lucifer-yellow and Adenocarcinoma

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

In addition to its antioxidant activity, beta-carotene (BC) is known to enhance gap junction intercellular communication (GJIC) by up-regulation of connexin 43 (Cx43), an action that may be important in its control of tumor growth. Surprisingly, two clinical trials on supplemental BC suggest that BC may increase lung cancer incidence in smokers. Recently, an animal study indicated that a very high dose of BC (50 mg/kg b.w./day for 5 days) decreases GJIC in rat liver, while a lower dose (5 mg/kg b.w./day) increases GJIC. It is unclear how high-doses of BC inhibit GJIC. In this study, we tested whether oxidized BC (OBC, obtained by heating BC at 60 degrees C in open air for 1 h) may inhibit GJIC. We incubated a human lung cancer cell line (A549) with OBC or BC at 2-10 microM for 5 days. Cell viability (by Trypan-blue assay), GJIC (by scrape-loading dye transfer) and Cx43 expression (by western blotting and immunocytochemical localization) were measured to investigate the effects of OBC and BC on GJIC and the possible mechanisms. The results show that OBC at concentrations lower than 10 microM did not significantly affect cell viability. However, OBC at 5 muM inhibited GJIC, whereas BC at 5 microM markedly increased GJIC. The loss of GJIC in A549 induced by OBC accompanied the aberrant localization and phosphorylation of connexin43 (Cx43). These changes in the expression of Cx43 induced by OBC were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. Thus, our results suggest that in vivo inhibition of GJIC by a high dose of BC on GJIC is, at least in part, attributable to the effect of OBC.

Topics: Adenocarcinoma; Antioxidants; beta Carotene; Blotting, Western; Cell Communication; Cell Line, Tumor; Connexin 43; Fluorescent Dyes; Gap Junctions; Humans; Immunohistochemistry; Indicators and Reagents; Isoquinolines; Lung Neoplasms; Oxidation-Reduction; Phosphorylation

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

We studied dendritic cell (DC) function in patients affected by pancreatic carcinoma, and the possibility of obtaining DC adequate for immunological treatment modalities.. Leucocytes were isolated from buffy coats obtained by autotransfusion of six patients undergoing pancreatico-duodenectomy. The leucocytes were cryopreserved and, after thawing, were purified by density gradient and/or plastic adhesion. They were then cultured in vitro in cytokine-enriched medium (granulocyte/macrophage-colony-stimulating factor + interleukin-4) with different sources of serum: 10% fetal calf serum (FCS), 2% autologous human serum or 2% pooled human AB serum.. The DC obtained were identical to those from healthy donors in terms of phenotype, antigen uptake capacity, capacity for antigen presentation and their capacity to mature after exposure to stimuli like CD40L. DC differentiated in human serum demonstrated more mature behaviour than did DC cultured in FCS but, after exposure to CD40L, this difference disappeared. In one patient soluble factors in serum were able to inhibit the capacity of DC to stimulate T cells.. It's possible to obtain DC from autotransfusion of patients with pancreatic carcinoma: these cells do not show evident quantitative or qualitative alterations, are able to present soluble antigen even when cultured in the presence of human serum and may be used in immunological tumour treatments.

Topics: Adenocarcinoma; Animals; Antigen Presentation; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Biological Factors; Blood Physiological Phenomena; Cattle; CD40 Ligand; Cell Differentiation; Cell Separation; Cells, Cultured; Coculture Techniques; Coloring Agents; Cryopreservation; Culture Media; Dendritic Cells; Dextrans; Endocytosis; Fetal Blood; Flow Cytometry; Fluorescein-5-isothiocyanate; Granulocyte-Macrophage Colony-Stimulating Factor; HLA Antigens; Humans; Immunophenotyping; Immunotherapy, Adoptive; Interleukin-4; Isoquinolines; Lymphocyte Activation; Membrane Glycoproteins; Pancreatic Neoplasms; Recombinant Proteins; Species Specificity; T-Lymphocytes; Tetanus Toxin

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Blotting, Southern; Blotting, Western; Cell Communication; Cells, Cultured; Connexin 43; Epithelial Cells; Female; Fluorescent Dyes; Gap Junctions; Humans; Immunohistochemistry; Isoquinolines; Mice; Ovarian Neoplasms; Ovary; Rats