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Colorectal cancer (CRC) is the fourth most common cause of malignant tumor death. The development of novel, more effective drugs is desperately needed to treat CRC. Zingiber officinale is believed to possess anticancer properties due to its flavonoids and phenols. Using Soxhlet (SOXT) and maceration (MACR) techniques, the present study aimed to evaluate the amounts of quercetin, gallic acid, rutin, naringin, and caffeic acid in ginger capsules of Z. officinale. High‐performance liquid chromatography (HPLC)/ultraviolet was used for separation and quantitation. In vitro toxicity evaluation of ginger capsules on the CRC cell line HT‐29 was also conducted to assess the anticancer activity of the supplement. The cell line HT‐29 (HTB‐38) colorectal adenocarcinoma was utilized for the antiproliferative effect of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide. Ginger herbal supplement extract at dosages of 200 and 100 μg had strong cytotoxic effects (IC50 < 50 μg/mL) on HT‐29 CRC cells via MACR. This extract is comparable to the SOXT extract, which has an IC50 of less than 50 μg/mL. The anticancer effect of ginger herbal supplement formulations against CRC lines was investigated, and the results obtained from both the MACR and SOXT extraction procedures were noteworthy. The quercetin content was the highest of all the extracts according to the HPLC data. [ABSTRACT FROM AUTHOR]

Background: Probiotic administration can be an effective treatment against intestinal inflammation. This study aimed to assess the potential effects of spores isolated from probiotic strains Bacillus subtilis natto and Bacillus coagulans Hammer on inflammation induced by lipopolysaccharide (LPS) in human colon epithelial cells in vitro. Materials and Methods: The viability of HT-29 cells treated with spores derived from B. subtilis natto and B. coagulans Hammer (MOI 10, 100, 1000), as well as LPS (10 µg/ml) was assessed. The anti-inflammatory effects of spores were examined on HT-29 cells that were pre-stimulated with LPS. The expression level of IL-6 and TLR4 genes in HT-29 cells was quantified after 24 h using RT-qPCR. Results: There was no significant reduction in the viability of HT-29 cells after exposure to LPS and various MOIs of probiotic spores. Stimulation of HT-29 cells with LPS significantly increased the expression level of IL-6 and TLR4 in comparison to control (P < 0.0001). Spores isolated from both probiotic strains, B. subtilis natto and B. coagulans Hammer, caused a significant reduction in the gene expression of IL-6 and TLR4 in HT-29 cells compared to LPS control (P < 0.0001). Conclusion: The findings of this study suggest that probiotics-derived spores may exert anti-inflammatory effects through interference with the LPS signaling pathway in colon cancer HT-29 cell line.

The shoots of Asparagus L. are consumed worldwide, although most species belonging to this genus have a restricted range, and several taxa remain unstudied. In this work, a total of four taxa from different locations were scrutinized and compared with cultivated A. officinalis. All shoots were screened for saponins via LC-MS, and in vitro antiproliferative activities against the HT-29 colorectal cancer cell line were assessed via the MTT assay. The total saponins (TS) contained in the crude extracts ranged from 710.0 (A. officinalis) to 1258.6 mg/100 g dw (A. acutifolius). The richness of the compounds detected in this work stands out; a total of 47 saponins have been detected and quantified in the edible parts (shoots) of five taxa of Asparagus. The structure of all the saponins found present skeletons of the furostane and spirostane type. In turn, the structures with a furostane skeleton are divided into unsaturated and dioxygenated types, both in the 20–22 position. The sum of dioscin and derivatives varied largely among the studied taxa, reaching the following percentages of TS: 27.11 (A. officinalis), 18.96 (A. aphyllus), 5.37 (A. acutifolius), and 0.59 (A. albus); while in A. horridus, this compound remains undetected. Aspachiosde A, D, and M varied largely among samples, while a total of seven aspaspirostanosides were characterized in the analyzed species. The hierarchical cluster analysis of the saponin profiles clearly separated the various taxa and demonstrated that the taxonomic position is more important than the place from which the samples were acquired. Thus, saponin profiles have chemotaxonomic significance in Asparagus taxa. The MTT assay showed dose- and time-dependent inhibitory effects of all saponins extracts on HT-29 cancer cells, and the strongest cell growth inhibition was exercised by A. albus and A. acutifolius (GI50 of 125 and 175 µg/mL). This work constitutes a whole approach to evaluating the saponins from the shoots of different Asparagus taxa and provides arguments for using them as functional foods. [ABSTRACT FROM AUTHOR]

The seeds of Boraginaceae species constitute a source of γ-linolenic acid (GLA, 18:3n-6)-rich oils, mainly those of Borago officinalis. However, there are many wild unexplored Boraginaceae taxa that are potentially GLA producers. This work scrutinizes the seeds of several Greek Boraginaceae species for fatty acids (FAs), phenolic compounds, antioxidant activity, and in vitro antiproliferative activity against HT-29 colorectal cancer cells. The FA profiles were checked by GC-FID, and GLA (FA% of total FA) highlights in Symphytum bulbosum (26.2), S. creticum (23.2), and Lithodora zahnii (17.4). The total FA content ranged from 9.2 in Alkanna methanaea to 27.3 g/100 g of seeds in Alkanna corcyrensis. The antioxidant activity of the water/methanol extracts, evaluated by the ABTS and DPPH methods, was in line with other GLA producer seeds. Phenolic acids were analyzed by HPLC-DAD and LC-MS, and rosmarinic acid ranged from 160.2 in S. bulbosum to 479.7 mg/100 g in L. zahnii. The MTT assay showed dose- and time-dependent inhibitory effects of the water/methanol extracts against HT-29 cancer cells, especially those of C. major and L. zahnii (GI50 of 600 and 375 μg/mL for 72 h-exposed cells). This work constitutes the first approach to evaluate the seeds of Boraginaceae taxa from Greece as functional oil providers. [ABSTRACT FROM AUTHOR]

Background: Probiotic administration can be an effective treatment against intestinal inflammation. This study aimed to assess the potential effects of spores isolated from probiotic strains Bacillus subtilis natto and Bacillus coagulans Hammer on inflammation induced by lipopolysaccharide (LPS) in human colon epithelial cells in vitro. Materials and Methods: The viability of HT-29 cells treated with spores derived from B. subtilis natto and B. coagulans Hammer (MOI 10, 100, 1000), as well as LPS (10 µg/ml) was assessed. The anti-inflammatory effects of spores were examined on HT-29 cells that were pre-stimulated with LPS. The expression level of IL-6 and TLR4 genes in HT-29 cells was quantified after 24 h using RT-qPCR. Results: There was no significant reduction in the viability of HT-29 cells after exposure to LPS and various MOIs of probiotic spores. Stimulation of HT-29 cells with LPS significantly increased the expression level of IL-6 and TLR4 in comparison to control (P < 0.0001). Spores isolated from both probiotic strains, B. subtilis natto and B. coagulans Hammer, caused a significant reduction in the gene expression of IL-6 and TLR4 in HT-29 cells compared to LPS control (P < 0.0001). Conclusion: The findings of this study suggest that probiotics-derived spores may exert anti-inflammatory effects through interference with the LPS signaling pathway in colon cancer HT-29 cell line. [ABSTRACT FROM AUTHOR]

