LS174T Cell Research Articles

Urolithin A-mediated augmentation of intestinal barrier function through elevated secretory mucin synthesis

Maintaining the mucus layer is crucial for the innate immune system. Urolithin A (Uro A) is a gut microbiota-derived metabolite; however, its effect on mucin production as a physical barrier remains unclear. This study aimed to elucidate the protective effects of Uro A on mucin production in the colon. In vivo experiments employing wild-type mice, NF-E2-related factor 2 (Nrf2)-deficient mice, and wild-type mice treated with an aryl hydrocarbon receptor (AhR) antagonist were conducted to investigate the physiological role of Uro A. Additionally, in vitro assays using mucin-producing cells (LS174T) were conducted to assess mucus production following Uro A treatment. We found that Uro A thickened murine colonic mucus via enhanced mucin 2 expression facilitated by Nrf2 and AhR signaling without altering tight junctions. Uro A reduced mucosal permeability in fluorescein isothiocyanate-dextran experiments and alleviated dextran sulfate sodium-induced colitis. Uro A treatment increased short-chain fatty acid-producing bacteria and propionic acid concentration. LS174T cell studies confirmed that Uro A promotes mucus production through the AhR and Nrf2 pathways. In conclusion, the enhanced intestinal mucus secretion induced by Uro A is mediated through the actions of Nrf-2 and AhR, which help maintain intestinal barrier function.

Targeting Colorectal Cancer: Unravelling the Transcriptomic Impact of Cisplatin and High-THC Cannabis Extract.

Cisplatin and other platinum-derived chemotherapy drugs have been used for the treatment of cancer for a long time and are often combined with other medications. Unfortunately, tumours often develop resistance to cisplatin, forcing scientists to look for alternatives or synergistic combinations with other drugs. In this work, we attempted to find a potential synergistic effect between cisplatin and cannabinoid delta-9-THC, as well as the high-THC Cannabis sativa extract, for the treatment of HT-29, HCT-116, and LS-174T colorectal cancer cell lines. However, we found that combinations of the high-THC cannabis extract with cisplatin worked antagonistically on the tested colorectal cancer cell lines. To elucidate the mechanisms of drug interactions and the distinct impacts of individual treatments, we conducted a comprehensive transcriptomic analysis of affected pathways within the colorectal cancer cell line HT-29. Our primary objective was to gain a deeper understanding of the underlying molecular mechanisms associated with each treatment modality and their potential interactions. Our findings revealed an antagonistic interaction between cisplatin and high-THC cannabis extract, which could be linked to alterations in gene transcription associated with cell death (BCL2, BAD, caspase 10), DNA repair pathways (Rad52), and cancer pathways related to drug resistance.

Glutathione Reductase Expression and Its Prognostic Significance in Colon Cancer.

Maintaining a balanced redox state within cells is crucial for the sustenance of life. The process involves continuous cytosolic disulfide reduction reactions to restore oxidized proteins to their reduced thiol forms. There are two main cellular antioxidant pathways-the thioredoxin (Trx) and glutathione (GSH)/glutaredoxin (Grx) systems. In the GSH/Grx system, glutathione reductase (GR; GSR) catalyses the reduction of GSH disulfide (GSSG) to its sulfhydryl form (GSH), which can then further reduce oxidized Grxs. GR is an essential enzyme that helps in maintaining the supply of reduced glutathione-GSH, which is a significant reducing thiol found in most cells and known for its antioxidant properties. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of GR protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of GR and tumour histological grade, depth of invasion, regional lymph node involvement, staging, and PCNA immunohistochemical expression. It was found that 95% of patients with stage I had low levels of GR expression, whereas 89% of patients with stage III had high levels of immunohistochemical expression. A high level of expression was also detected in the patients with stage II of the disease, where almost 63% were characterized by a high expression of GR. The Western blot method revealed that the highest level of expression was found in the LS 174T cell line, which corresponds to stage II. The results of our study indicate that the immunohistochemical expression of GR may act as an independent prognostic factor associated with colon adenocarcinoma patients' prognosis.

