Tetraiodothyroacetic Acid Research Articles

Thyroid hormone and P-glycoprotein in tumor cells.

P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3) at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

Abstract 5378: Actions of nanoparticulate tetraiodothyroacetic acid (Nanotetrac) on human prostate carcinoma xenograft growth, vascularity and integrin response to radiation

Abstract Tetraiodothyroacetic acid (tetrac) covalently bound to a nanoparticle (poly[lactic-co-glycolic acid]) (= Nanotetrac) has anti-cancer and anti-angiogenic properties that are initiated exclusively at a cell surface receptor on integrin αvβ3 of cancer cells and dividing blood vessel cells. The anti-cancer activities are pro-apoptotic by multiple mechanisms, disorder gene expression in multiple cancer cell survival pathways and are radiosensitizing. The anti-angiogenic effects involve disruption of actions of VEGF, bFGF, PDGF and EGF. In the present studies, susceptibility of human prostate cancer cell xenografts to activity of Nanotetrac was determined in grafts in the nude mouse or to the chick chorioallentoic membrane (CAM) model and radiosensitivity was estimated in the CAM system. Androgen-sensitive LNCaP cell xenografts in the CAM exposed to Nanotetrac (1 μg/CAM) showed 40% decreases in tumor weight and vascular branch points and 50% reduction in hemoglobin content at 7 d (P < 0.01). LNCaP xenograft volumes in the male mouse were reduced by 50% at 5 days (P < 0.01), approximating the volume of the tumor cell implant. In vivo imaging system (IVIS) scans of orthotopic androgen-independent PC3 cell xenografts in the nude mouse showed substantial reduction in tumor size vs. control (PBS) at 7 d (50% reduction) and at 17 d (75% reduction) post-implantation. Radiation studies in PC3 cell tumors established in the CAM system demonstrated that within 1 h of exposure to 1-2 Gy radiation, there was apparently defensive activation (assumption of ‘open conformation’) of αvβ3. Receptor activation response to radiation was blocked by Nanotetrac. We have shown in other cancer cell lines that Nanotetrac inhibits repair of radiation-induced double-stranded DNA breaks and propose that, based on the current observations, this DNA repair effect is due to inhibition of radiation-caused activation of αvβ3. In summary, examined in two models and acting at the cell surface, Nanotetrac substantially reduces volume/weight and vascularity of human prostate cancer xenografts. The agent also inhibited activation of integrin αvβ3 that is induced by radiation exposure and is seen to be a component of the radioresistance response. Citation Format: Shaker A. Mousa, Sudha Thangirala, Murat Yalcin, John T. Leith, Aleck Hercbergs, Hung-Yun Lin, Heng-Yuan Tang, Mary K. Luidens, Paul J. Davis. Actions of nanoparticulate tetraiodothyroacetic acid (Nanotetrac) on human prostate carcinoma xenograft growth, vascularity and integrin response to radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5378. doi:10.1158/1538-7445.AM2014-5378

Nanotetrac targets integrin αvβ3 on tumor cells to disorder cell defense pathways and block angiogenesis

The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3′-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells.

Thyroid hormone (3,5,3'-triiodothyronine, T3; L-thyroxine, T4) enhances cancer cell proliferation, invasion and angiogenesis via a discrete receptor located near the RGD recognition site on αvβ3 integrin. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation interfere with binding of T3/T4 to the integrin. This integrin is overexpressed in multiple myeloma (MM) and other cancers. MM cells interact with αvβ3 integrin to support growth and invasion. Matrix metalloproteinases (MMPs) are a family of enzymes active in tissue remodeling and cancer. The association between integrins and MMPs secretion and action is well established. In the current study, we examined the effects of thyroid hormone on myeloma cell adhesion, migration and MMP activity. We show that T3 and T4 increased myeloma adhesion to fibronectin and induced αvβ3 clustering. In addition, the hormones induced MMP-9 expression and activation via αvβ3 and MAPK induction. Bortezomib, a standard myeloma treatment, caused a decrease in activity/quantity of MMPs and thyroid hormone opposed this effect. RGD peptide and tetrac impaired the production of MMP-9 in cell lines and in primary BM cells from myeloma patients. In conclusion, thyroid hormone-dependent regulation via αvβ3 of myeloma cell adhesion and MMP-9 production may play a role in myeloma migration and progression.

Thyroid hormone inhibition in L6 myoblasts of IGF-I-mediated glucose uptake and proliferation: new roles for integrin αvβ3.

Thyroid hormones L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvβ3. Also insulin-like growth factor type I (IGF-I) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T4 and IGF-I in rat L6 myoblasts, we have examined integrin αvβ3-mediated modulatory actions of T4 on glucose uptake, measured through carrier-mediated 2-deoxy-[3H]-D-glucose uptake, and on cell proliferation stimulated by IGF-I, assessed by cell counting, [3H]-thymidine incorporation, and fluorescence-activated cell sorting analysis. IGF-I stimulated glucose transport and cell proliferation via the cell surface IGF-I receptor (IGFIR) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T4 caused little or no cell proliferation but prevented both glucose uptake and proliferative actions of IGF-I. These actions of T4 were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGFIR and the T4 receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvβ3, and the T4 metabolite tetraiodothyroacetic acid all blocked the inhibition by T4 of IGF-I-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-I and also showed a role for ERK1/2 in the actions of T4 that modified the effects of IGF-I. We conclude that thyroid hormone inhibits IGF-I-stimulated glucose uptake and cell proliferation in L6 myoblasts.

Tetraiodothyroacetic acid (tetrac) receptor on integrin αvβ3: tetrac blocks activation of the integrin response to tumor cell irradiation (974.6)

We have described a thyroid hormone‐tetrac receptor on plasma membrane integrin αvβ3 that mediates hormone analogue regulation of cancer cell proliferation and of tumor‐related angiogenesis (PJ Davis et al., Annu Rev Pharmacol Toxicol 51:99‐115, 2011). Liganded to this receptor, tetrac and nanoparticulate tetract (Nanotetrac) radiosensitize cancer cells (AH Hercbergs et al., Cell Cycle 10[2]:352‐357, 2011). One mechanism of radiosensitization is impairment of cellular repair of DNA double‐stranded breaks. We have defined in human prostate cancer (PC‐3) cells grown on the chick chorioallantoic membrane (CAM) the response of αvβ3 to ionizing radiation in the absence and presence of tetrac and Nanotetrac. PC‐3 grafts grow spherically in the CAM system and develop a core of hypoxic cells. Lysates of untreated PC‐3 cells contain about 6,900 αvβ3 molecules/cell (DuoSet R&D Systems) and ionizing radiation with 1‐10 Gy increased the number of αvβ3 moles/cell approximately 5‐fold over this radiation dose range. Investigated for state of activation (open conformation) by flow cytometry, the integrin was indeed activated 7‐fold over the 1‐10 Gy radiation dose exposure range, a protective response. Treatment of cells with tetrac or Nanotetrac (1‐2.5 μg/CAM) prevented the increase in cell number of αvβ3 molecules in response to radiation and blocked radiation‐induced activation of the integrin. Thus, radiosensitization of tumor cells in the CAM model that is conferred by tetrac formulations is complex. Inhibition by tetrac and Nanotetrac of the activation of integrin αvβ3 leads, via intracellular signaling mechanisms, to disordered repair in the nucleus of double‐stranded DNA breaks.

