids that contain 1-6 carbon atoms and are the primary products of the fermentation of undigested carbohydrates such as resistant starch, oligosaccharides and non-starch polysaccharides utilized by gut microbiota in the colon (10,11). Butyrate is one of the main SCFAs, accounting for ~20% of the total SCFAs (12). It acts as an energy source for the intestinal mucosa, and it regulates cell proliferation and differentiation (13). In total, 225 candidate bacteria potentially producing butyric acid were identified, with the majority belonging to the Firmicutes, Actinobacteria, Bacteroidetes, Fusobacteria and Proteobacteria groups (14). Changes in diet, misuse of antibiotics, ingestion of probiotics and gastrointestinal inflammation infections may alter the ecology and composition of the gut microbiota, thus affecting the production of SCFAs (15,16). Oral administration of Lactobacillus plantarum for 4 weeks revealed an increase in probiotics such as Bifidobacterium, a decrease in conditionally pathogenic bacteria such as Vibrio vulnificus and a significant increase in the level of SCFAs in adults (17). In addition, the temperature and pH levels of the intestinal tract also affect the synthesis of SCFAs (18).

SCFAs exist within the intestinal lumen as free anions (SCFA^-), exhibiting weak acidity and contributing to the reduction of colonic pH. The majority of SCFAs undergo absorption in the colon, and 5-10% are excreted in feces (19). The absorption of SCFAs by colonic epithelium is facilitated by four mechanisms: (i) Non-ionic diffusion (20); (ii) 1:1 exchange with intracellular bicarbonate (21); (iii) cellular entry through hydrogen-coupled monocarboxylate transporter; and (iv) cellular entry through sodium-coupled monocarboxylate transporter (SMCT) (22). Butyrate primarily functions as an energy substrate for intestinal epithelial cells, supplying ~70% of the energy requirements for colon cells (23). Downregulation of SMCT-1 expression within specific colorectal cancer (CRC) tissues results in reduced butyrate concentration or diminished butyrate uptake in colorectal tissues (24). Butyrate concentration varies in a biological gradient from the intestinal lumen to systemic circulation, reaching a maximum level of 100 mM in the cecum and proximal colon. However, the physiological significance of this change is currently unclear (25). Butyrate that is not utilized by enterocytes is transported by the portal vein to the liver and metabolized, where it mainly participates in gluconeogenesis, ketogenesis and triacylglycerol synthesis, and also exerts anti-inflammatory and antitumor effects through a variety of mechanisms (26).

At present, the assessment of intestinal SCFAs production and bioavailability predominantly relies on indirect measurements, primarily through the quantification of SCFAs concentrations in fecal samples. The main SCFAs detected within feces include acetate, propionate and butyrate. Factors contributing to elevated SCFAs levels in feces include high-fiber diets, reduced colonic transit time and proliferation of SCFAs-producing microbiota (27). In recent years, gas chromatography-mass spectrometry (GC-MS) has emerged as the prevalent methodology for SCFAs analysis. This technique leverages differences in the adsorption capacities of chromatographic columns for distinct SCFAs, enabling the direct assessment of the relative abundance of each SCFAs in a specimen. GC-MS offers a streamlined and expedited approach (28). Currently, multi-omics analysis is widely used, and the association between diseases and changes in the composition of gut microbiota and its metabolites can be revealed by analyzing the metagenomic and metabolomic differences in gut microbiota between patients.

In animal models, butyrate exhibits anti-inflammatory effects by activating GPCRs (92). GPCRs, also known as free fatty acid receptors (FFARs), can be dose-dependently activated by free fatty acids and are critical drug targets (34). Key receptors involved in the anti-inflammatory and immunomodulatory effects of SCFAs are GPR41 (FFAR3), GPR43 (FFAR2) and GPR109A (hydroxycarboxylic acid receptor 2) (93). These receptors are found in various cell types, with GPR43 predominantly expressed in immune cells; GPR41 mainly expressed in pancreas, spleen and adipose tissue; and GPR109A highly expressed in intestinal epithelial cells, macrophages, monocytes and neutrophils (94-96). All SCFAs activate GPR43 and GPR41, but only butyrate activates GPR109A (97).