Photoactive imaging and therapy for colorectal cancer using a CEA-Affimer conjugated Foslip nanoparticle.

Theranostic nanoparticles hold promise for simultaneous imaging and therapy in colorectal cancer. Carcinoembryonic antigen can be used as a target for these nanoparticles because it is overexpressed in most colorectal cancers. Affimer reagents are synthetic proteins capable of binding specific targets, with additional advantages over antibodies for targeting. We fabricated silica nanoparticles using a water-in-oil microemulsion technique, loaded them with the photosensitiser Foslip, and functionalised the surface with anti-CEA Affimers to facilitate fluorescence imaging and photodynamic therapy of colorectal cancer. CEA-specific fluorescence imaging and phototoxicity were quantified in colorectal cancer cell lines and a LS174T murine xenograft colorectal cancer model. Anti-CEA targeted nanoparticles exhibited CEA-specific fluorescence in the LoVo, LS174T and HCT116 cell lines when compared to control particles (p < 0.0001). No toxicity was observed in LS174T cancer mouse xenografts or other organs. Following photo-irradiation, the anti-CEA targeted particles caused significant cell death in LoVo (60%), LS174T (90%) and HCT116 (70%) compared to controls (p < 0.0001). Photodynamic therapy (PDT) at 24 h in vivo showed a 4-fold reduction in tumour volume compared to control mouse xenografts (p < 0.0001). This study demonstrates the efficacy of targeted fluorescence imaging and PDT using Foslip nanoparticles conjugated to anti-CEA Affimer nanoparticles in in vitro and in vivo colorectal cancer models.

124I-labeled anti-CD147 antibody for noninvasive detection of CD147-positive pan-cancers: construction and preclinical studies.

Extracellular matrix metalloproteinase inducer CD147 is a glycoprotein on the cell surface. There is minimal expression of CD147 in normal epithelial and fetal tissues, but it is highly expressed in a number of aggressive tumors. CD147 has been implicated in pan-cancer immunity and progression. With the development of CD147-targeting therapeutic strategy, accurate detection of CD147 expression in tumors and its changes during the therapy is necessary. In this study we constructed a novel radiotracer by labeling the anti-CD147 mAb with radionuclide 124/125I (124/125I-anti-CD147) for noninvasive detection of CD147 expression in pan-cancers, and characterized its physicochemical properties, affinity, metabolic characteristics, biodistribution and immunoPET imaging with 124I-IgG and 18F-FDG as controls. By examining the expression of CD147 in cancer cell lines, we found high CD147 expression in colon cancer cells LS174T, FADU human pharyngeal squamous cancer cells and 22RV1 human prostate cancer cells, and low expression of CD147 in human pancreatic cancer cells ASPC1 and human gastric cancer cells BGC823. 124/125I-anti-CD147 was prepared using N-bromine succinimide (NBS) as oxidant and purified by PD-10 column. Its radiochemical purity (RCP) was over 99% and maintained over 85% in saline or 5% human serum albumin (HSA) for more than 7 d; the RCP of 125I-anti-CD147 in blood was over 90% at 3 h post injection (p.i.) in healthy mice. The Kd value of 125I-anti-CD147 to CD147 protein was 6.344 nM, while that of 125I-IgG was over 100 nM. 125I-anti-CD147 showed much greater uptake in CD147 high-expression cancer cells compared to CD147 low-expression cancer cells. After intravenous injection in healthy mice, 125I-anti-CD147 showed high initial uptake in blood pool and liver, the uptake was decreased with time. The biological half-life of distribution and clearance phases in healthy mice were 0.63 h and 19.60 h, respectively. The effective dose of 124I-anti-CD147 was estimated as 0.104 mSv/MBq. We conducted immunoPET imaging in tumor-bearing mice, and demonstrated a significantly higher tumor-to-muscle ratio of 124I-anti-CD147 compared to that of 124I-IgG and 18F-FDG in CD147 (+) tumors. The expression levels of CD147 in cells and tumors were positively correlated with the maximum standardized uptake value (SUVmax) (P < 0.01). In conclusion, 124/125I-anti-CD147 displays high affinity to CD147, and represents potential for the imaging of CD147-positive tumors. The development of 124I-anti-CD147 may provide new insights into the regulation of tumor microenvironment and formulation of precision diagnosis and treatment programs for tumors.

