Colonic Epithelial Proliferation Research Articles

Involvement of autophagy and gut dysbiosis in ambient particulate matter-induced colonic inflammation

Ambient fine particulate matter (PM2.5), a vital environmental toxicant, not only adversely affects the cardiovascular and respiratory systems but also potentially exhibits an association with intestinal inflammation and colorectal cancer (CRC). The underlying molecular mechanisms of PM2.5 impacts on CRC are still unclear. In this study, we utilized collected ambient PM2.5 and standard reference material SRM2786 to investigate the toxic effects on the colon through in vivo chronic exposure mouse and in vitro cell culture models. We employed a chronic mouse exposure model to clarify the colonic injury and gut microbiome biomarkers. Prolonged exposure to PM2.5 via oropharyngeal aspiration led to a significant rise in colonic epithelial proliferation and reduced colon length in mice. It triggered characteristics indicative of gut microbiota dysbiosis linked to inflammatory bowel disease. The gut microbiome alternations may serve as a biomarker indicating the colonic health impacts of PM2.5 exposure. PM2.5 and SRM2786-induced cytotoxicity manifested as autophagy dysregulation-mediated abnormal proliferation, IL-8 production, p62/SQSTM1 accumulation, and lysosomal membrane damage in human colon cells WiDr and Caco-2. Both PM2.5 and SRM2786 exposures led to the accumulation of p62/SQSTM1 and compromised lysosomal membrane integrity, showing impaired autophagic flux in WiDr and Caco-2 cells. Finally, we examined the correlations between atmospheric PM2.5 data and biomarkers of colonic inflammation in human population. The serum level of IL-8 was significantly correlated with regional anthropogenic pollutants. In conclusion, our findings elucidate that ambient PM2.5 exhibits adverse effects on colon health manifested as inflammation, aberrant proliferation, and gut dysbiosis, potentially mediated through autophagy dysregulation, thereby highlighting the importance of further research on the impact of environmental pollutants on gastrointestinal health.

Hypusination Maintains Intestinal Homeostasis and Prevents Colitis and Carcinogenesis by Enhancing Aldehyde Detoxification

The amino acid hypusine, synthesized from the polyamine spermidine by the enzyme deoxyhypusine synthase (DHPS), is essential for the activity of eukaryotic translation initiation factor 5A (EIF5A). The role of hypusinated EIF5A (EIF5AHyp) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5AHyp in the gut epithelium in inflammation and carcinogenesis. We used human colon tissue messenger RNA samples and publicly available transcriptomic datasets, tissue microarrays, and patient-derived colon organoids. Mice with intestinal epithelial-specific deletion of Dhps were investigated at baseline and in models of colitis and colon carcinogenesis. We found that patients with ulcerative colitis and Crohn's disease exhibit reduced colon levels of DHPS messenger RNA and DHPS protein and reduced levels of EIF5AHyp. Similarly, colonic organoids from colitis patients also show down-regulated DHPS expression. Mice with intestinal epithelial-specific deletion of Dhps develop spontaneous colon hyperplasia, epithelial proliferation, crypt distortion, and inflammation. Furthermore, these mice are highly susceptible to experimental colitis and show exacerbated colon tumorigenesis when treated with a carcinogen. Transcriptomic and proteomic analysis on colonic epithelial cells demonstrated that loss of hypusination induces multiple pathways related to cancer and immune response. Moreover, we found that hypusination enhances translation of numerous enzymes involved in aldehyde detoxification, including glutathione S-transferases and aldehyde dehydrogenases. Accordingly, hypusination-deficient mice exhibit increased levels of aldehyde adducts in the colon, and their treatment with a scavenger of electrophiles reduces colitis. Hypusination in intestinal epithelial cells has a key role in the prevention of colitis and colorectal cancer, and enhancement of this pathway via supplementation of spermidine could have a therapeutic impact.

