Colonic Fibroblasts Research Articles

Magnetic Lateral Flow Immunoassay for Small Extracellular Vesicles Quantification: Application to Colorectal Cancer Biomarker Detection.

Colorectal cancer (CRC) is the third leading cause of cancer death and the fourth most common cancer in the world. Colonoscopy is the most sensitive test used for detection of CRC; however, their procedure is invasive and expensive for population mass screening. Currently, the fecal occult blood test has been widely used as a screening tool for CRC but displays low specificity. The lack of rapid and simple methods for mass screening makes the early diagnosis and therapy monitoring difficult. Extracellular vesicles (EVs) have emerged as a novel source of biomarkers due to their contents in proteins and miRNAs. Their detection would not require invasive techniques and could be considered as a liquid biopsy. Specifically, it has been demonstrated that the amount of CD147 expressed in circulating EVs is significant higher for CRC cell lines than for normal colon fibroblast cell lines. Moreover, CD147-containing EVs have been used as a biomarker to monitor response to therapy in patients with CRC. Therefore, this antigen could be used as a non-invasive biomarker for the detection and monitoring of CRC in combination with a Point-of-Care platform as, for example, Lateral Flow Immunoassays (LFIAs). Here, we propose the development of a quantitative lateral flow immunoassay test based on the use of magnetic nanoparticles as labels coupled to inductive sensor for the non-invasive detection of CRC by CD147-positive EVs. The results obtained for quantification of CD147 antigen embedded in EVs isolated from plasma sample have demonstrated that this device could be used as a Point-of-Care tool for CRC screening or therapy monitoring thanks to its rapid response and easy operation.

The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation.

Heterotypic interactions between newly transformed cells and normal surrounding cells define tumor’s fate in incipient carcinomas. Once homeostasis has been lost, normal resident fibroblasts become carcinoma-associated fibroblasts, conferring protumorogenic properties on these normal cells. Here we describe the IL1β-mediated interplay between cancer cells and normal colonic myofibroblasts (NCFs), which bestows differential sensitivity to cytotoxic drugs on tumor cells. We used NCFs, their conditioned media (CM), and cocultures with tumor cells to characterize the IL1β-mediated crosstalk between both cell types. We silenced IL1β in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1β is overexpressed in cocultured tumor cells. IL1β enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1β-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGFβ1-driven NCFs. IL1β induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1β-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1β-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells.

IL-33 is produced by colon fibroblasts and differentially regulated in acute and chronic murine colitis

IL-33 is upregulated in ulcerative colitis and has a protective role in chemically-induced acute murine colitis. We aimed to determine whether IL-33 influences Il10−/− chronic colitis and its cellular source in health and during colitis. Il10−/−Il33−/− and Il10−/−Il33+/+ littermates developed colitis of similar severity. Colon Il33 was induced in WT and Il10−/− mice exposed to DSS, but not in unchallenged Il10−/− mice with colitis. Il33-citrine reporter mice showed that Il33-citrine colocalized with α-smooth muscle actin+ myofibroblasts and vimentin+ fibroblasts in WT mice. Citrine+CD74+CD90hi inflammatory fibroblasts were increased with DSS treatment. IL-1β induced Il33 expression in colon myofibroblasts, but colon Il33 expression did not differ between DSS-treated WT and Il1r1−/− mice. In conclusion, deficiency of IL-33 does not alter the severity of chronic colitis in Il10−/− mice. Induction of Il33 upon DSS exposure in WT and Il10−/− mice, but not in unchallenged Il10−/− mice, suggests epithelial injury induces colon IL-33. Fibroblasts are the primary colonic source of IL-33 and IL-33-expressing CD90hiCD74+ fibroblasts are increased during DSS-induced colitis. IL-1β induces Il33 in colon myofibroblasts in vitro, but signaling through the IL-1R1 is not necessary for induction of IL-33 in DSS-induced colitis.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Colon Fibroblasts and Inflammation: Sparring Partners in Colorectal Cancer Initiation?