Cinnamaldehyde alleviates zearalenone-induced LS174T cell apoptosis, barrier dysfunction and mucin reduction through JNK/NF-κB signaling pathway

As a natural aldehyde organic compound, cinnamaldehyde (CA) is one of the main components of cinnamon essential oil with multiple bioactivities. In this study, we investigated the protective effects of CA on zearalenone (ZEA)-induced apoptosis, barrier dysfunction and mucin reduction, as well as underlying mechanisms in LS174T cells. In the present study, cells pre-treated with or without CA for 24 h were left untreated or subjected to ZEA for indicated time points Our results showed that 10 μM CA significantly prevented ZEA-induced cell viability decline, reversed ZEA-induced increase of the LDH level, cell cycle disruption and apoptosis in LS174T cells. Periodic acid-schiff (PAS) staining analysis showed that CA significantly alleviated the reduction of mucin secretion in LS174T cells caused by ZEA exposure. Western blot analysis showed that CA significantly reversed ZEA-induced reduction of the expression of mucin 2 (MUC2) and tight junction (TJ) proteins (claudin-1, claudin-3, ZO-1 and ZO-2) in LS174T cells. Notably, CA can significantly reduce the upregulation of the main effector of MAPK and NF-κB signaling pathways in LS174T cells. Further study showed that CA protects cells against ZEA-induced cellular damage through JNK/NF-κB signaling pathway in LS174T cells. Supplementation with CA might be an potential strategy to alleviate the damaging effect of ZEA on epithelial cells.

Shaoyao decoction restores the mucus layer in mice with DSS-induced colitis by regulating Notch signaling pathway.

Restoring the mucus layer is a potential strategy for treating ulcerative colitis (UC). Previous studies reported that a Chinese medicine formula Shaoyao Decoction (SYD) effectively improved UC. However, the role and mechanism of SYD in restoring the mucus layer are still vague. This study aimed to research the therapeutical effects and unravel the involved mechanism of SYD on DSS-evoked UC. First, the constituents of SYD were detected by UPLC-QTOF-MS/MS. Then, the DSS-induced UC model was introduced to investigate the pharmacologic action and molecular mechanism of SYD on UC. Pharmacodynamic indicators were assessed including body weight, colon length, ulcerations, disease activity index (DAI), inflammatory cytokines and histological parameters. To investigate the integrality and functions of the mucous layer, AB-PAS stain and UEA-1 stain were used to evaluate the completeness of mucous layer, as well as the maturation of goblet cells (GCs). The bacterial invasion was detected by fluorescence in situ hybridization. As to mechanism exploration, the expressions of Notch/Hes1 pathway were investigated by using agonists in lipopolysaccharides (LPS) -stimulated LS174T cell. After modeling in mice, SYD remarkedly ameliorated the symptoms of mouse colitis, the expression of pro-inflammatory factors declined, and increased IL-10 expression was observed in SYD-treated mice. Besides, SYD repaired the structure of the mucus layer and prevented bacterial invasion. Mechanism investigation discovered that SYD promoted GCs differentiation by inhibiting the Notch pathway, which was consistent with the results in LPS-challenged LS174cell. These findings demonstrated that SYD could restore the mucus layer to prevent UC via suppressing the Notch signaling pathway, which provided evidences for the UC treatment of SYD in the clinic.

