Tetraiodothyroacetic Acid Research Articles

Chemically-Modified Tetraiodothyroacetic Acid (Tetrac) Induces Cancer Cell Apoptosis and Facilitates Clearance of Apoptotic Debris (Efferocytosis)

Tetraiodothyroaetic acid (tetrac) is a derivative of L-thyroxine with anticancer properties. By multiple molecular mechanisms, tetrac and chemically-modified tetrac induce apoptosis in a variety of human cancer cells in vitro and in xenografts. The anticancer activities of tetrac are initiated at the thyroid hormone analogue receptor on the extracellular domain of plasma membrane integrin αvβ3 (PJ Davis et al., Physiol Rev 101:319-352, 2021). Induction of apoptosis in glioblastoma xenograft with chemically modified tetrac (P-bi-TAT) has yielded 90% in volume of grafts that continues after discontinuation of tetrac. In the present study, we show that human glioblastoma xenograft shrinkage in response to P-bi-TAT is associated with local appearance of phagocytic monocytes and clearance of apoptotic debris (efferocytosis). Primary culture xenograft of glioblastoma cells (GBM 052814, kindly provided by the University of Pittsburgh Medical Center, Department of Neurosurgery) and U87-luc (ATCC, Manassas, VA) xenografts were generated in 5-member groups of nude mice for each tumor cell type and for controls. Five days post-implantation, injection of animals was begun with PBS (control) or P-bi-TAT (10 mg/kg body weight). Injection was continued X21 days and animals were then maintained off-treatment for an additional 21 days. Tumors were harvested, formalin-fixed and slide-mounted, then analyzed by TUNEL assay for apoptosis and by anti-CD68 staining for monocytic macrophage content. Histologic analysis (H&E staining) was also carried out. TUNEL analysis and histopathology of both xenograft models revealed more than 90% apoptotic change with 21-days of P-bi-TAT treatment (P <0.001) and persistence of 40% apoptotic change 3 weeks post-discontinuation of drug (P<0.001 vs. end of treatment change). By H&E histology and CD68 analysis, monocytes accounted for more than 90% of the viable cells after 3 weeks’ drug treatment. Sixty percent of the end-of-treatment monocyte population persisted 3 weeks after discontinuation of P-bi-TAT (P <0.001). Histology revealed negligible cell debris after 3 weeks of drug treatment and at 3 weeks post-discontinuation of P-bi-TAT. Thus, the anticancer/pro-apoptotic action of tetrac-containing P-bi-TAT is associated with efferocytosis that contributes to the frank tumor shrinkage that results from P-bi-TAT treatment of human glioblastoma xenografts. This is the first documentation of efferocytosis regulated from the thyroid hormone analogue receptor on tumor cell integrin αvβ3.

New Thyrointegrin αvβ3 Antagonist with a Scalable Synthesis, Brain Penetration, and Potent Activity against Glioblastoma Multiforme.

We have previously reported that the αvβ3 inhibitor P-bi-TAT, a bifunctional version of the thyroid hormone metabolite tetraiodothyroacetic acid (tetrac) conjugated to polyethylene glycol (PEG) MW 4000, has excellent efficacy in a glioblastoma multiforme (GBM) mouse model. However, bioanalysis problems due to PEG polydispersity and large-scale synthesis issues led to a search for new molecules, culminating in the discovery of fb-PMT, a conjugate of tetrac and monodisperse PEG36, with a lipophilic 4-fluorobenzyl group at the opposite end of the PEG chain. fb-PMT reduces GBM tumor growth and viability by up to 98%, is suitable for large-scale synthesis, and is amenable to bioanalysis using mass spectrometry-based detection. We also showed that changes in lipophilicity at the opposite end of the PEG chain from the active tetrac component affected the proton NMR chemical shift of the tetrac moiety in D20 and brain levels of the compound after subcutaneous dosing.

