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Hirschsprung disease (HD) rarely presents as chronic constipation after the newborn period. At our institution, calretinin immunohistochemistry (CAL) is frequently requested by clinicians on rectal mucosal biopsies (RMBs) taken during colonoscopy in older children in whom suspicion for HD is low. We hypothesized that review of these biopsies would frequently reveal ganglion cells (GCs). We reviewed features of mucosal biopsies (November 2013 to September 2015) from children ≥1 year of age on which clinicians had requested CAL on at least one specimen. A total of 93 biopsies with paired CAL from 83 patients were suitable for study (ages 1-18 years, M:F 1.2). Submitted clinical indication was constipation in 62 patients (75%). GCs were found within or subjacent to muscularis mucosa in 63 biopsies (68%), 12 (19%) of which were designated from a specific anatomic site, eg, 2 or 3 cm. In 25 of 63 (40%) cases, GCs were identified on one of the first 3 sections (median 5th, range 1st-54th). Forty-six cases (73%) contained no or <0.5 mm of submucosa (SM, range 0-2 mm). All but one case (62/63, 98%) with identified GCs showed positive CAL staining; a single case showed equivocal staining. Among the 30 biopsies with no observed GCs, none (0%) had >1 mm of SM, and 21 (70%) had no SM. CAL was positive in 28 (93%) and equivocal/weak in 2 (7%); no additional work-up for HD was pursued. The data suggest that H&E sections of RMBs can exclude HD at a specified site in many cases and provide the basis for a future study examining the utility of CAL in RMBs without SM as a means for excluding HD.

Background: Fibulin-5 has recently been considered as a potential tumor suppressor in human cancers. Several studies have shown that it is down-regulated in a variety of tumor types and inhibits tumor growth and metastasis. In this study, the expression of fibulin-5 in colorectal cancer (CRC) and its clinical significance were assessed., Methods: Fibulin-5 expression was detected in 31 samples of surgically resected CRC and paired noncancerous tissues using western blot, qRT-PCR, and immunoblotting., Results: In this study, the expression levels of fibulin-5 protein and mRNA were down-regulated in CRC tissues as compared with those in paired noncancerous tissues. Low expression of fibulin-5 was significantly correlated with poor prognostic features including lymph node metastasis and advanced tumor-node-metastasis (TNM) tumor stage., Conclusions: Fibulin-5 is down-regulated in CRC and the reduced expression of fibulin-5 was correlated with malignant clinicopathological characteristics.

Background/aim: Matrix metalloproteinase 7 (MMP7) expression is highly associated with colorectal cancer and modulates tumour growth and invasion. Radiation injury induces inflammation with increases in MMP7 and in transforming growth factor beta (TGFβ). The aim of this study was to investigate the effect on MMP7 and TGFβ. expression in patients with rectal cancer undergoing different regimens of neoadjuvant radiotherapy (RT)., Patients and Methods: We studied 53 patients in three RT treatment groups receiving RT of 25 Gy, long-term RT 50 Gy and controls receiving no RT. Three biopsies were obtained from each patient during the treatments: before RT, after RT and after surgery. Tissue samples were formalin fixed, paraffin embedded and tissue microarrays were constructed and stained for MMP7 and TGFβ. Mann-Whitney U-tests and Wilcoxon Z-tests were used to determine differences between patients before and after RT, and after surgery, as well as between the RT groups., Results: In all three patient groups, increases of MMP7 and TGFβ expression were observed after surgery. MMP7 expression was significantly increased in patients receiving short-term RT but TGFβ expression was not affected by RT., Conclusion: 50 Gy Irradiation of rectal cancer gives less tumour activation of MMP7, whilst it is up-regulated by 25 Gy and surgery regardless of RT., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Pathogens and pesticides are likely to co-occur in honeybee hives, but much remains to be investigated regarding their potential interactions. Here, we first investigated the metabolisation kinetics of thiamethoxam in chronically fed honeybees. We show that thiamethoxam, at a dose of 0.25ng/bee/day, is quickly and effectively metabolised into clothianidin, throughout a 20day exposure period. Using a similar chronic exposure to pesticide, we then studied, in a separate experiment, the impact of thiamethoxam and Chronic bee paralysis virus (CBPV) co-exposure in honeybees. The honeybees were exposed to the virus by contact, mimicking the natural transmission route in the hive. We demonstrate that a high dose of thiamethoxam (5.0ng/bee/day) can cause a synergistic increase in mortality in co-exposed honeybees after 8 to 10days of exposure, with no increase in viral loads. At a lower dose (2.5ng/bee/day), there was no synergistic increase of mortality, but viral loads were significantly higher in naturally dead honeybees, compared with sacrificed honeybees exposed to the same conditions. These results show that the interactions between pathogens and pesticides in honeybees can be complex: increasing pesticide doses may not necessarily be linked to a rise in viral loads, suggesting that honeybee tolerance to the viral infection might change with pesticide exposure., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aims: Cullin 4A (CUL4A) is vital in cell survival, development, growth and cell cycle, it plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of CUL4A expression in colorectal cancer is unknown; in particular, the prognostic value of CUL4A combined with TP53 expression has not been explored., Methods: We analyzed the expression of CUL4A in both public database (Oncomine) and 180 cases of colorectal cancer and paired normal tissues by real-time polymerase chain reaction and western blotting. Colony formation, wound healing, migration and invasion assays and tumorigenesis in nude mice were used to explore the function of CUL4A in CRC proliferation and metastasis in vitro and in vivo. Markers of epithelial to mesenchymal transition (EMT) were evaluated by western blotting. Immunohistochemistry (IHC) was used to analyse the relationship between CUL4A expression and E-cadherin expression., Results: CUL4A and TP53 protein expression was significantly higher in cancerous tissues compared to normal tissues. Significant correlation between CUL4A and TP53 expression was observed. CUL4A expression was an independent prognostic factor for overall survival (OS) and disease-free survival (DFS). Interestingly, patients with tumors that had both CUL4A overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P < 0.001). Multivariate analysis showed that patients with both CUL4A+ and TP53+ positive tumors had extremely poor OS and DFS. Knockdown of CUL4A by a short interfering RNA (siRNA) significantly suppressed the progression of EMT, proliferation, migration, and invasion of colon cancer cells in vitro and tumor growth in vivo. ZEB1 silencing blocked CUL4A-driven these processes., Conclusion: CUL4A expression correlated positively with the prognosis of colorectal cancer. Mechanistically, ZEB1 was confirmed to mediate the function of CUL4A in regulating the EMT. The assessment of both CUL4A and mutant TP53 expression will be helpful in predicting colon cancer prognosis., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: Being grade 2 is a known risk factor for metastasis in rectal neuroendocrine tumors (R-NETs). We aimed to identify the efficacy of the Ki-67 labeling index (LI) in endoscopic biopsy specimens to predict the World Health Organization (WHO) grade of R-NETs., Methods: A total of 59 patients with 60 R-NETs (43 WHO grade 1 and 17 WHO grade 2), treated between October 2002 and December 2014, were retrospectively evaluated. The patients included in the study underwent biopsies followed by endoscopic submucosal resection with a ligation device, trans-anal full-thickness surgical resection, or radical surgery with lymph node dissection. The Ki-67 LI in the biopsy and resected specimens were compared between the 2 tumor grade groups, and the diagnostic sensitivity, specificity, and positive and negative predictive values for the detection of WHO grade 2 tumors were evaluated., Results: The sensitivity, specificity, and positive and negative predictive values of the Ki-67 LI in biopsy specimens for predicting grade 2 tumors were 53% (9 of 17), 95% (41 of 43), 100% (9 of 9), and 87% (41 of 47), respectively. Pearson's rank correlation coefficient between the Ki-67 LI in the biopsy and resected specimens was 0.92., Conclusions: The Ki-67 LI of the biopsy specimen is useful for determining the appropriate treatment for R-NETs., (© 2017 S. Karger AG, Basel.)