Dietary Fibers Differ in Their Effects on Large Bowel Epithelial Proliferation and Fecal Fermentation-Dependent Events in Rats

The effects of different fiber types and processing on putative protective mechanisms for colorectal cancer were evaluated. Rats were fed diets of similar nutrient balance containing either no added fiber or 10% fiber from various sources. The rate of distal colonic epithelial proliferation, measured by the metaphase arrest method, was dependent on fiber type; ranking of fibers from highest to lowest yielded the following order: methylcellulose > coarse wheat bran > fine wheat bran ≈ parboiled and extruded rice brans > no fiber (P = 0.012). Effect on stool output ranked identically. Ranking of effect on fecal pH, from most to least acidic was as follows: coarse wheat bran ≈ the rice brans > fine wheat bran > no fiber ≈ methylcellulose (P = 0.00001). Coarse wheat bran gave significantly higher fecal butyrate concentrations than did the rice brans, which in turn gave higher levels than fine wheat bran, methylcellulose and the no-fiber diet. Proximal colon epithelial proliferation was unaffected by diet although cecal short-chain fatty acid concentrations and pH were affected. Different fibers have different effects on events in the fecal environment and distal colonic epithelium. Putative protective events (increased output, low fecal pH, high butyrate, low proliferation) are not equally affected and are unlikely in themselves to allow prediction of the protective effect of a fiber.

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer.

Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer. We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3. After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC. YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.

Vitamin D3 and Lactobacillus rhamnosus GG/p40 Synergize to Protect Mice From Colitis by Promoting Vitamin D Receptor Expression and Epithelial Proliferation.

While vitamin D (VitD) levels are negatively correlated with inflammatory bowel disease (IBD) activity, VitD supplementation does not reduce IBD severity. The probiotic Lactobacillus rhamnosus GG (LGG), which secretes p40, can upregulate colonic VitD receptor (VDR) expression. We therefore evaluated synergy between VitD3 and LGG/p40 in the treatment of mouse colitis. A dextran sulfate sodium (DSS) colitis model was established in Vdr+/+ and Vdr-/- mice, and mice were treated with VitD3, LGG, or p40 alone or in combination for 7 to 14 days. Colitis severity was assessed by weight loss, disease activity index (DAI), colon length, histology, and inflammatory cytokine expression together with VDR expression, proliferation, and apoptosis. In vitro, VDR expression and cell viability were assessed in HCT116 cells after stimulation with p40. Total and nuclear VDR protein expression were lower in DSS-treated Vdr+/+ mice compared with control mice (P < .05). Compared with the DSS group, VitD3 + LGG alleviated colitis as assessed by significantly improved DAI and histological scores, increased colon length, decreased colonic Tnf, and increased Il10 expression together with increased colonic VDR gene and protein expression and increased Ki-67 proliferation index (P < .05). In Vdr-/- mice, VitD3 + LGG had no effect on DSS colitis. In Vdr+/+ mice, VitD3 + p40 also reduced colitis severity according to clinicopathological and immunological metrics and increased VDR expression and epithelial proliferation (P < .05). In HCT116 cells, p40 stimulation increased VDR protein expression and viability (P < .05). VitD3 and LGG/p40 synergistically improve the severity of colitis by increasing colonic VDR expression and promoting colonic epithelial proliferation.

Loss of Adhesion G‐Protein‐Coupled Receptor L2 Expression Impacts Colonic Epithelial Proliferation

BackgroundThe role of adhesion G‐protein‐coupled receptor L2 (ADGRL2, a.k.a. Latrophilin‐2) in human health has been understudied despite discoveries that the receptor is required for both heart development and neural synaptogenesis. This project tests the physiological function of ADGRL2 in the colon where our prior findings that ADGRL2 is lost in human colorectal cancer (CRC), downregulated during hyperproliferative recovery from murine DSS colitis, enriched in stem and progenitor cells, and is inversely correlated with growth arrest. This raises the intriguing possibility that ADGRL2 restricts stem cell expansion and therefore downregulation of ADGRL2 is required for stem‐cell driven regeneration and repair.MethodsThe effect of Adgrl2 on cell proliferation was assessed by histological staining for Ki‐67 antigen or for the thymidine analogue EdU (5‐ethynyl‐2’‐deoxyuridine, administered by intraperitoneal injection 2hr prior to harvest) in intestinal‐epithelial specific Adgrl2 knockout mice (Adgrl2 IE KO). Percent positive cells were quantified per crypt and per crypt region (crypt base, mid crypt, upper crypt) in Image J.Results/ConclusionsAdgrl2 IE KO mice have overall reduced numbers of Ki‐67+ cells (8.2% difference, p = 0.0016) in the colonic epithelium. This seems paradoxical given that we hypothesize a tumor suppressor role for ADGRL2. However, this effect is driven by a pronounced decrease in Ki‐67+ cells in the post‐mitotic upper crypt of Adgrl2 IE KO mice (15.2% difference, p = 0.0121) while percentages of Ki‐67+ cells in the more proliferative mid crypt and crypt base were determined to be roughly equal between groups, suggesting that the observed effect is likely not driven by a reduction in proliferating stem or progenitor cells but is perhaps due to increased cell cycle exit or terminal differentiation with Adgrl2 knockout. Given that Ki‐67 antigen is expressed in all active cell cycle phases, we used EdU labeling of S‐phase proliferating cells to further assess the impact of Adgrl2 loss on colonocyte proliferation. Proliferation is not impaired with loss of Adgrl2 and instead showed a trend towards increased EdU positive cells in the crypt base (6.7% increase, p= 0.0907). As expected, overall number of S‐phase cells in the upper crypt compartment was low for both Adgrl2 KO and WT animals, however a significant decrease in EdU positive cells was still detected in Adgrl2 KO mice (2.98% change, p=0.0500). Based on these data, the loss of Adgrl2 does not decrease proliferative capacity of the stem cell or progenitor niche, but does effect the distribution of active cycling or differentiating cells along the crypt axis, likely by promoting cell cycle exit or terminal differentiation. Further studies will investigate this possibility and the role of Adgrl2 loss on cell renewal in the context of disease. These data support our hypothesis that ADGRL2 modulates epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. Mechanistic studies on dysregulated growth and repair in the colon may also illuminate targets to prevent colitis‐associated cancer in IBD patients.

