Penta-O-galloyl-beta-D-glucose inhibits the invasion of mouse melanoma by suppressing metalloproteinase-9 through down-regulation of activator protein-1.
Penta-O-galloyl-beta-D-glucose (5GG) inhibited the invasion of highly metastatic mouse melanoma B16F10 cells in vitro, as demonstrated by transwell assay. Its ability to diminish the activity of matrix metalloproteinase (MMP) was demonstrated by zymographic assay. Our data showed 5GG could diminish the activity of MMP-9 more than that of MMP-2. The effect on MMP-9 was elicited in a dose- and time-dependent manner, with IC50 of 15 microM. Next, we analyzed the amounts of MMP-9 and MMP-2 protein in conditioned media and in the cells. The data indicated MMP-9 proteins were also suppressed by 5GG in the same manner. In accordance with these data above, the results of reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis showed a reduced level of MMP-9 mRNA. Furthermore, we studied transcription factor binding to MMP-2 and MMP-9 promoter regions by electrophoretic mobility shift assay (EMSA) in the nucleus. The results suggested that the transcription factor binding activities of Activator protein-1 (AP-1) and Sp-1 sites was mainly down-regulated by 5GG in the concentration range of 5-15 microM, but not that of nuclear factor kappaB (NF-kappaB), polioma enhancer activator 3 (PEA-3), and Activator protein-2 (AP-2) sites. The Western blot analysis of AP-1 nuclear protein showed a reduced level of c-Jun but not of c-Fos. In addition, the expression of Sp-1 and c-Jun protein was also suppressed. To elucidate whether the transcriptional activity of AP-1 or Sp-1 sites is more important, we transfected MMP-9/luciferase reporter vector, under MMP-9 promoter control, into the cells. We found that a decreased transcriptional activity of AP-1 sites is sufficient to reduce MMP-9 promoter activity. These results lead us to conclude that 5GG restricts the invasive ability of B16F10 mouse melanoma cells by reducing MMP-9 activity, by suppressing the transcriptional activity of AP-1 sites and the expression of c-Jun protein. The result may provide a potential mechanism for 5GG in cancer chemopreventive action.
Ho
Li-Lun
LL
Institute of Biochemistry, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan
Chen
Wei-Jen
WJ
Lin-Shiau
Shoei-Yn
SY
Lin
Jen-Kun
JK
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D047348:Hydrolyzable Tannins; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009361:Neoplasm Invasiveness; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013634:Tannins / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000096:biosynthesis* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D047348:Hydrolyzable Tannins; D061965:Matrix Metalloproteinase Inhibitors; D012333:RNA, Messenger; D013634:Tannins; D018808:Transcription Factor AP-1; C435084:pentagalloylglucose; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9