Heteronemin and Tetrac Induce Anti-Proliferation by Blocking EGFR-Mediated Signaling in Colorectal Cancer Cells.

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvß3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy.

In Vivo Clearance of Apoptotic Debris From Tumor Xenografts Exposed to Chemically Modified Tetrac: Is There a Role for Thyroid Hormone Analogues in Efferocytosis?

Apoptosis is induced in cancer cells and tumor xenografts by the thyroid hormone analogue tetraiodothyroacetic acid (tetrac) or chemically modified forms of tetrac. The effect is initiated at a hormone receptor on the extracellular domain of plasma membrane integrin αvß3. The tumor response to tetrac includes 80% reduction in size of glioblastoma xenograft in two weeks of treatment, with absence of residual apoptotic cancer cell debris; this is consistent with efferocytosis. The molecular basis for efferocytosis linked to tetrac is incompletely understood, but several factors are proposed to play roles. Tetrac-based anticancer agents are pro-apoptotic by multiple intrinsic and extrinsic pathways and differential effects on specific gene expression, e.g., downregulation of the X-linked inhibitor of apoptosis (XIAP) gene and upregulation of pro-apoptotic chemokine gene, CXCL10. Tetrac also enhances transcription of chemokine CXCR4, which is relevant to macrophage function. Tetrac may locally control the conformation of phagocyte plasma membrane integrin αvß3; this is a cell surface recognition system for apoptotic debris that contains phagocytosis signals. How tetrac may facilitate the catabolism of the engulfed apoptotic cell debris requires additional investigation.

Heteronemin and tetrac derivatives suppress non-small cell lung cancer growth via ERK1/2 inhibition.

The most common cancer, lung cancer, causes deaths worldwide. Most lung cancer patients have non-small cell lung carcinomas (NSCLCs) with a poor prognosis. The chemotherapies frequently cause resistance therefore search for new effective drugs for NSCLC patients is an urgent and essential issue. Deaminated thyroxine, tetraiodothyroacetic acid (tetrac), and its nano-analogue (NDAT) exhibit antiproliferative properties in several types of cancers. On the other hand, the most abundant secondary metabolite in the sponge Hippospongia sp., heteronemin, shows effective cytotoxic activity against different types of cancer cells. In the current study, we investigated the anticancer effects of heteronemin against two NSCLC cell lines, A549 and H1299 cells in vitro. Combined treatment with heteronemin and tetrac derivatives synergistically inhibited cancer cell growth and significantly modulated the ERK1/2 and STAT3 pathways in A549 cells but only ERK1/2 in H1299 cells. The combination treatments induce apoptosis via the caspases pathway in A549 cells but promote cell cycle arrest via CCND1 and PCNA inhibition in H1299 cells. In summary, these results suggest that combined treatment with heteronemin and tetrac derivatives could suppress signal transduction pathways essential for NSCLC cell growth. The synergetic effects can be used potentially as a therapeutic procedure for NSCLC patients.

Nano-Targeting of Thyrointegrin αvß3 Receptor in Solid Tumors and Impact on Radiosensitization.

Tetraiodothyroacetic acid is a ligand of thyrointegrin αvß3, a protein that is highly expressed in various solid tumors and surrounding neovascular regions. Its nano derivative, Nano-diamino-tetrac (NDAT), has anticancer properties in preclinical models, enhances radiosensitivity, and inhibits cancer cell growth in vitro after X-ray irradiation. Using a novel experimental system developed to deliver accurate radiation dose to tumors under sterile conditions, this study establishes NDAT's radiosensitizing effect in SUIT-2 pancreatic cancer and H1299 non-small cell lung carcinoma xenografts in athymic mice for tumor-targeted radiation. In this work, low-melting-point Lipowitz alloy was used to shield normal organs and allow accurate tumor-targeted irradiation. Over a three-week period, mice with SUIT-2 xenografts received daily NDAT treatment at different doses (0, 1, 3, or 10 mg/kg body weight) and tumor-targeted irradiation (1 or 5 Gy). Validation was performed with a test dose of 30 Gy to mice bearing SUIT-2 xenografts and resulted in more than 80% reduction in tumor weight, compared to nonirradiated tumor weight. The results of this work showed that NDAT had a radiosensitizing effect in a dose-dependent manner in decreasing tumor growth and viability. An enhanced anticancer effect of NDAT (1 mg/kg body weight) was observed in mice with H1299 xenografts receiving 5 Gy tumor-targeted irradiation, indicated by decreased tumor weight and increased necrosis, compared to nonirradiated tumors. This technique demonstrated accurate tumor-targeted irradiation with new shielding methodology, and combined with thyrointegrin antagonist NDAT treatment, showed anticancer efficacy in pancreatic cancer and non-small cell lung carcinoma.

