T. V. Do1, A. Dammalapati1, A. Hundal1, H. Jin1, R. Jaskula-Sztul1, H. Chen1 1Department Of Surgery,Madison, WI, USA
Introduction: Notch signaling involves in various aspects of mammalian biology such as cellular differentiation, cell-cycle regulation, metabolism and apoptosis. Among the four identified mammalian Notch receptors, the role of Notch1 and 3 as tumor suppressor in neuroendocrine tumor cells (NET) have been elucidated. Nevertheless, the function of Notch2 signaling still remains unclear in NETs. The aim of this study was to access the role of Notch2 in gastrointestinal (GI)NETs (carcinoids).
Methods: pcDNA4/V5-His plasmid ,containing constitutively expressed human active portion of Notch2 (NICD2), was transiently transfected into GI carcinoid (BON) cells. The same plasmid without NICD2 was used as a control. Transfection efficiency was assessed by co- transfection with plasmid expressing the green fluorescent protein (GFP). The expression of NICD2 was confirmed by quantitative RT-PCR and Western Blot. Next the functional activity of NICD2 was analyzed by measuring the degree of CBF-1 binding by luciferase reporter assay. Cell viability was then tested by MTT (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide) assay after 24, 48, 72 and 96 hours after transfection. To investigate the potential effects of NICD2 on BON cells proliferation, cell cycle and anti-apoptotic markers such as X-linked inhibitor of apoptosis (XIAP), cyclin D1, and c-Myc were examined by Western Blot analysis.
Results: The expression of NICD2 was detected over all time points of transfection on both mRNA and protein levels. CBF-1 binding assay showed increased luciferase activity indicating functional activation of NICD2. In contract to Notch1 and 3 which are tumor suppressive, NICD2 transfected cells did not reduce proliferation comparing to the cells transfected with control plasmid. Moreover, Western blot analysis showed an increase of anti- apoptotic markers XIAP, C-Myc, and Cyclin D1 with the overexpression of NICD2.
Conclusion: Carcinoid tumor cells have paucity Notch2. For the first time, we demonstrate the growth – promoting function of Notch2 receptor in carcinoid cancer with concomitant upturn of anti-apoptotic markers XIAP, c-Myc and cyclin D1. This opposing role of Notch2 isoform in NE cancer progression warrant further investigation.