Date on Master's Thesis/Doctoral Dissertation

4-2012

Document Type

Master's Thesis

Degree Name

M. Eng.

Department

Bioengineering

Committee Chair

Miller, Donald M.

Author's Keywords

VEGF; Lung cancer; Quadruplex; Cancer; Oligonucleotide

Subject

Lungs--Cancer--Genetic aspects; Oligonucleotides

Abstract

Vascular endothelial growth factor (VEGF), a commonly overexpressed oncogene in a variety of malignancies including non-small cell lung cancer (NSCLC), is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (fourstranded) DNA. The specific quadruplex forming sequence of interest is the 20 base pair polyG/polyC tract that codes for the VEGF promoter (VEGFq), which is located in the proximal promoter region upstream of the transcription initiation site. This oligonucleotide has been shown to have significant growth hindering effects when introduced in the NSCLC cell line, A549. To determine the biological role of VEGFq on non-small cell lung cancer in vitro, cells were treated with either VEGFq or the corresponding mutant sequence (MutVEGF), which lacks the runs of guanines necessary for quadruplex formation. Circular dichroism spectroscopy confirmed that VEGFq formed a parallel quadruplex, while the MutVEGF sequence did not form a quadruplex structure. It is hypothesized that the considerable growth inhibition is caused by hindering the VEGF signaling pathway. The VEGF signaling cascade, which is normally triggered by VEGF binding to FLK1 (VEGF Receptor 2), is unable to function adequately due to decreased VEGF protein levels. This causes a disruption in the phosphorylation of target proteins including ERK1/2 and AKT/PKB, which in turn decreases overall cell proliferation. This observed decline in overall cell proliferation corresponded to decreased levels of VEGF protein expression, indicating that there is a direct correlation between the treatment and the changes in proliferation. A single treatment of A549 NSCLC cells with VEGFq caused a significant dose and time-dependent decrease in cell viability after 3 and 6 days as determined by MTT assay. Parallel treatment of nontransformed human fibroblast cells with VEGFq showed no changes in growth, demonstrating the cancer specificity of VEGFq. Cell cycle analysis showed no changes in cell phases at 24, 48, 72 and 96 hours, indicating that VEGFq's effects due to a mechanism other than cell cycle arrest. Confocal microscopy and flow cytometry after 72 hours showed significant uptake and nuclear localization of VEGFq, but not MutVEGF. Boyden chamber invasion/migration measurement shows that the VEGFq treated cells have decreased cell movement, indicative of possible antiangiogenic effects as well. To determine the biological role of VEGFq on non-small cell lung cancer in vivo, A549 cells were injected into nude mice and grown for 10 days prior to daily IP injections of 10 mg/kg VEGFq or the control vehicle for 14 days. Tumor progression was physically measured using calipers three times a week. Fluorescent imaging was used to detect the presence, stability, and distribution of Alexa Fluor 750-labeled VEGFq after injection into the mouse to ensure localization in the targeted tumor. These results demonstrate that A549 cells treated with VEGF quadruplex-forming oligonucleotides experience a dramatic decrease in cell proliferation, suggesting that VEGFq may have significant therapeutic implications for the treatment of non-small cell lung cancer.

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