Background: Lateral lymph node dissection (LLND) is performed for advanced rectal cancers in Japan; however, it can cause sexual and urinary dysfunction. The incidence of lateral LN metastasis is estimated at 7-13.9%; therefore, excessive rectal surgery with LLND should be avoided, especially for prophylactic purposes. To identify the patients who require LLND, we examined metastases in perirectal LNs by using a one-step nucleic acid amplification (OSNA) assay to predict lateral LN metastases., Methods: Twenty-five patients who underwent surgery with bilateral LN dissection due to T3-T4 rectal cancers were prospectively included in this study. Twenty-two patients (88.0%) received preoperative chemotherapy. Among 1052 LNs from 25 patients (median 40 per case), 135 perirectal LNs (median 6 per patient) were divided into three pieces and analyzed by OSNA, reverse transcriptase-polymerase chain reaction for carcinoembryonic antigen mRNA, and pathological examination after surgery. These results were compared with the pathological diagnosis of lateral LNs., Results: Lateral LN metastases were present in 4 of 25 patients (16.0%). All of these patients were positive by OSNA for perirectal LN metastases. The OSNA assay had a sensitivity of 100%, specificity of 86%, positive predictive value of 57%, and negative predictive value (NPV) of 100% for predicting lateral LN metastases., Conclusions: The findings from this prospective study suggest that the OSNA assay of perirectal LNs may be useful for determining when LLND is necessary because of its high NPV, even in patients treated with preoperative chemotherapy.

The purpose of this experimental study was to evaluate the potential effects on the healing of colorectal anastomoses of the rectal administration of Ankaferd Blood Stopper (ABS). Thirty Wistar-Albino male rats were randomly separated into 3 groups. In the sham group, only laparotomy and colonic mobilization was performed. In the other 2 groups, colon transection and anastomosis were carried out. Saline (2 mL, 0.9% NaCl) was given rectally via a feeding tube for 10 days after the surgical procedure in the sham and control groups. In Group 3 (ABS group), the rats were treated with rectally administered ABS (2 mL/day) for 10 days. In all groups, after the measurement of bursting pressures, tissue samples were collected for the measurement of tissue hydroxyproline and prolidase levels, and for histopathological evaluation on postoperative day 11. The rectal administration of ABS showed positive effects on bursting pressures, tissue prolidase and hydroxyproline levels, and the histopathological findings of colonic anastomosis. The rectal application of ABS had positive effects on the healing of colorectal anastomosis. As a natural product, it may be used effectively and safely to achieve better healing results after colorectal anastomosis., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

MicroRNAs (miRNAs) and Transcription Factors (TFs) both influence messenger RNA (mRNA) expression, disrupting biological pathways involved in carcinogenesis and prognosis. As many miRNAs target multiple mRNAs, thus influencing a multitude of biological pathways, deciphering which miRNAs are important for cancer development and survival is difficult. In this study, we (i) determine associations between TF and survival (N = 168 colon cancer cases); (ii) identify miRNAs associated with TFs related to survival; and (iii) determine if factors derived from TF-specific miRNA principal component analysis (PCA) influence survival. Cox Proportional hazard models were run for each PCA factor to determine Hazard Ratios (HR) and 95% Confidence Intervals (CI) adjusting for age, center, and AJCC stage. Thirty TFs improved survival when differential expression increased; 27 of these were associated significantly with normal colonic mucosa expression of 65 unique miRNAs when an FDR q-value of <0.05 was applied. Five factors, comprising 21 miRNAs, altered survival in rectal cancer subjects; four of these five factors improved survival and one factor reduced survival. One factor comprising four miRNAs reduced survival in colon cancer subjects. In summary, our data suggest that expression of TFs and their related miRNAs influence survival after diagnosis with colorectal cancer., (© 2017 Wiley Periodicals, Inc.)

The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution. Here, using the in vivo non-human primate model, we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to antibodies for direct visualization and quantification of passively transferred antibodies in plasma, tissue, and in mucosal secretions. Antibodies were formulated with 1-2 fluorophores per antibody to minimally influence antibody function. Fluorophore conjugated Gamunex-C (pooled human IgG) were tested for binding to protein A, via surface plasmon resonance, and showed similar levels of binding when compared to unlabeled Gamunex-C. In order to assess the effect fluorophore labeling has on turnover and localization, rhesus macaques were IV infused with either labeled or unlabeled Gamunex-C. Plasma, vaginal Weck-Cel fluid, cervicovaginal mucus, and vaginal/rectal tissue biopsies were collected up to 8weeks. Similar turnover and biodistribution was observed between labeled and unlabeled antibodies, showing that the labeling process did not have an obvious deleterious effect on localization or turnover. Cy5 and Cy3 labeled antibodies were readily detected in the same pattern regardless of fluorophore. Tissue distribution was measured in macaque vaginal and rectal biopsies. The labeled antibody in macaque biopsies was found to have similar biodistribution pattern to endogenous antibodies in macaque and human tissues. In the vaginal and rectal mucosa, endogenous and infused antibodies were found primarily within the lamina propria. In the mucosal squamous epithelium of the vaginal vault, significant antibody was also observed in a striated pattern in the superficial, nonviable, stratum corneum. Endogenous antibody distribution in both human and macaque squamous tissues exhibited a similar pattern as seen with the labeled and unlabeled antibodies. This proof-of-principle study reveals that the labeled antibody is stable and physiologically similar relative to endogenous antibody setting the stage for future work to better understand the mechanisms of how antibodies reach unique anatomical sites. Direct visualization of fluorophore-conjugated antibodies following passive infusion can be utilized to assess the kinetics of biodistribution of infused antibodies and may be a useful approach to monitor and predict efficacy of therapeutic antibodies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mutations in the tumor suppressor p53 are highly prevalent in cancers and are known to influence the sensitivity of cells to various chemotherapeutics including the anti-cancer candidates 1,25-dihydrovitamin D3 [1,25D3] and metformin. Previous studies have demonstrated additive/synergistic anti-cancer effects of the 1,25D3-metformin combination in different models, however, the influence of p53 status on the efficacy of this regimen has not been investigated. The CRC colorectal cancer (CRC) cell lines HCT116 wild-type (wt), HCT116 p53-/-, and HT-29 (mutant; R273H) were employed, covering three different p53 variations. Synergistic effects of the combination were confirmed in all cell lines using MTT assay. Detailed evaluation of the combination's effects was performed, including on-line measurements of cellular metabolism (glycolysis/respiration) using a biosensor chip system, analyses of mitochondrial activity (membrane potential and ATP/ROS production), mRNA expression analysis of WNT/β-catenin pathway players, and a comprehensive proteomic screen using immunoblotting and ELISA microarrays. AMPK signaling was found to be more strongly induced in response to all treatments in HCT116 wt cells compared to other cell lines, an observation that was coupled to a stronger accumulation of intracellular ROS in response to metformin/combination, and finally an induction in autophagy, depicted by an increase in LC3II:LC3I ratio in combination-treated cells compared to mono-treatments. An induction in apoptotic signaling was observed in the other cell lines in response to the combination, illustrated by a decrease in expression of pro-survival Bcl2 family members. P53 status impacts cellular responses to the combination but does not hamper its anti-proliferative synergy., (© 2017 Wiley Periodicals, Inc.)

