Anchang Yuyang Decoction inhibits experimental colitis-related carcinogenesis by regulating PPAR signaling pathway and affecting metabolic homeostasis of host and microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammatory bowel disease (IBD) presents a risk of carcinogenesis, which escalates with the duration of IBD. Persistent histological inflammation is considered to be the driving factor of colitis carcinogenesis. Effective control of inflammation is helpful to prevent and treat colitis-related colorectal cancer (CAC). Anchang Yuyang Decoction (AYD), a traditional Chinese medicine (TCM) formula, is originated from the ancient prescription of TCM for treating colitis and colorectal cancer. AYD has demonstrated efficacy in treating IBD and potential anti-carcinogenic properties. AIM OF THE STUDY: This research aims to assess the therapeutic efficacy of AYD in ameliorating experimental colitis-related carcinogenesis induced by AOM/DSS. It further seeks to elucidate its potential mechanisms by integrating multiple omics sequencing approaches. MATERIALS AND METHODS: A rat model for colitis-related carcinogenesis was developed using azoxymethane (AOM)/dextran sulfate sodium (DSS). UPLC-MS identified AYD's chemical constituents. Rats were administered varying doses of AYD (18.37, 9.19 and 4.59 g/kg) orally for 53 days, with mesalazine as a positive control. The study evaluated anti-carcinogenic effects by examining adenoma number, adenoma load, abnormal crypt foci (ACF), histopathological damage, and tumor-related protein expression. Anti-inflammatory and reparative effects were assessed through body weight, disease activity index (DAI), colon length, spleen index, inflammatory cytokine levels, and tight junction protein expression. The effects on intestinal microbiota and host metabolism were explored through 16S rRNA sequencing, targeted short-chain fatty acid (SCFA) metabonomics, and non-targeted colon metabolomics. Potential AYD targets were identified through transcriptomic sequencing and validated by qRT-PCR and western blotting. RESULTS: AYD significantly reduced adenoma number, adenoma load, neoplasm-associated lesions, ACF, and tumor-related protein expression (e.g., p53, PCNA) in AOM/DSS-induced rats, thus impeding colitis-related carcinogenesis progression. AYD also alleviated histopathological damage and inflammation, promoting intestinal mucosal barrier repair. Furthermore, AYD modulated intestinal flora structure, enhanced SCFA production, and regulated colon metabolites. Transcriptomic sequencing revealed a significant impact on the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Subsequent qRT-PCR and western blotting experiments indicated AYD's influence in up-regulating PPAR-γ and down-regulating PPAR-α, PPAR-ß/δ, and related proteins (thrombomodulin [Thbd], fatty acid binding protein 5 [Fabp5], stearoyl-CoA desaturase 2 [Scd2], phospholipid transfer protein [Pltp]). CONCLUSIONS: This study demonstrates AYD's ability to inhibit experimental colitis-related carcinogenesis induced by AOM/DSS. Its mechanism likely involves modulation of the PPAR signaling pathway, impacting intestinal microbiota and host metabolic equilibrium.

Roseburia intestinalis sensitizes colorectal cancer to radiotherapy through the butyrate/OR51E1/RALB axis.

The radioresistant signature of colorectal cancer (CRC) hampers the clinical utility of radiotherapy. Here, we find that fecal microbiota transplantation (FMT) potentiates the tumoricidal effects of radiation and degrades the intertwined adverse events in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC mice. FMT cumulates Roseburia intestinalis (R. intestinalis) in the gastrointestinal tract. Oral gavage of R. intestinalis assembles at the CRC site and synthetizes butyrate, sensitizing CRC to radiation and alleviating intestinal toxicity in primary and CRC hepatic metastasis mouse models. R. intestinalis-derived butyrate activates OR51E1, a G-protein-coupled receptor overexpressing in patients with rectal cancer, facilitating radiogenic autophagy in CRC cells. OR51E1 shows a positive correlation with RALB in clinical rectal cancer tissues and CRC mouse model. Blockage of OR51E1/RALB signaling restrains butyrate-elicited autophagy in irradiated CRC cells. Our findings highlight that the gut commensal bacteria R. intestinalis motivates radiation-induced autophagy to accelerate CRC cell death through the butyrate/OR51E1/RALB axis and provide a promising radiosensitizer for CRC in a pre-clinical setting.

Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) Model of Colorectal Cancer.

Recent progress in developing new vaccination strategies against cancer requires the production of complex and reliable animal models reflecting the complexity of the tumors with their microenvironment. Mice can be considered a good source due to low cost and ease of being genetically modified, inoculated with tumor cell lines or treated by chemicals to induce different cancers. Despite significant limitations in modeling human cancer complexity, preclinical trials conducted in mice can efficiently contribute to understand molecular mechanisms of cancer, to closely resemble and follow carcinogenesis steps impossible to study into humans, and to test new anticancer therapies. In this chapter, we generally describe the different mouse models developed for cancer vaccines' preclinical trials. A particular focus is dedicated to a chemically-induced colorectal cancer model in use in our laboratories.

Mangiferin (mango) attenuates AOM-induced colorectal cancer in rat's colon by augmentation of apoptotic proteins and antioxidant mechanisms.

Mangiferin (MF) is a natural C-glucosylxantone compound that has many substantial curative potentials against numerous illnesses including cancers. The present study's goal is to appraise the chemo preventive possessions of MF on azoxymethane (AOM)-mediated colonic aberrant crypt foci (ACF) in rats. Rats clustered into 5 groups, negative control (A), inoculated subcutaneously with normal saline twice and nourished on 0.5% CMC; groups B-E injected twice with 15 mg/kg azoxymethane followed by ingestion of 0.5% CMC (B, cancer control); intraperitoneal inoculation of 35 mg/kg 5-fluorouracil (C, reference rats) or nourished on 30 mg/kg (D) and 60 mg/kg (E) of MF. Results of gross morphology of colorectal specimens showed significantly lower total colonic ACF incidence in MF-treated rats than that of cancer controls. The colon tissue examination of cancer control rats showed increased ACF availability with bizarrely elongated nuclei, stratified cells, and higher depletion of the submucosal glands compared to MF-treated rats. Mangiferin treatment caused increased regulation of pro-apoptotic (increased Bax) proteins and reduced the ß-catenin) proteins expression. Moreover, rats fed on MF had significantly higher glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and lower malondialdehyde (MDA) concentrations in their colonic tissue homogenates. Mangiferin supplementation significantly down-shifted pro-inflammatory cytokines (transforming growth factor-α and interleukine-6) and up-shifted anti-inflammatory cytokines (interleukine-10) based on serum analysis. The chemo-protective mechanistic of MF against AOM-induced ACF, shown by lower ACF values and colon tissue penetration, could be correlated with its positive modulation of apoptotic cascade, antioxidant enzymes, and inflammatory cytokines originating from AOM oxidative stress insults.

The role of bidirectional communication between the adipokines and the endogenous opioid system in an experimental mouse model of colitis-associated colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of death globally. Multiple factors may contribute to the pathogenesis of CRC, including the abnormalities in the functioning of the endogenous opioid system (EOS) or adiponectin-related signaling. The aim of our study was to evaluate if differences in the expression of opioid receptors (ORs) influence the development of CRC and if modulation of adiponectin receptors using AdipoRon, a selective AdipoR1 receptor agonist, affects colorectal carcinogenesis. METHODS: Naltrexone, an opioid receptor antagonist, was injected intraperitoneally every second day for 2 weeks, at the dose of 1 mg/kg in healthy Balb/C mice to induce changes in ORs expression. CRC was induced by a single intraperitoneal injection of azoxymethane (AOM) and the addition of dextran sodium sulfate (DSS) into drinking water in three-week cycles. The development of CRC was assessed using macro- and microscopic scoring and molecular analysis (RT qPCR, ELISA) after 14 weeks. RESULTS: Naltrexone significantly increased the mRNA expression of Oprm1, Oprd1, and Oprk1 in the mouse colon and in the brain (non-significantly). The pretreatment of mice with naltrexone aggravated the course of CRC (as indicated by tumor area, colon thickness, and spleen weight). The level of circulatory adiponectin was lowered in mice with CRC and increased in the colon as compared with healthy mice. The ß-endorphin level was increased in the plasma of mice with CRC and decreased in the colon as compared to healthy mice. AdipoRon, AdipoR1 agonist, worsened the CRC development, and pretreatment with naltrexone enhanced this negative effect in mice. CRC did not affect the expression of the Adipor1 gene, but the Adipor1 level was increased in mice pretreated with naltrexone (AOM/DSS and healthy mice). AdipoRon did not influence the expression of opioid receptors at the mRNA level in the colon of mice with CRC. The mRNA expression of Ptgs2, Il6, Nos2, Il1b, Il18, Gsdmd, and Rela was increased in mice with CRC as compared to the healthy colon. AdipoRon significantly decreased mRNA expression of Ptgs2, Il6, Il1b, and Il18 as compared to CRC mice. CONCLUSION: EOS and adiponectin-related signaling may play a role in the pathogenesis of CRC and these systems may present some additivity during carcinogenesis.

