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Well-standardized primary treatment and long-term management of differentiated thyroid carcinoma (DTC) include lowering or suppression of host thyrotropin (TSH) with exogenous L-thyroxine (T4). This treatment recognizes the trophic action of TSH on DTC cells. Suppression of endogenous TSH with T4 is continued in recurrent disease. However, T4 can induce proliferation of follicular and papillary thyroid carcinoma cell lines and of other human carcinoma cells. The proliferative mechanism is initiated at a cell surface receptor for T4 on integrin αvβ3, a receptor by which the hormone also inhibits p53-dependent apoptosis in tumor cells. In recurrent DTC with satisfactory suppression of endogenous TSH, we discuss here the possibility that the tumor is no longer TSH dependent and that T4 has become a critical growth factor for the cancer.

Acting via a cell surface receptor on integrin αvβ3, thyroid hormone is pro-angiogenic. Nongenomic mechanisms of actions of the hormone and hormone analogues at αvβ3 include modulation of activities of multiple vascular growth factor receptors and their ligands (vascular endothelial growth factor, basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor), as well as of angiogenic chemokines (CX3 family). Thyroid hormone also may increase activity of small molecules that support neovascularization (bradykinin, angiotensin II) and stimulate endothelial cell motility. Therapeutic angio-inhibition in the setting of cancer may be opposed by endogenous thyroid hormone, particularly when a single vascular growth factor is the treatment target. This may be a particular issue in management of aggressive or recurrent tumors. It is desirable to have access to chemotherapies that affect multiple steps in angiogenesis and to examine as alternatives in aggressive cancers the induction of subclinical hypothyroidism or use of antagonists of the αvβ3 thyroid hormone receptor that are under development.

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complex anti-angiogenic properties of tetrac and Nanotetrac caused us to search in the literature and in certain of our unpublished mRNA experiments for evidence that these agents affect the early inflammatory response, perhaps through actions on specific cytokines and chemokines., Results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.

The pathophysiology of brain damage that is common to ischemia-reperfusion injury and brain trauma include disodered neuronal and glial cell energetics, intracellular acidosis, calcium toxicity, extracellular excitotoxic glutamate accumulation, and dysfunction of the cytoskeleton and endoplasmic reticulum. The principal thyroid hormones, 3,5,3'-triiodo-l-thyronine (T(3)) and l-thyroxine (T(4)), have non-genomic and genomic actions that are relevant to repair of certain features of the pathophysiology of brain damage. The hormone can non-genomically repair intracellular H(+) accumulation by stimulation of the Na(+)/H(+) exchanger and can support desirably low [Ca(2+)](i.c.) by activation of plasma membrane Ca(2+)-ATPase. Thyroid hormone non-genomically stimulates astrocyte glutamate uptake, an action that protects both glial cells and neurons. The hormone supports the integrity of the microfilament cytoskeleton by its effect on actin. Several proteins linked to thyroid hormone action are also neuroprotective. For example, the hormone stimulates expression of the seladin-1 gene whose gene product is anti-apoptotic and is potentially protective in the setting of neurodegeneration. Transthyretin (TTR) is a serum transport protein for T(4) that is important to blood-brain barrier transfer of the hormone and TTR also has been found to be neuroprotective in the setting of ischemia. Finally, the interesting thyronamine derivatives of T(4) have been shown to protect against ischemic brain damage through their ability to induce hypothermia in the intact organism. Thus, thyroid hormone or hormone derivatives have experimental promise as neuroprotective agents.

