Menatetrenone, a vitamin K2 analogue, inhibits hepatocellular carcinoma cell growth by suppressing cyclin D1 expression through inhibition of nuclear factor kappaB activation.
OBJECTIVE
Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2-induced growth inhibition of HCC cells.


METHODS
HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor kappaB (NF-kappaB) activation was investigated by a NF-kappaB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IkappaB, and an in vitro kinase assay for IkappaB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1.


RESULTS
Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-kappaB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)-, TNF-alpha-, and interleukin (IL)-1-induced activation of NF-kappaB binding and transactivation. Concomitant with the suppression of NF-kappaB activation, vitamin K2 also inhibited the phosphorylation and degradation of IkappaBalpha and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment.


CONCLUSIONS
Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IkappaB/NF-kappaB pathway and might therefore be useful for treatment of HCC.
Ozaki
Iwata
I
Division of Hepatology and Metabolism, Department of Internal Medicine, and Health Administration Center, Saga Medical School, Saga University, Nabeshima, Saga, Japan. ozaki@cc.saga-u.ac.jp
Zhang
Hao
H
Mizuta
Toshihiko
T
Ide
Yasushi
Y
Eguchi
Yuichiro
Y
Yasutake
Tsutomu
T
Sakamaki
Toshiyuki
T
Pestell
Richard G
RG
Yamamoto
Kyosuke
K
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D019938:Cyclin D1 / Q000096:biosynthesis / Q000187:drug effects*; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D051550:I-kappa B Kinase / Q000187:drug effects / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D016328:NF-kappa B / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D024482:Vitamin K 2 / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; D016328:NF-kappa B; D024482:Vitamin K 2; D019938:Cyclin D1; C030814:menatetrenone; D051550:I-kappa B Kinase