Ephrin B3 exacerbates colitis and colitis-associated colorectal cancer.

Ephrin B3, a member of Eph/ephrin family, contributes to embryogenesis and carcinogenesis, but few studies have suggested whether this ligand has regulatory effect on colitis. This study was to determine whether ephrin B3 played a role in colitis and colonic carcinogenesis. Dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated carcinogenesis model was established in Efnb3-deficient (Efnb3-/-) mice. Label-free quantitative proteomics were performed to identify the Efnb3-regulated proteins. Our results showed that Efnb3 knock out reduced the symptoms of DSS-induced colitis, such as disease activity index (DAI), inflammatory factors release, and dysfunction of the intestinal barrier. Quantitative proteomics revealed that Efnb3 regulated 95 proteins which clustered in the platelet degranulation, response to elevated platelet cytosolic Ca2+, MAPK signaling for integrins such as ITGB4. Furthermore, ephrin B3 inactived ITGB4/AKT signal pathway and then promoted epithelial barrier dysfunction. Simultaneously, ephrin B3 promoted Gremlin-1/NF-κB signal pathway and thereby increased inflammatory factors release. In addition, the higher level of Efnb3 in colon cancer patients is correlated with worse survival. Efnb3-/- mice exhibited susceptibility to AOM/DSS-induced colorectal cancer. Our finding discovered that Efnb3 played an important role in the development of colitis and colitis-associated colorectal cancer. Efnb3 deficiency improved the intestinal barrier by ITGB4 and suppressed inflammation via Gremlin-1/NF-κB signal pathway, which may provide a novel therapeutic strategy for the treatment of colitis and colitis-associated colorectal cancer.

Gliclazide Reduces Colitis-Associated Colorectal Cancer Formation by Deceasing Colonic Inflammation and Regulating AMPK-NF-κB Signaling Pathway.

BACKGROUND: Gliclazide is a potential anti-cancer drug candidate for preventing carcinogenesis. However, the effect of gliclazide on colitis-associated colorectal cancer remains unknown. AIMS: We aimed to evaluate whether gliclazide plays a protective role in colitis-associated colorectal cancer and the underlying molecular mechanism. METHODS: The administration of azoxymethane (AOM) followed by dextran sulfate sodium (DSS) aimed to induce colitis-associated colorectal cancer in mice. C57BL mice were gavaged with gliclazide (6 mg/kg by gavage 5 days a week) for 12 weeks immediately following AOM administration. After sacrificing the mice, colon tissues were measured for tumor number and tumor burden. The proliferation- and inflammation-related molecular mechanisms were explored. RESULTS: The administration of gliclazide significantly reduced the tumor number and tumor burden in mice. Cell proliferation decreased in the gliclazide group compared with the control group, as indicated by reduced Ki-67 expression. Furthermore, gliclazide alleviated colonic inflammation, significantly decreased pro-inflammatory factor TNF-α levels and increased anti-inflammatory factor IL-10 levels in vivo. In vivo and vitro, it was shown that gliclazide increased the level of phospho-AMPK (p-AMPK) and inhibited NF-κB activity. Further studies demonstrated that the inhibition of NF-κB activity induced by gliclazide was mediated by p-AMPK in vitro. CONCLUSIONS: Gliclazide effectively alleviated colonic inflammation and prevented colonic carcinogenesis in an AOM-DSS mouse model by modulating the AMPK-NF-κB signaling pathway. Thus, gliclazide holds potential as a chemopreventive agent for colitis-associated colorectal cancer.

Anise (Pimpinella anisum L.) attenuates azoxymethane-induced colorectal cancer by antioxidant, anti-inflammatory, and anti-apoptotic pathways in rats.

