HT-29 Cells Research Articles

Protective Responses of the Intestinal Epithelial Cell Line HT-29 Cells Exposed to Dephosphorylated Salmonella Flagellin [RESEARCH NOTE

This study aimed to describe the effects of Salmonella Typhimurium flagellin (SFL) dephosphorylated by sweet potato purple acid phosphatase (PAP) on the protective responses of the intestinal epithelial cell line HT-29 cells. The enzyme was reported to display a broad substrate specificity for various organic phosphorylated conjugates and phosphoproteins. Dephosphorylation of SFL by sweet potato PAP decreased to 35% in the presence of 0.05 mM vanadate as compared with the negative control (p < 0.05). Intact SFL and the SFL treated with sweet potato PAP did not remarkably induce the activation of caspase-3 in HT-29 cells at all the tested levels of the substrate. Intact SFL maximally induced the release of IL (interleukin)-8 in HT-29 cells at 1000 ng/mL (p < 0.05). However, the SFL treated with the enzyme inhibited the release of IL-8 at over 100 ng/mL of the substrate as compared with intact SFL, resulting in an approximately 8-fold decrease even at 1000 ng/mL (p < 0.05). The SFL treated with the enzyme decreased the activation of the total ERK1/2 in the cells to 1.9 and 1.7–fold at 10 and 1000 ng/mL of the substrate, respectively, as compared with intact SFL (p < 0.05). In conclusion, sweet potato PAP could be a promising tool for controlling excessive inflammation during Salmonella infection in animal husbandry, and the enzyme could be a safe alternative that can overcome the drawbacks of chemotherapy.

Identification of FOXM1 as a novel protein biomarker and therapeutic target for colorectal cancer progression: Evidence from immune infiltration and bioinformatic analyses

Colorectal cancer (CRC) remains a major global health challenge, with its underlying molecular mechanisms, particularly the role of FOXM1, not yet fully understood. This study employed an integrated approach combining bioinformatics along with experimental validation to explore the role of FOXM1 in CRC. Using advanced computational tools and experimental techniques, we aimed to clarify the biological significance of FOXM1 and its potential impact on CRC progression and treatment. Bioinformatic analyses, including pan-cancer views, mRNA expression analysis, immune infiltrations, pathway enrichment, and functional annotations, highlighted the oncogenic potential of FOXM1 in CRC. Protein and gene expression analyses (western blot and qPCR) were conducted in HCT-116 and HT-29 cells. Platforms like GEPIA and UALCAN confirmed the diagnostic relevance of FOXM1, showing upregulated mRNA expression across various stages and metastasis. The influence of FOXM1 on immune cells, particularly CD4+, CD8+, and B cells, was significant, as revealed by immunohistochemistry. Protein-protein interaction analysis through STRING and CYTOSCAPE identified genes closely linked to FOXM1 in CRC. KEGG pathway enrichment suggested FOXM1's involvement in the p53 pathway, reinforcing its role in oncogenesis. Experimental validation confirmed elevated FOXM1 expression in HCT-116 and HT-29 cells. In summary, this study indicates that targeting FOXM1 could be a promising therapeutic strategy in CRC, emphasizing its importance in the molecular landscape of cancer progression.

Preparation, characterization, and ex vivo evaluation of isoxanthohumol nanosuspension

This study assessed the equilibrium solubility, oil–water distribution coefficient, and dissociation constant of Isoxanthohumol (IXN), and formulated IXN nanoparticles (IXN-Nps) using a micro media grinding method. The research characterized the particle size, polydispersity index, zeta potential, morphology, and structure of the nanoparticles, and evaluated the optimal cryoprotectant. Additionally, the study examined the toxicity and in vitro and in vivo release of IXN on HT-29 cells. IXN is classified as a Biopharmaceutical Classification System (BCS) II class drug with weak acidity. The average particle size of IXN-Nps is 249.500 nm, with a polydispersity index (PDI) of 0.149 and a zeta potential of −25.210 mV. The research identified 5 % mannitol as the optimal cryoprotectant. Compared to IXN, the half-maximal inhibitory concentration of IXN-Nps decreased to one-third, demonstrating a significant inhibitory effect on HT-29 colon cancer cells. The in vitro cumulative release rate of IXN-Nps within 24 h was 3.5 times higher than that of the IXN solution. In vivo pharmacokinetic results revealed that the oral bioavailability of IXN-Nps increased significantly by 2.8 times compared to the IXN solution. The correlation coefficient (r = 0.9227) exceeded the critical value for significance at the 0.01 (r = 0.834) level, indicating a strong correlation between in vivo and in vitro results. Consequently, the nanosuspension overcame the low solubility limitation of IXN and proved to be an effective method for enhancing the oral bioavailability of IXN

