In a previous study we demonstrated that retinoic acid (RA) decreased the growth only of thyroid carcinoma cell lines expressing RA receptor β (RAR β) and that decitabine (5-Aza-CdR) re-induced RARβ expression. The aim of this study was to analyze the effects induced by the combined treatment with RA and 5-Aza-CdR in the same thyroid cancer cell lines.
We studied the effect of 5-Aza-CdR 800 nM and RA 1 μM on the expression of thyroid specific genes and RARα, β and γ by quantitative RT-PCR and the effect of the two drugs on cell growth by cell counting, cytotoxicity, bromodeoxyuridine, apoptosis assays and FACS analysis.
After the combined treatment, we observed the induction of the RARβ mRNA expression in all cell lines, of NIS mRNA in ARO, FRO, WRO and TT, of TTF-1 in ARO, Tg in FRO and Pax-8 in WRO and TT. However, no cell line was able to actively take up 125I despite of NIS mRNA re-expression. Accordingly, immunofluorescence showed NIS protein expression only in the cytoplasm.
The combined treatment determined an inhibition of the growth curve in all cell lines: after 24 h in FRO and NPA, after 48 h in WRO, after 72 h in ARO and after a week in TT. We observed inhibition of DNA synthesis in NPA and WRO and apoptosis in ARO and, NPA and TT. Finally, FACS analysis showed a G0/G1 increase in FRO and WRO.
In conclusion, the combined treatment with 5-Aza-CdR and RA reduces the tumoral growth speed in vitro by means of apoptosis in ARO, NPA and TT and of inhibition of DNA synthesis in NPA and WRO. The combined treatment can also partially re-differentiate the analyzed thyroid cancer cell lines, inducing NIS mRNA expression. The cytoplasmic localization of NIS protein explains the inability of cells to take up radioiodine.