Herbal medicine is one of the most common fields explored for combating colon cancers, and Pimpinella anisum L. seeds (PAS) have been utilized widely as medicinal agents because of their increased essential oil (trans-anethole) contents. In this essence, our study investigates the toxic effect and chemoprotective potentials of PAS against azoxymethane (AOM)-induced colon cancer in rats. The toxicity trial for PAS conducted by clustering fifteen rats into three groups (five rats each): A, normal control had 10% Tween 20; B, ingested with 2 g/kg PAS; and C, supplemented with 4 g/kg PAS. The in vivo cancer trial was performed by using 30 rats (Sprague-Dawley) that were randomly adapted in five steel cages (six rats each): group A, normal controls received two subcutaneous injections of normal saline 0.09% and ingested orally 10% Tween 20; groups B-E, rats received two injections of 15 mg/kg of azoxymethane (AOM) subcutaneously in 2 weeks and treated orally with 10% Tween 20 (group B) or intraperitoneal injection of 5-fluorouracil (35 mg/kg) (group C), or orally given 200 mg/kg PAS (group D) and 400 mg/kg PAS (group E) for 8 weeks. After the scarification of rats, the colon tissues were dissected for gross and histopathological evaluations. The acute toxicity trial showed the absence of any toxic signs in rats even after 14 days of ingesting 4 g/kg of PAS. The chemoprotective experiment revealed significant inhibitory potentials (65.93%) of PAS (400 mg/kg) against aberrant crypto foci incidence that could be correlated with its positive modulation of the immunohistochemically proteins represented by a significant up-regulation of the Bax protein and a decrease of the Bcl-2 protein expressions in colon tissues. Furthermore, PAS-treated rats had notably lower oxidative stress in colon tissues evidenced by decreased MDA levels and increased antiradical defense enzymes (SOD, CAT, and GPx). The outcomes suggest 400 mg/kg PAS as a viable additive for the development of potential pharmaceuticals against colorectal cancer.

Klebsiella aerogenes exacerbates colon tumorigenesis in the AOM/DSS-induced C57BL/6J mouse.

Klebsiella aerogenes (K. aerogenes, KA) is a gram-negative opportunistic pathogen from the Klebsiella species and the Enterobacteriaceae family. However, the impact of K. aerogenes on colorectal cancer (CRC) remains uncertain. A colitis-associated tumorigenesis animal model was established by administering azoxymethane (AOM) and dextran sulfate sodium (DSS) to C57BL/6J mice. The concentration of K. aerogenes gavage in mice was 109 cfu. The study measured the following parameters: tumor formation (number and size), intestinal permeability (MUC2, ZO-1, and Occludin), colonic inflammation (TNF-α, IL-1ß, IL-6, and IL-10), proliferation and the fluctuation of the intestinal flora. Under the AOM/DSS-treated setting, K. aerogenes colonization worsened colitis by exacerbating intestinal inflammatory reaction and destroying the mucosal barrier. The intervention markedly augmented the quantity and dimensions of neoplasm in the AOM/DSS mice, stimulated cellular growth, and impeded cellular programmed cell death. In addition, K. aerogenes exacerbated the imbalance of the intestinal microbiota by elevating the abundance of Pseudomonas, Erysipelatoclostridium, Turicibacter, Rikenella, and Muribaculum and leading to a reduction in the abundance of Odoribacter, Alloprevotella, Roseburia, and Lachnospiraceae_NK4A136_group. The presence of K. aerogenes in AOM/DSS-treated mice promoted tumorigenesis, worsened intestinal inflammation, disrupted the intestinal barrier, and caused disturbance to the gut microbiota.

Murine models of colorectal cancer: the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colitis-associated cancer.