Naturally fermented dairy products are an important component of the human diet. They are a valuable source of nutrients as well as vitamins and minerals. Their importance as a source of probiotic bacterial strains should not be overlooked. A number of studies highlight the positive effects of species of the probiotic lactic acid bacteria on the intestinal microbiome and the overall homeostasis of the body, as well as a complementary treatment for some diseases. However, data on the effects on the intestinal epithelial cells of postmetabolites released by probiotic bacteria are incomplete. This is likely due to the fact that these effects are species- and strain-specific. In the present study, we investigated the effects of postmetabolites produced by a pre-selected candidate probiotic strain Limosilactobacillus fermentum on HT-29 intestinal epithelial cells. Our data showed a pronounced proliferative effect, evaluated by flow cytometry, quantification of the cell population and determination of the mitotic index. This was accompanied by the stabilization of the cell monolayer, measured by an increase in TEER (transepithelial electric resistance) and the reorganization of actin filaments. The data obtained are a clear indication of the positive effects that the products secreted by L. fermentum strain 53 have on intestinal epithelial cells. [ABSTRACT FROM AUTHOR]

The ice plant (Mesembryanthemum crystallinum L.) is a halophyte that could become an alternative crop because of its interest as a functional food and its adaptation to high‐saline soils. In this work, leaves from wild ice plants were compared with their cultivated counterparts in a soilless system at different salinities and light exposures for assessing growth parameters, moisture, fatty acid profiles, total carotenoids, phenolic compounds, vitamin C, antioxidant activity, and antiproliferative activity against the HT‐29 colorectal cancer cell line. Moisture ranged between 876 and 955 g kg−1, and wild plants contained higher proportions of α‐linolenic acid (58.7%–60.7% of total fatty acids) than cultivated ones (20.4%–36.6%). Vitamin C ranged between 819 and 1143 mg kg−1 fresh leaves. Higher salinity led to a larger production of carotenoids, whereas plant mass, total phenolic content, and antioxidant activity increased in plants grown using L8 NS1 and L8 AP67 lamps in comparison with white‐light ones. Phenolic profiles were assessed by LC coupled to a hybrid mass spectrometer Q‐Orbitrap. Total phenolic acid content was 3–4‐fold higher than that of flavonoids, and sinapic, p‐coumaric, gallic, 4‐hydroxybenzoic, and 2‐hydroxy‐4‐methoxybenzoic acids, as well as gallocatechin, occurred in all samples. Hydroalcoholic extracts of ice plant leaves showed dose‐ and time‐dependent antiproliferative activity against the HT‐29 human colorectal cancer cell line, and GI50 was between 920 and 977 μg mL−1 of plant extract. This work contributes to improving knowledge about the growth parameters, phytochemical profiles, and biological activities of wild and cultivated ice plants. [ABSTRACT FROM AUTHOR]

Objective To observe the effect of different concentrations of glutamine (Gln) on the radiosensitivity of colorectal cancer HT-29 cells and explore the possible mechanism. Methods According to different Gln concentrations, HT-29 cells at logarithmical growth were divided into control group (2 mmol/L, as the basal medium concentration group) and experimental groups Ⅰ, Ⅱ and Ⅲ (4, 6 and 8 mmol/L). After a 2-hour pre-treatment, all groups were exposed to 8 Gy irradiation of a Co-60 radiation source. CCK-8 assay and clonal formation assay were used respectively to explore the effects of different Gln concentrations on cell viability and cell radiosensitivity after irradiation. The level of reactive oxygen species (ROS) in each group was measured in 24 h after irradiation, and the apoptotic rate was detected with flow cytometry in 48 h after irradiation. The protein expression levels of Nrf2, HO-1, and cleaved-Caspase3 were determined by Western blotting. Results In 24 h after Gln intervention, the cell viability of experimental groups Ⅱ and Ⅲ of non-irradiated HT-29 cells was significantly higher than that of the control group and of experimental group Ⅰ (P < 0.05). In 24 h after radiation, the cell viability of each experimental group was significantly higher than that of the control group (P < 0.05). In 14 d after radiation, there were more clone formation in each experimental group than the control group (P < 0.05). The ROS level was significantly lower in each experimental group than the control group in 24 h after radiation (P < 0.05). After 48 h of radiation, the apoptotic rate was notably lower in each experimental group than the control group (P < 0.05). The expression level of Nrf2 in the experimental group Ⅰ was higher than that of the control group (P < 0.05), those of Nrf2 and HO-1 in the experimental groups Ⅱ and Ⅲ were higher than those of the control group and experimental group Ⅰ (P < 0.05). While the expression of cleaved-Caspase3 in the experimental groups Ⅱ and Ⅲ was lower than the control group and experimental group Ⅰ (P < 0.05), and it in the experimental group Ⅲ was lower than that of experimental group Ⅱ (P < 0.05). Conclusion Gln can significantly reduce the radiosensitivity of HT-29 cells, which is associated with its reducing oxidative stress damage and reducing cell apoptosis. Our results suggest that Gln might be detrimental to radiation therapy in patients with colorectal cancer.

Background: Colorectal cancer (CRC) is among the most common cancers worldwide. In spite of promising improvements, it has a relatively poor prognosis. Therefore, there is a crucial need for discovering novel and efficient therapies. This study aimed to investigate the potential of phycocyanin as a therapeutic agent for CRC, considering its antioxidant and anti-inflammatory properties. Methods: The study used HT-29 and HUVEC cell lines. Cells were exposed to different concentrations of phycocyanin for 24 h. The cytotoxic effects of the phycocyanin on HUVEC and HT-29 cells were evaluated using MTT. Following the treatment, RNA extraction was conducted using TRI reagent. Afterward, real-time PCR (RT-PCR) was performed on Bcl-2, Bax, Akt, PTEN, and ß-actin to study their expression levels. The phycocyanin-treated cells were also stained with Annexin V-FITC to detect any apoptosis and necrosis. Results: Following the MTT assay, the number of living HT-29 cells significantly decreased, with no toxic effect on HUVEC cells. RT-PCR results indicated a significant increase in PTEN and Bax expressions in HT-29 cells due to treatment with phycocyanin, while there was no change in the expression of Bcl-2 and AKT. Flow cytometry on HT-29 cells showed induction of apoptosis and necrosis in 8.79% and 2.9% of cells, respectively, while no necrosis or apoptosis was observed in normal cells. Conclusion: Phycocyanin has a potential for treating colon cancer by affecting AKT/PTEN and Bax/Bcl- 2 and inducing apoptotic genes (Bax and PTEN) while having minimal side effects. Therefore, it could be a promising therapeutic agent for colon cancer. [ABSTRACT FROM AUTHOR]