P925 Deep functional assessment of the colonic mucosa-associated microbiota in Crohn’s disease and health identifies pro- and anti- inflammatory secretomes and associated bacterial consortia. The ENIGMA study

Abstract Background Gut microbial dysbiosis and dysregulated host immune responses are key factors implicated in Crohn’s disease pathogenesis. However, the nexus between the mucosa-associated microbiota (MAM) and epithelial cell immune response(s) remains poorly understood. Here, we used in vitro cultures of LS174T human epithelial cells to examine the inflammatory tenor of the secretomes from colonic MAM recovered from healthy and Crohn’s disease subjects in Hong Kong (where Crohn’s disease incidence is low but increasing) and Australia (globally high incidence). Methods Biopsies from healthy (21 HKG, 20 AUS) and Crohn’s disease (30 HKG, 29 AUS) subjects were collected from the right colon and the MAM propagated from tissue using a habitat simulating medium. After 24h of growth, cultures were centrifuged, and the supernatant (secretome) fraction filter sterilised. For the immune assays, the LS174T cell line was transfected with recombinant lentivirus to provide an NF-κB:luc (luciferase) reporter gene fusion (Cellomics Technology, MD, USA) and aliquots used to inoculate 96-well microtiter plates and cultured for 24 h under standard conditions. Cultures were then challenged for 4 hours with 0.1 vol of a MAM secretome. Luc-bioluminescence was measured as a marker of NF-κB transcriptional activity from the individual wells and normalised to the reporter gene response from cells challenged with sterile, uninoculated bacterial medium. Results Across the 100 MAM secretomes tested, the median score for NF-ĸB activation was 104.4% relative to the medium control, suggesting most of the secretomes possessed a net neutral effect, with the 1st and 4th quartiles deemed stimulatory (&amp;gt;126% baseline activity) and suppressive (&amp;lt;92% baseline activity), respectively. There was no significant relationship found between a secretome’s inflammatory score and its corresponding α-diversity metric, nor was there any distinct clustering of the secretomes according to country of origin and/or case-control groups. However, the relative abundances of Roseburia spp. and Clostridium innocuum were significantly enriched in those consortia producing immunostimulatory secretomes. In contrast, the relative abundances of Erysipelatoclostridium ramosum, Ruminococcus gnavus, Parabacteroides distasonis, Blautia sp. CAG257 and Bacteroides xylanisolvens were enriched, and that of Coprococcus comes depleted, in those microbial consortia producing immunosuppressive secretomes. Conclusion Specific bacterial taxa drive the functional signature of the colonic MAM secretome in an immunostimulatory or suppressive direction. The ENIGMA study is supported by a grant from the Leona M. and Harry B. Helmsley Charitable Trust

Properties of Linum usitatissimum Seed Aqueous Extract Against Colon Cancer Cell Lines

Background: Colon cancer has been a rising health concern in the modern world for quite some time. Considering the harmful side effects of chemotherapy, the most common treatment for this cancer, new treatment methods are required to lessen the side effects of treatment on patients. Traditional and herbal medicine can help in this effort. Objectives: Our study investigated the antiproliferative and pro-apoptotic effects of Linum usitatissimum seed aqueous extract on colon cancer and the expression of apoptotic genes compared to normal cell lines to reduce chemotherapy's harmful and irreversible side effects on colon cancer patients. Methods: MTT assay was used to determine the cytotoxicity of our extract on LS174T and COLO205 cells. Cancer and normal cell lines were treated with 2, 4, 6, and 8 mg/mL of LU extract for 24 and 48 hours. RNA extraction was performed, and mRNAs were reverse transcribed to cDNAs using RT-PCR. qPCR was then performed to assess the expression of BAX and BAD genes. Results: MTT assay concluded that this extract has a good cytotoxic effect on LS174t and COLO205 cell lines and can decrease cell viability, while no significant decrease in cell viability was observed in normal cells. For qPCR assay, the cells were treated with 0.5, 1, and 2 mg/mL of LU extract for two days. The qPCR analysis revealed that the expression of pro-apoptotic BAX and BAD was significantly decreased following treatment with our extract, while the expression of these genes showed no significant changes in normal cell lines after the same treatment. Conclusions: These results show that the components within L. usitatissimum seed aqueous extract have antiproliferative and pro-apoptotic properties. This extract can successfully decrease the population of LS174t and COLO205 cells while increasing the expression of pro-apoptotic BAX and BAD in these cell lines. The results also demonstrate that normal non-cancerous cells are unharmed by this extract. These results hint at some potential anti-cancer properties of L. usitatissimum seed aqueous extract.

