title: Azacytidine induces cell cycle arrest and suppression of neuroendocrine markers in carcinoids.
abstract: Neuroendocrine tumors (NETs) hypersecrete neuropeptides that cause debilitating symptoms of carcinoid syndrome, including cardiac abnormalities. Surgical resection is the only potentially curative treatment for NETs; however, 90% of NE cancer patients are not candidates for surgery due to extensive hepatic sites involved with NETs. Recently, DNA methyltransferase inhibitors (DNMTI) such as azacytidine (AzaC) have shown efficacy in clinical treatments of hematological malignancies, but effects on NETs are not well-studied. We hypothesized that this novel class of drugs inhibits NET cell growth and decreases NE markers. Three carcinoid types-human midgut (CDNT2.5), pulmonary (H727), and gastrointestinal (BON)- were treated with AzaC (0-100uM) over 6 days. MTT Assays were used to measure cellular proliferation. Western blots were performed with antibodies against chromogranin A (CgA), Neuron-Specific Enolase (NSE), and Cyclin B1. Flow cytometric data was collected from AzaC-treated CNDT2.5 cells for DNA cell cycle analysis. Results showed that treatment of CDNT2.5, H727, and BON carcinoid cells with AzaC resulted in a dose-dependent reduction in tumor cell proliferation. Flow cytometric analysis showed that AzaC-treated cells accumulate in the G2 Phase of cell cycle. AzaC treatment led to: significant decreases in CgA and NSE, indicating that AzaC inhibits neuroendocrine markers; and significant increases in the levels of Cyclin B1, further supporting the flow cytometric data and conclusion that AzaC induces G2/M arrest. The data indicate that AzaC suppresses cell growth in three different carcinoid types, reduces neuroendocrine markers in CNDT2.5 cells, and inhibits cell proliferation by inducing G2/M phase arrest. The results suggest that DNMTIs may be a novel class of therapeutic agents that can effectively control tumor growth and the release of bioactive peptides in patients with NETs.
authors.affiliation: Endocrine Surgery Research Laboratory, Department of Surgery, University of Wisconsin Madison, Wisconsin, USA.
authors.forename: Vinita M
authors.initials: VM
authors.lastname: Alexander
authors.forename: Madhuchhanda
authors.initials: M
authors.lastname: Roy
authors.forename: Kristen A
authors.initials: KA
authors.lastname: Steffens
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Kunnimalaiyaan
authors.forename: Herbert
authors.initials: H
authors.lastname: Chen