Daekyu Sun

Daekyu Sun, PhD, believes that angiogenesis, the formation of new blood vessels, promotes tumor growth by providing oxygen and nutrients to proliferating cancerous cells. The switch to an angiogenic phenotype in cancer cells is often mediated by increased expression of the vascular endothelial growth factor (VEGF), which is a pluripotent cytokinine and angiogenic growth factor and is often transcriptionally activated by the transcription factor HIF-1 under hypoxic condition. Some of Dr. Sun’s early work has shown that the G-rich and C-rich strands could form specific G-quadruplex or i-motif structures, respectively, on the polypurine/polypyrimidine tract in the proximal promoter of these genes. That observation led his lab to explore a new therapeutic strategy to repress the transcriptional activation of the human VEGF and HIF-1ï gene with small molecules capable of binding selectively to non-canonical DNA structures formed within the promoter region of these genes.
Another aspect of his work focuses on understanding the chemical and biological mechanisms of the antineoplastic, DNA-damaging natural product leinamycin, in collaboration with Dr. Kent Gates (University of Missouri). Together, they proposed that the unusual DNA-damaging properties of leinamycin would represent a new biochemical route to potent anticancer activity. Thus, the experiments planned in this research are designed to relate leinamycin’s chemical and biochemical properties to its unique biological activity.
Other aspects of Dr. Sun’s research focus on understanding inducible resistant mechanisms in human cancer cells to DNA-damaging anticancer drugs, because the acquisition of chemoresistance toward chemotherapy in cancer cells remains one of the principal obstacles to the effective treatment of malignancies. Some early work showed that DNA ligase I levels are elevated after exposure to a variety of chemotherapeutic drugs, including cisplatin, ara-C, gemcitabine, and topotecan in various human cancer cells. Here the goal is to elucidate the molecular basis and the biological significance of induction of DNA repair genes in human cancer cells in response to major chemotherapeutics in order to identify potential targets appropriate for new anticancer agents that will enhance the lethal activity of many cancer chemotherapeutics.
Dna Cleavage Induced By Antitumor Antibiotic Leinamycin And Its Biological Consequences. Source: Bioorganic & Medicinal Chemistry
May 23rd, 2012 PMID: 22682923 Daekyu Sun
The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.<br /><br />
Heterogeneous Nuclear Ribonucleoprotein K And Nucleolin As Transcriptional Activators Of The Vascular Endothelial Growth Factor Promoter Through Interaction With Secondary Dna Structures. Source: Biochemistry
April 15th, 2011 PMID: 21466159 Daekyu Sun
The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single-stranded DNA, and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene.<br /><br />
Evidence Of The Formation Of G Quadruplex Structures In The Promoter Region Of The Human Vascular Endothelial Growth Factor Gene. Source: Nucleic Acids Research
October 18th, 2010 PMID: 20959293 Daekyu Sun
The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.<br /><br />
Characterization Of Dna Damage Induced By A Natural Product Antitumor Antibiotic Leinamycin In Human Cancer Cells. Source: Chemical Research In Toxicology
January 18th, 2010 PMID: 20017514 Daekyu Sun
Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC(50) values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.<br /><br />
Biochemical Techniques For The Characterization Of G Quadruplex Structures: Emsa, Dms Footprinting, And Dna Polymerase Stop Assay. Source: Methods In Molecular Biology (Clifton, N.J.)
December 16th, 2009 PMID: 20012416 Laurence Hurley Daekyu Sun
The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.<br><br>
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