Plasma, duodenal, and rectal tissue antiretroviral therapy (ART) drug concentrations, human immunodeficiency virus (HIV) RNA and HIV DNA copy numbers, and recovery of mucosal immunity were measured before and 9 months after initiation of 3 different ART regimens in 26 subjects. Plasma and tissue HIV RNA correlated at baseline and when 9-month declines were compared, suggesting that these compartments are tightly associated. Antiretroviral tissue:blood penetration ratios were above the 50% inhibitory concentration values in almost 100% of cases. There were no correlations between drug concentrations and HIV DNA/RNA. Importantly, no evidence was found for residual viral replication or deficient tissue drug penetration to account for delayed gastrointestinal-associated lymphoid tissue immune recovery., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Radiation-induced proctitis (RIP) is the most common clinical adverse effect for patients receiving radiotherapy as part of the standard course of treatment for ovarian, prostate, colon, and bladder cancers. RIP limits radiation dosage, interrupts treatment, and lowers patients' quality of life. A prophylactic treatment that protects the gastrointestinal tract from deleterious effects of radiotherapy will significantly improve patient quality of life and may allow for higher and more regular doses of radiation therapy. Semi-synthetic glycosaminoglycan (GAG), generated from the sulfation of hyaluronic acid, are anti-inflammatory but have difficulty achieving therapeutic levels in many tissues. To enhance the delivery of GAG, we created an in situ gelling rectal delivery system using silk-elastinlike protein polymers (SELPs). Using solutions of SELP 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit) with GAG GM-0111, we created an injectable delivery platform that transitioned in <5min from a liquid at room temperature to a hydrogel at body temperature. The hydrogels released 50% of their payload within 30min and enhanced the accumulation of GAG in the rectum compared to traditional enema-based delivery. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21days by 53±4%, 47±10%, and 12±6%, respectively. Matrix-mediated delivery of GAG by SELP represents an innovative method for more effective treatment of RIP and promises to improve quality of life of cancer patients by allowing higher radiotherapy doses with improved safety., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Growing evidence has supported that long non-coding RNAs (lncRNAs) could play vital roles in the development, progression, and prognosis of colorectal cancer (CRC). However, little is known about the clinical significance of BRAF-activated non-coding RNA (BANCR) in CRC. The aim of this study is to explore the clinical value of lncRNA BANCR in CRC patients., Methods: The expression of lncRNA BANCR was measured in 106 CRC tissues and 65 adjacent normal tissues using the quantitative real-time PCR., Results: The study showed that lncRNA BANCR was highly expressed in CRC tissues compared with adjacent normal tissues (P < 0.001). In addition, high expression of lncRNA BANCR was positively correlated with the lymph node metastasis (P < 0.001). Kaplan-Meier analysis showed that patients with high lncRNA BANCR expression had a shorter overall survival (OS) compared with the low lncRNA BANCR expression group (P = 0.001). Interestingly, for the group of patients with the lymph node metastasis, we found the similar result that high lncRNA BANCR expression was related to poor OS (P = 0.004). Furthermore, the multivariate Cox regression model analysis indicated that high expression of lncRNA BANCR was an independent poor prognostic factor in CRC patients (HR 2.24, 95% CI 1.22-4.16, P = 0.009)., Conclusions: Upregulation of lncRNA BANCR may be associated with the lymph node metastasis and poor survival of CRC. LncRNA BANCR could be served as a novel and useful biomarker for CRC lymph node metastasis and prognosis.

Small active RNA (saRNA)-induced gene activation (RNAa) is a novel strategy to treat cancer. Our previous work proved that the p21-saRNA-322 successfully hindered colorectal cancer growth by activating p21 gene. However, the barrier for successful saRNA therapy is lack of efficient drug delivery. In the present study, a rectal delivery system entitled p21-saRNA-322 encapsulated tumor-selective lipopolyplex (TSLPP-p21-saRNA-322) which consist of PEI/p21-saRNA-322 polyplex core and hyaluronan (HA) modulated lipid shell was developed to treat colorectal cancer. Our results showed that this system maintained at the rectum for more than 6 h and preferentially accumulated at tumor site. CD44 knock down experiment instructed that the superb cellular uptake of TSLPP-p21-saRNA-322 attributed to HA-CD44 recognition. An orthotopic model of bio-luminescence human colorectal cancer in mice was developed using microsurgery and TSLPP-p21-saRNA-322 demonstrated a superior antitumor efficacy in vitro and in vivo. Our results provide preclinical proof-of-concept for a novel method to treat colorectal cancer by rectal administration of TSLPP formulated p21-saRNA-322., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Patients with congenital anorectal malformation (ARM) often present with different degrees of defecation dysfunction severity following corrective operations. Therefore, studies on how to improve the postoperative defecation function of patients with ARM are of clinical importance. The present study investigated the expression of the HuD protein in the terminal rectum of ARM embryonic rats and explored the effect of HuD expression on the development of the intestinal nervous system. Pregnant Sprague Dawley rats were randomized into a control or ARM (induced by ethylene thiourea) group. The terminal rectums of the embryonic rats were obtained during pregnancy (20 days). The histological changes of the terminal rectum were observed using hematoxylin and eosin staining. The expression of the HuD protein was assessed by immunohistochemistry and western blot analysis. In the control group, the histological structure of the terminal rectum was well‑defined and a large number of submucosal and intermuscular neurons with a rich cytoplasm and strong neuritis were observed. In the ARM group, the histological layers were ill‑defined and the number of neurons was small. Immunohistochemistry and western blot analysis demonstrated that the concentration of the HuD protein in the ARM group was significantly lower compared with the control group (312.90±53.40:456.40±57.13; 0.24±0.05:0.45±0.06, P<0.05). HuD was abnormally expressed in the terminal rectum of the ARM embryonic rats and may be involved in the development and maturation of the enteric nervous system. The present study may provide a useful theoretical reference for the treatment of postoperative defecation dysfunction in patients with ARM.

Colorectal cancer (CRC) is one of the most frequent tumor types in Western countries. Approximately 20 % of patients show metastasis at the time of diagnosis, with the liver being one of the most affected organs. Transforming growth factor-beta (TGF-β) plays a regulatory role not only in the physiology of the normal colon but also in the development of CRC and its metastatic process. In this review, we analyze the molecular mechanisms leading to TGF-β dysregulation in tumor and stroma cells and the modification of the microenvironment that fosters CRC metastasis. Recent genomic studies have identified a CRC subtype with a mesenchymal and aggressive phenotype having TGF-β as a hub gene of this signature. Consistent with these findings, the inhibition of TGF-β signaling has been shown to impair experimental CRC metastasis to the liver. Based on these and other results conducted in various tumor types, the pharmaceutical industry has developed a variety of strategies to target TGF-β. We provide up-to-date information of these therapies, which are currently in preclinical or clinical trials.

Background: Although high-protein diets (HPDs) are frequently consumed for body-weight control, little is known about the consequences for gut microbiota composition and metabolic activity and for large intestine mucosal homeostasis. Moreover, the effects of HPDs according to the source of protein need to be considered in this context. Objective: The objective of this study was to evaluate the effects of the quantity and source of dietary protein on microbiota composition, bacterial metabolite production, and consequences for the large intestinal mucosa in humans. Design: A randomized, double-blind, parallel-design trial was conducted in 38 overweight individuals who received a 3-wk isocaloric supplementation with casein, soy protein, or maltodextrin as a control. Fecal and rectal biopsy-associated microbiota composition was analyzed by 16S ribosomal DNA sequencing. Fecal, urinary, and plasma metabolomes were assessed by 1 H-nuclear magnetic resonance. Mucosal transcriptome in rectal biopsies was determined with the use of microarrays. Results: HPDs did not alter the microbiota composition, but induced a shift in bacterial metabolism toward amino acid degradation with different metabolite profiles according to the protein source. Correlation analysis identified new potential bacterial taxa involved in amino acid degradation. Fecal water cytotoxicity was not modified by HPDs, but was associated with a specific microbiota and bacterial metabolite profile. Casein and soy protein HPDs did not induce inflammation, but differentially modified the expression of genes playing key roles in homeostatic processes in rectal mucosa, such as cell cycle or cell death. Conclusions: This human intervention study shows that the quantity and source of dietary proteins act as regulators of gut microbiota metabolite production and host gene expression in the rectal mucosa, raising new questions on the impact of HPDs on the large intestine mucosa homeostasis. This trial was registered at clinicaltrials.gov as NCT02351297., (© 2017 American Society for Nutrition.)