Background: Colorectal cancer (CRC) is the third most common cancer. It is a heterogeneous disease, including both hereditary and sporadic types of tumors. CRC results from complex interactions between various genetic and environmental factors. Inflammatory bowel disease is an important risk factor for developing CRC. Despite growing understanding of the CRC biology, preclinical models are still needed to investigate the etiology and pathogenesis of the disease, as well as to find new methods of treatment and prevention. Objectives: The purpose of this review is to describe existing murine models of CRC with a focus on the models of colitis-associated CRC. This manuscript could be relevant for experimental biologists and oncologists. Methodology: We checked PubMed and Google from 01/2018 to 05/2023 for reviews of CRC models. In addition, we searched PubMed from 01/2022 to 01/2023 for articles using the azoxymethane (AOM)/dextran sulfate sodium (DSS) CRC model. Results: Existing murine models of CRC include spontaneous, genetically engineered, transplantation, and chemically induced models. For the study of colitis-associated cancer (CAC), the AOM/DSS model is predominantly used. This model is very similar in histological and molecular characteristics to the human CAC, and is highly reproducible, inexpensive, and easy to use. Despite its popularity, the AOM/DSS model is not standardized, which makes it difficult to analyze and compare data from different studies. Conclusions: Each model demonstrates particular advantages and disadvantages, and allows to reproduce different subtypes or aspects of the pathogenesis of CRC.

Fecal microbiota transplanted from old mice promotes more colonic inflammation, proliferation, and tumor formation in azoxymethane-treated A/J mice than microbiota originating from young mice.

Aging is a strong risk factor for colorectal cancer (CRC). It is well established that gut microbial dysbiosis can play a role in the etiology of CRC. Although the composition of the gut microbial community changes with age and is reported to become more pro-inflammatory, it is unclear whether such changes are also pro-tumorigenic for the colon. To address this gap, we conducted fecal microbiota transplants (FMT) from young (DY, ~6 wk) and old (DO, ~72 wk) donor mice into young (8 wk) recipient mice that were pre-treated with antibiotics. After initiating tumorigenesis with azoxymethane, recipients were maintained for 19 wk during which time they received monthly FMT boosters. Compared to recipients of young donors (RY), recipients of old donors (RO) had an approximately 3-fold higher prevalence of histologically confirmed colon tumors (15.8 vs 50%, Chi2 P = .03), approximately 2-fold higher proliferating colonocytes as well as significantly elevated colonic IL-6, IL-1ß and Tnf-α. Transcriptomics analysis of the colonic mucosa revealed a striking upregulation of mitochondria-related genes in the RO mice, a finding corroborated by increased mitochondrial abundance. Amongst the differences in fecal microbiome observed between DY and DO mice, the genera Ruminoclostridium, Lachnoclostridium and Marvinbryantia were more abundant in DY mice while the genera Bacteroides and Akkermansia were more abundant in DO mice. Amongst recipients, Ruminoclostridium and Lachnoclostridium were higher in RY mice while Bacteroides was higher in RO mice. Differences in fecal microbiota were observed between young and old mice, some of which persisted upon transplant into recipient mice. Recipients of old donors displayed significantly higher colonic proliferation, inflammation and tumor abundance compared to recipients of young donors. These findings support an etiological role for altered gut microbial communities in the increased risk for CRC with increasing age and establishes that such risk can be transmitted between individuals.

A Novel Mouse Model of Intrahepatic Cholangiocarcinoma Induced by Azoxymethane.

Cholangiocarcinoma is the second most common primary cancer of the liver and has a poor prognosis. Various animal models, including carcinogen-induced and genetically engineered rodent models, have been established to clarify the mechanisms underlying cholangiocarcinoma development. In the present study, we developed a novel mouse model of malignant lesions in the biliary ducts induced by the administration of the carcinogen azoxymethane to obese C57BLKS/J-db/db mice. A histopathological analysis revealed that the biliary tract lesions in the liver appeared to be an intrahepatic cholangiocarcinoma with higher tumor incidence, shorter experimental duration, and a markedly increased incidence in obese mice. Molecular markers analyzed using a microarray and a qPCR indicated that the cancerous lesions originated from the cholangiocytes and developed in the inflamed livers. These findings indicated that this is a novel mouse model of intrahepatic cholangiocarcinoma in the context of steatohepatitis. This model can be used to provide a better understanding of the pathogenic mechanisms of cholangiocarcinoma and to develop novel therapeutic strategies for this malignancy.

Yogurt Prevents Colitis-Associated Colorectal Cancer in Mice.

