Colonic Fibroblasts Research Articles

Stromal SOX2 Upregulation Promotes Tumorigenesis through the Generation of a SFRP1/2-Expressing Cancer-Associated Fibroblast Population.

Cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. We show that the upregulation of SOX2 is central to this process, which is repressed by protein kinase Cζ (PKCζ). PKCζ deficiency activates the reprogramming of colonic fibroblasts to generate a predominant SOX2-dependent CAF population expressing the WNT regulator Sfrp2 as its top biomarker. SOX2 directly binds the Sfrp1/2 promoters, and the inactivation of Sox2 or Sfrp1/2 in CAFs impaired the induction of migration and invasion of colon cancer cells, as well as their tumorigenicity invivo. Importantly, recurrence-free and overall survival of colorectal cancer (CRC) patients negatively correlates with stromal PKCζ levels. Also, SOX2 expression in the stroma is associated with CRC T invasion and worse prognosis of recurrence-free survival. Therefore, the PKCζ-SOX2 axis emerges as a critical step in the control of CAF pro-tumorigenic potential.

Inositol-Requiring Kinase 1 Regulates Apoptosis via Inducing Endoplasmic Reticulum Stress in Colitis Epithelial Cells.

Endoplasmic reticulum stress (ERS) has been studied as critical factor during occurrence and development of ulcerative colitis (UC). However, the role of ERS in inflamed UC remains unclear. The purpose of this study was to analyze the role of inositol-requiring kinase 1 (IRE-1), a major regulator of ER, in regulating ERS and cell viability. In UC mucosa tissue, IRE-1, BiP, XBP-1s, CHOP caspase-12 and GADD34 mRNA were assayed by qRT-PCR. Then, human normal colon epithelial cell line (NCM-460) and colon fibroblast cell line (CCD-33Co) were cultured, and downregulated or upregulated IRE-1 expression. ERS was induced with 100ng/mL of Interleukin 6 (IL-6). CCK8 assay was performed to analyze cell proliferation. Flow cytometry analysis was conducted to detect the apoptosis. Western blot assay was used to examine ERS markers. IRE-1, BiP, XBP-1s, caspase-12 and CHOP mRNA were highly expressed in UC mucosa tissue, and the expression of GADD34 mRNA significantly decreased. These results show that ERS-induced unfolded protein response was enhanced in UC mucosa tissue. In cells, silencing the expression of IRE-1 could suppress cell proliferation and promote apoptosis through activating unfolded protein response, while the over-expression of IRE-1 had the opposite effect. IL-6 could induce ERS and cells apoptosis. Furthermore, we demonstrated that shRNA IRE-1 could enhance the inhibition of IL-6 on cells viability. Inhibition of IRE-1 enhanced unfolded protein response and cells apoptosis and IL-6-induced ERS and suggested that IRE-1 might be a potential target of UC.

The Effect of Carthamus tenuis Extracts on the Cell Proliferation of Different Tumor Cell Lines

Background: Carthamus tenuis is one of the medicinal plants that was used traditionally to treat skin diseases, hemorrhoids, abortion, infertility. It also showed an immunosuppressive role as well as antifungal, antibacterial, anti-inflammatory activity. Although this plant is widespread, there are few studies about its medical applications. Objectives: This study was done to explore the anticancer activity of this plant. Materials and Methods: The aerial parts of the plant were dried, grinded and extracted with hexane, ethyl acetate, and methanol. The extracts were applied in different concentrations to cell cultures of breast (MCF-7), colon (HT-29), prostate (PC-3) and colorectal (CaCo-2) cell lines and fibroblast (MRC- 5) was used as a control. The anticancer activity was evaluated by 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium (MTT) reduction assay that was measured by spectrophotometer. Results: The results showed that methanol extract significantly (p<0.05) have the highest inhibitory activity on MCF-7, HT-29, PC-3, and CaCo-2 with IC50; (25.52 μg/ml), (17.37 μg/ml), (25.77 μg/ml), (24.49 μg/ml), respectively. Followed by ethyl acetate extract that moderately inhibit cell growth of PC-3 and CaCo-2 with IC50; (28.99 μg/ml) and (21.45 μg/ml), respectively. n-hexane extract showed no significant inhibitory effect on all cell lines; IC50 (125.52 -152.34 μg/ml) when compared to Tamoxifen drug activity as a positive control. Conclusion: Results of this study showed the anticancer activity of the plant extracts in four different kinds of cancers that need further study.

