The oral-gut-circulatory axis: from homeostasis to colon cancer.

The human microbiota is widely recognized as providing crucial health benefits to its host, specifically by modulating immune homeostasis. Microbial imbalance, known as dysbiosis, is linked to several conditions in the body. The oral cavity and gut host the two largest microbial communities playing a major role in microbial-associated diseases. While the oral-gut axis has been previously explored, our review uniquely highlights the significance of incorporating the circulatory system into this axis. The interaction between immune cells, inflammatory factors, circulating bacteria, and microbial metabolites influences the homeostasis of both the oral and gut microbiota in a bidirectional manner. In this comprehensive review, we aim to describe the bacterial components of the oral-gut-circulatory axis in both health and disease, with a specific focus on colon cancer.

IL-1ß enhances cell viability and decreases 5-FU sensitivity in novel colon cancer cell lines derived from African American patients.

Background: In the U.S., African Americans (AAs) present with the highest incidence and mortality rates for Colorectal Cancer (CRC). When compared to Caucasian American (CA) patients, AAs also have reduced response to the first line standard of care chemotherapeutic agent 5-Fluorouracil (5-FU). Previously, we observed differential gene expression between the two populations, suggesting that colon tumors from AA patients display a decreased antitumor immune response and an increased expression of genes encoding proteins involved in inflammatory processes, such as Interleukin-1ß (IL-1ß). Here, we investigate the role of IL-1ß in modifying chemotherapeutic response and altering expression of proteins in novel AA and well-established CA colon cancer cell lines. Methods: RNA sequencing analysis was performed to detect expression of genes involved in inflammation in AA and CA colon cancer cells. The effects of IL-1ß on 5-FU response was evaluated by assessing cell viability (MTS assay) and apoptosis (flow cytometry analysis) following treatment with 5-FU alone or in combination with the cytokine. Further, we used an IL-1 receptor antagonist (IL-1Ra) to inhibit IL-1ß-induced effects on 5-FU sensitivity and NF-kB pathway activation. Results: AA colon cancer cell lines present significant increase in expression of genes IL1R2 (373-fold change (FC), IRAK1 (3.24 FC), IKBKB, (5.33 FC) NF-KB IA (5.95 FC), MYD88, (3.72 FC), IRAK3 (161 FC), TRAF5 (4.1 FC). A significant decrease in the response to 5-FU treatment, as well as a significant increase in phosphorylation of IκBα and secretion of IL-8, was seen following IL-1ß treatment, in both AA and CA cell lines. Finally, treatment with IL-1Ra was able to reverse the effects induced by IL-1ß, by increasing the cells sensitivity to 5-FU. IL-1Ra also inhibited phosphorylation of IκBα and IL-8 secretion. Conclusions: Our results suggest a differential expression of inflammatory genes and proteins that might regulate the different response to IL-1ß between AA and CA colon cancer cell lines. Our data also demonstrates that IL-1ß is involved in modulating 5-FU response in both AA and CA colon cancer cell lines. Further investigation of these mechanisms might help elucidate the differences seen in incidence, mortality and response to therapy in AA colon cancer patients.

Microbiome and colorectal carcinogenesis: Linked mechanisms and racial differences.

Various studies have shown the interplay between the intestinal microbiome, environmental factors, and genetic changes in colorectal cancer (CRC) development. In this review, we highlight the various gut and oral microbiota associated with CRC and colorectal adenomas, and their proposed molecular mechanisms in relation to the processes of "the hallmarks of cancer", and differences in microbial diversity and abundance between race/ethnicity. Patients with CRC showed increased levels of Bacteroides, Prevotella, Escherichia coli, enterotoxigenic Bacteroides fragilis, Streptococcus gallolyticus, Enterococcus faecalis, Fusobacterium nucleatum (F. nucleatum) and Clostridium difficile. Higher levels of Bacteroides have been found in African American (AA) compared to Caucasian American (CA) patients. Pro-inflammatory bacteria such as F. nucleatum and Enterobacter species were significantly higher in AAs. Also, AA patients have been shown to have decreased microbial diversity compared to CA patients. Some studies have shown that using microbiome profiles in conjunction with certain risk factors such as age, race and body mass index may help predict healthy colon vs one with adenomas or carcinomas. Periodontitis is one of the most common bacterial infections in humans and is more prevalent in Non-Hispanic-Blacks as compared to Non-Hispanic Whites. This condition causes increased systemic inflammation, immune dysregulation, gut microbiota dysbiosis and thereby possibly influencing colorectal carcinogenesis. Periodontal-associated bacteria such as Fusobacterium, Prevotella, Bacteroides and Porphyromonas have been found in CRC tissues and in feces of CRC patients. Therefore, a deeper understanding of the association between oral and gastrointestinal bacterial profile, in addition to identifying prevalent bacteria in patients with CRC and the differences observed in ethnicity/race, may play a pivotal role in predicting incidence, prognosis, and lead to the development of new treatments.