Design, synthesis, and biological evaluation of novel bifunctional thyrointegrin antagonists for neuroblastoma.

Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma membrane integrin αvß3. Dual targeting of the NET and integrin αvß3 receptors using a Drug-Drug Conjugate (DDC) might provide effective treatment strategy in the fight against neuroblastoma and other neuroendocrine tumors. In this work, we synthesized three dual-targeting BG-P400-TAT derivatives, dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400-PAT containing di-iodobenzene, di-methoxybenzene, and piperazine groups, respectively. These derivatives utilize to norepinephrine transporter (NET) and the integrin αvß3 receptor to simultaneously modulate both targets based on evaluation in a neuroblastoma animal model using the neuroblastoma SK-N-F1 cell line. Among the three synthesized agents, the piperazine substituted BG-P400-PAT exhibited potent integrin αvß3 antagonism and reduced neuroblastoma tumor growth and cancer cell viability by >90%. In conclusion, BG-P400-PAT and derivatives represent a potential therapeutic approach in the management of neuroblastoma.

Nano Diaminopropane tetrac and integrin αvß3 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 αvß3 has been recognized as a putative target for the treatment of several cancers. Thus, the characterization of αvß3 distribution in different human tumors is of substantial interest in tumor targeting and its suppression. In this study we evaluated the expression of integrin αvß3 in different cancer types to define the expression pattern in cancer model. Furthermore, we investigated the effect of novel αvß3 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 ß3 monomer expression. In vivo in tumor xenografts, similarly, NDAT downregulated αv and ß3. 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 αvß3 expression in various cancer types, which indicates its impact on the targetability and suppression of αvß3-associated tumor functions.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

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 αvß3. 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 αvß3, 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 αvß3 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-ß1 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-ß expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Dual Targeting of Norepinephrine Transporter (NET) Function and Thyrointegrin αvß3 Receptors in the Treatment of Neuroblastoma.

Therapeutic targeting of the norepinephrine transporter (NET) function with benzylguanidine (BG), conjugated with the high-affinity thyrointegrin αvß3 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 αvß3 receptors that are overexpressed in neuroblastoma and other neuroendocrine tumors. Various cancer cells and actively dividing tumor-endothelial cells express the thyrointegrin αvß3 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.

l-Thyroxin and the Nonclassical Thyroid Hormone TETRAC Are Potent Activators of PPARγ.

Thyroid hormones (THs) operate numerous physiological processes through modulation of the nuclear thyroid hormone receptors and several other proteins. We report direct activation of the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X receptor (RXR) by classical and nonclassical THs as another molecular activity of THs. The T4 metabolite TETRAC was the most active TH on PPARγ with nanomolar potency and binding affinity. We demonstrate that TETRAC promotes PPARγ/RXR signaling in cell-free, cellular, and in vivo settings. Simultaneous activation of the heterodimer partners PPARγ and RXR resulted in high dimer activation efficacy. Compared to fatty acids as known natural ligands of PPARγ and RXR, TETRAC differs markedly in its molecular structure and the PPARγ-TETRAC complex revealed a distinctive binding mode of the TH. Our observations suggest a potential connection of TH and PPAR signaling through overlapping ligand recognition and may hold implications for TH and PPAR pharmacology.

Competitive binding assays for measuring the binding affinity of thyroid-disrupting chemicals for integrin αvß3.

Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin αvß3. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin αvß3. 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 αvß3. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin αvß3 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 αvß3 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 αvß3.

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 αVß3. 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 αVß3. 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.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

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 αVß3 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 αvß3 to alter the leptin gene expression.

Thyroid Hormone Effects on Mesenchymal Stem Cell Biology in the Tumour Microenvironment.

Non-classical thyroid hormone signalling via cell surface receptor integrin αvß3, expressed on most cancer cells and proliferating endothelial cells, has been shown to drive tumour cell proliferation and survival, as well as angiogenesis. Tumours develop within a complex microenvironment that is composed of many different cell types, including mesenchymal stem cells. These multipotent progenitor cells actively home to growing tumours where they differentiate into cancer-associated fibroblast-like cells and blood vessel-stabilising pericytes and thus support the tumour's fibrovascular network. Integrin αvß3 expression on mesenchymal stem cells makes them susceptible to thyroid hormone stimulation. Indeed, our studies demonstrated - for the first time - that thyroid hormones stimulate the differentiation of mesenchymal stem cells towards a carcinoma-associated fibroblast-/pericyte-like and hypoxia-responsive, pro-angiogenic phenotype, characterised by the secretion of numerous paracrine pro-angiogenic factors, in addition to driving their migration, invasion, and recruitment to the tumour microenvironment in an experimental hepatocellular carcinoma model. The deaminated thyroid hormone metabolite tetrac, a specific inhibitor of thyroid hormone action at the integrin site, reverses these effects. The modulation of mesenchymal stem cell signalling and recruitment by thyroid hormones via integrin αvß3 adds a further layer to the multifaceted effects of thyroid hormones on tumour progression, with important implications for the management of cancer patients and suggests a novel mechanism for the anti-tumour activity of tetrac.

