Induction of apoptosis in PA-1 ovarian cancer cells by vitamin K2 is associated with an increase in the level of TR3/Nur77 and its accumulation in mitochondria and nuclei.
OBJECTIVE
We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2).


METHODS
Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment.


CONCLUSIONS
We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.
Sibayama-Imazu
Toshiko
T
Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan.
Fujisawa
Yukari
Y
Masuda
Yutaka
Y
Aiuchi
Toshihiro
T
Nakajo
Shigeo
S
Itabe
Hiroyuki
H
Nakaya
Kazuyasu
K
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering; D011987:Receptors, Steroid / Q000096:biosynthesis*; D011988:Receptors, Thyroid Hormone / Q000096:biosynthesis*; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation; D024482:Vitamin K 2 / Q000494:pharmacology*
C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D024482:Vitamin K 2