Colorectal cancer is a frequent malignancy, so its corresponding behaviors are essential to be understood deeply. This work was developed to explore the impacts of Livin on proliferation, migration, invasion, and epithelial-mesenchymal transition of HT-29 colorectal cancer cells. Colorectal cancer cells were extracted from pathological specimens collected from 80 colorectal cancer patients treated at our institution from June 2017 to June 2022. HT-29 human colorectal cancer cells were undertaken as the research materials. Real-time immunofluorescence polymerase chain reaction, Western blot, cell counting kit-8 experiments, cell scratch assays, and Transwell experiments were performed to assess the proliferation, migration, invasion, and epithelial-mesenchymal transition-related markers of the colorectal cancer cells. Livin expression in colorectal cancer cells increased greatly in contrast to that in healthy cells (p<0.05). Compared to normal HT-29, the overexpression of Livin promoted the proliferation, migration, and invasion abilities of colorectal cancer cells, exhibiting great differences (p<0.05). Moreover, levels of matrix metalloproteinase 2, vimentin, slug, and snail in colorectal cancer cells were sharply upregulated (p<0.05), while that of E-cadherin was remarkably downregulated (p<0.05) in contrast to the conditions in normal cells. The findings in this work revealed the crucial role of Livin in HT-29 colorectal cancer cells, suggesting its potential to be a target for colorectal cancer treatment. [ABSTRACT FROM AUTHOR]

Calostoma insigne puffball mushrooms are only found in forests with rich biodiversity in very few countries including Thailand, and their biofunctions remain largely unexplored. This study used the agar disk diffusion assay, the anti-glucosidase assay, and the 3, 4, 5-dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide (MTT) assay to evaluate the bioactive potential of these endangered puffball mushrooms. Internal transcribed spacer (ITS) gene analysis identified C. insigne, a puffball mushroom with green, globose, and spiny spores. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the polysaccharide structure while scanning electron microscopy (SEM) revealed a fiber-like network. The ethanolic gelatinous fruiting body extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging capacity (57.96%), a ferric ion-reducing antioxidant power (FRAP) value of 1.73 mg FeSO4/g, and α-glucosidase inhibition (73.18%). C. insigne cytotoxicity was effective towards HT-29 colon cancer cells using the MTT assay (IC50 of 770.6 µg/mL at 72 h) and also showed antiproliferative capacity (IC50 of 297.1 µg/mL). This puffball mushroom stimulated apoptotic genes and proteins (caspase-3, Bax, and p21) via an intrinsic apoptotic pathway in HT-29 cells. In the laboratory, the medium formula consisting of 20% potato, 2% sucrose, and 0.2% peptone was optimal to increase fungal mycelial biomass (2.74 g DW/100 mL), with propagation at pH 5.0 and 30 °C. Puffball mushrooms are consumed as local foods and also confer several potential health benefits, making them worthy of conservation for sustainable utilization. [ABSTRACT FROM AUTHOR]

Background: Apoptotic agents from natural products like phenolic compounds can be used effectively in the treatment of cancer. Chlorogenic acid (CGA) is one of the phenolic compounds in medicinal plants with anti-cancer properties. In this research, we aimed to explore the anti-cancer mode of action of CGA on colorectal cancer (CRC) cells in vitro conditions. Methods: HT-29 and HEK-293 cells were cultured after MTT assay for 24 h with CGA 100 µM, and without CGA. Then, flow cytometry assays and the expression of apoptosis-related genes including caspase 3 and 9, Bcl-2 and Bax, and cell cycle-related genes including P21, P53 and NF-κB at mRNA and protein levels were examined. Finally, we measured the amount of intracellular reactive oxygen species (ROS). Results: The cell viability of all two-cell lines decreased in a dose-dependent manner. Moreover, CGA induces cell cycle arrest in HT-29 cells by increasing the expression of P21 and P53. It also induces apoptosis in HT-29 cells by mitigating Bcl-2 and NF-κB expression and elevating caspase 3 and 9 expression and ROS levels. Conclusions: Considering the cytotoxicity and cell cycle arrest and induction of apoptosis in the colon cancer cell line by CGA, it can be concluded that CGA is a suitable option for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Marine cyanobacteria have been known to contain several unique bioactive compounds which have different therapeutic potentials. The current research focuses to identify the efficacy of Nostoc calcicola to counteract the harmful effects of free radicals and testing its anticancer activity against colorectal adenocarcinoma cells (HT-29). Results Methanol is used as a solvent for the extraction of bioactive metabolites from Nostoc calcicola followed by phytochemical screening representing the presence of flavonoids, phenols, tannins, saponins, and steroids to find out bioactive metabolites. Furthermore, evaluation of the extract efficacy revealed the profound ability of Nostoc calcicola to scavenge free radicals by neutralizing different reactive oxygen species. At 100 µg/mL concentration, it inhibited DPPH radicals (73.4%), enhanced phosphomolybdenum reduction (53.5%), displayed ferric-reducing power (55.1%), and finally the extract revealed remarkable hydroxyl radicals scavenging capacity (94.8%), compared to the standards. These compelling results emphasize the robust antioxidant potential of the Nostoc calcicola extract. In vitro, studies demonstrated the selective cytotoxic effects of methanol extracts of Nostoc calcicola on the HT-29 human colorectal cancer cell line, as indicated by IC50 values of 25 µg/mL for the extracts. Treatment with me Nostoc calcicola decreased the cell viability of HT-29 cells followed by consistent morphological changes leading to cytotoxicity. Nuclear condensation and DNA fragmentation were observed using AO/EtBr and DAPI staining. Flow cytometry analysis further confirmed the incidence of apoptosis during the S phase of the cell cycle. Furthermore, western blotting analysis confirmed the activation of caspase 9, a pivotal enzyme in the intrinsic apoptosis pathway, suggesting the ability of Nostoc calcicola to induce apoptosis in HT-29 colorectal cancer cells. Conclusion These findings underscore the potential of Nostoc calcicola as a valuable source of bioactive compounds with antioxidant and anticancer properties, warranting further investigation for their potential therapeutic applications in colorectal cancer. Graphical Abstract

Naturally fermented dairy products are an important component of the human diet. They are a valuable source of nutrients as well as vitamins and minerals. Their importance as a source of probiotic bacterial strains should not be overlooked. A number of studies highlight the positive effects of species of the probiotic lactic acid bacteria on the intestinal microbiome and the overall homeostasis of the body, as well as a complementary treatment for some diseases. However, data on the effects on the intestinal epithelial cells of postmetabolites released by probiotic bacteria are incomplete. This is likely due to the fact that these effects are species- and strain-specific. In the present study, we investigated the effects of postmetabolites produced by a pre-selected candidate probiotic strain Limosilactobacillus fermentum on HT-29 intestinal epithelial cells. Our data showed a pronounced proliferative effect, evaluated by flow cytometry, quantification of the cell population and determination of the mitotic index. This was accompanied by the stabilization of the cell monolayer, measured by an increase in TEER (transepithelial electric resistance) and the reorganization of actin filaments. The data obtained are a clear indication of the positive effects that the products secreted by L. fermentum strain 53 have on intestinal epithelial cells.

