The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents.
Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (-85 to -50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex-interactive agents, TMPyP4 and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design.
Sun
Daekyu
D
BIO5 Institute, Room 102, 1657 East Helen Street, Tucson, AZ 85721, USA. sun@pharmacy.arizona.edu
Liu
Wei-Jun
WJ
Guo
Kexiao
K
Rusche
Jadrian J
JJ
Ebbinghaus
Scot
S
Gokhale
Vijay
V
Hurley
Laurence H
LH
D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D015687:Cell Hypoxia; D002942:Circular Dichroism; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D016889:Endometrial Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D005260:Female; D054856:G-Quadruplexes / Q000187:drug effects*; D006801:Humans; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011166:Porphyrins / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018036:Selenium Compounds / Q000494:pharmacology*; D012689:Sequence Homology, Nucleic Acid; D013463:Sulfuric Acid Esters / Q000378:metabolism; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics*
C499730:5,10,15,20-(tetra(N-methyl-3-pyridyl))-26,28-diselenasapphyrin chloride; D011166:Porphyrins; D012333:RNA, Messenger; D018036:Selenium Compounds; D013463:Sulfuric Acid Esters; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C021096:tetra(4-N-methylpyridyl)porphine; D011189:Potassium Chloride; C007482:dimethyl sulfate