Tetraiodothyroacetic acid and its nanoformulation inhibit thyroid hormone stimulation of non-small cell lung cancer cells in vitro and its growth in xenografts
Introduction
Lung cancer remains a leading cause of death in the United States [1]. Approximately 60% of lung cancer patients present with advanced disease and the majority of newly diagnosed lung cancers are inoperable [2]. Recent advances in chemotherapy have yielded limited improvement in the prognosis for patients with lung cancer and the 5-year survival rate for all combined disease stages remains about 15%. For this reason, newer therapies such as those that inhibit actions of vascular or non-vascular growth factors [3], [4], [5] are of special interest in this group of patients. The two most prevalent histological types of lung carcinoma, categorized by the size and appearance of the malignant cells, are non-small cell (NSCLC) and small cell lung carcinoma [6]. The non-small cell type accounts for 80% of lung cancers. The principal NSCLC sub-types are squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma; these are grouped together because their prognosis and management are similar.
Receptors for thyroid hormone (l-thyroxine, T4, and 3,5,3′-triiodo-l-thyronine, T3) exist in the cell nucleus [7] to mediate genomic actions of the hormone, and nongenomic actions may be initiated at a cell surface receptor recently described on plasma membrane integrin αvβ3 [8]. The cell surface receptor is largely expressed on tumor cells and rapidly dividing blood vessel cells [8] and transduces thyroid hormone signals into an angiogenic response [8], [9], [10], [11] and into tumor cell proliferation [7], [12], [13]. The integrin-mediated proliferative response in tumor cells is generated by T4 at physiological total and free concentrations [8], whereas such responses to T3 appear to require supraphysiologic levels of the hormone [8]. The receptor site for thyroid hormone on integrin αvβ3 is proximal to the Arg-Gly-Asp (RGD) recognition site on the integrin [14] and this RGD domain facilitates interactions of the integrin with extracellular matrix proteins and growth factors [15], [16]. The RGD recognition site engages in crosstalk with receptors for vascular endothelial growth factor (VEGFR) [17] and basic fibroblast growth factor receptor (bFGFR) [18]. The pro-angiogenic activities of VEGF and bFGF are blocked by RGD peptides and by a T4 derivative, tetraiodothyroacetic acid (tetrac) [19], which is a thyroid hormone antagonist acting at the cell surface hormone receptor [7], [8], [20], [21]. However, the thyroid hormone antagonist tetrac acts on the integrin αvβ3, which is distinct from the RGD recognition site [20], [21], and can distinguish among thyroid hormone analogues, e.g., T3 vs. T4, and, downstream of the receptor, within the cell, can discretely activate extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) in response to specific signals [20]. Hormone activation of these two signal transduction mechanisms leads to different consequences [7], [21], [22], [23].
Tetrac blocks the binding of T4 and of T3 to the integrin receptor [8]. Tetrac has been shown to inhibit thyroid hormone-induced proliferation of a number of human cancer cell lines in vitro [13], [20], [24], [25]. Tetrac also acts to suppress angiogenesis in the absence of thyroid hormone [17]. This agent has been demonstrated to inhibit tumor growth and tumor-related angiogenesis in human cancer xenografts in the nude mouse [26], [27], [28]. Within the cell, tetrac may have thyromimetic actions [29]. To eliminate the latter undesirable effects, we have reformulated tetrac as a nanoparticle (tetrac-NP) in which the tetrac is covalently bonded by its outer ring hydroxyl group to biodegradable nanoparticles, limiting its entry into the nucleus [30]. This formulation acts exclusively at the integrin receptor for tetrac, T4, and T3. The fit of tetrac as a nanoparticle into the integrin receptor is somewhat different from that of unmodified tetrac, as shown by distinctive effects of tetrac and tetrac-NP on expression of cancer cell survival pathway genes [27], [31].
The purpose of the current investigation was to establish that T4 and T3 stimulate human NSCLC cell proliferation in vitro in a concentration-dependent manner and do so via the hormone receptor on integrin αvβ3. We show that unmodified tetrac and tetrac-NP block the proliferative responses of human NSCLC cells in vitro to T4 and T3. Tetrac and tetrac-NP have anti-tumor effects against NSCLC cells and are anti-angiogenic in xenografts in the chick chorioallantoic membrane (CAM) and in the nude mouse.
Section snippets
Cells and cell culture
Human non-small cell lung cancer (NSCLC) NCI-H1299 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured as instructed by the supplier, using complete growth RPMI medium supplemented with 10% FBS. Cells were cultured in a 5% CO2/air atmosphere at 37 °C to sub-confluence and then treated with 0.25% (w/v) trypsin/EDTA to affect cell release from the culture vessel. After cells were washed with culture medium, they were suspended in DMEM that was free of phenol red
Thyroid hormone stimulates cell proliferation of human non-small cell lung cancer cells
In a concentration-dependent manner, T4 induced proliferating-cell nuclear antigen (PCNA) accumulation in NCI-H1299 non-small cell lung carcinoma cells (Fig. 1A). Maximum effects were obtained at 10−8 to 10−7 M total hormone concentrations; in the medium/buffer systems we use, such concentrations yield physiological free T4 levels [8], [26]. T3 also increased PCNA abundance in NCI-H1299 cells (Fig. 1B), but the effective total hormone concentrations exceeded physiologic levels. Therefore, we
Discussion
In the present studies, T4 and T3 stimulated human non-small cell lung cancer H1299 cell proliferation in vitro in a concentration-dependent manner. This action was inhibited by either anti-integrin αvβ3 or tetrac and tetrac-NP. These observations support the concept that the cell surface receptor for iodothyronine at which tetrac is an inhibitor of binding of T4 and T3 is required for hormone action on tumor cells [22] and that physiological concentrations of T4 may be an endogenous growth
Conflict of interest statement
None declared.
Acknowledgements
We appreciate Dr. Kelly Keating for the outstanding editing of the manuscript.
Sources of support: This work was supported in part the Pharmaceutical Research Institute and by Charitable Leadership Foundation.
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