Butyrate exerts its anti-inflammatory and pro-immune effects primarily by activating GPCRs on immune and intestinal epithelial cells. For instance, butyric acid suppresses the expression of inducible nitric oxide synthase (iNOS), TNF-α, monocyte chemotactic protein-1, IL-6 and IL-; stimulates neutrophil migration; and modulates ROS production through the activation of GPR41 and GPR43 in macrophages, as well as GPR43 in neutrophils (98,99). Butyrate, via GPR43, promotes the production of IL-10 by microbiota antigen-specific Th1 cells (100). Additionally, butyrate induces the expansion of Tregs, and enhances the expression of IL-10 and IL-18 by activating GPR109A on macrophages and DCs (101,102). It is well-established that the breakdown of the intestinal mucosal barrier contributes to the development of CRC, and butyrate can enhance intestinal barrier function by activating GPCRs (103). On one hand, the combination of butyrate and GPR109A activates K⁺ outflow, leading to cell hyperpolarization. This activation, in turn, triggers the NLRP3 inflammasome, resulting in an increased production of IL-18 and the promotion of intestinal homeostasis (101,104). On the other hand, butyrate activates AMPK, facilitating tight junction assembly and stabilizing the intestinal environment. This occurs through the induction of hypoxia-inducing factor 1 by depleting O₂ levels (93). Additionally, butyrate plays a key role in the regulation of energy metabolism. By interacting with GPR43 and GPR41 receptors on the surface of intestinal endocrine L cells, butyrate promotes the secretion of glucagon-like peptide-1 and peptide tyrosine-tyrosine, which are essential for maintaining energy balance (105,106) (Fig. 3).

A partial overlap in the mechanisms through which butyric acid governs immune responses and inflammation within the context of GPR41, GPR43 and GPR109A exists (107). In general, GPCRs undergo phosphorylation, causing receptor internalization and desensitization. Activation of GPCRs subsequently triggers downstream signaling pathways, including MAPK, phospholipase C and NF-κB, alongside other cascades, thus promoting the secretion of chemokines and cytokines. GPCRs hold a pivotal role in the regulation of immunity and inflammation; the preservation of intestinal barrier integrity; and regulation of energy metabolism processes due to the diverse effects their transmitted signals exert on various cell types.

The level of histone acetylation is determined by the interplay between the functions of histone acetyltransferase, which promotes gene transcription, and HDAC, which inhibits it. HDAC is widely expressed in immune, endothelial and vascular smooth muscle cells, and its expression varies in different tumor cells (64). HDACIs were first developed as anticancer drugs. SCFAs, a type of natural HDACI, have been found to exhibit varying inhibitory efficiencies. Butyrate, in particular, has demonstrated the highest inhibitory efficiency (>80%), and its effective inhibitory concentration of HDAC is within the mM range (108,109). The following subsections summarize the genes whose expression is regulated by butyrate (Fig. 4).

SCFA receptors are not significantly expressed in T or B cells; thus, the role of butyrate is more reliant on HDACI activity. Inhibition of HDAC by butyric acid in T cells increases the acetylation of p70 S6 kinase and the phosphorylation of ribosomal protein S6; activates mTOR and STAT3; and promotes the differentiation of T lymphocytes into effector T cells (such as Th1 and Th2 cells) (110,111). Butyrate also promotes the differentiation of T lymphocytes into Tregs, increases the expression of Foxp3 in Tregs, and enhances the stability and activity of Foxp3. This process simultaneously promotes IL-10 production by naïve T cells while inhibiting excessive inflammation and immune responses (36,112).The inhibitory effect of butyric acid on B-cell HDAC activity is dose-dependent, and results in the promotion of B-cell class switching and antibody secretion. Butyric acid upregulates the expression of plasma cell differentiation-related genes such as Aicda, Xbp, Irf4, Prdm1 and Sdc1, and enhances the generation of post-switch transcripts expressed by IgG3, IgG1, IgG2b, IgG2a and IgA (39,40). In other immune cells, butyric acid downregulates the activity of NF-κB, thereby inhibiting its nuclear translocation and increasing the levels of IκB, which serves to ameliorate the regulation of prolonged inflammation. Furthermore, butyric acid reduces NO production in macrophages and intestinal myofibroblasts by modulating the expression of iNOS through the inhibition of the JAK/STAT1 signaling pathway (113). Butyrate inhibits NLRP3 inflammatory vesicle activation, and modulates immune cell infiltration in the pancreas by increasing the acetylation of histone H3K9, H3K14, H3K18 and H3K27 sites (114).

