Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells

Abstract

Anaplastic thyroid cancer (ATC) is a highly lethal undifferentiated malignancy without reliable therapies. Retinoic acid (RA) has been employed to promote redifferentiation of thyroid cancers by increasing their I¹³¹ uptake and radio-sensitivity, but its effect(s) on ATCs has not yet been ascertained. Likewise, resveratrol induces cancer redifferentiation but, also in this case, its effects on ATCs remain unknown. These issues have been addresses in the current study using three human ATC cell lines (THJ-11T, THJ-16T, and THJ-21T) through multiple experimental approaches. The results reveal that RA exerts a small inhibitory effect on these cell lines. In comparison with normally cultured cells, the total cell number in resveratrol-treated THJ-16T and THJ-21T cultures significantly decreased (p < 0.05), and this effect was accompanied by reduced Cyclin D1 immuno-labeling, increased apoptotic fractions, and distinct caspase-3 activation. Resveratrol failed to inhibit growth but enhanced RA sensitivity of THJ-11T cells, suppressed peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ), and upregulated cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor beta (RAR-β) expression. Increased thyroglobulin (Tg) and E-cadherin levels and appearance of membranous E-cadherin were evidenced in resveratrol-treated THJ-11T cells. Our results demonstrate for the first time: (1) the therapeutic value of resveratrol by itself or in combination with RA in the management of ATCs, (2) the capacity of resveratrol to overcome RA resistance in ATC cells by reprogramming CRABP2/RAR- and fatty acid-binding protein 5 (FABP5)/PPAR-β/δ-mediated RA signaling, and (3) the redifferentiating potential of resveratrol in ATC cells.

Keywords: CRABP2; anaplastic thyroid cancer; drug resistance; resveratrol; retinoic acid.

Figure 1

Lack of response of the three anaplastic thyroid cancer (ATC) cell lines to 10 µM retinoic acid (RA) treatment. (A) H/E staining (×40) and Cyclin D1 immunocytochemical staining (insets; ×40); (B) 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) cell proliferation assay; (C) flow cytometry. Control, without resveratrol treatment; RA-alone, 10 µM retinoic acid treatment. NS, without statistical significance (p > 0.05); the error bars, the mean ± standard deviation; , apoptosis peak; , G1 phase; , S phase; , G2 phase.

Figure 1

Lack of response of the three anaplastic thyroid cancer (ATC) cell lines to 10 µM retinoic acid (RA) treatment. (A) H/E staining (×40) and Cyclin D1 immunocytochemical staining (insets; ×40); (B) 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) cell proliferation assay; (C) flow cytometry. Control, without resveratrol treatment; RA-alone, 10 µM retinoic acid treatment. NS, without statistical significance (p > 0.05); the error bars, the mean ± standard deviation; , apoptosis peak; , G1 phase; , S phase; , G2 phase.

Figure 2

Different responses of the three ATC cell lines to resveratrol treatment. (A) H/E staining (×40) and Cyclin D1 immunocytochemical staining (insets; ×40) (B) MTT cell proliferation assay; (C) flow cytometry and Western blotting for pro-caspase-3 and active-caspase-3; (D) viable cell counting. *, with statistical significance (p < 0.05); the error bars, the mean ± standard deviation. Control, without resveratrol treatment; Res, 100 µM resveratrol treatment. NS, without statistical significance (p > 0.05); , apoptosis peak; , G1 phase; , S phase; , G2 phase.

Figure 2

Different responses of the three ATC cell lines to resveratrol treatment. (A) H/E staining (×40) and Cyclin D1 immunocytochemical staining (insets; ×40) (B) MTT cell proliferation assay; (C) flow cytometry and Western blotting for pro-caspase-3 and active-caspase-3; (D) viable cell counting. *, with statistical significance (p < 0.05); the error bars, the mean ± standard deviation. Control, without resveratrol treatment; Res, 100 µM resveratrol treatment. NS, without statistical significance (p > 0.05); , apoptosis peak; , G1 phase; , S phase; , G2 phase.

Figure 3

Resveratrol reverses the retinoic acid sensitivity of THJ-11T cells. (A) H/E staining (×40); (B) deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL) for apoptotic cell labeling (Green in color; ×40); (C) MTT cell proliferation assay; (D) viable cell counting; (E) immunocytochemical staining of thyroglobulin (Tg) and E-cadherin (×40). Control, cultured in 0.2% dimethylsulfoxide (DMSO)-containing medium; RA, retinoic acid treatment; Res, resveratrol treatment; Resveratrol plus RA, treated with a combination of 10 µM retinoic acid and 100 µM resveratrol for 48 h; Resveratrol-alone, 100 µM resveratrol; *, with statistical significance (p < 0.05); the error bars, the mean ± standard deviation. Arrows indicate the ports with higher magnification (×80) in the insets.

Figure 3

Resveratrol reverses the retinoic acid sensitivity of THJ-11T cells. (A) H/E staining (×40); (B) deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL) for apoptotic cell labeling (Green in color; ×40); (C) MTT cell proliferation assay; (D) viable cell counting; (E) immunocytochemical staining of thyroglobulin (Tg) and E-cadherin (×40). Control, cultured in 0.2% dimethylsulfoxide (DMSO)-containing medium; RA, retinoic acid treatment; Res, resveratrol treatment; Resveratrol plus RA, treated with a combination of 10 µM retinoic acid and 100 µM resveratrol for 48 h; Resveratrol-alone, 100 µM resveratrol; *, with statistical significance (p < 0.05); the error bars, the mean ± standard deviation. Arrows indicate the ports with higher magnification (×80) in the insets.

Figure 4

Differential expression of CRABP2 and FARP5 in THJ-11T cells in the absence or presence of 100 µM resveratrol. (A) Double immunofluorescent labeling (×40); (B) ImageJ (Version 1.0, National Institutes of Health, Bethesda, MD, USA)-based quantification of nuclear labeling of CRABP2 and FABP5; (C) Western blotting; (D) RT-PCR. Control, 0.2% DMSO-treated cells; Resveratrol-alone, 100 µM resveratrol treatment; Ratio, ratio between the levels of the target molecules and that of β-ACTIN/β-actin; *, with statistical significance (p < 0.05); NS, no statistical significance (p > 0.05); the error bars, the mean ± standard deviation.

Figure 5

Immunocytochemical and Western blotting demonstration of resveratrol-induced alterations of RAR-β and PPAR-β/δ expression and their intracellular distribution patterns in THJ-11T cells. (A) Immunocytochemical staining (×40); (B) ImageJ-based quantification of nuclear labeling of RAR-β and PPAR-β/δ; (C) Levels of RAR-β and PPAR-β/δ determined by Western blotting. Control, 0.2% DMSO-treated cells; Resveratrol, 48 h 100 µM resveratrol treatment. Arrows indicate the portions in higher magnification in the insets (×80). Ratio, ratio between the levels of the target molecules and that of β-ACTIN; *, with statistical significance (p < 0.05); NS, no statistical significance (p > 0.05); the error bars, the mean ± standard deviation. Gray values, nuclear RAR-β and PPAR-β/δ immunocytochemical staining densities.