Although an array of new therapeutics has emerged for the treatment of colorectal cancer, their use is significantly impacted by variability in patient response. Better pre-clinical models could substantially improve efficacy as it may allow stratification of patients into the correct treatment regime. Here we explore acute, ex vivo treatment of fresh, surgically resected human colorectal tumour biopsies as a novel pre-clinical model for identifying patient response to specific therapeutics. The MEK1/2 inhibitor, Selumetinib (AZD6244, ARRY-142886) was used as a tool compound. Firstly, we established an acute treatment protocol and demonstrated this protocol could differentiate phenotypic and pharmacodynamic responses to Selumetinib (0-3uM). We then used the protocol to evaluate Selumetinib response in tumours from 23 colon cancer patients. These studies revealed that the agent inhibited pERK1/2 phosphorylation in all tumours, caused a significant decrease in proliferation in 5/23 (22%) tumours, and that KRAS/BRAF mutant tumours were particularly sensitive to the anti-proliferative effects of the agent. These data are consistent with data from clinical trials of Selumetinib, suggesting that acute treatment of small tumour biopsies is worthy of further exploration as a pre-clinical model to evaluate colorectal cancer response to novel therapies.

Background: Terminal hindgut deformity is the leading digestive tract malformation, however, the etiology and pathogenesis remained unknown. To date, gene expression abnormalities were considered the primary cause of these diseases. miRNAs have been found to play an important role in regulating the expression of genes., Methods: A total of 24 pregnant rats were randomly divided into two groups. The experimental group (n=12) received 1% ethylenethiourea (125mg/kg) by gavage on gestational day 11, while the control group (n=12) received the same volume of distilled water. From each group, fetal rats were obtained by cesarean section on gestational day 16. For the extraction of total RNA, 1 cm rectum samples were obtained from four fetal rats that had similar weights. Chip hybridization was conducted after poly(A) and biotin were added to the RNA samples, and this was followed by washing, dyeing, and scanning of the chip. Differences identified in the miRNA expression profiles and the target gene analysis results were further analyzed to identify potential regulators of terminal hindgut development., Results: Compared with the control group, 111 miRNAs expressed in the terminal hindgut of the experimental group were up-regulated on gestational day 16, while 117 miRNAs were down-regulated. The ten miRNAs with the greatest differential expression profiles between the experimental and control samples were selected for target gene prediction, pathway analysis, and gene ontology analysis. A subset of these miRNAs was found to be closely related to rat fetus terminal hindgut growth and development. In addition, target gene analysis showed that miR-193 may have an important role in regulating a key gene in anorectal development, Hoxd13. This role was confirmed in a dual luciferase reporter assay when miR-193 was able to inhibit expression of a reporter gene under the control of the 3' untranslated region of the Hoxd13 gene in the human embryonic kidney cell line, 293T. Real-time PCR and Western blotting experiments further showed that the expression of Hoxd13 was significantly lower when miR-193 was highly expressed in rat intestinal epithelial cells. The differences in both sets of experiments were statistically significant compared with the negative control group (P<0.05)., Conclusion: These data support an important regulatory role for miRNAs in the expression of target genes during terminal hindgut development in fetal rats. In particular, miR-193-mediated inhibition of Hoxd13 was found to be significant in rat intestinal epithelial cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) risk is modulated by diet and there is convincing evidence of reduced risk with higher non-digestible carbohydrates (NDCs) consumption. Resistant starch (RS), a NDC, positively modulates the expression of oncogenic microRNAs, suggesting that this could be a mechanism through which NDCs protect against CRC. The present study aimed to investigate the effects of supplementation with two NDCs, RS, and polydextrose (PD), on microRNA expression in the macroscopically-normal human rectal epithelium using samples from the DISC Study, a randomized, double-blind, placebo-controlled dietary intervention. We screened 1008 miRNAs in pooled post-intervention rectal mucosal samples from participants allocated to the double placebo group and those supplemented with both RS and PD. A total of 111 miRNAs were up- or down-regulated by at least twofold in the RS + PD group compared with the control group. From these, eight were selected for quantification in individual participant samples by qPCR, and fold-change direction was consistent with the array for seven miRNAs. The inconsistency for miR-133b and the lower fold-change values observed for the seven miRNAs is probably because qPCR of individual participant samples is a more robust and sensitive method of quantification than the array. miR-32 expression was increased by approximately threefold (P = 0.033) in the rectal mucosa of participants supplemented with RS + PD compared with placebo. miR-32 is involved in the regulation of processes such as cell proliferation that are dysregulated in CRC. Furthermore, miR-32 may affect non-canonical NF-κB signaling via regulation of TRAF3 expression and consequently NIK stabilization., (© 2017 The Authors. Molecular Carcinogenesis Published by wiley periodicals, Inc.)

Synchrony of spontaneous Ca 2+ transients among venular mural cells (smooth muscle cells and pericytes) in visceral organs relies on the intercellular spread of L-type voltage-dependent Ca 2+ channel (LVDCC)-dependent depolarisations. However, the mechanisms underlying the synchrony of spontaneous Ca 2+ transients between arteriolar mural cells are less understood. The spontaneous intracellular Ca 2+ dynamics of arteriolar mural cells in the rat rectal submucosa were visualised by Cal-520 Ca 2+ imaging to analyse their synchrony. The mural cells in fine arterioles that had a rounded cell body with several extended processes developed spontaneous 'synchronous' Ca 2+ transients arising from Ca 2+ released from sarcoendoplasmic reticulum Ca 2+ stores. Gap junction blockers (3 μM carbenoxolone, 10 μM 18β-glycyrrhetinic acid), a Ca 2+ -activated Cl - channel (CaCC) blocker (100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or lowering extracellular Cl - concentration (from 134.4 to 12.4 mM) disrupted the synchrony of Ca 2+ transients between arteriolar mural cells. Blockers of T-type voltage-dependent Ca 2+ channels (TVDCCs, 1 μM mibefradil or ML218) or LVDCCs (1 μM nifedipine) reduced the Ca 2+ transient frequency or their area under curve (AUC), respectively. However, neither TVDCC nor LVDCC blockers disrupted the synchrony of Ca 2+ transients among arteriolar mural cells. This is in contrast with rectal venules in which nifedipine disrupted the synchrony of spontaneous Ca 2+ transients. Thus, spontaneous transient depolarisations arising from the opening of CaCCs may effectively spread to neighbouring arteriolar mural cells via gap junctions to maintain the Ca 2+ transient synchrony. Activation of TVDCCs appears to accelerate spontaneous Ca 2+ transients, while LVDCCs predominantly contribute to the duration of Ca 2+ transients.

B7-H3, an immunoregulatory protein, has been found highly expressed in several cancer types, and involved in cancer cell migration and invasion. Here, we investigated the role of B7-H3 in oxaliplatin resistance in colorectal cancer (CRC) cells. Transient silencing of B7-H3 enhanced oxaliplatin sensitivity by increasing oxaliplatin-induced DNA damage. The overexpression of B7-H3 increased oxaliplatin resistance reducing the formation of phosphorylated histone H2AX (γH2AX) loci. The silencing of X-ray repair cross complementing group 1 (XRCC1), upregulated in B7-H3 overexpressing cells, induced an increase in cell death following oxaliplatin treatment. Finally, the upregulation of XRCC1 expression induced by B7-H3 involved PI3K-AKT pathway. In conclusion, B7-H3 promotes the oxaliplatin resistance in CRC cells upregulating the expression of XRCC1 via PI3K-AKT pathway., (Copyright © 2017. Published by Elsevier Inc.)

Deregulated activation of RAS/extracellular signal-regulated kinase (ERK) signaling and defects in retinoic acid receptor (RAR) signaling are both implicated in many types of cancers. However, interrelationships between these alterations in regulating cancer cell fates have not been fully elucidated. Here, we show that RAS/ERK and RAR signaling pathways antagonistically interact with each other to regulate colorectal cancer (CRC) cell fates. We show that RAR signaling activation promotes spontaneous differentiation of CRC cells, while ERK activation suppresses it. Our microarray analyses identify genes whose expression levels are upregulated by RAR signaling. Notably, one of these genes, MKP4 , encoding a member of dual-specificity phosphatases for mitogen-activated protein (MAP) kinases, mediates ERK inactivation upon RAR activation, thereby promoting the differentiation of CRC cells. Moreover, our results also show that RA induction of RAR target genes is suppressed by the ERK pathway activation. This suppression results from the inhibition of RAR transcriptional activity, which is shown to be mediated through an RIP140/histone deacetylase (HDAC)-mediated mechanism. These results identify antagonistic interactions between RAS/ERK and RAR signaling in the cell fate decision of CRC cells and define their underlying molecular mechanisms., (Copyright © 2017 American Society for Microbiology.)