Ethanol-induced formation of colorectal tumours and precursors in a mouse model of Lynch syndrome.

Lynch syndrome (LS) confers inherited cancer predisposition due to germline mutations in a DNA mismatch repair (MMR) gene, e.g. MSH2. MMR is a repair pathway for removal of base mismatches and insertion/deletion loops caused by endogenous and exogenous factors. Loss of MMR through somatic alteration of the wild‐type allele in LS results in defective MMR (dMMR). Lifestyle/environmental factors can modify colorectal cancer risk in sporadic and LS patients. Ethanol and its metabolite acetaldehyde are classified as group one carcinogens, and acetaldehyde causes a range of DNA lesions. However, DNA repair pathways responsible for correcting most of such DNA lesions remain uncharacterised. We hypothesised that MMR plays a role in protecting colorectal epithelium from ethanol/acetaldehyde‐induced DNA damage. Here, an LS mouse model (intestinal epithelial conditional‐knockout for Msh2) was used to determine if there is a gene–environment interaction between dMMR and ethanol/acetaldehyde that accelerates colorectal tumourigenesis in LS. Mice underwent either long‐term ethanol treatment or water treatment. Most ethanol‐treated mice demonstrated colonic hyperproliferation and adenoma formation (with some invasive adenocarcinomas) within 6 months (15/23, 65%), compared with one colonic tumour after 15 months in water‐treated mice (1/23, 4%) (p < 0.0001, Fisher's exact test). A significantly greater number of dMMR colonic crypt foci precursors were observed in ethanol‐treated compared with water‐treated mice (p = 0.0029, Student's t‐test). Moreover, increased plasma acetaldehyde levels were detected in ethanol‐treated compared with water‐treated mice (p = 0.0019, Mann–Whitney U‐test), along with significantly increased DNA damage response in the colonic epithelium. Long‐term ethanol treatment was associated with significantly increased colonic epithelial proliferation and markedly reduced apoptosis in dMMR adenomas, consistent with enhanced survival of aberrant dMMR relative to MMR‐proficient colonic epithelium. In conclusion, there is strong evidence for a gene–environment interaction between dMMR and acetaldehyde, causing acceleration of dMMR‐driven colonic tumour formation in this LS model, indicating that advice to limit alcohol consumption should be considered for LS patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Symbiotic polyamine metabolism regulates epithelial proliferation and macrophage differentiation in the colon

