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Title: Investigating the effect of sutent on the IMR32 and SK-N-SH NB cell lines
Authors: Younes, Maroun Abi
Advisors: Nasr, Zeina 
Subjects: Neuroblastoma
Dissertations, Academic
University of Balamand--Dissertations
Issue Date: 2019
Neuroblastoma (NB) is the most common extracranial solid tumor arising in the first years of life, and is responsible for 15% of all pediatric cancer-related deaths. Its manifestation in the body is along the vertical axis, more specifically from the neck to the pelvis. NB is due to many chromosomal alterations and genetic complications leading to activation of cell survival pathways. Some of these complications include the amplification of the MYCN oncogene and the misexpression of receptor tyrosine kinases (RTKs) such as PDGF and VEGF. MYCN is present in 18% of NB patients, and is exclusive to high risk disease patients. On the other hand, the VEGF and PDGF pathways govern cell survival, proliferation, migration and invasion. Sutent, the multitargeted RTK inhibitor, inhibits these pathways potently. It is a competitive inhibitor for the ligand-binding sites in the RTKs, and does not lead to their degradation. Our aim is to check the phenotypic changes in two NB cell lines, IMR-32 MYCN-amplified and SK-N-SH MYCN non-amplified, after sutent treatment, by monitoring proliferation, migration, apoptosis and protein expression. We performed the WST-1 assay to test for changes in the proliferation potential of our cells, the wound healing assay to test for changes in migration, the PI/Annexin-V assay and flow cytometry to test for varying levels of apoptosis, and western blot analysis to check for differential protein expression in the IMR32 cell line. Sutent was able to significantly decrease the proliferation rate of IMR32 cells at the 5 μM concentration, but only had a slight effect on the SK-N-SH cell line. Targeting the VEGF and PDGF pathways for inhibition decreased the migration potential for both cell lines almost equally at low and high concentrations: at 0.2 μM, IMR32 and SK-N-SH cells had a 3-fold decrease in migration potential, while at 1 μM the IMR-32 cells had a further decrease and the SK-N-SH cells migration ceased. Sutent caused a 5-fold increase in the rate of late apoptotic cells in the IMR-32 cell line, with no significant effect on the SK-N-SH cell line. Furthermore, in the IMR-32 cell line, the levels of HSP90 protein increased by almost 40% in response to inhibiting the PDGF and VEGF pathways. We conclude that this change in phenotypic response to sutent is probably due to the difference in protein profiles between the two cell lines. Both cell lines express the PDGF receptors, and unlike the IMR-32 cells, SK-N-SH cells express only VEGFR2 and not VEGFR1. This leads us to hypothesize that proliferation and apoptosis might be governed by the VEGF through VEGFR1 in the MYCN-amplified cell line. The increase in HSP90 expression is probably a mechanism of overcompensation to restore the function of its client proteins, that are in this case the inhibited RTKs. Future research must focus on testing the expression levels of other highly tumorigenic proteins in response to PDGF and VEGF pathway inhibition, as well as identifying which of these pathways, if not both, control migration in IMR-32 and SK-N-SH cell lines.
Includes bibliographical references (p. 45-59).
Rights: This object is protected by copyright, and is made available here for research and educational purposes. Permission to reuse, publish, or reproduce the object beyond the personal and educational use exceptions must be obtained from the copyright holder
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Type: Thesis
Appears in Collections:UOB Theses and Projects

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