Sodium Iodide Symporter Expression Research Articles

Histone Deacetylase Inhibition Affects Sodium Iodide Symporter Expression and Induces 131I Cytotoxicity in Anaplastic Thyroid Cancer Cells

Anaplastic thyroid cancers (ATCs) represent only 1%-2% of all thyroid tumors, but they account for up to 50% of the mortality. Treatment of differentiated thyroid carcinomas is well standardized and the use of radioiodine represents an essential step; in contrast, there is no standardized therapeutic approach for anaplastic tumors and their prognosis is poor. The resistance of ATC to radioiodine treatment is principally due to the absence of expression of the sodium iodide symporter (NIS), mainly due to epigenetic silencing. The acetylation status of histones is involved in the epigenetic control of gene expression and is usually disrupted in advanced thyroid cancer. Histone deacetylase inhibitors have been demonstrated as potent anticancer drugs with several different effects on cell viability and differentiation. Stabilized ATC cell lines (BHT-101 and CAL-62) and primary cultures from patients who underwent thyroidectomy for ATC were treated with the histone deacetylase inhibitor LBH589. After treatment, we evaluated the expression and function of NIS. Gene expression was evaluated by real-time polymerase chain reaction (RT-PCR), NIS promoter activity was determined with a luciferase reporter assay, and protein expression was assessed through immunofluorescence. We tested the protein function by (125)I uptake and efflux experiments; finally the cytotoxic effect of (131)I was determined with a clonogenic assay. Our results demonstrate that treatment with LBH589 leads to NIS RNA expression as shown by RT-PCR and luciferase assay, and to protein expression as determined by immunofluorescence in vitro and by immunohistochemistry in xenograft tumors. Moreover, (125)I uptake and efflux experiments show the correct protein function and iodine retention, which translate into cytotoxicity effects, as demonstrated by a clonogenic assay with (131)I. This study supplies a new potential strategy for the treatment of ATC by modifying gene expression with the aim of inducing responsiveness towards radioiodine therapy.

Iodine-Induced Thyroid Blockade: Role of Selenium and Iodine in the Thyroid and Pituitary Glands

The purpose of this study was to determine the content of iodine and selenium in the thyroid and pituitary glands of rats under iodine-induced blockade of the thyroid gland. Electron probe microanalysis, wavelength-dispersive spectrometry, and point analysis were used in this investigation. We also determined the expression of sodium iodide symporter and caspase 32 in the thyroid and pituitary glands and the expression of thyroid-stimulating hormone in the pituitary. The samples for iodine analysis must be thoroughly dehydrated, and for this purpose, we developed a method that produced samples of constant mass with minimal loss of substrate (human thyroid gland was used for the investigation). Normal levels of iodine and selenium were found in the thyroid, pituitary, ovaries, testes hypothalamus, and pancreas of healthy rats. The levels of iodine and selenium in I- or Se-positive points and the percentage of positive points in most of these organs were similar to those of controls (basal level), except for the level of iodine in the thyroid gland and testes. Blockade of the thyroid gland changed the iodine level in iodine-positive points of the thyroid and the pituitary glands. On the sixth day of blockage, the iodine level in iodine-positive points of the thyroid gradually decreased to the basal level followed by an abrupt increase on the seventh day, implying a rebound effect. The opposite was found in the pituitary, in which the level of iodine in iodine-positive points increased during the first 6 days and then abruptly decreased on the seventh day. Expression of the thyroid-stimulating hormone in the pituitary decreased during the first 5 days but sharply increased on the sixth day, with a minimum level of iodine in the thyroid and maximum in the pituitary, before normalization of the iodine level in both glands preceding the rebound effect. The expression of sodium iodide symporter increased during the first 4 days of blockage and then decreased in both glands. The fluctuations of the thyroid-stimulating hormone in the pituitary gland reflected the changes of iodine in the thyroid gland more precisely than the changes of sodium iodide symporter. The selenium level in the selenium-positive points changed only in the pituitary, dropping to zero on the second and fifth day of the blockade. Simultaneously, the maximum induction of caspase 32 was observed in the pituitary gland. We believe that these results may help to clarify a role of the pituitary gland in the thyroid blockade.

SPECT/CT Evaluation of Unusual Physiologic Radioiodine Biodistributions: Pearls and Pitfalls in Image Interpretation

Radioiodine imaging has a well-established role in depicting metastatic disease after thyroidectomy in patients with well-differentiated thyroid cancer. Uptake of radioiodine in thyroid metastases depends on expression of sodium-iodide symporter (NIS) by tumor tissues. However, because radioiodine may also accumulate in normal structures and tissues, it is important to distinguish physiologic radioiodine activity from metastatic disease. Furthermore, secretions that contain radioiodine may also simulate pathologic uptake. A spectrum of physiologic distributions, normal variants, and benign mimics of disease have been described in the literature; yet, even when armed with a comprehensive knowledge of these patterns, interpreting radiologists and nuclear physicians may still encounter diagnostic uncertainty. Single-photon emission computed tomography (SPECT) with integrated computed tomography (CT) is a novel technology that, when applied to diagnostic iodine 123 or iodine 131 ((131)I) radioiodine scintigraphy, may accurately localize and help distinguish benign mimics of disease, with the potential to alter the management plan. SPECT/CT is increasingly being used with radioiodine scintigraphy to evaluate patients with thyroid cancer and shows promise for improving imaging specificity and reducing false-positive results.

Functional Radionuclide Imaging, In-Vitro Radioiodine Uptake Estimation and RT-PCR in the Evaluation of Sodium Iodide Symporter (NIS) Expression and Functionality in Breast Cancer: A Pilot Study

Breast cancer is a common malignancy in females, which is considered as a systemic disease, whose treatment involves combined modality including systemic as well as local treatment. Recent studies have shown that breast cancer also expresses Sodium Iodide Symporter (NIS) gene, like in the thyroid, which is the factor responsible for the uptake of iodide by the thyroid, enabling radioiodine therapy of thyroid disorders. This study aimed to evaluate various radionuclide imaging characteristics, in vitro radioiodine uptake (RAIU) and evaluation of NIS expression by using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) to explore sodium iodide symporter (NIS) expression and iodine uptake in breast cancer and to explor e whether radioiodine can be used for the diagnosis and treatment of breast cancer. Ways of differential regulation of NIS expression in breast cancer has also been explored. Female patients with palpable breast lump and histologically proven infiltrating duct carcinoma were taken up for the study, which included 50 females of mean age 49years. (range: 23-73years). The patients were categorized into different groups, depending on the type of the study performed. The uptake patterns in various imaging modalities were analyzed and compared with invitro and RT-PCR studies. 68% of breast cancer cases showed (99m)Tc-pertechnetate uptake at the initial images. This finding could partly be due to tumor vascularity, which is usually higher compared to the normal tissues. The uptake in the delayed imaging could be related to that due to NIS in the breast. Use of perchlorate or stable iodine did not alter the pertechnetate uptake pattern in breast tumor. Good correlation between (99m)Tc-pertechnetate and (99m)Tc-tetrofosmin uptake in breast cancer was demonstrated. In vitro radioactive iodine uptake in the breast tumor was significantly higher than that in the normal breast tissue. Only 42% of breast tumor samples studied using RT-PCR showed NIS expression. Correlation between (99m)Tc-pertechnetate uptake and NIS expression could not be well established. Further studies with higher dose of radioiodine and/or mechanisms of differentially blocking the thyroid are required to assess the feasibility of radioiodine therapy for breast cancer.

Prognostic Prediction of BRAFV600E and its Relationship with Sodium Iodide Symporter in Classic Variant of Papillary Thyroid Carcinomas

The aim of this study was to evaluate the prognosis of the classic variant of papillary thyroid carcinomas with the BRAF(V600E) mutation and 131I treatment failure in those tumors due to lower functional sodium iodide symporter expression. 109 papillary thyroid carcinomas were associated with clinicopathologic features. The BRAF(V600E) mutation was evaluated by direct sequencing and sodium iodide symporter protein was determined by immunohistochemistry. We found that the BRAF(V600E) mutation was significantly associated with the classic variant of papillary thyroid carcinomas and was independent of tumor size, the presence of extrathyroid invasion and lymph node metastasis, advanced TMN stages, and a high risk of disease recurrence. Moreover, the BRAF(V600E) mutation was associated with a statistically significant lower functional NIS protein expression in the classic variant of papillary thyroid carcinomas. However, those statistically significant relationships were not found in the follicular variant of papillary thyroid carcinomas. The BRAF(V600E) mutation might be associated with a more aggressive phenotype and a poor prognosis, causing less NIS-mediated 131I uptake due to a lower functional NIS protein expression in the classic variant of papillary thyroid carcinomas. Our current study appears to be valuable for predicting prognosis and is of important clinical significance for surgery and 131I treatment in patients with the classic variant of papillary thyroid carcinomas.

Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark.Our data demonstrate that in various thyroid cancer cell lines PARP inhibition increases NIS gene expression through a particular modulation of transcriptional regulatory mechanisms. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of thyroid cancer.

Preclinical efficacy of the oncolytic measles virus expressing the sodium iodide symporter in iodine non-avid anaplastic thyroid cancer: a novel therapeutic agent allowing noninvasive imaging and radioiodine therapy

Anaplastic thyroid cancer is an extremely aggressive disease resistant to radioiodine treatment because of loss of sodium iodide symporter (NIS) expression. To enhance prognosis of this fatal cancer, we validated the preclinical efficacy of measles virus (MV)-NIS, the vaccine strain of the oncolytic MV (MV-Edm), modified to include the NIS gene. Western blotting analysis confirmed that a panel of eight anaplastic thyroid cancer (ATC)-derived cell lines do not express NIS protein, but do express CD46, the MV receptor. In vitro cell death assays and in vivo xenograft studies demonstrate the oncolytic efficacy of MV-NIS in BHT-101 and KTC-3, ATC-derived cell lines. Radioactive iodine uptake along with single-photon emission computed tomography (SPECT)-computed tomography imaging of KTC-3 xenografts after (99)Tc(m) administration confirmed NIS expression in vitro and in vivo, respectively, after virus treatment. Adjuvant administration of RAI, to MV-NIS-treated KTC-3 tumors showed a trend for increased tumor cell killing. As current treatment for ATC is only palliative, and MV-NIS is currently Food and Drug Administration approved for human clinical trials in myeloma, our data indicate that targeting ATC with MV-NIS could prove to be a novel therapeutic strategy for effective treatment of iodine-resistant ATC and will expedite its testing in clinical trials for this aggressive disease.

Reacquisition of RAI uptake in RAI-refractory metastatic thyroid cancers by pretreatment with the MEK inhibitor selumetinib.

5509^ Background: Therapies for radioactive iodine (RAI)-refractory thyroid cancers of follicular origin (TC-FCO) are desperately needed. TC-FCO mouse models show that blocking mitogen-activated protein kinase (MAPK) signaling with a MAP kinase kinase 1/2 (MEK1/2) inhibitor increases sodium iodide symporter expression and iodine incorporation. This 20 patient (pt) pilot study aimed to evaluate if the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142866) can reverse RAI-refractoriness in TC-FCO pts (NCT00970359). Methods: RAI-refractory TC-FCO disease was defined as: 1) non-RAI-avid lesion/s on a diagnostic or post-therapy RAI scan, 2) RAI-avid lesion/s that was stable or increased in size after RAI therapy, or 3) fluorodeoxyglucose (FDG)-avid lesion/s by positron emission tomography (PET) scan. rhTSH-stimulated lesional dosimetry with 124I PET was performed prior to and after 4 weeks of treatment with selumetinib (75 mg orally bid). If the second 124I PET scan predicted a lesional RAI dose of > 2000 cGy, therapeutic RAI was administered while on the drug. Primary endpoints were to evaluate changes in tumor iodine uptake and RECIST response after RAI therapy; changes in serum thyroglobulin (TG) after RAI was a secondary endpoint. Results: 24 pts were enrolled, 22 eligible, and 20 evaluable. For the 20 evaluable pts, median age was 61 (range 44-77 yrs), 11 were men. 19 pts had tumors analyzed for BRAF and N-,K- RAS mutations. 8 pts had BRAF mutant (MUT), 11 BRAF wild-type (WT) tumors; 1 pt to be analyzed. Selumetinib increased 124I uptake in 12 of the 20 pts (4 of 8 BRAF MUT; 8 of the 12 other pts). The 8 of those 12 pts achieving sufficient iodine avidity to warrant RAI therapy included all 5 pts known to be NRAS MUT to date and 1 BRAF MUT pt. Of the 7 pts who have received RAI, 5 had partial responses (PRs); 2 stable disease (SD). Mean percent reduction in TG in this group (pre- vs 2 months post- RAI) was 91%. No > grade 2 CTCAE toxicities attributable to selumetinib were observed. 1 pt was diagnosed with myelodysplastic syndrome > 51 weeks after RAI (unrelated to selumetinib). Conclusions: Selumetinib can enhance iodine uptake in a subset of RAI-refractory TC-FCO and may be particularly effective for RAS MUT tumors.

