Thyrointegrin receptor

In HT-29 and HCT116 cells with varying K-RAS status, Tetrac (0.01-1 μM; 2-6 d) causes anti-proliferation[3]. In HT-29 and HCT116 cells, Tetrac (0.1 μM; 30 min) suppresses ERK1/2 activation[3]. THBS1 and CASP2 are promoted in expression while CCND1 and c-Myc are inhibited by Tetrac (0.1 μM; 24 h)[3].

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The naturally occurring T4 analogue tetrac blocks the pro-angiogenic actions of thyroid hormones at the integrin receptor, in addition to agonist-independent anti-angiogenic effects. Tetrac reduces endothelial cell proliferation, migration and tube formation through a reduction in the transcription of vascular growth factors/growth factor receptors, hypoxia-inducible factor-1α, pro-angiogenic cytokines and a number of other pro-angiogenic genes, while at the same time stimulating the expression of endogenous angiogenesis inhibitors. It further modulates vascular growth factor activity by disrupting the crosstalk between integrin αvβ3 and adjacent growth factor receptors. Moreover, tetrac disrupts thyroid hormone-stimulated tumour recruitment, differentiation and the pro-angiogenic signalling of tumour stroma-associated mesenchymal stem cells. Tetrac affects tumour-associated angiogenesis via multiple mechanisms and interferes with other cancer cell survival pathways. In conjunction with its low toxicity and high tissue selectivity, tetrac is a promising candidate for clinical application.[1]

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Both Tetrac and NDAT Bind to Cell Surface Integrin αvβ3 in Colorectal Cancer Cells. Tetrac and NDAT Induce Anti-proliferation in Colorectal Cancer Cells With Different K-RAS Status. NDAT and Tetrac Modulate Expression of Different Genes in Colorectal Cancer Cells. [3]
We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination. [3]

Tetrac (35 μg; po for 40 days) prevents the growth, inoculation, and expression of integrins in tumors in mice[4].
Tetrac Delayed the Onset of Ocular Melanoma in the Albino Mouse Model [4]
The B16F10 cells serve as a valid platform to examine the thyroid-hormone-αvβ3 axis in vivo, due to high expression of this specific integrin (Supplementary Figures S1A–D). In contrast, the B16LS9 cells express low levels of αvβ3 (Supplementary Figures S1E–H). We have recently established in a B16F10 ocular melanoma model that the hypothyroid environment enhances survival of mice inoculated with the B16F10 cells, while hyperthyroidism resulted with shorter survival. The unexpected observation that albino mice, when inoculated intraocularly with melanoma cells, do not develop metastasis and exhibit an extended survival, led us to exploit these models to study the potential of thyroid-hormone-αvβ3 inhibitors in delaying the onset of ocular melanoma. One such inhibitor that has been shown in numerous in vitro and in vivo studies to inhibit thyroid hormone binding to αvβ3 integrin is Tetrac and this agent was selected for the next study.
The subretinal space of the right eye of BALB/c mice was inoculated with aliquots of 102 B16F10 or B16LS9 cells/1 μL PBS (inoculation day or day 0) using a transconjunctival approach, as previously described. There were no cases of cell reflux following tumor inoculation and the subconjunctival space remained free of tumor cells. On the same day, each experimental tumor model was divided into mice given tap water (Control group) or drinking water containing Tetrac. The mice were monitored on a daily basis and the first sign of intraocular tumor growth was recorded. The experiment design, including the number of mice in each group is indicated in Figure 4. The mice were followed-up and recorded for tumor initiation for 37–40 days.
Mice were sacrificed after 90 days from study initiation, at which point eyes were enucleated and sent for pathological and immunohistochemical processing, including S100 (a melanoma marker) analysis and αvβ3 expression. Results indicate that in both the B16F10 (Figure 6A) and the B16LS9 (Figure 6B) cell models, the intraocular tumors were positive for S100 immunostaining, confirming the presence of melanoma cells. In accord with the flow-cytometry results, B16F10 (Figure 6A), but not B16LS9 (Figure 6B), were positively immunostained by the anti-integrin antibody. Lastly, in the B16F10 mice model, Tetrac treatment clearly indicated a reduced level of S-100 and integrin staining, suggesting an inhibitory effect on tumor inoculation, growth and integrin expression.