The shoots of Asparagus L. are consumed worldwide, although most species belonging to this genus have a restricted range, and several taxa remain unstudied. In this work, a total of four taxa from different locations were scrutinized and compared with cultivated A. officinalis. All shoots were screened for saponins via LC-MS, and in vitro antiproliferative activities against the HT-29 colorectal cancer cell line were assessed via the MTT assay. The total saponins (TS) contained in the crude extracts ranged from 710.0 (A. officinalis) to 1258.6 mg/100 g dw (A. acutifolius). The richness of the compounds detected in this work stands out; a total of 47 saponins have been detected and quantified in the edible parts (shoots) of five taxa of Asparagus. The structure of all the saponins found present skeletons of the furostane and spirostane type. In turn, the structures with a furostane skeleton are divided into unsaturated and dioxygenated types, both in the 20–22 position. The sum of dioscin and derivatives varied largely among the studied taxa, reaching the following percentages of TS: 27.11 (A. officinalis), 18.96 (A. aphyllus), 5.37 (A. acutifolius), and 0.59 (A. albus); while in A. horridus, this compound remains undetected. Aspachiosde A, D, and M varied largely among samples, while a total of seven aspaspirostanosides were characterized in the analyzed species. The hierarchical cluster analysis of the saponin profiles clearly separated the various taxa and demonstrated that the taxonomic position is more important than the place from which the samples were acquired. Thus, saponin profiles have chemotaxonomic significance in Asparagus taxa. The MTT assay showed dose- and time-dependent inhibitory effects of all saponins extracts on HT-29 cancer cells, and the strongest cell growth inhibition was exercised by A. albus and A. acutifolius (GI50 of 125 and 175 µg/mL). This work constitutes a whole approach to evaluating the saponins from the shoots of different Asparagus taxa and provides arguments for using them as functional foods.

The seeds of Boraginaceae species constitute a source of γ-linolenic acid (GLA, 18:3n-6)-rich oils, mainly those of Borago officinalis. However, there are many wild unexplored Boraginaceae taxa that are potentially GLA producers. This work scrutinizes the seeds of several Greek Boraginaceae species for fatty acids (FAs), phenolic compounds, antioxidant activity, and in vitro antiproliferative activity against HT-29 colorectal cancer cells. The FA profiles were checked by GC-FID, and GLA (FA% of total FA) highlights in Symphytum bulbosum (26.2), S. creticum (23.2), and Lithodora zahnii (17.4). The total FA content ranged from 9.2 in Alkanna methanaea to 27.3 g/100 g of seeds in Alkanna corcyrensis. The antioxidant activity of the water/methanol extracts, evaluated by the ABTS and DPPH methods, was in line with other GLA producer seeds. Phenolic acids were analyzed by HPLC-DAD and LC-MS, and rosmarinic acid ranged from 160.2 in S. bulbosum to 479.7 mg/100 g in L. zahnii. The MTT assay showed dose- and time-dependent inhibitory effects of the water/methanol extracts against HT-29 cancer cells, especially those of C. major and L. zahnii (GI50 of 600 and 375 μg/mL for 72 h-exposed cells). This work constitutes the first approach to evaluate the seeds of Boraginaceae taxa from Greece as functional oil providers.

The HT-29 cell line, derived from human colon cancer, is valuable for biological and cancer research applications. Early detection is crucial for improving the chances of survival, and researchers are introducing new techniques for accurate cancer diagnosis. This study introduces an efficient deep learning-based method for detecting and counting colorectal cancer cells (HT-29). The colorectal cancer cell line was procured from a company. Further, the cancer cells were cultured, and a transwell experiment was conducted in the lab to collect the dataset of colorectal cancer cell images via fluorescence microscopy. Of the 566 images, 80 % were allocated to the training set, and the remaining 20 % were assigned to the testing set. The HT-29 cell detection and counting in medical images is performed by integrating YOLOv2, ResNet-50, and ResNet-18 architectures. The accuracy achieved by ResNet-18 is 98.70 % and ResNet-50 is 96.66 %. The study achieves its primary objective by focusing on detecting and quantifying congested and overlapping colorectal cancer cells within the images. This innovative work constitutes a significant development in overlapping cancer cell detection and counting, paving the way for novel advancements and opening new avenues for research and clinical applications. Researchers can extend the study by exploring variations in ResNet and YOLO architectures to optimize object detection performance. Further investigation into real-time deployment strategies will enhance the practical applicability of these models.

Background: Marine cyanobacteria have been known to contain several unique bioactive compounds which have different therapeutic potentials. The current research focuses to identify the efficacy of Nostoc calcicola to counteract the harmful effects of free radicals and testing its anticancer activity against colorectal adenocarcinoma cells (HT-29). Results: Methanol is used as a solvent for the extraction of bioactive metabolites from Nostoc calcicola followed by phytochemical screening representing the presence of flavonoids, phenols, tannins, saponins, and steroids to find out bioactive metabolites. Furthermore, evaluation of the extract efficacy revealed the profound ability of Nostoc calcicola to scavenge free radicals by neutralizing different reactive oxygen species. At 100 µg/mL concentration, it inhibited DPPH radicals (73.4%), enhanced phosphomolybdenum reduction (53.5%), displayed ferric-reducing power (55.1%), and finally the extract revealed remarkable hydroxyl radicals scavenging capacity (94.8%), compared to the standards. These compelling results emphasize the robust antioxidant potential of the Nostoc calcicola extract. In vitro, studies demonstrated the selective cytotoxic effects of methanol extracts of Nostoc calcicola on the HT-29 human colorectal cancer cell line, as indicated by IC50 values of 25 µg/mL for the extracts. Treatment with me Nostoc calcicola decreased the cell viability of HT-29 cells followed by consistent morphological changes leading to cytotoxicity. Nuclear condensation and DNA fragmentation were observed using AO/EtBr and DAPI staining. Flow cytometry analysis further confirmed the incidence of apoptosis during the S phase of the cell cycle. Furthermore, western blotting analysis confirmed the activation of caspase 9, a pivotal enzyme in the intrinsic apoptosis pathway, suggesting the ability of Nostoc calcicola to induce apoptosis in HT-29 colorectal cancer cells. Conclusion: These findings underscore the potential of Nostoc calcicola as a valuable source of bioactive compounds with antioxidant and anticancer properties, warranting further investigation for their potential therapeutic applications in colorectal cancer. [ABSTRACT FROM AUTHOR]

Colon cancer is the fifth leading cause of cancer-related deaths worldwide. Stem cells have unique characteristics and are considered as a novel therapeutic platform for cancer. Sugen Kinase 269 (SgK269) is considered as an oncogenic scaffolding pseudo kinase which governs the rearranging of the cytoskeleton, cellular motility, and invasion. The aim of this study is to evaluate the expression of SgK269 in colon cancer patients and explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) on invasion and proliferation of colon cancer cells (HT-29) through analyzing SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2 signaling pathway. In this regard, we collected 30 samples from colon cancer patients and evaluated SgK269 expression using quantitative real-time PCR (qRT-PCR). Next, we employed a co-culture system using Transwell 6-well plates and after 72 h, tumor growth promotion and invasion were analyzed in hAMSCs-treated HT-29 cells through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway using qRT-PCR, western blot method, MTT assay, wound healing assay, and DAPI staining. Our results showed upregulation of SgK269 in colon cancer patients. Treatment of HT-29 colon cancer cells with hAMSCs secretome can inhibit SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway and the resulting suppression of cell invasion and proliferation. Our results suggest that SgK269 is an important target in colon cancer therapy and MSCs secretome may be an effective therapeutic approach to inhibit colon cancer cell invasion and proliferation through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway. [ABSTRACT FROM AUTHOR]

