Elsevier

Pharmacological Reports

Volume 66, Issue 4, August 2014, Pages 624-629
Pharmacological Reports

Original research article
Interferon alpha and rapamycin inhibit the growth of carcinoid and medullary thyroid cancer in vitro

https://doi.org/10.1016/j.pharep.2014.02.020Get rights and content

Abstract

Neuroendocrine tumors (NETs) are highly vascularized neoplasms characterized by rising incidence. Moreover, the neuroendocrine cells were shown to express vascular endothelial growth factor (VEGF) and VEGF receptors. Therefore, angiomodulators could be potentially a new group of drugs enhancing still unsatisfactory effectiveness of NET therapy.

The aim of this study was to assess the direct influence of angiomodulators: VEGF and five endogenous and exogenous antiangiogenic compounds (endostatin, interferon alpha [IFNα], rapamycin, JV1-36, semaxinib [SU5416]) on the growth of two NET cell lines: lung carcinoid H727 cell line and medullary thyroid cancer TT cell line in vitro.

IFNα and rapamycin induced the inhibitory effect on H727 and TT cell viability and proliferation, increasing apoptosis and arresting the cell cycle. Also semaxinib (10−5 M) inhibited proliferation of both cell lines. VEGF and endostatin did not influence the growth of H727 and TT cells.

The inhibitory effect of IFNα, rapamycin and semaxinib on carcinoid and medullary thyroid cancer growth was revealed in our in vitro study, although some other antiangiogenic agents did not directly influence H727 and TT cell growth. Thus, IFNα and mTOR inhibitors as multidirectionally acting drugs with antiangiogenic effect could be potentially efficient in treatment of neuroendocrine tumors and are worth further studies.

Introduction

Neuroendocrine tumors (NETs) are rare malignancies but their incidence has risen in the last years. NETs comprise a heterogeneous group of neoplasms with a wide spectrum of clinical behavior, which require specially tailored therapies. Although somatostatin analogs are routinely used to control hormone-mediated symptoms (carcinoid syndrome), the antitumor efficacy of traditional cytotoxic drugs is limited. These have led to the search for new targeted drugs based on the biological features of NETs, such as extraordinary vascularization with high expression of several proangiogenic molecules [1], [2]. Moreover, as the neuroendocrine cells were shown to secret VEGF [3], [4], [5] and express VEGFR [6], [7], the paracrine and autocrine action of VEGF is possible. The role of angiogenesis in neoplastic transformation was also suggested in medullary thyroid carcinoma (MTC) [8], [9], [10], [11], [12], which as a malignant tumor arising from the neuroendocrine calcitonin-producing parafollicular C cells is in broad understanding included to NETs. These facts suggest that antiangiogenic compounds could be potentially a group of drugs enhancing still unsatisfactory efficacy of NET therapy. They could act not only on endothelial cells, but also directly on neuroendocrine cells, which were shown to overexpress VEGF and VEGFR (vascular endothelial growth factor receptor) [5], [8], [11].

The aim of this paper was to study the direct influence of proangiogenic (VEGF) and antiangiogenic (rapamycin, interferon alpha, endostatin, JV1-36, SU5416) factors on the growth of two human neuroendocrine tumors cell lines: lung carcinoid H727 and medullary thyroid cancer TT line in vitro. We also decided to assess the influence of these substances on calcitonin secretion in TT cells. Calcitonin used in clinical practice as hormonal marker of MTC, was shown to be produced in TT cells [13].

Rapamycin, a naturally occurring macrolide antibiotic, was the first known inhibitor of PI3K/AKT/mTOR-pathway (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin-pathway), which takes part in regulation of cell growth, metabolism, angiogenesis and is frequently hyperactive in neoplasms [14]. Rapamycin was demonstrated to have immunosuppressive, antiangiogenic and antitumor activity and was proven to be effective in a number of neoplasms [14], [15], including neuroendocrine tumors [16]. In 2011, the FDA (Food and Drug Administration) approved an mTOR inhibitor – everolimus – for the treatment of progressive pancreatic neuroendocrine tumors (PNETs) (NCI; http://www.cancer.gov/cancertopics/druginfo/fda-everolimus).

JV1-36 belongs to GHRH (growth hormone releasing hormone) antagonists, which were shown to exert antiproliferative effects in different tumors. The mechanism of GHRH antagonist action is complex and apart from inhibition of IGFs (insulin-like growth factors) production, the suppression of VEGF and bFGF (basic fibroblast growth factor) expression in neoplasms is suggested [17].

SU5416 (semaxinib), a 3-substituted indolinone compound, was the first VEGFR tyrosine kinase inhibitor tested clinically as antiangiogenic treatment in cancer [18]. This strategy was continued in trials with another multitargeted, orally available tyrosine kinase inhibitor – sunitinib – (structurally related to semaxinib), which was approved by the FDA for treatment of several neoplasms, including PNETs (NCI; http://www.cancer.gov/cancertopics/druginfo/fda-sunitinib-malate).