Previously, we reported the discovery of a series of N-hydroxycinnamamide-based HDAC inhibitors, among which compound 11y exhibited high HDAC1/3 selectivity. In this current study, structural derivatization of 11y led to a new series of benzamide based HDAC inhibitors. Most of the compounds exhibited high HDACs inhibitory potency. Compound 11a (with 4-methoxybenzoyl as N-substituent in the cap and 4-(aminomethyl) benzoyl as the linker group) exhibited selectivity against HDAC1 to some extent, and showed potent antiproliferative activity against several tumor cell lines. In vivo studies revealed that compound 11a displayed potent oral antitumor activity in both hematological tumor cell U937 xenograft model and solid tumor cell HCT116 xenograft model with no obvious toxicity. Further modification of benzamide 3, 11a and 19 afforded new thienyl and phenyl compounds (50a, 50b, 63a, 63b and 63c) with dramatic HDAC1 and HDAC2 dual selectivity, and the fluorine containing compound 56, with moderate HDAC3 selectivity., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The gastrointestinal mucosa is an important site of HIV acquisition, viral replication, and pathogenesis. Immune cells in mucosal tissues frequently differ in phenotype and function from their non-mucosal counterparts. Although perforin-mediated cytotoxicity as measured in blood is a recognized correlate of HIV immune control, its role in gastrointestinal tissues is unknown. We sought to elucidate the cytotoxic features of rectal mucosal CD8 + T-cells in HIV infected and uninfected subjects. Perforin expression and lytic capacity were significantly reduced in rectal CD8 + T-cells compared with their blood counterparts, regardless of HIV clinical status; granzyme B (GrzB) was reduced to a lesser extent. Mucosal perforin and GrzB expression were higher in participants not on antiretroviral therapy compared with those on therapy and controls. Reduction in perforin and GrzB was not explained by differences in memory/effector subsets. Expression of T-bet and Eomesodermin was significantly lower in gut CD8 + T-cells compared with blood, and in vitro neutralization of TGF-β partially restored perforin expression in gut CD8 + T-cells. These findings suggest that rectal CD8 + T-cells are primarily non-cytotoxic, and phenotypically shaped by the tissue microenvironment. Further elucidation of rectal immune responses to HIV will inform the development of vaccines and immunotherapies targeted to mucosal tissues.

Acetylcholinesterase enzymatic histochemistry (AChE EHC), which highlights abnormal cholinergic nerves in the mucosa of aganglionic bowel, has been used for decades to evaluate rectal biopsies for Hirschsprung disease (HSCR). While useful diagnostically, AChE EHC is not compatible with conventional formalin-fixed and paraffin-embedded (FFPE) tissues and is not widely available. The choline transporter (ChT) is a putative alternative marker of cholinergic nerves. ChT immunohistochemistry (IHC) was investigated using FFPE biopsies and resections from patients with confirmed HSCR, as well as appropriate non-HSCR controls. ChT immunostaining was effective at identifying cases with HSCR and qualitatively similar to AChE EHC on frozen section. Among 3 pathologists, the diagnostic positive and negative predictive values based on ChT IHC ranged from 0.84-0.94 and 0.85-0.89, respectively, with good inter-observer agreement (Cohen kappa = 0.70-0.90). ChT IHC was useful in unusual scenarios in which calretinin (CR) IHC failed to correctly identify patients with HSCR. In 10 cases of short-segment HSCR, abnormal ChT+ mucosal innervation was present through the entire aganglionic segment and into portions of the TZ with submucosal nerve hypertrophy. In contrast, mucosal CR IHC was retained in the TZ and adjacent aganglionic bowel, which could lead to misinterpretation of a biopsy as ganglionic bowel. Indeed, 6 such patients were identified with paradoxical CR-positive mucosal innervation in their diagnostic biopsies. ChT IHC was interpreted as unequivocal HSCR in these cases, and HSCR was confirmed on resection. In summary, ChT IHC in FFPE tissue demonstrates high positive and negative predictive values for HSCR, is superior to CR IHC in a subset of cases, and can be incorporated into routine practice without the need for specialized techniques.

Anti-tumor immunity is a new line of research for the treatment of patients with solid tumors. In this field, negative regulators of the immune system called immune checkpoints play a key role in limiting antitumor immunologic responses. For this reason, immune checkpoint-inhibiting agents, such as those directed against cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death-1 receptor (PD1) and its ligand PD-L1, have been developed as antitumor drugs, producing interesting results in preclinical and clinical studies. We present an updated review of the biological background and clinical development of immune checkpoint inhibitors in colorectal cancer (CRC). Early trial results on PD1 and PD-L1 blockade appear promising, especially in CRC patients with microsatellite instability (MSI). Clinical trials are ongoing to confirm these preliminary results, evaluate combination strategies and identify biomarkers to predict which patients are most likely to benefit from, or show resistance to, the effects of checkpoint inhibition., Competing Interests: The authors declare no conflicts of interest.

Tumor necrosis factor-α (TNF-α), an important factor in systematic inflammation, is reportedly involved in several cancer types. The TNF-α -308 G>A (rs1800629) polymorphism in the promoter region influences TNF-α production. The association between TNF-α -308 G>A polymorphism and colorectal cancer (CRC) is not fully understood, especially the connections between TNF-α -308 G>A polymorphism and clinical features of CRC. In this study, TNF-α -308 G>A polymorphism was genotyped in 1140 individuals with or without CRC from Southwestern China. In case-control studies, we found no association between TNF-α -308 G>A polymorphism and CRC risk. Analysis of the correlations between TNF-α -308 G>A polymorphism and clinical features of CRC revealed that TNF-α -308 A allele was associated with higher body mass index (BMI) larger tumor size, and distant tumor metastasis in all CRC patients. Notably, rectal cancer (a subtype of CRC) patients with TNF-α -308 A allele had a very high risk of distant tumor metastasis [odds ratio (OR) = 4.481; 95% confidence interval (CI): 2.072-9.693; P = 0.00025]. The association between TNF-α -308 A allele and distant tumor metastasis remained even significant after adjusting all clinical characteristics (OR = 7.099; 95% CI: 2.482-20.301; P = 0.000256) in rectal cancer patients. Our results suggested that TNF-α -308 A allele was significantly associated with distant tumor metastasis in rectal cancer patients.

Colorectal cancer (CRC) is one of the most common cancers and has a high rate of morbidity and mortality worldwide. Very-low-density-lipoprotein receptor (VLDLR), a member of the low-density-lipoprotein receptor (LDLR) superfamily, is a multifunctional receptor that regulates cellular signaling by binding numerous ligands. Several studies reported the altered expression of VLDLR and suggested that VLDLR may play a critical role in tumor development by affecting cell proliferation and metastasis. However, the function of VLDLR and regulation of its expression by miRNAs have not been investigated in CRC. In the present study, we investigated the expression of VLDLR in CRC patients and found it to be significantly decreased in tumors in comparison with paired adjacent non-tumor tissues. Moreover, VLDLR over-expression inhibited the proliferation and migration of CRC cells. We also found that VLDLR expression was negatively regulated by miR-200c in CRC cells and that their expression levels were inversely correlated in CRC patients. These data suggest that VLDLR down-regulation mediated by the increased expression of miR-200c may be involved in the development of CRC., (© 2017 Wiley Periodicals, Inc.)