Expression of VEGF-A And COX-2 mRNA in non-steroidal anti-inflammatory drugs treated rat primary colonic fibroblast

Non-steroidal anti-inflammatory drugs (NSAIDs) is often used to shorten recovery time after surgery, including after colon anastomosis surgery. Studies showed that NSAIDs might involve in the development of colon anastomotic leakage. However, the effect of NSAIDs in colon anastomosis leakage is still a subject of controversy. Studies indicated that selectivity of COX-2 might have a role in the deleterious effect of NSAIDs in colon anastomosis. Disruption of VEGF-A by NSAIDs also suspected to be the culprit in the development of anastomosis leakage during NSAIDs treatment. This study aimed to investigate the NSAIDs effect toward VEGF-A and COX-2 mRNA in rat primary colonic fibroblast. The in vitro study was conducted using fibroblast isolated from rat colon. The isolated fibroblast was divided into 4 groups of treatment i.e.controlgroup, acetaminophen group, metamizole group, and ketorolacgroup. After 48 h of treatment, the cell was harvested and the RNA was isolated. The expression of VEGF-A and COX-2 mRNA was conducted using semi-quantitative PCR(sq-PCR). Both VEGF-A and COX-2 were not expressed in untreated rat colon fibroblast. However, VEGF-A mRNA washighly expressed in the ketorolacgroup. Interestingly, COX-2 mRNA couldbe seen in the ketorolac and metamizole groups but not in the acetaminophen group. The COX-2 mRNA expression wasthe highest in ketorolac treated rat colon fibroblast. It can be concluded that the effect of various kinds of NSAIDs towards VEGF-A and COX-2 mRNA expression of colon fibroblasts is different. This condition is duetotheir different inhibitory selectivity towards COX-1 and COX2.

Determining a correction coefficient for dielectric properties between in vivo and ex vivo tumor

This paper determines the possibility of generating a correlation or correction factor for the dielectric properties between in vivo and ex vivo tumor tissue measurements. The experiments performed include injecting the backs of C57BL/6 mice with colonic fibroblasts, which are derived from colonic tumors developed in the same species of mice. After the tumors grew to at least 9.5 mm in diameter, dielectric readings of the tumorous tissues and surrounding tissues are gathered for analysis. Data are collected using a network analyzer of the series E5071C and dielectric properties probe (85070E) over the microwave frequency range between 1-5 GHz. The results conclude that a general correction coefficient could be found ranging from 12.30 to 4.0 in terms of relative permittivity and from 0 to 1.44 in terms of effective conductivity.

Oral administration of curcumin ameliorates pulmonary fibrosis in mice through 15d-PGJ2-mediated induction of hepatocyte growth factor in the colon.

