The Role Of Cancer Stem Cells And Epithelial-Mesenchymal Transition In Drug Resistance And Metastasis Of Anaplastic Thyroid Carcinoma

Objective Drug resistance and metastasis are two main reasons for the failure of cancer treatment. It has been proved that cancer stem cells(CSCs) and epithelial-mesenchymal transition(EMT) play important roles. Current researches have found these two phenotypes may have some functional connections. Our previous study has proved the doxorubicin resistant anaplastic thyroid cell line consisted of higher proportion of side population (SP) cells, but the mechanism of the drug resistance is still unclear. To explore the relationship between thyroid CSCs and EMT, we use Fluorescent Activated Cell Sorting (FACS) by Hoechst 33342 dye to separate SP and non-SP cells from SW1736 cell line. Non-SP cells were treated with doxorubicin to establish a resistant cell line. Then compare the stem cell markers, EMT characteristics and drug resistance among SW1736, SP, non-SP and non-SP drug resistant cells. We further analyses whether the non-SP drug resistant cells own CSCs characteristics and compare them with parental non-SP cells in respect to EMT markers. The aim of this study is to clarify whether chemotherapy drugs can induce the transition of non-CSCs to acquired CSCs and explain the drug resistant mechanisms of anaplastic thyroid carcinoma, which may help to provide new therapeutic target for anaplastic thyroid carcinoma treatment.Methods Human anaplastic thyroid cancer cell line, SW1736, was stained with Hoechst 33342 dye and sorted for a small fraction of side population by FACS. Non-SP cells were treated with doxorubicin by increasing drug concentration gradually for 15 days to establish a resistant cell line. The clonal formation assay was performed to evaluate the self-renewal potential of SP and non-SP cells. To examine effects of doxorubicin doses and time on the proliferation of non-SP and non-SP drug resistant cells, they were treated with 0,0.001,0.01,0.05,0.08,1 and 2μg/ml doxorubicin for 48 or 72h, then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was adopted. Genes expression of stem cell markers-nestin and CD133, the gene related to cancer resisitance and relapse-ABCG2 (ATP-binding cassette superfamily G member 2) and some EMT associated genes-E-Cadherin, β-catenin, vimentin, Slug and N-cadherin, were compared by performing realtime PCR among SW1736, SP, non-SP and non-SP drug resistant cells. Proteins expression of β-catenin and vimentin were examined by western blot. SPSS 17.0 for Windows (Chicago, IL, USA) was used to perform the analyses, the statistical significance was determined by Student’s t-test and one-way ANOVA.Results The established human anaplastic thyroid cancer cell lines, SW1736, contained a small percentage of side populations. Non-SP cells were treated with doxorubicin for 15 days at final concentration of 0.05ug/ml to establish a resistant cell line. Non-SP drug resistant cells couldn’t generate SP cells, but just non-SP cells by FACS. Clonal formation assay revealed that thyroid cancer SP cells displayed markedly higher clonogenic potential than non-SP cells. MTT cell proliferation assay showed that both non-SP and non-SP drug resistant cells had lower survival rates after incubating for 72h than 48h. The half maximal inhibitory concentration (IC50) of non-SP drug resistant cells was obviously higher than non-SP cells. Among SP, non-SP and non-SP drug resistant cell, SP cells displayed higher gene expression of stem cell marker nestin, as well as ABC transporter genes ABCG2 and EMT related genes slug, β-catenin, vimentin and N-cadherin, than non-SP cells. The gene expression of slug was higher in non-SP drug resistant cells than non-SP cells.Compared to SP cells, non-SP drug resistant cells had higher levels of slug, but lower levels of ABCG2, nestin and N-cadherin. CD 133 only expressed rarely in SP cells, while E-cadherin was not detected among all cell types. The proteins expression of β-catenin and vimentin were higher in SP cells than non-SP cells, which was in accordance with gene results.Conclusions These findings suggest that the side population in thyroid cancer cell line highly expresses stem cells relevant and drug resistant genes, as well as EMT related genes and proteins. Seperated non-SP cells were treated with doxorubicin to establish drug resistant cells for the first time in thyroid carcinoma cell lines, and we find doxorubicin can’t make the transition of non-CSCs (namely non-SP) to acquired CSCs (namely SP), but can induce the aberrant activation of EMT. The proliferation of SP cells themselves may take the responsibility for the drug induced increasement of SP proportion. Consequently, these cancer stem-like cells may have significant impact on tumor drug resistance, recurrence and metastasis. The cancer therapy should focus on both CSCs and EMT signal pathway.