Integrin receptor mediated pH-responsive nano-hydrogel based on histidine-modified poly(aminoethyl methacrylamide) as targeted cisplatin delivery system

The aim of this project was to synthesize a novel nano-hydrogel (NH) and application of this nanomaterial to targeted delivery of cisplatin. The nano-hydrogel (NH) based on random copolymerization of aminoethyl methacrylamide cross-linked with polyethylene glycol dimethacrylate, was fabricated by reversible addition-fragmentation chain transfer method. l-histidine was conjugated to the backbone of the prepared structure. Then, the complexation of cisplatin with NHs was accomplished and tetraiodothyroacetic acid was conjugated to this structure. The NH structure was characterized by FT-IR and 1H NMR analyses. Particle sizes, ζ-potential, molecular weight, and morphology of NHs were determined. The concentrations of cisplatin during drug loading and release studies were quantified using ICP-OES. The cytotoxicity of NHs was evaluated by the MTT assay in HEK293 and HT29 cell lines. Finally, the plasma profiles of the developed NHs were evaluated in Sprague Dawley rats and pharmacokinetic parameters were calculated. The proposed nano-platform is capable of responding to pH changes in the tumor microenvironment and can actively target the cancerous cells more efficiently than free cisplatin in HT29 cells and potentially can serve as an anti-cancer agent. Also, the enhancement of pharmacokinetic parameters of cisplatin loaded NH, compared with free cisplatin, revealed that the development of NH as a carrier system for cisplatin could be promising. Suitable nano size, relatively good hemo-compatibility, pH-responsive drug release pattern, active targeting via integrin receptors, and improvement of pharmacokinetic parameters are the exquisite features in this NH formulation.

Nano Diaminopropane tetrac and integrin αvβ3 expression in different cancer types: Anti-cancer efficacy and Safety

Integrins are a family of heterodimeric plasma membrane glycoproteins, which regulate tumor growth, angiogenesis, migration, and metastasis. Integrin αvβ3 has been recognized as a putative target for the treatment of several cancers. Thus, the characterization of αvβ3 distribution in different human tumors is of substantial interest in tumor targeting and its suppression. In this study we evaluated the expression of integrin αvβ3 in different cancer types to define the expression pattern in cancer model. Furthermore, we investigated the effect of novel αvβ3 antagonist Diaminopropane Tetraiodothyroacetic acid conjugated to poly (lactic-co-glycolic acid) polymer and its nanoformulated form (NDAT), on different cancer cell lines both in vitro and in xenografts. In vitro, NDAT downregulated αv and β3 monomer expression. In vivo in tumor xenografts, similarly, NDAT downregulated αv and β3. Distinct reduction in tumor weight and viability was observed in glioblastoma xenografts treated with NDAT. Furthermore, NDAT was safe and tolerable in mice treated with high doses. In conclusion, NDAT is an effective and safe inhibitor of integrin αvβ3 expression in various cancer types, which indicates its impact on the targetability and suppression of αvβ3-associated tumor functions.

Novel Thyrointegrin αvβ3 Antagonist for the Effective Management of Acute Myeloid Leukemia

Acute myeloid leukemia is one of the most aggressive malignant hematological disorders on worldwide basis. More than 19,000 new cases are estimated in the United States in 2020 (1.1% of all new cancer cases). The estimated death rate from AML is 11,180 that represent 56% of the new cases (1.8% of all cancer death), with an overall 5-year survival rate of 27.4% [1]. Current treatment regimens for AML include traditional chemotherapy, radiotherapy, allogeneic hematopoietic cell transplantation and targeted therapies for specific mutations in limited numbers of AML patients, all of whom still suffer from adverse effects and relapse. New broad spectrum effective and safe treatment options are urgently needed for the different types of AML. Our strategy focused on developing a novel targeted therapy for the thyrointegrin αvβ3 receptors that are over-expressed in leukemic cells. This receptors support adhesion, engraftment, proliferation, invasion/metastasis, and angiogenesis functions of leukemic cells. The identified thyrointegrin αvβ3 antagonist fluorobenzyl Polyethylene glycol Mono-Triazole Tetraiodothyroacetic Acid (fb-PMT) binds with high affinity and specificity. Our study showed that fb-PMT effectively suppresses the functions of AML cell line and primary cell harboring FLT3-ITD after successful engraftment in transgenic NSG-S xenograft mouse models. Daily treatment with fb-PMT at doses ranging from 1-10 mg/kg, subcutaneously for 3-4 weeks were associated with leukemogenesis regression, suppression of cancer invasion and extended survival in both models. These findings were verified using IVIS scanning and histopathological examination to evaluate the engraftment of leukemic cells in the bone marrow and other organs including, spleen, liver, lung, and brain. Furthermore, fb-PMT at 3 and 10 mg/kg, s.c. daily for 3-4 weeks exhibited significant reduction (P&amp;lt;0.001) of leukemic cell burden 74% and &amp;gt;95%, respectively. Peripheral blood smears from fb-PMT treated animals were reversed back to normal with no blast cells along with normal cell counts. Bone marrow regain its normal maturation with abundant segmented neutrophil and megakaryocytes, representing complete hematological remission along with complete suppression of leukemic cell metastasis and invasion into different organs. To evaluate the relapse after treatment, 40 mice were maintained off treatment for further 2 weeks and IVIS scans and peripheral blood smear examination were performed on animals at end of first week and at sacrifice. The fb-PMT (10 mg/kg) off treatment has shown successful maintained remission after stopping the daily treatment as confirmed using blood smear examination, IVIS scan, and histopathological examination. Additionally, safety assessments in rodents and monkeys demonstrated safety and tolerability at multiple folds above the anticipated human doses. Lastly, our genome-wide microarray screen demonstrated that fb-PMT works through the Hedgehog pathway and Il-21 receptor down-regulation as well as downregulation of several genes including, IGF2, TWIST1 and FYN oncogene, angiopoietin 1 (ANGPT1), angiopoietin-like 2 (ANGPTL2) and PIM1 oncogene KIT, HRAS, INH2, BCL, AKT1, IDH2, CDK4/6, TIMP1, VEGF, EGFR and PD-L1, KDM6A, EZH2. Collectively, preclinical findings of fb-PMT warrant its clinical investigation for the effective and safe management of AML. Reference 1. Key Statistics for Acute Myeloid Leukemia (AML). 2020; Available from: https://www.cancer.org/cancer/acute-myeloid-leukemia/about/key-statistics.html. Figure 1 Disclosures Mousa: Vascular Vision Pharma Co.: Patents &amp; Royalties.

Actions of L-thyroxine (T4) and Tetraiodothyroacetic Acid (Tetrac) on Gene Expression in Thyroid Cancer Cells.

The clinical behavior of thyroid cancers is seen to reflect inherent transcriptional activities of mutated genes and trophic effects on tumors of circulating pituitary thyrotropin (TSH). The thyroid hormone, L-thyroxine (T4), has been shown to stimulate proliferation of a large number of different forms of cancer. This activity of T4 is mediated by a cell surface receptor on the extracellular domain of integrin αvβ3. In this brief review, we describe what is known about T4 as a circulating trophic factor for differentiated (papillary and follicular) thyroid cancers. Given T4′s cancer-stimulating activity in differentiated thyroid cancers, it was not surprising to find that genomic actions of T4 were anti-apoptotic. Transduction of the T4-generated signal at the integrin primarily involved mitogen-activated protein kinase (MAPK). In thyroid C cell-origin medullary carcinoma of the thyroid (MTC), effects of thyroid hormone analogues, such as tetraiodothyroacetic acid (tetrac), include pro-angiogenic and apoptosis-linked genes. Tetrac is an inhibitor of the actions of T4 at αvβ3, and it is assumed, but not yet proved, that the anti-angiogenic and pro-apoptotic actions of tetrac in MTC cells are matched by T4 effects that are pro-angiogenic and anti-apoptotic. We also note that papillary thyroid carcinoma cells may express the leptin receptor, and circulating leptin from adipocytes may stimulate tumor cell proliferation. Transcription was stimulated by leptin in anaplastic, papillary, and follicular carcinomas of genes involved in invasion, such as matrix metalloproteinases (MMPs). In summary, thyroid hormone analogues may act at their receptor on integrin αvβ3 in a variety of types of thyroid cancer to modulate transcription of genes relevant to tumor invasiveness, apoptosis, and angiogenesis. These effects are independent of TSH.

Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

Background: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. Methods: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3’,5,5’-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. Results: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-β1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. Conclusions: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-β expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Nongenomic Actions of Thyroid Hormone: The Integrin Component.

The extracellular domain of plasma membrane integrin αvβ3 contains a cell surface receptor for thyroid hormone analogues. The receptor is largely expressed and activated in tumor cells and rapidly dividing endothelial cells. The principal ligand for this receptor is l-thyroxine (T4), usually regarded only as a prohormone for 3,5,3'-triiodo-l-thyronine (T3), the hormone analogue that expresses thyroid hormone in the cell nucleus via nuclear receptors that are unrelated structurally to integrin αvβ3. At the integrin receptor for thyroid hormone, T4 regulates cancer and endothelial cell division, tumor cell defense pathways (such as anti-apoptosis), and angiogenesis and supports metastasis, radioresistance, and chemoresistance. The molecular mechanisms involve signal transduction via mitogen-activated protein kinase and phosphatidylinositol 3-kinase, differential expression of multiple genes related to the listed cell processes, and regulation of activities of other cell surface proteins, such as vascular growth factor receptors. Tetraiodothyroacetic acid (tetrac) is derived from T4 and competes with binding of T4 to the integrin. In the absence of T4, tetrac and chemically modified tetrac also have anticancer effects that culminate in altered gene transcription. Tumor xenografts are arrested by unmodified and chemically modified tetrac. The receptor requires further characterization in terms of contributions to nonmalignant cells, such as platelets and phagocytes. The integrin αvβ3 receptor for thyroid hormone offers a large panel of cellular actions that are relevant to cancer biology and that may be regulated by tetrac derivatives.

Dual Targeting of Norepinephrine Transporter (NET) Function and Thyrointegrin αvβ3 Receptors in the Treatment of Neuroblastoma.

Therapeutic targeting of the norepinephrine transporter (NET) function with benzylguanidine (BG), conjugated with the high-affinity thyrointegrin αvβ3 antagonist triazole tetraiodothyroacetic acid, TAT, via noncleavable bonding to poly(ethylene glycol) (PEG400) (P) might allow for effective treatment options in neuroblastoma. BG-P-TAT is a dual-targeting agent, targeting the NET function and the thyrointegrin αvβ3 receptors that are overexpressed in neuroblastoma and other neuroendocrine tumors. Various cancer cells and actively dividing tumor-endothelial cells express the thyrointegrin αvβ3 receptors. In this work, the novel compound BG-P-TAT was synthesized and evaluated in the neuroblastoma SK-N-FI cell line for improved targeting and to offer a new strategy for patients with neuroblastoma. BG-P-TAT demonstrated significant suppression of neuroblastoma tumor progression, growth, and viability in a dose-dependent manner. In conclusion, BG-P-TAT represents a potential lead candidate for the treatment of neuroblastoma and other neuroendocrine tumors.

A Mass Spectrometry-Based Panel of Nine Thyroid Hormone Metabolites in Human Serum.

While thyroxine (T4), 3,3',5-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals. Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards. The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9-11.3 ng/dL), 3-monoiodothyronine (0.06 --0.41 ng/dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3'-diiodothyronine (0.25--0.77 ng/dL), T3 (66.4--129.9 ng/dL), rT3 (15.0--64.1 ng/dL), T4 (4.3--10.0 µg/dL), triac/3,3',5-triiodothyroacetic acid (not detected), and tetrac/3,3',5,5'-tetraiodothyroacetic acid (2.2--27.2 ng/dL). A broad dynamic concentration range exists among the nine THMs. This method should help to develop a better understanding of the clinical relevance of other THMs, as well as an understanding of thyroid hormone metabolism in health and disease.

Competitive binding assays for measuring the binding affinity of thyroid-disrupting chemicals for integrin αvβ3

Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin αvβ3. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin αvβ3. Based on different probes, two assays were modified: a fluorescence competitive binding assay and a radioligand competitive binding assay. The chemicals tested included the known TH, 3,3′,5,5′-tetraiodo-l-thyronine (T4); a deaminated analog of T4, tetraiodothyroacetic acid (tetrac); and phthalate esters (PAEs). The relative binding potency of T4 was studied, and the concentration required to displace 50% of the ligands from their receptors (RIC50) of T4 was 4.9 × 105 and 9.7 × 104 nM for the fluorescence and radioligand competitive binding assays, respectively, suggesting that the radioligand competitive binding assay might be more sensitive for the evaluation of the binding affinity for integrin αvβ3. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin αvβ3 in the following order of potency: DnBP > DEHP > BBP tested by the radioligand competitive binding assay. A docking simulation of each of the three PAEs with integrin αvβ3 confirmed the calculated binding energies, which had a strong positive relationship with the log RIC20 values of the 3 PAEs (R = 0.99, p < 0.001). The present study shows that the established radioligand competitive binding assay could be used as a valuable tool for quantifying the affinity of TDCs for integrin αvβ3.

Development and characterization of a human cell line-based transactivation assay to assess thyroid EDCs

Thyroid hormones (THs) are one of the most important hormones, playing key roles in the regulation of various physiological functions. Although THs have important function in human, in vitro test methods based on human cells are currently insufficient to effectively screen and test TH-related endocrine disrupting chemicals (EDCs).We established a TH agonist TA assay using the adenocarcinomic human alveolar basal epithelial cell line A549 to test and screen potential TH agonists. To establish the TH agonist TA assay, a TRE-secNluc-IRES-EGFP reporter cassette was constructed and transfected into the A549 cell line using a retrovirus. We evaluated the TH agonistic properties of several chemicals which were tested by existing thyroid agonists testing method (OECD GD 207). Comparing the results of the TH agonist TA assay with the OECD GD 207, T3, T4, tiratricol, and tetrac (natural TH and 3,3′,5,5′-tetraiodothyroacetic acid derivatives), which are TH agonists according to the OECD GD 207, also tested positive in the TH agonist TA assay using the A549 cell line. These results suggested that the TH agonist TA assay developed in this study using a human cell line can provide the information, such as accuracy and specificity to TH agonistic properties of chemicals.

Triiodothyronine activated extranuclear pathways upregulate adiponectin and leptin in murine adipocytes

Adiponectin and leptin, important for metabolic regulation, are synthesized and secreted by adipose tissue and are influenced by triiodothyronine (T3) that activates the MAPK/ERK and integrin αVβ3 pathways, modulating gene expression. Adipocytes were treated with T3 (10 nM), for 1 h, in the absence or presence of PD98059 (PD) and tetraiodothyroacetic acid (Tetrac), which are pathways inhibitors. The cells were incubated with Adipo Red/Oil Red O reagents, and intracellular lipid accumulation [glycerol and triacylglycerol (TAG)], MTT, 8-hydroxideoxyguanosine (8-OH-dG), and mRNA and protein expression were assessed. T3 increased leptin mRNA and protein expression, and, in contrast, there was a decrease in the Tetrac + T3 group. Adiponectin mRNA expression was not altered by T3, though it had increased its protein expression, which was terminated by inhibitors PD + T3 and Tetrac + T3. However, T3 did not alter PPARγ protein expression, lipid accumulation, TAG, glycerol, and DNA damage, but PD + T3 and Tetrac + T3 reduced these parameters. T3 activated the MAPK/ERK pathway on adipocytes to modulate the adiponectin protein expression and integrin αvβ3 to alter the leptin gene expression.

