ECE2011 Poster Presentations Endocrine tumours and neoplasia (37 abstracts)
The demethylating agent 5-aza-2-deoxycytidine upregulates somatostatin type 2 receptor expression and enhances internalization of radiolabeled somatostatin analogue in human carcinoid tumour cells
M J Veenstra 1 , P M van Koetsveld 1 , W E Farrel 2 , F Dogan 1 , A M Waaijers 1 , D M Sprij-Mooij 1 , S W J Lamberts 1 , G Vitale 3, & L Hofland 1
1Division of Endocrinology, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands; 2Department Human Disease and Genomics Group, Institute of Science and Technology in Medicine, School of Medicine, Keele University, Stoke on Trent, Staffordshire ST4 7QB, UK; 3Department of Medical Sciences, University of Milan, Milan, Italy; 4IRCCS Istituto Auxologico Italiano, Milan, Italy.
Introduction: Neuroendocrine tumors (NET) are rare tumors originating from various types of neuroendocrine cells. The somatostatin receptor type 2 (sst2) is expressed in NET cells and is a target for therapy with somatostatin analogues.
Epigenetic changes including methylation of DNA at CpG dinucleotides, and particularly in the promoter region of genes can inhibit transcription. Methylation can be inhibited by cytidine analogues such as 5-aza-2-deoxycytidine (AZA). Since hypermethylation of the sst2 gene has been shown to influence sst2 expression we hypothesize that upregulation of sst2 expression by AZA would lead to increased uptake of [125I-Tyr3]octreotide.
Materials and methods: Cells of the human pancreatic carcinoid cell line BON were cultured 7 days with increasing doses of AZA to determine the effect on cell proliferation. To determine the effect of AZA on uptake of [125I-Tyr3]octreotide, cells were cultured 6 days at 3 conditions: untreated control and AZA 50 and 100 nM. Thereafter, the cells were plated and cultured without or with AZA for 2 days. Subsequently, an internalization experiment was performed using [125I-Tyr3]octreotide. Cells were subsequently collected for RT-qPCR of sst2. To determine the methylation status of the CpG island, methylation specific PCR and subsequent sequencing was performed on bisulfite treated DNA isolated from untreated cells.
Results: AZA induced a dose-dependent inhibition of cell proliferation with an IC50 value of 80 nM. AZA, at 50 and 100 nM, induced a 2- and 2.5-fold increased uptake of [125I-Tyr3]octreotide. RT-qPCR results showed a dose-dependent increase of sst2 mRNA expression. Partial CpG methylation of the sst2 gene was found in the CpG island, downstream of the transcription start site.
Discussion and conclusion: Measurement of internalized [125I-Tyr3]octreotide revealed increased dose dependent uptake after treatment with AZA, likely due to upregulation of sst2 expression. This indicates that either the region of the CpG island we determined to be methylated influences expression or there is an indirect mechanism causing upregulation of sst2.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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