Advisor(s)
J Bart Rose
Committee Member(s)
Karin Hardiman
Lyse Norian
Document Type
Thesis
Date of Award
1-1-2025
Degree Name by School
Master of Science (MS) College of Arts and Sciences
Abstract
INVESTIGATING THE ROLE OF NOTCH1 ON CELL VIABILITY IN PANCREATIC NEUROENDOCRINE TUMOR CELLS IN HYPOXIA ALEXIS KENNEDY MULTIDISCIPLINARY BIOMEDICAL SCIENCE ABSTRACT Pancreatic neuroendocrine tumors (PNETs) are rare neoplasms originating from neuroendocrine cells in the pancreas. Patients with advanced disease pose a complex challenge due to poor prognosis and limited treatment options. Notch signaling is a juxtracrine cell-cell communication mechanism whereby transmembrane Notch receptors (Notch 1-4) interact with neighboring cells through ligands binding, resulting in a cascade that regulates various developmental processes and cell fate decisions. Dysregulated Notch1 signaling is implicated in PNETs, affecting neuroendocrine tumor biology and potentially interacting with Hypoxia Inducible Factor (HIF) pathways in disease progression. We hypothesized Notch1 in PNET cells provides a survival advantage in hypoxia. The PNET cell line BON-1 and derivative Notch1 Knockout (N1KO) cells were grown under normoxic (21% O2) or hypoxic conditions (3% O2). N1KO was achieved using CRISPR-Cas9. Cellular viability was measured with MTT and CellTiter-Glo (CTG) viability assays, and apoptosis was analyzed by flow cytometry with Annexin V/7-AAD staining. HIF-2α expression was determined by immunofluorescence and immunohistochemistry after hypoxia. Statistical analysis was performed using t-tests in Microsoft Excel. After 48 hours of hypoxia exposure, CTG assay showed BON-1 cells maintained 84% +/- 10% viability, while N1KO cells showed 73% +/- 14% (p=0.04). In a 48h MTT assay, BON-1 cells exhibited increased viability of 110% +/- 5%, while N1KO viability dropped to 80% +/- 9% (p<0.001). Both lines had reduced viability at 72 hours, with a greater reduction in N1KO cells (BON-1: 52% +/- 14%, N1KO: 37% +/- 11%, p < 0.05). Flow cytometry revealed increased apoptosis and necrosis in both cell lines under hypoxia, with BON-1 showing higher survival than N1KO. Immunohistochemistry revealed increased nuclear expression of HIF-2α and HIF-1α in N1KO cells under normoxia, which was further confirmed by immunofluorescence. These findings demonstrate Notch1’s role in regulating cellular responses to hypoxia, influencing viability, apoptosis, and necrosis. The differential responses of BON-1 and N1KO cells suggest Notch1 promotes PNET cell survival under low oxygen and balances apoptosis and necrosis. Future studies should explore how Notch1 interacts with cell survival and metabolism regulators, potentially guiding therapeutic strategies for patients with PNETs.
Recommended Citation
Kennedy, Alexis, "Investigating The Role Of Notch1 On Cell Viability In Pancreatic Neuroendocrine Tumor Cells In Hypoxia" (2025). All ETDs from UAB. 6855.
https://digitalcommons.library.uab.edu/etd-collection/6855
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