Characterization of the effects of thymol derivatives on colorectal cancer spheroids.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies with a high mortality rate. In the last few years, attention has been focused on substances of natural origin with anticancer activity. One such substance is thymol and its derivatives, which have been shown to have an antitumor effect also against CRC cells. In our study, we focused on determining the biological and antibacterial effects of thymol and thymol derivatives. Analyses were performed on a 3D model of human colon carcinoma cell lines (HCT-116 and HT-29) - spheroids. The cytotoxic (MTT assay) and genotoxic effect (comet assay) of thymol and derivatives: acetic acid thymol ester and thymol ß-D-glucoside were determined. ROS levels (ROS-Glo™ H2O2 Assay) and total antioxidant status (Randox TAS Assay) were also monitored. Last but not least, we also detected the effect of the derivatives using a disk diffusion assay and determined the number of colonies on the plates on selected bacteria such as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Lactobacillus brevis, Lactobacillus pentosus and Weizmannia coagulans. The derivatives did not show a significant inhibitory effect on the growth of LAB bacteria (lactic acid bacteria) in contrast to thymol. Overall, thymol derivatives are cytotoxic, genotoxic and increase ROS levels. Among the derivatives tested, acetic acid thymol ester (IC50 ~ 0.2 µg/ml) was more effective. The second derivative tested (thymol ß-D-glucoside) was effective at higher concentrations than thymol. Our research confirmed that thymol derivatives have a toxic effect on the 3D model of intestinal tumor cells, while they do not have a toxic effect on selected intestinal bacteria. Thus, they could bring new significance to the prevention or treatment of CRC.

Chitin-glucan improves important pathophysiological features of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most frequent and debilitating conditions leading to gastroenterological referrals. However, recommended treatments remain limited, yielding only limited therapeutic gains. Chitin-glucan (CG) is a novel dietary prebiotic classically used in humans at a dosage of 1.5-3.0 g/d and is considered a safe food ingredient by the European Food Safety Authority. To provide an alternative approach to managing patients with IBS, we performed preclinical molecular, cellular, and animal studies to evaluate the role of chitin-glucan in the main pathophysiological mechanisms involved in IBS. AIM: To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models. METHODS: Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM. RESULTS: Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations. CONCLUSION: CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.

Isolation and characterization of two Staphylococcus aureus lytic bacteriophages "Huma" and "Simurgh".

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Development of a novel laboratory photodynamic therapy device: automated multi-mode LED system for optimum well-plate irradiation.

Photodynamic therapy (PDT) is a targeted treatment method that utilizes a photosensitizer (PS) to induce cytotoxicity in malignant and non-malignant tumors. Optimization of PDT requires investigation of the selectivity of PS for the target tissues, irradiating light source, irradiation wavelengths, fluence rate, fluence, illumination mode, and overall treatment plan. In this study, we developed the Multi-mode Automatized Well-plate PDT LED Laboratory Irradiation System (MAWPLIS), an innovative device that automates time-consuming well plate light dosage/PS dose measurement experiment. The careful control of LED current and temperature stabilization in the LED module allowed the system to achieve high optical output stability. The MAWPLIS was designed by integrating a 3-axis moving system and motion controller, a quick-switching LED controller unit equipped with interchangeable LED modules capable of employing multiple wavelengths, and a TEC system. The proposed system achieved high optical output stability (1 mW) within the range of 0-500 mW, high wavelength stability (5 nm) at 635 nm, and high temperature stability (0.2 °C) across all radiation modes. The system's validation involved in vitro analysis using 5-ALA across varying concentrations, incubation periods, light exposures, and wavelengths in HT-29 colon cancer and WI-38 human lung fibroblast cell lines. Specifically, a combination of 405 nm and 635 nm wavelengths was selected to demonstrate enhanced strategies for colon cancer cell eradication and system validation. The MAWPLIS system represents a significant advancement in photodynamic therapy (PDT) research, offering automation and standardization of time-intensive experiments, high stability and precision, and improved PDT efficacy through dual-wavelength integration.

