Dual action of a selective cyclooxygenase-2 inhibitor on vascular endothelial growth factor expression in human hepatocellular carcinoma cells: novel involvement of discoidin domain receptor 2.
OBJECTIVE
Vascular endothelial growth factor (VEGF) greatly contributes to the progression of hepatocellular carcinoma (HCC). It is reported that a selective cyclooxygenase-2 (COX-2) inhibitor inhibits cellular proliferation and may attenuate VEGF expression in HCC. We propose that different cascades in the VEGF pathway respond to COX-2 inhibition, depending on the cell types.


METHODS
The six human HCC cell lines--Hep3B, SNU387, SNU182, SNU423, SNU449, and PLC/PRF5--were cultured under normoxic and hypoxic conditions. Cells were treated with a selective COX-2 inhibitor (NS-398) and discoidin domain receptor 2 (DDR2) siRNA, and microarray analysis was performed.


RESULTS
NS-398 inhibited HCC proliferation and decreased the expression level of VEGF in HCC cells only under normoxia conditions. In hypoxia conditions, VEGF expression level in Hep3B cell was suppressed, while that in SNU387 cell was increased by NS-398 (P < 0.001). The NS-398-induced increase in VEGF expression in SNU387 cell was associated with the up-regulation of the DDR2 gene. NS-398-treated SNU series cells and PLC/PRF5 cells displayed a robust increase in DDR2 mRNA expression. Also, transfection with DDR2 siRNA decreased the VEGF expression level of SNU387, 423, 449 cells under hypoxia conditions (P < 0.05). In vivo chromatin immunoprecipitation assay demonstrated that NS-398 induces the enhancement of HIF-1α binding on VEGF promoter, leading to the increase in VEGF gene expression in hypoxic conditions. There is strong evidence that it is related to the DDR2 gene expression in SNU387 cells.


CONCLUSIONS
These findings disclose a novel cell-dependent regulatory mechanism of VEGF involving DDR2 gene in HCC cells.
Liver and Pancreatobiliary Cancer Research Branch, National Cancer Center, Goyang, South Korea.
Nam Oak
NO
Lee
Joong-Won
JW
Park
Jung Ahn
JA
Lee
Ju Hyun
JH
Shim
Sun-Young
SY
Kong
Kyung Tae
KT
Kim
Yeon-Su
YS
Lee
D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D048708:Cell Growth Processes / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D000070818:Discoidin Domain Receptors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009578:Nitrobenzenes / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology*; D014162:Transfection; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D052246:Cyclooxygenase 2 Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009578:Nitrobenzenes; D034741:RNA, Small Interfering; D011975:Receptors, Mitogen; D013449:Sulfonamides; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases