lucifer-yellow and Colonic-Neoplasms

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Cell Hypoxia; Cell Line, Tumor; Coculture Techniques; Colonic Neoplasms; Coloring Agents; Electric Impedance; Enterocytes; Gene Expression Regulation, Neoplastic; Goblet Cells; Heat-Shock Response; Humans; Intercellular Junctions; Isoquinolines; Mucins; Neoplasm Proteins; Oxidative Stress; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic

Dodecylmaltoside (DDM), an alkylglycoside showing tissue-permeability-enhancing properties, has been successful in improving nasal and ocular transport of poorly absorbed drugs. It was hypothesized that optimization of DDM concentration would improve the transport of insulin across epithelial monolayers without causing cell damage. Samples of markers and insulin were collected over a 6-h period and transepithelial electrical resistance was measured at concurrent time points to ascertain the effect of DDM on tight junctions. Samples were analyzed for lucifer yellow and insulin using reversed-phase high-performance liquid chromatography and for (3)[H]-mannitol by scintillation counting. A significant increase in the transport of markers and insulin was recorded in DDM-treated cells compared with controls. DDM enhanced the transport of markers and insulin in a concentration-dependent manner. Decreased transepithelial electrical resistance values confirmed that enhanced transport is caused by loosening of tight junctions. Cell recovery was >95% in 8 h indicating the potential of DDM as a penetration enhancer for clinical administration of insulin and other poorly absorbed drugs without causing cell damage.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Membrane Permeability; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Electric Impedance; Epithelial Cells; Fluorescent Dyes; Glycosides; Humans; Hypoglycemic Agents; Insulin; Isoquinolines; Time Factors

The possible role of tumor cell-derived factors in the regulation of gap junctional intercellular communication and proliferation of fibroblasts was studied in a model system of Balb/c 3T3 cells growing in tumor conditioned medium by Lucifer Yellow CH dye-transfer and BrdU incorporation assays. Six to 24 h incubation of Balb/c 3T3 cells in a medium conditioned by WiDr adenocarcinoma cells enhanced the gap junctional communication between the cells by 25-40% as revealed by intercellular transfer of a fluorescent dye Lucifer Yellow CH. Simultaneously the cell proliferation rates were examined and found to be reduced by 23% at 24 h treatment. Since adenocarcinoma cells are known to secrete different growth factor-like polypeptides into their conditioned medium, we suppose that tumors that produce these molecules might alter their host environment through the enhancement of cell-cell communication thereby facilitating the exchange of modulatory factors.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Bromodeoxyuridine; Cell Communication; Cell Division; Colonic Neoplasms; Culture Media, Conditioned; Fluorescent Dyes; Gap Junctions; Humans; Isoquinolines; Mice; Mice, Inbred BALB C; Tumor Cells, Cultured

Tumour-endothelial cell adhesion forms a key role in the establishment of distant metastases. This study examined the effect of gamma linolenic acid (GLA), an anti-cancer polyunsaturated fatty acid (PUFA), on both the gap junction communication of human vascular endothelial cells and tumour cell-endothelial interactions. By using scrape loading of Lucifer yellow dye, we showed that GLA at non-toxic levels increased Lucifer yellow transfer, indicating improved gap junction communication. The fatty acid also corrected the communication that was reduced by the mitogenic and motogenic factor HGF/SF. GLA inhibited the tyrosine phosphorylation of connexin-43, a protein that formed gap junction in this cell. When human tumour cells were added to quiescent or HGF/SF-activated endothelial cells, the presence of GLA reduced adhesion of tumour cells to the endothelium. It is concluded that GLA reduces tumour-endothelium adhesion, partly by improved gap junction communications of the endothelium.

Topics: Breast Neoplasms; Cell Adhesion; Cell Communication; Cell Line; Colonic Neoplasms; Connexin 43; Endothelium, Vascular; Fatty Acids; Fluorescent Dyes; gamma-Linolenic Acid; Gap Junctions; Hepatocyte Growth Factor; Humans; Isoquinolines; Kinetics; Phosphorylation; Precipitin Tests; Tumor Cells, Cultured