New treatment options are needed for medullary thyroid carcinoma (MTC), a highly metastasizing neuroendocrine tumor that is usually resistant to standard radiotherapy and chemotherapy. leading to increased function of the RET receptor tyrosine kinase. Oncogenic mutations of MEN2 are concentrated in a small sequence of the open reading frame and show striking correlations with the phenotype of the MEN2 variant. The most common Trametinib variant, MEN2A, is usually mainly caused by mutations in the cysteine-rich portion of the extracellular domain name (52% occur in codon 634), and additionally, predisposes to pheochromocytoma and to parathyroid hyperplasia. In MEN2W, the mutation is usually confined to one cytoplasmic amino acid substitution C Met918Thr. MEN2W has an earlier age of onset than MEN2A and a more aggressive course including pheochromocytoma, mucosal neuromas, megacolon and a Marfanoid habitus (3, 6, 7). MTCs do not respond to conventional therapies like radiation or chemotherapy, and up to 80% of patients present with nodal metastases at the time of diagnosis, so that surgical removal of all neoplastic tissue is usually the best option. Recurrence, however, is usually common, frequently with metastases in the bones, lungs, liver and brain. To date, no effective treatment for distant metastases in MTC has been found (1, 8), and the search for new treatment options is usually thus highly warranted. Roots of Boraginaceae genera and Bureau & Franchet on a cell line derived from multiple endocrine neoplasia syndrome type 2A (MEN2A). Additionally, we compared its active constituents acetylshikonin and ,-dimethylacrylshikonin to the unsubstituted shikonin derivative. Materials and methods Cell culture TT cells, obtained from the European Collection of Authenticated Cell Cultures (ECACC; Porton Down, Salisbury, UK), were cultivated in Hams F12 Nutrient Mixture (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS Platinum; PAA Laboratories, Pasching, Austria). Cells were passaged at approximately 80% confluence to an initial cell number of 2??105?cells/mL using trypsinCEDTA (PAA Laboratories). Normal human skin fibroblasts, HF-SAR (18), were cultured in EMEM (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 2?mM l-glutamine (PAA Laboratories) and 10% FBS. Cells were passaged to an initial cell number of 1??105?cells/mL using Accutase (PAA Laboratories). All cells were kept in a humidified 5% CO2 atmosphere at 37C. Shikonin derivatives As described previously (19, 20), AOM shikonin derivatives were isolated from Bureau & Franchet. Briefly, a petroleum ether extract (Ex lover) was prepared by exhaustive Soxhlet extraction and further fractionated using preparative HPLC. The main components were ,-dimethylacrylshikonin (DMAS, 38.2%) and acetylshikonin (AS, 24.5%) as identified by NMR spectroscopy. For further comparison of the activity, shikonin (SHK) was purchased from Sigma-Aldrich. Aliquots of the substances were freshly dissolved in DMSO (Sigma-Aldrich) every second week to make sure consistent bioactivity. Concentrations of DMSO after application of the compounds never exceeded 0.5%, which did not affect cell behavior as controlled by benchmark tests. Growth inhibition assay Aliquots (2?mL) of TT cells were seeded into 6-well dishes (Sarstedt, Wiener Neudorf, Austria) at 2??105?cells/mL. After allowing the cells to adhere overnight, seven different concentrations of each material were added. Seventy-two hours later, cells were detached with trypsinCEDTA (500?L, 3?min). Then, 1.5?mL of FBS-containing medium was added and cells were counted in triplicates using a CASY1 Cell Counter-top & Analyzer TTC (Sch?rfe System, Reutlingen, Philippines). All assays were performed with at least three different passage numbers and with medium made up of 10% FBS, but no antibiotics. IC50 values were calculated with Microsoft Excel 2010. Cell viability assay Cell viability of TT cells as well as normal human skin fibroblasts, HF-SAR, was assessed using the Cell Proliferation Reagent WST-1 (Roche Diagnostics). Cells were seeded into 96-well dishes in aliquots of 100?L with a cell density of 1??105?cells/mL and 3??104?cells/mL, respectively. After allowing the cells to adhere overnight, the medium was aspirated and replaced with medium supplemented with DMSO (solvent control) or different concentrations of shikonin derivatives as indicated. Samples were tested in 6 replicates after an incubation period of 72?h. Results are presented as percentage of solvent-treated control cells. Cell morphology Morphological changes occurring after application of shikonin were observed with a Nikon inverted microscope (Eclipse TE 300, Nikon). TT cells (2?mL) were seeded into 6-well dishes at a density of 2??105?cells/mL. After allowing them to adhere overnight, cells were incubated with shikonin (IC50) and cell morphology was Trametinib observed after 24 and 48?h. Images were taken with a Nikon 12-bit CCD camera (Nikon). Trametinib Monolayer wound-healing assay TT.