Podophyllic Aldehyde, a Podophyllotoxin Derivate, Elicits Different Cell Cycle Profiles Depending on the Tumor Cell Line: A Systematic Proteomic Analysis.

When new antitumor therapy drugs are discovered, it is essential to address new target molecules from the point of view of chemical structure and to carry out efficient and systematic evaluation. In the case of natural products and derived compounds, it is of special importance to investigate chemomodulation to further explore antitumoral pharmacological activities. In this work, the compound podophyllic aldehyde, a cyclolignan derived from the chemomodulation of the natural product podophyllotoxin, has been evaluated for its viability, influence on the cell cycle, and effects on intracellular signaling. We used functional proteomics characterization for the evaluation. Compared with the FDA-approved drug etoposide (another podophyllotoxin derivative), we found interesting results regarding the cytotoxicity of podophyllic aldehyde. In addition, we were able to observe the effect of mitotic arrest in the treated cells. The use of podophyllic aldehyde resulted in increased cytotoxicity in solid tumor cell lines, compared to etoposide, and blocked the cycle more successfully than etoposide. High-throughput analysis of the deregulated proteins revealed a selective antimitotic mechanism of action of podophyllic aldehyde in the HT-29 cell line, in contrast with other solid and hematological tumor lines. Also, the apoptotic profile of podophyllic aldehyde was deciphered. The cell death mechanism is activated independently of the cell cycle profile. The results of these targeted analyses have also shown a significant response to the signaling of kinases, key proteins involved in signaling cascades for cell proliferation or metastasis. Thanks to this comprehensive analysis of podophyllic aldehyde, remarkable cytotoxic, antimitotic, and other antitumoral features have been discovered that will repurpose this compound for further chemical transformations and antitumoral analysis.

Berberis vulgaris L. Root Extract as a Multi-Target Chemopreventive Agent against Colon Cancer Causing Apoptosis in Human Colon Adenocarcinoma Cell Lines.

Berberis vulgaris L. (Berberidaceae) is a shrub that has been widely used in European folk medicine as an anti-inflammatory and antimicrobial agent. The purpose of our study was to elucidate the mechanisms of the chemopreventive action of the plant's methanolic root extract (BVR) against colon cancer cells. Studies were conducted in human colon adenocarcinoma cell lines (LS180 and HT-29) and control colon epithelial CCD841 CoN cells. According to the MTT assay, after 48 h of cell exposure, the IC50 values were as follows: 4.3, 46.1, and 50.2 µg/mL for the LS180, HT-29, and CCD841 CoN cells, respectively, showing the greater sensitivity of the cancer cells to BVR. The Cell Death Detection ELISAPLUS kit demonstrated that BVR induced programmed cell death only against HT-29 cells. Nuclear double staining revealed the great proapoptotic BVR properties in HT-29 cells and subtle effect in LS180 cells. RT-qPCR with the relative quantification method showed significant changes in the expression of genes related to apoptosis in both the LS180 and HT-29 cells. The genes BCL2L1 (126.86-421.43%), BCL2L2 (240-286.02%), CASP3 (177.19-247.83%), and CASP9 (157.99-243.75%) had a significantly elevated expression, while BCL2 (25-52.03%) had a reduced expression compared to the untreated control. Furthermore, in a panel of antioxidant tests, BVR showed positive effects (63.93 ± 0.01, 122.92 ± 0.01, and 220.29 ± 0.02 mg Trolox equivalents (TE)/g in the DPPH•, ABTS•+, and ORAC assays, respectively). In the lipoxygenase (LOX) inhibition test, BVR revealed 62.60 ± 0.87% of enzyme inhibition. The chemical composition of BVR was determined using a UHPLC-UV-CAD-MS/MS analysis and confirmed the presence of several known alkaloids, including berberine, as well as other alkaloids and two derivatives of hydroxycinnamic acid (ferulic and sinapic acid hexosides). The results are very promising and encourage the use of BVR as a comprehensive chemopreventive agent (anti-inflammatory, antioxidant, and pro-apoptotic) in colorectal cancer, and were widely discussed alongside data from the literature.