Simple SummaryColorectal cancer (CRC) is the third most common cause of cancer-related death. Patients suffering inflammatory bowel disease have an increased risk of CRC. It is admitted that CRC found its origin within crypts of the colon mucosa, which host the intestinal stem cells (ISCs) responsible of the tissue renewal. ISC behavior is controlled by the fibroblasts that surround the crypt. During inflammation, the signals delivered by fibroblasts are altered, leading to stem cells’ dysregulation, possibly turning them into cancer-initiating cells. Here, we reviewed the interplays between the fibroblast and the ISCs, possibly leading to the initiation of CRC due to chronic inflammation.Colorectal cancer (CRC) is the third most common cause of cancer-related death. Significant improvements in CRC treatment have been made for the last 20 years, on one hand thanks to a better detection, allowing surgical resection of the incriminated area, and on the other hand, thanks to a better knowledge of CRC’s development allowing the improvement of drug strategies. Despite this crucial progress, CRC remains a public health issue. The current model for CRC initiation and progression is based on accumulation of sequential known genetic mutations in the colon epithelial cells’ genome leading to a loss of control over proliferation and survival. However, increasing evidence reveals that CRC initiation is more complex. Indeed, chronic inflammatory contexts, such as inflammatory bowel diseases, have been shown to increase the risk for CRC development in mice and humans. In this manuscript, we review whether colon fibroblasts can go from the main regulators of the ISC homeostasis, regulating not only the renewal process but also the epithelial cells’ differentiation occurring along the colon crypt, to the main player in the initiation of the colorectal cancer process due to chronic inflammation.

Synthesis of New Biscoumarin Derivatives, In Vitro Cholinesterase Inhibition, Molecular Modelling and Antiproliferative Effect in A549 Human Lung Carcinoma Cells.

A series of novel C4-C7-tethered biscoumarin derivatives (12a–e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC50 = 6.30 µM) and butyrylcholinesterase (BChE, IC50 = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood–brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer’s disease.

Antioxidant and cytotoxic activity studies of sulfur containing glycine imine derivatives MCF-7 and DLD-1 cell lines

Objective: To investigate the antioxidant and cytotoxic activities of sulfur-containing glycine imine derivatives MCF-7 (human breast adenocarcinoma) and DLD-1 (colorectal adenocarcinoma) cell lines. Methods: This study examined the antioxidant activities (25-200 µM) of sulfur-containing glycine imine derivatives via the DPPH, metal chelating and reduction methods. Furthermore the cytotoxic activity of MCF-7, MCF-12A (normal breast epithelial) and DLD-1, CCD-18CO (normal colon fibroblast) were examined with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and RTCA (Real-time Cell Analysis) assays. Results: The antioxidant assay of the metal chelating activity showed significant results (71, 77 and 40% respectively) as compared to knowing synthetic antioxidant (trolox; 95.45, EDTA; 97.06 %). Reducing activity was found to be very low compared to the standard compounds.Compounds were shown to be moderated by DPPH (2,2-Diphenyl-1-picrylhydrazyl) activity, and the IC50 value ranged from 91 to 150. The IC50 values (100 µM) of the MTT and RTCA analyses were similar. Conclusion: The study showed that the compounds had selective and significant antioxidant activities, and we also found that they had cytotoxic effects on MCF-7 and DLD-1 cells.

