Azoxymethane/dextran Sodium Sulfate Research Articles

The role and mechanism of action of RNF186 in colorectal cancer through negative regulation of NF-κB

Colorectal cancer (CRC) is one of the most common malignant gastrointestinal cancers worldwide. RING finger protein 186 (RNF186) is a member of the RING finger protein family. RNF186 has been reported to be involved in the regulation of the intestinal homeostasis through the regulation of endoplasmic reticulum (ER) stress in colonic epithelial cells. However, its role in CRC remains unclear. In this study, we found that colorectal tumours from human patients had decreased levels of RNF186. We demonstrated that overexpression of RNF186 suppressed the growth and migration of CRC-derived cell lines in vitro and inhibited tumour proliferation in vivo. Further, our findings indicated that forced expression of RNF186 inhibited nuclear factor-κB (NF-κB) activation by reducing the phosphorylation of NF-κB. In addition, our results showed that RNF186−/− mice exhibited significantly increased tumour burden compared to the wild type (WT) mice following treatment with azoxymethane/dextran sulfate sodium (AOM/DSS). Compared to WT mice, the percentage of Ki67 positive cells was increased in the RNF186−/− mice, indicating that RNF186 is crucial for intestinal cell proliferation during tumorigenesis. Taken together, our data suggest that RNF186 inhibits the development of CRC, and that this effect is mediated through the suppression of NF-κB activity.

Dietary Supplementation of Foxtail Millet Ameliorates Colitis-Associated Colorectal Cancer in Mice via Activation of Gut Receptors and Suppression of the STAT3 Pathway.

Coarse cereal intake has been reported to be associated with reduced risk of colorectal cancer. However, evidence from intervention studies is absent and the molecular basis of this phenomenon remains largely unexplored. This study sought to investigate the effects of foxtail millet and rice, two common staple grains in Asia, on the progression of colitis-associated colorectal cancer (CAC) and define the mechanism involved. In total, 40 BALB/c mice were randomized into four groups. The Normal and azoxymethane/dextran sodium sulfate (AOM/DSS) groups were supplied with an AIN-93G diet, while the millet- and rice-treated groups were supplied with a modified AIN-93G diet. Compared to the AOM/DSS-induced CAC mice supplemented with rice, an increased survival rate, suppressed tumor burden, and reduced disease activity index were observed in the millet-treated group. The levels of IL-6 and IL-17 were decreased in the millet-treated group compared to both the AOM/DSS and AOM/DSS + rice groups. Millet treatment inhibited the phosphorylation of STAT3 and the related signaling proteins involved in cell proliferation, survival and angiogenesis. These beneficial effects were mediated by the activation of gut receptors AHR and GPCRs via the microbial metabolites (indole derivates and short-chain fatty acids) of foxtail millet. Moreover, millet-treatment increased the abundance of Bifidobacterium and Bacteroidales_S24-7 compared to the rice-treated mice. This study could help researchers to develop better dietary patterns that work against inflammatory bowel disease (IBD) and for CAC patients.

17\u03b2-Estradiol supplementation changes gut microbiota diversity in intact and colorectal cancer-induced ICR male mice

The composition of the gut microbiota is influenced by sex hormones and colorectal cancer (CRC). Previously, we reported that 17β-estradiol (E2) inhibits azoxymethane/dextran sulfate sodium (AOM/DSS)-induced tumorigenesis in male mice. Here, we investigated whether the composition of the gut microbiota is different between male and female, and is regulated by estrogen as a secondary outcome of previous studies. We established four groups of mice based on the sex and estrogen status [ovariectomized (OVX) female and E2-treated male]. Additionally, three groups of males were established by treating them with AOM/DSS, and E2, after subjecting them to AOM/DSS treatment. The mice were sacrificed at 21 weeks old. The composition of the gut microbiota was analyzed using 16S rRNA metagenomics sequencing. We observed a significant increase in the microbial diversity (Chao1 index) in females, males supplemented with E2, and males treated with AOM/DSS/E2 compared with normal males. In normal physiological condition, sex difference and E2 treatment did not affect the ratio of Firmicutes/Bacteroidetes (F/B). However, in AOM/DSS-treated male mice, E2 supplementation showed significantly lower level of the F/B ratio. The ratio of commensal bacteria to opportunistic pathogens was higher in females and E2-treated males compared to normal males and females subjected to OVX. Unexpectedly, this ratio was higher in the AOM/DSS group than that determined in other males and the AOM/DSS/E2 group. Our findings suggest that estrogen alters the gut microbiota in ICR (CrljOri:CD1) mice, particularly AOM/DSS-treated males, by decreasing the F/B ratio and changing Shannon and Simpson index by supply of estrogen. This highlights another possibility that estrogen could cause changes in the gut microbiota, thereby reducing the risk of developing CRC.