Oral administration of curcumin has been shown to inhibit pulmonary fibrosis (PF) despite its extremely low bioavailability. In this study, we investigated the mechanisms underlying the anti-PF effect of curcumin in focus on intestinal endocrine. In bleomycin- and SiO2-treated mice, curcumin (75, 150 mg· kg-1 per day) exerted dose-dependent anti-PF effect when administered orally or rectally but not intravenously, implying an intestinal route was involved in the action of curcumin. We speculated that curcumin might promote the generation of gut-derived factors and the latter acted as a mediator subsequently entering the lungs to ameliorate fibrosis. We showed that oral administration of curcumin indeed significantly increased the expression of gut-derived hepatocyte growth factor (HGF) in colon tissues. Furthermore, in bleomycin-treated mice, the upregulated protein level of HGF in lungs by oral curcumin was highly correlated with its anti-PF effect, which was further confirmed by coadministration of c-Met inhibitor SU11274. Curcumin (5-40 μM) dose-dependently increased HGF expression in primary mouse fibroblasts, macrophages, CCD-18Co cells (fibroblast cell line), and RAW264.7 cells (monocyte-macrophage cell line), but not in primary colonic epithelial cells. In CCD-18Co cells and RAW264.7 cells, curcumin dose-dependently activated PPARγ and CREB, whereas PPARγ antagonist GW9662 (1 μM) or cAMP response element (CREB) inhibitor KG-501 (10 μM) significantly decreased the boosting effect of curcumin on HGF expression. Finally, we revealed that curcumin dose-dependently increased the production of 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) in CCD-18Co cells and RAW264.7 cells, which was a common upstream of the two transcription factors. Moreover, both the in vitro and in vivo effects of curcumin were diminished by coadministration of HPGDS-inhibitor-1, an inhibitor of 15d-PGJ2 generation. Together, curcumin promotes the expression of HGF in colonic fibroblasts and macrophages by activating PPARγ and CREB via an induction of 15d-PGJ2, and the HGF enters the lungs giving rise to an anti-PF effect.

Fibroblast-Derived Extracellular Vesicles Induce Colorectal Cancer Progression by Transmitting Amphiregulin.

Extracellular vesicles (EV), structures surrounded by a biological membrane, transport biologically active molecules, and represent a recently identified way of intercellular communication. Colorectal cancer (CRC), one of the most common cancer types in the Western countries, is composed of both tumor and stromal cells and the amount of stromal fibroblasts negatively correlates with patient survival. Here we show that normal colon fibroblasts (NCF) release EVs with a characteristic miRNA cargo profile when stimulated with TGFβ, one of the most important activating factors of fibroblasts, without a significant increase in the amount of secreted EVs. Importantly, fibroblast-derived EVs induce cell proliferation in epidermal growth factor (EGF)-dependent patient-derived organoids, one of the best current systems to model the intra-tumoral heterogeneity of human cancers. In contrast, fibroblast-derived EVs have no effect in 3D models where EGF is dispensible. This EV-induced cell proliferation did not depend on whether NCFs or cancer-associated fibroblasts were studied or on the pre-activation by TGFβ, suggesting that TGFβ-induced sorting of specific miRNAs into EVs does not play a major role in enhancing CRC proliferation. Mechanistically, we provide evidence that amphiregulin, transported by EVs, is a major factor in inducing CRC cell proliferation. We found that neutralization of EV-bound amphiregulin blocked the effects of the fibroblast-derived EVs. Collectively, our data suggest a novel mechanism for fibroblast-induced CRC cell proliferation, coupled to EV-associated amphiregulin.

Mast Cell Tryptase Promotes Inflammatory Bowel Disease-Induced Intestinal Fibrosis.

Intestinal fibrosis is the final pathological outcome of chronic intestinal inflammation without specific therapeutic drugs, which leads to ileus and surgical intervention. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM). The role of mast cells (MCs), which are members of the sentinel immune cell population, is unknown in intestinal fibrosis. In this study, we analyzed changes in MCs, tryptase proteins, and ECM components in human fibrotic and control patient intestines. We constructed dextran sodium sulfate-induced intestinal fibrosis models using wild-type mice, MC-reconstituted mice, and MC-deficient mice to explore the role of MCs and tryptase in intestinal fibrosis. The roles and mechanisms of MCs and tryptase on fibroblasts were evaluated using human MCs (HMC-1 and LAD-2), commercial tryptase proteins, human colon fibroblasts (CCD-18Co fibroblasts), the tryptase inhibitor APC366, and the protease-activated receptor-2 (PAR-2) antagonist ENMD-1068. Regardless of whether the colon was a human colon or a mouse colon, the fibrotic intestinal tissue had increased MC infiltration and a higher expression of ECM proteins or genes than that of the control group. The dextran sodium sulfate-induced intestinal fibrosis in MC-deficient mice was alleviated compared with that in wild-type mice. After MC reconstruction in MC-deficient mice, the alleviating effect disappeared. Tryptase, as a content stored in MC granules, was released into fibrotic intestinal tissues in the form of degranulation, resulting in an increased expression of tryptase. Compared with the control group, the tryptase inhibition group (the APC366 group) had reduced intestinal fibrosis. The CCD-18Co fibroblasts, when cocultured with MCs or treated with tryptase proteins, were activated to differentiate into myofibroblasts and secrete more ECM proteins (such as collagen and fibronectin). The underlying mechanism of fibroblast activation by tryptase was the activation of the PAR-2/Akt/mTOR pathway. We found that MC tryptase promotes inflammatory bowel disease-induced intestinal fibrosis. The underlying mechanism is that tryptase promotes the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/ mTOR pathway of fibroblasts.