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Compounds were added at 10 µg/10 µL to 200 µL Matrigel, which contains murine vascular growth factors, and injected subcutaneously in the mice, and the control group received Matrigel only at 200 µL + 10 µL vehicle/implant. There were four subcutaneous implants per mouse (2 implants on the right and 2 implants on left backsides of the animal). The Matrigel liquid plug at 4 °C became a solid matrix at 37 °C for the sustained release of the compounds over 14 days. At day 14 post-Matrigel implant, all animals were sacrificed and vessel formation was quantified by measuring the hemoglobin concentration in the Matrigel plug according to the Drabkin method27 using spectrophotometry. Briefly, Matrigel plugs were placed into a 0.5 mL tube containing double distilled water and then homogenized for 5–10 min. The samples were centrifuged at 1700×g for 10 min and the supernatants collected. Fifty μL of supernatant was mixed with 50 μL of Drabkin’s reagent and allowed to sit at room temperature for 15–30 min, after which it was placed in a 96-well plate and absorbance measured at 540 nm with a Microplate Manager ELISA reader. Hb concentration was expressed as mg/dL based on comparison with a standard curve. As shown in Fig. 2, in the Matrigel plug experiment, Tetrac and the 5 derivatives significantly inhibited angiogenesis and results are presented as mean ± standard error means (S.E.M.) or ±standard deviation (S.D.) as given in Figure legends. Control and experimental groups were compared and statistically analyzed with ANOVA and student’s t-test using SigmaPlot 10.0 software. Differences between control and experimental end points were considered statistically significant if P < 0.01. Tetrac analog compounds also blocked the FGF-induced angiogenesis in a comparable fashion to tetrac. Any modulations on either or both sides of tetrac did not greatly affect the antagonist effect of the parent compound. Anti-angiogenesis activity of 10a might be related to the presence of the alkyne function group on its structure, which may be modified in our further structure-activity studies. Also, anti-angiogenesis activity of compounds 10d and 10e were very interesting because of the presence of the phthalimide portion of their structures, which made them similar to thalidomide (Fig. 3), a known anti-angiogenesis inhibitor that can inhibit angiogenesis induced by angiogenic cytokines such as basic fibroblast growth factor (bFGF) in the rabbit cornea and VEGF in the corneas of mice. [5]

Cell Proliferation Assay[3]
Cell Types: HT-29 and HCT116 cells
Tested Concentrations: 0.01, 0.1, 1 μM
Incubation Duration: 0, 2, 4, 6 days
Experimental Results: Induced anti-proliferation of K- RAS wild-type colorectal cancer cells.

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Western Blot Analysis[3]
Cell Types: HT-29 and HCT116 cells
Tested Concentrations: 0.1 μM
Incubation Duration: 30 min
Experimental Results: Inhibited constitutively activated ERK1/2, and this inhibition can remove by anti-integrin αvβ3 antibody pretreatment.

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Anti-proliferative effects of Tetrac and NDAT were defined in a well-established perfusion bellows cell culture system. At the outset, 5 × 107 cells were seeded in perfusion bellows cell culture system and incubated at 37°C overnight. Then polymer flakes were harvested, trypsinized, and cells were collected and counted. The number of original cells attached to flakes was 0.5 × 107 cells/bottle. Cell cultures were refreshed with 1% stripped FBS-containing medium. Tetrac or NDAT was added in a medium bottle to the final concentrations indicated in the Results section. Specific concentration of tetrac and NDAT were chosen according to the physiological concentration of T4 (10−7 M) as described previously. The samples of cell-bearing flakes were then treated as indicated, and cells were harvested at timeframe indicated, trypsinized, and collected for counting. The cell cultures were refreshed with 10% hormone-stripped FBS containing medium. [3]

Animal/Disease Models: Wild-type male Balb/ C mice aged 8 weeks are inoculated with 102B16F10 or B16LS9 cells[4]
Doses: 35 μg per day
Route of Administration: Po (added to the drinking water) daily for 40 days
Experimental Results: Delayed the onset of ocular melanoma. diminished the S-100 and Integrain staining level in the B16F10 mice model.