Dynamic alterations in serum IgG N-glycan profiles in the development of colitis-associated colon Cancer in mouse model

BackgroundAlternative glycosylation of serum IgG has been shown to be closely associated with colorectal cancer (CRC). Currently, a dynamic study which can not only minimize the influence of genetic background, environment and other interfering factors during cancer development, but also focus on investigating carcinogenic characteristics of IgG glycan is lacking. MethodsSerum IgG N-glycans were characterized at four stages of CRC development by ultra-performance liquid chromatography in a typical colitis-related CRC mouse model induced by azoxymethane-dextran sodium sulfate. Furthermore, the expression of related glycosyltransferases in splenic B lymphocytes at the corresponding time was also assessed. ResultsThe relative abundance of seven IgG glycans, which can be classified as monoantennary, core fucose, sialic acid, galactose and bisecting, was changed during tumor growth. The abundance of some glycans was altered during the first stage of cancer induction. Correspondingly, the expression of glycosyltransferases in splenic B lymphocytes and different tissues in cancer groups was also decreased compared to that in controls. ConclusionsThis study represents the comprehensive analysis of IgG glycosylation in the dynamic process of colitis-associated CRC. To our knowledge, this is the first report that the expression of glycosyltransferases in mouse splenic B lymphocytes is consistent or inconsistent with the alterations of IgG N-glycans, and the variation tendency is tissue nonspecific. General SignificanceProviding a novel approach to identify the IgG glycans related to the development of CRC and laying a foundation for research on structure and function of glycans using mouse.

Quercetin Suppresses AOM/DSS-Induced Colon Carcinogenesis through Its Anti-Inflammation Effects in Mice.

Colorectal cancer (CRC) is the fourth leading cause of tumor-related deaths worldwide. In this study, we explored the in vivo effects of quercetin, a plant flavonol from the flavonoid group of polyphenols with antioxidant effects, on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Thirty mice were randomly assigned into three groups: the control group, the AOM/DSS group, and the quercetin+AOM/DSS group. CRC was induced by AOM injection and a solution of 2% DSS in the drinking water. In the AOM/DSS-induced colon cancer mice model, quercetin treatment dramatically reduced the number and size of colon tumors. In addition, quercetin significantly restored the leukocyte counts by decreasing the inflammation caused by AOM/DSS. We also observed that the expression of oxidative stress markers, such as lipid peroxide (LPO), nitric oxide (NO), superoxide dismutase (SOD), glucose-6-phosphate (G6PD), and glutathione (GSH), could be reduced by quercetin, suggesting that the anti-inflammatory function of quercetin comes from its antioxidant effect. Moreover, potential biomarkers were identified with serum metabolite profiling. Increased levels of 2-hydroxybutyrate, 2-aminobutyrate, and 2-oxobutyrate and decreased levels of gentian violet, indole-3-methyl acetate, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl were also found in the AOM/DSS-treated mice. However, quercetin treatment successfully decreased the levels of 2-hydroxybutyrate, 2-aminobutyrate, 2-oxobutyrate, endocannabinoids, and sphinganine and increased the levels of gentian violet, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl. Together, our data demonstrated that quercetin could maintain relatively potent antitumor activities against colorectal cancer in vivo through its anti-inflammation effect.

Caffeic Acid Phenethyl Ester Prevents Colitis-Associated Cancer by Inhibiting NLRP3 Inflammasome.

