ABSTRACT
Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZαADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII1 region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZαADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZαADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZαADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZαADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZαADAR1 suppresses the c-myc expression promoted by NHEIII1 region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.
Subject(s)
Adenosine Deaminase/chemistry , Adenosine Deaminase/metabolism , DNA, Z-Form/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Adenosine Deaminase/genetics , Amino Acid Sequence , Binding Sites , Circular Dichroism , G-Quadruplexes , Gene Expression Regulation , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Mutation , Protein Conformation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/metabolism , RNA-Binding ProteinsABSTRACT
Poly(ADP-ribose)polymerase-1 (PARP-1) enzyme is involved in the repair of DNA damages made by certain anticancer agents. It is suggested that PARP-1 inhibitors potentiate the cytotoxic effects and circumvent the resistance of DNA-modifying anticancer agents such as cisplatin. In this study, we conducted virtual screening of Korea Chemical Bank database targeting PARP-1 and identified several potent PARP-1 inhibitors with submicromolar IC50 values (77-79 nM). We then examined the chemosensitization of cisplatin by pre-treatment of PARP-1 inhibitors in cisplatin-resistant human gastric cancer cells. Our results show that PARP-1 inhibitors suppress the formation of poly(ADP-ribose) and enhance the cytotoxicity of cisplatin.
Subject(s)
Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Binding Sites , Catalytic Domain , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/therapeutic use , Databases, Factual , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Humans , Molecular Docking Simulation , Poly(ADP-ribose) Polymerases/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/enzymologyABSTRACT
The major obstacle of treating cancer patients is acquisition of chemoresistance, in which treated tumor cells become insensitive after chronic drug exposure. To study the mechanism of acquired cisplatin resistance, we established a cisplatin-resistant human gastric cancer cell line. The cisplatin-resistant cell line (YCC-3/R) was isolated after exposing the gastric cancer cell line, YCC-3, to a constant concentration (0.5 microg/mL) of cisplatin for 12 months. The expression of cell cycle regulatory proteins (p53, Bax, p21, p27) in the YCC-3/R were investigated by western blot analysis. The cisplatin treatment significantly down-regulated the p53 and p21 expression level, while up-regulated the p27 expression in the YCC-3/R cells compared to the parental cells. The Bax expression level was similar in both cells. These results suggest that the p27 dependent-cell cycle arrest may prevent cisplatin-induced apoptosis and give enough time to repair the DNA damage in the YCC-3/R cells.
Subject(s)
Cisplatin/pharmacology , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Drug Resistance, Neoplasm/physiology , Gene Expression Regulation, Neoplastic , Stomach Neoplasms/enzymology , Up-Regulation , Cell Line, Tumor , Cisplatin/therapeutic use , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Stomach Neoplasms/drug therapy , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
In an effort to develop dual PPARalpha/gamma activators with improved therapeutic efficacy, a series of diaryl alpha-ethoxy propanoic acid compounds comprising two aryl groups linked by rigid oxime ether or isoxazoline ring were designed and synthesized and their biological activities were examined. Most of the compounds possessing an oxime ether linker were more potent PPARgamma activators than the lead PPARalpha/gamma dual agonist, tesaglitazar in vitro. Compound 18, one of the derivatives with an oxime ether linker, was found to selectively transactivate PPARgamma (EC 50 = 0.028 microM) over PPARalpha (EC 50 = 7.22 microM) in vitro and lower blood glucose in db/ db mice more than muraglitazar after oral treatment for 11 days.
