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 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 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 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

<i>N</i>‑Hydroxycinnamide Derivatives of Osthole Presenting Genotoxicity and Cytotoxicity against Human Colon Adenocarcinoma Cells in Vitro and in Vivo

Osthole is extracted from the Chinese herbs <i>Cnidium monnieri</i> and <i>Angelica pubescens</i>, and it was found to have antitumor activity in vitro and in vivo. A series of osthole derivatives have been synthesized, and the <i>N</i>-hydroxycinnamide derivatives of osthole, WJ1376-1 and WJ1398-1 were found to have the greatest potential against human colon adenocarcinoma cells. In contrast to the parental osthole, both WJ1376-1 and WJ1398-1 were found to induce multinucleation and polyploidy by microscopic observation and flow cytometry. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3 related (ATR) kinase, which triggered activation of the checkpoint kinase 2 (Chk2) signaling pathway and then down regulated Cdc25 phosphatase and Cdc2/cyclin B kinase activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, which is associated with important processes during mitosis. The presence of a “comet” DNA fragment and phosphorylation of p53 at Ser 15 clearly indicated that DNA damage occurred with WJ1376-1 and WJ1398-1 treatment. WJ1376-1 and WJ1398-1 ultimately induced apoptosis as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 also showed a great effect in attenuating tumor growth without affecting the body weight of xenograft nude mice. Taken together, these results suggest that the toxic activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole, which can induce cell polyploidy and G₂/M cell cycle arrest in colon adenocarcinoma cells and may provide a potential therapeutic target for colon cancer treatment in the future

Binding of EV71 VLPs to immature human monocyte-derived DCs.

<p>For the analysis of concentration-dependent effects, human monocyte-derived DCs were incubated with 1 or 10<i> </i>µg/mL EV71 VLPs for 30<i> </i>min on ice to allow binding to the cell surface. For the analysis of time-dependent effects, DCs were incubated with 5<i> </i>µg/mL of EV71 VLPs for 30 or 120<i> </i>min on ice. Detection was facilitated using an anti-EV71 Ab and anti-mouse-IgG Ab (labeled with Alexa Fluor 488). Levels of fluorescence were determined using immunofluorescence confocal microscopy (A) and FACS analysis (B, C). (B) and (C) indicate the respective concentration-dependent and time-dependent effects of the binding of EV71 VLPs to immature human monocyte-derived DCs. The control cells (no VLPs; filled histogram) were stained with the same primary antibody (anti-EV71 Ab) followed by a secondary antibody. (D) and (E) indicate the bar diagram of mean fluorescence intensity (MFI) with statistics for concentration- and time-dependent binding of EV71 VLPs to DCs. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

TLR4 mediated the maturation of DCs induced by EV71 VLPs.

<p>(A) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR2, anti-TLR4, or IgG2a (as an isotype control) separately for 1<i> </i>h and were then challenged with EV71 VLPs (10<i> </i>µg/mL) for 16<i> </i>h. The control was not treated with EV71 VLPs. Supernatants from the cell cultures were collected to determine levels of IL-12 p70, IL-12 p40, and IL-10. Significant differences in the levels of cytokines produced by DCs treated with Abs and those treated with IgG2a are indicated by asterisks (*<i>p</i><0.05). (B) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR4 mAb or IgG2a (as an isotype control) separately for 1<i> </i>h and were then treated with 10<i> </i>µg/mL EV71 VLPs for 30<i> </i>min on ice to allow binding to the cell surface. Surface-bound EV71 VLPs were measured with flow cytometry through immunostaining with anti-EV71 monoclonal antibodies. The control cells (no VLPs; dotted line) were stained with the same primary antibody followed by a secondary antibody. The bar diagram shows the MFI with statistics for the anti-TLR4 mAb effect on binding of EV71 VLPs to DCs. (C) HEK293 cells were transfected with control plasmids, TLR4, or TLR4+MD2. The transfected cells were stimulated with medium alone, LPS (50<i> </i>ng/mL), or EV71 VLP (10<i> </i>µg/mL) for 24<i> </i>h, and IL-8 protein in the supernatants was measured by ELISA. This experiment was repeated three times with similar results. (D) Proteinase K-treated EV71 VLPs abrogated the production of IL-12 p70, IL-12 p40, and IL-10 by DCs. Human DCs were incubated with medium alone (Control), proteinase K (10<i> </i>ng/mL), EV71 VLPs (10<i> </i>µg/mL), proteinase K-treated EV71 VLPs, LPS (100<i> </i>ng/mL), or proteinase K-treated LPS for 24<i> </i>h. At the end of the incubation time, the production of IL-12 p70, IL-12 p40, and IL-10 was analyzed by ELISA. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

Induction of IκBα degradation and NF-κB activation by incubation of human DCs with EV71 VLPs.

<p>(A) Human DCs were treated with EV71 VLPs (10<i> </i>µg/mL) for the indicated times. Western blotting was used to analyze cytosolic fractions for degradation of IκBα. The lower panel shows anti-α-tubulin staining of the same blot and confirmed equal loading of all samples. (B) Human monocyte-derived DCs were not treated (Cont) or were treated with LPS (100<i> </i>ng/mL) or EV71 VLPs (5 or 10<i> </i>µg/mL) for 2<i> </i>h, and nuclear fractions were prepared. An electrophoretic mobility shift assay was used to assay NF-κB binding activity in the nuclear fractions. To assess the specificity of the binding, a 100-fold excess of cold NF-κB probe (a double-stranded NF-κB oligonucleotide, 5′-AGTTGA<u>GG<b>G</b>GACTTTCCC</u>AGGC-3′) or mutant probe (a double-stranded mutated oligonucleotide, 5′-AGTTGA<b>GG<u>C</u>GACTTTCCC</b>AGGC-3′) was added to the EV71 VLP condition. A double-stranded mutated probe, 5′-AGTTGA<u>GG<b>C</b>GACTTTCCC</u>AGGC-3′, was used to examine the specificity of binding of NF-κB to DNA (the underlined sequence is identical to the κB consensus sequence except for a <b>G</b>-to-<b>C</b> substitution in the NF-κB DNA binding motif). This experiment was repeated three times (from three different donors) with similar results.</p