OR16-06 Age-Associated Local GH Promotes Colon Neoplasia

Colon polyp and cancer frequency increase with age, yet little is known about age-related mechanisms underlying development of these neoplasms. Defective DNA damage response and accumulation of unrepaired DNA damage can trigger genomic instability and cellular transformation.Patients with acromegaly have a higher prevalence of colon polyps and, arguably, colon adenocarcinoma, while those with GH signaling deficiency do not develop cancer. We showed that APC+/- mice that all develop colon adenomas exhibit a significant decrease in the number and volume of colon tumors with deletion of the GH transcription factor Prop1. Further, DNA damage response triggered GH expression in colon cells, and GH, in turn, altered DNA damage repair, resulting in DNA damage accumulation and cell transformation both in vitro and in vivo. These findings prompted us to hypothesize that accumulated DNA damage in aging colon induces local GH, suppressing DNA damage repair and creating a milieu consistent with genomic instability favoring neoplastic development.In human colon tissue we now show increased expression of γH2AX, a marker of DNA breaks, associated with increased GH transcription (detected by RNA scope) and translation (assessed by immunohistochemistry) as well as GH induction in both human and murine colon after DNA damaging radiotherapy. In vitro studies support these results, showing GH induction in normal human colon epithelial cells, fibroblasts, and 3D human intestinal organoids after DNA damage. Of note, local GH was secreted in the medium, indicating a paracrine effect. Paracrine/autocrine GH expression in these cellular models resulted in suppression of p53, induction of EMT, and attenuation of DNA damage response, and accumulated unrepaired DNA damage in human colon cells and in human intestinal organoids. In vivo, colon cells infected with lentivirus expressing GH generated more metastases than did cells expressing control vector. Co-culturing of human normal colon fibroblasts expressing GH together with normal human colon cells led to increased motility and accumulation of DNA damage as well as increased proliferation of epithelial cells on a gut-on-a-chip microfluidic device, confirming paracrine GH effects. In an in vitro model of aging, culturing human intestinal organoids for up to 2 months resulted in decreased telomere length and increased GH mRNA and protein expression associated with suppressed DNA damage response evident by decreased phosphorylation of ATM and DNA-PKcs, both kinases involved in DNA repair, and DNA damage accumulation assessed by Comet assay. Suppression of GH in these aging organoids led to increased phospho-p53 and reduced DNA damage. Although somatotroph axis endocrine activity decreases with age, local GH induced in response to age-related DNA damage may trigger a “field change,” creating a milieu favorable for colon neoplastic development.

MON-443 SPECT/CT Localization of Incidental Diverticular Bleed After Radioiodine (i131) Therapy for Metastatic Thyroid Cancer