Integrin αvß3-Mediated Effects of Thyroid Hormones on Mesenchymal Stem Cells in Tumor Angiogenesis.

Background: Several clinical and experimental studies have implicated thyroid hormones in cancer progression. Cancer-relevant effects, including stimulation of tumor growth and new blood vessel formation by angiogenesis, are thought to be mediated by a nonclassical signaling pathway initiated through integrin αvß3 expressed on cancer cells and proliferating endothelium. In an earlier study, we established mesenchymal stem cells (MSCs), important contributors to the fibrovascular network of tumors, as new thyroid hormone-dependent targets. Here, we evaluated the effects of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) versus Tetrac, an integrin-specific inhibitor of thyroid hormone action, on MSCs in tumor angiogenesis. Methods: Modulation of the expression and secretion of angiogenesis-relevant factors by thyroid hormones in primary human MSCs and their effect on endothelial cell tube formation were tested in vitro. We further engineered MSCs to express the sodium iodide symporter (NIS) reporter gene under control of a hypoxia-responsive promoter and the vascular endothelial growth factor (VEGF) promoter to test effects on these pathways in vitro and, for VEGF, in vivo in an orthotopic hepatocellular carcinoma (HCC) xenograft mouse model by positron emission tomography imaging. Results: T3 and T4 increased the expression of pro-angiogenic genes in MSCs and NIS-mediated radioiodide uptake in both NIS reporter MSC lines in the presence of HCC cell-conditioned medium. Supernatant from thyroid hormone-treated MSCs significantly enhanced endothelial cell tube formation. Tetrac and/or inhibitors of signaling pathways downstream of the integrin reversed all these effects. Tumoral radioiodide uptake in vivo demonstrated successful recruitment of MSCs to tumors and VEGF promoter-driven NIS expression. Hyperthyroid mice showed an increased radioiodide uptake compared with euthyroid mice, while tracer uptake was markedly reduced in hypothyroid and Tetrac-treated mice. Conclusions: Our data suggest that thyroid hormones influence angiogenic signaling in MSCs via integrin αvß3 and further substantiate the anti-angiogenic activity of Tetrac in the tumor microenvironment.

Triazole Modified Tetraiodothyroacetic Acid Conjugated to Polyethylene Glycol: High Affinity Thyrointegrin αvß3 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 αvß3's binding pocket (thyrointegrin αvß3 receptors). We have enhanced the biological activity of a tetrac-based inhibitor via significantly improving its αvß3 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 αvß3 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.

Novel thyroid hormones.

The field of thyroid hormone signaling has grown more complex in recent years. In particular, it has been suggested that some thyroid hormone derivatives, tentatively named "novel thyroid hormones" or "active thyroid hormone metabolites", may act as independent chemical messengers. They include 3,5-diiodothyronine (T2), 3-iodothyronamine (T1AM), and several iodothyroacetic acids, i.e., 3,5,3',5'-thyroacetic acid (TA4), 3,5,3'-thyroacetic acid (TA3), and 3-thyroacetic acid (TA1). We summarize the present knowledge on these compounds, namely their biosynthetic pathways, endogenous levels, molecular targets, and the functional effects elicited in experimental preparations or intact animals after exogenous administration. Their physiological and pathophysiological role is discussed, and potential therapeutic applications are outlined. The requirements needed to qualify these substances as chemical messengers must still be validated, although promising evidence has been collected. At present, the best candidate to the role of independent chemical messenger appears to be T1AM, and its most interesting effects concern metabolism and brain function. The responses elicited in experimental animals have suggested potential therapeutic applications. TA3 has an established role in thyroid hormone resistance syndromes, and is under investigation in Allen-Herndon-Dudley syndrome. Other potential targets are represented by obesity and dyslipidemia (for T2 and T1AM); dementia and degenerative brain disease (for T1AM and TA1); cancer (for T1AM and TA4). Another intriguing and unexplored question is the potential relevance of these metabolites in the clinical picture of hypothyroidism and in the response to replacement therapy.

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 αvß3) 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 αvß3 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.