N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.
Reactive oxygen species-mediated attack of the acyl chains of polyunsaturated fatty acids and triglycerides leads to the formation of lipid hydroperoxides. Lipid hydroperoxides are subject to nonenzymatic Fenton chemistry producing a variety of reactive aldehydes that covalently modify proteins in a reaction referred to as protein carbonylation. Given the significant content of triglycerides in fat tissue, adipose proteins are among the most heavily carbonylated. The laboratory has utilized two methodologies for the detection of protein carbonylation in tissue- and cell-based samples. The first utilizes biotin coupled to a hydrazide moiety and takes advantage of the numerous biotin detection systems. The second method utilizes an anti 4-hydroxy- -2,3-nonenal (4-HNE)-directed antibody that can detect both 4-HNE and the corresponding 4-oxo derivative when the samples are reduced. Using such methods, we have evaluated the profile of carbonylated proteins in epididymal white adipose tissue and 3T3-L1 adipocytes using both methods. In addition, we have investigated the effects of two antidiabetic drugs, pioglitazone and metformin, on protein carbonylation in 3T3-L1 adipocytes. Overall, the biotin hydrazide method is rapid, inexpensive, and easy to use, but its usefulness is limited because it detects a wide variety of carbonylated derivatives, which makes assignments of individual proteins difficult. Compared to the biotin hydrazide method, the anti-HNE antibody method detects specific proteins more readily but identifies only a subset of carbonylated proteins. As such, the combination of both methods allows for a comprehensive evaluation of protein carbonylation plus provides a means towards identification of specific carbonylation targets.
Lichens are fascinating symbiotic organisms, biosynthesizing a broad spectrum of interesting secondary metabolites and polysaccharides. A considerable number of them have been found to exert biological activities, such as antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative, and cytotoxic effects. Only a very low percentage of “lichen substances” have been actually screened for their biological activities and their potential therapeutic applications in medicine. This is due to difficulties to obtain large quantities of lichens from nature, isolated lichen fungi and algae from cultures for extractions. Ten years ago, we have started to bypass these problems by introducing first traditional and then by exploring novel microbiological techniques and advanced molecular tools for our culture experiments. “Case studies” with selected cultured mycobionts and photobionts, accumulating considerable quantities of a focused compound, have been performed as tests for large-scale culturing, to be able to utilize facilities like phytotrons and bioreactors (small-scale bioreactors) for future approaches. Further studies have focused on the chemical identification of the metabolites from cultures and the genetic characterization of lichen PKS genes (Polyketide synthase genes). Another interesting group of lichen metabolites is cell wall polysaccharides. All lichen species investigated so far produce these polymers in considerable amounts and many of them have been shown to exhibit antitumor, immunostimulating, antiviral as well as other types of biological activity. Lichens polysaccharides are mainly of the following structural types: α-glucans (isolichenan, nigeran, pseudonigeran, and pullulan), β-glucans (lichenan, pustulan, laminaran, and lentinan-type glucan), galactomannans, and complex heteroglycans (galactoglucomannan, galactomannoglucan, rhamnopyranosylgalactofuranan, and glucomannan). Investigations on lichen polysaccharides were carried out using material extracted from the entire thallus with no mention of the origin of component polymers (fungal partner or photobiont). In order to understand the contribution of the symbiotic partners to the polysaccharide present in the lichen thallus, the carbohydrates produced by some aposymbiotically cultured mycobionts and photobionts ( , , and ) were analyzed. The studies demonstrated that most of the polysaccharides previously found in the symbiotic thalli were also produced by the aposymbiotically cultivated fungal partner, while there were no similarities between the polysaccharides extracted from the photobiont with those from the respective lichen. Surprisingly, the photobionts synthesized very interesting polysaccharides, such as β-galactofuranan, mannogalactofuranan, rhamnopyranosylgalactofuranan, and an -methylated mannogalactan. One of them was biologically active, having activity on murine peritoneal macrophages.