Long-lasting inflammation in the intestinal tract renders individuals susceptible to colitis-associated cancer (CAC). The NOD-like receptor protein 3 (NLRP3) inflammasome plays a key role in the progression of inflammatory bowel disease and CAC. Therefore, identifying effective drugs that prevent CAC by targeting NLRP3 inflammasome is of great interest. Here, we aimed to evaluate the anti-inflammatory effect of caffeic acid phenethyl ester (CAPE) on bone marrow-derived macrophages (BMDMs), THP-1 cells, and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon cancer mouse model. We also investigated the anti-tumor mechanism of CAPE. We found that CAPE decreased NLRP3 inflammasome activation in BMDMs and THP-1 cells and protected mice from colorectal cancer induced by AOM/DSS. CAPE regulated NLRP3 at the post-transcriptional level by inhibiting reactive oxygen species (ROS) production. However, CAPE did not affect NLRP3 or IL-1β transcription, but instead enhanced NLRP3 binding to ubiquitin molecules, promoting NLRP3 ubiquitination, and contributing to the anti-tumor effect in the AOM/DSS mouse model. Moreover, CAPE suppressed the interaction between NLRP3 and CSN5 but enhanced that between NLRP3 and Cullin1 both in vivo and in vitro. Altogether, our findings demonstrate that CAPE prevents CAC by post-transcriptionally inhibiting NLRP3 inflammasome. Thus, CAPE may be an effective candidate for reducing the risk of CAC in patients with inflammatory bowel disease.

Inhibition of Aurora-A recruited CD8+ T cells infiltration via mediating IL-10 production of cancer cells

Abstract It has been shown that approximately 10% of infiltrated CD8+ T cells could be the tumor-specific intratumoral CD8+ T cells in colorectal cancer. While, inhibition of Aurora-A, a member of serine/threonine Aurora kinase family, by alisertib promoted the percentage of infiltrated intratumoral CD8+ T cells. We therefore assumed that targeting Aurora-A could be a therapeutic strategy for recruitment of intratumoral CD8+ T cells. In CT-26 tumor model, blockade of Aurora-A with alisertib slowed the tumor growth in association with an increased infiltration, activation and expansion of intratumoral CD8+ T cells. We further analyzed the role of Aurora-A in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model, and found that AURKA deficiency significantly decreased tumor formation, in parallel with an increase in infiltration of intratumoral CD8+ T cells. Gene expression profiling indicated that Il10ra expression was obviously increased in intratumoral CD8+ T cells sorted from alisertib-treated tumors. Antibody-mediated blockade of IL-10Ra dramatically attenuated the anti-tumor ability of alisertib in association with reducing the percentage of intratumoral CD8+ T cells. IL-10Ra was the key mediator of IL-10. We herein examined the levels of IL-10 and found that the levels of IL-10 in the serum of AURKA deficiency mice treated with AOM/DSS were elevated. Additionally, IL-10 obviously promoted migration of CD8+ T cells in vitro. Blockade of Aurora-A increased the transcriptional levels of IL-10. In conclusion, inhibition of Aurora-A could be useful for recruiting infiltration of intratumoral CD8+ T cell by mediating IL-10 production.

Gadolinium-Loaded Solid Lipid Nanoparticles for Colorectal Tumor in MR Colonography.

The current study aims to investigate the possibility of using solid lipid nanoparticles (SLNs)-enhanced magnetic resonance (MR) colonography to diagnose colorectal cancer. Gd-FITC-SLNs were synthesized by loading gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and fluorescein isothiocyanate (FITC) simultaneously. Twenty mice received azoxymethane/dextran sulfate sodium (AOM/DSS) to induce adenocarcinoma of the colon and were divided into 4 groups, and 5 in per group. MR colonography were performed at different time periods before and after enema or intravenous injection of Gd-FITC-SLNs or Gd-DTPA. The results demonstrated SNR (signal-to-noise ratio) significantly increased from 1.56- to 1.76-fold within the colorectal tumors after the enema of Gd-FITC-SLNs (p < 0.001). No differences in SNR were observed after the enema of Gd-DTPA (p > 0.05). Besides, SNR increased from 1.54- to 1.72-fold within the colorectal tumors after the intravenous injection of Gd-FITC-SLNs (p < 0.001) while SNR increased from 1.39to 1.57-fold within the colorectal tumors after the injection of Gd-DTPA (p < 0.001). In addition, SNR within colorectal tumors significantly increased ranging from 20th to 140th min, and lasted for about 120 min (p < 0.05) after the enema of Gd-FITC-SLNs and SNR within colorectal tumors also significantly increased ranging from 0th hour to 8th hour, lasted for about 8 hour (p < 0.05) after the injection of Gd-FITC-SLNs. However, after the injection of Gd-DTPA, SNR within colorectal tumors significantly increased only ranging from 0th min to 20th min after administration (p < 0.01). Furthermore, hematoxylin and eosin (H&E) staining revealed that all mice developed adenocarcinoma of the colon. In summary, it is feasible by using Gd-FITC-SLNs in MR colonography to diagnose colorectal cancer. Enema of Gd-FITC-SLNs can provide marked enhancement of colorectal tumors quickly, and safer while intravenous injection of Gd-FITC-SLNs can provide a long-lasting enhancement of colorectal tumors in MR colonography. These findings present a potential clinical application of Gd-FITC-SLNs on MR colonography.