Thyroid hormone induced angiogenesis through the integrin αvβ3/protein kinase D/histone deacetylase 5 signaling pathway

Thyroid hormone is reported to induce angiogenesis, which is mediated by the membrane receptor integrin αvβ3, but the precise signaling pathway is still not very clear. Recently, studies have shown that protein kinase D (PKD) regulates the recycling of integrin αvβ3, which is required for cell migration. Moreover, phosphorylated PKD stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in endothelial cells. As a potent pro-angiogenic growth factor, basic fibroblast growth factor (bFGF (FGF2)) is a downstream target gene of HDAC5. Therefore, we examined the hypothesis that a novel signaling pathway through integrin αvβ3/PKD/HDAC5 might contribute to thyroxine (T4)-induced angiogenesis. We selected human umbilical vein endothelial cells (HUVECs) for treatment. Angiogenesis was assessed using wound-healing and tubulogenesis assays. Signaling molecules, including phosphorylated PKD and HDAC5, were measured by western blotting. bFGF mRNA was analyzed by real-time PCR. Our results showed that T4 (100 nmol/l) stimulated the migration and formation of tube-like structures of HUVECs, whereas tetraiodothyroacetic acid (Tetrac, 100 nmol/l) inhibited T4-induced cell migration. Importantly, T4 promoted the phosphorylation of PKD and HDAC5. These effects were inhibited respectively by Tetrac, PKC inhibitor (2.5 μmol/l) and PKD siRNA. Meanwhile, T4 could promote the cytoplasmic accumulation of phosphorylated HDAC5 in HUVECs. In addition, bFGF mRNA expression in HUVECs significantly increased within 2 h of T4 treatment, but was decreased by Tetrac. Our findings indicate that T4 increases the expression of bFGF mRNA via the integrin αvβ3/PKD/HDAC5 signaling pathway, which plays an important role in angiogenesis.

Tetraiodothyroacetic acid-conjugated PLGA nanoparticles: a nanomedicine approach to treat drug-resistant breast cancer.

The aim was to evaluate tetraiodothyroacetic acid (tetrac), a thyroid hormone analog of L-thyroxin, conjugated to poly(lactic-co-glycolic acid) nanoparticles (T-PLGA-NPs) both in vitro and in vivo for the treatment of drug-resistant breast cancer. The uptake of tetrac and T-PLGA-NPs in doxorubicin-resistant MCF7 (MCF7-Dx) cells was evaluated using confocal microscopy. Cell proliferation assays and a chick chorioallantoic membrane model of FGF2-induced angiogenesis were used to evaluate the anticancer effects of T-PLGA-NPs. In vivo efficacy was examined in a MCF7-Dx orthotopic tumor BALBc nude mouse model. T-PLGA-NPs were restricted from entering into the cell nucleus, and T-PLGA-NPs inhibited angiogenesis by 100% compared with 60% by free tetrac. T-PLGA-NPs enhanced inhibition of tumor-cell proliferation at a low-dose equivalent of free tetrac. In vivo treatment with either tetrac or T-PLGA-NPs resulted in a three- to five-fold inhibition of tumor weight. T-PLGA-NPs have high potential as anticancer agents, with possible applications in the treatment of drug-resistant cancer.

Thyroid hormones differentially regulate phosphorylation of ERK and Akt via integrin αvβ3 receptor in undifferentiated and differentiated PC‐12 cells

The effects of 3,5,3'-triiodo-l-thyronine (T3) and l-thyroxine (T4) on the integrin αvβ3 receptor of thyroid hormones (TH) were investigated in pheochromocytoma PC-12 cells. Differentiation was induced by treatment of PC-12 cells with fisetin and the levels of phosphorylated extracellular signal-regulated kinase (ERK) and Akt in cytoplasm, as well as the content of FoxO6 transcription factor in nuclei was analysed in undifferentiated and differentiated conditions. We have found that in undifferentiated PC-12 cells, tetraiodothyroacetic acid (TETRAC), a known inhibitor of binding of T4 and T3 to plasma membrane integrin αvβ3 receptor inhibits T4-dependent phosphorylation of ERK, whereas in differentiated PC-12 cells, TETRAC abolishes the effect of T3. In undifferentiated PC-12 cells, both TH increase the level of p-Akt, and this enhancement is not sensitive to TETRAC. In differentiated PC-12 cells, both TH increase the level of p-Akt; however, only T3-dependent activation of Akt is sensitive to the TETRAC. Furthermore, our results have shown that in differentiated PC-12 cells, the expression of FoxO6 was higher than in undifferentiated PC-12 cells, and this elevation has not changed under the action of TH. Only in undifferentiated PC-12 cells the T3-dependent expression of FoxO6 was sensitive to the TETRAC. We propose that PC-12 cells contain integrin αvβ3 receptor, which T3 and T3/T4 sites are differentially regulated by TH in undifferentiated and differentiated conditions.

