P. aeruginosa augments irradiation injury via 15-lipoxygenase-catalyzed generation of 15-HpETE-PE and induction of theft-ferroptosis.

Total body irradiation (TBI) targets sensitive bone marrow hematopoietic cells and gut epithelial cells, causing their death and inducing a state of immunodeficiency combined with intestinal dysbiosis and nonproductive immune responses. We found enhanced Pseudomonas aeruginosa (PAO1) colonization of the gut leading to host cell death and strikingly decreased survival of irradiated mice. The PAO1-driven pathogenic mechanism includes theft-ferroptosis realized via (a) curbing of the host antiferroptotic system, GSH/GPx4, and (b) employing bacterial 15-lipoxygenase to generate proferroptotic signal - 15-hydroperoxy-arachidonoyl-PE (15-HpETE-PE) - in the intestines of irradiated and PAO1-infected mice. Global redox phospholipidomics of the ileum revealed that lysophospholipids and oxidized phospholipids, particularly oxidized phosphatidylethanolamine (PEox), represented the major factors that contributed to the pathogenic changes induced by total body irradiation and infection by PAO1. A lipoxygenase inhibitor, baicalein, significantly attenuated animal lethality, PAO1 colonization, intestinal epithelial cell death, and generation of ferroptotic PEox signals. Opportunistic PAO1 mechanisms included stimulation of the antiinflammatory lipoxin A4, production and suppression of the proinflammatory hepoxilin A3, and leukotriene B4. Unearthing complex PAO1 pathogenic/virulence mechanisms, including effects on the host anti/proinflammatory responses, lipid metabolism, and ferroptotic cell death, points toward potentially new therapeutic and radiomitigative targets.

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

Estudio in vitro de la viabilidad de células Caco-2 en presencia de componentes del aceite esencial de Allium spp / In vitro study of the viability of Caco-2 cells in presence of two compound of Allium spp essential oil

El aceite esencial de los componentes del género Allium, principalmente ajo y cebolla, presenta propiedades antioxidantes y antibacterianas debidas a la presencia de compuestos azufrados en su composición. La industria alimentaria ha comenzado a desarrollar nuevos sistemas de envasado activo a partir de polímeros seleccionados, a los que se incorporan aceites esenciales que, por sus propiedades, contribuyen a aumentar la vida útil de los alimentos perecederos. En este sentido, se hace necesario evaluar la seguridad asociada al uso de estas sustancias en envases alimentarios que van a estar en contacto con el consumidor a través del alimento. El objetivo del presente estudio fue determinar la citotoxicidad producida por dipropil sulfuro y dipropil disulfuro, dos de los componentes del aceite esencial de ajo y cebolla, en la línea celular Caco-2, células humanas procedentes de carcinoma de colon. Los biomarcadores ensayados fueron el contenido total de proteínas, la captación de rojo neutro y la reducción de la sal de tetrazolio (3-(4,5-dimetiltiazol-2- il)-5-(3-carboximetoxifenil)-2-(4sulfofenil)-2H-tetrazolio). Las células fueron expuestas durante 2, 4 y 8 h a concentraciones comprendidas entre 0 y 200 μM. Los resultados no mostraron diferencias significativas frente al control para ninguno de los tres marcadores, lo que demuestra que bajo las condiciones de los ensayos ambos compuestos azufrados no son citotóxicos para esta línea celular gastrointestinal y podrían ser útiles en la industria alimentaria para desarrollar envases activos (AU)

Allium spp. essential oil, mainly from garlic and onion, possesses different beneficial properties, for example antioxidant and antimicrobial effects, due to the presence of sulfur compounds. Food industry is developing new active packaging systems that include the essential oil of garlic in their structure, in order to improve the shelf-life of perishable products. Therefore it is necessary to evaluate the safety associated with the use of these substances in food packaging that will be in contact with the consumer through food. The aim of our study was to evaluate in vitro the cytotoxicity of dipropyl sulfide and dipropyl disulfide. For this purpose, we used the human Caco-2 cell line, from human small intestinal mucosa carcinoma. The assayed cytotoxicity biomarkers were the total protein content, neutral red uptake and reduction of the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboximethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. Cells were exposed to dipropyl sulfide and dipropyl disulfide in concentrations between 0-200 μM for 2, 4 and 8 h. After periods of exposure, no alterations were observed in any of the biomarkers assayed. These results suggest that both organosulfur compounds are safety options for food industry and could be a choice in the development of active packaging. (AU)

[Anti-microRNA-221 enhances radiosensitivity of colorectal carcinoma cells by up-regulating PTEN].

