Western blotting analysis of NIS protein expression in M14, HepG2 and MKN-7 cells after treatment with the MEK, Akt and HDAC inhibitors.

<p>Cells were treated for 30 hrs with combination use of RDEA119, perifosine, and SAHA at the concentrations described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#pone-0031729-g001" target="_blank">Fig. 1</a>, followed by standard Western blotting analysis of cell lysates using the specific primary antibody against NIS. The expression level of ß-actin was analyzed in parallel for quality control. “Control”, treatment of cells with vehicle; “R+P+S”, treatment of cells with combined use of RDEA119, perifosine and SAHA.</p

Effects of the treatment with MEK, Akt and HDAC inhibitors on histone acetylation at the NIS promoter in M14, HepG2 and MKN-7 cells.

<p>Cells were treated with SAHA alone or SAHA in combination with RDEA119 and perifosine as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#pone-0031729-g002" target="_blank">Fig. 2</a>. Histone acetylation levels at the three regions (P1, P2, and P3) that comprise the minimal essential promoter of the <i>NIS</i> gene were analyzed by chromatin immunoprecipitation (ChIP) analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#s4" target="_blank">Materials and Methods</a>. Acetylation status of both histone H3 and H4 was examined using specific antibodies for ChIP. Non-specific IgG antibodies were used as control. The results for M14, HepG2 and MKN-7 cells are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#pone-0031729-g004" target="_blank">Figs. 4a, 4b and 4c</a>, respectively. For each cell, the upper panel shows the actual results of PCR on the DNA fragments obtained by ChIP using non-specific control IgG, anti-acetylated H3 (Anti Ac-H3) antibody or anti-acetylated H4 (Anti Ac-H4) antibody. Identical amounts of pre-immunoprecipitation cell lysates from different treatment conditions were used to start the immunoprecipitation. An aliquot of pre-immunoprecipitation cell lysate was directly used to isolate DNA as “Input” control. The PCR results reflect the histone acetylation levels. Presented in the lower panel for each cell is a bar graph showing quantitatively the acetylation levels of H3 and H4 at the three regions of <i>NIS</i> promoter based on densitometric measurements of the upper panel. The results are normalized by dividing the corresponding input signals and are presented as the ratio of the indicated treatment over the control. “Control”, treatment of cells with vehicle; “SAHA”, treatment of cells with SAHA alone; “R+P+S”, treatment of cells with combined use of RDEA119, perifosine and SAHA.</p

NIS mRNA expression in different human cancer cells after drug treatments (mean±SD).^*

<p>*The data in the table represent folds of increase in NIS expression over the control treatment with DMSO/PBS as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#s4" target="_blank">Materials and Methods</a>. RD, RDEA119; PE, perifosine; SA, SAHA.</p

Induction of radioiodine uptake in M14, HepG2 and MKN-7 cells by treatment with the MEK, Akt and HDAC inhibitors.

<p>Cells were treated with RDEA119, perifosine and SAHA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#pone-0031729-g002" target="_blank">Fig. 2</a>, followed by incubation with Na¹²⁵I for 1 hr. Parallel cells were additionally treated with the NIS blocker NaClO₄ to obtain non-specific radioiodine uptake/binding with the cells. Cells were then washed, lysed, and measured for radioactivity. Cell radioiodine uptake is presented as cpm/10⁶ cells (on the y-axis of the figure) after correction for the non-specific radioiodine binding. Detailed experimental procedures are as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031729#s4" target="_blank">Materials and Methods</a>. “Control”, treatment of cells with vehicle; “R+P+S”, treatment of cells with combination use of RDEA119, perifosine and SAHA. ** In comparison with control, p<0.01.</p

Target effects of inhibitors of the MAP kinase and PI3K/Akt pathways and HDAC in various non-thyroid human cancer cells.

<p>The MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA were used to respectively target these signaling pathways or molecules. Cells were treated for 30 hrs with 0.5 µM RDEA119, 5 µM perifosine or 0.5 µM SAHA as indicated. DMSO or PBS was used in parallel as the vehicle control. Cells were lysed for Western blotting after treatments to reveal the levels of phosphorylated ERK (p-ERK), phosphorylated Akt (p-Akt), and acetylated histone (Ac-His) with specific antibodies. “+”, treatment with the indicated inhibitor; “−”, treatment with vehicle.</p

Effects of a history of thyroid cancer on breast cancer-specific mortality—Deaths per 1000 person-years and hazard ratios.

Effects of a history of thyroid cancer on breast cancer-specific mortality—Deaths per 1000 person-years and hazard ratios.</p

Kaplan-Meier analyses of the differential protective effects of a history of TC on BC-specific survival between young and old patients.

A, comparisons of the effects of a history of TC on BC-specific survival curves in BC/TC and matched BC-only patients between the group aged B, comparisons of the effects of a history of TC on BC-specific survival curves in BC-1st and matched BC-only patients between the group aged C, comparisons of the effects of a history of TC on BC-specific survival curves in TC-1st and matched BC-only patients between the group aged BC, breast cancer; TC, thyroid cancer; BC-only, patients only with a diagnosis of breast cancer and without a history of thyroid cancer; BC/TC, breast cancer patients also with a history of thyroid cancer diagnosed any time—either before or after the diagnosis of breast cancer; BC-1st, breast cancer was diagnosed first, followed by diagnosis of thyroid cancer; TC-1st, thyroid cancer was diagnosed first, followed by diagnosis of breast cancer.</p

Comparison of clinicopathological characteristics of breast cancer in various clinical settings.

Comparison of clinicopathological characteristics of breast cancer in various clinical settings.</p

Kaplan-Meier analysis of the effect of a history of TC on BC-specific and overall survivals of patients in various settings.

A, comparison of BC-specific survival curves between BC/TC and matched BC-only patients; B, comparison of BC-specific survival curves between BC-1st and matched BC-only patients; C, comparison of overall survival curves between BC-1st and matched BC-only patients. D, comparison of BC-specific survival curves between TC-1st and matched BC-only patients. BC, breast cancer; TC, thyroid cancer; BC-only, patients only with a diagnosis of breast cancer and without a history of thyroid cancer; BC/TC, breast cancer patients also with a history of thyroid cancer diagnosed any time—either before or after the diagnosis of breast cancer; BC-1st, breast cancer was diagnosed first, followed by the diagnosis of thyroid cancer; TC-1st, thyroid cancer was diagnosed first, followed by the diagnosis of breast cancer.</p

Differential protective effects of a history of thyroid cancer on breast cancer-specific mortality in patients at age<50years and age≥50years at the diagnosis of breast cancer.

Differential protective effects of a history of thyroid cancer on breast cancer-specific mortality in patients at age<50years and age≥50years at the diagnosis of breast cancer.</p