Inherited defects of thyroid hormone-cell-membrane transport

This review summarizes the most significant findings over the last year regarding human and animal models deficient in thyroid hormone cell-membrane transporters (THCMTs). Although several THCMTs have been modelled in genetically engineered mice, the only THCMT defect known in humans is that caused by mutations in the monocarboxylate transporter 8 (MCT8) gene. The importance of several amino acid residues has been assessed in vitro to further our understanding on the structure-function of the MCT8. The administration of the thyromimetic compound, diiodothyropropionic acid, has been tested in patients with MCT8 gene mutations, following studies of its use in mice. Another thyroid hormone analogue, 3,3',5,5'-tetraiodothyroacetic acid, was tested in Mct8-deficient mice. The phenotypes of L-type aminoacid transporter 2 and organic anion transporting polypeptide 1C1 deficiencies have been studied in mouse models. Mct8/organic anion transporting polypeptide 1C1 double knockout mice have been shown to manifest neurodevelopmental deficits. Zebrafish is emerging as another vertebrate model that may be useful to study the role of Mct8 in brain development. Studies on the pathogenesis and therapy of MCT8 deficiency are in progress, and new vertebrate models that are suitable to study the neurological consequences of the syndrome are being explored.

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

We synthesized and evaluated (64)Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin αVβ3. Tetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with (64)Cu at 40 °C for 1 h and purified using a PD-10 column. (64)Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/(64)Cu-DOTA-liposomes or (64)Cu-DOTA-liposomes. HAEC binding studies exhibited that tetrac/(64)Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while (64)Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/(64)Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of (64)Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/(64)Cu-DOTA-liposomes and (64)Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively. These results demonstrate that tetrac/(64)Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to (64)Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/(64)Cu-DOTA-liposomes.

Small molecule hormone or hormone-like ligands of integrin αVβ3: implications for cancer cell behavior.

Integrins are heterodimeric structural components of the plasma membrane whose ligands include a large number of extracellular matrix (ECM) proteins. The ligands contain Arg-Gly-Asp (RGD) sequences that enable recognition of ECM proteins by as many as eight integrins, but other distinguishing features of the proteins permit the integrins to generate intracellular signals specific to the ECM molecules. Recently, integrin αvβ3 has been shown to have a panel of previously unappreciated small molecule receptor sites for thyroid hormone and hormone analogues, for dihydrotestosterone, and for resveratrol, a polyphenol that has certain estrogen-like features. These binding sites are close to the RGD recognition site of αvβ3. The thyroid hormone receptor site on the extracellular domain of αvβ3 contains two domains with discrete functions in terms of intracellular protein trafficking and gene expression. Occupancy of the receptor by a deaminated thyroid hormone analogue, tetraiodothyroacetic acid (tetrac), prevents cell responses to agonist thyroid hormones (L-thyroxine; 3, 5, 3'-triiodo-L-thyronine) and modulates expression of a number of cancer cell survival pathway genes in an up- or downregulation pattern coherent to induction of cell death. The small molecule thyroid hormone receptor on the integrin also regulates activity of five vascular growth factor receptors and/or their ligands, providing control of angiogenesis via specific pharmacologic regulation of this thyroid hormone receptor. The resveratrol receptor induces programmed cancer cell death via p53, even when the latter has undergone specific mutations. There is also evidence for the presence of several receptors on integrin αvβ3 for authentic steroids, including a dihydrotestosterone site that supports proliferation of breast cancer cells that lack nuclear androgen and estrogen receptors. The existence of these small molecule hormone receptors on an integrin with a remarkably complex functional profile defines novel pharmacologic options via individual small molecule receptor manipulation for control of cancer cell behavior. This refinement of up-down control at the level of discrete receptors is not a function of the use of αvβ3 antibody or RGD peptides that occlude regions of the integrin.