Cell cycle arrest is a key regulatory mechanism in tumor cell proliferation (115). Butyrate has been found to induce cell cycle arrest in the G0/G1 phase in a dose-dependent manner in vitro in numerous tumors, including colon, liver, lung and bladder cancer, and G1 phase arrest may be one of the mechanisms by which butyrate induces apoptosis (67). Butyrate alters the expression of certain genes that regulate the cell cycle and apoptosis by increasing the level of histone acetylation, such as p21^WAF1/CIP1, c-Myc, c-Myb, cyclin-dependent kinase (CDK) 6, cyclin D1 and cyclin D3, while this effect is not exhibited in other SCFAs (116). Butyrate upregulates p21 protein expression via the p53 and Sp1/Sp3 pathways, and p21 protein induces G1-phase arrest in tumor cells through loss of function upon binding to CDK/cyclin (117,118). In addition to directly inhibiting the expression of p21^WAF1/CIP1, butyrate inhibits the degradation of splicing factor, arginine/serine-rich 2 (SRSF2) through HDAC6 inhibition, which results in the accumulation of acetylated and non-phosphorylated forms of SRSF2, thus promoting the activation of p21^WAF1/CIP1 transcription (119). In addition, butyrate might further induce cell cycle arrest via the PI3K-Akt pathway, and thus cause cell death (120).

Butyrate was found to modulate the Bcl-2 family by disrupting the mitochondrial transmembrane potential, activating the caspase cascade amplification reaction and inducing apoptosis in tumor cells (121). Bcl-2 serves as the most important regulator of mitochondrial membrane permeability (MMP), which is localized at the outer membrane of mitochondria. Alterations in MMP lead to a decrease in mitochondrial transmembrane potential (ΔΨm). In the presence of butyrate, Bcl-2 expression is inhibited and the mitochondrial permeability transition pore opens, leading to a decrease in ΔΨm until it is lost, thus disrupting the structure of the inner and outer membranes, and ultimately disintegrating the mitochondrion (122,123). Subsequently, mitochondria release cytochrome c (Cyt c), which forms apoptotic vesicles alongside apoptotic protease activating factor-1 (Apaf-1) and caspase-9 in the presence of ATP, thus activating caspase-3, initiating a cascade reaction and inducing apoptosis in tumor cells (124,125). Furthermore, in breast cancer cells, butyrate-induced apoptosis is accompanied by elevated ROS levels and caspase activity (126). Such mechanism suggests that ROS can induce mitochondrial membrane damage, release Cyt c from damaged mitochondria, and enhance apoptosis via the Cyt c/caspase-3 pathway (127).

Butyrate, through the regulation of a complex molecular network that induces apoptosis, exerts inhibitory effects on tumor cell proliferation, invasion and metastasis. For example, 666 differentially expressed mRNAs and 30 differentially expressed long nc RNAs (lncRNAs) were involved in the inhibition of CRC by butyrate. By constructing protein-protein interaction network and competing endogenous RNA networks based on differentially expressed mRNAs and lncRNAs, a series of differentially expressed genes related to the prognosis of CRC were found (66). Butyrate-treated lymphoma cell lines demonstrated increased expression levels of microRNA (miRNA or miR)-101, miR-143 and miR-145, and these differential miRNAs were involved in migration, proliferation and apoptotic processes in lymphoma cells (128). Among the previous studies on the role of ncRNAs in butyrate on tumors, miRNA-related studies are the most extensive. miRNAs are typically 20 nucleotides in length, and they are processed by sequential shearing of precursor transcripts. miRNAs have emerged as important regulators of tumorigenesis, and some miRNAs are novel targets for HDACI. Butyrate, by targeting miRNAs, induces apoptosis, and inhibits tumor cell proliferation, migration and invasion, which is associated with the diverse roles of individual miRNAs within the organism (Table I) (129-134). The construction of RNA predictive molecular networks should be the focus of future research.