Design, synthesis and biological evaluation of novel TMPRSS2-PROTACs with florosubstituted 4-guanidino-N-phenylbenzamide derivative ligands

Transmembrane Serine Protease 2 (TMPRSS2) plays a critical role in tumorigenesis and progression, making its degradation a promising therapeutic strategy. In this study, we designed and synthesized TMPRSS2-PROTACs, including VPOT64 and VPOT76, based on camostat. Both compounds exhibited superior inhibitory effects on HT-29 colorectal and Calu-3 lung cancer cells compared to paclitaxel. Notably, VPOT76 effectively degraded TMPRSS2, significantly inhibiting the proliferation of TMPRSS2-positive HT-29 cells and inducing apoptosis with an IC50 of 0.39 ± 0.01 μM. Flow cytometry analysis demonstrated that VPOT76 increased early apoptotic cells in a dose-dependent manner and caused G2 phase arrest at 0.8 μM. Colony formation assays showed that VPOT76 inhibited HT-29 colony formation, even at low concentrations, further confirming its antiproliferative effect. Additionally, wound healing assays indicated that VPOT76 reduced the migration of HT-29 cells after 48 h, suggesting its potential to impair tumor cell invasion and metastasis. These findings highlight the multifaceted anticancer activities of VPOT76, including apoptosis induction, cell cycle arrest, colony formation inhibition, and migration suppression. Overall, this study establishes VPOT76 as a potent TMPRSS2-degrading PROTAC with strong therapeutic potential, laying the groundwork for further development of TMPRSS2-targeting treatments for colorectal and other cancers.

Development of new LSM-83177 analogues as anti-tumor agents against colorectal cancer targeting p53-MDM2 interaction

LSM-83177, a phenoxy acetic acid derivative, is a small molecule reported for its promising anti-tumor properties. Via inhibiting the interaction between MDM2 and p53, LSM-83177 can elevate the active p53 levels within cells, thereby promoting apoptosis and inhibiting tumor growth. Also, LSM-83177 has been shown to inhibit GST activity in colorectal cancer HT29 cells. In the current work, novel LSM-83177 hydrazone analogs 5a-f, 7a-b, 10a-e, and 13a-b have been designed according to the structure features of LSM-83177 and their binding mode in the active site of MDM2. The anti-cancer activity of the newly synthesized analogs is evaluated against the HT29 cell line. The most potent compounds, 7a and 10a, showed IC50 = 12.48 and 10.44 µg/ml, respectively, when compared with Cisplatin (IC50 = 11.32 µg/ml) as a reference drug. Compounds 7a and 10a were introduced for further inspection for p53-MDM2 protein–protein interaction, where they displayed IC50 values of 3.65 and 11.08 µg/ml, respectively. Furthermore, hydrazones 7a and 10a increased the p-53 expression levels by 3.22– and 4.25-fold, respectively; in addition, they effectively reduced the GST expression levels in HT29 cancer cells with 0.56- and 0.30-fold increments in comparison to the untreated control.

Designing sustainable antioxidant and anti-inflammatory ingredients from seaweeds for functional gummies