Aim: To enhance oral bioavailability and chemotherapeutic efficacy of naringenin (NG) by fabricating the NG-encapsulated Soluthin-maltodextrin-based nanocarrier (NC) system., Materials & Methods: NG-encapsulated nanocarriers (NG/NCs) were developed, and in vitro physicochemically characterized. Furthermore, Wistar rats were used to evaluate the pharmacokinetic profile. Furthermore, in vitro and in vivo colorectal cancer efficacy was evaluated in BALB/c mice-bearing colon-26 cells., Results: The NG/NCs demonstrated favorable mean particle size (176 ± 2.35 nm) and percent entrapment efficiency (70.83 ± 4.55%), respectively. The oral bioavailability was found to be approximately 116-fold higher and in vitro cytotoxicity exhibited approximately 21-fold reduction as compared with pure NG. Moreover, optimized NG/NCs demonstrated significant tumor suppression compared with pure NG in vivo., Conclusion: The NG/NCs would be an efficient formulation for enhancing oral bioavailability and chemotherapeutic efficacy of NG.

The aberrant expression of miR-30d has been reported in several types of human malignancies. However, its biological function in colorectal cancer (CRC) remains largely unknown. In this study, we identified that miR‑30d was significantly downregulated in CRC tissues compared to that observed in normal controls as detected by RT‑qPCR analysis. Downregulation of miR‑30d was significantly associated with aggressive clinicopathological parameters including tumor differentiation, invasive depth, TNM stage, lymph node metastasis, distant metastasis, and poor prognosis. Furthermore, functional analysis revealed that overexpression of miR‑30d significantly inhibited cell proliferation, caused cell cycle arrest at the G0/G1 phase, suppressed cell migration and invasion, induced cell apoptosis in vitro, and decreased tumor growth in a xenograft mouse model. Bioinformatic analysis and dual‑luciferase reporter assay revealed that liver receptor homologue‑1 (LRH‑1) is a direct target of miR‑30d in CRC cells. Rescue assay showed that LRH‑1 overexpression could restore the inhibitory effect of miR‑30d on CRC cells. In addition, miR‑30d overexpression suppressed the activation of key components of the Wnt/β‑catenin signaling pathway, β‑catenin, c‑Myc and cyclin D1, which contributed to the inhibition of CRC development. Thus, our findings suggest that miR‑30d functions as a tumor suppressor against CRC development and miR‑30d/LRH‑1/Wnt signaling may be novel potential targets for CRC treatment.

Objectives: Tenofovir alafenamide, a prodrug of tenofovir, produces higher PBMC concentrations of tenofovir diphosphate (tenofovir-dp) than tenofovir disoproxil fumarate. To understand tenofovir alafenamide's mucosal tissue distribution and its implications for pre-exposure prophylaxis, we characterized tenofovir-dp in female genital tract (FGT) and lower gastrointestinal (GI) tissues., Methods: Healthy seronegative women were given 5, 10 or 25 mg of tenofovir alafenamide ( n  =   8/group). Each participant provided plasma, PBMC and cervical, vaginal and rectal tissue samples over 14 days. Plasma, cell lysate and tissue homogenate concentrations were analysed by LC-MS/MS. Dose proportionality was declared in plasma and PBMCs if the natural log AUC versus natural log dose regression line 90% CI was within 0.57-1.43. In vitro tenofovir-dp formation was assessed in PBMCs and ectocervical (Ect1/E6E7) and vaginal (VK2/E6E7) cells incubated in 0.5 and 10 μM tenofovir alafenamide or tenofovir. clinicaltrials.gov: NCT02357602., Results: Following single doses of 5, 10 and 25 mg, median (IQR) tenofovir plasma AUC 0-14 days was 52.8 (49.5-59.6), 78.1 (68.2-86.9) and 169.7 (131.2-211.4) ng·h/mL and tenofovir-dp PBMC AUC 0-14 days was 2268 (1519-4090), 4584 (3113-5734) and 9306 (6891-10785) fmol·h/10 6 cells, respectively. Tenofovir was quantifiable in 52% and 92% of FGT and GI tissues, whereas tenofovir-dp was quantifiable in only 5% and 19% of FGT and GI tissues, respectively. Plasma tenofovir and PBMC tenofovir-dp were dose proportional (90% CI = 0.87-1.15 and 0.62-1.02, respectively). In vitro tenofovir-dp was 1.7-17-fold higher in epithelial cells than PBMCs., Conclusions: After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To explore the expression of R-spondin family in colorectal cancer tissues and adjacent tissues, and to evaluate its relationship with clinic-pathological stage.
 Methods: A total of 64 samples of colorectal cancer tissues and adjacent tissues were collected from the patients, who received radical surgery in Xiangya Hospital, Central South University between January 2014 and August 2014. The mRNA and protein expression levels of R-spondin 1-4 and β-catenin in the colorectal cancer tissues and adjacent tissues were detected by qRT-PCR and immunohistochemistry. The relationship between the expression level of R-spondin 1-4 and the clinic-pathological factors were analyzed to explore the correlation between the expression level of R-spondin 1-4 and β-catenin in colorectal cancer.
 Results: Compared with the adjacent tissues, the mRNA and protein expression levels of R-spondin 1 were elevated in the colorectal cancer tissues (P<0.05). The mRNA and protein expression levels of R-spondin 2-4 were increased in the colorectal cancer tissues than those in the normal tissues (P<0.05), but there was no significant difference between the colorectal cancer tissues and adjacent tissues (P>0.05). The expression level of R-spondin 1 was positively correlated with the nuclear expression of β-catenin in the colorectal cancer tissues (r=0.6307, P<0.05).
 Conclusion: Compared with the adjacent tissues, the mRNA and protein expression levels of R-spondin 1 are significantly elevated in the colorectal cancer tissues. R-spondin 1 may play a role in promoting carcinogenesis by regulating the activity of β-catenin in the downstream of Wnt signaling pathway.

The underlying molecular mechanisms for anastomotic leakage (AL) after colorectal surgery are unknown and there are no therapeutics for AL prevention. Our aim was to correlate endogenous matrix metalloproteinase (MMP) activity, collagen concentration, and collagen/MMP/cytokine mRNA levels with anatomic location in human colorectal tissue. We enrolled 22 patients in this prospective study: 7 underwent elective laparoscopic sigmoid resection and 15 underwent low anterior resection for colorectal cancer. Full-thickness intestinal tissue rings from anastomoses constructed with a circular stapler were used for the determination of the MMP activity, tissue collagen concentration and mRNA levels. COL1A1 (p = 0.017) and COL3A1 (p = 0.0013) mRNA levels were lower in rectal tissue than in colonic samples. Neither MMP activities nor collagen concentrations differed significantly between the two anatomic locations. By elucidating the factors responsible for the decreased collagen production we may identify specific molecular targets in AL prophylaxis., (© 2017 by the Wound Healing Society.)

Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca 2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca 2+ , Ca 2+ -calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca 2+ release. Thus, Ca 2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

Background: Evidence indicates that most cases of colorectal carcinoma (CRC) develop from adenoma. A previous study demonstrated that mitochondrial Tu translation elongation factor (TUFM) might serve as an independent prognostic factor for colorectal cancer. However, the expression and function of TUFM in the normal-adenoma-cancer sequence have not been reported. In this study, we investigated the clinicopathologic significance of TUFM and p53 expression for the normal-adenoma-carcinoma sequence in colorectal epithelia and evaluated the roles of TUFM during the progression of colorectal tumors., Methods: Paraffin-embedded specimens from 261 colorectal normal mucosa samples, 157 adenomas, and 104 early carcinomas were analyzed for TUFM and p53 expression by immunohistochemistry., Results: Expression of TUFM and p53 was significantly increased during the colorectal normal-adenoma-carcinoma sequence (all P < 0.05). The expression of TUFM and p53 was associated with histologic type of adenomas (P = 0.028; P = 0.001) and grade of dysplasia (all P = 0.001). Expression of TUFM was positively correlated with that of p53 (r = 0.319, P = 0.001)., Conclusions: Upregulated TUFM expression may play an important role in the transformation from colorectal normal mucosa to carcinoma through adenoma. Combined immunohistochemical detection of TUFM and p53 may be useful for evaluating the biological behavior of colorectal adenoma.