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Experimental Groups and Inoculation of Tumor Cells [4]
For model optimization in the C57Bl/6 mice, the subretinal space (i.e., the choroid) of each mouse's right eye was first inoculated with aliquots of 5 × 105 B16F10 cells (n = 8 mice). Next, the same cells were inoculated at decreasing concentration (104, 103, and 102 cells, total 15 mice, n = 5 for each concentration). For assessing the Balb/C Albino mice model, the subretinal space of each mouse's right eye was inoculated with aliquots of 102B16F10 or B16LS9 cells/1 μL PBS (n = 5 for each cell type), using a transconjunctival approach as previously described, allowing the inoculated cells to remain in the eye. Mice were anesthetized with a mixture of ketamine and xylazine (120 mg/kg ketamine, 10 mg/kg xylazine), and the experimental eye was desensitized by a drop of oxybuprocaine. Under a dissecting microscope, a 30-gauge needle was inserted ~1 mm posterior to the limbus through the conjunctiva and sclera and into the subretinal space. The tip of a 10 μL glass syringe with a 32-gauge blunt needle was introduced into the subretinal space via the needle track, and a 1 μL suspension of tumor cells was then injected into the eyes of the animals. No cells were inoculated until the needle tip was inside the eye, no tumor cell reflux occurred, and the subconjunctival space remained free of tumor cells. For the final interventional study, the subretinal space of each Balb/C Albino mouse's right eye was inoculated with aliquots of 102B16F10 or B16LS9 cells in 1 μL PBS. For each experimental model, mice were given drinking water with 35 μg Tetrac per day (n = 16 mice in the B16F10 model and n = 16 in the B16LS9 model), whereas the control group mice were given only polyethylene glycol dissolved in water (n = 15 mice in the B16F10 model and n = 13 mice in the B16LS9 model). Drinking water was exchanged on daily basis.
Tetrac was dissolved in 0.04 N KOH 4% propylene glycol (PG) solution to a concentration of 1 mg/1 mL.

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To investigate the structure-activity relationships, compounds 10a–10e were tested for their ability to block angiogenesis in comparison to Tetrac. Inhibition of angiogenesis was measured in an in vivo study with an assay that exposed test angiogenesis-inducing and inhibitory compounds to cold liquid Matrigel® Matrix, a mix of several proteins that, after subcutaneous injection into mice, solidified and permitted the formation of new blood vessels. Female C57/B6 mice aged 5–6 weeks, with body weights of 20 g, were maintained under specific pathogen-free conditions and housed 4 animals per cage, under controlled conditions of temperature (20–24 °C) and humidity (60–70%) and a 12 h light/dark cycle. Water and food were provided ad libitum. Mice were allowed to acclimatize for 5 d prior to the start of experiments. Mice were dived into 7 groups (3 mice per group): control, Tetrac, and 5 tetrac derivatives.

[1]. Tetrac as an anti-angiogenic agent in cancer. Endocr Relat Cancer. 2019 Jun 1; 26(6):R287-R304.

[2]. Nongenomic Actions of Thyroid Hormone: the Integrin Component. Physiol Rev. 2020 Jun 25.

[3]. Tetrac and NDAT Induce Anti-proliferation via Integrin αvβ3 in Colorectal Cancers With Different K-RAS Status. Front Endocrinol (Lausanne). 2019 Mar 12; 10:130.

[4]. Tetrac Delayed the Onset of Ocular Melanoma in an Orthotopic Mouse Model. Front Endocrinol (Lausanne). 2019 Jan 8; 9:775.

[5]. Synthesis of new analogs of tetraiodothyroacetic acid (tetrac) as novel angiogenesis inhibitors for treatment of cancer. Bioorg Med Chem Lett. 2018 Apr 15;28(7):1223-1227.