Atlantic wakame (AW) and sea lettuce (SL) are edible seaweeds commonly produced in Asia and Europe, typically consumed as fresh or dried biomass. Because of the short shelf life, developing new post-harvest treatments, such as the extraction of bioactive compounds, is essential for establishing reliable macroalgae supply chains for food applications. This study aims to develop a sustainable and functional seaweed-based food ingredient rich in bioactive compounds for functional food applications. The results indicated that ultrasound-assisted extraction using 70% ethanol for 10 minutes was optimal for both SL (410±23 mg of gallic acid equivalent [GAE] per kg) and AW (666±63 mg GAE/kg). Both extracts exhibited chemical antioxidant activity and inhibited the protection of human plasma against chemically induced oxidation. SL extract did not exhibit free radical scavenging activity (DPPH). Importantly, no cytotoxicity (IC50>250 µg/mL) was observed in human-derived THP-1, HUVEC, HT-29, and HepG2 cell lines, ensuring the relative safety of these extracts for potential food technology applications. AW and SL extracts (100 µg/mL) reduced intracellular reactive oxygen species generation in lipopolysaccharide (LPS)-challenged THP-1 cells by 66% and 30%, respectively, highlighting the higher antioxidant activity of AW extract. AW and SL extracts did not modulate TNF-α and IL-6 secretion to show anti-inflammatory activity. Gummies infused with AW and SL extracts had a higher total phenolic content than commercial products, but their antioxidant activity was lower. Sensory evaluation revealed that gummies containing seaweed extracts received lower scores for colour, taste, overall impression, and purchase intention than those without extracts.

A customizable and low-cost 3D-printed transwell device coupled with 3D cell culture for permeability assay

The permeability-based assay is commonly used to assess intestinal barrier function, and it relies on using a transwell insert as an essential compartment. The device consists of a semipermeable membrane that is attached at the bottom of the insert and splits the system into the apical and basolateral compartments. However, commercial inserts are standardized with different pore sizes based on the application and offer only a flat plane of two-dimensional cell culture. Herein, we present a simple, low-cost 3D-printed transwell device and a robust method to functionalize the inserts for paper-based 3D cell culture. This 3D-printed device was fabricated from a polylactic acid (PLA) filament, and a paper membrane used to support HT-29 cells for intestinal permeability assessment. A device showed good biocompatibility when culturing HT-29 cells for 48 and 72 h with 97 % and 98 % cell viability, respectively. Together with fluorescence images, cells were attached directly to the microfiber networks of a Matrigel-functionalized paper, indicating that the functionalized paper is biocompatible and bioactive. Furthermore, in a more appropriate culture microenvironment, SEM analyses revealed cellular features differentiating into mucus-secreting cells, evidenced by the formation of microvilli on the cell surface, which was further confirmed by immunofluorescence staining of villin-1. To demonstrate the usability of the 3D-printed transwell device, intestinal permeability was assessed using both chemical and biological stimulation treatments. The permeability results employing FITC-dextran validated the association between a different level of relative fluorescence intensity unit (RFU) and the orange color of live cells by CellTrackerTM. As a result, this 3D-printed transwell device provides a straightforward and cost-effective method for manufacturing a device for customization in many laboratory settings, making it a feasible alternative to marketed transwell devices that do not allow for customization.

Abstract A001: HNRNPH1 regulates the alternative splicing of transcripts expressed by cancer-associated genes

Abstract The RNA binding protein HNRNPH1 is a critical regulator of alternative splicing (AS). We have previously demonstrated that HNRNPH1 mediates an exon exclusion event required for the expression of the mRNA encoding the fusion oncoprotein expressed in a subset (∼37%) of Ewing sarcomas. Furthermore, studies by others have shown that HNRNPH1 regulates a mutually exclusive exon (MXE) AS event (exon-18a versus exon-18b) that defines the expression of TCF3 transcript variants, which, when dysregulated, promotes tumorigenesis in Burkitt’s lymphoma. In this study, we have interrogated the transcriptome-wide effects of depleting HNRNPH1 by RNAi to further explore its regulation of transcripts expressed by cancer-associated genes. We performed whole transcriptome RNA sequencing (RNAseq; short-read, Illumina; long-read, PacBio IsoSeq) of two cell lines (HEK-293T and HT-1080) post 48-hour transfection with a control siRNA (siNeg) or an siRNA targeting HNRNPH1 (siHNRNPH1). We then assembled a workflow to quantify changes in individual AS events (percent-spliced-in, ΔPSI=±0.1) using rMATS and MAJIQ, and differential transcript expression (DTE) using RSEM (pValue≤0.05) and EBSeq (FDR≤0.05). RNAseq analyses were validated using PCR assays. The rMATS analysis revealed skipped exons (SE) as the most frequent event effected in HEK-293T and HT-1080 cells following HNRNPH1 silencing, with 1069 SE or MXE events from 607 genes common to both. Gene annotation (Metascape) of these 607 genes identified 49 as cancer-associated. Consistent with previous studies, we detected a significant change in the TCF3-exon-18a/18b MXE event using rMATS (decreased PSI for 18a) and MAJIQ (negative ΔPSI for 18a and positive ΔPSI for 18b) following HNRNPH1 silencing in both cell lines. Furthermore, DTE revealed a corresponding decrease in 18a-containing TCF3-transcripts and an increase in 18b-containing TCF3-transcripts, validating our analytical pipeline. Next, we extended our analysis to uncharacterized HNRNPH1-regulated AS events, beginning with examination of transcripts encoding the mitotic kinase AURKA. Previous studies have shown that AS of AURKA transcripts localizes to the untranslated regions (UTRs), particularly the 5’UTR. However, regulators of these AS events are unknown, and it is unclear how these UTR variants affect AURKA protein expression. Long-read RNAseq of AURKA mRNAs from both cell lines revealed extensive 5’UTR AS, and the rMATS and MAJIQ analyses showed decreased PSI and negative ΔPSI for a specific 5’UTR-exon following HNRNPH1 silencing. DTE revealed corresponding decreases in abundance of AURKA transcripts containing this exon (AURKA-203, -205, -209, and -213) upon HNRNPH1 depletion, which we confirmed with variant-specific RT-PCR. Furthermore, immunoblot analysis showed a decrease in AURKA protein expression following the silencing of HNRNPH1. Our assessment of AURKA AS suggests HNRNPH1 mediates the inclusion of a specific AURKA-5’UTR-exon, providing evidence for a previously uncharacterized post-transcriptional regulatory mechanism of AURKA expression. Citation Format: Tayvia Brownmiller, Patricio Pichling, Tamara L Jones, Ioannis Grammatikakis, Soumya Sundara Rajan, Erica C Pehrsson. HNRNPH1 regulates the alternative splicing of transcripts expressed by cancer-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A001.

Mechanism of Resveratrol on LPS/ATP-induced pyroptosis and inflammatory response in HT29 cells

Pyroptosis plays an important role in maintenance of intestinal homeostasis, the abnormal activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome can promote the event and development of ulcerative colitis (UC). Its protective effects such as inhibiting pyroptosis in various inflammation-related diseases have been demonstrated, but whether resveratrol (RES) can also alleviate the progression of the disease by inhibiting pyroptosis in UC and the mechanism have rarely been studied. In this study, lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP) was used to induce HT29 human colon cancer cells to construct an intestinal epithelial cell pyroptosis and inflammation model in vitro to investigate the anti-inflammatory effect of RES, reveal the regulatory mechanism of RES on pyroptosis, and provide a new theoretical basis for the treatment of UC. In vitro experiences, HT29 cells were dividing into control group, LPS/ATP group, RES low-dose group, RES high-dose group, NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), and LPS/ATP+PDTC group. The mRNA expressions of pyroptosis-related indicators such as NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1(CASP1), IL-18, IL-1β, and inflammatory factors such as TNF-α and IL-6 were detected by qRT-PCR. The protein expressions of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β, NF-κB-p65 in the nucleus, and IκBα and p-IκBα in the cytoplasm were detected by Western blot. Immunofluorescence saw the distribution and expression of NLRP3, ASC and NF-κB-p65 protein in each group. The morphology and degree of pyroptosis in each group were observed by transmission electron microscope. The results showed that compared with the control group, the pyroptosis-related proteins including NLRP3, ASC, CASP1, IL-18, IL-1β, and inflammatory factors including TNF-α and IL-6 in the LPS/ATP group were increased, and LPS/ATP activated the activity of NF-κB signaling pathway. Compared with the LPS/ATP group, RES downregulated the expression of pyroptosis-related proteins and inflammatory factors in HT29 cells, and inhibited the activation of the NF-κB signaling pathway in HT29 cells pyroptosis. RES down-regulates the pyroptosis of HT29 cells induced by LPS/ATP and the expression of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β and inflammatory factors TNF-α and IL-6 in the inflammatory response and inhibits the occurrence of pyroptosis. The mechanism is related to the inhibition of NF-κB pathway activity.

