agar and Thyroid-Neoplasms

Anaplastic thyroid cancers (ATC) are aggressive tumors, which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy, and most patients die within a few months of diagnosis. In the present study, we evaluated the in vitro effects on ATC cells of VX-680, an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy were tested on the ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time- and dose-dependent manner, with the IC50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with > or =4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion, our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment.

Topics: Agar; Apoptosis; Aurora Kinases; Cell Cycle; Cell Division; Cell Line, Tumor; Enzyme Inhibitors; Histones; Humans; Microtubule-Associated Proteins; Phosphorylation; Piperazines; Ploidies; Protein Serine-Threonine Kinases; Thyroid Gland; Thyroid Neoplasms

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4(100)/20(10) plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G(1) phase of the cell cycle was greatly increased and the proportion in the S/G(2)-M phases greatly reduced, suggesting that plitidepsin blocks G(1)-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4(100)/20(10) plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4(100)/20(10) plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is pe

Topics: Adult; Agar; Aged; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Depsipeptides; Dose-Response Relationship, Drug; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, p53; HeLa Cells; Histones; Humans; Immunoblotting; Male; Middle Aged; Models, Statistical; Peptides, Cyclic; Thyroid Gland; Thyroid Neoplasms; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Beta-thymosins are structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. We have recently shown that the TB10 gene is overexpressed in human thyroid carcinoma cell lines and tissues, particularly in undifferentiated thyroid carcinomas, but expressed at an undetectable level in normal thyroid cells. The precise role of thymosin beta-10 (TB10) activity in maintaining the malignant phenotype of thyroid cell lines is unknown. To investigate TB10 function and relevance in a model system we used an antisense methodology to suppress TB10 protein synthesis in two human thyroid carcinoma cell lines (NPA and ARO). The growth in soft agar of NPA and ARO cells carrying a TB10 construct in an antisense orientation was significantly reduced. Conversely, anchorage-dependent growth was unchanged in NPA and ARO cells carrying the TB10 construct in a sense orientation or carrying the backbone vector. TB10 expression also affected actin organization. In fact, stress fibers were long and thick in ARO cells in which TB10 expression was suppressed by the antisense construct. Conversely, they were scarce and short in the vector-transfected ARO cells. These data suggest that TB10 plays a critical role in the regulation of anchorage-independent growth and assembly of actin filaments.

Topics: Agar; Antisense Elements (Genetics); Carcinoma, Papillary; Cell Adhesion; Cell Division; Culture Media; Gene Expression Regulation, Neoplastic; Humans; Phenotype; Stress Fibers; Thymosin; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured

Topics: Abdominal Neoplasms; Agar; Aged; Antigens, Neoplasm; Breast Neoplasms; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Immune Sera; Immunity, Cellular; Kidney Neoplasms; Liposarcoma; Lymphocytes; Male; Melanoma; Methods; Middle Aged; Neoplasms; Prostatic Neoplasms; Rectal Neoplasms; Thyroid Neoplasms