Geminin is overexpressed in human pancreatic cancer and downregulated by the bioflavanoid apigenin in pancreatic cancer cell lines.
Pancreatic adeniocarcinoma is among the deadliest of human cancers. Apigenin, an antitumor flavonoid, inhibits pancreatic cancer cell proliferation in vitro. Geminin is a recently identified novel protein that plays a critical role in preventing abnormal DNA replication by binding to and inhibiting the essential replication factor Cdt1. Microarray analysis identified geminin to be downregulated in pancreatic cancer cells treated with apigenin. Therefore, we investigated the effects of apigenin on geminin expression and other proteins involved in replication (Cdc6, Cdt1, and MCM7) in pancreatic cancer cell lines CD18 and S2013. Real time RT-PCR and western blotting analysis showed that geminin expression is downregulated by apigenin at both mRNA and protein levels. Furthermore, treatment of cells with proteosome inhibitor MG132 reversed the downregulation of geminin by apigenin, supporting our hypothesis that the degradation pathway is another mechanism by which apigenin affects geminin expression. Apigenin treatment also resulted in downregulation of Cdc6 at both mRNA and protein levels. However, Cdt1 and MCM7 expression was not affected in apigenin-treated cells. The effect of apigenin treatment on geminin promoter activity was measured by transient transfection of Hela cells with a reporter gene, demonstrating that apigenin inhibited geminin promoter activity. Geminin expression was also evaluated in human pancreatic tissue (n = 15) by immunohistochemistry and showed that geminin is overexpressed in human pancreatic cancer compared to normal adjacent pancreatic tissue. In conclusion, our studies demonstrated that geminin is overexpressed in human pancreatic cancer and downregulated by apigenin which may contribute to the antitumor effect of this natural flavonoid.
Salabat
Mohammad R
MR
Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Melstrom
Laleh G
LG
Strouch
Matthew J
MJ
Ding
Xian-Zhong
XZ
Milam
Benjamin M
BM
Ujiki
Michael B
MB
Chen
Catherine
C
Pelling
Jill C
JC
Rao
Sambasiva
S
Grippo
Paul J
PJ
McGarry
Thomas J
TJ
Bentrem
David J
DJ
D047310:Apigenin / Q000494:pharmacology*; D001688:Biological Products / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects*; D064248:Geminin; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000235:genetics
D001688:Biological Products; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D007976:Leupeptins; D009687:Nuclear Proteins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D047310:Apigenin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde