Selenium bioavailability modulates the sensitivity of thyroid cells to iodide excess.

INTRODUCTION: Iodide is an essential micronutrient for the synthesis of thyroid hormones and its imbalance is involved in the origin of different thyroid pathological processes. Selenium (Se) is another essential trace element that contributes to thyroid preservation through the control of the redox homeostasis. Different studies have demonstrated that sodium-iodide-symporter (NIS) is downregulated in the presence of iodide excess and Se supplementation reverses this effect. We also demonstrated that NOX4-derived ROS are involved in NIS repression induced by iodide excess. The aim of this study was to investigate how Se bioavailability is decisive in the sensitivity to iodide excess on a differentiated rat thyroid cell line (FRTL-5). RESULTS: We demonstrated that siRNA-mediated silencing of Nox4 suppressed AKT phosphorylation induced by iodide excess. Iodide increases TGF-ß1 mRNA expression, AKT phosphorylation, ROS levels and decreases GPX1 and TXRND1 mRNAs expression while Se reversed these effects. Furthermore, iodide induced Nrf2 transcriptional activity only in Se-supplemented cultures, suggesting that Se positively influences Nrf2 activation and selenoenzyme response in FRTL-5. Se, also inhibited NF-κB phosphorylation induced by iodide excess. In addition, we found that iodide excess decreased total phosphatase activity and PTP1B and PTEN mRNA expression. Se supply restored only PTEN mRNA expression. Finally, we studied the 2-α-iodohexadecanal (2-IHD) effects since it has been proposed as intermediary of iodide action on thyroid autoregulation. 2-IHD stimulated PI3K/AKT activity and reduced NIS expression by a ROS-independent mechanism. Also, we found that 2-IHD increased TGF-ß1 mRNA and TGF-ß inhibitor (SB431542) reverses the 2-IHD inhibitory effect on NIS mRNA expression, suggesting that TGF-ß1 signaling pathway could be involved. Although Se reduced 2-IHD-induced TGFB1 levels, it could not reverse its inhibitory effect on NIS expression. CONCLUSION: Our study suggests that Se bioavailability may improve the expression of antioxidant genes through the activation of Nrf2, interfere in PI3K/AKT signaling and NIS expression by redox modulation.

Participation of NADPH 4 oxidase in thyroid regulation.

Different factors are involved in thyroid function and proliferation such as thyrotropin (TSH), insulin, growth factors, iodide, etc. TSH and IGF1/insulin increase proliferation rate and stimulate genes involved in thyroid differentiation. In the present study, we analyse the physiological regulation of NOX4 expression by TSH, insulin and iodine, and the role of NOX4 on thyroid genes expression. Differentiated rat thyroid cells (FRTL-5) were incubated in the presence or absence of TSH/insulin and TTF2, PAX8, TPO, NIS, NOX4, TGFß1, FOXO1/3 mRNA levels were examined by Real Time PCR. We showed that TSH and insulin repress NOX4 expression and appears to be inversely correlated with some thyroid genes. SiRNA targeted knockdown of NOX4 increased mRNA levels of TGFß1, TPO, PAX8, TTF2, FOXO1 and FOXO3. A PI3K inhibitor (LY294002), increases the expression of NIS, TTF2 and FOXO1/3, however PI3K/AKT pathway does not regulate NOX4 expression. We observed that iodine increased NOX4 expression and knockdown of NOX4 reduced ROS and reversed the inhibitory effect of iodine on NIS, TPO, PAX8 and TTF2 expression. Our findings provide strong evidence that NOX4 could be a novel signaling modulator of TSH/insulin pathway and would have a critical role in the autoregulatory mechanism induced by iodine.

Radiosensitivity enhancement of human thyroid carcinoma cells by the inhibitors of histone deacetylase sodium butyrate and valproic acid.

Radiotherapy is one of the leading treatments for clinical cancer therapy. External beam radiotherapy has been proposed as an adjuvant treatment for patients bearing differentiated thyroid cancer refractory to conventional therapy. Our purpose was to study the combined effect of HDAC inhibitors (HDACi) and ionizing irradiation in thyroid cancer cell lines (Nthy-ori 3-1, WRO, TPC-1 and 8505c). HDACi radiosensitized thyroid cancer cells as evidenced by the reduction of survival fraction, whereas they had no effect in the normal cells. HDACi enhanced radiation-induced cell death in WRO cells. Gamma-H2AX foci number increased and persisted long after ionizing exposure in the HDACi-treated cells (WRO and TPC-1). Moreover, the expression of the repair-related gene Ku80 was differentially modulated only in the cancer cells, by the compounds at the protein and/or mRNA levels. We present in vitro evidence that HDACi can enhance the radiosensitivity of human thyroid cancer cells.

NFE2-Related Transcription Factor 2 Coordinates Antioxidant Defense with Thyroglobulin Production and Iodination in the Thyroid Gland.

BACKGROUND: The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis. METHODS: Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation. RESULTS: The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide. CONCLUSIONS: Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.

Regulation of NADPH oxidase NOX4 by delta iodolactone (IL-δ) in thyroid cancer cells.

INTRODUCTION: Iodine is not used only by the thyroid to synthesize thyroid hormones but also directly influences a number of thyroid parameters such as thyroid proliferation and function. Several iodinated lipids, biosynthesized by the thyroid, were postulated as intermediaries in the action of iodide. Among these, iodolactone (IL-δ) and 2-iodohexadecanal (2-IHDA) have shown to inhibit several thyroid parameters. The antiproliferative effect of IL-δ is not restricted to the thyroid gland. IL-δ exhibits anti-tumor properties in breast cancer, neuroblastoma, glioblastoma, melanoma and lung carcinoma cells suggesting that IL-δ could be used as a chemotherapeutic agent. Moreover in a colon cancer cell line (HT-29), IL-δ induced cell death, and this effect was mediated by reactive oxygen species (ROS) generation. The aim of the present study was to analyze the sources of reactive oxygen species induced by IL-δ and to explore the contribution of ROS induced by IL-δ on cell proliferation and apoptosis. METHODOLOGY AND RESULTS: Cancer thyroid follicular (WRO) and papilar (TPC-1) cells lines were treated with IL-δ. Proliferation and apoptosis was analyzed. IL-δ caused a significant loss of cell viability on WRO and TPC-1 cells in a concentration dependent manner and induced apoptosis after 3 h of treatment. Furthermore, IL-δ (10 µM) increased ROS production (39% WRO and 20% TPC-1). The concomitant treatment of WRO and TPC-1 cells with Trolox or NAC plus IL-δ abrogated the augment of ROS induced by IL-δ exposure. Additionally Trolox and NAC reversed the effect of IL-δ on cell proliferation and apoptosis. Only in WRO cells IL-δ upregulates NADPH oxidase NOX4 expression, and siRNA targeted knock-down of NOX4 attenuates ROS production, apoptosis (p < 0.05) and the inhibitory effect of IL-δ on cell proliferation and PCNA expression (p < 0.05). CONCLUSIONS: The antiproliferative and pro-apoptotic effect of IL-δ is mediated by different mechanisms and pathway involving different sources of ROS generation depending on the cellular context.

Effects of 2-iodohexadecanal in the physiology of thyroid cells.

Iodide has direct effects on thyroid function. Several iodinated lipids are biosynthesized by the thyroid and they were postulated as intermediaries in the action of iodide. Among them, 2-iodohexadecanal (2-IHDA) has been identified and proposed to play a role in thyroid autoregulation. The aim of this study was to compare the effect of iodide and 2-IHDA on thyroid cell physiology. For this purpose, FRTL-5 thyroid cells were incubated with the two compounds during 24 or 48 h and several thyroid parameters were evaluated such as: iodide uptake, intracellular calcium and H2O2 levels. To further explore the molecular mechanism involved in 2-IHDA action, transcript and protein levels of genes involved in thyroid hormone biosynthesis, as well as the transcriptional expression of these genes were evaluated in the presence of iodide and 2-IHDA. The results obtained indicate that 2-IHDA reproduces the action of excess iodide on the "Wolff-Chaikoff" effect as well as on thyroid specific genes transcription supporting its role in thyroid autoregulation.

2-Iodohexadecanal inhibits thyroid cell growth in part through the induction of let-7f microRNA.

UNLABELLED: It is well known that pituitary TSH exerts the major task in the regulation of thyroid function. However, this gland is capable of certain degree of autonomy, independently of TSH control. Iodine plays an important role in thyroid physiology and biochemistry. The thyroid is capable of producing different iodolipids such as 2-iodohexadecanal (2-IHDA). It was shown that this iodolipid mimic some of the inhibitory effects of excess iodide on several thyroid parameters. OBJECTIVES: To identify the miRNAs regulated by 2-IHDA in rat thyroid cells and likely characterize their role in thyroid cell proliferation and function. RESULTS: FRTL-5 cells were grown in the presence of TSH and treated with 2-IHDA. Among the miRNAs up-regulated by 2-IHDA we focused on miR-let-7f and miR-138. When we transfected the miRNAs, miR-let-7f but not miR-138 overexpression inhibited proliferation of FRTL 5 cells, while miR-let-7f inhibition restored cell growth in 2-IHDA treated cultures. Analysis of cell cycle by flow cytometric DNA analysis revealed that miR-let-7f inhibition reduced the percentage of 2-IHDA treated cells in G1 phase and an increased of the percentage of cells in S phase was observed upon anti-let-7f transfection. The expresion of Cyclin D1 and Cyclin D3 were reduced after the transfection of miR-let-7f and miR-138, respectively. In in vivo studies we observed that miR-let-7f and miR-138 were up regulated by 2-IHDA during goiter involution. CONCLUSION: These results suggest that the inhibitory effects of 2-IHDA on FRTL-5 thyroid cell proliferation are mediated in part through the induction of let-7f microRNA.

Inhibitory effects of 2-iodohexadecanal on FRTL-5 thyroid cells proliferation.

UNLABELLED: Although thyroid gland function is mainly under the control of pituitary TSH, other factors, such as iodine, play a role in this process. The thyroid is capable of producing different iodolipids such as 6-iodo-deltalactone and 2-iodohexadecanal (2-IHDA). It was shown that these iodolipids mimic some of the inhibitory effects of excess iodide on several thyroid parameters. OBJECTIVES: To study the effect of 2-IHDA on cell proliferation and apoptosis in FRTL-5 cells. RESULTS: FRTL-5 cells were grown in the presence of TSH and treated with increasing concentrations of KI and 2-IHDA (0.5, 5, 10 and 33 µM) for 24, 48 and 72 h. Whereas KI inhibited cell proliferation only at 33 µM after 72 h of treatment, 2-IHDA inhibited in a time and concentration dependent manner. Analysis of cell cycle by flow cytometric DNA analysis revealed an accumulation of cells in G1 phase induced by 2-IHDA. The expression of cyclin A, cyclin D1 and cyclin D3 were reduced after treatment with 2-IHDA whereas CDK4 and CDK6 proteins were not modified. 2-IHDA induced a dynamic change in cytoplasmic to nuclear accumulation of p21 and p27 causing these proteins to be accumulated mostly in the nucleus. We also observed evidence of a pro-apoptotic effect of 2-IHDA at highest concentrations. No significant effect of KI was observed. CONCLUSION: These results suggest that the inhibitory effects of 2-IHDA on FRTL-5 thyroid cell proliferation are mediated by cell cycle arrest in G1/S phase and cell death by apoptosis.

6 Iodo-δ-lactone: a derivative of arachidonic acid with antitumor effects in HT-29 colon cancer cells.

BACKGROUND: IL-δ (5-hydroxy-6 iodo-8,11,14-eicosatrienoic delta lactone) an iodinated arachidonic acid (AA) derivative, is one of the iodolipids biosynthesized by the thyroid. Although IL-δ regulates several thyroid parameters such as cell proliferation and goiter growth it was found that this iodolipid inhibits the growth of other non thyroid cell lines. OBJECTIVES: To study the effect of IL-δ on cell proliferation and apoptosis in the colon cancer cell line HT-29. RESULTS: Treatment with IL-δ reduced cell viability in a concentration-dependent manner: 1µM 20%, 5µM 25%, 10µM 31%, 50µM 47% and caused a significant decrease of PCNA expression (25%). IL-δ had pro-apoptotic effects, evidenced by morphological features of programmed cell death such as pyknosis, karyorrhexis, cell shrinkage and cell blebbing observed by fluorescence microscopy, and an increase in caspase-3 activity and in Bax/Bcl-2 ratio (2.5 after 3h of treatment). Furthermore, IL-δ increased ROS production (30%) and lipid peroxidation levels (19%), suggesting that apoptosis could be a result of increased oxidative stress. A maximum increase in c-fos and c-jun protein expression in response to IL-δ was observed 1h after initiation of the treatment. IL-δ also induced a tumour growth delay of 70% compared to the control group in NIH nude mice implanted with HT-29 cells. CONCLUSION: Our study shows that IL-δ inhibits cell growth and induces apoptosis in the colon cancer cell line, HT-29 and opens the possibility that IL-δ could be a potential useful chemotherapy agent.