Chlamydia is the most common bacterial sexually transmitted infection among men who have sex with men. Repeat infection following treatment with 1g azithromycin is common and treatment failure of up to 22% has been reported. This study measured the pharmacokinetics of azithromycin in rectal tissue in men following a single 1g dose to assess whether azithromycin reaches the rectal site in adequate concentrations to kill chlamydia. Ten healthy men took a single oral dose of 1g azithromycin and provided nine self-collected swabs and one blood sample over 14 days. Participant demographics, medications, sexual behaviour, treatment side effects, lubricant use and douching practices were recorded with each swab. Drug concentration over time was determined using liquid chromatography-mass spectrometry and total exposure (AUC0-∞) was estimated from the concentration-time profiles. Following 1g of azithromycin, rectal concentrations peaked after a median of 24 hours (median 133mcg/g) and remained above the minimum inhibitory concentration for chlamydia (0.125mcg/mL) for at least 14 days in all men. AUC0-∞ was the highest ever reported in human tissue (13103((mcg/g).hr)). Tissue concentrations were not associated with weight (mg/kg), but data suggest that increased gastric pH could increase azithromycin levels and diarrhoea or use of water-based lubricants could decrease concentrations. High and sustained concentrations of azithromycin were found in rectal tissue following a single 1g dose suggesting that inadequate concentrations are unlikely to cause treatment failure. Factors effecting absorption (pH and diarrhoea) or drug depletion (douching and water-based lubricants) may be more important determinants of concentrations in situ.

Metastasis is one of the most decisive factors influencing CRC patient prognosis and current studies suggest that a molecular mechanism known as EMT broadly regulates cancer metastasis. NR2F2 is a key molecule in the development of CRC, but the roles and underlying mechanisms of NR2F2 in TGF-β induced EMT in CRC remain largely unknown. In the current study, we were interested to examine the role of NR2F2 in the TGF-β-induced EMT in CRC. Here, we found NR2F2 was upregulated in CRC cells and promotes TGF-β-induced EMT in CRC. Using comparative miRNA profiling TGF-β pre-treated CRC cells in which NR2F2 had been knocked down with that of control cells, we identified miR-21 as a commonly downregulated miRNA in HT29 cells treated with TGF-β and NR2F2 siRNA, and its downregulation inhibiting migration and invasion of CRC cells. Moreover, we found NR2F2 could transcriptional activated miR-21 expression by binding to miR-21 promoter in HT29 by ChIP and luciferase assay. In the last, our data demonstrated that Smad7 was the direct target of miR-21 in CRC cells. Thus, NR2F2 could promote TGF-β-induced EMT and inhibit Smad7 expression via transactivation of miR-21, and NR2F2 may be a new common therapeutic target for CRC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The TGF-β signaling pathway is involved in regulation of cell growth, angiogenesis, and metastasis. We test the hypothesis that genetic variation in the TGF-β signaling pathway alters miRNA expression.We use data from 1188 colorectal cancer cases to evaluate associations between 80 SNPs in 21 genes.Seven variants eIF4E rs12498533, NFκB1 rs230510, TGFB1 rs4803455, TGFBR1 rs1571590 and rs6478974, SMAD3 rs3743343, and RUNX1 rs8134179 were associated with expression level of miRNAs in normal colorectal mucosa. RUNX2 rs12333172 and BMPR1B rs13134042 were associated with miRNAs in normal colon mucosa; eIF4EBP3 rs250425, SMAD3 rs12904944, SMAD7 rs3736242, and PTEN rs532678 were associated with miRNA expression in normal rectal mucosa. Evaluation of the differential expression between carcinoma and normal mucosa showed that SMAD3 rs12708491 and rs2414937, NFκB1 rs230510 and rs3821958, and RUNX3 rs6672420 were associated with several miRNAs for colorectal carcinoma. Evaluation of site-specific differential miRNA expression showed that BMPR1B rs2120834, BMPR2 rs2228545, and eIF4EBP3 rs250425 were associated with differential miRNA expression in colon tissue and SMAD3 rs12901071, rs1498506, and rs2414937, BMPR2 rs2228545, and RUNX2 rs2819854, altered differential miRNA expression in rectal tissue.These data support the importance of the TGF-β signaling pathway to the carcinogenic process, possibly through their influence on miRNA expression levels.

Background: Intestinal fibrosis is characterized by abnormal production and deposition of extracellular matrix (ECM) proteins by activated myofibroblasts. The main progenitor cells of activated myofibroblasts are the fibroblasts and the epithelial cells, the latter through the epithelial-mesenchymal transition (EMT)., Aim: To evaluate the action of the new PPAR-γ modulator, GED-0507-34 Levo (GED) on the expression of EMT associated and regulatory proteins such as TGF-β, Smad3, E-cadherin, Snail, ZEB1, β-catenin, and GSK-3β, in a mouse model of DSS-induced intestinal fibrosis., Methods: Chronic colitis and fibrosis were induced by oral administration of 2.5% DSS (w/v) for 6 weeks. GW9662 (GW), a selective PPAR-γ inhibitor, was also administered by intraperitoneal injection at the dose of 1 mg/kg/day combined with GED treatment. All drugs were administered at the beginning of the second cycle of DSS (day 12). 65 mice were randomly divided into five groups (H2O as controls n = 10, H2O+GED n = 10, DSS n = 15, DSS+GED n = 15, DSS+GED+GW n = 15). The colon was excised for macroscopic examination and histological and morphometric analyses. The level of expression of molecules involved in EMT and fibrosis, like TGF-β, Smad3, E-cadherin, Snail, ZEB1, β-catenin, GSK-3β and PPAR-γ, was assessed by immunohistochemistry, immunofluorescence, western blot and Real Time PCR., Results: GED improved the DSS-induced chronic colitis and fibrosis. GED was able to reduce the expression of the main fibrosis markers (α-SMA, collagen I-III and fibronectin) as well as the pivotal pro-fibrotic molecules IL-13, TGF-β and Smad3, while it increased the anti-fibrotic PPAR-γ. All these GED effects were nullified by co-administration of GW with GED. Furthermore, GED was able to normalize the expression levels of E-cadherin and β-catenin and upregulated GSK-3β, that are all known to be involved both in EMT and fibrosis., Conclusions: The DSS-induced intestinal fibrosis was improved by the new PPAR-γ modulator GED-0507-34 Levo through the modulation of EMT mediators and pro-fibrotic molecules and through GSK-3β induction.

The hypothesis of the local recurrence of preneoplastic lesions was first put forward in the 1950s. Disease recurrence may result from an inherent imbalance in cell proliferation that promotes carcinogenesis in apparently normal mucosa. Our review sheds light on how early preneoplastic lesions could be used to diagnose relapsed preneoplastic and, developing neoplastic lesions. We focus in detail on the clinical-pathological and molecular features of adenoma subtypes and their role in relapsed adenoma and their development into colorectal carcinoma. Moreover, we include the data available on microbiota and its metabolites and their role in recurrence. We strongly believe that a significant improvement could be achieved in colorectal screening by introducing personalized endoscopic surveillance for polyp-bearing patients on the basis of the presence of molecular markers that are predictive of recurrence.