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

Effects of fibers and gut microbiota on low-grade inflammatory human disease

Obesity represents a major public health problem, as it leads to metabolic disorders involved in the development of type 2 diabetes, fatty liver diseases, cardiovascular diseases and cancer (1). The Western lifestyle, including over-feeding of highly refined diets and sedentary behavior, is associated with systemic low-grade inflammation, responsible for chronic degenerative disease. Moreover, animal and human data have demonstrated the impact of high-fat diet feeding on the onset of metabolic endotoxemia. This latter, being characterized by increase blood lipopolysaccharide (LPS) levels, triggers low-grade inflammation and contributes to the development of obesity. The gut microbiota are a candidate sources of plasma LPS. Indeed, LPS is a major component of the outer membrane in Gram-negative bacteria and its plasma levels are correlated with changes in intestinal microbiota where the Gram-negative to Grampositive ratio is increased by high fat feeding (1). Not surprising that in humans, obesity is characterized by dysbiosis, i.e. imbalances in the composition and function of the intestinal microbes. Currently, many studies are being conducted to assess the contribution of intestinal microflora metabolites [mainly short-chain fatty acids (SCFAs)] to the organism’s homeostasis. Dietary fibers have been promoted for their health benefits and high-fiber intake has been reported as beneficial in numerous chronic diseases (2). However, we have to keep in mind that consumption of dietary fiber significantly alters the composition of the gut microbiota. For instance, addition of fibers to the diet increases the abundance of butyrate-producing bacteria Roseburia species and Eubacterium rectale , whereas a lowfiber diet is associated to a major decrease in this group, with Faecalibacterium prausnitzii becoming the main butyrate-producer. Hence, a greater understanding of the interaction between dietary fibers and the intestinal microbiota could represent a mean of maintaining or improving the community of gut microorganisms, particularly when dysbiosis exists. In addition, although various data indicate that increased SCFAs production positively affects the host by exerting antiobesity and antidiabetic effects, some in vitro and in vivo studies have indicated that overproduction or accumulation of SCFAs in the bowel may lead to obesity and cancer development, owing to increased energy accumulation and elevation of the inflammatory state. As correctly pointed out in the recent paper by Wen and Schwabe , more experimental and epidemiologic data are needed to completely elucidate the link among dietary fibers, gut microbiota and the onset of low-grade inflammatory diseases (3). However, the authors commented about the recent study by Singh et al. (4) and based only on these results stated that soluble fibers could be associated to liver disease and hepatocellular carcinoma. Specifically, it will be a great achievement to shed light on the influence that SCFAs exert on the intestinal environment as well as on distal tissues and organs. SCFAs, mainly acetate, propionate and butyrate, are organic acid produced by intestinal microbial fermentation of dietary fibers. Various mechanisms have been highlighted to justify the biological effects of butyrate: (I) inhibition of histone deacetylase (HDAC); (II) downregulation of neuropilin-1 (NRP-1); (III) activation of G-protein-coupled receptors, GPR41, GPR43 and GPR109A, and of the peroxisome proliferator-activated receptor γ (PPARγ); (IV) modulation of canonical Wnt signaling pathway; (V) activation of AIM2 and NLRP3 inflammasomes, enhancing production of interleukin-18 (IL-18) and epithelial barrier function; (VI) differentiation of T lymphocytes into effector (Te) and regulatory (T reg) T cells; and (VII) modulation of T lymphocytes and dendritic cell functions (5). In the meantime, SCFAs can reach the liver through the portal vein and directly promote hepatic triglyceride accumulation and gluconeogenesis. In addition, various data have demonstrated that butyrate appears to have “paradoxical” effects on colonic epithelial proliferation due to the fact that the response of proliferative cells to butyrate are affected by the dose of butyrate, the physiological conditions under which the study was performed and the phenotype of the target cells (3). Butyrate can instead suppress proliferation in hyperproliferation situations (i.e., colon cancers cells), or stimulate proliferation in impaired energy status (i.e., atrophic colon) or having no effect in normal tissues. Similarly, butyrate can promote or inhibit apoptosis (1).

Chemopreventive properties of young green barley extracts in in vitro model of colon cancer

Young green barley is the most valuable source of nutrients and bioactive substances. It has a broad spectrum of health-promoting properties such as antioxidant, anti-inflammatory, hypoglycaemic, anti-depressant, anti-atherosclerotic and anticancer. The presented study is an attempt to extend this knowledge with particular emphasis on the possibility of using green barley in colon cancer prevention. Extracts were prepared on the basis of two commercial products: ground dried barley grass (YGB INT) and powder of young green barley juice (YGB GW). Their influence on colon epithelial cells (CCD841 CoN) viability and proliferation were analyzed by LDH and MTT assays. Anticancer properties of extracts were screened on colon cancer cell lines (LS180, HT-29) by MTT and BrdU assays. Changes in cells morphology induced by extracts were investigated after May-Grünwald-Giemsa staining. Tested extracts were not toxic against CCD841 CoN and did not affected their proliferation or morphology (LDH test, MTT test, microscopy observation). The MTT revealed that extracts significantly inhibited proliferation of colon cancer cells in a dose-dependent manner. Results of BrdU test confirmed antiproliferative properties of extracts, but opposite to MTT test, indicated YGB GW as a better anticancer agent. Light microscopy observation proved the data obtained from both MTT and BrdU tests and additionally suggested the ability of the extracts to induce necrosis in LS180 and HT-29 cells. The study demonstrated that YGB extracts specifically inhibit proliferation of colon cancer cells without any undesirable effect on colon epithelial cells. Obtained results will provide a rationele for the future development of dietary supplements which could be beneficial in colon cancer chemoprevention.

