Combined Treatment with Troglitazone and Lovastatin Inhibited Epidermal Growth Factor-Induced Migration through the Downregulation of Cysteine-Rich Protein 61 in Human Anaplastic Thyroid Cancer Cells

<div><p>Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.</p></div

Effects of troglitazone and lovastatin on cell viability in ATC and fibroblast cells.

<p><b>A.</b> SW1736 human ATC cells were treated with the indicated concentrations of troglitazone and lovastatin for 24 h. <b>B.</b> WI-38 human fibroblast cells were cotreated with troglitazone (5 μM) and lovastatin (1 μM) for 24 h. Cell viability was determined using the crystal violet assay. The data are presented as the mean ± SEM of 3 independent experiments. *, <i>p</i> < 0.05, compared with the control group. Con, control group; Trog, troglitazone; Lova, lovastatin; and T/L, combined treatment with troglitazone and lovastatin.</p

Effects of combined treatment with troglitazone and lovastatin on EGF-induced phosphorylation of ERK, CREB, and EGFR in ATC cells.

<p><b>A–C.</b> SW1736 cells were treated with troglitazone (5 μM) and lovastatin (1 μM) for 4 h and subsequently treated wtih EGF (10 ng/mL) for (A) 15 min, (B) 30 min, or (C) 2 min. Total protein was collected to detect the phosphorylation levels of (A) ERK, (B) CREB, and (C) EGFR by performing western blot analysis. Trog, troglitazone; Lova, lovastatin; and T/L, combined treatment with troglitazone and lovastatin.</p

Proposed model of the effects of combined treatment with troglitazone and lovastatin on the inhibition of EGF-induced cell migration in ATC cells.

<p>EGF treatment increases Cyr61 expression through the ERK/CREB signal pathways and then promotes cell migration. Combined treatment with troglitazone and lovastatin downregulates CREB activity by inhibiting the ERK and other unknown signaling pathways, eventually reducing Cyr61 expression and cell migration in ATC cells. Trog, troglitazone; Lova, lovastatin.</p

Effects of combined treatment with troglitazone and lovastatin on EGF-induced cell migration in ATC cells.

<p><b>A.</b> SW1736 cells were treated with troglitazone (5 μM) and/or lovastatin (1 μM) for 7 h, and the migrated cells were detected using a wound healing assay. <b>B.</b> The SW1736 cells were preincubated with troglitazone (5 μM) and/or lovastatin (1 μM) for 0.5 h before EGF treatment. After 7 h, the migrated cells were detected using the wound healing assay. <b>C.</b> The SW1736 cells were cotreated with troglitazone (5 μM) and lovastatin (1 μM) for 4 h, and the cells were then seeded in the upper Transwell chamber and EGF (20 ng/mL) was added to the lower chamber as a chemoattractant for cell migration. After 18 h of incubation, the transmigrated cells were stained and counted. All data are presented as the mean ± SE of 3 independent experiments. *<i>p</i> < 0.05, compared with the control group; ^#, <i>p</i> < 0.05. Con, control group; Trog, troglitazone; Lova, lovastatin; and T/L, combined treatment with troglitazone and lovastatin.</p

Effects of combined treatment with troglitazone and lovastatin on EGF-induced stress-fiber formation in ATC cells.

<p><b>A–B.</b> SW1736 cells were cotreated with troglitazone (5 μM) and lovastatin (1 μM) for 3.5 h and subsequently treated with EGF (10 ng/mL) for 1 h. The cells were fixed, and immunocytochemistry staining was performed using phalloidin (red) to detect actin polymerization and DAPI staining was performed to detect nuclei (blue). <b>A.</b> The average number of filopodia per cell was determined by counting 30 cells. <b>B.</b> The figure depicts representative images. The arrows indicate the membrane ruffles and filopodia, and the representative regular membrane (open arrow) and filopodia (closed arrow) are shown. Con, control group and T/L, combined treatment with troglitazone and lovastatin.</p