Fluorescent imaging using novel conjugated polymeric nanoparticles-affimer probes in complex in vitro models of colorectal cancer.

We developed a carcinoembryonic antigen (CEA) conjugated polymer nanoparticle (CPN510-CEA-Af) probe to target CEA-expressing CRC cells in vitro. Its efficacy was evaluated in 2D and 3D cultures of LS174T, LoVo, and HT29 CRC cell lines. CPN510-CEA-Af produced greater fluorescent signal intensity than unconjugated particles in both 2D cells and 3D spheriods, indicating its potential as a probe for image-guided colorectal cancer surgery.

Apoptotic and Necroptotic Mediators are Differentially Expressed in Mucinous and Non-Mucinous Colorectal Cancer.

BackgroundMucinous colorectal cancer (CRC) represents 10% of all CRC and is associated with chemotherapy resistance. This study aimed to determine expression of apoptosis and necroptosis mediators in mucinous CRC.MethodsRNA gene expression data were extracted from TCGA. Protein levels in 14 mucinous and 39 non-mucinous tumors were measured by multiplexed immunofluorescence. Levels of apoptosis and necroptosis signalling proteins were analysed in SW1463 (mucinous rectal), SW837 (non-mucinous rectal), LS174T (mucinous colon) and HCT116 (non-mucinous colon) cell lines by western blot. Cell death was investigated by flow cytometry measurement of propidium iodide stained cells.ResultsHigh cleaved-Caspase 3 expression was noted in resected mucinous tumors. Western blot identified alterations in apoptosis proteins in mucinous CRC, most prominently downregulation of Bcl-xL protein levels (p=0.029) which was also observed at the mRNA level in patients by analysis of TCGA gene expression data (p<0.001). Treatment with 5-FU did not significantly elevate cell death in mucinous cells, while non-mucinous cells showed robust cell death responses. However, 5-FU-induced phosphorylation of MLKL in mucinous cancer cells, suggestive of a switch to necroptotic cell death signaling.ConclusionApoptotic and necroptotic mediators are differentially expressed in mucinous and non-mucinous colorectal cancers and represent targets for investigation of cell death mechanisms in the mucinous subtype.

Establishment of a luciferase-based method for measuring cancer cell adhesion and proliferation

BackgroundMethods measuring cell proliferation and adhesion are widely used but each hold limitations. We, therefore, introduce novel methods for measuring cell proliferation and adhesion based on CRISPR-modified cancer cell lines secreting luciferase to the growth media. Materials and methodsUsing CRISPR genome editing, we generated stable luciferase-secreting LS174T, HCT 116, Caco-2, and PANC-1 cell lines. The modified cells were seeded, and luciferase activity was measured in the media and compared to Coulter counter cell counts and iCELLigence impedance assay to evaluate the value of the secreted luciferase activities as a measurement for adhesion and proliferation. ResultsOur results demonstrate that luciferase secreted into the media can be used quantifying cell proliferation and adhesion. The adhesion luciferase assay and the iCELLigence impedance assay showed similar results with increased significant difference observed in the luciferase assays. The luciferase proliferation assay showed increased growth following increased serum concentrations in all cell lines vs. only two cell lines in the iCELLigence impedance assay. ConclusionsOur results show that the luciferase adhesion and proliferation assays are reliable methods for measuring adhesion and proliferation. The luciferase assays have advantages over existing assays as they are highly sensitive, easy to perform, non-invasive and suitable as high-throughput measurements.

Health Benefits of Postbiotics Produced by E. coli Nissle 1917 in Functional Yogurt Enriched with Cape Gooseberry (Physalis peruviana L.)