Molecular Mechanisms of Actions of Formulations of the Thyroid Hormone Analogue, Tetrac, on the Inflammatory Response

Background. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation (Nanotetrac) act at a cell surface receptor to block angiogenesis and tumor cell proliferation. Objective. The complex anti-angiogenic properties of tetrac and Nanotetrac caused us to search in the literature and in certain of our unpublished mRNA experiments for evidence that these agents affect the early inflammatory response, perhaps through actions on specific cytokines and chemokines. Results and Discussion. Tetrac and Nanotetrac inhibit expression in tumor cells of cytokine genes, e.g., specific interleukins, and chemokine genes, such as fractalkine (CX3CL1), and chemokine receptor genes (CX3CR1) that have been identified as high priority targets in the development of inflammation-suppressant drugs. The possibility is also examined that tetrac formulations have an effect on the function of inflammatory cells.

Response of human pancreatic cancer cell xenografts to tetraiodothyroacetic acid nanoparticles.

Tetraiodothyroacetic acid (tetrac) and its nanoparticle formulation (Tetrac NP) act at an integrin cell surface receptor to inhibit tumor cell proliferation and tumor-related angiogenesis. Human pancreatic cancer cell (PANC-1 and MPanc96) xenografts were established in nude mice, and the effects of tetrac versus Tetrac NP on tumor growth and tumor angiogenesis were determined. The in vitro effects of tetrac and Tetrac NP were also determined by reverse transcription polymerase chain reaction or immunoblot on gene expression or gene products relevant to cell cycle arrest, apoptosis, or angiogenesis. Tetrac and Tetrac NP reduced both PANC-1 tumor mass by 45-55 % and PANC-1 tumor hemoglobin content, a marker of angiogenesis, by 50-60 % (*P < 0.05) in treated groups vs. controls by treatment day 15. Comparable results were obtained with tetrac and Tetrac NP in suppressing tumor growth and tumor angiogenesis in MPanc96 xenografts. In vitro studies showed that tetrac and Tetrac NP caused accumulation of pro-apoptotic protein BcLx-s. Tetrac NP was more effective than tetrac in increasing cellular abundance of mRNAs of pro-apoptotic p53 and p21 and anti-angiogenesis thrombospondin 1 protein in PANC-1 and MPanc96 cancer cell lines. Tetrac NP noticeably decreased expression of EGFR and of anti-apoptosis gene XIAP; tetrac did not affect EGFR and increased XIAP mRNA in both MPanc96 and PANC-1. In conclusion, tetrac or Tetrac NP effectively inhibited human pancreatic xenograft growth and tumor angiogenesis via a plasma membrane receptor that downstream modulated cellular abundance of proteins or mRNAs relevant to apoptosis and angiogenesis.

Tetrac Can Replace Thyroid Hormone During Brain Development in Mouse Mutants Deficient in the Thyroid Hormone Transporter Mct8