3,3',5,5'-Tetraiodothyroacetic acid (TETA) is a monocarboxylic acid, meaning thyroacetic acid has four iodine substituents at the 3, 3', 5, and 5' positions. It is a thyroid hormone, a human metabolite, and an inducer of apoptosis. It is an iodophenol, a 2-halophenol, a monocarboxylic acid, and an aromatic ether. Thyroid hormones T3 and T4 have been shown to be pro-angiogenic hormones, playing a significant role in cancer treatment. Endogenous circulating hormone levels may contribute to promoting cancer progression and limiting the efficacy of anticancer therapies, but clinical data remain unclear. The ability of thyroid hormones to regulate angiogenesis is mediated through a non-classical mechanism originating from the cell surface receptor integrin αvβ3. This integrin is primarily expressed in tumor cells, proliferating endothelial cells, and tumor stromal-associated cells, highlighting its potential importance in angiogenesis and tumor biology. The thyroid hormone/integrin αvβ3 signaling pathway activates intracellular pathways typically associated with angiogenesis, mediated by classical pro-angiogenic molecules such as vascular endothelial growth factor (VEGF). In addition to its agonist-independent anti-angiogenic effects, the naturally occurring T4 analog tetrahydropyridine (Tetrac) can also block the pro-angiogenic effects of thyroid hormones on integrin receptors. Tetrahydropyridine reduces the proliferation, migration and tubular formation of endothelial cells by reducing the transcription of angiogenic factor/growth factor receptor, hypoxia-inducible factor-1α, pro-angiogenic cytokines and many other pro-angiogenic genes, while stimulating the expression of endogenous angiogenesis inhibitors. It further modulates angiogenic factor activity by disrupting the interaction between integrin αvβ3 and neighboring growth factor receptors. In addition, tetrahydropyridine can interfere with thyroid hormone-stimulated tumor recruitment, differentiation and pro-angiogenic signal transduction of tumor matrix-associated mesenchymal stem cells. Tetrahydropyridine affects tumor-associated angiogenesis through multiple mechanisms and interferes with other cancer cell survival pathways. Combined with its low toxicity and high tissue selectivity, tetrahydropyridine is a promising candidate drug for clinical application. [1]

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The extracellular domain of plasma membrane integrin αvβ3 contains a cell surface receptor for a thyroid hormone analog. This receptor is highly expressed and activated in tumor cells and rapidly dividing endothelial cells. Its primary ligand is levothyroxine (T4), often considered a precursor hormone of 3,5,3'-triiodo-levothyroxine (T3). T3 is a hormone analog that expresses thyroid hormones in the nucleus via a nuclear receptor independent of the structure of integrin αvβ3. Upon binding to the thyroid hormone integrin receptor, T4 regulates the division of cancer cells and endothelial cells, tumor cell defense pathways (e.g., anti-apoptosis), and angiogenesis, and promotes tumor metastasis, radioresistance, and chemoresistance. Its molecular mechanisms involve signal transduction via mitogen-activated protein kinases and phosphatidylinositol 3-kinases, differential expression of multiple genes associated with these cellular processes, and regulation of the activity of other cell surface proteins (e.g., angiogenesis factor receptors). Tetraiodothyroacetic acid (Tetrac) is a derivative of T4 that competitively binds to integrins. In the absence of T4, Tetraiodothyroacetic acid (Tetrac) and its chemically modified derivatives also exhibit anticancer activity, ultimately leading to alterations in gene transcription. Both unmodified and chemically modified Tetrac inhibited the growth of xenograft tumors. The mechanism of action of this receptor on non-malignant cells (such as platelets and phagocytes) needs further investigation. The integrin αvβ3 receptor of thyroid hormones has a variety of cellular activities related to cancer biology, which may be regulated by Tetrac derivatives. [2]

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Colorectal cancer is a serious medical problem in Taiwan. New and effective treatments are urgently needed. Screening for promising anticancer drugs and transitioning from preclinical studies to clinical trials is often challenging. Deaminothyroid hormone analogs (tetraiodothyroacetic acid, Tetrac) and their nanoparticle analogs (NDAT) have been shown to have antiproliferative activity in vitro and in xenograft models of various tumors, including colorectal cancer. However, the mechanism by which tetrahydropyridine (terac) and nidatetinib (NDAT) induce the inhibition of colorectal cancer cell proliferation has not been fully elucidated. We investigated the mechanism of action of tetrahydropyridine and nidatetinib in colorectal cancer cells using a perfusion corrugated cell culture system. The system can efficiently and on a large scale screen the mechanism of action of drugs on tumor cells. Although the expression of integrin αvβ3 in K-RAS wild-type colorectal cancer HT-29 cells is much lower than that in K-RAS mutant HCT116 cells, HT-29 cells are more sensitive to both tetrahydropyridine and nitratetinib. The results also showed that both tetrahydropyridine and nitratetinib can bind to integrin αvβ3 on the surface of tumor cells, and the two drugs may have different anti-proliferative mechanisms in colorectal cancer cells. K-RAS status seems to play an important role in the drug resistance that may be encountered when using this combination therapy. [3]

