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Original Article
Azacytidine Induces cell cycle arrest and suppression of
neuroendocrine markers in carcinoids
Vinita M. Alexander, Madhuchhanda Roy, Kristen A. Steffens, Muthusamy Kunnimalaiyaan, Herbert Chen
Endocrine Surgery Research Laboratory, Department of Surgery, University of Wisconsin, Madison, Wisconsin,
USA
Received February 16, 2010; accepted March, 2010; available online March, 2010
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. 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. AzaC suppresses cell growth in three different carcinoid types, reduces neuroendocrine markers,
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. (AJTR1002003).
Key words: Azacytidine, neuroendocrine tumors, chromogranina A, neuron specific enolase – G2/M arrest
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Address all correspondence to:
Herbert Chen, MD
H4/722 Clinical Science Center
600 Highland Avenue
Madison, WI 53792-7375
Tel: (608) 263-1387
Fax: (608) 263-7652
E-mail: chen@surgery.wisc.edu
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