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 is a monocarboxylic acid that is thyroacetic acid carrying four iodo substituents at positions 3, 3', 5 and 5'. It has a role as a thyroid hormone, a human metabolite and an apoptosis inducer. It is an iodophenol, a 2-halophenol, a monocarboxylic acid and an aromatic ether.

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The thyroid hormones T3 and T4 have emerged as pro-angiogenic hormones with important implications for cancer management. Endogenous circulating hormone levels may help stimulate cancer progression and limit the effectiveness of anticancer therapy, though clinical data remain inconclusive. The capacity of thyroid hormones to modulate angiogenesis is mediated through non-canonical mechanisms initiated at the cell surface receptor integrin αvβ3. This integrin is predominantly expressed on tumour cells, proliferating endothelial cells and tumour stroma-associated cells, emphasising its potential relevance in angiogenesis and tumour biology. Thyroid hormone/integrin αvβ3 signalling results in the activation of intracellular pathways that are commonly associated with angiogenesis and are mediated through classical pro-angiogenic molecules such as vascular endothelial growth factor. 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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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. [2]

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Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. 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]

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Ocular melanoma research, the most common primary intraocular malignancy in adults, is hindered by limited in vivo models. In a series of experiments using melanoma cells injected intraocularly into mouse eyes, we developed a model for ocular melanoma. Inoculation of 5 × 105 B16F10 cells led to rapid tumor growth, extensive lung metastasis, and limited animal survival, while injection of 102 cells was sufficient for intraocular tumors to grow with extended survival. In order to improve tumor visualization, 102 melanoma cells (B16F10 or B16LS9) were inoculated into Balb/C albino mouse eyes. These mice developed intraocular tumors that did not metastasize and exhibited extended survival. Next, we studied the therapeutic potential of inhibitor of the thyroid hormones-αvβ3 integrin signaling pathway in ocular melanoma. By utilizing tetraiodothyroacetic acid (tetrac), a thyroid hormone derivative, a delay in tumor onset in the B16F10 (integrin+) arm was observed, compared to the untreated group, while in the B16LS9 cells (integrin-) a similar rate of tumor onset was noticed in both experimental and control groups. In summary, following an optimization process, the mouse ocular melanoma model was developed. The models exhibited an extended therapeutic window and can be utilized as a platform for investigating various drugs and other treatment modalities.[4]

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In the angiogenesis process, integrins, which are members of a family of cell surface transmembrane receptors, play a critical role particularly in blood vessel formation and the local release of vascular growth factors. Thyroid hormones such as l-thyroxine (T4) and 3,5,3'-triiodo-l-thyronine (T3) promote angiogenesis and tumor cell proliferation via integrin αvβ3 receptor. At or near an arginine-glycine-aspartate (RGD) recognition site on the binding pocket of integrin αvβ3, tetraiodothyroacetic acid (tetrac, a deaminated derivative of T4) is a thyrointegrin receptor antagonist and blocks the actions of T3 and T4 as well as different growth factors-mediated angiogenesis. In this study, we synthesized novel tetrac analogs by modifying the phenolic moiety of tetrac and tested them for their anti-angiogenesis activity using a Matrigel plug model for angiogenesis in mice. Pharmacological activity results showed that tetrac can accommodate numerous modifications and maintain its anti-angiogenesis activity.
In summary, to find new angiogenesis inhibitors we synthesized new tetrac analogs by modifying the phenolic moiety of tetrac. We analyzed their structure-activity relationships and tested for anti-angiogenesis activity using a mouse Matrigel model for angiogenesis. Design and development of novel angiogenesis inhibitors has been validated as a target in several tumor types. Our results showed that phthalimide-modified tetrac analogs 10d and 10e have higher anti-angiogenesis activity than the other analogs generated and this might be due to the presence of the phthalimide portion of their structure, similar to thalidomide. Our further studies will require additional bioanalytical studies (LC/MS/MS) to see the exact contribution of the phthalimide portion to the in vivo activity in the tetrac combination. In order to increase the water solubility of these new materials, we also plan to conjugate these anti-angiogenesis molecules to PLGA-based NPs. [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.)