Nuclear receptors for thyroid hormone and steroids are members of a receptor superfamily with similar molecular organization, but discrete transcriptional functions that define genomic actions of these nonpeptide hormones. Nongenomic actions of thyroid hormone and estrogens and androgens are initiated outside the nucleus, at receptors in the plasma membrane or in cytoplasm; these actions are largely regarded to be unique to the respective hormones. However, there is an increasing number of descriptions of overlapping nongenomic and genomic effects of thyroid hormone and estrogens and testosterone. These effects are concentrated in tumor cells, where, for example, estrogens and thyroid hormone have similar mitogen-activate protein kinase (MAPK)-dependent proliferative actions on ERα-positive human breast cancer cells, and where dihydrotestosterone also can stimulate proliferation. Steroids and thyroid hormone have similar anti-apoptotic effects in certain tumors. But thyroid hormone and steroids also have overlapping or interacting nongenomic and genomic actions in heart and brain cells. These various effects of thyroid hormone and estrogens and androgens are reviewed here and their possible clinical consequences are enumerated., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Integrin αvβ3 is a heterodimeric structural protein of the plasma membrane that bears a cell surface receptor for thyroid hormone. The functions of this receptor are distinct from those of the classical nuclear receptor (TR) for thyroid hormone. The integrin is expressed primarily by cancer cells, dividing endothelial and vascular smooth muscle cells, and osteoclasts. The hormone receptor on αvβ3 enables L-thyroxine (T(4)) and 3, 5, 3'-triiodo-L-thyronine (T(3)) to stimulate cancer cell proliferation and angiogenesis and to regulate the activity of certain membrane ion pumps. Bound to the receptor, the hormone ligand also stimulates protein trafficking within the cell. A deaminated derivative of T(4), tetraiodothyroacetic acid (tetrac), blocks binding and actions of T(4) and T(3) at the receptor on αvβ3; tetrac also has anti-proliferative actions at the integrin thyroid hormone receptor beyond the effects of antagonizing actions of agonist thyroid hormone analogues at the receptor. The structure-activity relationships of hormone analogues at the receptor have been computer-modeled and indicate that the receptor includes a site that binds T(3) and a site that binds both T(4) and T(3). Mathematical modeling of the kinetics of hormone-binding also suggests the existence of two sites. Cell proliferation is modulated from the T(4)/T(3) site. Tetrac has been re-formulated as a nanoparticle (nanotetrac) that acts exclusively at the αvβ3 receptor and does not enter cells. Nanotetrac disrupts expression of genes in multiple cancer cell survival pathways. The tetrac formulations block human cancer cell proliferation in vitro and in tumor xenografts. Nanotetrac and tetrac inhibit the pro-angiogenic actions in vitro of vascular endothelial growth factor, basic fibroblast factor, and other growth factors. Thus, the receptor described on integrin αvβ3 for T(4) and T(3), the function of which is materially affected by tetrac and nanotetrac, provides insight into tumor cell biology and vascular biology.

Plasma membrane integrin αvβ3 is a cell surface receptor for thyroid hormone at which nongenomic actions are initiated. L-thyroxine (T₄) and 3,3',5-triiodo-L-thyronine (T₃) promote angiogenesis and tumor cell proliferation via the receptor. Tetraiodothyroacetic acid (tetrac), a deaminated T₄ derivative, blocks the nongenomic proliferative and proangiogenic actions of T₄ and T₃. Acting at the integrin independently of T₄ and T₃, tetrac and a novel nanoparticulate formulation of tetrac that acts exclusively at the cell surface have oncologically desirable antiproliferative actions on multiple tumor cell survival pathway genes. These agents also block the angiogenic activity of vascular growth factors. Volume and vascular support of xenografts of human pancreatic, kidney, lung, and breast cancers are downregulated by tetrac formulations. The integrin αvβ3 receptor site for thyroid hormone selectively regulates signal transduction pathways and distinguishes between unmodified tetrac and the nanoparticulate formulation. The receptor also mediates nongenomic thyroid hormone effects on plasma membrane ion transporters and on intracellular protein trafficking.

