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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

Journal of Cancer Research and Clinical Oncology volume 134, pages 803–812 (2008)Cite this article

Abstract

Purpose

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K2 (VK2; menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK2.

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.

Results and conclusions

We found that PA-1 cells were the most sensitive to VK2 (IC50 = 5.0 ± 0.7 μM), while SK-OV-3 cells were resistant to VK2. Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 μM VK2. In the VK2-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 VK2-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 VK2 and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

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Abbreviations

BSA:

Bovine serum albumin

CHX:

Cycloheximide

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HM:

Heavy membrane

JNK:

c-Jun N-terminal kinase

NGFI-B:

Nerve growth factor induced clone B

OX:

Cytochrome oxidase subunit IV

PBS:

Phosphate-buffered saline

PMSF:

Phenylmethylsulfonyl fluoride

ROS:

Reactive oxygen species

siRNA:

Small interfering RNA

TBS:

Tris-buffered saline

TPA:

Tetradecanoylphorbol-1,3-acetate

VK2 :

Vitamin K2

XTT:

Sodium-3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate

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Affiliations

  1. Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan

    Toshiko Sibayama-Imazu, Yukari Fujisawa, Shigeo Nakajo, Hiroyuki Itabe & Kazuyasu Nakaya

  2. Department of Molecular Physiopathology, Showa Pharmaceutical University, Tokyo, Japan

    Yutaka Masuda

  3. Analytical Center, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan

    Toshihiro Aiuchi

Authors
  1. Toshiko Sibayama-Imazu
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  2. Yukari Fujisawa
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  3. Yutaka Masuda
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  4. Toshihiro Aiuchi
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  5. Shigeo Nakajo
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  6. Hiroyuki Itabe
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  7. Kazuyasu Nakaya
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Corresponding author

Correspondence to Hiroyuki Itabe.

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Sibayama-Imazu, T., Fujisawa, Y., Masuda, Y. et al. 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. J Cancer Res Clin Oncol 134, 803–812 (2008). https://doi.org/10.1007/s00432-007-0349-z

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  • DOI: https://doi.org/10.1007/s00432-007-0349-z

Keywords

  • Apoptosis
  • Vitamin K2
  • TR3
  • JNK
  • Ovarian cancer cells