Differential Regulation by Glucocorticoids of Mouse Proα2(I) and Proα1(III) DNA-Binding Proteins

Multiple procollagen-specific DNA-binding proteins were identified for the α2(I) and the αl(III) procollagen promotor containing gene fragments. The proteins are genespecific, differing in their relative molecular weights and relative binding specificities. A major finding was the increased DNA-binding with specificity for the procollagen promoter containing DNAs by several nonhistone nuclear proteins in mouse embryo fibroblasts treated with dexamethasone. The previously reported coordinate decrease of type I and type III procollagens by glucocorticoids may involve differential regulation by glucocorticoids of procollagen gene specific DNA-binding proteins.     

Prognostic Significance and Correlation with Survival of bcl-2 and TGF-β RII in Colon Cancer

Bcl-2 and TGF-beta receptors type II (RII) in colon carcinomas were studied in a series of 113 patients, to determine their prognostic significance and to correlate their expression with other prognostic indicators. Bcl-2 expression in the tumor cells showed a reverse relation with tumor size (P = 0.018), histological grade (P = 0.04), and stage (P = 0.013). Univariate survival analysis using the log rank test showed that the survival of patients with bcl-2-positive tumors was significantly better than the survival of patients with bcl-2-negative tumors (P = 0.02). However, when entered into a multivariate analysis model, it was not found to be of independent prognostic significance. TGF-beta RII expression was correlated with stage (P = 0.03), while no statistically significant correlation was found between TGF-beta RII expression and histological grade or survival. In conclusion, these results provide additional evidence for the role of bcl-2 and TGF-beta RII in carcinogenesis of the colon, while they do not support the use of these factors as prognostic markers in patients with colon cancer.     

Deguelin, an Akt Inhibitor, Down-Regulates NF-κB Signaling and Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in Mice

Background

Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to have an anti-tumor effect on several cancers.

Aims

This study was performed to elucidate the effect of deguelin on apoptotic pathways related to NF-κB signaling in colon cancer cells and on the anti-tumor effect in colon cancer xenograft mice.

Methods

We studied COLO 205 and HCT116 cells in the presence or absence of deguelin. NF-κB signaling was examined by real-time RT-PCR for interleukin (IL)-8, by Western blotting for IκB phosphorylation/degradation, and by the electrophoretic mobility shift assay. Cell death was determined by the MTT assay, and apoptosis by Annexin V-FITC staining and caspase-3 activity. We also assessed the expression of antiapoptotic and proapoptotic factors by use of RT-PCR. In colon cancer xenograft mice, we evaluated the effect of deguelin on inoculated tumor growth, and apoptotic index was measured by the in vivo TUNEL assay.

Results

Deguelin significantly inhibited IL-8 gene expression, IκB phosphorylation/degradation, and DNA binding activity of NF-κB in colon cancer cells. Deguelin induced cell death and apoptosis in colon cancer cells in a dose and time-dependent manner. Deguelin down-regulated expression of NF-κB-mediated antiapoptotic factors such as cFLIP, Bcl-2, and Bcl-X_L. In the colon cancer xenograft model, the volume of the tumor treated with deguelin was significantly lower than that of the control, and the apoptotic index for deguelin-treated mice was much higher.

Conclusion

Deguelin might be a potential therapeutic agent for treatment of colorectal cancer.     

Functional Expression of μ-Opioid Receptors in the Human Colon Cancer Cell Line,HT-29, and their Localization in Human Colon

We have investigated the functional expression of μ-opioid receptors (MORs) in the human colon cancer cell line, HT-29. As revealed by immunocytochemistry, immunoreactivity was present in both the cytoplasm and nuclei of the cells. Challenge with morphine for 24 h (1 nM to 1 μM) barely affected cell proliferation, while the secretion of urokinase type plasminogen activator (a protease involved in invasion/metastasis) was markedly augmented by a concentration of 0.1 μM. Human colon cancer tissue from 14 consecutively operated patients was investigated by immunohistochemistry. MORs were found in the nuclei of colonocytes and immune cells of the lamina propria in tumor-free tissue. In tumor tissue, immunoreactivity was found in the membrane and often in the nuclei of tumor cells. The current findings suggest that morphine administration could affect tumor progression by interfering with, for example, invasive properties. Our demonstration of a nuclear expression of the MORs appears to be a novel finding.     