The monocarboxylate transporter 8 (MCT8) plays a critical role in mediating the uptake of thyroid hormones (THs) into the brain. In patients, inactivating mutations in the MCT8 gene are associated with a severe form of psychomotor retardation and abnormal serum TH levels. Here, we evaluate the therapeutic potential of the TH analog 3,5,3',5'-tetraiodothyroacetic acid (tetrac) as a replacement for T(4) in brain development. Using COS1 cells transfected with TH transporter and deiodinase constructs, we could show that tetrac, albeit not being transported by MCT8, can be metabolized to the TH receptor active compound 3,3',5-triiodothyroacetic acid (triac) by type 2 deiodinase and inactivated by type 3 deiodinase. Triac in turn is capable of replacing T(3) in primary murine cerebellar cultures where it potently stimulates Purkinje cell development. In vivo effects of tetrac were assessed in congenital hypothyroid Pax8-knockout (KO) and Mct8/Pax8 double-KO mice as well as in Mct8-KO and wild-type animals after daily injection of tetrac (400 ng/g body weight) during the first postnatal weeks. This treatment was sufficient to promote TH-dependent neuronal differentiation in the cerebellum, cerebral cortex, and striatum but was ineffective in suppressing hypothalamic TRH expression. In contrast, TSH transcript levels in the pituitary were strongly down-regulated in response to tetrac. Based on our findings we propose that tetrac administration offers the opportunity to provide neurons during the postnatal stage with a potent TH receptor agonist, thereby eventually reducing the neurological damage in patients with MCT8 mutations without deteriorating the thyrotoxic situation in peripheral tissues.

The Stimulative Effect of T3 and T4 on Human Myocardial Endothelial Cell Proliferation, Migration and Angiogenesis

Angiogenesis, the process involving the growth of new blood vessels from pre-existing vessels, may be a target for combating diseases characterized by either poor vascularisation or abnormal vasculature as in cardiovascular diseases. Thyroid hormones (THs) are strong proangiogenic factors whose action starts at the plasma membrane integrin αvβ3 protein transducing rapid nongenomic signals on tumor thyroid cells. The tetraiodothyroacetic acid (Tetrac) inhibits the binding of TH to integrin receptor αVs3 blocking angiogenesis. Growing evidences suggest that, also in heart, Triiodothyronine (T3) and Thyroxine (T4) triggered nongenomic pathways through their binding to integrin receptor αVβ3 inducing capillary proliferation. The angiogenic activity of T3 and T4 has been studied by the chick choriallontoic endothelial cell microtubule assay and the human dermal microvascular endothelial cells microtubule assay. The aim of this work was to evaluate the direct stimulative activity of T3 and T4 on human cardiac microvascular endothelial cell (HMVEC-C) proliferation, migration and tube formation, employing Tetrac as inhibitor. Our in vitro study indicates that T3 and T4 directly stimulate angiogenesis in HMVEC-C observed as capillary density, cell proliferation and cell migration. In all models, Tetrac (5 μM) inhibited the proangiogenic effect of T3 and T4 suggesting its integrin-mediated action. Sq RT-PCR assay revealed that T3, and in less extent T4, increased the expression of angiogenic genes such as angiopoietin-1 (Angpt-1), angio-associated migratory cell protein (AAMP) and vascular endothelial growth factor (VEGF), whereas when the cells were pre-incubated with Tetrac this effect was abolished. In conclusion, our results show that THs stimulate angiogenesis, suggesting a potential therapeutic role aimed at increasing capillary density in cardiac diseases.

Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Modulation of diazepam-insensitive GABAA receptors by micromolar concentrations of thyroxine and related compounds in vitro

The effects of thyroxine and its related compounds on the benzodiazepine-insensitive γ-aminobutyric acid type A (GABAA) receptors were studied. Thyroxine at micromolar concentrations potentiated the 3H-Ro15-4513 binding to rat brain membranes in-vitro in the thalamus, striatum, cortex and hippocampus, but not in cerebellum. In the thalamus, the rank order of potency was the following: 3,3′,5,5′-tetraiodothyroacetic acid (TETRAC)>L-thyroxine>3,5-diiodo-l-thyronine (3,5-T2). TETRAC induced a slight potentiation of flumazenil binding to diazepam-sensitive GABAA receptors in the thalamus and striatum while no effect was found in cortex and hippocampus. Consequently, we examined whether these compounds could exert their modulatory effect on the currents mediated by benzodiazepine-insensitive GABAA receptors. The diazepam-insensitive GABAA receptor-mediated currents were recorded from acutely isolated rat ventrobasal thalamic neurons by applying low concentrations of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). TETRAC and thyroxine at low μM concentrations potentiated the THIP-evoked currents, although 3,5-T2 had no effect on the THIP-induced currents. Ethanol had no effect on the enhancing effects of TETRAC. TETRAC itself evoked GABAA receptor-mediated currents at high concentrations beyond 30μM. Although the effects of TETRAC and thyroxine were observed at non-physiological concentrations of hormones, the present results might lead to new lead structures with specificity to diazepam-insensitive GABAA receptor subtypes.