Huaier Polysaccharide Alleviates Dextran Sulphate Sodium Salt-Induced Colitis by Inhibiting Inflammation and Oxidative Stress, Maintaining the Intestinal Barrier, and Modulating Gut Microbiota.

The incidence of ulcerative colitis (UC) is increasing annually, and UC has a serious impact on patients' lives. Polysaccharides have gained attention as potential drug candidates for treating ulcerative colitis (UC) in recent years. Huaier (Trametes robiniophila Murr) is a fungus that has been used clinically for more than 1000 years, and its bioactive polysaccharide components have been reported to possess immunomodulatory effects, antitumour potential, and renoprotective effects. In this study, we aimed to examine the protective effects and mechanisms of Huaier polysaccharide (HP) against UC. Based on the H2O2-induced oxidative stress model in HT-29 cells and the dextran sulphate sodium salt (DSS)-induced UC model, we demonstrated that Huaier polysaccharides significantly alleviated DSS-induced colitis (weight loss, elevated disease activity index (DAI) scores, and colonic shortening). In addition, HP inhibited oxidative stress and inflammation and alleviated DSS-induced intestinal barrier damage. It also significantly promoted the expression of the mucin Muc2. Furthermore, HP reduced the abundance of harmful bacteria Escherichia-Shigella and promoted the abundance of beneficial bacteria Muribaculaceae_unclassified, Anaerotruncus, and Ruminococcaceae_unclassified to regulate the intestinal flora disturbance caused by DSS. Nontargeted metabolomics revealed that HP intervention would modulate metabolism by promoting levels of 3-hydroxybutyric acid, phosphatidylcholine (PC), and phosphatidylethanolamine (PE). These results demonstrated that HP had the ability to mitigate DSS-induced UC by suppressing oxidative stress and inflammation, maintaining the intestinal barrier, and modulating the intestinal flora. These findings will expand our knowledge of how HP functions and offer a theoretical foundation for using HP as a potential prebiotic to prevent UC.

Structure-Inherent Tumor-Targeted IR-783 for Near-Infrared Fluorescence-Guided Photothermal Therapy.

IR-783, a commercially available near-infrared (NIR) heptamethine cyanine dye, has been used for selective tumor imaging in breast, prostate, cervical, and brain cancers in vitro and in vivo. Although the molecular mechanism behind the structure-inherent tumor targeting of IR-783 has not been well-demonstrated, IR-783 has unique properties such as a good water solubility and low cytotoxicity compared with other commercial heptamethine cyanine dyes. The goal of this study is to evaluate the phototherapeutic efficacy of IR-783 as a tumor-targeted photothermal agent in human colorectal cancer xenografts. The results demonstrate that IR-783 shows both the subcellular localization in HT-29 cancer cells and preferential accumulation in HT-29 xenografted tumors 24 h after its intravenous administration. Furthermore, the IR-783 dye reveals the superior capability to convert NIR light into heat energy under 808 nm NIR laser irradiation in vitro and in vivo, thereby inducing cancer cell death. Taken together, these findings suggest that water-soluble anionic IR-783 can be used as a bifunctional phototherapeutic agent for the targeted imaging and photothermal therapy (PTT) of colorectal cancer. Therefore, this work provides a simple and effective approach to develop biocompatible, hydrophilic, and tumor-targetable PTT agents for targeted cancer phototherapy.

Additive Cytotoxic and Colony-Formation Inhibitory Effects of Aspirin and Metformin on PI3KCA-Mutant Colorectal Cancer Cells.