Aim: To test the feasibility of conducting parallel analyses of circulating T-cells in blood and intratumoural T-cells in colorectal cancer. A pre-operative 'liquid biopsy' to determine immune status would facilitate clinical decision-making., Materials and Methods: A total of 18 patients with stage I-III colorectal cancer (CRC) were included. Blood was analyzed for T-cell type (CD3 + , CD4 + and CD8 + ) and count using flow cytometry. Intratumoural T-cells were stained using immunohistochemistry and quantified by digital pathology. Tumour location was defined as invasive front (IF) or tumour center (TC)., Results: The number of CD3 + and CD4 + T-cells in pre-surgical blood samples correlated with the number of CD3 + T-cells found in the IF (Spearman ϱ=0.558, p<0.05 and 0.598, p<0.01 respectively) and CD3 + in the TC (ϱ=0.496, p<0.05, and ϱ=0.637, p<0.01, respectively). A strong correlation was found between CD4 + cells in blood and CD8 + T-cells found in the TC and IF (ϱ=0.602 and ϱ=0.591, p<0.01)., Conclusion: There is a correlation between blood CD3 + and CD4 + T-cells and the T-cells found at the TC and IF., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy., Design: We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints., Results: At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirin's pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=-0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention., Conclusions: We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice., Trial Number: NCT00468910., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. The majority of studies to date focused on genetic mutations and epigenetic changes that drive the CRC carcinogenesis process. Xenobiotic transporters play an important role in safeguarding our body from external toxic substances. These transporters lining the gastrointestinal tract protect us from dietary carcinogens. This study aimed to investigate the downregulation of an efflux transporter ABCG2 in CRC versus normal colon mucosa, so as to shed light on its relevance to CRC initiation and progression. We found that ABCG2 expression is at least 50-fold lower in adenomatous polyps and colon carcinoma specimens obtained from CRC patients than in their matched pair of adjacent normal colon mucosa. The underlying mechanism(s) for ABCG2 under-expression in CRC is currently not known. To this end, aberrant promoter methylation of ABCG2 has been reported to cause its repression in a few cancer types including renal carcinoma and multiple myeloma. In this study, miR-203 was found to be downregulated in all polyps and CRC specimens, relative to adjacent normal colon mucosa. We demonstrated that the de novo DNA methyltransferase DNMT3b is a direct target of miR-203. Importantly, by relieving the repression on DNMT3b, the lower expression of miR-203 in CRC caused ABCG2 promoter methylation and remarkable lower ABCG2 expression in colon cancer cell lines and the patient CRC specimens. The restoration of ABCG2 function via modulating this new microRNA-methylation mechanism in precancerous cells may represent an attractive strategy to delay the carcinogenesis process. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

T cell immunoglobulin mucin-3 (Tim-3) has previously been implicated in the immune response and tumor biology. Colorectal carcinoma (CRC) is a malignancy, which is closely associated with inflammation. However, the role of Tim‑3 in the progression of CRC remains to be fully elucidated. The present study aimed to investigate the role of Tim‑3 in the progressive activities of human CRC. A total of 30 clinical CRC tissues and their adjacent tissues were collected. Slides from another 112 cases that underwent CRC surgical resection were also obtained. The protein and mRNA levels of Tim‑3 in the clinical tissues and in CRC cell lines were initially examined using western blot and reverse transcription‑quantitative polymerase chain reaction analyses, respectively. Immunohistochemical staining was performed to detect Tim‑3 in the CRC samples. Specific small interfering (si)RNA against Tim‑3 (siTim‑3) was synthesized to knock down the expression of Tim‑3, and the subsequent effects of Tim‑3 knockdown on cell proliferation, migration and invasion were assessed. The data obtained showed that Tim‑3 was expressed at high levels in the CRC tissues, compared with the non‑cancerous tissues. The expression of Tim‑3 in the clinical tissues was significantly associated with tumor size (P=0.007), tumor‑node‑metastasis staging (P<0.0001) and distant metastasis (P<0.0001). Knockdown of Tim‑3 significantly reduced the cell proliferative rate of HCT116 and HT‑29 cells. Wound closure activity was also inhibited by knockdown of Tim‑3 in these two cell lines, and the migration and invasive abilities of these two cell lines were consistently decreased following knockdown of Tim‑3. Taken together, Tim‑3 was found to be a critical mediator in the progression of CRC and may serve as a potential therapeutic target for the treatment of CRC.

Methyl jasmonate is found universally in the plant kingdom and functions to regulate plant growth and development, as well as in stress responses through signal transduction pathways. The present study aimed to investigate the anticancer effect of methyl jasmonate on SW620 human colorectal cancer cells and its potential underlying mechanism. SW620 cells were treated with 0, 0.5, 0.75, 1.5 and 2.0 mM methyl jasmonate for 12, 24 and 48 h. Methyl jasmonate was shown to be able to inhibit cell growth and induce apoptosis of SW620 cells in a concentration and time‑dependent manner, whilst promoting an increase in caspase‑3 protein expression. Compared with control, the anticancer effect of methyl jasmonate inhibited Enhancer of zeste homolog 2 (EZH2) protein expression and activated microRNA (miR)‑101 expression in SW620 cells. However, knockdown of miR‑101 suppresses methyl jasmonate‑induced cell growth inhibition, activation of caspase‑3 expression and inhibition of EZH2 expression in SW620 cells. These results demonstrate that methyl jasmonate induced the apoptosis of human colorectal cancer cells via downregulation of EZH2 expression by miR‑101.

As the most common type of cancer and the second leading cause of cancer-associated mortality, colorectal cancer (CRC) has received increasing attention. The aim of the present study was to investigate the mechanisms of CRC by analyzing the microarray dataset, GSE32323. The GSE32323 dataset was downloaded from the Gene Expression Omnibus, and included 17 pairs of matched cancer and normal colorectal tissue samples. The differentially expressed genes (DEGs) were screened using the Linear Models for Microarray Data package and a search of CRC genes, also denoted as seed genes, was performed using the Online Mendelian Inheritance in Man database. Subsequently, the protein‑protein interaction (PPI) network was downloaded from the Search Tool for the Retrieval of Interacting Genes database and the sub‑network (CRC.PPI) of the DEGs and seed genes were obtained. In addition, the top 50 nodes with highest affinity scores in the CRC.PPI were identified using random walk with restart analysis. The potential functions of the DEGs included in the top 50 nodes were analyzed using the Database for Annotation, Visualization and Integrated Discovery online tool. Using the Drug Gene Interaction database, drug‑gene interaction analysis was performed to identify antineoplastic drug interacts with genes. A total of 1,640 DEGs between the CRC and normal samples were screened. The obtained seed genes included cyclin D1 (CCND1) and aurora kinase A (AURKA). The enriched functions for the 31 DEGs in the PPI network of the top 50 nodes were predominantly associated with cell cycle. The DEGs may function in CRC by interacting with other genes in the PPI network of the top 50 nodes, for example, DEP domain‑containing MTOR‑interacting protein (DEPTOR)‑CCND1, AURKA‑breast carcinoma amplified sequence‑1 (BCAS1), CCND1‑BCAS1, CCND1‑neural precursor cell expressed developmentally downregulated 9 (NEDD9) and CCND1‑mitogen‑activated protein kinase kinase 2 (MAP2K2). Only three DEGs (CCND1, AURKA and DEPTOR) had interactions with their corresponding antineoplastic drugs. Taken together, DEPTOR, AURKA, CCND1, BCAS1, NEDD9 and MAP2K2 may act in CRC.

Scutellarin is a flavonoid isolated from a medicinal herb Scutellaria barbata D. Don and exerts therapeutic effects on cardiovascular diseases. However, it remains unclear whether Scutellarin exhibits anti‑tumor actions on human colon cancer. The current study aimed to investigate whether Scutellarin produces antiproliferative and pro‑apoptotic effects on HCT‑116 human colon cancer cells and to elucidate the mechanisms involved. Human colon cancer cells were exposed to different concentrations of Scutellarin, and cellular growth and apoptosis were evaluated by MTT assay, terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling (TUNEL) staining, western blot analysis and other assays. A cell viability assay demonstrated that Scutellarin treatment reduced the viability of HCT‑116 cells in a dose‑ and time‑dependent manner. TUNEL staining demonstrated that Scutellarin also induced apoptotic changes in HCT‑116 cells. The expression level of the anti‑apoptotic protein, Bcl‑2 apoptosis regulator (Bcl‑2), was reduced by Scutellarin in HCT‑116 cells, whereas the expression Bcl‑2 associated X apoptosis regulator (Bax) and the activation of caspase‑3 protein were increased by Scutellarin treatment. Further investigation revealed that Scutellarin significantly increased the phosphorylation of p53 protein in HCT‑116 cells. Additionally, suppression of p53 using a specific inhibitor, pifithrin‑α, abrogated the pro‑apoptotic effects of Scutellarin in HCT‑116 cells. Collectively, Scutellarin reduced the viability and induced apoptosis of human colon carcinoma cells, potentially by regulating p53 and Bcl‑2/Bax expression. These data suggested that Scutellarin may be useful as a promising anti‑tumor drug for treating colon cancer.