SCOPE: Epidemiological studies indicate an inverse correlation between yogurt consumption and colorectal cancer (CRC), but whether there is a cause-and-effect relationship has not yet been validated. This study aims to investigate the effects and possible mechanisms of yogurt on colitis-associated colorectal cancer (CAC) in mice. METHODS AND RESULTS: Experimental CAC is induced by azoxymethane (AOM, 10 mg kg-1 , ip) followed by three cycles of dextran sulfate sodium (DSS, 3%) treatment. Colitis is induced by adding DSS (3%) in drinking water for 5 days. Primary mouse macrophages are isolated for mechanistic studies. Data clearly show that yogurt (15 g kg-1 body weight) significantly reduces the multiplicity of colonic neoplasms by 38.83% in mice. Yogurt protects mice from colitis dependent on lactate receptor GPR81. The deficiency of Gpr81 exacerbates colitis and CAC in mice. Further investigation reveals that GPR81 may be dispensable for gut barrier function but essential for colonic mucosal repair. d-lactate in yogurt can activate GPR81 to suppress proinflammatory macrophage polarization, thereby facilitating inflammatory resolution after colonic injury and consequently suppressing CAC progression. CONCLUSION: Yogurt effectively protects against colitis-associated colorectal tumorigenesis in mice, and this study provides a rationale for introducing yogurt supplementation to patients with chronic inflammatory bowel diseases.

Aqueous extracts of Ocimum gratissimum mitigate colitis and protect against AOM/DSS-induced colorectal cancer in mice.

In this study, we explored the in vivo effects of Ocimum gratissimum aqueous extracts (OGE) on colorectal cancer (CRC) development provoked by azoxymethane/dextran sodium sulfate (AOM/DSS). The results showed a significant reduction in the tumor load and tumor number for the OGEH group that received continued administration of OGE compared to the AOM/DSS group, with P values of <0.01, but this was not observed in the OGEHs group that received separated administration of OGE. All groups except the control group exhibited aberrant crypt foci (ACF) and adenocarcinoma of lesion pathology in colon, and both conditions were significantly reduced in the OGEH group (P < 0.01) as compared to the AOM/DSS group. Subsequent investigation into whether OGE exhibits eliminative effects on DSS-induced severe colitis (SC) in mice showed that the disease activity index score was significantly reduced in the OGE-treated groups (P < 0.01), also colon colitis histological score was reversed. These data suggest that OGE may be potentially effective in preventing CRC when administered throughout the promotional stages of carcinogenesis by inhibiting inflammatory SC.

Traditional Medicine Pien Tze Huang Suppresses Colorectal Tumorigenesis Through Restoring Gut Microbiota and Metabolites.

BACKGROUND & AIMS: Pien Tze Huang (PZH) is a well-established traditional medicine with beneficial effects against inflammation and cancer. We aimed to explore the chemopreventive effect of PZH in colorectal cancer (CRC) through modulating gut microbiota. METHODS: CRC mouse models were established by azoxymethane plus dextran sulfate sodium treatment or in Apcmin/+ mice treated with or without PZH (270 mg/kg and 540 mg/kg). Gut barrier function was determined by means of intestinal permeability assays and transmission electron microscopy. Fecal microbiota and metabolites were analyzed by means of metagenomic sequencing and liquid chromatography mass spectrometry, respectively. Germ-free mice or antibiotic-treated mice were used as models of microbiota depletion. RESULTS: PZH inhibited colorectal tumorigenesis in azoxymethane plus dextran sulfate sodium-treated mice and in Apcmin/+ mice in a dose-dependent manner. PZH treatment altered the gut microbiota profile, with an increased abundance of probiotics Pseudobutyrivibrio xylanivorans and Eubacterium limosum, while pathogenic bacteria Aeromonas veronii, Campylobacter jejuni, Collinsella aerofaciens, and Peptoniphilus harei were depleted. In addition, PZH increased beneficial metabolites taurine and hypotaurine, bile acids, and unsaturated fatty acids, and significantly restored gut barrier function. Transcriptomic profiling revealed that PZH inhibited PI3K-Akt, interleukin-17, tumor necrosis factor, and cytokine-chemokine signaling. Notably, the chemopreventive effect of PZH involved both microbiota-dependent and -independent mechanisms. Fecal microbiota transplantation from PZH-treated mice to germ-free mice partly recapitulated the chemopreventive effects of PZH. PZH components ginsenoside-F2 and ginsenoside-Re demonstrated inhibitory effects on CRC cells and primary organoids, and PZH also inhibited tumorigenesis in azoxymethane plus dextran sulfate sodium-treated germ-free mice. CONCLUSIONS: PZH manipulated gut microbiota and metabolites toward a more favorable profile, improved gut barrier function, and suppressed oncogenic and pro-inflammatory pathways, thereby suppressing colorectal carcinogenesis.