Isorhamnetin has gained research interest for its anti-inflammatory, anti-proliferative and chemoprotective properties. In this study, human colon adenocarcinoma cells were cultured in the presence or absence of different isorhamnetin concentrations (5–150 μM) for 24 h or 48 h of cultivation to explore the impact on several parameters of viability/proliferation (mitochondrial function using an MTT test, metabolic activity, cell membrane integrity and lysosomal activity using a triple test). The intracellular generation of superoxide radicals using an NBT test and ELISA analysis was performed to observe the biosynthesis of interleukin 8 (IL-8) in cells stimulated with zymosan, as well as in basal conditions. The antiproliferative activity of isorhamnetin was demonstrated by significantly reduced values of mitochondrial and metabolic activity, integrity of cell membranes and lysosomal activity. Its high prooxidant potential was reflected by the significantly elevated generation of superoxides even in cells with low viability status. The anti-inflammatory effect of isorhamnetin was evident due to decreased IL-8 production, and the most significant decline in IL-8 concentration was observed after 24 h treatment in cells with induced inflammation. We demonstrated that isorhamnetin can suppress the proliferation of HT-29 cells, and this effect was correlated with pro-oxidative and anti-inflammatory activity of isorhamnetin. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract with the second highest mortality rate globally. Piper nigrum is a widely used traditional medicinal plant, exhibiting antitumor activity against various tumor cells. At present, research on the effect of Piper nigrum on CRC is limited to in vitro cytotoxicity, lacking comprehensive mechanism investigations. This study aimed to explore the inhibitory effect and mechanism of Piper nigrum extract (PNE) on HT-29 cells. Firstly, we identified the chemical components of PNE. Then, MTT assay, colony formation assay, JC-1 staining, and flow cytometry were used to analyze the effect of PNE on HT-29 cells in vitro. A xenograft model, histopathological examination, immunohistochemistry, and western blot were used to evaluate the tumor growth inhibitory activity and mechanism of PNE in vivo. The results indicated that PNE could inhibit cell proliferation and colony formation, reduce mitochondrial membrane potential, induce cell apoptosis in vitro, and inhibit tumor growth in vivo. Furthermore, PNE could regulate p53 and its downstream proteins, and subsequently activate the caspase-3 pathway. In summary, PNE probably induced apoptosis of HT-29 cells through the mitochondrial pathway mediated by p53. All these results suggested that PNE might be a potential natural-origin anti-CRC drug candidate. [ABSTRACT FROM AUTHOR]

Wild Asparagus shoots are consumed worldwide, although most species remain understudied. In this work, a total of four wild Asparagus species were collected from different locations and analyzed compared with farmed A. officinalis. Shoots were screened for (i) phenolic compounds by HPLC-DAD and LC-MS; (ii) total phenolic acids and total flavonoid content by the Folin–Ciocalteu and aluminum chloride methods; (iii) vitamin C by HPLC-DAD; (iv) antioxidant activity by the DPPH and ABTS•+ methods; and (v) the in vitro antiproliferative activities against HT-29 colorectal cancer cells by the MTT assay. Phenolics ranged from 107.5 (A. aphyllus) to 605.4 mg/100 g dry weight (dw) (A. horridus). Vitamin C ranged from 15.8 (A. acutifolius) to 22.7 mg/100 g fresh weight (fw) (A. officinalis). The antioxidant activity was similar in all species, standing out in A. officinalis with 5.94 (DPPH) and 4.64 (ABTS) mmol TE/100 g dw. Among phenolics, rutin reached the highest values (574 mg/100 g dw in A. officinalis), followed by quercetin, nicotiflorin, asterin, and narcissin. The MTT assay revealed the inhibitory effects of ethanol extracts against HT-29 cancer cells, highlighting the cell growth inhibition exercised by A. albus (300 µg/mL after 72 h exposure to cells). This work improves knowledge on the phytochemicals and bioactivities of the shoots of wild Asparagus species and confirms their suitability for use as functional foods. [ABSTRACT FROM AUTHOR]

Abstract Backgrounds The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. Results p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. Conclusions This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Abstract Background Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA. Results C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj

L. paracasei subsp. paracasei X12 was obtained from traditional cheese produced in northwestern China. In this study, we showed that whole peptidoglycan (WPG), extracted from L. paracasei subsp. paracasei X12, inhibited proliferation and induced apoptosis in HT-29 cells in a dose-dependent manner. In addition, WPG-induced apoptosis was associated with the loss of mitochondrial membrane potential (Ψm), the release of cytochrome c (Cyto-C) from mitochondrialto cytosolic spaces, activation of Caspase 3, and accumulation of intracellular reactive oxygen species (ROS). Finally, semi-quantitative RT-PCR showed that these events were accompanied by upregulation of proapoptotic genes (Bax or Bad) and downregulation of antiapoptotic genes (Bcl-xl). Taken together, our results demonstrated that WPG induced apoptosis in HT-29 cells through activation of the mitochondrial pathway. WPG exerted only minor toxicity upon noncancerous cells and therefore might be used as a natural agent in the treatment of cancer in future. [ABSTRACT FROM AUTHOR]

Objective To initially investigate the effects of curcumin analog WH-8 on the proliferation and autophagy of human colon cancer HT-29 cells. Methods Different concentrations of WH-8 were used to act on HT-29 cells, and MTT method was used to investigate its inhibitory effect on cell growth; fluorescence staining and cell imaging techniques were used to investigate the changes of autophagic vesicles induced by WH-8; MTT method was used to detect the effect of WH-8 on cell growth inhibition rate after pretreatment with autophagy-specific inhibitor 3-MA; western blot was used to investigate the expression of autophagy characteristic proteins [Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] in WH-8 cells. Results WH-8 inhibited the proliferation of HT-29 cells in a time-dose dependent manner, with an IC50 value of (12. 5±1. 3) μmol/L in 48 hours; WH-8 at a certain concentration could induce an increase in autophagic vesicles in cells; 3-MA could reverse the growth inhibition of WH-8 on human colon cacner HT-29 cells, and the difference is statistically sigificant (P＜0. 05); WH-8 could induce the up-regulation of the expression of Beclin-1, and promote the transformation from LC3 Ⅰ to LC3 Ⅱ within a certain period of time. Conclusion The curcumin analog WH-8 could inhibit the proliferation of HT-29 cells and induce autophagy. [ABSTRACT FROM AUTHOR]

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immuneprivileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective antichagasic drugs. [ABSTRACT FROM AUTHOR]

Calostoma insigne puffball mushrooms are only found in forests with rich biodiversity in very few countries including Thailand, and their biofunctions remain largely unexplored. This study used the agar disk diffusion assay, the anti-glucosidase assay, and the 3, 4, 5-dimethylthiazol-2-yl-2-5-diphenyltetrazolium bromide (MTT) assay to evaluate the bioactive potential of these endangered puffball mushrooms. Internal transcribed spacer (ITS) gene analysis identified C. insigne, a puffball mushroom with green, globose, and spiny spores. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the polysaccharide structure while scanning electron microscopy (SEM) revealed a fiber-like network. The ethanolic gelatinous fruiting body extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging capacity (57.96%), a ferric ion-reducing antioxidant power (FRAP) value of 1.73 mg FeSO4/g, and α-glucosidase inhibition (73.18%). C. insigne cytotoxicity was effective towards HT-29 colon cancer cells using the MTT assay (IC50 of 770.6 µg/mL at 72 h) and also showed antiproliferative capacity (IC50 of 297.1 µg/mL). This puffball mushroom stimulated apoptotic genes and proteins (caspase-3, Bax, and p21) via an intrinsic apoptotic pathway in HT-29 cells. In the laboratory, the medium formula consisting of 20% potato, 2% sucrose, and 0.2% peptone was optimal to increase fungal mycelial biomass (2.74 g DW/100 mL), with propagation at pH 5.0 and 30 °C. Puffball mushrooms are consumed as local foods and also confer several potential health benefits, making them worthy of conservation for sustainable utilization.