Differential contributions of the gut microbiota and metabolome to pathomechanisms in ulcerative colitis: an in vitro analysis

ABSTRACT The gut microbiota has been implicated in onset and progression of ulcerative colitis (UC). Here, we assess potential causal involvement of the microbiota and -associated fecal water (FW) metabolome in altering key functional parameters of the colonic epithelium. Fecal samples were collected from N = 51 healthy controls (HC), N = 36 patients with active UC (UC-A), and N = 41 subjects in remission N = 41 (UC-R). Using in vitro incubation experiments, the FW metabolome’s impact on butyrate oxidation rates/gene expression and cell death (cytotoxicity) of HT-29 cells, cytokine production by PBMC, and barrier integrity of Caco2 monolayers was evaluated. The FW metabolome from patients and individuals hosting the Bacteroides 2 (Bact2) enterotype (69% of UC-A, 31% of UC-R, 3% of HC), characterized by lower levels of median- and short-chain fatty acids and furan compounds, left butyrate oxidation rates unaltered but affected associated gene expression profiles. UC patients/Bact2-carriers’ FW lowered PBMC IL-8 production and increased IL-1β production. Patients’ FW increased cytotoxicity, associated with sulfide compound levels. Bact2 carriers’ FW, displaying higher levels of bile acids, lowered barrier function upon incubation of monolayers. The FW metabolome of patients and individuals hosting a dysbiotic microbiota could contribute to the disruption of functional processes of the colonic epithelium as observed in UC.

Enrichment of cancer stem cell subpopulation alters the glycogene expression profile of colorectal cancer cells.

Colorectal cancer (CRC) has a high mortality rate, resulting from the processes of metastasis and disease recurrence. Cancer stem cells (CSCs) are believed to be crucial for both processes, as they ensure the maintenance of the tumor bulk, in addition to being intrinsically resistant to conventional therapies. Thus, the present study aimed to investigate glycobiomarkers in colorectal cancer stem cell subpopulations. For this purpose, a sphere formation assay was standardized for CACO-2 and HT-29 cell lines, which were monitored through gene expression analysis of five known CSC markers (CD24, CD44, ALDH1, LGR5, and PROM1). Compared to the parental condition (2D), a reduction in CD24 expression was seen in CACO-2, while in HT-29 an increase in the expression levels of ALDH1, LGR5, and PROM1 was observed. Regarding glycogenes, eight of them (ST3GAL1, OGT, OGA, MGAT5, GFAT1, GFAT2, B4GALT1 e B3GNT2) have had their expression monitored. An increase in B3GNT2, OGT, and OGA was observed in the HT-29 sphere condition. On the other hand, no change in the glycogenes expression was observed in CACO-2. In silico correlation analyses (CSCs markers versus glycogenes) using TCGA data from colon and rectum carcinoma samples showed a weak positive correlation between LGR5 vs OGA expression regardless of the sample location. In addition, an increase in the expression of LGR5, OGA, and OGT as well as a decrease in the expression of ALDH1 were observed in colon carcinoma samples when compared to the adjacent normal tissue. Interestingly, greater OGA expression resulted in both lower overall survival of colon carcinoma patients and lower disease-free survival of rectum carcinoma patients. Therefore, our data indicates that OGA expression correlates with CSC markers and directly impacts the survival of colorectal carcinoma patients.

Whey proteins from camel's milk have higher in vitro wound-healing effect than whey proteins from cow's milk.

The effectiveness of whey obtained by either enzyme (sweet) or acid treatment on wound healing remains unclear. This study investigated the effectiveness of camel and bovine whey prepared enzymatically (CSW and BSW) or by pH reduction (CAW and BAW). After removing the cream from milk, HCl or rennet was used to remove casein, resulting in acid or sweet whey, respectively, followed by lactose removal using dialysis. Casein removal was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Wound-healing activity was measured in vitro on HT-29 cells by scratch assay. All four whey samples (0-1000 mg L-1) were applied on the cells, and the closure of the cell-free scratched areas was monitored for 48 h. All whey samples increased the cell migration significantly (P < 0.05) to help close the cell-free areas as an indication of wound healing compared to the negative control. However, the closure amounts between the highest dose (1000 mg L-1) and the control were not significantly different (P > 0.05). Acid whey samples significantly (P < 0.05) elevated the closure speed compared to the sweet whey samples. The highest closure percentage (64.69%) was achieved after treatment with 10 mg L-1 CAW for 48 h. Between the sweet whey samples, BSW was significantly (P < 0.05) more effective in closing the cell-free zone compared to CSW. This study investigated the wound-healing potential of camel and bovine whey in vitro by comparing their effects on HT-29 cell migration. CAW showed the greatest activity and may find uses as a treatment agent in wound healing. © 2024 Society of Chemical Industry.