Prevention by Intrarectal 5-Aminosalicylic Acid of <Emphasis Type="Italic">N</Emphasis>-Methylnitrosourea–Induced Colon Cancer in F344 Rats

PURPOSE: The protective effect of 5-aminosalicylic acid against colon carcinogenesis was investigated. METHODS: Eighty female F344 rats aged seven weeks received an intrarectal dose of 2 mg N-methylnitrosourea dissolved in 0.5 ml of water three times weekly for five weeks to induce colon cancer. Beginning at 6 weeks after the last dose of N-methylnitrosourea, the rats were treated with an intrarectal dose of 1 mg 5-aminosalicylic acid suspended in 0.5 ml of vehicle solution (0.3 percent water solution of methylcellulose) three times weekly for 15 weeks. RESULTS: Colon cancer incidence and mean number of tumors per rat at the end of the 15-week treatment period were significantly lower and smaller in the 5-aminosalicylic acid–treated group (10 percent and 0.2) than in the vehicle-treated (80 percent and 1.6) and untreated (68 percent and 1.1) control groups. However, the mean numbers of tumors per tumor-bearing rat were comparable: 1.5, 2, and 1.6. No distinct differences among the groups were observed in the tumor pathology with respect to their location (within 0–10 cm proximal to the anus), shape (plaque shaped or polypoid), size (<10 mm in diameter), invasion (restricted to the mucosa or submucosa), or histologic type (differentiated adenocarcinoma). CONCLUSION: Our results indicate that 5-aminosalicylic acid administered directly into the colonic lumen strongly suppresses the promotion stage of colon carcinogenesis.     

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics.MHC class I molecules are ligands for the antigen receptors of CD8⁺ T cells and natural killer cells. The assembly and folding of MHC class I molecules with antigenic peptides take place within the endoplasmic reticulum (ER) and are facilitated by a multiprotein complex called the peptide loading complex (PLC) (reviewed in 1, 2). Structurally, the PLC involves the association of MHC class I heterodimers with the transporter associated with antigen processing (TAP), an interaction bridged, via several protein–protein interactions, within the ER lumen. Tapasin interacts with TAP via its transmembrane domain and with MHC class I via its ER luminal domains. MHC class I molecules also interact with the glycan-binding chaperone calreticulin through a conserved glycan on the α2-domain of the MHC class I heavy chain (3, 4). The thiol oxidoreductase ERp57, which functions as a cellular cochaperone for calreticulin (CRT), forms a disulfide-linked heterodimer with tapasin (reviewed in 1). In this manner, by bridging interactions with MHC class I and tapasin, respectively, calreticulin and ERp57 stabilize the binding of MHC class I molecules to tapasin–TAP complexes (reviewed in 1, 2). Cellular deficiencies in calreticulin and ERp57 destabilize MHC class I interactions with other components of the PLC (5–8). Furthermore, peptide binding to MHC class I has been shown to destabilize MHC class I–tapasin interactions (9, 10), but how other cellular factors influence PLC stability is largely uncharacterized.In this investigation, we show that ATP destabilizes MHC class I interactions with PLC components. ATP is a known regulator of substrate interactions with several cellular chaperones. Calreticulin, like several other chaperones, is known to interact with ATP (8, 11, 12). However, the location of the calreticulin-ATP–binding site is unknown, as is the influence of ATP on calreticulin binding to cellular substrates, including MHC class I. In this study, using computational methods validated by experimental approaches, we identify residues within the globular domain of calreticulin that are important for ATP binding and ATPase activity. Based on further investigations into the functional effects of calreticulin mutants with deficiencies in ATP interactions, we elucidate a key role for ATP in the regulation of PLC dynamics and the interaction of calreticulin with other cellular proteins.     

Regulation of insulin gene expression by cytokines and cell–cell interactions in mouse medullary thymic epithelial cells

Aims/hypothesis  

The expression of tissue-specific self-antigens in the thymus is essential for self-tolerance. Genetic susceptibility to type 1 diabetes correlates inversely with thymic insulin expression and, in mice, lowered levels of this expression result in T cell responses against insulin. This study was undertaken to examine whether thymic insulin expression is regulated by the same metabolic stimuli as in beta cells or by different inputs, possibly of an immune nature.     

Effect of Citric Acid and Citric Acid–Sucrose Mixtures on Swallowing in Neurogenic Oropharyngeal Dysphagia

The ability of sour and sweet–sour mixtures to improve swallowing in 11 nursing home residents with neurogenic oropharyngeal dysphagia was investigated using fiberoptic endoscopic evaluation of swallowing. Citric acid (2.7%) significantly reduced aspiration and penetration compared with water. Teaspoon delivery of liquids significantly reduced aspiration and penetration compared with natural cup drinking. Subjects tended to appropriately self-regulate the cup volume they consumed after the first trial. A significant increase in spontaneous dry swallows was observed after both taste stimuli. The mechanisms for improved swallowing due to citric acid are not understood but may be due to increased gustatory and trigeminal stimulation of acid to the brainstem in neurologically impaired subjects. This research was conducted in partial fulfillment of the requirements for a PhD by CAP at Cornell University.     

ZNF281 Promotes Growth and Invasion of Pancreatic Cancer Cells by Activating Wnt/β-Catenin Signaling

Digestive Diseases and Sciences - Zinc finger protein 281 (ZNF281) has been identified to be involved in embryonic stem cell differentiation and tissue development. Also, ZNF281 was found in...     