The active fraction of Garcinia yunnanensis suppresses the progression of colorectal carcinoma by interfering with tumorassociated macrophage-associated M2 macrophage polarization in vivo and in vitro.

Colorectal cancer (CRC) is the third most common solid tumor worldwide and has shown resistance to several immunotherapies, particularly immune checkpoint blockade therapy, which is effective in many other types of cancer. Our previous studies indicated that the active fraction of Garcinia yunnanensis (YTE-17), had potent anticancer activities by regulating multiple signaling pathways. However, knowledge regarding the mechanism and effect of YTE-17 in the prevention of CRC is limited. This study tested the effects of YTE-17 on colon cancer development in vivo by using two murine models: the carcigenic azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC model and a genetically induced model using ApcMin/+ mice. Here, the tumor load, tumor number, histology, and even some oncogenes were used to evaluate the effect of YTE-17. The intragastric administration of YTE-17 for 12weeks significantly decreased CRC incidence, tumor number and size, immunity, and some tumor-associated macrophage (TAM) markers, including CD206, Arg-1, IL-10, and TGF-β. Importantly, the macrophages depletion by clodronate (CEL) also played a role in reducing the tumor burden and inhibiting tumor development, which were not affected by YTE-17 in the ApcMin/+ mice. Moreover, the YTE-17 treatment attenuated CRC cell growth in a co-culture system in the presence of macrophages. Consistently, YTE-17 effectively reduced the tumor burden and macrophage infiltration and enhanced immunity in the AOM/DSS and ApcMin/+ colon tumor models. Altogether, we demonstrate that macrophages in the microenvironment may contribute to the development and progression of CRC cells and propose YTE-17 as a new potential drug option for the treatment of CRC.

NUDT7 Loss Promotes KrasG12D CRC Development

Studies have suggested that dysregulation of peroxisomal lipid metabolism might play an important role in colorectal cancer (CRC) development. Here, we found that KrasG12D-driven CRC tumors demonstrate dysfunctional peroxisomal β-oxidation and identified Nudt7 (peroxisomal coenzyme A diphosphatase NUDT7) as one of responsible peroxisomal genes. In KrasG12D-driven CRC tumors, the expression level of Nudt7 was significantly decreased. Treatment of azoxymethane/dextran sulfate sodium (AOM/DSS) into Nudt7 knockout (Nudt7−/−) mice significantly induced lipid accumulation and the expression levels of CRC-related genes whereas xenografting of Nudt7-overexpressed LS-174T cells into mice significantly reduced lipid accumulation and the expression levels of CRC-related genes. Ingenuity pathway analysis of microarray using the colon of Nudt7−/− and Nudt7+/+ mice treated with AOM/DSS suggested Wnt signaling as one of activated signaling pathways in Nudt7−/− colons. Upregulated levels of β-catenin were observed in the colons of KrasG12D and AOM/DSS-treated Nudt7−/− mice and downstream targets of β-catenin such as Myc, Ccdn1, and Nos2, were also significantly increased in the colon of Nudt7−/− mice. We observed an increased level of palmitic acid in the colon of Nudt7−/− mice and attachment of palmitic acid-conjugated chitosan patch into the colon of mice induced the expression levels of β-catenin and CRC-related genes. Overall, our data reveal a novel role for peroxisomal NUDT7 in KrasG12D-driven CRC development.

Chemopreventive Effect of the Germinated Oat and its Phenolic-AVA Extract in Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) Model of Colon Carcinogenesis in Mice.