In models of thyroid hormone-induced cardiac hypertrophy, there is appropriate, supportive angiogenesis. Twenty years ago in one such model, angiogenesis in response to the hormone was observed before hypertrophy developed and it is now understood that iodothyronines induce neovascularization in a variety of settings, including the heart, ischemic striated muscle and tumor beds. The molecular mechanism of the proangiogenic action of thyroid hormone is both nongenomic and genomic. It is initiated nongenomically at the cell surface receptor for the hormone on integrin alphavbeta3. Kinase transduction of the hormone signal and, ultimately, transcription of several anagiogenesis-relevant genes result. The genes include basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). In addition, the integrin receptor for thyroid hormone (l-thyroxine, T(4), and 3, 5, 3'-triiodo-l-thyronine, T(3)) engages in crosstalk with the VEGF and bFGF receptors. Occlusion with tetraiodothyroacetic acid (tetrac) of the hormone receptor on the integrin in the absence of T(4) and T(3) suppresses the angiogenic effects of VEGF and bFGF. Tetrac also blocks the proangiogenic actions of T(4) and T(3). Other thyroid hormone analogues that are angiogenic include diiodothyropropionic acid (DITPA) and the nuclear thyroid hormone receptor-beta-selective agonist, GC-1. Thyroid hormone sustains angiogenesis and coronary blood flow about infarcted heart tissue in experimental models and blocks deleterious heart remodeling that otherwise is predictable in such tissue. The hormone may also induce expression of the hypoxia-inducible factor 1alpha (HIF1alpha) gene, a transcription factor important to coronary artery collateralization in the setting of hypoxia. The hormone also causes transcription of the matrix Gla protein (MGP) gene that opposes vascular smooth muscle calcification., (Copyright 2009. Published by Elsevier Inc.)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin alphavbeta3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Constitutively expressed cyclooxygenase-2 (COX-2) is a marker of tumor cell aggressiveness. Inducible COX-2 has also been described in cancer cells and localizes in the cancer cell nucleus, where formation of a complex of mitogen-activated protein kinase (MAPK) and COX-2 is antecedent to p53-dependent apoptosis. The stilbene resveratrol is a model pharmacologic activator of this pro-apoptotic mechanism. Physiological concentrations of thyroid hormone are anti-apoptotic in several types of tumor cells. A mechanism by which the hormone is anti-apoptotic is disruption of the nuclear MAPK-COX-2 complex. We review here the apoptosis-relevant effects of resveratrol and thyroid hormone and then speculate about the significance of convergence of these actions in cancer cells in the intact organism. Clinical activity of resveratrol may be modulated by normal tissue levels of endogenous thyroid hormone, and hypothyroidism in the cancer patient -- whether spontaneous or induced by chemotherapeutic agents -- may permit full expression of the apoptotic activity of the administered stilbene. Chronic pharmacologic inhibition of COX-2 may oppose the pro-apoptotic effect of resveratrol.

3,5,3'-Triiodo-l-thyronine (T(3)), but not l-thyroxine (T(4)), activated Src kinase and, downstream, phosphatidylinositol 3-kinase (PI3-kinase) by means of an alpha(v)beta(3) integrin receptor on human glioblastoma U-87 MG cells. Although both T(3) and T(4) stimulated extracellular signal-regulated kinase (ERK) 1/2, activated ERK1/2 did not contribute to T(3)-induced Src kinase or PI3-kinase activation, and an inhibitor of PI3-kinase, LY-294002, did not block activation of ERK1/2 by physiological concentrations of T(3) and T(4). Thus the PI3-kinase, Src kinase, and ERK1/2 signaling cascades are parallel pathways in T(3)-treated U-87 MG cells. T(3) and T(4) both caused proliferation of U-87 MG cells; these effects were blocked by the ERK1/2 inhibitor PD-98059 but not by LY-294002. Small-interfering RNA knockdown of PI3-kinase confirmed that PI3-kinase was not involved in the proliferative action of T(3) on U-87 MG cells. PI3-kinase-dependent actions of T(3) in these cells included shuttling of nuclear thyroid hormone receptor-alpha (TRalpha) from cytoplasm to nucleus and accumulation of hypoxia-inducible factor (HIF)-1alpha mRNA; LY-294002 inhibited these actions. Results of studies involving alpha(v)beta(3) receptor antagonists tetraiodothyroacetic acid (tetrac) and Arg-Gly-Asp (RGD) peptide, together with mathematical modeling of the kinetics of displacement of radiolabeled T(3) from the integrin by unlabeled T(3) and by unlabeled T(4), are consistent with the presence of two iodothyronine receptor domains on the integrin. A model proposes that one site binds T(3) exclusively, activates PI3-kinase via Src kinase, and stimulates TRalpha trafficking and HIF-1alpha gene expression. Tetrac and RGD peptide both inhibit T(3) action at this site. The second site binds T(4) and T(3), and, via this receptor, the iodothyronines stimulate ERK1/2-dependent tumor cell proliferation. T(3) action here is inhibited by tetrac alone, but the effect of T(4) is blocked by both tetrac and the RGD peptide.