OBJECTIVE: To investigate the effect of knocking-down microRNA-221 (miR-221) expression on the radiosensitivity of human colorectal carcinoma cells. METHODS: Human colorectal carcinoma-derived cell line Caco2 was transfected with miR-221 antisense oligonucleotides (anti-miR-221) via Lipofectamine 2000. Real-time quantitative PCR was performed to detect the expression of miR-221 and PTEN mRNA in Caco2 cells. The changes in the protein expression of PTEN in the transfected cells were detected by Western blotting. The cell death after transfection and irradiation was detected by flow cytometry. RESULTS: Transfection with anti-miR-221 caused a significant reduction in miR-221 expression (P<0.05) and up-regulated PTEN protein expression (P<0.05) in Caco2 cells. The percentage of cell death was significantly increased in anti-miR-221 group and anti-miR-221 with irradiation group (P<0.01). Anti-miR-221 significantly enhanced the radiosensitivity of Caco2 cells, which was partially reversed by PTEN-siRNA. CONCLUSION: Anti-miR-221 can enhance the radiosensitivity of colorectal carcinoma cells by up-regulating the expression of PTEN.

Substance P accelerates intestinal tissue regeneration after gamma-irradiation-induced damage.

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Bcl-x(L) antisense oligonucleotides radiosensitise colon cancer cells.

Advanced colon cancer is a malignancy with poor response to various treatment modalities including ionising radiation (IR) and chemotherapy. Both IR and chemotherapeutic agents have been shown to act by inducing apoptosis, a type of cell death antagonised by the Bcl-x(L) gene product. Since approximately 60% of human colon cancers express Bcl-x(L), it was the aim of this study to explore the potential of Bcl-x(L) antisense oligonucleotides as a novel radiosensitisation strategy. Caco-2 colon cancer cells were treated with Bcl-x(L) antisense oligonucleotides in combination with IR or cisplatin, and Bcl-x(L) protein expression, apoptosis, cell viability and clonogenic survival were examined. Bcl-x(L) antisense oligonucleotide specifically reduced the Bcl-x(L) protein level by almost 50% in Caco-2 cells. The decreased threshold for the induction of apoptosis resulted in a 300% increase of apoptosis after IR or cisplatin treatment and led to a 60% reduction of cell proliferation beyond response rates achieved with IR. These data suggest that Bcl-x(L) is an important factor contributing to the treatment resistance of human colon cancer. Specific reduction of Bcl-x(L) protein levels by antisense oligonucleotides qualifies as a promising therapeutic strategy for colon cancer that may help overcome resistance and improve clinical outcome in this malignancy.

In vitro transport and uptake of protohypericin and hypericin in the Caco-2 model.

The intestinal absorption characteristics of protohypericin, a protonaphthodianthrone present in Hypericum extract, were studied and compared with those of hypericin. The Caco-2 model was used as a model of the intestinal mucosa to assess transepithelial transport and cell uptake. The experimental work was performed in specific light conditions that prevented both the photoconversion of protohypericin into hypericin and the photosensitization of the cells. Following application of the individual compounds (80-200 microM) to the apical side of the monolayers, the appearance in the basolateral compartment was found to be very low (<0.5%/5 h), but was comparable for both compounds. A lag-time of 2-3 h was observed, suggesting gradual saturation of binding sites on the membrane or inside the cells. Uptake experiments of protohypericin and hypericin by Caco-2 cells revealed a very significant cellular accumulation (4-8%); uptake was characterised by saturation after 3 h. The findings of this study suggest that protohypericin has comparable absorption characteristics as hypericin and may contribute to the beneficial effect of Hypericum extract after oral dosing.