Background: Radioiodine therapy for patients with metastatic papillary and follicular thyroid cancer status post total thyroidectomy improves overall survival and is standard of care. Physiologic and pathologic biodistribution of I131 is dependent on tissue sodium iodine symporter expression with nonspecific radioiodine distribution seen secondary to physiologic routes of excretion. Clinical Case: Whole body scintigraphy (WBS) was performed seven days after I131 therapy in a 37-year-old male treated for metastatic papillary thyroid cancer with extension to a thyroglossal duct cyst at the time of surgery. His post-procedural course was uncomplicated with the exception of self-limited hematochezia six days after ablation. WBS images demonstrated a focus more superiorly in the neck concordant with metastatic thyroglossal duct cyst involvement. Activity was also present in the small and large bowel distribution reflecting normal routes of radioiodine excretion. WBS images depicted a discrete focus of radioiodine activity in the right lower quadrant, which SPECT/CT further localizes as activity in the sigmoid colon. Non-contrast CT images demonstrate an associated linear pattern of hyperdense hyperattenuation in the colonic lumen consistent with fresh blood. It is well established that the physiologic and pathologic biodistribution of I131 is dependent on tissue sodium-iodine symporter expression with additional distribution secondary to normal routes of excretion. Nonspecific radioiodine localization has been described at sites of inflammation secondary to vasodilation and increased vascular permeability resulting in leakage and accumulation in tissues. Sodium-iodine expression is found in many tissues, including salivary glands, lactating mammary glands, gastric mucosa, thymus, and small bowel; however, they are not expressed in the colon, nasopharyngeal mucosa, or orbital fibroblasts. I131 localization has been previously described within numerous body diverticula, such as Zenker’s and Meckel’s diverticula; however, radioiodine activity in association with an acute colonic diverticular bleed has not been reported. Conclusion: We propose that the isolated focus of radioiodine activity in the sigmoid colon associated with transient self-limited bleed could be either related to preexisting diverticulitis leading to I131 accumulation secondary to hyperemia, increased vascular capillary permeability, and subsequent aggravation of colonic mucosa resulting in a transient bleed or due to incidental nonspecific pooling of radioiodine in a colonic diverticulum resulting in secondary mucosal irritation with subsequent minor bleeding. To our knowledge this is the first reported case of SPECT/CT localization of radioiodine to an isolated colonic diverticular bleed in a patient status post I131 therapy for metastatic papillary thyroid cancer.

Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells.

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role.

Protective effects of zinc L-carnosine against hydrogen peroxide-induced DNA damage and micronucleus formation in CCD-18co human colon fibroblast cells

Zinc L-carnosine (ZnC) is a chelated compound of zinc and L-carnosine. The present study aims to determine the protective effects of ZnC against hydrogen peroxide (H2O2)-induced oxidative stress and genomic damage in CCD-18co human normal colon fibroblast cells. Generally, cells were pretreated with ZnC (0–100 µM) for 24 h before challenged with 20 µM of H2O2 for 1 h to induce oxidative damage. Results showed that pretreatment with ZnC was able to reduce the intracellular ROS level in CCD-18co cells after being challenged with H2O2. Moreover, pretreatment with ZnC demonstrated protection from H2O2-induced DNA strand breaks and micronucleus formation. Our current findings revealed that pretreatment with ZnC could induce the activation of MTF-1 signaling pathway and expression of metallothionein (MT) in a dose-dependent manner. However, ZnC did not have any effects on Nrf2 signaling pathway and the expression of glutathione, superoxide dismutase 1, and glutamate-cysteine ligase catalytic subunit (GCLC). Furthermore, pretreatment with ZnC did not induce the expression of OGG1 and PARP-1 in CCD-18co cells, suggesting that these two DNA repairing enzymes are not related to the genoprotective effects of ZnC. Since the expression of MT has been demonstrated to protect cells from oxidative DNA damage induced by various genotoxic agents, the genoprotective effects of ZnC might be due to the ability of ZnC to induce the expression of MT. In conclusion, ZnC pretreatment was able to protect CCD-18co cells from H2O2-induced genomic damage via the activation of the MTF-1 signalling pathway and the induction of MT expression.

Endomorphin-2 Analog Inhibits the Growth of DLD-1 and RKO Human Colon Cancer Cells by Inducing Cell Apoptosis.