Oxidative stress cytotoxicity induced by platinum-doped magnesia nanoparticles in cancer cells

The aim of this study was to prepare, characterize, and determine the in vitro anticancer effects of platinum-doped magnesia (Pt/MgO) nanoparticles. The chemical compositions, functional groups, and size of nanoparticles were determined using X-ray diffraction, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, transmission electron microscopy, and scanning electron microscopy. Pt/MgO nanoparticles were cuboid and in the nanosize range of 30–50 nm. The cytotoxicity of Pt/MgO nanoparticles was determined via the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide assay on the human lung and colonic cancer cells (A549 and HT29 respectively) and normal human lung and colonic fibroblasts cells (MRC-5 and CCD-18Co repectively). The Pt/MgO nanoparticles were relatively innocuous to normal cells. Pt/MgO nanoparticles downregulated Bcl-2 and upregulated Bax and p53 tumor suppressor proteins in the cancer cells. Pt/MgO nanoparticles also induced production of reactive oxygen species, decreased cellular glutathione level, and increased lipid peroxidation. Thus, the anticancer effects of Pt/MgO nanoparticles were attributed to the induction of oxidative stress and apoptosis. The study showed the potential of Pt/MgO nanoparticles as an anti-cancer compound.

Soluble Fraction from Lysate of a High Concentration Multi-Strain Probiotic Formulation Inhibits TGF-β1-Induced Intestinal Fibrosis on CCD-18Co Cells.

Fibrosis is a severe complication of chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Current strategies are not fully effective in treating fibrosis; therefore, innovative anti-fibrotic approaches are urgently needed. TGF-β1 plays a central role in the fibrotic process by inducing myofibroblast differentiation and excessive extracellular matrix (ECM) protein deposition. Here, we explored the potential anti-fibrotic impact of two high concentration multi-strain probiotic formulations on TGF-β1-activated human intestinal colonic myofibroblast CCD-18Co. Human colonic fibroblast CCD-18Co cells were cultured in the presence of TGF-β1 to develop a fibrotic phenotype. Cell viability and growth were measured using the Trypan Blue dye exclusion test. The collagen-I, α-SMA, and pSmad2/3 expression levels were evaluated by Western blot analysis. Fibrosis markers were also analyzed by immunofluorescence and microscopy. The levels of TGF-β1 in the culture medium were assessed by ELISA. The effects of commercially available probiotic products VSL#3® and Vivomixx® were evaluated as the soluble fraction of bacterial lysates. The results suggested that the soluble fraction of Vivomixx® formulation, but not VSL#3®, was able to antagonize the pro-fibrotic effects of TGF-β1 on CCD-18Co cells, being able to prevent all of the cellular and molecular parameters that are related to the fibrotic phenotype. The mechanism underlying the observed effect appeared to be associated with inhibition of the TGF-β1/Smad signaling pathway. To our knowledge, this study provides the first experimental evidence that Vivomixx® could be considered to be a promising candidate against intestinal fibrosis, being able to antagonize TGF-β1 pro-fibrotic effects. The differences that were observed in our fibrosis model between the two probiotics used could be attributable to the different number of strains in different proportions.

Cytotoxic Activities of Methanol Extract and Compounds of Porodaedalea pini Against Colorectal Cancer

Porodaedalea pini is a medicinally important mushroom with antioxidant, cytotoxic, immunostimulating, antitumor, antiviral and immunomodulating activities. Therefore, in this study, P. pini methanol extract and isolated compounds from the methanol extract were tested for cytotoxic activities against DLD-1 (colorectal cancer) and CCD-18Co (human colon fibroblast cell line) by using Alamar Blue assay. Cytotoxic activity on DLD-1 was decreased in the order of P. pini methanol extract> 4-(3,4-dihydroxyphenyl)but-3-en-2-one (3)> pinoresinol (2)> ergosta-7,24(28)-dien-3β-ol (1). P. pini methanol extract was determined to have the best cytotoxic activity with the lowest IC50 value on DLD-1 (IC50: 25.33±0.29 µg/mL) and the highest IC50 value on CCD-18Co (434.30±1.45 µg/mL). Within the scope of the findings, it is thought that P. pini mushroom can be used as a new and natural agent in the treatment of colorectal cancer.

(E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) induces cytoprotection in CCD18-Co human colon fibroblast cells through Nrf2/ARE pathway activation

Cytoprotection involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important preventive strategy for normal cells against carcinogenesis. In our previous study, the chemopreventive potential of (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been elucidated through its cytoprotective effects against DNA and mitochondrial damages in the human colon fibroblast CCD-18Co cell model. Therefore this study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection and the involvement of the Nrf2/ARE pathway. The cells were pretreated with BK3C231 before exposure to carcinogen 4-nitroquinoline N-oxide (4NQO). BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), as well as restoring the expression of glutamate-cysteine ligase catalytic subunit (GCLC) back to the basal level. Furthermore, dissociation of Nrf2 from its inhibitory protein, Keap1, and ARE promoter activity were upregulated in cells pretreated with BK3C231. Taken together, our findings suggest that BK3C231 exerts cytoprotection by activating the Nrf2 signaling pathway which leads to ARE-mediated upregulation of cytoprotective proteins. This study provides new mechanistic insights into BK3C231 chemopreventive activities and highlights the importance of stilbene derivatives upon development as a potential chemopreventive agent.

A Direct and Sensitive Method for Determination of 5-Fluorouracil in Colorectal Cancer Cells: Evaluating the Effect of Stromal Cell on Drug Resistance of Cancer Cells.

Fibroblasts in the stroma play a critical role in tumor evolution. In this study, we assessed the influence of colonic fibroblasts on colon cancer cells treated with 5-fluorouracil (5-FU), and mouse colon cancer cell lines MC38 and colonic fibroblasts NIH3T3 were used in this study. A sensitive and rapid UHPLC-MS/MS method for the quantitation of 5-FU from the cell and their medium has been successfully developed and validated. The cells were lysed with methanol, and the mixture was evaporated and then redissolved to extract intracellular 5-FU. The analysis was performed on UHPLC-MS/MS using an Atlantis T3-C18 column (3 μm, 2. 1 ∗ 100 mm) and gradient elution with acetonitrile and 0.1% formic acid in water. Method validation included the following parameters: the matrix effect range 88.82%–93.64% and the recovery range 93.52%–94.56%. The intraday and interday precision and accuracy were <11% and within ±6%, and the stability, specificity, carry-over, dilution effect, and linearity all conformed to the criteria. The method was applied to detect the concentration of 5-FU inside cells and cell culture medium. The preliminary results present that NIH3T3 could enhance the drug resistance of MC38 to 5-FU with a decreased intracellular concentration of 5-FU in MC38, which showed a positive relationship with NIH3T3 number.

Green Curmin reduces pro-inflammatory cytokines and fibroblast-associated colon cancer migration

Introduction: Green Curmin is a soluble curcumin extract product made in Thailand and reported to reduce gastric inflammation. Inflammatory cytokines including IL-1α, IL-6 and TNF-β play a crucial role in cancer cell migration, which is a major process towards poor prognosis. Objective: To investigate the effects of Green Curmin on colon cancer cell growth and migration, which involve the pro-inflammatory cytokines. Method: Curcuma longa, L. were extracted and named as Green Curmin. The cytotoxicity of the Green Curmin-treated HCT116-colon cancer cells was obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The effect of the extract on cancer migration was investigated with wound healing assay. In addition, fibroblast-associated cancer migration was confirmed by using Transwell migration assay. Pro-inflammatory cytokines were also determined. Results: Green Curmin had cytotoxicity in the induced-cancer cell apoptosis at concentrations of 1 and 5 mg/ml. However, at concentrations of 0.05 and 0.1 mg/ml (non-toxicity dose), andreduced colon cancer migration and fibroblast associated-cancer migration. They also suppressed pro-inflammatory cytokines expression including IL-1α, IL-6 and TNF-β in colon cancer cells and IL-6 in fibroblast cells. Conclusion: Green Curmin suppressed pro-inflammatory cytokines and reduced fibroblast associated-cancer migration.

Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-β1 synthesis in indirect co-cultures with normal colon fibroblasts.

Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-β1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-β1. The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-β1, while the NEP downregulation decreased the synthesis of TGF-β1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-β1 production. The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-β1 synthesis during paracrine interactions with NCFs.