Tetraiodothyroacetic acid-tagged liposomes for enhanced delivery of anticancer drug to tumor tissue via integrin receptor

Nanoparticles have demonstrated potential for promoting drug delivery to tumor sites and enhancing uptake. Here, we report tetraiodothyroacetic acid (tetrac) as a promising new targeting moiety for delivery of anticancer drugs to tumor tissues. Tetrac, an antagonist that blocks the binding of thyroid hormone to integrin αvβ3, was covalently linked to the activated end of pegylated lipid and used to formulate tetrac-tagged pegylated liposomes (TPL). After incubating with TPL for 9h, cellular accumulation efficiency into A375 human melanoma cells, which express integrin αvβ3 at high density, was high (98.5%±0.5% of cells), whereas that in KB cells, which express integrin at a very low density, was much lower (35.1%±4.5%). Molecular imaging revealed that TPL preferentially distributed to tumor tissues after systemic administration in mice, where as non-targeting pegylated liposomes were distributed to tumors at background levels. Treatment with the alkyl lysophospholipid anticancer drug edelfosine, encapsulated in TPL, significantly reduced the survival of A375 tumor cells compared to cells treated with edelfosine in pegylated liposomes or with lysophosphatidylcholine encapsulated in TPL. Moreover, intravenous administration of edelfosine in TPL significantly reduced the growth of tumors and prolonged the survival of A375-xenografted mice, providing 100% protection for up to 50days and some protection until 66days (0% survival endpoint). In contrast, no untreated mice or mice treated with edelfosine-loaded pegylated liposomes survived up to 50 or 48days, respectively, after tumor inoculation. These results suggest the potential of tetrac as a new ligand moiety for enhancing the delivery of anticancer drug-loaded nanoparticles to tumors and enhancing the therapeutic efficacy of encapsulated anticancer drugs.

Triiodothyronine [T3]-induced hypothyroxinemia: Response and survival in a compassionate care cancer patient population.

e19573 Background: Increased survival under chemically induced hypothyroidism has been reported in cancer patients with poor prognosis, but the associated morbidity can deter patient compliance. Acting via a receptor site upon the plasma membrane avβ3 integrin, L-thyroxine (T4) in physiological concentrations non-genomically activates mitogenesis and neo-angiogenesis of cancer cells and proliferating vascular endothelium. Occlusion of the receptor by tetraiodothyroacetic acid (tetrac), a T4 derivative, induces apoptosis, inhibits angiogenesis and enhances chemotherapy and radiation. 3, 5, 3’-Triiodo-L-thyronine (T3) in physiological concentrations is significantly less mitogenic than T4 and inhibits pituitary release of thyrotropin (TSH). Absence of TSH, but with T3 support, depletes circulating T4 without resultant hypothyroidism. Methods: 21 cancer patients at dispersed clinical sites with stage 4 solid tumors and projected limited survival received T3 [n=10] only or with methimazole (MT) [n=9]. Serum TSH, free T4 (FT4) and T3 concentrations were obtained initially, then monthly. FT4 levels below lower normal range were achieved in all patients within 3 to 12 weeks. Results: See Table. Conclusions: T3 treatment with resultant hypothyroxinemia in poor prognosis cancer patients was associated with favorable response rates in 17/19 subjects. Two others with aggressive tumors had extended survival on long-term T3. [Table: see text] [Table: see text] [Table: see text]