Discovery of Thiazole Carboxamides as Novel Vanin-1 Inhibitors for Inflammatory Bowel Disease Treatment.

Inflammatory bowel disease (IBD) is a clinically heterogeneous disease demanding more therapeutic targets and intervention strategies. Vanin-1, an oxidative stress-regulating protein, has emerged as a promising target for alleviating inflammation and oxidative stress. In this study, a series of thiazole carboxamide derivatives as vanin-1 inhibitors were designed and synthesized. The preferred compound, X17, demonstrated potent inhibition against vanin-1 at the protein, HT-29 cell, and tissue levels, whose binding mode with the target was confirmed via the cocrystal structure. X17 achieved a high bioavailability of 81% in rats, accompanied by concentration-dependent inhibition of serum vanin-1. In a DSS-induced mouse colitis model, X17 exhibited potent anti-inflammatory and antioxidant activities, repressing the inflammatory factor expressions and myeloperoxidase activity, elevating the colonic glutathione reserve, and restoring the intestinal barrier. Collectively, these findings depict the discovery of a potent vanin-1 inhibitor, providing an opportunity for further drug candidate development for treating IBD.

Design, synthesis, and cytotoxic activity of 2-amino-1,4-naphthoquinone-benzamide derivatives as apoptosis inducers

A new series of 2-amino-1,4-naphthoquinone-benzamides 5a-n was designed based on previously reported potent cytotoxic agents. These compounds were synthesized from the reaction of 1,4-naphthoquinone, 4-aminobenzoic acid, and appropriate amine derivatives in good yields. Cytotoxic activities of the target compounds 5a-n were evaluated against three cancer cell lines MDA-MB-231, SUIT-2, and HT-29 by MTT assay and the obtained in vitro data. All newly synthesized compounds were more potent than positive control cisplatin against MDA-MB-231 cell line and less potent than this control against SUIT-2 cell line. Moreover, most of the synthesized compounds were more potent than cisplatin against HT-29 cell line. Among the synthesized compounds, compound 5e was the most potent cytotoxic agent against all the three evaluated cell lines. Moreover, cell cycle analysis showed that derivatives 5f and 5l dose-dependently increase the percentage of sub-G1 cells. Induction of apoptosis as an important mechanism of anti-cancer drugs was confirmed morphologically in the most potent new compounds 5e, 5f, 5g, and 5l by Hoechst 33,258 staining.

Hybrid Molecules Containing Methotrexate, Vitamin D, and Platinum Derivatives: Synthesis, Characterization, In Vitro Cytotoxicity, In Silico ADME Docking, Molecular Docking and Dynamics.

Designing hybrid-based drugs is one promising strategy for developing effective anticancer drugs that explore combination therapy to enhance treatment efficacy, overcome the development of drug resistance, and lower treatment duration. Bisphosphonates and Vitamin D are commonly administered drugs for the treatment of bone diseases and the prevention of bone metastases. Platinum-based and methotrexate are widely used anticancer drugs in clinics. However, their use is hampered by adverse side effects. Hybrid-based compounds containing either bisphosphonate, vitamin D, platinum-based, or methotrexate were synthesized and characterized using FTIR, 1H-,31P, 13C-NMR, and UHPLC-HRMS which confirmed their successful synthesis. The hydroxyapatite bone binding assay revealed a promising percentage binding affinity of the bisphosphonate hybrid compounds. In vitro cytotoxicity assays on MCF-7 and HT-29 cell lines revealed a promising cytotoxic effect of hybrid 19 at 50 and 100 μg/mL on HT-29 and hybrid 15 on MCF-7 at 100 μg/mL. Molecular docking and dynamics simulation analysis revealed a binding affinity of -9.70 kcal/mol for hybrid 15 against Human 3 alpha-hydroxysteroid dehydrogenase type 3, showing its capability to inhibit Human 3 alpha-hydroxysteroid dehydrogenase type 3. The Swiss ADME, ProTox-II, GUSAR (General Unrestricted Structure-Activity Relationships), and molecular docking and dynamics studies revealed that these compounds are promising anticancer compounds.