Case–Control Study on Prednisolone Combined With Ursodeoxycholic Acid and Azathioprine in Pure Primary Biliary Cirrhosis With High Levels of Immunoglobulin G and Transaminases: Efficacy and Safety Analysis

To the best of our knowledge, this is the first study to address the use of glucocorticoids in the comparatively special population of pure primary biliary cirrhosis (PBC) patients who have high levels of immunoglobulin G (IgG) and transaminases but do not have PBC-autoimmune hepatitis overlap syndrome. Ursodeoxycholic acid (UDCA) is now assumed to be the standard therapy for PBC patients. However, patients treated with UDCA still have a risk of progression to cirrhosis and end-stage liver disease. The most recent European Association for the Study of the Liver guidelines of 2009 declared that further studies on glucocorticoid therapy in this disease should be a priority. Therefore, we designed this 3-year longitudinal retrospective study, which might provide deep insight into the treatment for PBC.The aim of this study was to assess whether the combination of prednisolone, UDCA, and azathioprine was superior to UDCA alone in these PBC patients.Sixty patients were enrolled in this study. Thirty-one patients underwent UDCA monotherapy, and 29 patients were treated with prednisolone, UDCA, and azathioprine. We analyzed their biochemistries, immune parameters, liver synthetic function, and noninvasive assessments of liver fibrosis, as well as treatment efficacy and adverse effects at baseline and at 1, 3, 6, 12, 24, and 36 months.Alkaline phosphatase (ALP), γ-glutamyl transpeptidase, alanine aminotransferase, and aspartate aminotransferase levels and the aspartate aminotransferase-to-platelet ratio index (APRI) and S-index improved dramatically in both groups, whereas IgG levels only decreased in the combination group (all P < 0.05). Albumin (ALB) levels decreased in the UDCA group but increased with the combination treatment at 36 months. Significant differences between the 2 groups were observed at 36 months in ALP (P = 0.005), IgG (P = 0.002), ALB (P = 0.002), APRI (P = 0.015), and S-index (P = 0.020). Prednisolone combined with UDCA and azathioprine showed a higher efficacy based on our new criteria.The combination of prednisolone, UDCA, and azathioprine is superior to UDCA alone for the treatment of pure PBC patients with high levels of IgG and transaminases. Side effects were minimal or absent.     

Homocysteine Induces Oxidative–Nitrative Stress in Heart of Rats: Prevention by Folic Acid

Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and thrombosis; however, the mechanisms by which homocysteine triggers these dysfunctions are not fully understood. In the present study, we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely thiobarbituric acid reactive substances, an index of lipid peroxidation, 2′,7′-dichlorofluorescein (H₂DCF) oxidation, activities of antioxidant enzymes named superoxide dismutase and catalase, as well as nitrite levels in heart of young rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3–0.6 μmol/g body weight) and/or folic acid (0.011 μmol/g body weight) from their 6th to the 28th day of life. Controls and treated rats were killed 1 h and/or 12 h after the last injection. Results showed that chronic homocysteine administration increases lipid peroxidation and reactive species production and decreases enzymatic antioxidant defenses and nitrite levels in the heart of young rats killed 1 h, but not 12 h after the last injection of homocysteine. Folic acid concurrent administration prevented homocysteine effects probable by its antioxidant properties. Our data indicate that oxidative stress is elicited by chronic hyperhomocystenemia, a mechanism that may contribute, at least in part, to the cardiovascular alterations characteristic of hyperhomocysteinemic patients. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in cardiovascular alterations caused by homocysteine.     

Regulation of cell–matrix contacts and β-catenin signaling in VSMC by integrin-linked kinase: implications for intimal thickening

Vascular smooth muscle cell (VSMC) proliferation and migration is responsible for intimal thickening that occurs in restenosis and atherosclerosis. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in signaling pathways involved in cell proliferation and migration. We studied the involvement of ILK in intimal thickening. ILK expression was significantly increased in two models of intimal thickening: balloon-injured rat carotid arteries and human saphenous vein organ cultures. Over-expression of a dominant negative ILK (DN-ILK) significantly reduced intimal thickening by approximately 50% in human saphenous vein organ cultures, demonstrating an important role in intimal thickening. ILK protein and activity was reduced on laminin and up-regulated on fibronectin, indicating ILK protein expression is modulated by extracellular matrix composition. Inhibition of ILK by siRNA knockdown and DN-ILK significantly decreased VSMC proliferation and migration while wild type ILK significantly increased proliferation and migration on laminin, confirming an essential role of ILK in both processes. Localization of paxillin and vinculin and protein levels of FAK and phospho-FAK indicated that inhibition of ILK reduced focal adhesion formation. Additionally, inhibition of ILK significantly attenuated the presence of the cell-cell complex proteins N-cadherin and beta-catenin, and beta-catenin signaling. We therefore suggest ILK modulates VSMC proliferation and migration at least in part by acting as a molecular scaffold in focal adhesions as well as modulating the stability of cell-cell contact proteins and beta-catenin signaling. In summary, ILK plays an important role in intimal thickening by modulating VSMC proliferation and migration via regulation of cell-matrix and cell-cell contacts and beta-catenin signaling.