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Abstract
Background
Induction of re-differentiation is necessary for the proper treatment of patients with recurrent or metastatic differentiated thyroid cancer (DTC) because cancer cells show de-differentiation in about 30% of these patients. In this study, we evaluated the expression of thyroid specific genes after treatment with various agents to induce re-differentiation in the follicular thyroid cancer cell line FTC-133.
Methods
FTC-133 cells were treated with U0126, LY294002, trichostatin A, retinoic acid (RA), 5’-azacytidine and α-lipoic acid (ALA). We evaluated mRNA expression of thyroid specific genes, thyroglobulin (Tg), sodium iodine symporter (NIS), PAX-8 and TTF-1 by reverse transcriptase polymerase chain reaction (PCR). Quantified expression of Tg mRNA was also evaluated by real-time PCR.
Results
The expression of Tg mRNA increased after 48 h of treatment with 0.1 uM RA and the expression of Tg mRNA and TTF-1 mRNA increased after 48–72 h of treatment with ALA (10∼100 uM). There was no change in thyroid specific gene expression by the other agents. Increased expression of Tg mRNA was confirmed by real-time PCR (1.3 times by 10 uM ALA and 3.6 times by 100 uM ALA). There was no basal NIS mRNA expression in FTC-133 cells and none of the tested agents induced expression of NIS mRNA. There was no change in phosphorylation of AMPK1-α after ALA treatment of FTC-133 cells.
Conclusion
ALA increases mRNA expression of Tg and TTF-1 of FTC-133 thyroid cancer cells and these effects are not mediated by activation of AMP kinase. The finding that ALA could be a potential re-differentiation inducing agent in thyroid cancer cells is novel. Further studies are needed to elucidate the mechanism of induction of re-differentiation. Furthermore, the effect of ALA on NIS expression and on iodine uptake should be evaluated using diverse thyroid cancer cell lines.
References
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Fig. 1.
Cell survival curves measured by cell counting kit-8 after 48 hours of treatment with re-differentiation agents in FTC-133 cells. U0126; selective inhibitor of MEK1/2, LY294002; selective inhibitor of PI3K, TSA; trichostatin A. U0126 (A), LY294002 (B), TSA (C), Retinoic acid (D), 5-Azacytidine (E), a-Lipoic acid (F).
Fig. 2.
Results of reverse transcriptase polymerase chain reaction after 48 hours of treatment with re-differentiation agents in FTC-133 cells. GAPDH, glyceraldehydes 3-phosphate dehydrogenase; Tg, thyroglobulin; PAX-8, paired box 8; TTF-1, thyroid transcription factor-1; NIS, sodium iodine symporter; (+), positive control (human thyroid tissue); (−), negative control (no template); C, control without vehicle; U0126, selective inhibitor of MEK1/2; LY294002, selective inhibitor of PI3K; TSA, trichostatin A; RA, retinoic acid; AZT, 5’-azacytidine. U0126 (A), LY294002 (B), TSA (C), RA (D), AZT (E).
Fig. 3.
Results of reverse transcriptase polymerase chain reaction after 72 hours of treatment with α-lipoic acids in FTC-133 cells. GAPDH, glyceraldehydes 3-phosphate dehydrogenase; Tg, thyroglobulin; PAX-8, paired box 8; TTF-1, thyroid transcription factor-1; NIS, sodium iodine symporter; (+), positive control (human thyroid tissue); (−), negative control (no template); C, control without vehicle.
Fig. 4.
Results of real-time polymerase chain reaction after 72 hours of treatment with α-lipoic acids in FTC-133 cells.
Fig. 5.
Western blotting of phospho-5-AMP-activated protein kinase (AMPK)-1α and total-AMPK1-α protein after treatment of α-lipoic acids during 30∼60 minutes (A) and 24–48 hours (B) in FTC-133 cells.
Table 1.
Primers and conditions of reverse transcriptase polymerase chain reaction for thyroid specific genes in FTC-133 cell
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