Immune-Related Gene Expression and Cytokine Secretion Is Reduced Among African American Colon Cancer Patients.

Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. Here, we investigated if differences in tumor immunology could be contributive to disparities observed between these populations. Methods: We examined gene expression of tumor and non-tumor adjacent tissues from AA and CA by whole transcriptome sequencing, and generated scores for immune cell populations by NanoString. In addition, we utilized "The Cancer Genome Atlas" (TCGA) database from AA and CA as a validation cohort. Finally, we measured the secretion of cytokines characteristic of effector T helper cell (Th) subsets by ELISA using plasma from each AA and CA participant. Results: Colon tumors from AA patients showed significant fold-change increase in gene expression when compared to CA for FOXP3 (6.22 vs. 3.22), IL1B (103 vs. 11.4) and IL8 (220 vs. 28.9) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and the immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). TCGA data validated our observed higher gene expression of GZMB and PDL1 in CA patients when compared to AA. Notably, our observations on immune cell populations show that AA tumors have significantly higher number of exhausted CD8+ cells (p < 0.01), mast cells (p < 0.02) and increased T regulatory cells when compared to CA. AA colon cancer patients differed from CA in cytokine production patterns in plasma (i.e., reduced IL-12). Conclusions: Our study demonstrates significant differences of the immunological profiles of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense mechanisms in terms of gene expression, recruitment of immune cells and systemic secretion of cytokines. As such, these immune differences could be mitigated through population-specific therapeutic approaches.

EZH2 Downregulation Augments the Effect of Irradiation in Reducing Pancreatic Cancer Cell Proliferation in vitro.

OBJECTIVE: Pancreatic ductal carcinoma has a 5-year survival rate of 9%. This makes it the 4th leading cause of cancer-related death in the United States. Advanced-stage diagnosis and limited treatment options contribute to poor prognosis. Thus, there is an urgent need for new approaches to treatment. Enhancer of Zeste 2 (EZH2), a catalytic subunit of the multi-protein histone methyltransferase, known as the polycomb repressive complex, has been implicated in carcinogenesis. E2H2's downregulation has been shown to have a therapeutic effect in B cell lymphomas. MATERIALS AND METHODS: We examined the effect of EZH2 downregulation in combination with irradiation on the proliferation and apoptosis of pancreatic cancer cells (PANC-1 and MIA PaCa-2) in vitro. EZH2 downregulation was accomplished by treatment of cells with small interfering RNA (siRNA) or EPZ. To do this, cell survival was assessed over a 96 hr (short-term) by ATP measurement and immunohistochemical assessment of Phosphohistone 3 (PHH3), Ki-67 and CC3 over two weeks (long-term) by clonogenic assay. RESULTS: EZH2 downregulation resulted in the decreased proliferation of PANC-1 and MIA PaCa-2 cells within short-term assays with maximal reduction at 72 hr. Irradiation reduced cell proliferation beginning at 96 hr and continued over the long-term. Irradiation with EZH2 downregulation reduced cell proliferation between 48 and 72 hr. This combined treatment reduced cell proliferation by 3 to 14% as compared to those treated with irradiation alone at two weeks. PANC-1 and MIA PaCa-2 cells exhibited similar responses to EZH2 downregulation and irradiation, but to different degrees. siRNA or EPZ were equally effective in EZH2 downregulation. CONCLUSIONS: EZH2 downregulation in combination with irradiation reduces PANC-1 and MIA PaCa-2 cell proliferation more than irradiation alone. This study affirms the role of EZH2 downregulation for radiosensitization in pancreatic cancer treatment.

Evaluation of the feasibility of intrapancreatic delivery of drug-loaded microparticles via EUS-guided fine needle injection using a swine model.