A Bifidobacterial pilus-associated protein promotes colonic epithelial proliferation.

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad2003 pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.

Mo1071 - Butyrate and Proteolytic Bioactive Form of Wnt-5A Regulate Colonic Epithelial Proliferation and Development

Mo1071 - Butyrate and Proteolytic Bioactive Form of Wnt-5A Regulate Colonic Epithelial Proliferation and Development

Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development

Reduced short-chain fatty acids (SCFAs) have been reported in patients with ulcerative colitis, and increased intake of dietary fiber has shown to be clinically beneficial for colitis. Whether SCFAs suppress tumorigenesis in colitis-associated colorectal cancer remains unknown. The chemopreventive effect of SCFAs in colitis-associated colorectal cancer was evaluated in this study. Model of colitis-associated colorectal cancer in male BALB/c mice was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). SCFAs mix (67.5 mM acetate, 40 mM butyrate, 25.9 mM propionate) was administered in drink water during the study period. Macroscopic and histological studies were performed to examine the colorectal inflammation and tumorigenesis in AOM/DSS-induced mice treated with or without SCFA mix. The effects of SCFAs mix on colonic epithelial cellular proliferation were also assessed using Ki67 immunohistochemistry and TUNEL staining. The administration of SCFAs mix significantly reduced the tumor incidence and size in mice with AOM/DSS-induced colitis associated colorectal cancer. SCFAs mix protected from AOM/DSS-induced colorectal cancer by improving colon inflammation and disease activity index score as well as suppressing the expression of proinflammatory cytokines including IL-6, TNF-α and IL-17. A decrease in cell proliferation markers and an increase in TUNEL-positive tumor epithelial cells were also demonstrated in AOM/DSS mice treated with SCFAs mix. SCFAs mix administration prevented development of tumor and attenuated the colonic inflammation in a mouse model of colitis-associated colorectal cancer. SCFAs mix may be a potential agent in the prevention and treatment of colitis-associated colorectal cancer.

Indoleamine 2,3-dioxygenase expression regulates the survival and proliferation of Fusobacterium nucleatum in THP-1-derived macrophages

Fusobacterium nucleatum (Fn) is a tumor-associated obligate anaerobic bacterium, which has a role in the progression of colorectal cancer (CRC). Fn can invade and promote colon epithelial cells proliferation. However, how Fn survives and proliferates in its host cells remains largely unknown. In this study, we aimed to determine the molecular mechanisms underlying the morphology, survival, and proliferation of Fn in THP-1-derived macrophages (dTHP1). For the first time, we found that Fn is a facultative intracellular bacterium that can survive and limited proliferate in dTHP1 cells up to 72 h, and a live Fn infection can inhibit apoptosis of dTHP1 cells by activating the PI3K and ERK pathways. Both Fn bacteria and dTHP1 cells exhibit obvious morphological changes during infection. In addition, Infection of Fn-induced indoleamine 2,3-dioxygenase (IDO) expression by TNF-α-dependent and LPS-dependent pathway in a time-dependent and dose-dependent manner, and the IDO-induced low tryptophan and high kynurenine environment inhibited the intracellular multiplication of Fn in dTHP1 cells. IDO expression further impaired the function of peripheral blood lymphocytes, permitting the escape of Fn-infected macrophages from cell death. IDO inhibition abrogated this effect caused by Fn and relieved immune suppression. In conclusion, we identified IDO as an important player mediating intracellular Fn proliferation in macrophages, and inhibition of IDO may aggravate infection in Fn-associated tumor immunotherapy.

The influence of genetic ablation of tumor necrosis factor‐α on the colonic Wnt pathway cascade under an obese state