Butyrate inhibits glucose transporter protein 1 expression and promotes hexokinase activity, leading to improved mitochondrial function and oxidative metabolism in lung cancer cells (135). Additionally, butyrate reverses the Warburg effect by blocking the activation of the pyruvate dehydrogenase complex via sirtuin 3 (136).

In summary, butyrate plays an important role by inhibiting HDAC. Butyrate can be readily received by any cell type via SMCT-1 and subsequently inhibit HDAC (24), which is the basic of the butyrate's regulatory role in cells that exhibit low expression of GPCRs. In such cells, butyrate enhances target gene histone acetylation by inhibiting HDAC activity, thus changing chromatin from a dense repressive structure to a relaxed transcriptionally activated structure, which contributes to the binding of transcription factors such as STAT3 and Foxp3 to DNA and initiates gene transcription. The present study reviewed the extensive inhibitory impact of butyrate on HDAC without an in-depth exploration of specific HDAC subtypes. Subsequent studies should consider selectively targeting different HDAC subtypes to elucidate whether butyrate manifests its antitumor efficacy through particular HDAC subtypes.

Adjuvant treatments for tumors often involve dietary supplementation with SCFAs. Fiber consumption alters the composition of gut microbiome to a greater extent than other dietary factors and increases the number of butyrate-producing bacteria (15). Direct butyrate supplementation (via capsules or enemas) differs from endogenous butyrate production that is not generated in discrete, punctuated boluses, but is rather coupled to fermentation, which is diurnal and rhythmic (137). Butyrate can selectively modulate gut bacteria. After the resistant starch diet was increased in rats, culturable Lactobacillus and Bifidobacterium were found to increase, while Enterobacterium decreased (138). While promising results have been observed in cellular and animal experiments, the efficacy in humans has been less consistent. Blindly increasing dietary fiber intake does not consistently yield desired outcomes. Clinical trials predominantly center on CRC, melanoma, breast cancer and gastric cancer. These studies investigate the connection between SCFAs, disease progression and prognosis, including the impact of resistant starch and probiotics on gut microbiota composition, SCFAs content and outcome of patients (Table II). However, these studies lack specificity regarding SCFAs types, and their objectives encompass diverse aspects such as treatment and prognosis. Variability in internal and external environments of individuals, timing, dosage and SCFAs sources may introduce bias into the results of note; clinical studies lack uniform criteria for evaluating effects, and challenges remain regarding assessing the impact of dietary fiber and supplementation of SCFAs.

Clostridium butyricum is an anaerobic bacterium classified as a probiotic due to its production of butyric acid (139). CBM588, a live bacterial product, includes Clostridium butyricum, which was originally identified by Chikaji Miyairi in 1933 in healthy human feces. This non-pathogenic and non-toxic bacterium has gained widespread use in Japan, Korea, China and Europe (140). Administration of CBM588 in combination with antitumor therapy may result in increased efficacy. A retrospective study revealed a significant and positive association between CBM588 and PFS and overall survival in patients with NSCLC receiving ICIs (141). The beneficial effect of CBM588 was even more pronounced in patients receiving antibiotic therapy (142). In an open, single-center study (NCT03829111), CBM588 significantly prolonged PFS in patients with metastatic renal cancer who had been treated with nivolumab-ipilimumab and increased the response rate to treatment (143). CBM588 exerts direct antitumor effects by inducing polymorphonuclear neutrophils in the bladder to release substantial quantities of apoptosis-inducing ligands, thus achieving antitumor effects (144). Moreover, CBM588 modulates the structure and composition of gut microbiota, leading to a lower incidence of colitis-associated colon cancer (145). These findings strongly support the hypothesis that CBM588 may be a safe and efficient treatment for oncological diseases. Clinical trials related to Clostridium butyricum and oncology therapy are summarized in Table III and additional information can be found in the ClinicalTrials. gov database.