Human malignancies are one of the major health-related issues throughout the world and are anticipated to rise in the future. Despite huge investments made in anticancer drug development, limited success has been obtained and the average number of FDA approvals per year is declining. So, an increasing interest in drug repurposing exists. Metformin (MET) and aspirin (ASP) possess anticancer properties. This work aims to test the effect of these two drugs in combination on colorectal cancer (CRC) cells in vitro. The effects of MET and/or ASP on cell proliferation, viability, migratory ability, anchorage-independent growth ability (colony formation), and nutrient uptake were determined in two (HT-29 and Caco-2) human CRC cell lines. Individually, MET and ASP possessed antiproliferative, cytotoxic, and antimigratory effects and reduced colony formation in HT-29 cells (BRAF- and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PI3KCA)-mutant), although MET did not affect either 3H-deoxy-D-glucose or 14C-butyrate uptake and lactate production, and ASP caused only a small decrease in 14C-butyrate uptake. Moreover, in these cells, the combination of MET and ASP resulted in a tendency to an increase in the cytotoxic effect and in a potentiation of the inhibitory effect on colony formation, although no additive antiproliferative and antimigratory effects, and no effect on nutrient uptake and lactate production were observed. In contrast, MET and ASP, both individually and in combination, were almost devoid of effects on Caco-2 cells (BRAF- and PI3KCA-wild type). We suggest that inhibition of PI3K is the common mechanism involved in the anti-CRC effect of both MET, ASP and their combination and, therefore, that the combination of MET + ASP may especially benefit PI3KCA-mutant CRC cases, which currently have a poor prognostic.

Identification of LPCAT1 as a key biomarker for Crohn's disease based on bioinformatics and machine learnings and experimental verification.

Epithelial-mesenchymal transition (EMT) plays a crucial role in regulating inflammatory responses and fibrosis formation. This study aims to explore the molecular mechanisms of EMT-related genes in Crohn's disease (CD) through bioinformatics methods and identify potential key biomarkers. In our research, we identified differentially expressed genes (DEGs) related to EMT based on the GSE52746 dataset and the gene set in the GeneCards database. Key genes were identified through Lasso-cox and Random Forest and validated using the external dataset GSE10616. Immune infiltration analysis showed that Lysophosphatidylcholine acyltransferase 1 (LPCAT1) was positively correlated with Neutrophils and Macrophages M1. The Gene Set Enrichment Analysis (GSEA) results for LPCAT1 showed associations with celladhesionmolecules and ECM receptor interaction. Additionally, a lncRNA-miRNA-mRNA ceRNA network was constructed. Finally, we validated that knocking down LPCAT1 could inhibit the release of inflammatory factors, EMT, and the elevation of fibrosis indices as well as the activation of NF-κB signaling pathway in LPS-induced HT-29 cells. LPCAT1 plays an important role in the occurrence and development of CD and may become a new biomarker.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

BACKGROUND: Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). METHODS: The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. RESULTS: It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. CONCLUSION: The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.

Bifidobacterium longum and Chlorella sorokiniana Combination Modulates IFN-γ, IL-10, and SOCS3 in Rotavirus-Infected Cells.

Rotavirus is the main cause of acute diarrhea in children up to five years of age. In this regard, probiotics are commonly used to treat or prevent gastroenteritis including viral infections. The anti-rotavirus effect of Bifidobacterium longum and Chlorella sorokiniana, by reducing viral infectivity and improving IFN-type I response, has been previously reported. The present study aimed to study the effect of B. longum and/or C. sorokiniana on modulating the antiviral cellular immune response mediated by IFN-γ, IL-10, SOCS3, STAT1, and STAT2 genes in rotavirus-infected cells. To determine the mRNA relative expression of these genes, HT-29 cells were treated with B. longum and C. sorokiniana alone or in combination, followed by rotavirus infection. In addition, infected cells were treated with B. longum and/or C. sorokiniana. Cellular RNA was purified, used for cDNA synthesis, and amplified by qPCR. Our results demonstrated that the combination of B. longum and C. sorokiniana stimulates the antiviral cellular immune response by upregulating IFN-γ and may block pro-inflammatory cytokines by upregulating IL-10 and SOCS3. The results of our study indicated that B. longum, C. sorokiniana, or their combination improve antiviral cellular immune response and might modulate pro-inflammatory responses.

Cytotoxic, acute oral toxicity, genotoxic and mutagenic assessment of the essential oil from fresh leaves of Croton argyrophyllus (Kunth.).

ETHNOPHARMACOLOGICAL RELEVANCE: Croton argyrophyllus Kunth., commonly known as "marmeleiro" or "cassetinga," is widely distributed in the Brazilian Northeast region. Its leaves and flowers are used in traditional medicine as tranquilizers to treat flu and headaches. AIM OF THE STUDY: This study was conducted to determine the chemical composition and toxicological safety of essential oil from C. argyrophyllus leaves using in vitro and in vivo models. MATERIALS AND METHODS: The chemical composition of the essential oil was determined using a gas chromatograph coupled to a mass spectrometer. Cytotoxicity was tested in the HeLa, HT-29, and MCF-7 cell lines derived from human cells (Homo sapiens) and Vero cell lines derived from monkeys (Cercopithecus aethiops) using the MTT method. Acute toxicity, genotoxicity. Mutagenicity tests were performed in Swiss mice (Mus musculus), which were administered essential oil orally in a single dose of 2000 mg/kg by gavage. RESULTS: The main components of the essential oil were p-mentha-2-en-1-ol, α-terpineol, ß-caryophyllene, and ß-elemene. The essential oil exhibited more than 90% cytotoxicity in all cell lines tested. No deaths or behavioral, hematological, or biochemical changes were observed in mice, revealing no acute toxicity. In genotoxic and mutagenic analyses, there was no increase in micronuclei in polychromatic erythrocytes or in the damage and index in the comet assay. CONCLUSIONS: The essential oil was cytotoxic towards the tested cell lines but did not exert toxic effects or promote DNA damage when administered orally at a single dose of 2000 mg/kg in mice.

Isoprenoid flavonoids isolated from Sophora davidii and their activities induces apoptosis and autophagy in HT29 cells.

Four previously undescribed isoprenoid flavonoids (2-5) were isolated from Sophora davidii, along with five known analogues. The structures of the compounds were established through comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and absolute configurations determined by theoretical calculations, including ECD and NMR calculation. The cytotoxic effects of the isolated compounds on human HT29 colon cancer cells were evaluated using the MTT assay, compound 1 exhibited cytotoxicity against human HT29 colon cancer cells with an IC50 value of 8.39 ± 0.09 µM. Studies conducted with compound 1 in HT29 cells demonstrated that it may induce apoptosis and autophagy in HT29 by promoting the phosphorylation of P38 MAPK and inhibiting the phosphorylation of Erk MAPK.

An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

N-methyl-D-aspartate receptors and thymoquinone induce apoptosis and alteration in mitochondria in colorectal cancer cells.

Colon cancer is on the rise in both men and women. In addition to traditional treatment methods, herbal treatments from complementary and alternative medicine are actively followed. Naturally derived from plants, thymoquinone (TQ) has drawn a lot of attention in the field of cancer treatment. MK-801, an N-methyl-D-aspartate agonist, is used to improve memory and plasticity, but it has also lately been explored as a potential cancer treatment. This study aimed to determine the roles of N-Methyl-D-Aspartate agonists and Thymoquinone on mitochondria and apoptosis. HT-29 cells were treated with different TQ and MK-801 concentrations. We analyzed cell viability, apoptosis, and alteration of mitochondria. Cell viability significantly decreased depending on doses of TQ and MK-801. Apoptosis and mitochondrial dysfunctions induced by low and high doses of TQ and MK-801. Our study emphasizes the need for further safety evaluation of MK-801 due to the potential toxicity risk of TQ and MK-801. Optimal and toxic doses of TQ and MK-801 were determined for the treatment of colon cancer. It should be considered as a possibility that colon cancer can be treated with TQ and MK-801.

Overexpression of ZNF169 promotes the growth and proliferation of colorectal cancer cells via the upregulation of ANKZF1.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The 5­year survival rate of patients diagnosed with the early stages of the disease is markedly higher than that of patients in the advanced stages. Therefore, identifying novel biomarkers and drug targets for CRC is critical for clinical practice. Zinc finger protein 169 (ZNF169) is a crucial transcription factor, and its role in CRC remains to be explored. The present study aimed to investigate the clinical relevance, function and underlying mechanisms of ZNF169 in CRC growth and proliferation. The Cancer Genome Atlas (TCGA) database was utilized to analyze the clinical relevance of ZNF169 in patients with CRC. Immunohistochemical staining was performed on tissue samples from patients with CRC to detect the expression of ZNF169. The HCT­116, HT­29 and RKO cell lines were employed for in vitro experiments. The overexpression and knockdown of ZNF169 were achieved by transfecting the cells with lentivirus and small interfering RNAs, respectively. Cell Counting Kit­8, colony formation and EdU staining assays were applied to investigate the function of ZNF169 in CRC cells. Dual luciferase activity and chromatin immunoprecipitation (ChIP)­quantitative PCR (qPCR) assays were performed to identify the regulatory effects of ZNF169 on the ankyrin repeat and zinc­finger domain­containing 1 (ANKZF1; also known as ZNF744) gene. Reverse transcription­quantitative PCR and western blot analysis were performed to measure mRNA and protein expression, respectively. The analysis of TCGA data revealed a positive correlation between ZNF169 and ANKZF1, with the overexpression of ANKZF1 being associated with a poor prognosis of patients with CRC. The experimental results demonstrated that ZNF169 was expression upregulated in CRC tissue compared with that in normal colon tissue. Gain­of­function and loss­of­function experiments revealed that ZNF169 enhanced the intensity of EdU staining, promoting the growth and proliferation of CRC cells. Furthermore, the overexpression of ZNF169 potentiated the transcriptional activity of the ANKZF1 gene, while the knockdown of ZNF169 produced the opposite results. ChIP­qPCR confirmed the interaction between ZNF169 and the promoter sequence of ANKZF1. Rescue experiments revealed that ZNF169 accelerated CRC cell growth and proliferation through the upregulation of ANKZF1. Furthermore, there was a positive correlation identified between ZNF169 and ANKZF1, and upregulation of ANKZF1 expression was associated with the poor prognosis of patients with CRC. On the whole, the present study demonstrates that ZNF169 contributes to CRC malignancy by potentiating the expression of ANKZF1. Thus, the regulation of ZNF169 and/or ANKZF1 expression may represent a viable strategy for the treatment patients with CRC with a high expression of ZNF169.

10-Secocycloartane (=9,19-cyclo-9,10-secolanostane) triterpenoid saponins: Huangqiyenins M-X from Astragalus membranaceus (Fisch.) Bge.

Phytochemical investigations of the leaves of Astragalus membranaceus (Fisch.) Bge. have led to the isolation of 12 undescribed triterpenoid saponins named huangqiyenins M-X. The structures of the undescribed compounds were determined using NMR and HRESIMS data. The cytotoxicity of these compounds against the RKO and HT-29 colon cancer cell lines was evaluated. Among these compounds, huangqiyenin W exhibited the highest cytotoxic activity against RKO colon cancer cells, whereas huangqiyenin Q and W showed moderate cytotoxic activity against HT-29 colon cancer cells. The network pharmacology results indicated that STAT3, IL-2 and CXCR1 are the correlated targets of huangqiyenin W against colon cancer, with AGE-RAGE and Th17 cell differentiation as the key signaling pathways.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

BACKGROUND: Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. METHODS: Epithelial activation and/or barrier effects upon exposure to 2-50 µg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 µg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. RESULTS: Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. CONCLUSIONS: Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.