Obesity is an established risk factor for colorectal cancer (CRC), but the mechanism(s) responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low‐grade colonic inflammatory microenvironment, which facilitates the development of CRC. Our prior study suggested tumor necrosis factor alpha (TNF‐α) might elevate the Wnt‐signaling, and thereby promote the colonic tumorigenesis. In the present study, we observed that several colonic inflammatory cytokines (TNF‐α, IL‐1β, IFNg) and Wnt pathway specific genes (e.g. Axin2) were upregulated in an obese state (p &lt; 0.05), whereas the genetic ablation of TNF‐α suppressed the elevation of Wnt‐signaling induced by dietary obesity, as indicated by decreased levels of phopho‐GSK3β, and active β‐Catenin (p &lt; 0.05), two key components in Wnt pathway cascade. A Wnt pathway specific gene expression profiling further demonstrated that the genetic abolishment of TNF‐α attenuated aberrations of several genes within the colonic Wnt pathway cascade including the over‐expressed Axin2, a readout of the Wnt pathway (p &lt; 0.05). Colonic epithelial proliferation (Ki‐67), which is partially under the control of Wnt signaling, was also decreased by the blockade of TNF‐in obese mice (p &lt; 0.05). These results indicate that obesity‐driven inflammation plays a role in the regulation of the tumorigenic Wnt‐signaling pathway, which contributes to the development of obesity‐associated CRC.Support or Funding InformationSupported in part by a USDA/NIFA grant, a USDA/Hatch grant and a HNRCA competitive intramural pilot grant to ZL

Is Metformin a Therapeutic Paradigm for Colorectal Cancer: Insight into the Molecular Pathway?

Colorectal cancer (CRC) remains one of the major leading causes of cancer related morbidity and mortality. Apart from the conventional anti-neoplastic agents, metformin, a biguanide anti-diabetic agent, has recently found to have anti-cancer property. Several studies observed the effect of metformin towards its anti-cancer effect on colon or colorectal cancer in diabetic patients. However, only a few studies showed its effect on colorectal cancer in relation to the non-diabetic status. The present review aimed to highlight the insight into the molecular pathway of metformin towards colorectal cancer in the absence of diabetes mellitus. In CRC-independent of diabetes mellitus, highly deregulation of PI3K/AKT pathway is found which activates the downstream mammalian target of rapamycin (mTOR). Metformin inhibits cancer growth in colon by suppressing the colonic epithelial proliferation by inhibiting the mTOR pathway. Metformin exerts its anti-neoplastic effects by acting on tumour suppressor pathway via activating the adenosine monophosphate.activated protein kinase (AMPK) signaling pathway. Metformin interrupts the glucose metabolism by activating the AMPK. Metformin reduces tumour cell growth and metastasis by activating the p53 tumour suppressor gene. In addition to its therapeutic benefits, metformin is easily accessible, cost effective with better tolerance to the patients compared to the chemotherapeutic agents. This review summarised modern findings on the therapeutic applications of metformin on the colorectal cancer with no evidences of diabetes mellitus.

MCM2 expression in serrated polyps demonstrates aberrant cellular proliferation

In normal colonic epithelium, the proliferative zone is limited to the lower half of the colonic crypt. Evaluating the changes in the colonic epithelial proliferation can be useful in understanding pathophysiology of various diseases. Our aim was to investigate the proliferative compartment of serrated polyps (SPs) using MCM2, a protein involved in DNA replication, and assess for changes along the SP spectrum. Immunohistochemistry was performed on serrated polyps (16 microvesicular-type hyperplastic polyps (HP), 58 sessile serrated adenomas (SSA), 7 SSAs with dysplasia) and 6 sections of normal colon using anti-MCM2 antibody. Multiple sections of normal colon showed the following pattern for MCM2 and Ki-67 staining: positive nuclear staining of the lower half of the colonic crypts and/or slightly expanded to the lower two-thirds of the crypt. By MCM2, SPs show expansion of the proliferative compartments; 81.3% of HPs and 100% of SSAs showed some degree of full crypt MCM2 staining. SSAs with dysplasia showed consistent diffuse polyp staining. Aberrant staining in adjacent normal mucosa was also seen in SSAs with dysplasia and in a subset of non-dysplastic SSAs. By using MCM2, we show that serrated polyps exhibit changes in proliferation during progression along the pathway. HPs and SSAs show a similar highly proliferative profile. Aberrant proliferative cell staining patterns in adjacent normal colonic mucosa as seen in SSAs with dysplasia and a subset of SSAs suggest a field effect phenomenon. This indicates that changes in the colonic micro-environment may promote adenoma morphogenesis and predisposition to malignancy.

Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.

BVES Loss Is Protective in Radiation Enteritis

Blood Vessel Epicardial Substance (BVES) is a tight-junction associated protein that is expressed in the intestine and regulates epithelial-to-mesenchymal transition. We have previously demonstrated that BVES regulates colonic epithelial proliferation and differentiation, with BVES loss resulting in hyperactive WNT signaling, but its role in the small intestine (SI) remains unexplored. To determine the role of BVES in SI integrity we used radiation-induced injury modeling.