CaMKII Mediates TGFβ1-Induced Fibroblasts Activation and Its Cross Talk with Colon Cancer Cells.

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. TGFβ1 has been the mostly accepted factor to fuel normal fibroblasts transformation into CAFs. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to play an important role in fibroblasts activation induced by TGFβ1. The aim of this study is to investigate the potential role of CaMKII in TGFβ1-induced fibroblasts activation and CAF-like differentiation. Cross talk between CaMKII-dependent fibroblasts and colon cancer in colon cancer progression also was addressed RESULTS: Immunostaining demonstrated that in colon cancer stroma, CaMKII overexpressed in stromal CAFs. In vitro, TGFβ1 increased CAF markers expression in human colon fibroblasts CCD-18Co, but not in CaMKII depletion fibroblasts. CaMKII knockdown by CaMKII shRNA significantly inhibited TGFβ1-induced fibroblasts activation and CAF-like differentiation. Smad3, AKT, and MAPK were targeted in TGFβ1-CaMKII-mediated pathway. Human colon cancer cell line HCT-116 activated fibroblasts directly, whereas CaMKII depletion dragged CCD-18Co fibroblasts undergoing CAF-associated trans-differentiation. Furthermore, increased proliferation, migration, and invasion of colon cancer cells were stimulated when co-cultured with normal fibroblasts, but not with CaMKII depletion fibroblasts. These findings provide evidence that CaMKII is a critical mediator in TGFβ1-induced fibroblasts activation and is involved in the cross talk with colon cancer cells. CaMKII is a potentially effective target for future treatment of colon cancer.

Pyrazolo[3,4-d]pyrimidine-based dual EGFR T790M/HER2 inhibitors: Design, synthesis, structure-activity relationship and biological activity as potential antitumor and anticonvulsant agents.

A new series of pyrazolo[3,4-d]pyrimidine/triazine hybrids 6a-r was designed as antitumor and anticonvulsant agents. All the prepared compounds were evaluated against colon (HCT-116), breast (MCF-7) and normal human fibroblast (WI38) cell lines. The most potent derivatives against HCT-116 and MCF-7cells were 6o and 6q, with IC50=4.80 and 6.50nM, respectively, when compared to lapatinib, the reference drug (IC50=12.00 and 21.00nM, on HCT-116 and MCF-7, sequentially). All other derivatives exhibited good to moderate cytotoxic activity. Four compounds 6f, 6j, 6o and 6q were evaluated for their EGFR T790M/HER2 inhibitory activity. They revealed 81.81-65.70% and 86.66-54.49% inhibitory activity against EGFR T790M and HER2 in a sequent. The most potent derivatives 6o and 6q were further estimated for cell cycle analysis showing pre G1 apoptotic activity and cell growth arrest at G2/M phase. Apoptotic marker proteins expression levels (caspase-3/7/9, Bax and Bcl-2) were measured for 6o and 6q. They showed pro-apoptotic effect by increasing caspase-3/7/9 protein levels and Bax/Bcl-2 ratio. Moreover, anticonvulsant activity for the prepared compounds 6a-r were evaluated invivo using lithium-pilocarpine mice model of Status Epilepticus. EEG changes where recorded and MDA, GSH, GABA and glutamate were measured in brain tissue of different groups. All tested compounds revealed variable anti-epileptic effects, the most potent compounds were 6b and 6m. Also 6d, 6e, 6h, 6i, 6k, 6l and 6n compounds exhibited good anti-seizure activity, while compound 6j showed the lower activity. The rest of compounds displayed a neutral activity.