Sodium Iodide Symporter and Phosphatase and Tensin Homolog Deleted on Chromosome Ten Expression in Cholangiocarcinoma Analysis with Clinicopathological Parameters

Background/AimsThis study was performed to investigate the correlation of sodium iodide symporter (NIS) expression with the functionality and loss of phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression in human cholangiocarcinoma (CCA).MethodsImmunohistochemistry for the expression of NIS and PTEN was performed in 60 biopsy specimens of CCA. The clinicopathological parameters were retrospectively identified from medical records. The expression pattern of NIS and loss of PTEN expression were analyzed in association with the clinicopathological characteristics, including survival.ResultsNormal biliary trees displayed NIS expression, but hepatocytes did not. NIS expression was divided into two patterns: cytoplasmic and membranous. Fifty-nine cases, all except for one case, displayed NIS expression in tumor cells. Twenty-two cases (33.3%) were mixed pattern, and 39 cases (65.05%) were cytoplasmic pattern; the pure membranous pattern was not noted. There was no association between the NIS expression pattern and clinicopathological parameters, including age, sex, differentiation grade, T stage and tumor, node, metastasis stage (p>0.05). The survival rates were similar among various NIS expression patterns. Normal hepatocytes and biliary trees exhibited PTEN expression in the nucleus and cytoplasm. CCA cells displayed nuclear staining. Thirty-six (60.0%) of 60 cases displayed a loss of PTEN expression. The loss of PTEN expression was observed in the advanced T-stage group (p=0.0036), but there was no association between the loss of PTEN expression and other clinicopathological parameters (p>0.05). No association between the loss of PTEN expression and survival was noted.ConclusionsNIS is expressed in most types of human CCA. The expression pattern suggests a role in cancer development. PTEN loss expression is common in the context of human CCA, especially in the advanced T stage.

Cyclic AMP–Response Element Modulator Inhibits the Promoter Activity of the Sodium Iodide Symporter Gene in Thyroid Cancer Cells

Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells. Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines. Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines. These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

The pituitary tumor transforming gene in thyroid cancer.

The pituitary tumor transforming gene (PTTG) is a multifunctional proto-oncogene that is over-expressed in various tumors including thyroid carcinomas, where it is a prognostic indicator of tumor recurrence. PTTG has potent transforming capabilities in vitro and in vivo, and many studies have investigated the potential mechanisms by which PTTG contributes to tumorigenesis. As the human securin, PTTG is involved in critical mechanisms of cell cycle regulation, whereby aberrant expression induces aneuploidy. PTTG may further contribute to tumorigenesis through its role in DNA damage response pathways and via complex interactions with hormones and growth factors. Furthermore, PTTG over-expression negatively impacts upon the efficacy of radioiodine therapy in thyroid cancer, through repression of expression and function of the sodium iodide symporter. Given its various roles at all disease stages, PTTG appears to be an important oncogene in thyroid cancer. This review discusses the current knowledge of PTTG with particular focus on its role in thyroid cancer.

