Browsing by Author "Diaz, Heli"

Background Protein called ?-catenin has the key role in the Wnt signaling pathway which induces cell division and growth. The disruption of the ?-catenin degradation can lead to uncontrolled cell division and development of colon cancer due to ?-catenin´s ability to enhance the expression of the proto-oncogenes in Wnt singaling pathway. Cells control ?-catenin by degradation and phosphorylation. Phosphorylated ?-catenin at serine675 and serine552 residues increases proto-oncogene expression, and thereby promotes colon cancer formation. At present, only few research articles have addressed whether diet can affect the phosphorylation status of ?-catenin in the intestinal mucosa. Objective The objective of the master´s thesis was to develop fluorescence-based western blotting method to analyze phosphorylated ?-catenin forms from colon cancer cell and intestinal mucosa tissue samples. Optimized method was used to analyze phospho-Ser675- and phospho-Ser552-?-catenin from samples derived from two dietary studies conducted with ApcMin mice. The aim of this was to determine whether dietary components have effect on phosphorylation of ?-catenin in the intestinal mucosa tissue and whether phosphorylated ?-catenin correlated with intestinal adenoma number and size. Materials and methods Fluorescence-based western blotting method was developed to analyze all the ?-catenin phosphorylation forms that can be detected by using commercially available antibodies (Ser33/37/Thr41, Thr41/Ser45, Ser552, Ser675 and Tyr654). Phospho-?-catenin was measured from samples obtained from colon cancer cells lines and intestinal mucosa tissue of ApcMin mice (a model of colon cancer). Optimized method was used to analyze ?-catenin, phospho-Ser675- and phospho-Ser552-?-catenin levels from normal intestinal mucosa samples of ApcMin mice originated from two dietary studies. In the first study mice were fed 0,8 % plant stanol diet and in the second study mice were fed western type diet which contained high levels of fat and saturated fat and low levels of calcium and vitamin D3. The aim of the two studies was to determine whether plant stanol and western type diet have effect on tumor formation. Statistical analyses were made by using Mann-Whitney U test and Spearman correlation (PASW Statistics 18.0 software). Results By using fluorescence-based western blotting method phospho-Ser675- and phospho-Ser552-?-catenin were detected in cell and mouse samples as well as phospho-Ser33/37/Thr41-?-catenin in cell samples. The method could not be optimized for the detection of phospho-Tyr654- and phospho-Thr41/Ser45-?-catenin in cell and mouse samples and phospho-Ser33/37/Thr41-?-catenin in mouse samples. However, phospho-Thr41/Ser45-?-catenin was detected in cell samples but not in mouse samples by using the chemiluminescence-based western blotting method. The adenoma number was significantly higher in mice fed plant stanol diet than in control mice (p=0,002). Plant stanol diet resulted in significantly higher levels of ?-catenin (p=0,043) and phospho-Ser675-?-catenin (p=0,027) compared with control diet. Although, phospho-Ser552-?-catenin followed the same trend as phospho-Ser675-?-catenin, the levels of phospho-Ser552-?-catenin did not reach statistical significance between plant stanol and control group (p=0,077). Mice consumed western type diet had significantly higher number of adenomas than control mice (p=0,002). The levels of ?-catenin and phosphorylated ?-catenin (Ser552 and Ser675) were consistently higher in western type diet mice than control mice but there were no significant differences between the groups (p=0,165, p=0,198, p=0,440). Conclusions Fluorescence-based western blotting method is more reliable for analyzing ?-catenin phosphorylation forms than chemiluminescence-based western blotting method: unlike chemiluminescence-based western blotting method, fluorescence-based western blotting method can allow the detection ?-catenin and phospho-?-catenin at the same time. Based on results, diet can affect ?-catenin phosphorylation. Especially plant stanol diet increased ?-catenin fosforylation at residues serine675 as well as serine552 but less significantly in the intestinal mucosa. Phosphorylation of serine675 residue inhibits ?-catenin degradation which seems to have increased the cytosolic level of ?-catenin. Phosphorylation of serine552 residue has probably induced ?-catenin to translocate from the cytoplasm to the nucleus. Inside the nucleus ?-catenin may have promoted tumor formation by increasing the expression of proto-oncogenes.