Vitamin D receptor inhibits EMT via regulation of the epithelial mitochondrial function in intestinal fibrosis

We previously showed that the vitamin D receptor (VDR) plays a crucial role in acute inflammatory bowel disease and that intestinal fibrosis is a common complication of Crohn’s disease (CD). Epithelial–mesenchymal transition (EMT) is an important hallmark of fibrogenesis through which epithelial cells lose their epithelial phenotype and transform into mesenchymal cells. It is known that the VDR plays an essential role in epithelial integrity and mitochondrial function, but its role in intestinal fibrosis remains unknown. Here, we investigated whether the VDR is involved in epithelial mitochondrial dysfunction that results in EMT in intestinal fibrosis. Using human CD samples, intestine-specific VDR-KO mice, and fibroblast cellular models, we showed that the expression of the VDR was significantly lower in intestinal stenotic areas than in nonstenotic areas in patients with chronic CD. Genetic deletion of the VDR in the intestinal epithelium exacerbated intestinal fibrosis in mice administered with dextran sulfate sodium or 2,4,6-trinitrobenzene sulfonic acid, two experimental colitis inducers. In addition, we found that vitamin D dietary intervention regulated intestinal fibrosis by modulating the intestinal expression of the VDR. Mechanistically, knocking down the VDR in both CCD-18Co cells and human primary colonic fibroblasts promoted fibroblast activation, whereas VDR overexpression or VDR agonist administration inhibited fibroblast activation. Further analysis illustrated that the VDR inhibited EMT in the HT29 cell model and that mitochondrial dysfunction mediated epithelial integrity and barrier function in VDR-deficient epithelial cells. Together, our data for the first time demonstrate that VDR activation alleviates intestinal fibrosis by inhibiting fibroblast activation and epithelial mitochondria-mediated EMT.

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

Heat Shock Factor 1-dependent extracellular matrix remodeling mediates the transition from chronic intestinal inflammation to colon cancer

In the colon, long-term exposure to chronic inflammation drives colitis-associated colon cancer (CAC) in patients with inflammatory bowel disease. While the causal and clinical links are well established, molecular understanding of how chronic inflammation leads to the development of colon cancer is lacking. Here we deconstruct the evolving microenvironment of CAC by measuring proteomic changes and extracellular matrix (ECM) organization over time in a mouse model of CAC. We detect early changes in ECM structure and composition, and report a crucial role for the transcriptional regulator heat shock factor 1 (HSF1) in orchestrating these events. Loss of HSF1 abrogates ECM assembly by colon fibroblasts in cell-culture, prevents inflammation-induced ECM remodeling in mice and inhibits progression to CAC. Establishing relevance to human disease, we find high activation of stromal HSF1 in CAC patients, and detect the HSF1-dependent proteomic ECM signature in human colorectal cancer. Thus, HSF1-dependent ECM remodeling plays a crucial role in mediating inflammation-driven colon cancer.

Tropinone-Derived Alkaloids as Potent Anticancer Agents: Synthesis, Tyrosinase Inhibition, Mechanism of Action, DFT Calculation, and Molecular Docking Studies.

A new series of hybrid compounds with tropinone and thiazole rings in the structure was designed and synthesized as potential anticancer agents. They were tested against human multiple myeloma (RPMI 8226), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), and mouse skin melanoma (B16-F10) cell lines. Toxicity was tested on human normal skin fibroblasts (HSF) and normal colon fibroblasts (CCD-18Co). The growth inhibition mechanism of the most active derivative was analyzed through investigation of its effect on the distribution of cell cycle phases and ability to induce apoptosis and necrosis in RPMI 8226 and A549 cancer cells. The tyrosinase inhibitory potential was assessed, followed by molecular docking studies. Compounds 3a–3h show high anticancer activity against MDA-MB-231 and B16-F10 cell lines with IC50 values of 1.51–3.03 µM. Moreover, the cytotoxic activity of the investigated compounds against HSF and CCD-18Co cells was 8–70 times lower than against the cancer cells or no toxicity was shown in our tests, with derivative 3a being particularly successful. The mechanism of action of compound 3a in RPMI 8226 cell was shown to be through induction of cell death through apoptosis. The derivatives show ability to inhibit the tyrosinase activity with a mixed mechanism of inhibition. The final molecular docking results showed for IC50 distinct correlation with experiment.