The consumption of fruits, vegetables, nuts, legumes, and whole grains has been associated with a lower risk of colorectal cancer (CRC) due to the content of natural compounds with antioxidant and anticancer activities. The oat (Avena sativa L.) is a unique source of avenanthramides (AVAs), among other compounds, with chemopreventive effects. In addition, oat germination has shown enhanced nutraceutical and phytochemical properties. Therefore, our objective was to evaluate the chemopreventive effect of the sprouted oat (SO) and its phenolic-AVA extract (AVA) in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced CRC mouse model. Turquesa oat seeds were germinated (five days at 25 °C and 60% relative humidity) and, after 16 weeks of administration, animals in the SO- and AVA-treated groups had a significantly lower inflammation grade and tumor (38–50%) and adenocarcinoma (38–63%) incidence compared to those of the AOM+DSS group (80%). Although both treatments normalized colonic GST and NQO1 activities as well as erythrocyte GSH levels, and significantly reduced cecal and colonic β-GA, thus indicating an improvement in the intestinal parameters, the inflammatory states, and the redox states of the animals, SO exerted a superior chemopreventive effect, probably due to the synergistic effects of multiple compounds. Our results indicate that oats retain their biological properties even after the germination process.

Exopolysaccharides isolated from Rhizopus nigricans induced colon cancer cell apoptosis in vitro and in vivo via activating the AMPK pathway.

Colorectal cancer (CRC) is a leading cause of cancer-related human deaths. The exopolysaccharide (EPS1-1), isolated from Rhizopus nigricans, has been described as exhibiting anti-tumor and pro-apoptotic activity against CRC, although the underlying mechanism is poorly understood. Herein, we investigate how EPS1-1 induces apoptosis of CRC cells in vitro and in vivo. Our results show that, in vitro, EPS1-1 suppressed cell growth and facilitated apoptosis in a dose- and time-dependent manner by activating the AMP-activated protein kinase (AMPK) pathway in mouse colon cancer CT26 cells. However, treatment with small interfering RNAs (siRNAs) targeting AMPKα or with compound C, an AMPK inhibitor, interfered with the pro-apoptosis effects of EPS1-1. We also show that EPS1-1 initiated the release of reactive oxygen species (ROS) and liver kinase B1 (LKB1), both of which are necessary signals for AMPK activation. Furthermore, EPS1-1-mediated apoptosis is regulated by inactivation of mammalian target of rapamycin complex 1 (mTORC1) and activation of the jun-NH2 kinase (JNK)-p53 signaling axis dependent on AMPK activation. In vivo, azoxymethane/dextran sulfate sodium (AOM/DSS)-treated CRC mice, when administered EPS1-1, exhibited activation of the AMPK pathway, inhibition of mTORC1, and accumulation of p53 in tumor tissues. Collectively, these findings suggest that EPS1-1-induced apoptosis relies on the activation of the AMPK pathway. The present study provides evidence suggesting that EPS1-1 may be an effective target for development of novel CRC therapeutic agents.

Continuity of Tumor Microenvironmental Suppression in AOM/DSS Mice by Fucoxanthin May Be Able to Track With Salivary Glycine.

Fucoxanthin (Fx) is a potent anticancer carotenoid, demonstrated by mouse cancer models. We recently showed the decrease of salivary glycine could represent an attenuation of tumor microenvironment (TME) formation in an azoxymethane/dextran sodium sulfate (AOM/DSS) colon cancer mouse model. However, it remains unclear whether the salivary glycine is an indicator for continuous TME suppression of Fx in the mice. In the present study, we time-dependently analyzed salivary metabolites in AOM/DSS mice, and investigated candidate markers to evaluate the continuous inhibition of colonic TME formation and carcinogenesis in the mice with and without Fx. Fx attenuated the incidence and/or multiplicity of colonic lesions developed in AOM/DSS mice. The number of apoptosis-like cleaved caspase-3high cells was significantly increased, and colonic cancer stem cell-like CD44high/EpCAMhigh cells and cancer-associated fibroblast-like αSMAhigh cells were significantly decreased in colon mucosal tissue by Fx administration. Salivary glycine at 4, 11 and 14 weeks after the final DSS exposure in the Fx-treated mice showed successful and consecutive decreases of 0.5-, 0.4- and 0.7-fold respectively compared to that of control mice. Salivary glycine is a valuable indicator that could evaluate sustained efficacy of cancer chemopreventive effects of Fx in AOM/DSS mice.

Mucoadhesive-to-penetrating controllable peptosomes-in-microspheres co-loaded with anti-miR-31 oligonucleotide and Curcumin for targeted colorectal cancer therapy.