Modulation of sodium iodide symporter expression and function by LY294002, Akti-1/2 and Rapamycin in thyroid cells

The selective increase of Na(+)/I(-) symporter (NIS)-mediated active iodide uptake in thyroid cells allows the use of radioiodine I(131) for diagnosis and targeted treatment of thyroid cancers. However, NIS-mediated radioiodine accumulation is often reduced in thyroid cancers due to decreased NIS expression/function. As PI3K signaling is overactivated in many thyroid tumors, we investigated the effects of inhibitors for PI3K, Akt, or mTORC1 as well as their interplay on NIS modulation in thyroid cells under chronic TSH stimulation. PI3K inhibition by LY294002 increased NIS-mediated radioiodide uptake (RAIU) mainly through upregulation of NIS expression, however, mTORC1 inhibition by Rapamycin did not increase NIS-mediated RAIU despite increased NIS protein levels. In comparison, Akt inhibition by Akti-1/2 did not increase NIS protein levels, yet markedly increased NIS-mediated RAIU by decreasing iodide efflux rate and increasing iodide transport rate and iodide affinity of NIS. The effects of Akti-1/2 on NIS-mediated RAIU are not detected in nonthyroid cells, implying that Akti-1/2 or its derivatives may represent potential pharmacological reagents to selectively increase thyroidal radioiodine accumulation and therapeutic efficacy.

Hesperetin Activates The Notch1 Signaling Cascade, Induces Cellular Differentiation, And Causes Apoptosis In Anaplastic Thyroid Cancer

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. In addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). To determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. In addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

Hesperetin Activates the Notch1 Signaling Cascade, Induces Cellular Differentiation, and Causes Apoptosis in Anaplastic Thyroid Cancer

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. in addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). to determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. in addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

Glucose Tolerance Evaluation in Graves Patients Treated with Methimazole and Radioiodine

The manuscript, “Evaluation of Glucose Tolerance in Methimazole and Radioiodine Treated Graves’ Patients,” by Kiani J et al. (1) compared glucose tolerance in Graves’ disease patients treated with methimazole to those treated with radioiodine. The results indicated that radioiodine therapy had no adverse effects on glucose tolerance or insulin resistance. This study has an impact on whether particular attention should be paid to diabetes mellitus in patients after radioiodine therapy. The conclusion is reasonable, but the design of this study raises several questions concerning glucose tolerance, beyond simply damage to the pancreas caused by radioiodine. First, not only the present state of thyroid function but also its functional changes throughout each treatment for Graves' disease may affect glucose tolerance. Without knowing the patient’s glucose tolerance before receiving radioiodine, the exact change is difficult to evaluate in each individual. Second, the level of sodium iodide symporter (NIS) expression in the pancreas remains controversial. One report demonstrated strong NIS immunoreactivity in pancreatic islet cells (2). Another report, however, did not find any evidence of NIS immunoreactivity in the pancreas (3). This discrepancy may be caused by the anti-NIS antibodies used in each study. Regardless, in both studies, the NIS expression level in the pancreas was quite low compared to the level in the thyroid gland. Given that TSH stimulates radioiodine uptake in thyroid tissue but not in extrathyroidal tissue because of the absence of TSH receptors, radioiodine accumulation in the pancreas is inhibited in the presence of the thyroid gland. To decrease this interference with radioiodine uptake, more efficient radioiodine accumulation in the pancreas may be one way to administer high doses of radioiodine as pointed out by the authors. Alternatively, thyroid-cancer patients who undergo a total thyroidectomy followed by remnant ablation may be good candidates for evaluation of glucose tolerance in patients after radioiodine therapy (1). In this situation, physiological accumulation of radioiodine can be identified by scintigraphy, and, when present, suggests possible subsequent damage in the tissues. Although it is rare to detect clear radioiodine accumulation in the pancreas of thyroid-cancer patients after radioiodine therapy or development Copyright c 2011 Kowsar M. P. Co. All rights reserved.