Triazole Modified Tetraiodothyroacetic Acid Conjugated to Polyethylene Glycol: High Affinity Thyrointegrin αvβ3 Antagonist with Potent Anticancer Activities in Glioblastoma Multiforme.

Discovery of bioactive molecules that target integrins has implicated their role in tumor angiogenesis, tumor growth, metastasis, and other pathological angiogenesis processes. Integrins are members of a family of cell surface receptors that play a critical role in the angiogenesis process. Tetraiodothyroacetic acid (tetrac), a deaminated derivative of l-thyroxine (T4), is a "thyrointegrin" antagonist that blocks the actions of l-triiodothyronine (T3) and T4 with an interaction site that is located at or near the RGD recognition site identified on integrin αvβ3's binding pocket (thyrointegrin αvβ3 receptors). We have enhanced the biological activity of a tetrac-based inhibitor via significantly improving its αvβ3 receptor binding affinity by introducing a triazole ring on the outer ring of tetrac and covalently conjugating to polymer to increase the product's hydrophilicity via PEGylation. The product, P-bi-TAT, was restricted from nuclear translocation and demonstrated high blood brain barrier permeability and retention in contrast to the non-PEG conjugated derivative. Results of biological activity indicated that this macromolecule new chemical entity P-bi-TAT has greater than 400-fold potent integrin αvβ3 affinity versus the parent compound tetrac and has potent anticancer/anti-angiogenesis efficacy against glioblastoma multiforme (GBM). P-bi-TAT administered subcutaneously once daily for 21 days at 1-10 mg/kg mouse body weight resulted in a dose-dependent suppression of GBM tumor growth and viability as monitored with IVIS imaging (P < 0.001). GBM tumors had >95% volume loss and maximal loss of GBM cell viability during the 21 days ON-treatment experiment as well as in the 21 days ON followed by 21 days OFF-treatment experiment (P < 0.001). In conclusion, P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM.

Synthesis and characterization of 64Cu- and Cy5.5-labeled tetraiodothyroacetic acid derivatives for tumor angiogenesis imaging

It was previously reported that tetraiodothyroacetic acid (tetrac) inhibits angiogenesis by binding to the cell surface receptor for thyroid hormone on integrin αVβ3. Therefore, we synthesized and evaluated two 64Cu-labeled tetrac derivatives and a Cy5.5-labeled tetrac derivative for tumor angiogenesis imaging. Tetrac was structurally modified to conjugate with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) via its hydroxy or carboxylic acid end, and the resulting DOTA-conjugated tetrac derivatives were then labeled with 64Cu. Tetrac was also conjugated with Cy5.5 via its carboxylic acid end. All three tetrac derivatives (1–3) exhibited greater inhibitory activity than tetrac against endothelial cell tube formation. The U87MG cell binding of [64Cu]2 showed a time-dependent increase over 24 h and it was inhibited by 38% at 4 h in the presence of tetrac, indicating specificity of [64Cu]2 to the thyroid hormone receptor site on integrin αVβ3. Positron emission tomography (PET) images of U87MG tumor-bearing mice injected with [64Cu]1 and [64Cu]2 revealed that high radioactivity accumulated in the tumors, and that the tumor uptake and tumor-to-nontarget uptake ratio were higher in small tumors than in large tumors. In addition, the Cy5.5-labeled tetrac derivative (3) displayed a strong near-infrared (NIR) signal in the tumors. Taken together, these results suggest that these ligands hold promise as imaging agents for visualization of tumor angiogenesis.

Molecular Functions of Thyroid Hormone Signaling in Regulation of Cancer Progression and Anti-Apoptosis.