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Ocular melanoma is the most common primary intraocular malignant tumor in adults, but its research has been hampered by the limited number of in vivo models. We established an ocular melanoma model by a series of experiments involving intraocular injection of melanoma cells into the eyes of mice. Inoculation with 5 × 10⁵ B16F10 cells led to rapid tumor growth, widespread lung metastasis, and shortened animal survival, while inoculation with 10² cells was sufficient to induce intraocular tumor growth and prolong animal survival. To improve tumor visualization, we inoculated 10² melanoma cells (B16F10 or B16LS9) into the eyes of Balb/C albino mice. These mice developed intraocular tumors without metastasis and had prolonged survival. Next, we investigated the potential of inhibitors of the thyroid hormone-αvβ3 integrin signaling pathway in the treatment of ocular melanoma. By using the thyroid hormone derivative tetraiodothyronine (Tetrac), tumor development was delayed in the B16F10 (integrin-positive) cell group compared to the untreated group; while in the B16LS9 (integrin-negative) cell group, the tumor incidence was similar in the experimental and control groups. In conclusion, after optimization, we established a mouse model of ocular melanoma. This model has a broad therapeutic window and can serve as a platform for studying various drugs and other treatment methods. [4]

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In angiogenesis, integrins, as members of the cell surface transmembrane receptor family, play a crucial role in angiogenesis and the local release of angiogenic factors. Thyroid hormones, such as levothyroxine (T4) and 3,5,3'-triiodothyronine (T3), promote angiogenesis and tumor cell proliferation through the integrin αvβ3 receptor. Tetraiodothyronine (TET, a deamination derivative of T4) is located at or near the arginine-glycine-aspartate (RGD) recognition site in the integrin αvβ3 binding pocket and is a thyroid integrin receptor antagonist that blocks the effects of T3 and T4 as well as angiogenesis mediated by various growth factors. In this study, a series of novel TTE analogues were synthesized by modifying the phenolic hydroxyl group of TTE and their anti-angiogenic activity was tested using a mouse Matrigel angiogenesis model. Pharmacological activity results showed that TTE can be modified in various ways while maintaining its anti-angiogenic activity. In summary, in order to find new angiogenesis inhibitors, we synthesized a series of novel tetraiodothyronine analogs by modifying the phenolic hydroxyl group of tetraiodothyronine. We analyzed their structure-activity relationship and tested their anti-angiogenic activity using a mouse Matrigel angiogenesis model. The design and development of novel angiogenesis inhibitors have been proven to be therapeutic targets for a variety of tumor types. Our results showed that the phthalimide-modified tetraacetyl analogs 10d and 10e had higher anti-angiogenic activity than other synthesized analogs, which may be due to the presence of the phthalimide moiety in their structure, similar to thalidomide. Further bioanalyses (LC/MS/MS) are needed in our follow-up studies to determine the exact contribution of the phthalimide moiety to the in vivo activity of the tetraacetyl combination. To improve the water solubility of these new materials, we also plan to couple these anti-angiogenic molecules with PLGA-based nanoparticles. [5]

C14H8I4O4

747.8288

747.66

67-30-1

65552

White to off-white solid powder

2.727g/cm3

544.8ºC at 760 mmHg

230 °C

283.3ºC

1.801

5.23

2

4

4

22

373

0

IC1C([H])=C(C([H])=C(C=1OC1C([H])=C(C(=C(C=1[H])I)O[H])I)I)C([H])([H])C(=O)O[H]

PPJYSSNKSXAVDB-UHFFFAOYSA-N

InChI=1S/C14H8I4O4/c15-8-4-7(5-9(16)13(8)21)22-14-10(17)1-6(2-11(14)18)3-12(19)20/h1-2,4-5,21H,3H2,(H,19,20)

2-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetic acid

Tetrac; 67-30-1; Tetraiodothyroacetic acid; 3,3',5,5'-TETRAIODOTHYROACETIC ACID; UNII-PA7UX1FFYQ; T4-acetic acid; 2-(4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl)acetic acid; Benzeneacetic acid, 4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (133.72 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.34 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)