Purpose: Radiotherapy is one of the most important therapeutic options used to treat cancers.Radiation effects can be improved using nanoparticles and chemotherapeutic drugs asradiosensitizing agents. The aim of the present study was to evaluate the effects of folic acidconjugatedgold nanoparticles (GNP-F) in combination with doxorubicin (DOX) and x-Rayirradiation in colorectal cancer (CRC) cell line (HT-29).Methods: The cell viability assay (WST-1) was performed to study the cytotoxic effects ofdifferent concentrations of DOX and GNP-F after 24 and 48 hours treatments. Then, the effectsof the GNP-F, X-Ray irradiation, and DOX drug in single and combined treatments wereexamined after 24 and 48 hours treatment with effective doses. Likewise, the caspase 3 geneexpression ratio and the caspase 3 activity were assessed after 48 h treatment. Moreover, themalondialdehyde (MDA) level was determined in treated and untreated cells.Results: When GNP-F (at a concentration of 70 μM) was combined with X-ray irradiation (2Gy) and DOX drug, induced more cytotoxic effects compared to the control group. The resultsof cell viability assay showed that GNP-F + X-Ray in combination with a low concentrationof DOX (0.25 × IC50) enhanced the cytotoxic effects of cells compared to related singletreatments. Caspase 3 gene expression ratio and caspase 3 activity increased in double andtriple combination treatments in comparison with the single groups. Moreover, the MDA levelincreased in triple combination compared to the single treatments.Conclusion: Our findings confirmed the potential anti-cancer effects of the GNP-F and DOX incombination with X-Ray irradiation in CRC cells.

Berry consumption is increasing worldwide due to their high content of bioactive compounds. However, such fruits have a very short shelf life. To avoid this drawback and to offer an effective alternative for its consumption at any time of the year, an agglomerated berry powder mix (APB) was developed. The aim of this work was to evaluate the stability of APB during a 6-months-period storage at 3 temperatures. The stability of APB was determined by moisture, aw, antioxidant activity, total phenolics, total anthocyanins, vitamin C, color, phenolic profiles, and MTT assay. APB showed differences in antioxidant activity between 0 and 6 months. It experimented non-enzymatic browning, which was more remarkable at 35 °C. APB at time 0 exhibited growth inhibitory effects against HT-29 human cancer cells. Most properties were significantly modified by storage temperature and time, which induces a significant decreasing of bioactive compounds.

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immune-privileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective anti-chagasic drugs.

Probiotics have been investigated to improve the universal rotavirus (RV) vaccination as well as to ameliorate the RV infection. However, underlying mechanisms how probiotics mediate beneficial effects needs more investigation. Thus, in the present study, we used polarized HT-29 cells to assess the anti-RV properties of Gram-positive, (Lactobacillus acidophilus, Lacticaseibacillus rhamnosus GG, and Bifidobacterium subsp. Lactis Bb12) and Gram negative, (Escherichia coli Nissle 1917) probiotics and study their underlying mechanisms. Our results showed that pre-treatment of HT-29 cells for 4 h with probiotics, significantly reduced (p < 0.05) human RV replication and this effect was most pronounced for E. coli Nissle followed by L. acidophilus and L. rhamnosus GG. Strikingly, only pre-treatment with live bacteria or their supernatants demonstrated anti-RV properties. Except Gram negative E. coli Nissle, the Gram-positive probiotics tested did not bind to RV. Ingenuity pathway analysis of tight junction (TJ)- and innate immune-associated genes indicated that E. coli Nissle or E. coli Nissle + RV treatments improved cell–cell adhesion and cell contact, while L. acidophilus or L. acidophilus + RV treatments also activated cell–cell contact but inhibited cell movement functions. RV alone inhibited migration of cells event. Additionally, E. coli Nissle activated pathways such as the innate immune and inflammatory responses via production of TNF, while RV infection activated NK cells and inflammatory responses. In conclusion, E. coli Nissle's ability to bind RV, modulate expression of TJ events, innate immune and inflammatory responses, via specific upstream regulators may explain superior anti-RV properties of E. coli Nissle. Therefore, prophylactic use of E. coli Nissle might help to reduce the RV disease burden in infants in endemic areas. [ABSTRACT FROM AUTHOR]

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon. Aim. This study aimed to determine whether E. coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔlngA) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. Methods. A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔlngA (ECBLΔlngA). The point substitutions were performed in the pJETlngAFMU073332 vector. Results. Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells. Conclusion. In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence. [ABSTRACT FROM AUTHOR]

Seeds of most Arecaceae species are an underutilized raw material that can constitute a source of nutritionally relevant compounds. In this work, seeds of 24 Arecaceae taxa were analyzed for fatty acids (FAs) by GC-FID, for phenolics by HPLC-DAD and LC-MS, and for their antitumor activity against the HT-29 colorectal cancer cell line by the MTT assay. Lauric, oleic, and linoleic acids were the prominent FAs. Cocoseae species contained total FAs at 28.0–68.3 g/100 g seeds, and in other species total FAs were from 1.2 (Livistona saribus) to 9.9 g/100 g (Washingtonia robusta). Sabal domingensis, Chamaerops humilis, and Phoenix dactylifera var. Medjool had unsaturated/saturated FA ratios of 1.65, 1.33–1.78, and 1.31, respectively, and contained 7.4, 5.5–6.3, and 6.4 g FAs/100 g seeds, respectively. Thus, they could be used as raw materials for healthy oilseed production. Phenolics ranged between 39 (Livistona fulva) and 246 mg/100 g (Sabal palmetto), and of these, caffeic acid, catechin, dactylifric acid, and rutin had the highest values. (-)-Epicatechin was identified in most seed extracts by LC-MS. Hydroalcoholic extracts from five species showed a dose-dependent inhibitory effect on HT-20 cells growth at 72 h (GI50 at 1533–1968 µg/mL). Overall, Arecaceae seeds could be considered as a cheap source of health-promoting compounds. [ABSTRACT FROM AUTHOR]

Backgrounds: The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. Results: p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. Conclusions: This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40. [ABSTRACT FROM AUTHOR]

Prebiotics and probiotics have gained much attention in the pursuit of a healthy life. Recently, postbiotics have been spotlighted as next-generation compounds that can improve health. Postbiotics are designated into non-viable, inactivated, and ghost probiotics, and linked to several health benefits for the gut, immune system, and various other aspects of health. This study investigated the anti-proliferation effects of postbiotics against HT-29 cells, a colon cancer cell line. The postbiotics were produced by the ultrasonication method from two Lactobacillus strains (Lactobacillus sp. La1, and La2) and designated to Pobt-La1 and Pobt-La2, respectively, and non-viability was confirmed on the plate media. The anti-proliferation effect was concentration-dependent. The HT-29 cells showed viabilities of 39% and 49% when treated with 300 µL/mL of Pobt-La1 and Pobt-La2, respectively. During observation of the morphological changes of HT-29 cells when treated with IC50, a cell nucleus was not observed but cell condensation was observed. Moreover, in comparison with the control group, a reduced number of cells were observed. Based on these results, it considered that the postbiotic compounds from Lactobacillus La1 and La2 could provide crucial information in the development of anticancer research. Through further research, it would be beneficial to investigate the possibility of using these postbiotics (Pobt-La1 and -La2) as an anticancer drug. [ABSTRACT FROM AUTHOR]