BackgroundIn developed countries, colon cancer is a leading cause of cancer-associated mortality. Dietary changes have resulted in an increased incidence of colon cancer in Asia. This study aimed to investigate the effects of the structural analog of endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) on human colon cancer cells in vitro.Material/MethodsHuman DLD-1 and RKO colon cancer cells and CCD-18Co normal human colonic fibroblasts were treated with increasing doses of the structural analog of endomorphin-2. Cells underwent the MTT assay, fluorescence confocal flow cytometry, and Hoechst 33258 staining to investigate cell proliferation, the cell cycle, and apoptosis. Western blot was used to measure the expression levels of poly(ADP-ribose) polymerase-1 (PARP-1), cytochrome c, caspase-3, and caspase-9. The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescence method measured reactive oxygen species (ROS).ResultsCell proliferation of DLD-1 and RKO cells was inhibited by the endomorphin-2 analog in a dose-dependent manner, and a 100 μM dose reduced DLD-1 and RKO cell proliferation by 28% and 23%, respectively, at 72 h. Endomorphin-2 analog induced cell apoptosis and the generation of ROS, activated caspase-3 and caspase-9, and increased the levels of p53 and cytochrome c release, and down-regulated of Akt activation in DLD-1 and RKO cells in a dose-dependent manner. Treatment of the DLD-1 and RKO cells with the endomorphin-2 analog increased the expression of Bax and reduced the expression of Bcl-2.ConclusionsEndomorphin-2 analog inhibited colon cancer cell proliferation, activated apoptosis, and down-regulated Akt phosphorylation of human DLD-1 and RKO colon cancer cells in vitro in a dose-dependent manner.

Recapitulation of Hypoxic Tumor–stroma Microenvironment to Study Photodynamic Therapy Implications

Tumor microenvironment (TME) is a dynamic ecosystem where fibroblasts are recruited in order to provide a niche to support growth and, in some extent, to promote therapeutic resistance. However, the role of fibroblasts in stimulating or impairing photodynamic therapy (PDT) outcome has not yet been fully addressed. PDT is based on interactions between light, oxygen and photosensitizer, leading to phototoxic reactions that culminate in cell death. In this study, we demonstrated the consequences of a hypoxic stromal phenotype on tumor mass for exploring PDT response. We mimicked TME complexity implementing colon cancer cells and fibroblasts 3D cultures called spheroids. Using hypoxia reporting lines, we verified that homotypic spheroids exhibited a size-dependent transcriptional HIF-1 activity. When cocultured, fibroblasts were localized in the hypoxic core. In homotypic stromal spheroids, the distribution of the endogenous photosensitizer PpIX was homogeneous while decreased in hypoxic areas of tumor 3D cultures. When monocultured, fibroblasts were more efficient to produce PpIX from its prodrug Me-ALA. Interestingly, the cross talk between cancer cells and fibroblasts attenuated PpIX accumulation and conferred tumor PDT resistance when compared to homotypic 3D cultures. Overall, our data suggest that stroma and tumor act in an integrated, reciprocal fashion which could ultimately influence on therapeutic response.

Zinc L-Carnosine Protects CCD-18co Cells from L-Buthionine Sulfoximine-Induced Oxidative Stress via the Induction of Metallothionein and Superoxide Dismutase 1 Expression.

Zinc L-carnosine (ZnC) is the chelate form of zinc and L-carnosine and is one of the zinc supplements available in the market. This study aims to determine the protective effects of ZnC against L-buthionine sulfoximine (BSO)-induced oxidative stress in CCD-18co human normal colon fibroblast cell line. CCD-18co cells were pretreated with ZnC (0-100μM) for 24h before the induction of oxidative stress by BSO (1mM) for another 24h. Results from this present study demonstrated that ZnC up to the concentration of 100μM was not cytotoxic to CCD-18co cells. Induction with BSO significantly increased the intracellular reactive oxygen species (ROS) levels and reduced the intracellular glutathione (GSH) levels in CCD-18co cells. Pretreatment with ZnC was able to attenuate the increment in intracellular ROS level in CCD-18co cells significantly in a concentration-dependent manner. However, ZnC did not have any effects on intracellular GSH levels and Nrf2 activation. Mechanistically, pretreatment with ZnC was able to upregulate the expression of metallothionein (MT) and superoxide dismutase 1 (SOD1) in CCD-18co cells. Results from dual-luciferase reporter gene assay reported that ZnC was able to increase the MRE-mediated relative luciferase activities in a concentration-dependent manner, suggesting that the induction of MT expression by ZnC was due to the activation of MTF-1 signaling pathway. Taken together, our current findings suggest that ZnC can protect CCD-18co cells from BSO-induced oxidative stress via the induction of MT and SOD1 expression.