Background and study aims Patients with pancreatic cancer often have locally advanced or metastatic disease and are not candidates for curative surgery. Polymer-based microparticles (MPs) represent a drug delivery system that offers sustained release of a chemotherapeutic drug after intralesional injection for local tumor management. The aim of this study was to determine the feasibility of endoscopic ultrasound-guided fine-needle injection (EUS-FNI) of drug-loaded MPs tagged with a fluorophore and fiducial markers for locating the injection site. Secondary aims were to determine the tissue-specific effects of MPs. Methods Five pigs underwent EUS with selection of an injection site within the pancreas that was marked by placing fiducial markers prior to the MPs injection. EUS-FNI of either blank microparticles (BMPs), containing no drug, or gemcitabine-loaded microparticles (GMPs) was performed. A saline flush containing Spot Endoscopic Marker was used to expel any residual MPs in the needle shaft and tattoo the injection site. Results A green fluorescent protein flashlight was used to successfully identify the site of MP injection sites in the dissected pancreas. Frozen sections of pig pancreas demonstrated a defined deposit, confirming the delivery of the MPs. Finally, fluorescence microscopy showed activation of caspase-mediated cell death in pancreases of animals that received injections of GMPs. Conclusions This pilot study demonstrated that fiducial marker placement and pancreatic EUS-FNI of MPs was successful in all pigs with no animals demonstrating pancreatitis. Further studies are needed to determine the role for intralesional injection of drug-loaded MPs in borderline resectable or unresectable pancreatic cancer.

Establishment of three novel cell lines derived from African American patients with colorectal carcinoma: A unique tool for assessing racial health disparity.

The incidence and mortality rates of colorectal carcinoma (CRC) are higher among African Americans (AAs) compared with Caucasian Americans (CAs). To assess the molecular properties associated with racial health disparity, three cell lines derived from colorectal tumors of three AA subjects were established. Cellular and molecular characterization of the cell lines designated CHTN06, SB501 and SB521 was performed using standard technologies, including immunofluorescence, electron microscopy, karyotyping, reverse transcription-polymerase chain reaction, ELISA and immunoblot analysis. The histology and morphology of CHTN06 xenografts were examined by hematoxylin and eosin staining. A total of three AA CRC cell lines derived from primary tumors were established and characterized. These cell lines were successfully cultured without immortalization and were found to be tumorigenic as mouse xenografts. In the present study, immunoblotting and immunofluorescence confirmed the expression of proteins known to be dysregulated in CRC, such as p53, DNA mismatch repair proteins and villin-1. Oncogenic miRNAs (i.e., miR-17, miR-21, miR-182, miR-210 and miR-222) were overexpressed in the AA CRC lines compared with the CA CRC lines (HT-29, HCT116 and SW480). Additionally, the AA CRC cell lines exhibited a differential inflammatory profile compared with HT-29 (CA CRC cell line); specifically noted was IL-8 secretion in response to inflammatory stimuli. In conclusion, three novel cell lines derived from AA CRC tissues were generated. These cell lines were characterized as epithelial in nature and exhibited differential expression of several miRNAs and inflammatory responses compared with commercially available cell lines of CA origin. The CRC cell lines CHTN06, SB501 and SB521 represent novel tools that may be used to provide diverse in vitro and in vivo models for studying CRC and racial health disparity.

Rapidly progressive course of Trypanosoma cruzi infection in mice heterozygous for hexamethylene bis-acetamide inducible 1 (Hexim1) gene.

Infection with Trypanosoma cruzi causes Chagas disease and results in myocardial inflammation and cardiomyopathy. Downregulated Hexim1 expression, as in Hexim1+/- mice, reduces cardiac inflammation and fibrosis following ischemic stress. We asked whether reduced expression of Hexim1 would also afford protection against T. cruzi-induced cardiomyopathy. C57BL/6J (wild type - WT) and Hexim1+/- mice were infected with sub-lethal doses of T. cruzi (Brazil strain), and cardiac function, serologic markers of inflammation and tissue pathology were examined. Infected Hexim1+/- mice had compromised cardiac function, altered expression of both pro- and anti-inflammatory cytokines, and increased inflammation and fibrosis. Cardiac failure was evidenced by severely diminished heart rate, compensatory increase in respiratory rate, and abnormally high left ventricular mass with severe transmural inflammation. Lungs displayed intense peribronchial inflammation and fibrosis extending into the parenchyma. We also observed Smad3-serine208 phosphorylation in hearts and lungs of infected mice, suggesting increased TGF-ß signaling pathway activity. This was more pronounced in Hexim1+/- mice and correlated with increased fibrosis in these tissues. Conspicuous splenomegaly in the Hexim1+/- mice most likely resulted from the observed extensive white pulp expansion. T. cruzi infection induced colonic dilatation and marked villous atrophy in both the WT and Hexim1+/- mice but more so in the latter. The profound exacerbation of pathologic findings suggests a protective role for Hexim1 in T. cruzi infection.

Identifying predictive features of Clostridium difficile infection recurrence before, during, and after primary antibiotic treatment.