Cancer-fighting needs more effective and targeted drugs, desirably with least side-effects and from natural bases. The assessment and synergistic actions of multiple anticancer drug candidates were investigated. Fucoidan (Fu) was extracted from Sargassum cinereum, which was attained from the Saudi Red-Sea coast. The extract of Ganoderma lucidum or Reishi mushroom (Re) was achieved, intermixed with Fu solution, and they were used for reducing/decorating/capping selenium nanoparticles (SeNPs). The infrared analysis of produced/synthesized agents emphasized their biochemical structures and interactions. The decorated Fu/Re/SeNPs had negative (−30.6 mV) zeta potential and the SeNPs mean particle size was 6.5 nm. The transmission imaging of SeNPs indicated their spherical profiles and well-dispersion within Fu/Re composite. The anticancer potentiality of Fu/Re and Fu/Re/SeNPs against human colorectal adenocarcinoma cells was investigated using MTT, 4′,6-diamidino-2-phenylindole staining and comet assay. Both composites exhibited potent bioactivities toward adenocarcinoma cells; the reduced cells survival was detected with IC50 of 57.82 and 16.43 µg·mL−1 for Fu/Re and Fu/Re/SeNPs, respectively. The constrained apoptosis was notably observed from treated cells with the composites; substantial molecular damages were also verified via the comet assay, especially in Fu/Re/SeNPs treated cells. The innovative innocuous composite from Fu/Re/SeNPs is highly recommended to deactivate/destruct colorectal adenocarcinoma cells.

Protein aggregation is a common characteristic of several human diseases such as Alzheimer’s disease. Recent evidence has indicated that the aggregation of peptides such as p53 is also marked in cancer cells. The aim of this study was to correlate Thioflavin T (ThT) data with different cellular viability assays (Neutral Red and MTT) in SH-SY5Y neuroblastoma cells and HT-29 colon cancer cells treated with doxorubicin, a classical antineoplastic agent. We also studied the effects of the well-known peptide Aβ42 on the aggregation process in these cells. Our data suggest that both cancer cell lines are responsive to doxorubicin and formed aggregates, highlighting a relationship between ThT and cellular viability methodologies. We observed that lower values of cell viability corresponded with pronounced aggregation. Thus, these results indicated that the ThT methodology used in cells may complement the cell viability assays. In addition, this methodology may be of interest to evaluate the role of protein aggregation in other cancer cells.

Isorhamnetin has gained research interest for its anti-inflammatory, anti-proliferative and chemoprotective properties. In this study, human colon adenocarcinoma cells were cultured in the presence or absence of different isorhamnetin concentrations (5–150 μM) for 24 h or 48 h of cultivation to explore the impact on several parameters of viability/proliferation (mitochondrial function using an MTT test, metabolic activity, cell membrane integrity and lysosomal activity using a triple test). The intracellular generation of superoxide radicals using an NBT test and ELISA analysis was performed to observe the biosynthesis of interleukin 8 (IL-8) in cells stimulated with zymosan, as well as in basal conditions. The antiproliferative activity of isorhamnetin was demonstrated by significantly reduced values of mitochondrial and metabolic activity, integrity of cell membranes and lysosomal activity. Its high prooxidant potential was reflected by the significantly elevated generation of superoxides even in cells with low viability status. The anti-inflammatory effect of isorhamnetin was evident due to decreased IL-8 production, and the most significant decline in IL-8 concentration was observed after 24 h treatment in cells with induced inflammation. We demonstrated that isorhamnetin can suppress the proliferation of HT-29 cells, and this effect was correlated with pro-oxidative and anti-inflammatory activity of isorhamnetin.

Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract with the second highest mortality rate globally. Piper nigrum is a widely used traditional medicinal plant, exhibiting antitumor activity against various tumor cells. At present, research on the effect of Piper nigrum on CRC is limited to in vitro cytotoxicity, lacking comprehensive mechanism investigations. This study aimed to explore the inhibitory effect and mechanism of Piper nigrum extract (PNE) on HT-29 cells. Firstly, we identified the chemical components of PNE. Then, MTT assay, colony formation assay, JC-1 staining, and flow cytometry were used to analyze the effect of PNE on HT-29 cells in vitro. A xenograft model, histopathological examination, immunohistochemistry, and western blot were used to evaluate the tumor growth inhibitory activity and mechanism of PNE in vivo. The results indicated that PNE could inhibit cell proliferation and colony formation, reduce mitochondrial membrane potential, induce cell apoptosis in vitro, and inhibit tumor growth in vivo. Furthermore, PNE could regulate p53 and its downstream proteins, and subsequently activate the caspase-3 pathway. In summary, PNE probably induced apoptosis of HT-29 cells through the mitochondrial pathway mediated by p53. All these results suggested that PNE might be a potential natural-origin anti-CRC drug candidate.

Novel pyrazolo[3,4-d] pyrimidine derivatives bearing carbon-aryl(heteryl)idene moieties were synthesized via a condensation reaction of 5-aminopyrazoles and cyclic lactams. The preparation of the target compounds employed bioisosterism, where a pyrazole ring was a major replacement. Fifteen target compounds were investigated for their antiproliferative activity on five human cancer cell lines; derivative (E)− 1-methyl-9-(3,4,5-trimethoxybenzylidene)− 6,7,8,9-tetrahydropyrazolo[3,4-d]pyrido[1,2-a]pyrimidin-4(1H)-one (10k) showed the highest activity (IC50 value (0.03–1.6 μM), on selected cell lines. Results of an in vivo experiment on an HT-29 xenograft nude mouse model also confirmed that 10k inhibited tumor growth. The proposed anticancer mechanism of 10k in HT-29 and HCT-116 cells was that 10k caused G2/M phase arrest in cancer cells and decreased the mitochondrial membrane potential (Δψmt). Additional studies were conducted on HUVEC, where 10k significantly inhibited HUVEC cell migration, adhesion, and tube formation activity. Molecular modeling studies revealed that 10k forms hydrogen bonds with cys-919 of vascular endothelial growth factor receptor 2 (VEGFR-2) and inhibit VEGFR-2 kinase activity. Moreover, tubulin polymerization assay results showed that 10k formed hydrogen bonds with Asn-101 and Gln-11 of tubulin. Furthermore, it could change the aberration of microtubule arrangements in HUVEC and inhibit tubulin polymerization. These results indicate that the main anticancer activity of 10k may be mediated by anti-vascular effects and inhibition of tubulin polymerization in pre-clinical trials.

