Oncology/Endocrine
Leflunomide suppresses growth in human medullary thyroid cancer cells

https://doi.org/10.1016/j.jss.2013.05.089Get rights and content

Abstract

Background

Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. Leflunomide (LFN) is a disease-modifying antirheumatic drug approved for the treatment of rheumatoid arthritis, and its active metabolite teriflunomide has been identified as a potential anticancer drug. In this study we investigated the ability of LFN to similarly act as an anticancer drug by examining the effects of LFN treatment on MTC cells.

Methods

Human MTC-TT cells were treated with LFN (25–150 μmol/L) and Western blotting was performed to measure levels of neuroendocrine markers. MTT assays were used to assess the effect of LFN treatment on cellular proliferation.

Results

LFN treatment downregulated neuroendocrine markers ASCL1 and chromogranin A. Importantly, LFN significantly inhibited the growth of MTC cells in a dose-dependent manner.

Conclusions

Treatment with LFN decreased neuroendocrine tumor marker expression and reduced the cell proliferation in MTC cells. As the safety of LFN in human beings is well established, a clinical trial using this drug to treat patients with advanced MTC may be warranted.

Introduction

Medullary thyroid cancer (MTC) is a neuroendocrine tumor (NET) that arises from the calcitonin-secreting parafollicular C cells of the thyroid gland. MTC is the third most common type of thyroid cancer, accounting for approximately 3%–5% of all cases [1]. The natural history of MTC is characterized by early lymph node metastasis [2]. In advanced cases, MTC can invade local structures such as the trachea and jugular vein, and metastasize to distant organs such as the liver and lungs [3], [4]. Surgery is potentially curative, but complete resection is often not possible due to disseminated disease at diagnosis. Furthermore, conventional treatments such as external beam radiation and cytotoxic chemotherapy are less effective in MTC. Therefore, new therapeutic approaches to MTC are needed [3].

Leflunomide, originally reported to be effective in rheumatoid arthritis (LFN; Arava, SU-101), has proved to be remarkably safe in patients [1], [2]. Approved by the Food and Drug Administration in 1998, LFN is nearly completely converted to its main active metabolite, teriflunomide (TFN), in first-pass metabolism [1], [3]. Recently, TFN in the form of oral tablets has been approved for the treatment of patients with relapsing forms of multiple sclerosis. A number of preclinical studies have reported the ability of LFN and TFN to inhibit the proliferation of a variety of human malignancies, including myeloma, carcinoids, and colon cancer [3], [4], [5]. While both drugs are capable of inducing apoptosis in some cell lines, they are also capable of initiating cell cycle arrest alone, which is consistent with the in vivo safety profile for LFN [3], [4].

The growing focus on the antitumor applications of LFN stimulated interest in potential effects in NETs. These cancers characteristically express high levels of chromogranin A (CgA), an acidic glycopeptide co-secreted with hormones and considered a clinical marker of disease. High serum levels of CgA have been associated with a poor clinical prognosis [3], [6]. The basic helix-loop-helix transcription factor, achaete-scute complex-like 1 (ASCL1), is also highly expressed in NETs. ASCL1 is important in the development of normal neuroendocrine cells. However, it is highly expressed in medullary thyroid cancer and small cell lung cancer and its expression is associated with poor prognosis in NETs [7].

We have earlier reported the activity of LFN and TFN in suppressing carcinoid tumors [3]. Earlier reports show that LFN and its active metabolite, TFN, are capable of suppressing the growth of human carcinoid cells in vitro by inducing cell cycle arrest, and that this finding can be replicated in vivo. Furthermore, we have observed that these compounds can inhibit the cellular expression of CgA and ASCL1, well-characterized markers of NETs [3].

However, the effect of these compounds on MTC is not known. Based on our earlier findings on carcinoids, we hypothesized that LFN may also have anti-MTC effects. In this study, we describe for the first time the effects of LFN on NET marker expression and cancer cell growth in human MTC-TT cells.

Section snippets

Cell culture and treatment

Human MTC-TT cells were obtained from American Type Culture Collection (Manassas, VA) and maintained as previously described [3], [8]. Cells were plated and allowed to adhere overnight. LFN at concentrations ranging from 0–150 μmol/L was then added, and cells were incubated for 48 or 96 h. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, ST Louis, Missouri), the solvent for LFN, was used as control.

Cell proliferation assay

The cells were then plated in 24-well culture plates, treated with varying concentrations of LFN, and

LFN suppresses MTC-TT proliferation

We have reported that treatment of carcinoid tumors with LFN or TFN resulted in significant reduction of cell proliferation. Therefore we are interested in the effect of LFN on MTC-TT cell proliferation. MTT assays revealed that exposure to LFN concentrations ≥75 μM resulted in significant dose-dependent reduction in cell proliferation. Compared with control media and DMSO, 75 μM, 100 μM, 125 μM, and 150 μM LFN treatments for 4 d reduced growth by 0.7%, 7.4%, 13.4%, 14.6%, and 22.2%,

Discussion

MTC is a rare aggressive thyroid cancer. In patients with disseminated disease, conventional chemotherapy has minimal efficacy and carries considerable toxicity to normal cells [9]. This lack of efficacy is shared by other adjuvant therapies as well as palliative treatments like octreotide, to which patients rapidly become resistant [3]. The need for novel targeted therapies is therefore clear. In the current study we report the results of treatment with the disease-modifying antirheumatic drug

Acknowledgment

Funding was provided by NIH R01 CA 12115 American Cancer Society Research Scholars Grant; American Cancer Society MEN2 Thyroid Cancer Professorship; and Ministry of Higher Education of Saudi Arabia, and KFSH&RC, Riyadh, Saudi Arabia (fellowship and financial support to A. Alhefdhi).

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    1

    Amal Alhefdhi and Jocelyn F. Burke contributed equally to completion of the manuscript.

    2

    Current affiliation: Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin.

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