Endostatin is a proteolytic fragment of collagen XVII. It is an endogenous inhibitor of angiogenesis acting by blocking the binding of VEGF to VEGFR [19] and down-regulating VEGF expression in tissues [20]. Endostatin was also shown to exert an antineoplastic action [21], [22]. Although endostatin suppressed the growth of pancreatic neuroendocrine tumor in transgenic RT2 (RIP-Tag2) mice [23], the clinical study did not confirm the efficacy of endostatin in PNET treatment [24].

Interferon alpha (IFNα) is a pleiotropic cytokine with proven antitumor activity triggered in the direct (through prolongation of the cell cycle time and enhancement of apoptosis in malignant cells) and indirect (interaction with other cytokines, immunomodulatory and antiangiogenic influence) mechanism [25]. IFNα as an angiogenesis inhibitor was first reported in 1980 [26] and then was confirmed by other authors [19], [25]. The clinical use of IFNα includes treatment of neuroendocrine tumors, renal cell carcinoma, melanoma, and some other neoplasms [25].

Section snippets

Cell line and culture condition

The human lung carcinoid cell line H727 and the human medullary thyroid cancer cell line TT obtained from the American Type Culture Collection (ATCC) were used in the experiments. The cells were routinely grown as a monolayer in a humidified incubator at 37 °C with 5% CO2 in RPMI medium (ATCC) for H727 line or DMEM medium for TT line, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin solution (Sigma) and 10% fetal bovine serum (Biochrom). The cells were passaged every 7 days with

Lung carcinoid H727 cell line

IFNα at the highest concentrations inhibited the H727 growth in 72 h culture, up to 87–92% of the control group in the MTT method (Fig. 1) and up to 81–93% of the control group in the BrdU incorporation method (Fig. 1). IFNα at the concentration of 105 U/ml increased apoptosis (2.33% vs. 0.86% in the control group) and necrosis (13.39% vs. 4.65% in the control group) of the H727 cells in 72 h culture (Table 1) and induced also G0/G1 phase arrest (Fig. 2, Table 2).

Rapamycin at all studied

Discussion

The role of angiogenesis and antiangiogenic therapy in NET is quite well established in pancreatic tumors [1]. However, it has been less frequently studied in other types of NETs. Therefore, in this paper we decided to explore the direct influence of several proangiogenic and antiangiogenic compounds on the growth of two human neuroendocrine tumor cell lines: lung carcinoid H727 and medullary thyroid cancer TT line. These cell lines seem to be a good in vitro model for studying the influence of

Conflict of interest

The authors declare that they have no conflicts of interest.

Funding

This work has been supported with the contribution of the Medical University of Lodz, Grant No. 502-03/1-153-03/502-14-114.

References (42)

  • J. Zhang et al.

    Elevated expression of vascular endothelial growth factor correlates with increased angiogenesis and decreased progression-free survival among patients with low-grade neuroendocrine tumors

    Cancer

    (2007)
  • G. Van Buren et al.

    The development and characterization of a human midgut carcinoid cell line

    Clin Cancer Res

    (2007)
  • C. Capp et al.

    Increased expression of vascular endothelial growth factor and its receptors, VEGFR-1 and VEGFR-2, in medullary thyroid carcinoma

    Thyroid

    (2010)
  • G. Fontanini et al.

    Microvessel count: an indicator of poor outcome in medullary thyroid carcinoma but not in other types of thyroid carcinoma

    Mod Pathol

    (1996)
  • J. Komorowski et al.

    Matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases and angiogenic cytokines in peripheral blood of patients with thyroid cancer

    Thyroid

    (2002)
  • C. Rodríguez-Antona et al.

    Overexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis

    Endocr Relat Cancer

    (2010)
  • D. Vitagliano et al.

    The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells

    Endocr Relat Cancer

    (2010)
  • I.R. Konings et al.

    The applicability of mTOR inhibition in solid tumors

    Curr Cancer Drug Targets

    (2009)
  • N.Z. Khokhar et al.

    Emerging roles for mammalian target of rapamycin inhibitors in the treatment of solid tumors and hematological malignancies

    Cur Opin Oncol

    (2011)
  • J. Capdevila et al.

    Innovations therapy: mammalian target of rapamycin (mTOR) inhibitors for the treatment of neuroendocrine tumors

    Cancer Metastasis Rev

    (2011)
  • A. Siejka et al.

    Antineoplastic action of growth hormone-releasing hormone (GHRH) antagonists

    Recent Pat Anticancer Drug Discov

    (2012)
  • 1

    E. Motylewska and H. Lawnicka contributed equally to this work.

    View full text