Background: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA. Results: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-β as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). Conclusion: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs. [ABSTRACT FROM AUTHOR]

The discovery of new oral antidiabetic drugs remains a priority in medicine. This research aimed to evaluate the activity of the flavonoid baicalein and its natural glucuronide baicalin, compared to the antidiabetic drug metformin, as potential antiglycation, anti–radical, and anti-α–glucosidase agents, in order to assess their potential role in counteracting hyperglycemia-induced tissue damage. The study considered: (i) the BSA assay, to detect the formation of advanced glycation end products (AGEs), (ii) the GK peptide–ribose assay, which evaluates the cross–linking between the peptide and ribose, and (iii) the carbonyl content assay to detect the total carbonyl content, as a biomarker of tissue damage. In addition, to obtain a reliable picture of the antiglycation capacity of the investigated compounds, DPPH scavenging and oxygen radical absorbance capacity (ORAC) assays were performed. Furthermore, the anti–α–glucosidase activity of baicalein and baicalin was detected. Furthermore, to estimate cell permeability, preliminarily, the cytotoxicity of baicalein and baicalin was evaluated in HT–29 human colon adenocarcinoma cells using the MTT assay. Successively, the ability of the compounds to pass through the cytoplasmic membranes of HT–29 cells was detected as a permeability screen to predict in vivo absorption, showing that baicalein passes into cells even if it is quickly modified in various metabolites, being its main derivative baicalin. Otherwise, baicalin per se did not pass through cell membranes. Data show that baicalein is the most active compound in reducing glycation, α-glucosidase activity, and free radicals, while baicalin exhibited similar activities, but did not inhibit the enzyme α–glucosidase. [ABSTRACT FROM AUTHOR]

Purpose: Radiotherapy is one of the most important therapeutic options used to treat cancers. Radiation effects can be improved using nanoparticles and chemotherapeutic drugs as radiosensitizing agents. The aim of the present study was to evaluate the effects of folic acidconjugated gold nanoparticles (GNP-F) in combination with doxorubicin (DOX) and x-Ray irradiation in colorectal cancer (CRC) cell line (HT-29). Methods: The cell viability assay (WST-1) was performed to study the cytotoxic effects of different concentrations of DOX and GNP-F after 24 and 48 hours treatments. Then, the effects of the GNP-F, X-Ray irradiation, and DOX drug in single and combined treatments were examined after 24 and 48 hours treatment with effective doses. Likewise, the caspase 3 gene expression ratio and the caspase 3 activity were assessed after 48 h treatment. Moreover, the malondialdehyde (MDA) level was determined in treated and untreated cells. Results: When GNP-F (at a concentration of 70 μM) was combined with X-ray irradiation (2 Gy) and DOX drug, induced more cytotoxic effects compared to the control group. The results of cell viability assay showed that GNP-F + X-Ray in combination with a low concentration of DOX (0.25 x IC50) enhanced the cytotoxic effects of cells compared to related single treatments. Caspase 3 gene expression ratio and caspase 3 activity increased in double and triple combination treatments in comparison with the single groups. Moreover, the MDA level increased in triple combination compared to the single treatments. Conclusion: Our findings confirmed the potential anti-cancer effects of the GNP-F and DOX in combination with X-Ray irradiation in CRC cells. [ABSTRACT FROM AUTHOR]

For several decades, people have been searching for natural substances of plant origin that, when introduced into the diet, could strengthen immunity, have anticancer properties, and support conventional therapy. The development of agriculture with the implementation of various plant cultivation systems, apart from the economic aspect, results in the search for such cultivation conditions that would contribute to obtaining the most beneficial product for health. Therefore, the aim of our research is as follows: (a) to compare the antiproliferative activity and the ability to induce apoptosis of HT-29 cells by extracts from blueberry fruits deriving from different types of cultivation systems (conventional, organic, and biodynamic); (b) to examine whether the interaction of extracts with anticancer drugs used in the treatment of colorectal cancer is influenced by the type of cultivation, and (c) to investigate whether extracts obtained from fruits from subsequent years of cultivation retain the same biological activity. The results of our study are promising but inconclusive. A statistically significant difference occurred in only one of the two years of the study. The greatest inhibition of proliferation is observed for biodynamic cultivation compared to organic cultivation, while the highest levels of apoptosis and necrosis of HT-29 cells are induced by blueberry fruit extracts obtained from organic cultivation. The complementary effect of the extracts on the inhibition of HT-29 cell proliferation by anticancer drugs (5-FU and Erbitux) is not demonstrated. The induction of apoptosis by 5-FU is not enhanced by blueberry extracts, in contrast to necrosis. The level of apoptosis and necrosis induced by Erbitux is potentiated, but no dependence on crop type is shown. Blueberry fruit extracts from two consecutive years of cultivation did not maintain the same activity. A plausible reason for the variability in the composition and biological activity of fruit extracts obtained from two years of cultivation is the varying environmental conditions. [ABSTRACT FROM AUTHOR]

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Novel thiazepine-based hybrids (9a-d) were designed and synthesized to create lead molecules with exceptional anti-colon cancer efficacy. Analytical methods, including IR, NMR, and HR-MS, characterized the synthesized compounds. The in vitro colorectal study was carried out to compare the biological activity of newly developed compounds with the computational data. The tested compounds induced cytotoxicity in HT-29 cells for both 24h and 48h in a dose-dependent manner. However, compound 9a induced cytotoxicity at much higher concentrations compared to the rest of the compounds. 9b and 9c caused 50% cell death (compared to the untreated cells) at a dose of ~ 50µM and 40 µM in case of 24-hour exposure, respectively. On the contrary, for 48h exposure, both 9b and 9c induced 50% cell death concerning untreated cells at a dose of around ~20 µM, whereas 9d exhibited 50% cell death at 5 µM in the case of 48 h exposure. In silico ADMET was also carried out to understand the pharmacokinetics and safety profiles of the drug candidates. We found some of the critical targets of these compounds, which eventually will be integral to exploring the mechanistic actions of these compounds in colon cancer., (© 2024 Wiley‐VCH GmbH.)

A Lacticaseibacillus rhamnosus GG-derived protein, p75, is one of the key molecules exhibiting probiotic activity. However, the molecular mechanism and transcriptional response of p75 in human intestinal epithelial cells are not completely understood. To gain a deeper understanding of its potential probiotic action, this study investigated genome-wide responses of HT-29 cells to stimulation by spore-displayed p75 (CotG-p75) through a transcriptome analysis based on RNA sequencing. Analysis of RNA-seq data showed significant changes of gene expression in HT-29 cells stimulated by CotG-p75 compared to the control. A total of 189 up-regulated and 314 down-regulated genes was found as differentially expressed genes. Gene ontology enrichment analysis revealed that a large number of activated genes was involved in biological processes, such as epithelial cell differentiation, development, and regulation of cell proliferation. A gene–gene interaction network analysis showed that several DEGs, including AREG, EREG, HBEGF, EPGN, FASLG, GLI2, CDKN1A, FOSL1, MYC, SERPINE1, TNFSF10, BCL6, FLG, IVL, SPRR1A, SPRR1B, SPRR3, and MUC5AC, might play a critical role in these biological processes. RNA-seq results for selected genes were verified by reverse transcription-quantitative polymerase chain reaction. Overall, these results provide extensive knowledge about the transcriptional responses of HT-29 cells to stimulation by CotG-p75. This study showed that CotG-p75 can contribute to cell survival and epithelial development in human intestinal epithelial cells. [ABSTRACT FROM AUTHOR]