Elsevier

Lung Cancer

Volume 76, Issue 1, April 2012, Pages 39-45
Lung Cancer

Tetraiodothyroacetic acid and its nanoformulation inhibit thyroid hormone stimulation of non-small cell lung cancer cells in vitro and its growth in xenografts

https://doi.org/10.1016/j.lungcan.2011.10.003Get rights and content

Abstract

Thyroid hormone stimulates cell proliferation of several types of cancers and stimulates cancer-relevant angiogenesis. In the present study, we investigated the proliferative effect of thyroid hormone and the anti-proliferative and anti-angiogenic action of its nano-derivative, tetrac-NP, on human non-small cell lung cancer (NSCLC) H1299 cells in vitro and in xenografts. The anti-proliferative activity of unmodified tetrac and tetrac-NP against human H1299 cells was determined in three models: (a) cultured H1299 cells in vitro, (b) tumor cell implants in the fertilized chick chorioallantoic membrane (CAM) system and (c) xenografts in the nude mouse. An integrin αvβ3 antibody inhibited thyroid hormone-induced cell proliferation in vitro, as did unmodified tetrac and tetrac-NP. Pharmacologic inhibition of the mitogen-activated protein kinase pathway also blocked NSCLC cell proliferation in response to thyroid hormone. Tetrac and tetrac-NP arrested tumor growth and tumor-related angiogenesis in H1299 cells grown in the CAM model and both agents prevented chick embryo mortality. Xenografts of H1299 cells were established in nude mice (n = 8, treatment and control groups) and when tumor volumes reached 250–300 mm3, tetrac (1 mg/kg) or tetrac-NP (1 mg tetrac as the nanoparticle/kg) were administered intraperitoneally every 2 days. Tetrac and tetrac-NP significantly suppressed tumor growth and angiogenesis. Thus, both tetrac and tetrac-NP effectively arrest human NSCLC tumor cell proliferation in vitro and in the CAM assay and in murine xenograft models.

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.

References (46)

  • C.X. Song et al.

    Formulation and characterization of biodegradable nanoparticles for intravascular local drug delivery

    J Control Release

    (1997)
  • American Lung Association (Epidemiology and Statistics Unit). Trends Lung Cancer Morb Mortal 2010 (March). Available...
  • D.N. Carney

    Chemotherapy in the management of subjects with inoperable non-small cell lung cancer

    Semin Oncol

    (1996)
  • W.D. Travis et al.

    Lung cancer

    Cancer

    (1995)
  • S.Y. Cheng et al.

    Molecular aspects of thyroid hormone actions

    Endocr Rev

    (2010)
  • J.J. Bergh et al.

    Integrin alpha v beta3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis

    Endocrinology

    (2005)
  • F.B. Davis et al.

    Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface

    Circ Res

    (2005)
  • S.A. Mousa et al.

    Proangiogenesis action of the thyroid hormone analog 3,5-diiodothyropropionic acid (DITPA) is initiated at the cell surface and is integrin mediated

    Endocrinology

    (2006)
  • F.B. Davis et al.

    Acting via a cell surface receptor, thyroid hormone is a growth factor for glioma cells

    Cancer Res

    (2006)
  • C.G. Gahmberg et al.

    Regulation of integrin activity and signaling

    Biochem Biophys Acta

    (2009)
  • B. Masson-Gadais et al.

    αvβ3, requirement for VEGFR2-mediated activation of SAPK2/p38 and Hsp90-dependent phosphorylation of focal adhesion kinase in endothelial cells activated by VEGF

    Cell Stress Chaperones

    (2003)
  • S.A. Mousa et al.

    Tetraiodothyroacetic acid, a small molecule integrin ligand, blocks angiogenesis induced by vascular endothelial growth factor and basic fibroblast growth factor

    Angiogenesis

    (2008)
  • H.Y. Lin et al.

    l-Thyroxine vs. 3,5,3′-triiodo-l-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

    Am J Physiol Cell Physiol

    (2009)
  • Cited by (71)

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

      2022, Food and Chemical Toxicology
      Citation Excerpt :

      Thus, finding new effective drugs for NSCLC patients is a necessary and important issue. Our research showed that thyroid hormones, especially thyroxine, can promote NSCLC cell proliferation (Meng et al., 2011; Mousa et al., 2012; Lin et al., 2013). The effect of thyroxine-induced cancer cell proliferation is via interactions between thyroxine and the αvβ3 cell surface integrin (Lin et al., 2013).

    • Effects of thyroxine on apoptosis and proliferation of mammary tumors: Thyroxine effects on mammary carcinogenesis

      2021, Molecular and Cellular Endocrinology
      Citation Excerpt :

      On the other hand, hyperthyroidism is associated with an increased incidence and aggressiveness of breast cancer (Glushakov et al., 2013). In vitro and xenografts studies have demonstrated that 3,3,5-triiodo-L-thyronine (T3) and L-thyroxine (T4) are proliferative factors for several human cancer cell lines (Mousa et al., 2012; Yalcin et al, 2010, 2013). Thyroid hormones exert their actions through genomic and/or non-genomic signaling pathways.

    • The chorioallantoic membrane as a bio-barrier model for the evaluation of nanoscale drug delivery systems for tumour therapy

      2021, Advanced Drug Delivery Reviews
      Citation Excerpt :

      They obtained tumour regression, pronounced destruction of the tumour vasculature and a selective inhibition of the metastatic growth by administering doxorubicin encapsulated in vascular-targeted and GD2-targeted (disialoganglioside receptor) liposomes. In 2012, Mousa et al. also used one of the few DDSs in their treatment [147]. They examined the relationship between the thyroid hormone stimulation of non-small cell lung cancer cells and their growth.

    • Lactobacillus acidophilus and Bifidobacterium longum exhibit antiproliferation, anti-angiogenesis of gastric and bladder cancer: Impact of COX2 inhibition

      2020, PharmaNutrition
      Citation Excerpt :

      In our study of the effect of LA and BL on tumor growth in the ex vivo CAM tumor model, we confirmed the effective role of LA and BL on the reduction of AGS but not on J253 tumor weight. We previously showed that the CAM model is a valuable tool to analyze tumor growth and angiogenesis with natural and other compounds [44–46]. We observed that both probiotic bacteria decreased hemoglobin levels of AGS tumors in a dose-dependent way, and the inhibition of blood vessel formation decreased the blood supply, leading to tumor weight reduction [47].

    View all citing articles on Scopus
    View full text