Butyrate has not shown side effects or drug toxicity in previous animal studies. However, its brief half-life and unpleasant odor render it unsuitable for direct use as a chemotherapeutic agent (146). Alternative viable strategies involve the utilization of oral butyrate analogs, derivatives, or administration techniques at nanometer level to enhance bioavailability and mitigate olfactory irritation (147,148). Furthermore, butyrate not only alleviates the side effects associated with conventional chemotherapeutic agents such as oxaliplatin, irinotecan and 5-fluorouracil (149-151), but it also enhances the efficacy of both chemotherapy and immunotherapy (128). This has increased the interest in research on butyrate as an adjuvant therapy. Notably, certain conventional drugs can act synergistically with oncological treatments by influencing the body's butyrate concentration. For example, metformin has been demonstrated to enhance the biosynthesis of butyrate while concurrently inhibiting the progression of CRC (152), while safranin has been observed to augment the prevalence of butyrate-producing bacterial strains such as Allobaculum, Butyricoccus and Phascolarctobacter. This augmentation contributes to the increased production of butyrate, which has proven efficacious in preventing the recurrence of colorectal adenoma (153). The identification of effective biomarkers offers a potential solution to the challenge of heterogeneous response rates observed in immunotherapy across different tumor types. Considering the non-invasive nature of fecal examination, butyrate is anticipated to serve as an independent predictor of immunotherapeutic response for a diverse spectrum of malignancies. This could encompass the successful stratification of responders and non-responders to immune checkpoint inhibition through metabolomics analyses, or as a therapeutic target for improving the efficacy of ICIs in the treatment of specific tumors. In order to produce more accurate pharmacokinetic, absorption, distribution and metabolic data, organoids can simulate human-specific metabolic pathways, cell interactions and tissue responses more effectively (154). In a study linking butyric acid to colon cancer organoids, butyrate enhanced the efficacy of radiotherapy while protecting the normal mucosa, thus minimizing the associated toxicity of radiotherapy. Butyrate significantly enhanced radiation-induced cell death and enhanced treatment effects compared with administration of radiation alone. Notably, butyrate did not increase radiation-induced cell death and improved regeneration capacity after irradiation in normal organoids (155,156). Butyrate can increase the uptake of serotonin by mouse ileal organoids and alleviate gastrointestinal reactions by providing energy to cancer patients given radiation therapy (157). In liver organoids, butyrate could reduce drug-induced hepatotoxicity by improve metabolic activation (158).

Another alternative that may promote the generation of butyric acid and combat cancer is FMT. FMT decreases the diversity of gut microbiota in patients with colon cancer and promotes the growth of gastrointestinal tumors in mice (159). Conversely, FMT in wild-type mice alleviates CRC induced by dextran sulfate sodium or azoxymethane (160). Restoring the intestinal flora disturbed by FOLFOX (5-fluorouracil, leucovorin and oxaliplatin) therapy in mice receiving healthy donor feces decreased diarrhea and intestinal mucosal inflammation (161). FMT offers a wide range of potential applications in tumor therapy because it is the most direct method of altering the microbiome and directly increasing the bacteria that produce butyrate. However, FMT has the potential to disperse hazardous bacteria. Certain FMT materials have been linked to the production of autoimmune disorders in animal studies due to their microbiome (162). Thus, FMT donors should undergo thorough screening, questioning regarding their family history and previous illness, as well as fecal testing for potential parasites in order to minimize these risks.

The role of butyrate in helping to develop personalized anticancer strategies cannot be overstated. However, each of the aforementioned therapeutic approaches necessitates meticulous evaluation within patient cohorts prior to their clinical implementation. This rigorous scrutiny will not only advance the current understandings of human pathophysiology but also increase the current knowledge about complex metabolite-target group interactions and therapeutic associations, and such insights are instrumental in the refinement of clinical treatment protocols.