A novel mechanism for the inhibition of hyaluronan biosynthesis by 4-methylumbelliferone.
Specific inhibitors of hyaluronan (HA) biosynthesis can be valuable therapeutic agents to prevent cancer invasion and metastasis. We have found previously that 4-methylumbelliferone (MU) inhibits HA synthesis in human skin fibroblasts and in group C Streptococcus. In this paper, the inhibition mechanism in mammalian cells was investigated using rat 3Y1 fibroblasts stably expressing HA synthase (HAS) 2. Exposure of the transfectants to the inhibitor resulted in significant reduction of HA biosynthesis and matrix formation. The evaluation of HAS transcripts and analysis of cell-free HA synthesis demonstrated the post-transcriptional suppression of HAS activity by MU. Most interesting, the post-transcriptional suppression of HAS activity was also observed using p-nitrophenol, a well known substrate for UDP-glucuronyltransferases (UGT). We investigated whether the inhibition was exerted by the glucuronidation of MU using both high pressure liquid chromatography and TLC analyses. The production of MU-glucuronic acid (GlcUA) was consistent with the inhibition of HA synthesis in HAS transfectants. MU-GlcUA was also detected at a similar level in control cells, suggesting that the glucuronidation was mediated by an endogenous UGT. Elevated levels of UGT significantly enhanced the inhibitory effects of MU. In contrast, the inhibition by MU was diminished to the control level when an excess of UDP-GlcUA was added to the cell-free HA synthesis system. We propose a novel mechanism for the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UGT resulting in a depletion of UDP-GlcUA.
Kakizaki
Ikuko
I
Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Kojima
Kaoru
K
Takagaki
Keiichi
K
Endo
Masahiko
M
Kannagi
Reiji
R
Ito
Masaki
M
Maruo
Yoshihiro
Y
Sato
Hiroshi
H
Yasuda
Tadashi
T
Mita
Satoka
S
Kimata
Koji
K
Itano
Naoki
N
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D006923:Hymecromone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014166:Transferases / Q000235:genetics
D004791:Enzyme Inhibitors; D020719:Glucuronides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006923:Hymecromone; D006820:Hyaluronic Acid; D014166:Transferases; D014453:Glucuronosyltransferase; C491097:Has2 protein, rat; D000076002:Hyaluronan Synthases