Effects of nutraceuticals on anaplastic thyroid cancer cells

Abstract

Purpose: The anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer with a high mortality rate. Since nutraceuticals may exert beneficial effects on tumor biology, here, effects of four of these compounds [resveratrol, genistein, curcumin and epigallocatechin-3-gallate (EGCG)] on ATC cell lines were investigated.

Methods: Two ATC-derived cell lines were used: SW1736 and 8505C. Cell viability and in vitro aggressiveness was tested by MTT and soft agar assays. Apoptosis was investigated by Western Blot, using an anti-cleaved-PARP antibody. mRNA and miRNA levels were quantified by real-time PCR.

Results: All tested nutraceuticals caused in both cell lines decrease of cell viability and increase of apoptosis. In contrast, only curcumin reduced in vitro aggressiveness in both SW1736 and 8505C cell lines, while genistein and EGCG determined a reduction of colony formation only in 8505C cells. Effects on genes related to the thyroid-differentiated phenotype were also tested: resveratrol and genistein administration determined the increment of almost all tested mRNAs in both cell lines. Instead curcumin and EGCG treatments had opposite effects in the two cell lines, causing the increment of almost all the mRNAs in 8505C cells and their reduction in SW1736. Finally, effects of nutraceuticals on levels of several miRNAs, known as important in thyroid cancer progression (hsa-miR-221, hsa-miR-222, hsa-miR-21, hsa-miR-146b, hsa-miR-204), were tested. Curcumin induced a strong and significant reduction of all miR analyzed, except for has-miR-204, in both cell lines.

Conclusions: Altogether, our results clearly indicate the anti-cancer proprieties of curcumin, suggesting the promising use of this nutraceutical in ATC treatment. Resveratrol, genistein and EGCG have heterogeneous effects on molecular features of ATC cells.

Keywords: Curcumin; EGCG, ATC; Genistein; Nutraceuticals; Resveratrol.

Fig. 1

Nutraceuticals effects on cell viability. SW1736 (a) and 8505C (b) cells were treated with DMSO, resveratrol, curcumin, genistein or EGCG at different doses (0.5, 1, 10 and 50 μM) and different time (24, 48 or 72 h) and cell viability was analyzed by MTT assay. All experiments were run in sixfold

Fig. 2

Nutraceuticals effects on apoptosis. a SW1736 and 8505C cells were treated with 50 μM DMSO, resveratrol, curcumin, genistein or EGCG for 48 h. Cleaved PARP protein levels were evaluated as apoptosis marker by Western Blotting, as described in “Materials and Methods” section. For each cell line, the results were normalized against actin and expressed in arbitrary unit. b Densitometric analysis of cleaved PARP fraction levels obtained with Western blot assay in SW1736 and 8505C cells. Data are representative of 3 independent experiments and results are shown as mean ± SD. *P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t test

Fig. 3

Nutraceuticals effects on tumor aggressiveness. SW1736 and 8505C cells were treated with 50 μM DMSO, resveratrol, curcumin, genistein or EGCG for 48 h. Tumor aggressiveness were evaluated as the clonogenic ability of ATC cells, by soft agar assay. The histogram represents the number of colonies per cell line after the treatments. *P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t test. Data are representative of 3 independent experiments

Fig. 4

Expression levels of thyroid-specific genes in ATC cell lines after nutraceuticals treatment. SW1736 and 8505C were treated with 50 μM DMSO, resveratrol, curcumin, genistein or EGCG for 48 h. PAX8, TPO, TG, NIS, TSHR, TTF1 and TTF2 mRNA levels were evaluated by qPCR. All samples were run in triplicate and expressed as fold change against DMSO control. *P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t test. Data are representative of 3 independent experiments

Fig. 5

miRNAs modulation in ATC cell lines after nutraceuticals treatment. SW1736 and 8505C were treated with 50 μM DMSO, resveratrol, curcumin, genistein or EGCG for 48 h. miR-146b-5p, miR-221-3p, miR-222-3p, miR-21-5p, miR-204-5p levels were evaluated after treatments by qPCR. The relative miRNA expression levels were calculated applying the comparative ΔΔC _t method and are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t test