Several physiological processes, including cellular growth, embryonic development, differentiation, metabolism and proliferation, are modulated by genomic and nongenomic actions of thyroid hormones (TH). Several intracellular and extracellular candidate proteins are regulated by THs. 3,3,5-Triiodo-L-thyronine (T3) can interact with nuclear thyroid hormone receptors (TR) to modulate transcriptional activities via thyroid hormone response elements (TRE) in the regulatory regions of target genes or bind receptor molecules showing no structural homology to TRs, such as the cell surface receptor site on integrin αvβ3. Additionally, L-thyroxine (T4) binding to integrin αvβ3 is reported to induce gene expression through initiating non-genomic actions, further influencing angiogenesis and cell proliferation. Notably, thyroid hormones not only regulate the physiological processes of normal cells but also stimulate cancer cell proliferation via dysregulation of molecular and signaling pathways. Clinical hypothyroidism is associated with delayed cancer growth. Conversely, hyperthyroidism is correlated with cancer prevalence in various tumor types, including breast, thyroid, lung, brain, liver and colorectal cancer. In specific types of cancer, both nuclear thyroid hormone receptor isoforms and those on the extracellular domain of integrin αvβ3 are high risk factors and considered potential therapeutic targets. In addition, thyroid hormone analogs showing substantial thyromimetic activity, including triiodothyroacetic acid (Triac), an acetic acid metabolite of T3, and tetraiodothyroacetic acid (Tetrac), a derivative of T4, have been shown to reduce risk of cancer progression, enhance therapeutic effects and suppress cancer recurrence. Here, we have reviewed recent studies focusing on the roles of THs and TRs in five cancer types and further discussed the potential therapeutic applications and underlying molecular mechanisms of THs.

Abstract 1300: Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme

Abstract Background: The proliferative and pro-angiogenic actions mediated by L-thyroxine, pro-inflammatory and all known growth factors in glioblastoma multiforme (GBM) are initiated at cell surface thyrointegrin αvβ3 receptors for thyroid hormone on the extracellular domain of integrin αvβ3. Thyrointegrin αvβ3 receptors are over-expressed on cancer and rapidly dividing blood vessel cells, but quiescent on normal cells. A macromolecule Polyethylene glycol-conjugated bi-TriAzole Tetraiodothyroacetic acid (P-bi-TAT) acts with high affinity (Ki 3.1 nM) and specificity for the thyrointegrin αvβ3 receptors without any significant nuclear translocation. Methods: In the present studies, three primary human GBM cells and U87 glioma cell line were implanted orthotopically or subcutaneously (s.c.) into nude mice that were treated daily for up to 21 days with P-bi-TAT at different doses. Additionally, preclinical Pharmacokinetic and Safety assessments were carried out in multiple species. Results: Pharmacokinetic profiles indicated once-a-day dosing, and safety assessment studies demonstrated high safety and tolerability at 100-fold over the anticipated human equivalent dose. Fluorescence labeled P-bi-TAT administered s.c. demonstrated high biodistribution to GBM tumor in the brain that was comparable to peripherally implanted tumor. P-bi-TAT administered daily for 10-21 days s.c. at 0.3-10 mg/kg resulted in a dose-dependent suppression of GBM tumor growth and viability monitored with IVIS imaging (p &amp;lt;0.001). Histopathological analysis of tumors revealed 95% loss of the vascularity of treated tumors at 10 days (p &amp;lt;0.001) along with extensive cellular necrosis and apoptosis, without intratumoral hemorrhage. GBM tumors had a 97% volume loss and maximal loss of GBM cell viability during a subsequent 22 days off-treatment period (p &amp;lt; 0.001). Genomic micro-array studies with human primary GBM revealed that over 30 pathways relevant to the progression of GBM are modulated by P-bi-TAT. Conclusions: P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM. Citation Format: Shaker A. Mousa, Thangirala Sudha, Kavitha Godugu, Mehdi Rajabi, Stewart Sell, Paul J. Davis. Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1300.

