Effect of DNA damage response by quinazolinone analogue HMJ-38 on human umbilical vein endothelial cells: evidence for γH2A.X and DNA-PK-dependent pathway.
The present study aims to explore the mechanism of quinazolinone analogue HMJ-38-induced DNA damage in endothelial cells in vitro. We attempt to evaluate the antiangiogenetic response utilizing human umbilical vein endothelial cells (HUVECs). Herein, the results demonstrated that HMJ-38 incubation triggered DNA damage behavior and showed a longer DNA migration in HUVECs based on the comet assay and the analysis of DNA agarose gel electrophoresis to contact DNA smears. We further gained to determine a marker of DNA double strand breaks, phosphorylated histone H2A.X (Ser139) (γH2A.X), in HMJ-38-treated HUVECs by flow cytometry and Western blotting assay. We consider that HMJ-38 has caused an increase in γH2A.X, and DNA damage seemed to mediate through DNA-dependent serine/threonine protein kinase (DNA-PK) binding to Ku70/Ku80 as well as advanced activated p-Akt (Ser473) and stimulated phosphorylated glycogen synthase kinase-3β (p-GSK-3β) conditions in HUVECs. Importantly, the effect of above DNA damage response was prevented by N-acetyl-l-cysteine (a reactive oxygen species scavenger), and NU7026 (a DNA-PK inhibitor) could attenuate DNA-PK catalytic subunit and phosphorylation of H2A.X on Ser139 expression in comparison with HMJ-38 alone treated HUVECs. Therefore, HMJ-38-provoked DNA damage stress in HUVECs probably led to the activation of γH2A.X/DNA-PK/GSK-3β signaling. In summary, our novel finding provides more information addressing the pharmacological approach of newly synthesized HMJ-38 for further development and therapeutic application in antiangiogenetic effect of cancer chemotherapy.
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
J-H
JH
Chiang
Department of Pharmacology, China Medical University, Taichung, Taiwan.
J-S
JS
Yang
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
C-C
CC
Lu
School of Pharmacy, China Medical University, Taichung, Taiwan.
M-J
MJ
Hour
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.
K-C
KC
Liu
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.
J-H
JH
Lin
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.
T-H
TH
Lee
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Department of Biotechnology, Asia University, Taichung, Taiwan jgchung@mail.cmu.edu.tw.
J-G
JG
Chung
D020533:Angiogenesis Inhibitors / Q000633:toxicity*; D034961:Antigens, Nuclear / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000187:drug effects; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006657:Histones / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011759:Pyrrolidines / Q000633:toxicity*; D052999:Quinazolinones / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
C416653:2-(3'-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone; D020533:Angiogenesis Inhibitors; D034961:Antigens, Nuclear; D000975:Antioxidants; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011759:Pyrrolidines; D052999:Quinazolinones; D017382:Reactive Oxygen Species; D051747:DNA-Activated Protein Kinase; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C497263:PRKDC protein, human; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen
DNA damage; DNA-PK; HMJ-38; human umbilical vein endothelial cells; γH2A.X