Changes in the activities of antimicrobial, antitumor, and antioxidant properties of postbiotics (YCG) are related to changes in the composition of phenolic compounds. Antimicrobial activity was found to be highest in postbiotic (YCG-7) against P. aeruginosa, S. aureus, and E. faecalis with an MIC of 3.1 µg/mL. YCG-7 revealed the most cytotoxicity against LS-174T and PC-3 cell lines with an IC50 of 5.78 and 6.56 µg/mL, respectively. YCG-7 was far more effective for scavenging free radicals in the NO• and DPPH assays with a scavenging activity of 70.73% and 85.6%, respectively. YCG-7’s total phenolic acid content is up to eightfold higher compared with control. Escherichia coli Nissle 1917 retained high viable counts during refrigerated storage, particularly in YCG (&gt;108 cells g−1) revealing a potential prebiotic activity of Cape gooseberry juice. EcN affected the phenolic profile of the YCG. Pyrogallol, p-coumaric acid, ellagic acid, 4-hydroxybenzoic acid, salicylic acid, gallic acid, vanillic acid, o-coumaric acid, caffeic acid, catechol, syringic acid, and rutin were the predominant phenolic compounds in YCG-7 or YCG-15. Chlorogenic, rosmarinic, cinnamic acid, naringin, and kaempferol were degraded by EcN in YCG-7 and YCG-15. The YCG had significantly higher sensory scores for appearance, smoothness, sourness, mouthfeel, and overall acceptance. These results provide the basis to target the functional benefits of YCG for further human health applications.

In Vivo Evaluation of Sgc8-c Aptamer as a Molecular Imaging Probe for Colon Cancer in a Mouse Xenograft Model.

Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications.

Initial assessment of Id protein antagonist (aHLH) shows anti-proliferative activity on HEK293T, LS174T and HCT116 cells

Id proteins (1–4) which are dominant-negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors function in a variety of cellular processes. They bind to the ubiquitously expressed ‘E box’ protein of the class A bHLH proteins (E12, E47, E2–2, and HEB). Each Id protein has distinct preferences and specificities for their E protein targets. A mutant version of E47 protein, an Id protein antagonist known as aHLH which is defective in DNA binding functions is known to suppress growth in human carcinoma cell lines. This work investigates the effects of the Id protein antagonist aHLH on growth and on pro-apoptotic functions in vitro using immortalized (highly tumorigenic) transformed human embryonic kidney cells (HEK293T), human epithelial colon adenocarcinoma cell line (LS174T) and human epithelial colon carcinoma cell line (HCT116). Plasmid encoding the aHLH proteins was transiently over-expressed in cultured HEK293T, LS174T and HCT116. Cell viability assay with MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was performed. Apoptotic and cell cycle assays were also performed on the transfected cells thereafter. Here, the results demonstrate that Id inhibition by aHLH impairs cell viability, inhibits cell proliferation and induces apoptosis in HEK293T, LS174T and HCT116 cell lines. These findings on the role of aHLH in cell growth and survival could help manipulate their functions as targets for developing anti-cancer agents and could also serve as molecular markers of human cancers.

Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer.

Simple SummaryWhole-genome sequencing and bioinformatics analysis on unique colorectal cancer families revealed two attractive candidate predisposition genes, CYBA and TRPM4, each with a loss-of-function variant. Supported by our functional studies, we suggest that the two gene defects mechanistically involve intestinal barrier integrity through reactive oxygen species and mucus biology, which converges in chronic bowel inflammation, a known risk factor for colorectal cancer.Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25–1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48–12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN

PurposeG-protein coupled receptor (GPR 34) has been found to play important roles in some cancers and regulates the proliferation, apoptosis, and migration of these cancer cells. However, the mechanisms underlying how GPR34 functions to regulate growth and proliferation of colorectal cancer cells remains to be clarified.MethodsWe employed stable GPR34 knockdown LS174T cell models, GPR34 Mab blocking, a CCK-8 kit, and a colony formation assay to characterize the effect of GPR34 on the proliferation of LS174T in vitro and xenograft tumor growth in vivo. The mRNA level of GPR34 was detected by RT-PCR in tumor tissues and adjacent normal tissues from 34 CRC patients.ResultsBased on RT-PCR results, GPR34 exhibited high level in tumor samples compared with adjacent normal samples. Increased expression of GPR34 is more associated with poor prognosis of CRC as shown in The Cancer Genome Atlas (TCGA) dataset by Kaplan–Meier survival analysis. Furthermore, we showed that GPR34 knockdown inhibited the proliferation of LS174T colon cancer cells and related xenograft tumor growth. Searching for the distinct molecular mechanism, we identified several contributors to proliferation of LS174T colon cancer cells: PI3K subunits/PTEN, PDK1/AKT, and Src/Raf/Ras/ERK. GPR34 knockdown inhibited the proliferation of LS174T cells by upregulating expression of PTEN, and downregulating expression of PI3K subunits p110-beta.ConclusionOur findings provide direct evidence that GPR34 regulates the proliferation of LS174T cells and the growth of LS174T tumor xenografts by regulating different pathways. High expression of GPR34 mRNA could then be used to predict poor prognosis of CRC.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Wogonoside: Anti-human colon cancer activities and survey of HMG-CoA ‎reductase inhibition properties with molecular modeling