Discovery of cancer-preventive juices reactivating RB functions.

BACKGROUND: Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired. METHODS: We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo. RESULTS: Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs. CONCLUSIONS: RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Chemopreventive effect of a milk whey by-product derived from Buffalo (Bubalus bubalis) in protecting from colorectal carcinogenesis.

BACKGROUND: Several studies show that natural foods are a source of compounds with anticancer properties that affect the gut microbiota and its metabolites. In the present study, we investigate the effect of a delactosed buffalo milk whey by-product (DMW) on colorectal carcinogenesis. METHODS: The effect of DMW on colorectal carcinoma (CRC) was investigated in the established mouse model of azoxymethane (AOM)-induced colon carcinoma, which closely resembles the human clinical condition of CRC. The effect of DMW on CRC immortalized cell lines was also evaluated to further identify the antineoplastic mechanism of action. RESULTS: Pretreatment of AOM-treated mice with DMW significantly (P < 0.05) reduced the percentage of mice bearing both aberrant crypt foci with more than four crypts (which are early precancerous lesions that progress to CRC) and tumors. In addition, DMW completely counteracted the effect of AOM on protein expression of caspase-9, cleaved caspase-3 and poly ADP-ribose polymerase in colonic tissue. Administration of DMW alone (i.e. without AOM) resulted in changes in the composition of the gut microbiota, leading to enrichment or depletion of genera associated with health and disease, respectively. DMW was also able to restore AOM-induced changes in specific genera of the gut microbiota. Specifically, DMW reduced the genera Atopobiaceae, Ruminococcus 1 and Lachnospiraceae XPB1014 and increased the genera Parabacteroides and Candidatus Saccharimonas, which were increased and reduced, respectively, by AOM. Blood levels of butyric acid and cancer diagnostic markers (5-methylcytidine and glycerophosphocholine), which were increased by AOM treatment, were reduced by DMW. Furthermore, DMW exerted cytotoxic effects on two human CRC cell lines (HCT116 and HT29) and these effects were associated with the induction of apoptotic signaling. CONCLUSIONS: Our results suggest that DMW exerts chemopreventive effects and restores the gut microbiota in AOM-induced CRC, and induces cytotoxic effect on CRC cells. DMW could be an important dietary supplement to support a healthy gut microbiota and reduce the prevalence of CRC in humans. Video Abstract.

Astragaloside IV inhibits AOM/DSS-induced colitis-associated tumorigenesis via activation of PPARγ signaling in mice.

BACKGROUND: Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD), resulting from long-term inflammation in the intestines. The primary cause of CAC is the imbalance of oxidative metabolism in intestinal cells, triggered by excessive reactive oxygen (ROS) and nitrogen (NO) species production due to prolonged intestinal inflammation. This imbalance leads to genomic instability caused by DNA damage, eventually resulting in the development of intestinal cancer. Previous studies have demonstrated that astragaloside IV is effective in treating dextran sulfate sodium salt (DSS)-induced colitis, but there is currently no relevant research on its efficacy in treating CAC. METHODS: To investigate the effect of astragaloside IV against CAC and the underlying mechanism, C57 mice were treated with (20, 40, 80 mg/kg) astragaloside IV while CAC was induced by intraperitoneal injection of 10 mg/kg azoxymethane (AOM) and ad libitum consumption of 2% dextran sulfate sodium salt (DSS). We re-verified the activating effects of astragaloside IV on PPARγ signaling in IEC-6 cells, which were reversed by GW9662 (the PPARγ inhibitor). RESULTS: Our results showed that astragaloside IV significantly improved AOM/DSS-induced CAC mice by inhibiting colonic shortening, preventing intestinal mucosal damage, reducing the number of tumors and, the expression of Ki67 protein. In addition, astragaloside IV could activate PPARγ signaling, which not only promoted the expression of Nrf2 and HO-1, restored the level of SOD, CAT and GSH, but also inhibited the expression of iNOS and reduced the production of NO in the intestine and IEC-6 cells. And this effect could be reversed by GW9662 in vitro. Astragaloside IV thus decreased the level of ROS and NO in the intestinal tract of mice, as well as reduced the damage of DNA, and therefore inhibited the occurrence of CAC. CONCLUSION: Astragaloside IV can activate PPARγ signaling in intestinal epithelial cells and reduces DNA damage caused by intestinal inflammation, thereby inhibiting colon tumourigenesis. The novelty of this study is to use PPARγ as the target to inhibit DNA damage to prevent the occurrence of CAC.