Expression of Sodium–Iodide Symporter and TSH Receptor in Subclinical Metastatic Lymph Nodes of Papillary Thyroid Microcarcinoma

The clinical significance of the subclinical lymph node (LN) metastasis in clinically node-negative (cN0) papillary thyroid microcarcinoma (PTMC) has been debated. We investigated the expression of sodium-iodide symporter (NIS) and thyroid-stimulating hormone receptor (TSHR) in the subclinical metastatic LNs of PTMC, which are crucial prerequisites for the response to radioactive iodine treatment. Among 149 consecutive patients who received total thyroidectomy in conjunction with prophylactic central neck dissection for cN0 PTMC from October 2005 to December 2007, 20 who had single PTMC and subclinical LN metastasis (cN0, pN1, single PTMC) were included. Immunohistochemical staining was performed with anti-human NIS antibody and anti-human TSHR antibody in 20 primary tumors and 52 metastatic LNs. NIS and TSHR expression was detected in 19 (95%) and 18 (90%) of 20 PTMCs, respectively. NIS and TSHR expression were also detected in 50 (96.2%) and 39 (75%) of 52 metastatic LNs, respectively. In 85% of patients, the presence of NIS expression in primary PTMCs was concordant with that in corresponding metastatic LNs. Intensities of NIS and TSHR expression were diverse. In 6 of 12 cases of multiple metastatic LNs, the metastatic LNs showed heterogeneous intensities of NIS expression. The presence of NIS and TSHR expression was observed with high frequency in both PTMCs and corresponding subclinical metastatic LNs. However, the intensity of NIS and TSHR expression was diverse. Multiple metastatic LNs from single primary tumor focus could have heterogeneous intensity of NIS expression.

The Urokinase Plasminogen Activator System in Metastatic Papillary Thyroid Carcinoma: A Potential Therapeutic Target

Although distant metastases occur in a minority of papillary thyroid carcinoma (PTC) patients, their impact on disease course and survival rates is profound, with aggregate 10-yr survival rates of 40% (1). Limited treatment options further complicate management of metastatic PTC. Radioiodine (RAI) remains the standard of care for most distant metastases in PTC. Of particular concern, however, is the significant prevalence of PTC metastases unable to trap RAI; such metastases are termed RAI-refractory (RAIR). Because thyroid cancer loses differentiated features of normal thyroid tissue, one feature commonly lost is sodium iodide symporter expression, which is the entire basis for the use of RAI therapy in thyroid malignancies. This phenomenon can occur in a heterogeneous manner: in a given patient, RAI uptake can vary between individual metastases, and it can even vary within a metastasis at the cellular level.

Use of sodium iodide symporter expression in differentiated thyroid carcinomas

We aimed to investigate the use of NIS mRNA and protein expression as a diagnostic and/or prognostic marker in patients with differentiated thyroid cancer (DTC). This is a case-control study. We studied 397 thyroid nodules tissue samples, including 224 papillary thyroid carcinomas (PTCs), 41 follicular carcinomas, 58 nodular goiters, 56 follicular adenomas and 18 normal tissues assembled in a tissue microarray. NIS protein was identified using a monoclonal antibody that labelled only the follicular cell basolateral membrane of all 397 tissue samples. In addition, NIS mRNA was quantified in 145 DTC patients and 85 PTC cases were screened for BRAF(V600E) mutation. We found low NIS mRNA expression and low or negative NIS protein expression in most DTC. NIS expression was lower in DTC patients over 45 years old and in tumours larger than 2 cm. There was a tendency for lower NIS expression in advanced stages and patients presenting recurrences. All 13 DTC patients who succumbed to the disease were NIS negative at immunohistochemistry and had very low mRNA expression. NIS expression was lower in PTC presenting BRAF(V600E) mutation. However, neither NIS immunohistochemical analysis nor NIS mRNA quantified expression could identify individuals with poor prognosis. Our data suggest that NIS expression may help characterize patients' risk and individuals with a poor response to therapy, but is not useful as a diagnostic or prognostic marker, reinforcing the current concept that an appropriate management of DTC patient is the most important and modifiable prognostic factor.