Background: Accumulating evidences indicate that nanomedicines greatly decrease the side effects and enhance the efficacy of colorectal cancer (CRC) treatment. In particular, the use of rectal delivery of nanomedicines, with advantages such as fast therapeutic effects and a diminishing hepatic first-pass effect, is currently emerging.Method: We established a CRC targeted delivery system, in which α-lactalbumin peptosomes (PSs) co-loaded with a microRNA (miR)-31 inhibitor (miR-31i) and curcumin (Cur) were encapsuslated in thiolated TEMPO oxidized Konjac glucomannan (sOKGM) microspheres, referred as sOKGM-PS-miR-31i/Cur. The CRC targeting capability, drug release profiles, mucoadhesive-to-penetrating properties and therapeutic efficacy of sOKGM-PS-miR-31i/Cur delivery system were evaluated in colorectal cancer cells and azoxymethane-dextran sodium (AOM-DSS) induced tumor models.Results: sOKGM-PS-miR-31i/Cur delivery system were stable in the harsh gastrointestinal environment after rectal or oral administration; and were also mucoadhesive due to disulfide bond interactions with the colonic mucus layer, resulting in an enhanced drug retention and local bioavailability in the colon. Concomitantly, the released PS-miR-31i/Cur PSs from the microsphere was mucus-penetrating, efficiently passing through the colonic mucus layer, and allowed Cur and miR-31i specifically target to colon tumor cells with the guide of CD133 targeting peptides. Consequently, rectal delivery of sOKGM-PS-miR-31i/Cur microspheres suppressed tumor growth in an azoxymethane-dextran sodium sulfate (AOM-DSS)-induced tumor model.Conclusion: sOKGM-PS-miR-31i/Cur microspheres are effective rectal delivery system with combined advantages of mucoadhesive and mucus-penetrating properties, representing a potent and viable therapeutic approach for CRC.

Selective targeting of the TLR4 co-receptor, MD2, prevents colon cancer growth and lung metastasis.

Toll-like receptor (TLR) signaling is an emerging pathway in tumor cell invasion and metastasis. Myeloid differentiation protein-2 (MD2) contributes to ligand recognition and activation of TLRs in response to exogenous microbial insults or endogenous agents. We hypothesized that blocking MD2 using a specific inhibitor would prevent TLR4-mediated inflammatory responses and metastatic cancer growth. Here, we report that a MD2 inhibitor, L6H21, inhibited migration and invasion of LPS-activated colon cancer CT26.WT cells. These activities were accompanied by inhibition of nuclear factor-κB (NF-κB) activation, and thereby inhibition of the production of pro-inflammatory cytokines and adhesive molecules in colon cancer cells. Furthermore, L6H21 inhibited CT26.WT metastasis to the lung in BALB/c mice as well as suppressed colitis-induced colon cancer induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Taken together, our results demonstrated that L6H21 suppressed tumor invasion and metastasis through blocking TLR4-MD2/NF-κB signaling axis. These findings reveal that inhibition of MD2 may be an important target for the development of colon cancer therapies.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Azoxymethane-dextran sulfate sodium induces colorectal cacner mouse model

Colorectal cancer is one of the most common malignancies around the world that seriously affects human life and health. Most studies used cell line experiments to investigate the characteristics of colorectal cancer, but the foundation of colorectalcancer (CRC) in situ mouse model would better imitate the occurrence the process of CRC, and improve the credibility of basic experiments greatly. In the present, azoxymethane/dextran sulfate sodium (AOM/DSS)-induced cancer model is widely use in the building of CRC animal model, but there are quite differences exist in the quantities of research. As a result, this review will concisely introduce the process and assessment of the AOM/DSS-induced mouse CRC model in order to provide a comprehensive reference for the one does this ailment model. Key words: Azoxymethane/dextran sulfate sodium; Inflammation-induced cancer; Model, mouse; Assessment of model

Hyaluronic Acid Binding to TLR4 Promotes Proliferation and Blocks Apoptosis in Colon Cancer.