IntroductionThe biological activities and interactions of wogonoside in the presence of HMG-COA reductase were investigated using the molecular docking study as a versatile theoretical approach. Wogonoside showed a considerable binding affinity to the enzyme with a docking score of -7.582 kcal/mol.Material and methodsThe in vitro cytotoxic and anti- colon ‎ carcinoma effects of biologically synthesized Wogonoside ‎against GP5d, MDST8‎, HCA-46‎, HT115, LS174T, and COLO 320DM cancer cell lines were ‎assessed.ResultsThe results indicated that the compound makes hydrophobic contacts with essential residues of the catalytic domain of the enzyme. Therefore, wogonosid could be considered as a potential inhibitor for HMG-COA reductase. The IC50 of the Wogonoside were 105, 198, 173, 382, 71, and 183 µg/mL against GP5d, ‎MDST8‎, HCA-46‎, HT115, LS174T, and COLO 320DM cancer cell lines. The anti-colon‎ ‎carcinoma properties of the Wogonoside could significantly remove GP5d, MDST8‎, HCA-46‎, ‎HT115, LS174T, and COLO 320DM cancer cell lines in a time and concentration-dependent ‎manner by MTT assay. ‎ConclusionsIt appears that the anti-human colorectal carcinoma effect of recent nanoparticles is due to their antioxidant effects. We have obtained results for the HMG-CoA Reductase enzyme at the micromolar level. In our study, inhibition result of on HMG-CoA reductase showed lower values 28.70±4.73 micromolar.

Let-7a Inhibits Tumor Metastasis by Regulating TGF-β/Smad Signaling in the Colorectal Adenocarcinoma Cell Line LS-174T.

Colorectal adenocarcinoma has a poor prognosis due to its propensity for metastasis. It has been experimentally demonstrated that the microRNA (miRNA) let-7a can effectively inhibit tumor proliferation and metastasis by regulating the transforming growth factor (TGF)-β signaling pathway; however, limited research has been conducted in the area of on colorectal cancer. Herein, we aimed to clarify the role and regulation of let-7a in a colorectal adenocarcinoma cell line (LS-174T). LS-174T cells were transfected to express let-7a. Let-7a miRNA expression was detected by quantitative real-time polymerase chain reaction (RT-qPCR). Cell growth was assessed by methyl thiazolyl tetrazolium (MTT) assay; invasion and migration were examined by Matrigel invasion and wound healing assays. The expression levels of matrix metalloproteinase (MMP)-2, phosphorylated Drosophila mothers against decapentaplegic 2 (p-SMAD2), and TGF-β1 were analyzed by western blotting. The mRNA expression levels of TGFB1 were also analyzed by RT-qPCR. Overexpression of let-7a resulted in significant inhibition of LS-174T cell proliferation in vitro. The invasion and migration abilities of the cells overexpressing let-7a were decreased, compared to the control group and miR-negative control group. Transfection of LS-174T cells with let-7a resulted in down-regulation of MMP-2, as well as of TGF-β1 and p-SMAD2 protein expression. Moreover, TGF-β1 mRNA levels were reduced following let-7a overexpression. Let-7a inhibited the growth and metastasis of colonic mucinous adenocarcinoma cells, at least partially, by regulating the TGF-β/Smad signaling pathway.