BACKGROUND: Colonization by the pathogen Clostridium difficile often occurs in the background of a disrupted microbial community. Identifying specific organisms conferring resistance to invasion by C. difficile is desirable because diagnostic and therapeutic strategies based on the human microbiota have the potential to provide more precision to the management and treatment of Clostridium difficile infection (CDI) and its recurrence. METHODS: We conducted a longitudinal study of adult patients diagnosed with their first CDI. We investigated the dynamics of the gut microbiota during antibiotic treatment, and we used microbial or demographic features at the time of diagnosis, or after treatment, to predict CDI recurrence. To check the validity of the predictions, a meta-analysis using a previously published dataset was performed. RESULTS: We observed that patients' microbiota "before" antibiotic treatment was predictive of disease relapse, but surprisingly, post-antibiotic microbial community is indistinguishable between patients that recur or not. At the individual OTU level, we identified Veillonella dispar as a candidate organism for preventing CDI recurrence; however, we did not detect a corresponding signal in the conducted meta-analysis. CONCLUSION: Although in our patient population, a candidate organism was identified for negatively predicting CDI recurrence, results suggest the need for larger cohort studies that include patients with diverse demographic characteristics to generalize species that robustly confer colonization resistance against C. difficile and accurately predict disease relapse.

Characterization of Trypanosoma cruzi infectivity, proliferation, and cytokine patterns in gut and pancreatic epithelial cells maintained in vitro.

Trypanosoma cruzi infects all nucleated cells in both humans and experimental animals. As a prelude to our studies of T. cruzi pathogenesis in the gastrointestinal system, we have initiated in vitro cultures of gut (Caco-2 and HT-29) and pancreatic (Panc-1) epithelial cells. We show that along with primary human fibroblasts, all three cell lines are susceptible to infection and support proliferation of T. cruzi. Infection with T. cruzi modified dramatically the cytokines elaborated by these cells. Substantially greater quantities of IL-5 and TGF-ß1 were produced by fibroblasts and Caco-2 and Panc-1 cells, whereas secretion of IFN-γ and TNF-α was greatly reduced in all three cell types. Since these cells are not known to be the primary sources of IFN-γ, we examined IFN-γ mRNA expression in these cells. Both Caco-2 and Panc-1 cells were found to express IFN-γ mRNA, validating its secretion. These findings may provide insight into signaling pathways that mediate innate immunity to T. cruzi and pathogenesis of gastrointestinal and pancreatic alterations in Chagas disease.

Biochemical and biological analyses of Rgr RalGEF oncogene.

The Ras superfamily of GTP-binding proteins is involved in many cellular processes, including cell proliferation, movement, and morphology. One such member, Ral GTPase, activates downstream signaling molecules after a conversion to the active state on GTP binding. The RalGDS-related (Rgr) oncogene belongs to the RalGDS family of guanine nucleotide exchange factors (GEFs). RalGEFs activate Ral by stimulating the dissociation of GDP, allowing the binding of GTP and the initiation of downstream signaling events by Ral effectors. Rgr was first identified as a fusion between the rabbit homolog of the Rad 23 gene and the Rgr gene in a rabbit squamous cell carcinoma. The Rgr portion of the fusion was demonstrated to contain the oncogenic activity. The human form of the Rgr oncogene was identified recently, and expression was detected in human T-cell malignancies. This chapter describes the analysis of rabbit and human Rgr function using various methods. These assays may be used for the study of oncogene function in other systems.

Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation.

The A549 Taxol-resistant cell lines, A549-T12 and A549-T24, were isolated in our laboratory, and are dependent on Taxol for normal growth. The microtubules in these cells displayed increased dynamicity in the absence of Taxol. In the present study, a heterozygous point mutation in Kalpha1-tubulin was discovered at alpha379 (Ser to Ser/Arg). Although Taxol binds to beta-tubulin in the microtubule, sequencing of beta-tubulin class I did not reveal any mutations. The expression of the alpha-tubulin mutation was demonstrated using high-resolution isoelectric focusing and two-dimensional gel analysis. Both the wild-type and mutant tubulin were expressed in the Taxol-resistant cell lines. The region of alpha-tubulin that encompasses alpha379 is near the COOH terminus that has been proposed as a site of interaction with microtubule-associated protein (MAP) 4 and stathmin, a tubulin-interacting protein. In the Taxol-resistant cells, the active nonphosphorylated form of stathmin was increased approximately 2-fold, whereas the inactive phosphorylated forms were barely detected. The inactive phosphorylated forms of MAP4 were increased in the A549-T12 and A549-T24 cell lines. We hypothesize that these changes in tubulin/MAPs that result in increased microtubule instability may be related to the alpha-tubulin mutation and are compensated for by the stabilizing properties of Taxol.