Original research articleInterferon alpha and rapamycin inhibit the growth of carcinoid and medullary thyroid cancer in vitro
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.
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E. Motylewska and H. Lawnicka contributed equally to this work.