Introduction: In CV-1 cells, shuttling from cytoplasm to nucleus of the nuclear thyroid hormone receptor-beta1 (TRbeta1, TR) is shown in this report to be regulated by extracellular thyroid hormone at a hormone receptor on cell surface integrin alphav3., Methods: The receptor was introduced into cells as a GFP-TR1 chimera and intracellular movement of the receptor was monitored by confocal microscopy of cells treated with L-thyroxine (T(4))., Results and Discussion: TR-GFP translocation in the presence of T(4) requires activation of extracellular-regulated protein kinases 1/2 (ERK1/2). Inhibition of T(4)-binding to alphavbeta3 with anti-alphavbeta3 or Arg-Gly-Asp (RGD) peptide blocks T(4)-stimulated GFP-TR nuclear translocation, as do the hormone-binding inhibitor tetraiodothyroacetic acid (tetrac) and the ERK1/2 inhibitor, PD98059. TR1 is an ERK1/2 substrate., Conclusions: Via a nongenomic mechanism initiated at plasma membrane integrin v3, T(4)-activated ERK1/2 and TR1 move transiently in an immunoprecipitable complex to the nuclei of T(4)-treated cells.

The alpha-helix stabilizing solvent 2,2,2-trifluoroethanol (TFE) is frequently used as a medium for determining the average alpha-helicity of polypeptides by CD spectroscopy. CD spectra measured in solutions containing 10, 15, 20, 50, and 90% (vol/vol) TFE are presented for 5 peptides that were selected to demonstrate possible variations in the effect of TFE concentration on the secondary structure. The analysis is extended to 6 further peptides whose CD spectra as measured in TFE are documented in the literature. The observed alpha-helicity at a high TFE concentration is compared with the alpha-helicity determined by a structure prediction method that combines conformational filtering [S. Vajda, (1993) Journal of Molecular Biology, Vol. 229, pp. 125-145], and a Monte Carlo simulation [J. Figge et al. (1993) Protein Science, Vol. 2, pp. 155-164]. For the set of 11 peptides we find a correlation of 0.84 between the predicted [theta]222 values and the corresponding values observed by CD spectroscopy in a high concentration of TFE (p < 0.01). Although we generally find a good correlation at high TFE concentration between observed and predicted alpha-helicity, there are several peptides that do not follow the predicted behavior. An analysis of the TFE titration curves in one such case revealed that TFE can induce a sharp transition from a partial beta-sheet conformation to an alpha-helical conformation as the TFE concentration is increased above a critical value.

Recent improvements in circular dichroism (CD) instrumentation now allow investigators to obtain highly reliable and reproducible CD spectra in the far-UV range to near 180 nm. These advances, coupled with new computer software for spectral interpretation, allow accurate calculations of secondary structural content in proteins and polypeptides. CD is particularly reliable for the calculation of alpha-helical content. We have utilized these features to determine the propensity of alpha-helix formation in highly purified synthetic peptides corresponding to segments from proteins. We obtain CD spectra of the peptides in 90% 2,2,2-trifluoroethanol (90% TFE; an alpha-helix promoting solvent) and in 2 mM sodium dodecyl sulfate (2 mM SDS; a beta-sheet promoting solvent) to assess helix stability in these different chemical environments. Using this methodology, we demonstrate that a peptide corresponding to a biologically active segment of the human estrogen receptor forms a stable alpha-helix in both environments. In contrast, peptide segments of equal length from other steroid receptors are alpha-helical in TFE but not in 2 mM SDS. These results show that the conformation of a peptide is a function of both its amino acid sequence and the local chemical environment.