A Short-Term Model of Colitis-Associated Colorectal Cancer That Suggests Initial Tumor Development and the Characteristics of Cancer Stem Cells.

The mechanisms underlying the transition from colitis-associated inflammation to carcinogenesis and the cell origin of cancer formation are still unclear. The azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model reproduces human colitis-associated colorectal cancer. To elucidate the mechanisms of cancer development and dynamics of the linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr)-positive cells, we explored the early stages of colitis-associated colorectal cancer in AOM/DSS mice. The AOM/DSS mice were sacrificed at 4 to 6 weeks following AOM administration. To analyze the initial lesions, immunofluorescence staining for the following markers was performed: ß-catenin, Ki67, CDK4, Sox9, Bmi1, cyclin D1, and pSmad2/3L-Thr. Micro-neoplastic lesions were flat and unrecognizable, and the uni-cryptal ones were either open to the surfaces or hidden within the mucosae. These neoplastic cells overexpressed ß-catenin, Sox9, Ki67, and Cyclin D1 and had large basophilic nuclei in the immature atypical cells. In both the lesions, pSmad2/3L-Thr-positive cells were scattered and showed immunohistochemical co-localization with ß-catenin, CDK4, and Bmi1 but never with Ki67. More ß-catenin-positive neoplastic cells of both lesions were detected at the top compared to the base or center of the mucosae. We confirmed initial lesions in the colitis-associated colorectal cancer model mice and observed results that suggest that pSmad2/3L-Thr is a biomarker for tissue stem cells and cancer stem cells.

Altered gut microbiota of obesity subjects promotes colorectal carcinogenesis in mice.

BACKGROUND: Obesity is a risk factor for colorectal cancer (CRC). The role of gut microbiota in mediating the cancer-promoting effect of obesity is unknown. METHODS: Azoxymethane (AOM)-treated, ApcMin/+ and germ-free mice were gavaged with feces from obese individuals and control subjects respectively. The colonic tumor load and number were recorded at the endpoint in two carcinogenic models. The gut microbiota composition and colonic transcriptome were assessed by metagenomic sequencing and RNA sequencing, respectively. The anticancer effects of bacteria depleted in fecal samples of obese individuals were validated. FINDINGS: Conventional AOM-treated and ApcMin/+ mice receiving feces from obese individuals showed significantly increased colon tumor formation compared with those receiving feces from control subjects. AOM-treated mice receiving feces from obese individuals showed impaired intestinal barrier function and significant upregulation of pro-inflammatory cytokines and activation of oncogenic Wnt signaling pathway. Consistently, transferring feces from obese individuals to germ-free mice led to increased colonic cell proliferation, intestinal barrier function impairment, and induction of oncogenic and proinflammatory gene expression. Moreover, germ-free mice transplanted with feces from obese human donors had increased abundance of potential pathobiont Alistipes finegoldii, and reduced abundance of commensals Bacteroides vulgatus and Akkermansia muciniphila compared with those receiving feces from human donors with normal body mass index (BMI). Validation experiments showed that B. vulgatus and A. muciniphila demonstrated anti-proliferative effects in CRC, while A. finegoldii promoted CRC tumor growth. INTERPRETATION: Our results supported the role of obesity-associated microbiota in colorectal carcinogenesis and identified putative bacterial candidates that may mediate its mechanisms. Microbiota modulation in obese individuals may provide new approaches to prevent or treat obesity-related cancers including CRC. FUNDING: This work was funded by National Key Research and Development Program of China (2020YFA0509200/2020YFA0509203), National Natural Science Foundation of China (81922082), RGC Theme-based Research Scheme Hong Kong (T21-705/20-N), RGC Research Impact Fund Hong Kong (R4632-21F), RGC-CRF Hong Kong (C4039-19GF and C7065-18GF), RGC-GRF Hong Kong (14110819, 14111621), and NTU Start-Up Grant (021337-00001).