Hyaluronic acid (HA), a constituent of the extracellular matrix, promotes colorectal cancer growth. CD44 is a relevant HA receptor in this context. However, HA is also a ligand for TLR4, a receptor of significance in colorectal cancer. In this study, we examine the relative contribution of HA interactions with CD44 and TLR4 in colon tumorigenesis. Colorectal cancer models included ApcMin/+ mice, azoxymethane/dextran sodium sulfate (AOM-DSS), and CT26 tumor isografts. We used knockout mice and CT26 colorectal cancer cells with CRISPR knockdown of CD44 and TLR4. HA activity was modulated by PEP1 (a 12-mer peptide that blocks HA from binding its receptors), hyaluronidase (which promotes HA degradation), or 4-MU (HA synthesis inhibitor). Blockade of HA binding via PEP1 decreased growth in all colorectal cancer models and in cell culture. The effects were significant in WT and with CD44 deletion, but not with TLR4 deletion. In the AOM-DSS model, mice deficient in CD44 or TLR4 had fewer tumors. CD44- and TLR4-deficient CT26 isografts grew more slowly, exhibiting decreased tumor cell proliferation and increased apoptosis. In vitro, endogenous HA blocked LPS binding to TLR4 suggesting that HA is a relevant TLR4 ligand in colon cancer. Finally, PEP1 enhanced tumor radiation sensitivity in the isograft model. Together, these results indicate that HA binding to TLR4, as well as CD44, plays a key role in colon tumorigenesis. These findings also raise the possibility that an agent that blocks HA binding, such as PEP1, may be useful as an adjuvant therapy in colon cancer.

Protective Role of Fecal Microbiota Transplantation on Colitis and Colitis-Associated Colon Cancer in Mice Is Associated With Treg Cells.

Colitis-associated cancer (CAC) is the most serious outcome of inflammatory bowel disease, which has an alteration of commensal intestinal microbiota. However, the role of intestinal microbiota on CAC progression is not well-understood. Fecal microbiota transplantation (FMT) was used for treating murine azoxymethane–dextran sodium sulfate (AOM-DSS) model of CAC. Composition of gut microbiota during FMT treatment was analyzed. RT-PCR and ELISA were used to detect the inflammatory factors, and immunofluorescence was applied to examine the phospho-nuclear factor (NF)-κB p65/p100 and Ki67-positive cells in the colons. In addition, flow cytometry was performed to analyze the immune cell after FMT treatment. Rehabilitation of the intestinal microbiota by FMT restored both the ratio and diversity of microbiota during CAC progression. Remarkably, a favorable morphometric outcome characterized by decreased tumor load and size was observed in CAC mice with FMT treatment. In addition, an anti-inflammatory function of FMT was demonstrated by decreasing pro-inflammatory factors but increasing anti-inflammatory factors through inhibiting canonical NF-κB activity and cellular proliferation in colons of CAC mice. The expression of CD4+CD25+Foxp3+ regulatory T cells (Tregs) was significantly increased after FMT treatment in CAC mice, but not T helper (Th)1/2/17 cells. Our study aids in the understanding of CAC pathogenesis and reveals a previously unrecognized role for FMT in the treatment of CAC through restoring the intestinal microbiota and inducing regulatory T cells.

Antitumor properties of triptolide: phenotype regulation of macrophage differentiation

ABSTRACTTumor-associated macrophages (TAMs), which generally exhibit an M2-like phenotype, play a critical role in tumor development. Triptolide exerts a unique bioactive spectrum of anticancer activities. The aim of this study was to determine whether triptolide has any effect on the activation of TAMs and the production of tumor-promoting mediators. ICR-1 mice with azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumors and BALB/c mice co-inoculated with 4T1 cells and M2-polarized RAW264.7 cells were used to examine whether the inhibitory effect of triptolide on tumor progression was mediated by the targeting of TAMs. Real-time PCR, Western blot, immunofluorescence staining, and flow cytometry assays were performed to determine the expression of cell surface markers and cytokine production. The results showed that triptolide inhibited macrophage differentiation toward the M2 phenotype and abolished M2 macrophage-mediated tumor progression. Furthermore, triptolide inhibited the expression of M2 markers, such as CD206, Arginase 1, and CD204, and inhibited the secretion of anti-inflammatory cytokines. Thus our study indicated that triptolide selectively inhibited the functions of M2-polarized macrophages and TAMs, and this inhibitory effect of triptolide on TAM viability, differentiation, and cytokine production might elucidate the major mechanisms underlying its antitumor activity. Our findings provide important information for the potential clinical application of triptolide in cancer therapy.