Effects Of Nevirapine On Cell Proliferation,Differentiation And Uptake Of Radioiodide In Human Anaplastic Thyroid Carcinoma Cells

ObjectiveIt is quite often that the anaplastic thyroid carcinoma has metastasized at the time of diagnosed, as a result of its high proliferation characteristic of the cells so that the conventional therapeutic approaches such as chemotherapy, surgical operation and radiotherapy do not effect well on it. Besides, the sodium/iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (Tg), thyrotropin receptor (TSHR) are called thyroid-specific genes, but the expression of them is down-regulaed even deleted, especially the expression of NIS, result in the anaplastic thyroid carcinoma cells inability to concentrate radioiodide, which ordinarily renders the anaplatic thyroid carcinoma unresponsive to the treatment of radioiodide.Endogenous reverse transcriptase (RT) is primally encoded by retrotransposons and endogenous retroviruses, participating many pathophysiology processes. RT-encoding genes are expressed at low levels in differentiated tissues, in contrast, high expression is detected in germ cells, embryonic tissues, undifferentiated and transformed cells, which suggesting that there might be a direct relationship between RT and cell proliferation and differentiation. RT inhibitors have the ability to inhibit cell proliferation and induce cell differentiation by way of inhibiting the RT activity.Our study aims to observe cell proliferation and the expression of NISmRNA and TSHRmRNA in anaplastic thyroid carcinoma FRO cells treated with nevirapine (a non-nucleoside reverse transcriptase inhibitors), then iodine uptake assay was performed to compare the variation of radioiodide uptake rates afer nevirapine treated. Finally, to investigate the effects of nevirapine on cell proliferation, differentiation and uptake of radioiodide in human anaplastic thyroid carcinoma cells.Methods1. Cell proliferation-inhibiting assay:MTT was used to determine the inhibition effect of different concentrations of nevirapine(0,100,200,350,500umol/L) on cell proliferation of FRO cells.2. Cytometry was used to observe the reversibility of FRO cell proliferation after nevirapine treatment.3. Hoechst33258fluorescein stain was performed to observe apoptosis, trypan bule was used to determine the cell vitality of FRO cells treatment by different concentrations of nevirapine(0,200,350,500umol/L).4. Inverted phase contrast microscope to obseve the morphologic changes of FRO cells induced by nevirapine.5. Real-time PCR was used to analysis the expression of NISmRNA, TSHRmRNA in FRO and BHP cells after nevirapine treatment.6. RT-PCR and real-time PCR was used to analysis the expression of NISmRNA and TSHRmRNA in FRO cells stimulated with TSH.7. Iodine uptake assay was performed to compare the changes before and after nevirapine therapy.Results1. The proliferation of FRO cells was inhibited after nevirapine treatment, and this phenomena become more significant as the concentration of nevirapine increased.2. Nevirapine induced a reversible inhibition of cell growth in FRO cells.3. FRO cells exposed to nevirapine within the concentration of350umol/L did not exhibit any significant apoptos, and there was no significant effect on cell vitality under this concentration. 4. Nevirapine induced morphological features of cell differentiation in FRO cells.5. The expression of NISmRNA and TSHRmRNA of FRO cells elevated in comparison with control(P<0.01and P<0.05). However, there was no statistical significance of the expression of NISmRNA and TSHRmRNA in BHP cells comparison with control(P>0.05).6. The expression of NISmRNA up-regulated significantly after TSH induction in nevirapine pretreated FRO cells(P<0.05).7. The radioactive counting of FRO cells before and after nevirapine therapy was respectively [(5.34±0.93)X103cpm/106cells]å'Œ[(6.76±0.60)×103cpm/106cells], there was no statistical significance of radioactive counting before and after nevirapine treatment(P>0.05).ConclusionThe proliferation of FRO cells can be inhibited by nevirapine and the inhibition was reversible. Moreovre, nevirapine induced morphological features of cell differentiation. FRO cells exposed to nevirapine exhibited an up-regulation of NISmRNA and TSHRmRNA expression, and TSH can stimulate the expression of NISmRNA in FRO cells pretreated with nevirapine. These data suggest that nevirapine is able to re-establish TSH/TSHR signaling, and restore the ability of TSH to up-regulate NIS expression. It is a pity that we did not observe any increase of radioiodide uptake in FRO cells.