The VEGF quadruplex-forming sequence inhibits lung cancer cell growth.

VEGF;Lung cancer;Quadruplex;Cancer;Oligonucleotide

Edward David Muench, 1987-

University:University of Louisville

Department:Bioengineering

关键词: VEGF;    Lung cancer;    Quadruplex;    Cancer;    Oligonucleotide;   

Others  :  https://ir.library.louisville.edu/cgi/viewcontent.cgi?article=2021&context=etd

美国|英语

来源: The Universite of Louisville's Institutional Repository

【 摘 要 】

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