The Co2+ Complex of [7‐Hydroxy‐4‐methyl‐8‐coumarinyl]glycine as a Nanocatalyst for the Synthesis and Biological Evaluation of New Mannich Bases of Benzimidazoles and Benzothiazoles

AbstractIn this paper, a series of novel Mannich bases of benzimidazoles and benzothiazoles were synthesized via a one‐pot two steps reaction of 1,2‐phenylenediamines or 2‐aminothiophenols with several in situ synthesized Mannich bases of aldehydes in the presence of nanostructured Co2+ complex of [7‐hydroxy‐4‐methyl‐8‐coumarinyl]glycine ([Co(MCG)(H2O)3] as a reusable and bioactive catalyst. To the best of our knowledge, it is the first report on the application of nanostructured coumarin‐based metal complexes [M(MCG)(H2O)3] for the synthesis of organic compounds. The nanostructured coumarin‐based cobalt complex as a catalyst could be reused up to six times without a decrease in its catalytic activity. The cytotoxic activities of synthesized benzimidazoles were investigated against the colon cancer cell line (HT‐29) and fibroblasts cell lines (HFF) as a non‐cancerous cell line, at 24 and 72 hours after incubation using 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium (MTT) assay.

Wu-Mei-Wan ameliorates chronic colitis-associated intestinal fibrosis through inhibiting fibroblast activation

Ethnopharmacological relevanceWu-Mei-Wan (WMW), a classic traditional Chinese herb medicine, is one of the most important formulations to treat digestive diseases from ancient times to the present. Previous study showed that WMW has satisfactory curative effects on experimental colitis, which motivating the application of WMW on colitis-associated complications. Aim of the studyIntestinal fibrosis is usually considered to be a common complication of inflammatory bowel disease (IBD), particularly Crohn's disease (CD). Currently, no effective preventive measures or medical therapies are available for that. This work was designed to evaluate the effect and related mechanism of WMW on chronic colitis-associated intestinal fibrosis mice model. Materials and methodsThe chronic colitis-associated intestinal fibrosis mice model was established by weekly intrarectal injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The mice survival rate, disease activity index (DAI), colon length and histological score were examined to assess the therapeutic effect of WMW. Masson's trichrome staining, hydroxyproline assay, immunohistochemical staining and western blot analysis were used to evaluate fibrosis level. Colon inflammation was determined by ELISA and immunofluorescence staining. Immunofluorescence staining was used to evaluate fibroblasts proliferation and epithelial to mesenchymal transition (EMT), and the expression of key molecules in fibrosis was analyzed by western blot. ResultsHere we showed that WMW alleviates chronic colitis with improved survival rate, DAI, colon length and histological score. WMW inhibited the progression of intestinal fibrosis, decreased the expression of various fibrosis markers, such as α-SMA, collagen I, MMP-9 and fibronectin. In addition, WMW treatment reduced cytokines IL-6 and IFN-γ, and downregulated proinflammatory NF-κBp65 and STAT3 signaling pathways. Importantly, administration of WMW led to the inhibition of colon fibroblast proliferation and EMT, which are important mediators during fibrosis. Several key profibrotic pathways, including TGF-β/Smad and Wnt/β-catenin pathways, were downregulated by WMW treatment. ConclusionOur work demonstrated that WMW can prevent intestinal fibrosis and the mechanisms involved may be related to the inhibition of colon fibroblasts activation.