Antiproliferative and Morphological Effects of Fenretinide Lipid Nanosystems in Colon Adenocarcinoma Cells

Objective: Colon adenocarcinoma is characterized by the downregulation of the retinoic acid receptor, making natural retinoids such as all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid effective in treatment and chemoprevention due to their ability to increase RARβ expression. However, major limitations to their use include tolerability and acquired resistance. In this study, we evaluated fenretinide, a semisynthetic derivative of all-trans retinoic acid, in an HT-29 cell line. Fenretinide was evaluated both as a free drug and encapsulated in self-assembling phosphatidylcholine nanosystems with the aim of increasing the aqueous solubility and cell availability of the drug. Methods: Fenretinide was encapsulated in lipid nanosystems obtained in water by the dispersion of an amphiphilic mixture of phospholipids, glyceryl tributyrate and polysorbate 80. The physico-chemical characterization of the nanosystems was carried out by dynamic light scattering and spectrophotometry. The biological activity was evaluated by quantitative phase imaging microscopy, MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Results: Fenretinide in phosphatidylcholine nanosystems was more active than free fenretinide in inhibiting HT-29 cells’ proliferation, as indicated by quantitative phase imaging data. Indeed, encapsulated fenretinide increased duplication time, decreased dry mass and decreased the rate of cell growth more efficiently than fenretinide. Moreover, encapsulated fenretinide effectively decreased the motility of the cells that survived the treatment. Conclusions: The results indicate that the proposed nanosystems can be considered a valuable alternative to natural retinoids in the chemoprevention and treatment of colorectal cancer. This is due to the favorable pharmacologic characteristics of fenretinide in colorectal cancer and the improved drug activity provided by nanoencapsulation.

Development of protective egg yolk immunoglobulins (IgY) targeting CfaB, LTB, and EtpA recombinant proteins of Enterotoxigenic Escherichia coli (ETEC) for inhibiting toxin activity and bacterial adherence.

Enterotoxigenic Escherichia coli (ETEC) stands as a prevalent bacterial cause of global diarrheal incidents. ETEC's primary virulence factors encompass the B subunit of the Heat Labile Enterotoxin, along with the adhesion factors CfaB and EtpA. In this study, we isolated IgY antibodies against the three virulence factors individually, in pairs, and as triple cocktails. The in vitro efficacy of these IgY antibodies was examined, focusing on inhibiting heat-labile enterotoxin (LT) toxin cytotoxicity and impeding ETEC adherence to HT29 cells. Assessing the impact of IgY-treated bacteria on intestinal epithelial cells utilized the standard ileal loop method. Results demonstrated that the anti-LTB IgY antibody at 125µg/ml and IgY antibodies from double and tertiary cocktails at 200µg/ml effectively inhibited LT toxin attachment to the Y1 cell line. Pre-incubation of HT29 intestinal cells with specific IgYs reduced bacterial attachment by 59.7%. In the ileal loop test, toxin neutralization with specific IgYs curtailed the toxin's function in the intestine, leading to a 74.8% reduction in fluid accumulation compared to control loops. These findings suggest that egg yolk immunoglobulins against recombinant proteins LTB, CfaB, and EtpA, either individually or in combination, hold promise as prophylactic antibodies to impede the functioning of ETEC bacteria.