PP#149 - Preliminary study on 64Cu-labeled tetraiodothyroacetic acid derivative for tumor angiogenesis imaging

PP#149 - Preliminary study on 64Cu-labeled tetraiodothyroacetic acid derivative for tumor angiogenesis imaging

RETRACTED ARTICLE: Pharmacokinetics, Biodistribution, and Anti-Angiogenesis Efficacy of Diamino Propane Tetraiodothyroacetic Acid-conjugated Biodegradable Polymeric Nanoparticle

The anti-angiogenic agent, diamino propane tetraiodothyroacetic acid (DAT), is a thyro-integrin (integrin αvβ3) antagonist anticancer agent that works via genetic and nongenetic actions. Tetraiodothyroacetic acid (tetrac) and DAT as thyroid hormone derivatives influence gene expression after they transport across cellular membranes. To restrict the action of DAT to the integrin αvβ3 receptors on the cell surface, we used DAT-conjugated PLGA nanoparticles (NDAT) in an active targeting mode to bind to these receptors. Preparation and characterization of NDAT is described, and both in vitro and in vivo experiments were done to compare DAT to NDAT. Intracellular uptake and distribution of DAT and NDAT in U87 glioblastoma cells were evaluated using confocal microscopy and showed that DAT reached the nucleus, but NDAT was restricted from the nucleus. Pharmacokinetic studies using LC-MS/MS analysis in male C57BL/6 mice showed that administration of NDAT improved the area under the drug concentration curve AUC(0–48 h) by 4-fold at a dose of 3 mg/kg when compared with DAT, and Cmax of NDAT (4363 ng/mL) was 8-fold greater than that of DAT (548 ng/mL). Biodistribution studies in the mice showed that the concentrations of NDAT were higher than DAT/Cremophor EL micelles in heart, lung, liver, spleen, and kidney. In another mouse model using female NCr nude homozygous mice with U87 xenografts, tumor growth was significantly decreased at doses of 1 and 3 mg/kg of NDAT. In the chick chorioallantoic membrane (CAM) assay used to measure angiogenesis, DAT (500 ng/CAM) resulted in 48% inhibition of angiogenesis levels. In comparison, NDAT at low dose (50 ng/CAM) showed 45% inhibition of angiogenesis levels. Our investigation of NDAT bridges the study of polymeric nanoparticles and anti-angiogenic agents and offers new insight for the rational design of anti-angiogenic agents.

Thyroxine promotes lung cancer growth in an orthotopic mouse model.

Thyroid hormones are important for physiology and homeostasis. In addition to nuclear thyroid hormone receptors, the plasma membrane protein integrin αvβ3 has been recognized as a receptor for both thyroxine (T4) and triiodothyronine (T3). Here, we studied whether thyroid hormone promotes growth of murine lung cancer via αvβ3 in vivo. Murine Lewis lung carcinoma cells (3LL), stably transfected with luciferase, were injected into mouse lungs. Tumor growth in untreated mice was compared to hypothyroid mice and hypothyroid mice treated with T3 or T4 with or without the αvβ3 inhibitor 3,5,3',5'-tetraiodothyroacetic acid (Tetrac). Tumor progression was determined by serial in vivo imaging of bioluminescence emitted from the tumor. Tumor weight was recorded at the end of the experiment. Neoangiogenesis was determined by immunohistochemistry for CD31. Tumor growth was reduced in hypothyroidism and increased by T4 treatment. Strikingly, only T4 but not T3 treatment promoted tumor growth. This T4 effect was abrogated by the αvβ3 inhibitor Tetrac. Tumor weight and neoangiogenesis were also significantly increased only in T4-treated mice. The T4 effect on tumor weight and neoangiogenesis was abolished by Tetrac. In vitro, T4 did not stimulate 3LL cell proliferation or signaling pathway activation. We conclude that T4 promotes lung cancer growth in this orthotopic mouse model. The tumor-promoting effect is mediated via the plasma membrane integrin αvβ3 and increased neoangiogenesis rather than direct stimulation of 3LL cells. These data suggest that such effects of levothyroxine may need to be considered in cancer patients on T4 substitution.