DOP85 The role of fibroblasts in the pathogenesis of Crohn’s disease-associated fistulas and in mesenchymal stem cell therapy

Abstract Background Perianal fistulas are a severe and frequent complication in Crohn’s disease (CD) patients, significantly affecting their quality of life. High recurrence rates, incomplete fistula healing and non-responding patients make the treatment challenging. Despite some novel insights, current knowledge about the pathogenesis of fistula formation is still limited. Fibroblasts are abundantly present in CD fistulas and were recently reported to regulate Th1 cell activity in inflammatory bowel disease (IBD) (Beswick EJ et al. 2018). We hypothesised that fibroblasts act as the key drivers of fistula pathogenesis by regulating inflammatory cell recruitment. Methods Fibroblasts were isolated from curettage material of perianal CD fistulas, non-CD-associated fistulas, and the normal colon (obtained at surgery for colorectal cancer at least 10 cm away from the primary tumour). For all experiments, fibroblast populations were identified by qPCR and flow cytometry analysis first. Then gene expression profiling via mRNA sequencing of cultured fistula- and colon-derived fibroblasts was performed. The characterisation was complemented by flow cytometry analysis. Furthermore, the interaction of fistula-derived fibroblasts with regulatory T cells (Tregs) was studied in co-culture experiments. Results mRNA sequencing analysis revealed no significant differences in gene expression between perianal CD fistulas and non-CD-associated fistulas. However, comparing fistula-derived samples to fibroblasts derived from the normal colon, we found over 6000 significantly differentially expressed genes. Among the 20 most significant differentially expressed genes between CD fistulas and colon fibroblasts were genes related to epithelial-mesenchymal transition, cell migration or the NF-κB signalling pathway. Furthermore, co-culture experiments with CD fistula-derived fibroblasts, revealed increased proliferation rates of CD25+FoxP3+ Treg cells. In addition, more CD4+ T cells differentiated into Treg cells after exposure to CD fistula fibroblasts. Conclusion Our data indicate a pathogenic role of fibroblasts located in the fistula tract. Their gene expression profile markedly differed from fibroblasts derived from the normal part of the colon. Based on our data, there is no difference between CD-associated and non-CD-associated fistula fibroblasts. CD fistula-derived fibroblasts seem to increase both the proliferation of Treg cells, and the differentiation of CD4+ T cells into Treg cells, indicating an important immunoregulatory role of fibroblasts. Ongoing experiments should reveal which cytokines and chemokines are involved in this process.

Comparison of metamizole and paracetamol effects on colonic anastomosis and fibroblast activities in Wistar rats

BackgroundLeakage following colorectal anastomosis surgery causes various complications associated with high morbidity and mortality, especially in pediatric patients. It might be caused by the use of non-steroidal anti-inflammatory drugs (NSAIDs) as postoperative analgesics. This study aimed to compare the effect of metamizole and paracetamol on colonic anastomosis and fibroblast activities, including proliferation, migration, and collagen synthesis, in Wistar rats.MethodsRats were divided into control, paracetamol and metamizole groups. The colonic anastomosis was evaluated by determining the integrity of the muscle layers, the formation of granulation tissue, and mucosal anastomosis. Fibroblast activities were analyzed by measuring the proliferation, migration, and collagen synthesis.ResultsMetamizole caused more damage to muscle layer integrity, more inhibition of granulation tissue formation in the anastomosis area and lower mucosal anastomosis compared with paracetamol and control groups. Metamizole had a higher cytotoxic effect than paracetamol, which suppressed the proliferation and migration of fibroblasts. Furthermore, both drugs did not affect the synthesis of collagen.ConclusionMetamizole shows worse effects on the integrity of muscle layers, inhibition of granulation tissue formation, mucosal anastomosis, fibroblast proliferation, and migration, but not collagen synthesis, than paracetamol in Wistar rat intestines following colonic anastomosis. These findings might indicate that paracetamol is safer than metamizole as analgesic following colonic anastomosis.

Bovine HDL and Dual Domain HDL-Mimetic Peptides Inhibit Tumor Development in Mice.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28–2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.