The implication of TET1, miR-200, and miR-494 expression with tumor formation in colorectal cancer: through targeting Wnt signaling

ObjectiveColorectal cancer (CRC) is a diverse and multifaceted disease characterized by genetic and epigenetic changes that contribute to tumor initiation and progression. CRC pathophysiology has been linked to the deregulation of the Wnt signaling pathway and the ten-eleven translocation (TET) DNA demethylases. This study aimed to evaluate the expression level of selective miRNAs (miR-200 and miR-494), TET1, and Wnt1 in colorectal polyps, actual colorectal tumors, and normal adjacent tissues. We also evaluated the effect of 5-aza cytidine on the expression level of TET1 and wnt1 in the HT29 cell line.Materials and methodsIn this study, we assessed TET1 and Wnt1 expression in 5-azacytidine-treated HT29 cells, a demethylating agent commonly used in cancer therapy. Additionally, we enrolled 114 individuals who underwent radical surgical colon resection, including 47 with cancerous tissues and 67 with polyps. We utilized qRT-PCR to measure miR-200, miR-494, TET1, and Wnt1 mRNA levels in colorectal polyps, actual colorectal tumors, and normal adjacent tissues.ResultsOur study revealed that TET1 expression was notably lower in both polyps and CRC tissue compared to adjacent normal tissue, with higher TET1 expression in tumors than polyps. We also observed significant differences in miR-200 and miR-494 expression in tumor samples compared to adjacent normal tissue. Our in vitro experiments revealed that 5-azacytidine administration increased TET1 and decreased Wnt1 expression in CRC cell lines. This suggests that DNA-demethylating drugs may have a therapeutic role in modifying TET1 and Wnt signaling in the development of CRC.ConclusionsOverall, our findings shed light on the intricate interactions between TET1, Wnt1, and specific miRNAs in colorectal cancer (CRC) and their potential implications for diagnosis and treatment.

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99

This study aimed to evaluate the safety profile and beneficial effects of Heyndrickxia coagulans strain BC99 (BC99) for potential use in functional foods and pharmaceuticals. We began with whole genome sequencing of BC99, followed by a comprehensive safety assessment comprising genome analysis, hemolysis, cytotoxicity, antibiotic susceptibility tests, and cell adhesion and tolerance studies, along with acute and subacute oral toxicity studies in animal models. BC99 was isolated from a well-characterized collection originating from the feces of a healthy infant. Our results indicated no hemolytic activity on Columbia blood agar plates and broad antibiotic sensitivity, including to gentamicin, ampicillin, chloramphenicol, ciprofloxacin, and others. Cytotoxicity testing confirmed no adverse effects on HT-29 cells and significant adhesive properties to intestinal epithelial cells. Tolerance tests demonstrated over 90% viability of BC99 under simulated gastrointestinal conditions. In vivo studies in mice and rats confirmed the absence of adverse effects following oral administration. Collectively, these findings support BC99’s robust tolerance to gastrointestinal environments, strong adhesion capabilities, and a broad spectrum of antibiotic resistance, underlining its potential as a safe and effective agent for gut microbiota modulation and host health enhancement.

Co-Colorectal cancer stem cells employ the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia

Colorectal cancer (CRC) ranks as China’s second most common cancer and fifth top cancer death cause. The study highlights the role of Natural Killer (NK) cells in targeting cancer stem cells (CSCs) that evade immune responses in CRC. Colorectal cancer stem cells (CCSCs) were stem from HT-29 cells and co-cultured with NK cells under normoxic or hypoxic conditions. The impact of this co-culture was evaluated using CCK8 assays for NK cell viability, ELISA for cytokine level changes, and flow cytometry for assessing NK cell apoptosis and activation. Comprehensive metabolomic and transcriptomic analyses were also performed to identify key genes and metabolites involved in the interaction between CCSCs and NK cells Co-culture of CCSCs with NK cells under hypoxia reduced NK cytotoxicity, increased NK apoptosis, and altered cytokine secretion by decreasing IFN-γ and TNF-α levels while increasing IL-6. Transcriptomic and metabolomic analysis identified 4 genes (FADS1, ALDH3A2, GCSH, MTCL1) and 3 metabolites (glyoxylic acid, spermine, DDA) as significant. Interfering with FADS1 counteracted the suppression of IFN-γ and TNF-α induced by CSC cells. Curiously, this inhibition caused by si-FADS1 could be neutralized by the addition of exogenous DDA. Co-culturing with NK cells notably increased spermine levels. Exogenous spermine resulted in a significant reduction in HT-29 cell death rates at 32 µM, 64 µM, and 128 µM, compared to NK cells without spermine. Our research explored CCSCs employed the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia.